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| <?xml version="1.0" encoding="UTF-8"?><!DOCTYPE article PUBLIC "-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD v1.1d1 20130915//EN" "JATS-archivearticle1.dtd"><article article-type="research-article" dtd-version="1.1d1" xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"><front><journal-meta><journal-id journal-id-type="nlm-ta">elife</journal-id><journal-id journal-id-type="hwp">eLife</journal-id><journal-id journal-id-type="publisher-id">eLife</journal-id><journal-title-group><journal-title>eLife</journal-title></journal-title-group><issn publication-format="electronic">2050-084X</issn><publisher><publisher-name>eLife Sciences Publications, Ltd</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="publisher-id">00857</article-id><article-id pub-id-type="doi">10.7554/eLife.00857</article-id><article-categories><subj-group subj-group-type="display-channel"><subject>Research article</subject></subj-group><subj-group subj-group-type="heading"><subject>Biophysics and structural biology</subject></subj-group><subj-group subj-group-type="heading"><subject>Human biology and medicine</subject></subj-group></article-categories><title-group><article-title>Structure-based discovery of fiber-binding compounds that reduce the cytotoxicity of amyloid beta</article-title></title-group><contrib-group><contrib contrib-type="author" equal-contrib="yes" id="author-5053"><name><surname>Jiang</surname><given-names>Lin</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="equal-contrib">†</xref><xref ref-type="other" rid="par-1"/><xref ref-type="other" rid="par-3"/><xref ref-type="fn" rid="con1"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" equal-contrib="yes" id="author-5069"><name><surname>Liu</surname><given-names>Cong</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="equal-contrib">†</xref><xref ref-type="other" rid="par-1"/><xref ref-type="fn" rid="con2"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-5070"><name><surname>Leibly</surname><given-names>David</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con3"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-5071"><name><surname>Landau</surname><given-names>Meytal</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="pa1">‡a</xref><xref ref-type="fn" rid="con4"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-5072"><name><surname>Zhao</surname><given-names>Minglei</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="pa2">‡b</xref><xref ref-type="fn" rid="con5"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-5073"><name><surname>Hughes</surname><given-names>Michael P</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con6"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" corresp="yes" id="author-5056"><name><surname>Eisenberg</surname><given-names>David S</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1">*</xref><xref ref-type="other" rid="par-1"/><xref ref-type="other" rid="par-2"/><xref ref-type="other" rid="par-3"/><xref ref-type="fn" rid="con7"/><xref ref-type="fn" rid="conf1"/></contrib><aff id="aff1"><institution content-type="dept">Departments of Chemistry and Biochemistry and Biological Chemistry</institution>, <institution>Howard Hughes Medical Institute, UCLA–DOE Institute for Genomics and Proteomics, University of California, Los Angeles</institution>, <addr-line><named-content content-type="city">Los Angeles</named-content></addr-line>, <country>United States</country></aff></contrib-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Kuriyan</surname><given-names>John</given-names></name><role>Reviewing editor</role><aff><institution>Howard Hughes Medical Institute, University of California, Berkeley</institution>, <country>United States</country></aff></contrib></contrib-group><author-notes><corresp id="cor1"><label>*</label>For correspondence: <email>david@mbi.ucla.edu</email></corresp><fn fn-type="con" id="equal-contrib"><label>†</label><p>These authors contributed equally to this work</p></fn><fn fn-type="present-address" id="pa1"><label>a</label><p>Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel</p></fn><fn fn-type="present-address" id="pa2"><label>b</label><p>Department of Molecular and Cellular Physiology, Stanford University, Stanford, United States</p></fn></author-notes><pub-date date-type="pub" publication-format="electronic"><day>16</day><month>07</month><year>2013</year></pub-date><pub-date pub-type="collection"><year>2013</year></pub-date><volume>2</volume><elocation-id>e00857</elocation-id><history><date date-type="received"><day>19</day><month>04</month><year>2013</year></date><date date-type="accepted"><day>10</day><month>06</month><year>2013</year></date></history><permissions><copyright-statement>© 2013, Jiang et al</copyright-statement><copyright-year>2013</copyright-year><copyright-holder>Jiang et al</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/3.0/"><license-p>This article is distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/3.0/">Creative Commons Attribution License</ext-link>, which permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><self-uri content-type="pdf" xlink:href="elife00857.pdf"/><related-article ext-link-type="doi" id="ra1" related-article-type="commentary" xlink:href="10.7554/eLife.01089"/><abstract><object-id pub-id-type="doi">10.7554/eLife.00857.001</object-id><p>Amyloid protein aggregates are associated with dozens of devastating diseases including Alzheimer’s, Parkinson’s, ALS, and diabetes type 2. While structure-based discovery of compounds has been effective in combating numerous infectious and metabolic diseases, ignorance of amyloid structure has hindered similar approaches to amyloid disease. Here we show that knowledge of the atomic structure of one of the adhesive, steric-zipper segments of the amyloid-beta (Aβ) protein of Alzheimer’s disease, when coupled with computational methods, identifies eight diverse but mainly flat compounds and three compound derivatives that reduce Aβ cytotoxicity against mammalian cells by up to 90%. Although these compounds bind to Aβ fibers, they do not reduce fiber formation of Aβ. Structure-activity relationship studies of the fiber-binding compounds and their derivatives suggest that compound binding increases fiber stability and decreases fiber toxicity, perhaps by shifting the equilibrium of Aβ from oligomers to fibers.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00857.001">http://dx.doi.org/10.7554/eLife.00857.001</ext-link></p></abstract><abstract abstract-type="executive-summary"><object-id pub-id-type="doi">10.7554/eLife.00857.002</object-id><title>eLife digest</title><p>Alzheimer’s disease is the most common form of dementia, estimated to affect roughly five million people in the United States, and its incidence is steadily increasing as the population ages. A pathological hallmark of Alzheimer’s disease is the presence in the brain of aggregates of two proteins: tangles of a protein called tau; and fibers and smaller units (oligomers) of a peptide called amyloid beta.</p><p>Many attempts have been made to screen libraries of natural and synthetic compounds to identify substances that might prevent the aggregation and toxicity of amyloid. Such studies revealed that polyphenols found in green tea and in the spice turmeric can inhibit the formation of amyloid fibrils. Moreover, a number of dyes reduce the toxic effects of amyloid on cells, although significant side effects prevent these from being used as drugs.</p><p>Structure-based drug design, in which the structure of a target protein is used to help identify compounds that will interact with it, has been used to generate therapeutic agents for a number of diseases. Here, Jiang et al. report the first application of this technique in the hunt for compounds that inhibit the cytotoxicity of amyloid beta. Using the known atomic structure of the protein in complex with a dye, Jiang et al. performed a computational screen of 18,000 compounds in search of those that are likely to bind effectively.</p><p>The compounds that showed the strongest predicted binding were then tested for their ability to interfere with the aggregation of amyloid beta and to protect cells grown in culture from its toxic effects. Compounds that reduced toxicity did not reduce the abundance of protein aggregates, but they appear to increase the stability of fibrils. This is consistent with other evidence suggesting that small, soluble forms (oligomers) of amyloid beta that break free from the fibrils may be the toxic agent in Alzheimer’s disease, rather than the fibrils themselves.</p><p>In addition to uncovering compounds with therapeutic potential in Alzheimer’s disease, this work presents a new approach for identifying proteins that bind to amyloid fibrils. Given that amyloid accumulation is a feature of many other diseases, including Parkinson’s disease, Huntington’s disease and type 2 diabetes, the approach could have broad therapeutic applications.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00857.002">http://dx.doi.org/10.7554/eLife.00857.002</ext-link></p></abstract><kwd-group kwd-group-type="author-keywords"><title>Author keywords</title><kwd>amyloid fiber</kwd><kwd>computational biology</kwd><kwd>drug discovery</kwd><kwd>Alzheimer's disease</kwd><kwd>ligand docking</kwd></kwd-group><kwd-group kwd-group-type="research-organism"><title>Research organism</title><kwd>Other</kwd></kwd-group><funding-group><award-group id="par-1"><funding-source><institution-wrap><institution>National Institutes of Health</institution></institution-wrap></funding-source><award-id>AG029430</award-id><principal-award-recipient><name><surname>Jiang</surname><given-names>Lin</given-names></name><name><surname>Liu</surname><given-names>Cong</given-names></name><name><surname>Eisenberg</surname><given-names>David S</given-names></name></principal-award-recipient></award-group><award-group id="par-2"><funding-source><institution-wrap><institution>US Department of Energy</institution></institution-wrap></funding-source><award-id>DE-FC02-02ER63421</award-id><principal-award-recipient><name><surname>Eisenberg</surname><given-names>David S</given-names></name></principal-award-recipient></award-group><award-group id="par-3"><funding-source><institution-wrap><institution>Howard Hughes Medical Institute</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Jiang</surname><given-names>Lin</given-names></name><name><surname>Eisenberg</surname><given-names>David S</given-names></name></principal-award-recipient></award-group><funding-statement>The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.</funding-statement></funding-group><custom-meta-group><custom-meta><meta-name>elife-xml-version</meta-name><meta-value>2</meta-value></custom-meta><custom-meta specific-use="meta-only"><meta-name>Author impact statement</meta-name><meta-value>By using structural methods to screen compounds for their ability to interact with amyloid beta, researchers have identified small molecules that stabilize amyloid fibers and reduce the toxic effect of smaller aggregates on cells.</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="s1" sec-type="intro"><title>Introduction</title><p>Protein aggregates, both amyloid fibers and smaller amyloid oligomers, have been implicated in the pathology of Alzheimer’s and other neurodegeneration diseases (<xref ref-type="bibr" rid="bib7">Chiti and Dobson, 2006</xref>; <xref ref-type="bibr" rid="bib15">Eisenberg and Jucker, 2012</xref>). The increasing prevalence of Alzheimer’s disease in our aging societies, the associated tragedy for patients and their families, and the mounting economic burden for governments have all stimulated intense research into chemical interventions for this condition. Much work has been focused on screening compounds that prevent aggregation and the associated cytotoxicity of the amyloid β-peptide (Aβ) (reviews by <xref ref-type="bibr" rid="bib51">Sacchettini and Kelly, 2002</xref>; <xref ref-type="bibr" rid="bib2">Bartolini and Andrisano, 2010</xref>; <xref ref-type="bibr" rid="bib22">Hard and Lendel, 2012</xref>).</p><p>Screens have often focused on natural products from plants and lichens. These include polyphenols, such as epigallocatechin gallate (EGCG) from green tea (<xref ref-type="bibr" rid="bib14">Ehrnhoefer et al., 2008</xref>) and curcumin from the spice turmeric (<xref ref-type="bibr" rid="bib60">Yang et al., 2005</xref>). These natural polyphenolic compounds show inhibition on the fibrillation of a variety of amyloid proteins, including Aβ40 as well as α-synuclein, IAPP and PrP (<xref ref-type="bibr" rid="bib50">Porat et al., 2006</xref>; <xref ref-type="bibr" rid="bib11">Dasilva et al., 2010</xref>; <xref ref-type="bibr" rid="bib47">Ono et al., 2012</xref>). Several dyes have also been found to ameliorate amyloid toxicity. Orcein from lichens appears to diminish toxic oligomers and enhance fiber formation (<xref ref-type="bibr" rid="bib3">Bieschke et al., 2011</xref>). Congo red, thioflavin T and their analogs, commonly used as staining reagents for amyloid detection, exhibit ameliorative effects on neurodegenerative disorders, such as Alzheimer’s, Parkinson’s, Huntington’s, and prion diseases (<xref ref-type="bibr" rid="bib20">Frid et al., 2007</xref>; <xref ref-type="bibr" rid="bib1">Alavez et al., 2011</xref>), however their application is limited by significant side effects (<xref ref-type="bibr" rid="bib27">Klunk et al., 2004</xref>).</p><p>Additional screens have identified a variety of molecules, including proteins (<xref ref-type="bibr" rid="bib16">Evans et al., 2006</xref>), antibodies (<xref ref-type="bibr" rid="bib26">Kayed et al., 2003</xref>; <xref ref-type="bibr" rid="bib33">Ladiwala et al., 2012</xref>), synthetic peptide mimetics (<xref ref-type="bibr" rid="bib18">Findeis, 2002</xref>; <xref ref-type="bibr" rid="bib28">Kokkoni et al., 2006</xref>; <xref ref-type="bibr" rid="bib56">Takahashi and Mihara, 2008</xref>; <xref ref-type="bibr" rid="bib6">Cheng et al., 2012</xref>) and small molecules (<xref ref-type="bibr" rid="bib58">Wood et al., 1996</xref>; <xref ref-type="bibr" rid="bib57">Williams et al., 2005</xref>; <xref ref-type="bibr" rid="bib42">McLaurin et al., 2006</xref>; <xref ref-type="bibr" rid="bib45">Necula et al., 2007</xref>; <xref ref-type="bibr" rid="bib2">Bartolini and Andrisano, 2010</xref>; <xref ref-type="bibr" rid="bib13">De Felice et al., 2001</xref>; <xref ref-type="bibr" rid="bib34">Ladiwala et al., 2011</xref>; <xref ref-type="bibr" rid="bib22">Hard and Lendel, 2012</xref>; <xref ref-type="bibr" rid="bib31">Kroth et al., 2012</xref>), that inhibit Aβ fibrillogenesis and/or Aβ-associated cytotoxicity in vitro. While most efforts have targeted the deposition of Aβ fibers as the hallmark of Alzheimer’s, smaller amyloid oligomers are now receiving greater attention as the possible toxic entities in Alzheimer’s and other neurodegenerative diseases (<xref ref-type="bibr" rid="bib23">Hartley et al., 1999</xref>; <xref ref-type="bibr" rid="bib8">Cleary et al., 2005</xref>; <xref ref-type="bibr" rid="bib55">Silveira et al., 2005</xref>). Furthermore, emerging evidence suggests that mature, end-stage amyloid fibers may serve as a reservoir, prone to releasing toxic oligomer (<xref ref-type="bibr" rid="bib59">Xue et al., 2009</xref>; <xref ref-type="bibr" rid="bib10">Cremades et al., 2012</xref>; <xref ref-type="bibr" rid="bib30">Krishnan et al., 2012</xref>; <xref ref-type="bibr" rid="bib54">Shahnawaz and Soto, 2012</xref>). Recent screens have identified compounds that reduce Aβ cytotoxicity, without interfering with Aβ fibrillation (<xref ref-type="bibr" rid="bib5">Chen et al., 2010</xref>) or promoting the formation of stable Aβ aggregates (<xref ref-type="bibr" rid="bib3">Bieschke et al., 2011</xref>).</p><p>Structural information about protein targets often aids drug development, so here we take a structure-based approach, combined with computational screening, to discover amyloid interacting compounds that reduce amyloid toxicity. This approach has been enabled by the determination of atomic structures of the adhesive segments of amyloid fibers, termed steric zippers (<xref ref-type="bibr" rid="bib46">Nelson et al., 2005</xref>), and of solid state NMR-based structures of amyloid fibers (such as full-length Aβ fibers [<xref ref-type="bibr" rid="bib41">Luhrs et al., 2005</xref>; <xref ref-type="bibr" rid="bib48">Petkova et al., 2005</xref>] and the HET-s prion domain complexed with Congo Red [<xref ref-type="bibr" rid="bib53">Schutz et al., 2011</xref>]). The steric zipper structures reveal a common motif for the spine of amyloid fibers, in which a pair of fibrillar β-sheets is held together by the side-chain interdigitation (<xref ref-type="bibr" rid="bib52">Sawaya et al., 2007</xref>). We focus on Aβ, a peptide of 39–42 residues cleaved from the Amyloid precursor protein (APP) associated with Alzheimer’s, as a target for inhibitor discovery. The segment Aβ<sub>16–21</sub> with the sequence KLVFFA is an amyloid-forming peptide, which packs in a steric zipper form, and has been identified as the spine of the full-length Aβ fiber (<xref ref-type="bibr" rid="bib41">Luhrs et al., 2005</xref>; <xref ref-type="bibr" rid="bib49">Petkova et al., 2006</xref>; <xref ref-type="bibr" rid="bib9">Colletier et al., 2011</xref>). Co-crystal structures have been determined for small molecules in complex with the fibrillar β-sheets of Aβ<sub>16–21</sub> (<xref ref-type="bibr" rid="bib36">Landau et al., 2011</xref>). One of these structures—Aβ<sub>16–21</sub> with the dye Orange G—reveals the specific pattern of hydrogen bonds and apolar interactions between orange G and the steric zipper: the negatively charged dye binds specifically to lysine side chains of adjacent sheets, and its planar aromatic portion packs against apolar residues (phenylalanine and valine) of adjacent sheets. By creating a tight, low energy interface across several β-strands within fiber core, this fiber-binding molecule appears to stabilize the fiber structure. With this atomic structure as a basis, we are able to screen for small molecular compounds that bind to amyloid fibers, stabilizing them and possibly reducing amyloid toxicity. Applying our structure-based screening procedure, we screen computationally for compounds that bind to Aβ fibers, termed BAFs (Binders of Amyloid Fibers) and then experimentally test their effects on Aβ aggregation and cytotoxicity.</p></sec><sec id="s2" sec-type="results"><title>Results</title><sec id="s2-1"><title>Structure-based screening procedure</title><p>We have devised a structure-based procedure for the identification of small molecules that bind to amyloid and affect amyloid toxicity (<xref ref-type="fig" rid="fig1">Figure 1</xref>). The procedure starts from a co-crystal structure of a ligand bound to an amyloidogenic segment of Aβ (<xref ref-type="bibr" rid="bib36">Landau et al., 2011</xref>), the dye orange G bound to the fiber-like crystal structure of KLVFFA(Aβ<sub>16–21</sub>) segment. This structure reveals the chemical environment or ‘<italic>pharmacophore</italic>’ presented by the ligand binding site of this Aβ segment, that is, orange G binds to stacked β-sheets of Aβ. Knowledge of the amyloid pharmacophore (<xref ref-type="fig" rid="fig1">Figure 1A</xref>) permitted us to screen for compounds that could be expected to bind in this chemical environment, possibly stabilizing amyloid fibers.<fig-group><fig id="fig1" position="float"><object-id pub-id-type="doi">10.7554/eLife.00857.003</object-id><label>Figure 1.</label><caption><title>Structure-based identification of small compound inhibitors of Aβ toxicity.</title><p>In step (<bold>A</bold>) the crystal structure (<xref ref-type="bibr" rid="bib36">Landau et al., 2011</xref>) is determined of a complex of an amyloidogenic segment of Aβ (in this case residues 16-KLVFFA-21 of the spine of the Aβ fiber) with an amyloid-binding Ligand X (in this case orange G), revealing aspects of the pharmacophore for Ligand X. Prior to step (<bold>B</bold>) a large library of available compounds is selected for computational docking (∼18,000 purchasable compounds in this case). In step (<bold>B</bold>) computational docking is applied to test the compatibility of each member of the library for the pharmacophore of the amyloidogenic segment defined in step (<bold>A</bold>). In step (<bold>C</bold>), the top scoring members of the library are tested for compatibility of binding within a full-length Aβ fiber (in this case the 400 top scoring members were tested on a solid state NMR-derived model of an Aβ fiber, pdb entry 2LMO) (<xref ref-type="bibr" rid="bib49">Petkova et al., 2006</xref>). The representative models from steps <bold>B</bold> and <bold>C</bold> are shown in <xref ref-type="fig" rid="fig1s1 fig1s2">Figure 1<bold>—</bold>figure supplements 1 and 2</xref>. In step (<bold>D</bold>), the compounds are ranked by tightest binding energy and best shape complementarity for the pharmacophore. In step (<bold>E</bold>), the top-ranking compounds (25 in this case) are selected for experimental characterization and validation, including NMR assessment of binding, EM assays of their effects on fiber formation, and cell viability assays for their effects on Aβ cytotoxicity. In step (<bold>F</bold>), new compounds (9 in this case) and compound derivatives (17 in this case) are selected for an additional cycle of computational and experimental testing, based on their similarity to the lead compounds from the initial cycle.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00857.003">http://dx.doi.org/10.7554/eLife.00857.003</ext-link></p></caption><graphic xlink:href="elife00857f001"/></fig><fig id="fig1s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00857.004</object-id><label>Figure 1—figure supplement 1.</label><caption><title>Structural models of the representative BAFs and orange G docked to the side of the KLVFFA(Aβ<sub>16–21</sub>) fiber.</title><p>In step (<bold>B</bold>) (<xref ref-type="fig" rid="fig1">Figure 1</xref>), a large library of ∼18 thousand commercially available compounds were docked onto the Aβ<sub>16–21</sub> fiber, and ∼400 top ranking compounds, whose binding energy and shape complementary score are better than the control molecule orange G, were selected for the next docking step. The models of representative BAFs docked on single beta-sheet of Aβ<sub>16–21</sub> fiber are compared to that of orange G. (<bold>A</bold>). A side view of the compound BAF1 (in green sticks) docked on the Aβ<sub>16–21</sub> fiber (in a grey color) with a predicted binding energy of −8.4 kcal/mol. (<bold>B</bold>). A side view of BAF8 (in cyan sticks) docked on the Aβ<sub>16–21</sub> fiber with a predicted binding energy of −12 kcal/mol. (<bold>C</bold>). A side view of orange G (in orange sticks) docked on the Aβ<sub>16–21</sub> fiber with a predicted binding energy of −8.0 kcal/mol. The charge interactions between the compounds and Lysine residues of Aβ<sub>16–21</sub> fiber are highlighted by black lines.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00857.004">http://dx.doi.org/10.7554/eLife.00857.004</ext-link></p></caption><graphic xlink:href="elife00857fs001"/></fig><fig id="fig1s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00857.005</object-id><label>Figure 1—figure supplement 2.</label><caption><title>Structural models of the representative BAFs and orange G docked onto the full-length Aβ fiber.</title><p>In step (<bold>C</bold>) (<xref ref-type="fig" rid="fig1">Figure 1</xref>), the top-ranking compounds after the first docking step were further filtered by docking onto full-length Aβ fiber model (pdb entry 2LMO) (<xref ref-type="bibr" rid="bib49">Petkova et al., 2006</xref>). The models of representative BAFs docked onto Aβ fiber are compared to that of orange G. (<bold>A</bold>–<bold>C</bold>). A top view of the compounds (BAF1, BAF8 and orange G) docked onto Aβ fiber (in a light yellow color). (<bold>D</bold>–<bold>F</bold>). A side view of the same compounds docked onto Aβ fiber. (<bold>A</bold> and <bold>D</bold>). BAF1 (in a green color) binds to the side of Aβ fiber (in a light yellow color) with a predicted binding energy of −10 kcal/mol. (<bold>B</bold> and <bold>E</bold>). BAF8 (in a cyan color) binds to the side of Aβ with a predicted binding energy of −12 kcal/mol. (<bold>C</bold> and <bold>F)</bold>. Orange G (in an orange color) binds to the side of Aβ fiber with a predicted binding energy of −9 kcal/mol. The charge interactions between the compounds and Lysine residues are highlighted by black lines.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00857.005">http://dx.doi.org/10.7554/eLife.00857.005</ext-link></p></caption><graphic xlink:href="elife00857fs002"/></fig><fig id="fig1s3" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00857.006</object-id><label>Figure 1—figure supplement 3.</label><caption><title>Alternative binding modes of BAF1 with the Aβ full-length fibers.</title><p>When identifying BAFs by two steps of computational docking (<xref ref-type="fig" rid="fig2">Figure 2A</xref> as well as step <bold>B</bold> and <bold>C</bold> in <xref ref-type="fig" rid="fig1">Figure 1</xref>), most models of the second docking step (docking to full-length Aβ fiber in step (<bold>C</bold>) retained their binding modes found in the previous docking step (docking to Aβ<sub>16–21</sub> fiber in step (<bold>B</bold>). Interestingly, docking of BAF1 onto full-length Aβ fiber not only recapitulated the initial binding mode found in previous Aβ<sub>16–21</sub> docking step but also revealed the different binding mode with comparable binding energies. Two examples of those alternative binding modes are shown in (<bold>A</bold> and <bold>B</bold>). In both modes, BAF1 tends to use its polar (hydroxyl) group to interact with the charged residues Glu22 of Aβ and use its non-polar (aromatic) portion to pack against the hydrophobic residues Phe20 of Aβ full fibers.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00857.006">http://dx.doi.org/10.7554/eLife.00857.006</ext-link></p></caption><graphic xlink:href="elife00857fs003"/></fig></fig-group></p></sec><sec id="s2-2"><title>Construction of compound libraries for computational screening</title><p>For assembling the compounds in our screening library, we sought three characteristics: (a) commercially available compounds since we intended to follow the in silico screening with experimental validation; (b) compounds with known three-dimensional structures such that our screening would be as realistic as possible; (c) generally flat compounds able to bind to the β-sheets of the steric zipper, as does orange G. Some ∼11,000 compounds having the first two characteristics (CSD-ZINC set) were selected as the intersection of molecules found both in the Cambridge Structure Database (<ext-link ext-link-type="uri" xlink:href="http://www.ccdc.cam.ac.uk">http://www.ccdc.cam.ac.uk</ext-link>) and the Zinc Database of purchasable compounds (<ext-link ext-link-type="uri" xlink:href="http://zinc.docking.org/">http://zinc.docking.org/</ext-link>) (<xref ref-type="bibr" rid="bib25">Irwin and Shoichet, 2005</xref>). This CSD-ZINC set spans a variety of structural shapes and molecular properties. A second set of ∼7000 compounds, the Flat Compound Set, was gathered from the ZINC database to include molecules expected to bind to the flat surface of a steric zipper. The members of this set contain multiple aromatic rings or one aromatic ring with additional planar groups.</p></sec><sec id="s2-3"><title>Computational screening of compounds that bind to Aβ fibers</title><p>Computational screening was carried out with the RosettaLigand program (<xref ref-type="bibr" rid="bib12">Davis and Baker, 2009</xref>), after adapting its docking approach to carry out high-throughput screening (<xref ref-type="fig" rid="fig2">Figure 2</xref>). The conformational flexibilities of ligand and protein side chains are in a ‘near-native’ perturbation fashion, meaning that the fine sampling of conformations was restrained to be close to the starting conformation. A balance was achieved between extensive sampling and the speed required for screening a large compound library by fine sampling of side chain and ligand torsion angles only around their starting conformations, as illustrated by sticks in <xref ref-type="fig" rid="fig2">Figure 2C</xref>.<fig-group><fig id="fig2" position="float"><object-id pub-id-type="doi">10.7554/eLife.00857.007</object-id><label>Figure 2.</label><caption><title>Computational screening for fiber-binding compounds.</title><p>(<bold>A</bold>). Outline of our procedure for structure-based screening. We prepare two sets of compounds (shown in the upper left) for screening against both types of fibers shown in the upper right. Compound Set 1 is the intersection of the ZINC Database of purchasable compounds with the Cambridge structural database (CSD) of known structures. Set 2 consists of other flat aromatic and multiple conjugated compounds found in the ZINC Database. The full description of each computational step is in ‘Materials and methods’. (<bold>B</bold>). Distribution of calculated binding energies for the compound libraries of Sets 1 and 2. Those top-ranking compounds have better predicted binding energy than orange G. Structural comparison of docked models of such compound BAF8 and orange-G is discussed in the <xref ref-type="fig" rid="fig2s1">Figure 2<bold>—</bold>figure supplement 1</xref>. Notice the starred bins which suggest that some members of Set 2, containing flat compounds, tend to be among the top scoring compounds, presumably having the tightest binding to the flat fiber surface. (<bold>C</bold>). The conformational ensemble of a compound representative shown docked onto the Aβ<sub>16–21</sub> fiber structure. (<bold>D</bold>). A model of BAF8 docked onto an NMR-derived model of full-length Aβ fiber. Notice that the apolar ring structure of the compound binds to the relatively flat apolar (gray) surface of the fiber, and the polar moieties of the compound (red) form hydrogen bonds to the polar groups of the fiber (yellow). The stereo view of BAF8 model is shown in <xref ref-type="fig" rid="fig2s2">Figure 2<bold>—</bold>figure supplement 2</xref>.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00857.007">http://dx.doi.org/10.7554/eLife.00857.007</ext-link></p></caption><graphic xlink:href="elife00857f002"/></fig><fig id="fig2s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00857.008</object-id><label>Figure 2—figure supplement 1.</label><caption><title>Structural comparison between docked models of BAF8 and orange G.</title><p>BAF8 has a chemical structure similar to orange G (top panels). The comparison of the shape complimentary at binding interfaces reveals that BAF8 binds more tightly to the side of fibers than orange G. (<bold>A</bold>). A top view of the docked model of BAF8 (in a cyan color) with the predicted binding energy of −12 kcal/mol highlights the tight shape complementary at the fiber-ligand interface. (<bold>B</bold>). A top view of the docked model of orange G (in an orange color) with the predicted binding energy of −8 kcal/mol shows a poorly packed interface with cavities.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00857.008">http://dx.doi.org/10.7554/eLife.00857.008</ext-link></p></caption><graphic xlink:href="elife00857fs004"/></fig><fig id="fig2s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00857.009</object-id><label>Figure 2—figure supplement 2.</label><caption><title>Stereo view of the structural model of BAF8 with Aβ fiber.</title><p>A wall-eyed stereo view of BAF8 (<xref ref-type="fig" rid="fig2">Figure 2D</xref>) (in cyan sticks) docked to the side of an Aβ<sub>16–21</sub> fiber (light yellow) reveals good non-polar and polar interaction across binding interfaces. The hydrophobic binding site for the aromatic portion of BAF8 is indicated by grey mesh surfaces to highlight the good shape complementary. The polar interaction of hydrogen bonds between the charged residues Lys 16 of Aβ and the polar portion of BAF8 are indicated by black thick lines.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00857.009">http://dx.doi.org/10.7554/eLife.00857.009</ext-link></p></caption><graphic xlink:href="elife00857fs005"/></fig></fig-group></p><p>In the screening steps of computational docking (<xref ref-type="fig" rid="fig2">Figure 2A</xref>), a library of ∼18,000 purchasable compounds (Sets 1 and 2) was scanned computationally for structural compatibility with the pharmacophore (ligand binding site) presented by a single sheet of the Aβ<sub>16–21</sub> steric zipper. Structural compatibility was assessed by a combination of binding energy (<xref ref-type="bibr" rid="bib43">Meiler and Baker, 2006</xref>) and steric complementarity (<xref ref-type="bibr" rid="bib37">Lawrence and Colman, 1993</xref>). After computational docking, the distribution of calculated binding energies suggests that, statistically the flat compounds from Set 2 fit more snugly on the flat surfaces of Aβ<sub>16–21</sub> fibers than those with diverse shapes in Set 1 (<xref ref-type="fig" rid="fig2">Figure 2B</xref>). The best scoring compounds were screened further by requiring that each is also structurally compatible with the solid-state NMR-derived model of the Aβ full-length fiber structure (<xref ref-type="bibr" rid="bib49">Petkova et al., 2006</xref>) (<xref ref-type="fig" rid="fig1">Figure 1C</xref> and <xref ref-type="fig" rid="fig1s3">Figure 1—figure supplement 3</xref>).</p></sec><sec id="s2-4"><title>Experimental characterization of BAFs</title><p>After in silico screening of a library of ∼18,000 purchasable compounds, twenty-five of the top-ranking compounds all with better scores for binding energy and steric complementarity than orange G (<xref ref-type="fig" rid="fig1">Figure 1D</xref>, <xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1</xref>), were selected for experimental validation. First these 25 compounds were tested for their ability to protect mammalian cells from Aβ toxicity (<xref ref-type="fig" rid="fig1">Figure 1E</xref>, <xref ref-type="table" rid="tbl1 tbl2">Tables 1 and 2</xref>), and five of them were found to reduce the toxic effects of Aβ. These five were tested for binding to both Aβ<sub>1–42</sub> and Aβ<sub>16–21</sub> fibers by NMR. Two were found to have tighter binding than orange G, and the others gave insufficient NMR signals for detection. To expand this set of the five compounds, a second cycle of inhibitor discovery was performed. From the computed positions of the five compounds, a refined pharmacophore was inferred (<xref ref-type="fig" rid="fig1">Figure 1F</xref>), and used in the next cycle of screening. Added to the compound set were nine additional compounds apparently related to the five lead compounds from the initial cycle, plus 17 chemical derivatives of compounds (<xref ref-type="table" rid="tbl1 tbl3">Tables 1 and 3</xref>). The second cycle produced three additional compounds and three compound derivatives that also protected the mammalian cells from Aβ fibers. One of these compounds was confirmed by NMR to bind to Aβ fibers. The detailed description of those experimental results is as follows.<table-wrap id="tbl1" position="float"><object-id pub-id-type="doi">10.7554/eLife.00857.010</object-id><label>Table 1.</label><caption><p>List of all tested BAF compounds</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00857.010">http://dx.doi.org/10.7554/eLife.00857.010</ext-link></p></caption><table frame="hsides" rules="groups"><thead><tr><th>Compound</th><th>Molecular formula</th><th>Molecular weight<xref ref-type="table-fn" rid="tblfn1">*</xref></th><th>Sources/purchasing</th><th>Rescuing percentage (%)</th><th>ZINC entry</th></tr></thead><tbody><tr><td>BAF1</td><td>C<sub>20</sub>H<sub>8</sub>Br<sub>4</sub>O<sub>5</sub></td><td align="char" char=".">648</td><td>Sigma-Aldrich</td><td align="char" char="plusmn">44 ± 7</td><td>ZINC04261875</td></tr><tr><td>BAF2</td><td>C<sub>19</sub>H<sub>14</sub>O<sub>5</sub>S</td><td align="char" char=".">354</td><td>Sigma-Aldrich</td><td align="char" char="plusmn">4 ± 3</td><td>ZINC03860918</td></tr><tr><td>BAF3</td><td>C<sub>16</sub>H<sub>13</sub>NO<sub>3</sub></td><td align="char" char=".">267</td><td>Ryan Scientific</td><td align="char" char="plusmn">4 ± 5</td><td>ZINC04289063</td></tr><tr><td>BAF4</td><td>C<sub>24</sub>H<sub>16</sub>N<sub>2</sub>O<sub>6</sub></td><td align="char" char=".">428</td><td>Aldrich</td><td align="char" char="plusmn">88 ± 22</td><td>ZINC13346907</td></tr><tr><td>BAF5</td><td>C<sub>16</sub>H<sub>7</sub>Na<sub>3</sub>O<sub>10</sub>S<sub>3</sub></td><td align="char" char=".">524</td><td>Sigma-Aldrich</td><td align="char" char="plusmn">11 ± 7</td><td>ZINC03594314</td></tr><tr><td>BAF6</td><td>C<sub>26</sub>H<sub>20</sub>N<sub>2</sub></td><td align="char" char=".">360</td><td>Alfa-Aesar</td><td align="char" char="plusmn">5 ± 7</td><td>ZINC08078162</td></tr><tr><td>BAF7</td><td>C<sub>18</sub>H<sub>12</sub>N<sub>6</sub></td><td align="char" char=".">312</td><td>Alfa-Aesar</td><td align="char" char="plusmn">2 ± 2</td><td>ZINC00039221</td></tr><tr><td>BAF8</td><td>C<sub>17</sub>H<sub>14</sub>N<sub>2</sub>O<sub>5</sub>S</td><td align="char" char=".">358</td><td>Sigma-Aldrich</td><td align="char" char="plusmn">23 ± 11</td><td>ZINC12358966</td></tr><tr><td>BAF9</td><td>C<sub>19</sub>H<sub>13</sub>N<sub>3</sub>O<sub>4</sub>S</td><td align="char" char=".">379</td><td>NCI plated 2007<xref ref-type="table-fn" rid="tblfn2">†</xref></td><td align="char" char="plusmn">−3 ± 22</td><td>ZINC03954432</td></tr><tr><td>BAF10</td><td>C<sub>17</sub>H<sub>13</sub>NO<sub>3</sub></td><td align="char" char=".">279</td><td>NCI plated 2007</td><td align="char" char="plusmn">3 ± 5</td><td>ZINC00105108</td></tr><tr><td>BAF11</td><td>C<sub>20</sub>H<sub>13</sub>N<sub>2</sub>O<sub>5</sub>S</td><td align="char" char=".">393</td><td>NCI plated 2007</td><td align="char" char="plusmn">48 ± 12</td><td>ZINC04521479</td></tr><tr><td>BAF12</td><td>C<sub>13</sub>H<sub>8</sub>Br<sub>3</sub>NO</td><td align="char" char=".">434</td><td>NCI plated 2007</td><td align="char" char="plusmn">38 ± 6</td><td>ZINC12428965</td></tr><tr><td>BAF13</td><td>C<sub>19</sub>H<sub>16</sub>ClNO<sub>4</sub></td><td align="char" char=".">358</td><td>Sigma-Aldrich</td><td align="char" char="plusmn">0 ± 2</td><td>ZINC00601283</td></tr><tr><td>BAF14</td><td>C<sub>10</sub>H<sub>6</sub>S<sub>2</sub>O<sub>8</sub></td><td align="char" char=".">318</td><td>Sigma-Aldrich</td><td align="char" char="plusmn">3 ± 3</td><td>ZINC01532215</td></tr><tr><td>BAF15</td><td>C<sub>23</sub>H<sub>28</sub>O<sub>8</sub></td><td align="char" char=".">432</td><td>Sigma-Aldrich</td><td align="char" char="plusmn">13 ± 4</td><td>ZINC00630328</td></tr><tr><td>BAF16</td><td>C<sub>19</sub>H<sub>19</sub>NO<sub>5</sub></td><td align="char" char=".">341</td><td>Sigma-Aldrich</td><td align="char" char="plusmn">5 ± 8</td><td>ZINC28616347</td></tr><tr><td>BAF17</td><td>C<sub>23</sub>H<sub>25</sub>N<sub>5</sub>O<sub>2</sub></td><td align="char" char=".">404</td><td>Sigma-Aldrich</td><td align="char" char="plusmn">6 ± 3</td><td>ZINC00579168</td></tr><tr><td>BAF18</td><td>C<sub>24</sub>H<sub>16</sub>O<sub>2</sub></td><td align="char" char=".">336</td><td>ChemDiv</td><td align="char" char="plusmn">6 ± 2</td><td>ZINC02168932</td></tr><tr><td>BAF19</td><td>C<sub>18</sub>H<sub>14</sub>N<sub>2</sub>O<sub>6</sub></td><td align="char" char=".">354</td><td>ChemDiv</td><td align="char" char="plusmn">3 ± 4</td><td>ZINC01507439</td></tr><tr><td>BAF20</td><td>C<sub>25</sub>H<sub>19</sub>N<sub>5</sub>OS</td><td align="char" char=".">438</td><td>ChemDiv</td><td align="char" char="plusmn">8 ± 4</td><td>ZINC15859747</td></tr><tr><td>BAF21</td><td>C<sub>19</sub>H<sub>14</sub>Br<sub>2</sub>O</td><td align="char" char=".">418</td><td>ChemDiv</td><td align="char" char="plusmn">6 ± 3</td><td>ZINC38206526</td></tr><tr><td>BAF22</td><td>C<sub>21</sub>H<sub>16</sub>N<sub>2</sub>O<sub>3</sub>S<sub>2</sub></td><td align="char" char=".">408</td><td>Life Chemicals</td><td align="char" char="plusmn">3 ± 5</td><td>ZINC04496365</td></tr><tr><td>BAF23</td><td>C<sub>16</sub>H<sub>11</sub>ClO<sub>5</sub>S</td><td align="char" char=".">351</td><td>Enamine Ltd</td><td align="char" char="plusmn">3 ± 5</td><td>ZINC02649996</td></tr><tr><td>BAF24</td><td>C<sub>23</sub>H<sub>19</sub>NO<sub>3</sub></td><td align="char" char=".">357</td><td>Sigma-Aldrich</td><td align="char" char="plusmn">16 ± 5</td><td>ZINC03953119</td></tr><tr><td>BAF25</td><td>C<sub>14</sub>H<sub>8</sub>Cl<sub>2</sub>N<sub>4</sub></td><td align="char" char=".">303</td><td>Sigma-Aldrich</td><td align="char" char="plusmn">4 ± 3</td><td>ZINC00403224</td></tr><tr><td>BAF26</td><td>C<sub>17</sub>H<sub>10</sub>O<sub>4</sub></td><td align="char" char=".">278</td><td>Aldrich</td><td align="char" char="plusmn">46 ± 23</td><td>ZINC05770717</td></tr><tr><td>BAF27</td><td>C<sub>21</sub>H<sub>16</sub>BrN<sub>3</sub>O<sub>6</sub></td><td align="char" char=".">486</td><td>ChemBridge</td><td align="char" char="plusmn">4 ± 1</td><td>ZINC01208856</td></tr><tr><td>BAF28</td><td>C<sub>17</sub>H<sub>12</sub>N<sub>2</sub>O<sub>3</sub></td><td align="char" char=".">292</td><td>ChemBridge</td><td align="char" char="plusmn">2 ± 4</td><td>ZINC00061083</td></tr><tr><td>BAF29</td><td>C<sub>22</sub>H<sub>10</sub>N<sub>4</sub>O<sub>2</sub></td><td align="char" char=".">362</td><td>ChemBridge</td><td align="char" char="plusmn">1 ± 5</td><td>ZINC00639061</td></tr><tr><td>BAF30</td><td>C<sub>14</sub>H<sub>8</sub>O<sub>5</sub></td><td align="char" char=".">256</td><td>Aldrich</td><td align="char" char="plusmn">18 ± 13</td><td>ZINC03870461</td></tr><tr><td>BAF31</td><td>C<sub>19</sub>H<sub>21</sub>NO<sub>3</sub></td><td align="char" char=".">311</td><td>Sigma</td><td align="char" char="plusmn">84 ± 12</td><td>ZINC00011665</td></tr><tr><td>BAF32</td><td>C<sub>15</sub>H<sub>14</sub>O<sub>7</sub></td><td align="char" char=".">306</td><td>Sigma-Aldrich</td><td align="char" char="plusmn">15 ± 9</td><td>ZINC03870336</td></tr><tr><td>BAF33</td><td>C<sub>27</sub>H<sub>33</sub>N<sub>3</sub>O<sub>8</sub></td><td align="char" char=".">528</td><td>Sigma-Aldrich</td><td align="char" char="plusmn">7 ± 2</td><td>SIGMA-R2253<xref ref-type="table-fn" rid="tblfn4">§</xref></td></tr><tr><td>BAF34</td><td>C<sub>30</sub>H<sub>16</sub>N<sub>4</sub>O<sub>14</sub>S<sub>4</sub></td><td align="char" char=".">785</td><td>Aldrich</td><td><xref ref-type="table-fn" rid="tblfn3">‡</xref></td><td>ALDRICH-S432830<xref ref-type="table-fn" rid="tblfn4">§</xref></td></tr><tr><td>orange G</td><td>C<sub>16</sub>H<sub>12</sub>N<sub>2</sub>O<sub>7</sub>S<sub>2</sub></td><td align="char" char=".">408</td><td>Sigma-Aldrich</td><td align="char" char="plusmn">−2 ± 8</td><td>ZINC04261935</td></tr></tbody></table><table-wrap-foot><fn><p>The 25 compounds (BAF1-25) are from the first round, and the nine compounds (BAF26-34) are from the second round. Another set of the 17 derivatives of the BAFs are shown in <xref ref-type="table" rid="tbl3">Table 3</xref>.</p></fn><fn id="tblfn1"><label>*</label><p>Molecular weight (anhydrous basis) excluding the salt and water molecules.</p></fn><fn id="tblfn2"><label>†</label><p>National Cancer Institute (NCI) free compound library (<ext-link ext-link-type="uri" xlink:href="http://dtp.nci.nih.gov/">http://dtp.nci.nih.gov/</ext-link>).</p></fn><fn id="tblfn3"><label>‡</label><p>Toxicity results of BAF34 were not consistent among several independent replica experiments, possibly due to impurity and the high molecular weight of the compound.</p></fn><fn id="tblfn4"><label>§</label><p>ZINC entry of the compound is not applicable, and the catalog number from Sigma-Aldrich is provided.</p></fn></table-wrap-foot></table-wrap><table-wrap id="tbl2" position="float"><object-id pub-id-type="doi">10.7554/eLife.00857.011</object-id><label>Table 2.</label><caption><p>Detailed list of the active BAF compounds</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00857.011">http://dx.doi.org/10.7554/eLife.00857.011</ext-link></p></caption><table frame="hsides" rules="groups"><thead><tr><th rowspan="2">Compound</th><th rowspan="2">Molecular formula</th><th rowspan="2">Molecular weight<xref ref-type="table-fn" rid="tblfn5">*</xref></th><th rowspan="2">Sources/companies</th><th rowspan="2">Purity</th><th colspan="2">Rescuing percentage<xref ref-type="table-fn" rid="tblfn8">§</xref> (%)</th><th rowspan="2">ZINC entry code<xref ref-type="table-fn" rid="tblfn9">¶</xref></th><th rowspan="2">SMILES string</th></tr><tr><th>PC12</th><th>Hela</th></tr></thead><tbody><tr><td>BAF1</td><td>C<sub>20</sub>H<sub>8</sub>Br<sub>4</sub>O<sub>5</sub></td><td align="char" char=".">647.9</td><td>Sigma-Aldrich</td><td>∼99%</td><td align="char" char="plusmn">38 ± 11</td><td align="char" char="plusmn">44 ± 7</td><td>ZINC04261875</td><td>c1ccc2c(c1)C(=O)OC23c4ccc(c(c4Oc5c3ccc(c5Br)O)Br)O</td></tr><tr><td>BAF4</td><td>C<sub>24</sub>H<sub>16</sub>N<sub>2</sub>O<sub>6</sub></td><td align="char" char=".">428.4</td><td>Aldrich</td><td>≥95%</td><td align="char" char="plusmn">85 ± 18</td><td align="char" char="plusmn">88 ± 22</td><td>ZINC13346907</td><td>c1cc(c(cc1O)O)c2cc3c(cc2N)oc-4cc(=O)c(cc4n3)c5ccc(cc5O)O</td></tr><tr><td>BAF8</td><td>C<sub>17</sub>H<sub>14</sub>N<sub>2</sub>O<sub>5</sub>S</td><td align="char" char=".">358.4</td><td>Sigma-Aldrich</td><td>≥90%</td><td align="char" char="plusmn">26 ± 12</td><td align="char" char="plusmn">23 ± 11</td><td>ZINC12358966</td><td>Cc1ccc(c(c1)/N=N/c2c3ccccc3c(cc2O)S(=O)(=O)[O-])O</td></tr><tr><td>BAF11</td><td>C<sub>20</sub>H<sub>13</sub>N<sub>2</sub>O<sub>5</sub>S</td><td align="char" char=".">393.5</td><td>NCI plated 2007</td><td><xref ref-type="table-fn" rid="tblfn6">†</xref></td><td align="char" char="plusmn">51 ± 11</td><td align="char" char="plusmn">48 ± 12</td><td>ZINC04521479</td><td>c1ccc2c(c1)ccc(c2O)/N=N/c3c4ccccc4c(cc3O)S(=O)(=O)[O-]</td></tr><tr><td>BAF12</td><td>C<sub>13</sub>H<sub>8</sub>Br<sub>3</sub>NO</td><td align="char" char=".">433.9</td><td>NCI plated 2007</td><td><xref ref-type="table-fn" rid="tblfn6">†</xref></td><td align="char" char="plusmn">19 ± 6</td><td align="char" char="plusmn">38 ± 6</td><td>ZINC12428965</td><td>c1cc(ccc1/N=C/c2cc(cc(c2O)Br)Br)Br</td></tr><tr><td>BAF26</td><td>C<sub>17</sub>H<sub>10</sub>O<sub>4</sub></td><td align="char" char=".">278.3</td><td>Aldrich</td><td><xref ref-type="table-fn" rid="tblfn7">‡</xref></td><td align="char" char="plusmn">60 ± 21</td><td align="char" char="plusmn">46 ± 23</td><td>ZINC05770717</td><td>c12c(cc(cc1)C(=O)C=O)Cc1c2ccc(c1)C(=O)C=O</td></tr><tr><td>BAF30</td><td>C<sub>14</sub>H<sub>8</sub>O<sub>5</sub></td><td align="char" char=".">256.2</td><td>Aldrich</td><td><xref ref-type="table-fn" rid="tblfn6">‡</xref></td><td align="char" char="plusmn">37 ± 18</td><td align="char" char="plusmn">18 ± 13</td><td>ZINC03870461</td><td>c1cc2c(cc1O)C(=O)c3c(ccc(c3O)O)C2=O</td></tr><tr><td>BAF31</td><td>C<sub>19</sub>H<sub>21</sub>NO<sub>3</sub></td><td align="char" char=".">311.4</td><td>Sigma</td><td>≥98%</td><td align="char" char="plusmn">92 ± 22</td><td align="char" char="plusmn">84 ± 12</td><td>ZINC03874841</td><td>CCCN1CCC2=C3C1CC4=C(C3=CC(=C2)O)C(=C(C=C4)O)O</td></tr></tbody></table><table-wrap-foot><fn><p>BAFs 1, 4, 8, 11, 12 are from the first round. BAFs 26, 30, 31 are from the second round.</p></fn><fn id="tblfn5"><label>*</label><p>Molecular weight (anhydrous basis) excluding the salt and water molecules.</p></fn><fn id="tblfn6"><label>†</label><p>With the standard of NCI free compound library.</p></fn><fn id="tblfn7"><label>‡</label><p>Analytical data for Aldrich<sup>CPR</sup> products are not available.</p></fn><fn id="tblfn8"><label>§</label><p>Rescue percentage is a scaled cell survival rate.</p></fn><fn id="tblfn9"><label>¶</label><p>Entry code for the ZINC database (<ext-link ext-link-type="uri" xlink:href="http://zinc.docking.org">http://zinc.docking.org</ext-link>).</p></fn></table-wrap-foot></table-wrap><table-wrap id="tbl3" position="float"><object-id pub-id-type="doi">10.7554/eLife.00857.012</object-id><label>Table 3.</label><caption><p>List of the representative BAFs 11, 30, 31 and their derivatives</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00857.012">http://dx.doi.org/10.7554/eLife.00857.012</ext-link></p></caption><table frame="hsides" rules="groups"><thead><tr><th>Compound</th><th>Molecular formula</th><th>Molecular weight</th><th>Description</th><th>Toxicity inhibition (%)</th><th>ZINC entry/catalog no.</th></tr></thead><tbody><tr><td>BAF31</td><td>C<sub>19</sub>H<sub>21</sub>NO<sub>3</sub></td><td align="char" char=".">311</td><td/><td align="char" char="plusmn">84 ± 12</td><td>ZINC03874841</td></tr><tr><td> BAF31ΔOH</td><td>C<sub>19</sub>H<sub>21</sub>NO<sub>2</sub></td><td align="char" char=".">295</td><td>remove one hydroxyl (OH)</td><td align="char" char="plusmn">15 ± 2</td><td>ZINC03874841</td></tr><tr><td>BAF30</td><td>C<sub>14</sub>H<sub>8</sub>O<sub>5</sub></td><td align="char" char=".">256</td><td/><td align="char" char="plusmn">18 ± 13</td><td>ZINC03870461</td></tr><tr><td> BAF30αR</td><td>C<sub>22</sub>H<sub>20</sub>O<sub>13</sub></td><td align="char" char=".">492</td><td>add additional R group away from binding interface</td><td align="char" char="plusmn">20 ± 10</td><td>ZINC28095922</td></tr><tr><td> BAF30σOH<sup>A</sup>αOH</td><td>C<sub>14</sub>H<sub>8</sub>O<sub>6</sub></td><td align="char" char=".">272</td><td>change one OH (A) position; add another OH</td><td align="char" char="plusmn">9 ± 9</td><td>ZINC03874832</td></tr><tr><td>BAF30σOH<sup>A</sup>ΔOH<sup>B</sup>αCOO</td><td>C<sub>15</sub>H<sub>8</sub>O<sub>6</sub></td><td align="char" char=".">284</td><td>move one OH (A) position; delete an OH from loc B; add a carboxyl</td><td align="char" char="plusmn">9 ± 3</td><td>ZINC04098704</td></tr><tr><td> BAF30σOH<sup>AB</sup>αCH<sub>3</sub></td><td>C<sub>15</sub>H<sub>10</sub>O<sub>5</sub></td><td align="char" char=".">270</td><td>move two OH (AB) positions; add a methyl</td><td align="char" char="plusmn">6 ± 3</td><td>ZINC03824868</td></tr><tr><td>BAF11</td><td>C<sub>20</sub>H<sub>13</sub>N<sub>2</sub>O<sub>5</sub>S</td><td align="char" char=".">393</td><td/><td align="char" char="plusmn">48 ± 12</td><td>ZINC04521479</td></tr><tr><td> BAF11<sup>ISO</sup></td><td>C<sub>20</sub>H<sub>13</sub>N<sub>2</sub>O<sub>5</sub>S</td><td align="char" char=".">393</td><td>isomer form of BAF11</td><td align="char" char="plusmn">33 ± 5</td><td>ZINC12405071</td></tr><tr><td> BAF11σR1</td><td>C<sub>20</sub>H<sub>14</sub>N<sub>4</sub>O<sub>8</sub>S<sub>2</sub></td><td align="char" char=".">502</td><td>change the aromatic group</td><td align="char" char="plusmn">35 ± 9</td><td>ZINC25558261</td></tr><tr><td> BAF11σR2 (BAF8)</td><td>C<sub>17</sub>H<sub>14</sub>N<sub>2</sub>O<sub>5</sub>S</td><td align="char" char=".">358</td><td>change the aromatic group</td><td align="char" char="plusmn">22 ± 11</td><td>ZINC12358966</td></tr><tr><td> BAF11σR3</td><td>C<sub>16</sub>H<sub>12</sub>N<sub>2</sub>O<sub>6</sub>S</td><td align="char" char=".">360</td><td>change the aromatic group</td><td align="char" char="plusmn">28 ± 4</td><td>ZINC04900892</td></tr><tr><td> BAF11αNO<sub>2</sub><sup>-</sup></td><td>C<sub>20</sub>H<sub>12</sub>N<sub>3</sub>O<sub>7</sub>S</td><td align="char" char=".">438</td><td>add charged group (nitro)</td><td align="char" char="plusmn">15 ± 6</td><td>ZINC16218542</td></tr><tr><td> BAF11<sup>ISO</sup>αCOO<sup>-</sup></td><td>C<sub>21</sub>H<sub>12</sub>N<sub>2</sub>O<sub>7</sub>S</td><td align="char" char=".">436</td><td>BAF11 isomer; add charged group (carboxyl)</td><td align="char" char="plusmn">6 ± 5</td><td>ZINC03861030</td></tr><tr><td> BAF11<sup>ISO</sup>αSO<sub>3</sub><sup>-</sup></td><td>C<sub>20</sub>H<sub>11</sub>N<sub>2</sub>O<sub>11</sub>S<sub>3</sub></td><td align="char" char=".">552</td><td>BAF11 isomer; add charged group (sulfate)</td><td align="char" char="plusmn">2 ± 5</td><td>SIGMA-33936</td></tr><tr><td> BAF11ΔOHσR</td><td>C<sub>20</sub>H<sub>14</sub>N<sub>2</sub>O<sub>4</sub>S</td><td align="char" char=".">378</td><td>remove an OH;change the position of the aromatic group</td><td align="char" char="plusmn">15 ± 6</td><td>ZINC04803992</td></tr><tr><td> BAF11ΔOHαSO<sub>3</sub><sup>−</sup></td><td>C<sub>20</sub>H<sub>14</sub>N<sub>2</sub>O<sub>7</sub>S<sub>2</sub></td><td align="char" char=".">458</td><td>remove an OH; add sulfate group</td><td align="char" char="plusmn">12 ± 3</td><td>ZINC03954029</td></tr><tr><td> BAF11ΔOHαR</td><td>C<sub>20</sub>H<sub>18</sub>N<sub>4</sub>O<sub>5</sub>S</td><td align="char" char=".">426</td><td>remove an OH; add additional group to the aromatic ring</td><td align="char" char="plusmn">12 ± 6</td><td>ZINC04416667</td></tr><tr><td> BAF11σOHαR1</td><td>C<sub>24</sub>H<sub>20</sub>N<sub>4</sub>O<sub>4</sub>S</td><td align="char" char=".">461</td><td>swap the position of the OH and aromatics</td><td align="char" char="plusmn">5 ± 5</td><td>ZINC04804174</td></tr><tr><td> BAF11σOHαR2</td><td>C<sub>16</sub>H<sub>19</sub>N<sub>3</sub>O<sub>5</sub>S</td><td align="char" char=".">365</td><td>swap the position of the OH and aromatics</td><td align="char" char="plusmn">4 ± 6</td><td>ZINC17378758</td></tr></tbody></table></table-wrap></p></sec><sec id="s2-5"><title>Inhibition of Aβ<sub>1–42</sub> toxicity by BAFs</title><p>Having identified compounds that bind Aβ fibers, by a structure-based procedure, we tested their effects on the cytotoxicity of Aβ<sub>1–42</sub> fiber against two mammalian cell lines: PC12 and HeLa (<xref ref-type="fig" rid="fig3">Figure 3</xref>). Five BAFs—1,4,8,11, and 12—in the initial cycle and three additional BAFs—26, 30, and 31—from the second cycle, with diversified chemical structures shown in <xref ref-type="fig" rid="fig4">Figure 4</xref>, significantly increased both PC12 and HeLa cell survival after 24 hr incubation with Aβ<sub>1–42</sub> (0.5 µM) at concentration of 2.5 µM, while the BAFs alone had little or no effect on cell survival (<xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1</xref>). Three BAFs—11, 26, and 31—showed clear dose-response profiles in their protection of both PC12 and HeLa cells (<xref ref-type="fig" rid="fig3">Figure 3B</xref>). Among them, the two best BAFs—26 and 31—were tested and did not affect the cytotoxicity of amyloid fibers other than Aβ (<xref ref-type="fig" rid="fig3s2">Figure 3—figure supplement 2</xref>). Although all of these BAFs provide protection against Aβ toxicity, none diminish the amount of Aβ fibers in electron micrographs (<xref ref-type="fig" rid="fig3">Figure 3C</xref>).<fig-group><fig id="fig3" position="float"><object-id pub-id-type="doi">10.7554/eLife.00857.013</object-id><label>Figure 3.</label><caption><title>Experimental characterization of compounds that bind to amyloid fibers.</title><p>Our newly discovered BAFs diminish Aβ<sub>1–42</sub> toxicity without significantly reducing Aβ<sub>1–42</sub> fibrillation. (<bold>A</bold>). Eight BAFs reduce Aβ toxicity in mammalian cell lines (PC12 in orange; HeLa in green). These identified compounds with diversified chemical structures are quite different from orange G, whose co-crystal structure with an amyloid segment is the basis of our approach (<xref ref-type="fig" rid="fig4">Figure 4</xref> and <xref ref-type="table" rid="tbl2">Table 2</xref>). For each compound, 2 to 4 repeats of each independent experiment were performed. For each experimental repeat, four replicates per sample per concentration were tested. The symbol * indicates a p<0.1; the symbol ** indicates a p<0.01 and the symbol *** indicates a p<0.001. The student’s <italic>t</italic>-test and p-value analysis are in <xref ref-type="table" rid="tbl4">Table 4</xref>. (<bold>B</bold>). The representative BAFs—31, 26, and 11—inhibit Aβ cyto-toxicity in a dose-dependent manner. (<bold>C</bold>). Transmission electron microscopy (TEM) images of Aβ fibers alone and Aβ fibers with the BAFs, the same samples prepared for cell viability assay. All 8 BAFs that diminish Aβ toxicity do not noticeably diminish Aβ fibrillation. Scale bars indicate 200 nm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00857.013">http://dx.doi.org/10.7554/eLife.00857.013</ext-link></p></caption><graphic xlink:href="elife00857f003"/></fig><fig id="fig3s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00857.014</object-id><label>Figure 3—figure supplement 1.</label><caption><title>The BAFs alone exhibit little or no toxicity on mammalian cell lines.</title><p>Incubating cells with or without BAFs for 24 hours caused little or no change for cell viability of both PC12 and HeLa. The error bars are calculated from four experiment replicates.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00857.014">http://dx.doi.org/10.7554/eLife.00857.014</ext-link></p></caption><graphic xlink:href="elife00857fs006"/></fig><fig id="fig3s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00857.015</object-id><label>Figure 3—figure supplement 2.</label><caption><title>BAFs cannot reduce the cytotoxicity of amyloid fibers formed by IAPP and α-synuclein, as much as those fibers formed by Aβ.</title><p>The final concentration of IAPP is 1 µM and α-synuclein is 2 µM. The molar ratio of amyloid fibers and BAFs is 1:1. BAFs (26 and 31), which significantly reduces Aβ toxicity (<xref ref-type="fig" rid="fig3">Figure 3</xref>), cannot rescue the toxicity of IAPP and α-synuclein, suggesting that the toxicity alleviating effect of BAFs are specific to the fibers for which they were designed.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00857.015">http://dx.doi.org/10.7554/eLife.00857.015</ext-link></p></caption><graphic xlink:href="elife00857fs007"/></fig></fig-group><table-wrap id="tbl4" position="float"><object-id pub-id-type="doi">10.7554/eLife.00857.016</object-id><label>Table 4.</label><caption><p>Student’s <italic>t</italic>-test and p value analysis suggests that BAFs reduce the cytotoxicity of Aβ fibers significantly</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00857.016">http://dx.doi.org/10.7554/eLife.00857.016</ext-link></p></caption><table frame="hsides" rules="groups"><thead><tr><th rowspan="2"/><th rowspan="2">Average of cell viability (n = 4)</th><th rowspan="2">SD(σ)</th><th colspan="2">Comparison to Aβ fiber alone</th></tr><tr><th><italic>t</italic> value</th><th>p value</th></tr></thead><tbody><tr><td colspan="5">HeLa cell line</td></tr><tr><td> Aβ fiber alone</td><td align="char" char=".">0.40</td><td align="char" char=".">0.05</td><td>/</td><td>/</td></tr><tr><td> BAF1</td><td align="char" char=".">0.66</td><td align="char" char=".">0.04</td><td align="char" char=".">8.4</td><td>5E-05</td></tr><tr><td> BAF4</td><td align="char" char=".">0.93</td><td align="char" char=".">0.13</td><td align="char" char=".">7.4</td><td>1E-4</td></tr><tr><td> BAF8</td><td align="char" char=".">0.54</td><td align="char" char=".">0.06</td><td align="char" char=".">3.3</td><td>1E-2</td></tr><tr><td> BAF11</td><td align="char" char=".">0.69</td><td align="char" char=".">0.07</td><td align="char" char=".">6.6</td><td>2E-04</td></tr><tr><td> BAF12</td><td align="char" char=".">0.63</td><td align="char" char=".">0.04</td><td align="char" char=".">7.6</td><td>1E-04</td></tr><tr><td> BAF26</td><td align="char" char=".">0.68</td><td align="char" char=".">0.14</td><td align="char" char=".">3.8</td><td>5E-3</td></tr><tr><td> BAF30</td><td align="char" char=".">0.51</td><td align="char" char=".">0.08</td><td align="char" char=".">2.3</td><td>4E-2</td></tr><tr><td> BAF31</td><td align="char" char=".">0.91</td><td align="char" char=".">0.07</td><td align="char" char=".">11.5</td><td>7E-06</td></tr><tr><td colspan="5">PC12 cell line</td></tr><tr><td> Aβ fiber alone</td><td align="char" char=".">0.37</td><td align="char" char=".">0.07</td><td>/</td><td>/</td></tr><tr><td> BAF1</td><td align="char" char=".">0.61</td><td align="char" char=".">0.07</td><td align="char" char=".">4.9</td><td>1E-3</td></tr><tr><td> BAF4</td><td align="char" char=".">0.90</td><td align="char" char=".">0.11</td><td align="char" char=".">8.0</td><td>7E-05</td></tr><tr><td> BAF8</td><td align="char" char=".">0.53</td><td align="char" char=".">0.07</td><td align="char" char=".">3.2</td><td>1E-2</td></tr><tr><td> BAF11</td><td align="char" char=".">0.69</td><td align="char" char=".">0.07</td><td align="char" char=".">6.5</td><td>2E-4</td></tr><tr><td> BAF12</td><td align="char" char=".">0.49</td><td align="char" char=".">0.04</td><td align="char" char=".">2.9</td><td>2E-2</td></tr><tr><td> BAF26</td><td align="char" char=".">0.74</td><td align="char" char=".">0.13</td><td align="char" char=".">5.0</td><td>1E-3</td></tr><tr><td> BAF30</td><td align="char" char=".">0.60</td><td align="char" char=".">0.11</td><td align="char" char=".">3.5</td><td>8E-3</td></tr><tr><td> BAF31</td><td align="char" char=".">0.95</td><td align="char" char=".">0.14</td><td align="char" char=".">7.4</td><td>1E-4</td></tr></tbody></table><table-wrap-foot><fn><p>The Student’s T-test and p-value are based on the comparison to Aβ fiber alone.</p></fn></table-wrap-foot></table-wrap><fig id="fig4" position="float"><object-id pub-id-type="doi">10.7554/eLife.00857.017</object-id><label>Figure 4.</label><caption><title>Diversified chemical structures of 8 active BAF compounds that reduce Aβ toxicity.</title><p>Orange G in an orange box is also displayed for comparison.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00857.017">http://dx.doi.org/10.7554/eLife.00857.017</ext-link></p></caption><graphic xlink:href="elife00857f004"/></fig></p></sec><sec id="s2-6"><title>Validation of compound binding by NMR titration</title><p>Promising candidate binders from in silico screening and toxicity tests were validated by titration of Aβ fibers into solutions of each compound, as monitored by NMR signals of aromatic protons of the compound (<xref ref-type="fig" rid="fig5">Figure 5</xref>). The proton resonances of the freely rotating compounds disappear as the compound binds to the fibers. By increasing the amount of fibers, an apparent Kd for compound binding can be estimated. From in silico screening, all tested BAF compounds are calculated to bind more tightly to Aβ fibers than orange G. In NMR studies, the apparent Kd of orange G binding to Aβ<sub>16–21</sub> fibers was found to be 43 ± 21 µM, whereas the apparent Kd of BAF1 binding to Aβ<sub>16–21</sub> fibers is 12 ± 7 µM. BAFs were found to bind to both Aβ<sub>16–21</sub> fibers and Aβ<sub>1–42</sub> fibers. <xref ref-type="fig" rid="fig5">Figure 5F</xref> shows a notable correlation between the calculated binding energies and the reduction in NMR peak areas upon Aβ binding. That is, all BAFs with predicted binding energy better than orange G also reduce NMR peak areas more than orange G. On the other hand, BAF31ΔOH, a derivative of BAF31 by removal of a key hydroxyl group essential for binding, exhibits both a worse calculated binding energy and a diminished reduction of NMR peak upon titration of Aβ<sub>1–42</sub> fibers.<fig-group><fig id="fig5" position="float"><object-id pub-id-type="doi">10.7554/eLife.00857.018</object-id><label>Figure 5.</label><caption><title>NMR evidence for binding of compounds to both Aβ<sub>16–21</sub> and Aβ<sub>1–42</sub> fibers.</title><p>NMR binding experiments were performed on BAF compounds and the dye orange G. By monitoring the aromatic regions of the <sup>1</sup>H NMR spectra of BAFs 1, 8, and 31, these compounds were shown to bind to both Aβ<sub>16–21</sub> and Aβ<sub>1–42</sub> fibers more tightly than does orange G. As shown in (<bold>A</bold> and <bold>B</bold>), BAF1 binds to Aβ<sub>16–21</sub> fibers with affinity stronger than orange G. The determination of binding parameters for Aβ<sub>16–21</sub> fibers is detailed in <xref ref-type="table" rid="tbl5">Table 5</xref> and <xref ref-type="fig" rid="fig5s1 fig5s3">Figure 5—figure supplements 1 and 3</xref>. In panel (<bold>A</bold>)<bold>,</bold> the <sup>1</sup>H NMR spectrum of compound BAF1 (at 100 μM) is shown as a function of increasing concentration of Aβ<sub>16–21</sub> fibers (0–500 μM, as monomer). The insert shows the area decrease of BAF1 NMR peaks as a function of Aβ<sub>16–21</sub> concentration, and the red curve fitting the data defines an apparent Kd of 12 ± 7 µM. In panel (<bold>B</bold>), the NMR spectrum of orange G (50 μM) is plotted against increasing concentration of Aβ<sub>16–21</sub> fibers (0–950 μM), giving an apparent Kd of 43 ± 21 µM. In (<bold>C</bold>, <bold>D</bold> and <bold>E</bold>), BAFs 1 and 8 both bind to Aβ<sub>1–42</sub> fibers more strongly than orange G. Notice that the molar ratio of BAFs to Aβ<sub>1–42</sub> fibers is comparable to that used in cell toxicity assays (<xref ref-type="fig" rid="fig3">Figure 3</xref>). (<bold>F</bold>). The calculated binding energies of BAFs—1, 8, and 31—to Aβ<sub>1–42</sub> fibers are compared to the decreases in NMR peak of these compounds upon their binding to full-length Aβ fibers. These three BAFs have higher affinities and a larger NMR peak reduction than orange G while the ‘knock-out’ derivative with removal of key interactions (BAF31ΔOH) discussed below has a weaker calculated affinity and a smaller NMR peak reduction than orange G. We observe good correlation between computed energies and experimental data from NMR.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00857.018">http://dx.doi.org/10.7554/eLife.00857.018</ext-link></p></caption><graphic xlink:href="elife00857f005"/></fig><fig id="fig5s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00857.019</object-id><label>Figure 5—figure supplement 1.</label><caption><title>NMR peak assignment of BAF1 with Aβ<sub>16–21</sub> fiber.</title><p>The 1D 1H NMR spectrum shows the aromatic proton regions of BAF1 upon the titration of Aβ<sub>16–21</sub> fibers shown in <xref ref-type="fig" rid="fig5">Figure 5A</xref>. The insert is the chemical structure of BAF1 with the color-labeled aromatic proton observed in the NMR spectrum. The arrows with different colors indicate the proton assignment for NMR peaks.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00857.019">http://dx.doi.org/10.7554/eLife.00857.019</ext-link></p></caption><graphic xlink:href="elife00857fs008"/></fig><fig id="fig5s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00857.020</object-id><label>Figure 5—figure supplement 2.</label><caption><title>NMR peak assignment of the control compound orange G with Aβ<sub>16–21</sub> fiber.</title><p>The 1D 1H NMR spectrum shows the aromatic proton regions of orange G against the increasing concentrations of Aβ<sub>16–21</sub> fibers shown in <xref ref-type="fig" rid="fig5">Figure 5B</xref>. The insert is the chemical structure of orange G with the highlighted label of the aromatic proton shown in the NMR spectrum. The arrows with different colors indicate the proton assignment for NMR peaks.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00857.020">http://dx.doi.org/10.7554/eLife.00857.020</ext-link></p></caption><graphic xlink:href="elife00857fs009"/></fig><fig id="fig5s3" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00857.021</object-id><label>Figure 5—figure supplement 3.</label><caption><title>NMR titration of BAF8 with Aβ<sub>16–21</sub> fibers.</title><p>To validate our computation methods, NMR titration experiments were performed. (<bold>A</bold>) One representative peak of aromatic protons of the 1D 1H NMR spectra of the compound BAF8 (at 100µM) upon Aβ<sub>16–21</sub> fibers titration (0–500 µM, monomer equivalent). (<bold>B</bold>) Fitting curve upon the area decrease of BAF8 NMR peaks as a function of fiber concentration. The apparent Kd of BAF8 (24 ± 5 µM) is lower than that of orange-G (<xref ref-type="fig" rid="fig4">Figure 4B</xref>), indicating the tighter binding affinity of BAF8 to Aβ<sub>16–21</sub> fibers.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00857.021">http://dx.doi.org/10.7554/eLife.00857.021</ext-link></p></caption><graphic xlink:href="elife00857fs010"/></fig></fig-group><table-wrap id="tbl5" position="float"><object-id pub-id-type="doi">10.7554/eLife.00857.022</object-id><label>Table 5.</label><caption><p>Predicted binding energy and experimental measurement of the binding of two BAFs and orange G against both Aβ<sub>16–21</sub> (KLVFFA) and full-length Aβ fibers</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00857.022">http://dx.doi.org/10.7554/eLife.00857.022</ext-link></p></caption><table frame="hsides" rules="groups"><thead><tr><th rowspan="2"/><th colspan="2">Binding to KLVFFA fiber</th><th colspan="2">Binding to Aβ fiber</th></tr><tr><th>Predicted binding energy (kcal/mol)</th><th>NMR Kd (µM)</th><th>Predicted binding energy (kcal/mol)</th><th>NMR peak reduction (%)</th></tr></thead><tbody><tr><td>BAF1</td><td align="char" char=".">−8</td><td align="char" char=".">12</td><td align="char" char=".">−10</td><td align="char" char=".">8</td></tr><tr><td>BAF8</td><td align="char" char=".">−12</td><td align="char" char=".">24</td><td align="char" char=".">−12</td><td align="char" char=".">13</td></tr><tr><td>orange G</td><td align="char" char=".">−8</td><td align="char" char=".">43</td><td align="char" char=".">−9</td><td align="char" char=".">6</td></tr></tbody></table><table-wrap-foot><fn><p>The determination of the binding parameters with KLVFFA fiber is detailed in <xref ref-type="table" rid="tbl6">Table 6</xref>.</p></fn></table-wrap-foot></table-wrap><table-wrap id="tbl6" position="float"><object-id pub-id-type="doi">10.7554/eLife.00857.023</object-id><label>Table 6.</label><caption><p>Comparison of the measured binding parameters of the representative BAFs with orange G by NMR titrations</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00857.023">http://dx.doi.org/10.7554/eLife.00857.023</ext-link></p></caption><table frame="hsides" rules="groups"><thead><tr><th>Compound</th><th>Predicted binding energy (kcal/mol)</th><th>f<sub>max</sub></th><th>Kd (µM)</th></tr></thead><tbody><tr><td>BAF1</td><td align="char" char=".">−8</td><td align="char" char="plusmn">0.47 ± 0.04</td><td align="char" char="plusmn">12 ± 7</td></tr><tr><td>BAF8</td><td align="char" char=".">−12</td><td align="char" char="plusmn">0.82 ± 0.04</td><td align="char" char="plusmn">24 ± 5</td></tr><tr><td>Orange-G</td><td align="char" char=".">−8</td><td align="char" char="plusmn">0.46 ± 0.06</td><td align="char" char="plusmn">43 ± 21</td></tr></tbody></table><table-wrap-foot><fn><p>The second column lists the predicted binding energy for each top docked model of BAF compounds with KLVFFA fiber, and the binding energy of Orange-G with KLVFFA fiber were also calculated for comparison. Our computational method identified the BAF with better fit to the binding interface than Orange-G. We then used NMR titration to determine the binding affinity. Our previous mass spectrometric analyses of the crystal of the Orange-G with KLVFFA fibers have suggested a binding ratio of compound:fiber with the range of 1:1 to 1:10 (<xref ref-type="bibr" rid="bib36">Landau et al., 2011</xref>). Together with our structural models and single binding site assumption, we estimated the binding ratio to be 1:3. Accordingly, calculated NMR binding parameters are listed in the table. The third column f<sub>max</sub> is the maximum fraction of NMR signal decrease of compound upon binding saturation (‘Materials and methods’).</p></fn></table-wrap-foot></table-wrap></p></sec><sec id="s2-7"><title>Structure-activity relationship studies of the Aβ pharmacorphore</title><p>Based on the lead compounds found in the initial cycle of the procedure, we carried out a second cycle to expand our understanding of the Aβ pharmacorphore. BAF11 (<xref ref-type="fig" rid="fig6">Figure 6A</xref>), one of the lead compounds in the initial cycle, was used to perform structure-activity relationship studies. Twelve derivatives of BAF11 were scanned to pinpoint the essential apolar and polar interactions for the pharmacorphore refinement (<xref ref-type="fig" rid="fig6">Figure 6B</xref>, <xref ref-type="fig" rid="fig6s1">Figure 6—figure supplement 1</xref>). These derivatives are grouped in five classes, whose effects on Aβ toxicity have been tested (<xref ref-type="fig" rid="fig6">Figure 6C</xref>). Classes I and II assess the polar region of BAF11, which makes hydrogen bonds to charged Lys16 ladders of the Aβ fiber: the deletion of the hydroxyl group (Class I) significantly decreased the inhibition of toxicity; the swapping of the hydroxyl group with the aromatic tail (Class II) almost abolished inhibition of toxicity. Classes III, IV, and V focused on the aromatic moieties of BAF11: altering the sizes of aromatic groups (Class III) showed little change in inhibition of toxicity while adding charged or polar groups within aromatic region (Classes IV and V) resulted in a significant decrease of inhibition of toxicity. These differences among BAF11 derivatives in inhibition of toxicity (<xref ref-type="fig" rid="fig6">Figure 6C</xref>) further validated our structure-based approach and provided guidelines for the refinement of Aβ pharmacophore.<fig-group><fig id="fig6" position="float"><object-id pub-id-type="doi">10.7554/eLife.00857.024</object-id><label>Figure 6.</label><caption><title>Refinement of the Aβ pharmacorphore based on studies of BAF11.</title><p>(<bold>A</bold>) Atomic model of BAF11 from the initial cycle docked on the full-length Aβ fiber, viewed in perpendicular to the fiber axis (left panel) and down the fiber axis (right panel). BAF11 is shown as a cyan stick model, whose polar groups form hydrogen bonds (green thick lines) to Lys16 of Aβ. The extensive non-polar interactions arise from the flat aromatic rings of BAF11 packing against the hydrophobic surface formed by Val18 and Phe20 of Aβ. (<bold>B</bold>) Schematic representation of the polar and nonpolar interactions of BAF11 and its derivatives modeled on the Aβ fiber (in orange and light brown). In the process of the Aβ pharmacophore refinement, five different classes (I–V) of BAF11 derivatives were introduced into the second cycle of screening, to expand the BAF set and to assess the specificity of the compounds identified in the initial cycle. The full description and chemical structure of each derivative are in <xref ref-type="table" rid="tbl3">Table 3</xref> and <xref ref-type="fig" rid="fig5s1">Figure 5—figure supplement 1</xref>. (<bold>C</bold>) Comparison of the toxicity inhibition (defined in ‘Materials and methods’) among five types of BAF11 derivatives after 24 hr incubation with Aβ (0.5 µM). Notice that all changes to BAF11 which remove binding groups diminish its effectiveness as an inhibitor of toxicity.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00857.024">http://dx.doi.org/10.7554/eLife.00857.024</ext-link></p></caption><graphic xlink:href="elife00857f006"/></fig><fig id="fig6s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00857.025</object-id><label>Figure 6—figure supplement 1.</label><caption><title>Chemical structures of the lead compound BAF11 and its derivatives.</title><p>12 derivatives of the lead compound BAF11 were included to expand the set during the refinement of amyloid pharmacophore (<xref ref-type="fig" rid="fig5">Figure 6C</xref>). (<bold>A</bold>) Chemical structures of BAF11 derivatives. A magenta open circle indicates the deletion of the important hydroxyl group. A green open circle indicates the missing of aromatic atoms in hydrophobic region of BAF11. The red color in chemical structures indicates the addition of atoms or groups to BAF11. The full description of each derivative is in <xref ref-type="table" rid="tbl3">Table 3</xref>. (<bold>B</bold>) Comparison of toxicity inhibition among BAF11 derivatives after 24 hour incubation with Aβ fibers.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00857.025">http://dx.doi.org/10.7554/eLife.00857.025</ext-link></p></caption><graphic xlink:href="elife00857fs011"/></fig></fig-group></p><p>In the second cycle, nine new compounds were derived from the refined pharmacophore (<xref ref-type="fig" rid="fig7">Figure 7</xref>). Three of them detoxified Aβ in cell survival assay. BAF31, the best inhibitor which protected mammalian cells from Aβ toxicity in the second cycle, increased cell survival from the 40% induced by Aβ alone to >90% (<xref ref-type="fig" rid="fig3">Figure 3</xref>). A derivative of BAF31, BAF31ΔOH, lacking the hydroxyl group believed to bind to the Lys residue of the Aβ fiber (shown by the magenta oval in <xref ref-type="fig" rid="fig8">Figure 8B</xref>), is calculated no longer to bind to the Aβ fiber. NMR and cell viability assessments indicated that BAF31ΔOH binds much less strongly to Aβ fibers than BAF31 itself and shows significantly reduced power to inhibit toxicity (<xref ref-type="fig" rid="fig8">Figure 8E</xref>). Similarly, the detoxifying profile of derivatives of another inhibitor, BAF30, validated the key interactions of BAF30 across the binding interface (<xref ref-type="fig" rid="fig9">Figure 9</xref>). Our conclusion is that the NMR binding and toxicity results for the BAF derivatives studied are consistent with our model for the pharmacophore of Aβ (<xref ref-type="fig" rid="fig10">Figure 10</xref>).<fig id="fig7" position="float"><object-id pub-id-type="doi">10.7554/eLife.00857.026</object-id><label>Figure 7.</label><caption><title>New BAFs derived from the refined amyloid pharmacophore.</title><p>(<bold>A</bold>). Amyloid pharmacophore based on the structural overlay of active BAFs and derivatives. The overlay of the lead compounds from the initial round (BAF4, BAF8, and BAF11) elucidated the consensus of polar and nonpolar interactions at fiber binding interfaces, which sheds light on the amyloid pharmacophore. The amyloid pharmacophore was further refined by iterative approaches of computational docking and experimental testing. The derivatives of those lead compounds were tested to explore the essential role of those consensus interactions, and the differences of binding patterns and toxicity inhibition effects of the BAF derivatives can provide a guideline for the further refinement of amyloid pharmacophore. (<bold>B</bold>). New BAFs were ‘designed’ based on the refined pharmacophore. One successful example, BAF31 (green sticks) derived from the pharmacophore (grey sticks), showed the enhanced capability of inhibiting Aβ toxicity (<xref ref-type="fig" rid="fig8">Figure 8C</xref>). The success of developing enhanced binder from pre-defined pharmacophore highlights the important role of iterative docking/test approach in structure-based drug development.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00857.026">http://dx.doi.org/10.7554/eLife.00857.026</ext-link></p></caption><graphic xlink:href="elife00857f007"/></fig><fig-group><fig id="fig8" position="float"><object-id pub-id-type="doi">10.7554/eLife.00857.027</object-id><label>Figure 8.</label><caption><title>Elimination of one key hydrogen bond from BAF31 causes both the loss of NMR binding to Aβ fibers and the decrease in inhibition of Aβ cyto-toxicity.</title><p>(<bold>A</bold>) Atomic model of the new inhibitor BAF31 (our most tightly binding BAF) derived from the refined pharmacophore (<xref ref-type="fig" rid="fig7">Figure 7</xref>, <xref ref-type="fig" rid="fig1">Figure 1F</xref>) in the second cycle, viewed perpendicular to the fiber axis on the left and down the fiber axis on the right. In panel (<bold>B</bold>), one important hydroxyl group forming hydrogen bonds to Lys16 residue of Aβ is highlighted by a magenta circle. (<bold>C</bold>) A representative NMR band (left panel) of mixture of Aβ fiber with the compound BAF31 compares with that (right panel) of Aβ fiber the derivative BAF31ΔOH which omits that important hydroxyl group. Their full NMR spectrums showing the same trend are shown in <xref ref-type="fig" rid="fig8s1">Figure 8—figure supplement 1</xref>. (<bold>D</bold>) Cell survival rates after 24 hr incubation with Aβ (0.5 µM), the molar ratio (1:5) of Aβ and the compound is comparable with the ratio in NMR binding experiment (<bold>C</bold>). (<bold>E</bold>) Notably, the elimination of one hydrogen bond from BAF31 (the derivative BAF31ΔOH) causes both the marked decrease in inhibition of Aβ toxicity to HeLa cells (<bold>D</bold>) and the loss of NMR binding to Aβ fibers (<bold>C</bold>).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00857.027">http://dx.doi.org/10.7554/eLife.00857.027</ext-link></p></caption><graphic xlink:href="elife00857f008"/></fig><fig id="fig8s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00857.028</object-id><label>Figure 8—figure supplement 1.</label><caption><title>NMR titration of BAF31 and its derivative with the Aβ<sub>1–42</sub> fiber.</title><p>(<bold>A</bold>). 1D 1H NMR spectrum of BAF31 (100 µM) without (in black) and with Aβ<sub>1–42</sub> fiber (12.5 µM monomer equivalent, in a green color). The magnified peaks are shown in the right panel to highlight the peak differences. (<bold>B</bold>). NMR spectrum of its derivative BAF31ΔOH (100 µM) when BAF31 is modified by the removal of a key hydroxyl group, without or with Aβ<sub>1–42</sub> fiber (0 µM, 12.5 µM). The significant difference in NMR signal reduction between the BAF31 and BAF31ΔOH further validates the model of BAF31docked onto Aβ fibers.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00857.028">http://dx.doi.org/10.7554/eLife.00857.028</ext-link></p></caption><graphic xlink:href="elife00857fs012"/></fig></fig-group><fig id="fig9" position="float"><object-id pub-id-type="doi">10.7554/eLife.00857.029</object-id><label>Figure 9.</label><caption><title>Analysis of the lead compound BAF30 and its derivatives.</title><p>Structural models of BAF30 (green sticks) docked on Aβ fiber structure (in a light yellow color) are shown in (<bold>A</bold> and <bold>B</bold>). The important polar (black hydrogen bonds) interaction between BAF30 and single β-sheet of Aβ fiber, as well as shape complimentary between the aromatic rings of BAF30 and the hydrophobic patches of Aβ fiber are highlighted respectively. Schematic representation of the polar and nonpolar interactions of BAF30 with Aβ fiber is shown in panel (<bold>C</bold>). The magenta circles highlight two important hydroxyl groups which are absent in BAF30 derivatives. (<bold>D</bold>). The chemical structure of each derivative is listed. The dark blue open circles indicate the deletion of the important hydroxyl group. The red color in chemical structures indicates the addition of atoms or groups to BAF30. (<bold>E</bold>). HeLa cell survival rates in the presence of Aβ (0.5 µM monomer equivalent) and BAF30 or the derivatives are compared. The hydrogen bonds between BAF30 and Lys16 residues of Aβ fiber are important for binding of Aβ fiber and inhibition of Aβ toxicity. With additional groups at the opposite side of hydrogen binding sites, the derivative BAF30αR showed little change in toxicity inhibition. However, two BAF30 derivatives (σOH<sup>A</sup>αOH and σOH<sup>A</sup>ΔOH<sup>B</sup>αCOO), which alter or delete the two important hydroxyl groups (magenta circles in panel <bold>C</bold>) of BAF30 that form hydrogen bonds to Lys16, showed a significant decrease in the toxicity inhibition. Furthermore, when BAF30 was modified by shifting both hydroxyl groups (<bold>A</bold> and <bold>B</bold>) to their neighboring positions, the derivative BAF30σOHABαCH<sub>3</sub> almost lost the inhibition of Aβ toxicity. The rescuing percentage (%) is defined in ‘Materials and methods’.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00857.029">http://dx.doi.org/10.7554/eLife.00857.029</ext-link></p></caption><graphic xlink:href="elife00857f009"/></fig><fig id="fig10" position="float"><object-id pub-id-type="doi">10.7554/eLife.00857.030</object-id><label>Figure 10.</label><caption><title>General rule of the essential interactions between BAFs and Aβ fiber can be derived from structure-based screening of Aβ toxicity inhibitor.</title><p>The carbonyl group is used to represent the H-bond acceptor (or negative charge) of BAFs, and the naphthalene ring is used to represent the planar aromatic portion of BAFs. Based on the rounds of computing search and experimental test, the detailed description about essential interactions and geometrical parameters are in ‘Materials and methods’.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00857.030">http://dx.doi.org/10.7554/eLife.00857.030</ext-link></p></caption><graphic xlink:href="elife00857f010"/></fig></p></sec></sec><sec id="s3" sec-type="discussion"><title>Discussion</title><sec id="s3-1"><title>Structure-based discovery of compounds that bind amyloid fibers</title><p>Amyloid fibers differ fundamentally in structure from the enzymes and signaling proteins that are the traditional targets in structure based design of binding compounds, and thus their pharmacophores might be expected to differ fundamentally as would the types of compounds that bind. In general, the binding sites of the traditional targets are often concave pockets; in contrast, the surfaces of amyloid fibers are flat and repetitive along the fiber axis, without well-defined surface cavities. The widely used ligand-docking software, such as DOCK (<xref ref-type="bibr" rid="bib17">Ewing et al., 2001</xref>), or AutoDock (<xref ref-type="bibr" rid="bib44">Morris et al., 2009</xref>), is intended to fit well-defined protein pockets rather than shallow grooves at flat fiber surfaces.</p><p>Consequently we have adapted the RosettaLigand program (<xref ref-type="bibr" rid="bib12">Davis and Baker, 2009</xref>) for docking a library of commercially available compounds onto the flat surface of amyloid fibers. Similarly to other software packages, RosettaLigand scores each candidate compound for its energetic fit to its binding site. The initial site is chosen near that occupied by a bound compound, as determined in a crystal structure. The conformational flexibilities of ligand and protein side chains are modeled in a ‘near-native’ perturbation fashion (‘Materials and methods’), meaning that the fine sampling of conformations was restrained to be close to the starting conformation. To find the position along the flat fibrillar surface of greatest binding energy for each candidate compound, our screening approach leverages the rotamer repacking algorithm (<xref ref-type="bibr" rid="bib39">Leaver-Fay et al., 2011</xref>) and Rosetta energy function (<xref ref-type="bibr" rid="bib32">Kuhlman and Baker, 2000</xref>) to account for flexibility of protein side chains and ligand, which is critical in modeling of such shallow grooves on the fiber surface.</p><p>Our procedure identified 34 BAF compounds predicted to bind to Aβ fibers, among which eight BAFs diminish the toxicity of the fibers in mammalian cells. We suggest that the same procedure can be used to discover other compounds that reduce the toxicity of Aβ fibers, starting from other co-crystal structures of Aβ segments with other bound ligands. Similarly, the same procedure can be applied to the discovery of compounds that bind to other amyloid proteins, for use as either toxicity inhibitors or imaging agents for amyloid diagnosis.</p></sec><sec id="s3-2"><title>Mechanism of inhibition of Aβ toxicity</title><p>Our observation is that our tightest binding BAFs all diminish the toxicity of Aβ fibers, and yet do not substantially diminish the amount of fibers. Further study will be required to understand the molecular mechanism underlying the inhibition of Aβ toxicity, but here we offer the following hypothesis.</p><p>Emerging evidence suggest<bold>s</bold> that amyloid oligomers, rather than amyloid fibers, are toxic entities (<xref ref-type="bibr" rid="bib23">Hartley et al., 1999</xref>; <xref ref-type="bibr" rid="bib8">Cleary et al., 2005</xref>; <xref ref-type="bibr" rid="bib55">Silveira et al., 2005</xref>), and that perhaps toxic oligomers can be released from amyloid fibers (<xref ref-type="bibr" rid="bib59">Xue et al., 2009</xref>; <xref ref-type="bibr" rid="bib10">Cremades et al., 2012</xref>; <xref ref-type="bibr" rid="bib30">Krishnan et al., 2012</xref>; <xref ref-type="bibr" rid="bib54">Shahnawaz and Soto, 2012</xref>). By binding to fibers, BAFs stabilize them, thereby shifting the equilibrium of Aβ molecules from smaller, toxic entities towards the fibrillar state. The BAF compounds in their computationally docked sites on Aβ fibers contact several (as few as three and as many as six) adjacent β-strands of the fiber. By creating a low energy binding interface across several fiber strands, the BAFs apparently stabilize the Aβ fibers from breaking into smaller entities.</p><p>From previous studies, we expect BAFs to bind to amyloid fibers rather than oligomers. In recent work (<xref ref-type="bibr" rid="bib35">Laganowsky et al., 2012</xref>; <xref ref-type="bibr" rid="bib40">Liu et al., 2012</xref>), we proposed that amyloid forming proteins can enter either of two distinct aggregation pathways, which are separated by an energy barrier. One pathway leads to in-register fibers in which every β-strand lies directly above or below an identical strand in the fiber. The other pathway leads to out-of-register oligomers in which antiparallel β-strands are sheared relative to one another and roll into a β-barrel. We found that three out-of-register amyloid-like structures exhibit cytotoxicity (<xref ref-type="bibr" rid="bib35">Laganowsky et al., 2012</xref>; <xref ref-type="bibr" rid="bib40">Liu et al., 2012</xref>), which tend to be transient, equilibrating eventually into in-register fibers. In our approach, we search for BAFs based on in-register β-sheets rather than out-of-register β-strands found in toxic oligomeric structures, to which our BAFs are not expected to bind (<xref ref-type="fig" rid="fig11">Figure 11</xref>). We speculate that BAFs stabilize the in-register fibers revealed by our steric zippers, relative to out-of-register toxic oligomers, thereby shifting the equilibrium from toxic oligomers towards fibers (<xref ref-type="fig" rid="fig12">Figure 12</xref>). Supporting this is our result that diminished toxicity accompanies compound binding.<fig-group><fig id="fig11" position="float"><object-id pub-id-type="doi">10.7554/eLife.00857.031</object-id><label>Figure 11.</label><caption><title>BAFs are designed to bind to in-register β-sheets, rather than out-of-register β-sheets.</title><p>As illustrated in (<bold>A</bold>), BAFs bind to in-register β-sheets. Our structure-based approach searches for BAFs based on in-register β-sheets in Aβ fibers. These BAFs are predicted to bind along the flat hydrophobic surfaces of the fibers and are anchored by polar sidechains of Lysine residues. The Cβ distances between the Lys residues interacting with the BAFs are ∼9.6 Å following the stacked arrangement of in-register β-sheets. Orange G, as well as screened BAFs, favorably interact with the in-register fiber and are compatible with the geometry of the Lys residues aligned in in-register β-sheets. As illustrated in (<bold>B</bold>), BAFs cannot bind to out-of-register β-sheets. The estimation of Cβ distance between the lysine residues, based on three out-of-register β-sheets structures previously determined (<xref ref-type="bibr" rid="bib35">Laganowsky et al., 2012</xref>; <xref ref-type="bibr" rid="bib40">Liu et al., 2012</xref>), ranges from 11 Å to 14 Å, quite different from the ∼9.6 Å measured in in-register β-sheet. We speculate that the BAFs are unable to bind to out-of-register β-sheets, and this difference accounts for the diminished toxicity that accompanies compound binding. Supporting this is our in vitro cell toxicity tests (<xref ref-type="table" rid="tbl7">Table 7</xref> and <xref ref-type="fig" rid="fig11s1">Figure 11—figure supplement 1</xref>).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00857.031">http://dx.doi.org/10.7554/eLife.00857.031</ext-link></p></caption><graphic xlink:href="elife00857f011"/></fig><fig id="fig11s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00857.032</object-id><label>Figure 11—figure supplement 1.</label><caption><title>Active BAFs show no or little effects on the cyto-toxicity of pre-formed Aβ oligomers.</title><p>To assess if BAFs inhibit Aβ toxicity by directly interfering with toxic Aβ oligomers, four BAFs —1,11,26,31—, showing the inhibition to Aβ toxicity, were incubated with pre-formed Aβ oligomer and then tested by MTT cell viability assay using HeLa cell line. None of the BAFs significantly reduces toxicity of pre-formed Aβ oligomer. Aβ oligomer was prepared by incubating purified Aβ<sub>1–42</sub> in PBS for 4 hr at 37°C at the concentration of 5 µM without agitation. Pre-formed Aβ oligomer was mixed with four different BAFs (Aβ<sub>1–42</sub>/BAFs = 1:1 molar ratio) and further incubated for 15 min to allow potential binding of BAFs to pre-formed oligomer. The final concentration of Aβ oligomer as monomer is 0.5 µM, the same as what we test Aβ toxicity in MTT cell viability assay.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00857.032">http://dx.doi.org/10.7554/eLife.00857.032</ext-link></p></caption><graphic xlink:href="elife00857fs013"/></fig></fig-group><table-wrap id="tbl7" position="float"><object-id pub-id-type="doi">10.7554/eLife.00857.033</object-id><label>Table 7.</label><caption><p>BAFs reduce Aβ cyto-toxicity by targeting fibers rather than oligomers.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00857.033">http://dx.doi.org/10.7554/eLife.00857.033</ext-link></p></caption><table frame="hsides" rules="groups"><thead><tr><th>Compound</th><th>Inhibition to the cyto-toxicity of Abeta oligomers (%)</th><th>Inhibition to the cyto-toxicity of Abeta fibers (%)</th></tr></thead><tbody><tr><td>BAF1</td><td align="char" char="plusmn">−4 ± 6</td><td align="char" char="plusmn">36 ± 9</td></tr><tr><td>BAF11</td><td align="char" char="plusmn">−9 ± 7</td><td align="char" char="plusmn">7 ± 7</td></tr><tr><td>BAF26</td><td align="char" char="plusmn">−6 ± 6</td><td align="char" char="plusmn">26 ± 7</td></tr><tr><td>BAF31</td><td align="char" char="plusmn">−17 ± 15</td><td align="char" char="plusmn">58 ± 7</td></tr></tbody></table><table-wrap-foot><fn><p>The BAF inhibitions of toxicity from either Aβ oligomer or fibers are compared. Four BAFs, which reduce the toxicity of Aβ fibers, show no inhibitory effects to Aβ oligomer toxicity at the equal molar ratio of BAF to Aβ. The inhibition (%) are calculated using the same method defined in ‘Materials and methods’. The toxicity assay of Aβ oligomer is described in <xref ref-type="fig" rid="fig11s1">Figure 11—figure supplement 1</xref>. The toxicity assay of Aβ fiber is the same as that described in <xref ref-type="fig" rid="fig3">Figure 3</xref>.</p></fn></table-wrap-foot></table-wrap><fig id="fig12" position="float"><object-id pub-id-type="doi">10.7554/eLife.00857.034</object-id><label>Figure 12.</label><caption><title>Proposed mechanism of how compound binding increases fiber stability and decreases fiber toxicity.</title><p>BAFs (green) bind to the side of amyloid fibers, stabilizing the fiber, and shifting the equilibrium from smaller and more toxic oligomers towards fibers. This shift in equilibrium reduces amyloid toxicity.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00857.034">http://dx.doi.org/10.7554/eLife.00857.034</ext-link></p></caption><graphic xlink:href="elife00857f012"/></fig></p></sec><sec id="s3-3"><title>BAFs strengthen the hypothesis that Aβ<sub>16–21</sub> fibers reflect essential features of full Aβ fibers</title><p>The identification of BAFs starts with the atomic structure of orange G bound within the fiber-like crystals of Aβ<sub>16–21</sub>, because as yet there is no high-resolution atomic structure available for ligands bound to full-length Aβ fibers. Nevertheless, we found that BAFs diminish toxicity of full-length Aβ fibers. This finding suggests that the steric zipper structure of Aβ<sub>16–21</sub> fibers recapitulates some of the essential structural features of full-length Aβ fibers. We are currently attempting cocrystallization of BAFs with Aβ<sub>16–21</sub> and other steric zipper structures. We speculate that coupled with computational methods, other steric zipper structures could enable the discovery of the lead compounds for inhibitors of other toxic amyloid entities.</p></sec></sec><sec id="s4" sec-type="materials|methods"><title>Materials and methods</title><sec id="s4-1"><title>Computational procedures</title><sec id="s4-1-1"><title>Two choices of compound libraries for structure-based screening</title><p>We generated two sets of purchasable compounds to be screened via the computational docking:<list list-type="order"><list-item><p>Cambridge Structure Database (CSD) set. 102,236 organic compounds, whose crystal structures have R-factor better than 0.1, were extracted from the Cambridge Structure Database (version 5.32 November 2010) using ConQuest. The SMILES string of each structure was then used to locate its purchasing information among the ZINC purchasable set (<ext-link ext-link-type="uri" xlink:href="http://zinc.docking.org/">http://zinc.docking.org/</ext-link>) (<xref ref-type="bibr" rid="bib25">Irwin and Shoichet, 2005</xref>) by OpenBabel package (<ext-link ext-link-type="uri" xlink:href="http://openbabel.org/">http://openbabel.org/</ext-link>) (<xref ref-type="bibr" rid="bib21">Guha et al., 2006</xref>). The fast index table of all SMILES strings of the ZINC purchasable set was generated to allow the fast search of each CSD structure against ZINC purchasable set. CSD structures that failed in locating their purchasing information (i.e., without any hit in searching against ZINC purchasable set) were omitted. A library of 13,918 structures from CSD representing 11,057 compounds were finally compiled, whose purchasing information is annotated by ZINC purchasable database. The complete list of CSD/ZINC entries of these compounds in this CSD set can be found in <xref ref-type="supplementary-material" rid="SD1-data">Supplementary file 1</xref>.</p></list-item><list-item><p>Flat Compound (FC) set. A library of 6589 compounds containing phenol and less than three freely rotatable bonds were extracted from the ZINC database (<ext-link ext-link-type="uri" xlink:href="http://zinc.docking.org/">http://zinc.docking.org/</ext-link>) (<xref ref-type="bibr" rid="bib25">Irwin and Shoichet, 2005</xref>). Those compounds have a common feature of planar aromatic ring, resulting in a ‘flat’ compound. The flat compound library includes compounds with similar chemical structures to naturally fiber-binding molecules, for instance, Thioflavin-T (ThT), Congo red, Green tea epigallocatechin-3-gallate (EGCG), and Curcumin. It also includes many natural phenols, such as gallic acid, ferulic acid, coumaric acid, propyl gallate, epicatechin, epigallocatechin, etc. The complete list of ZINC entries of these compounds in this FC set can be found in <xref ref-type="supplementary-material" rid="SD2-data">Supplementary file 2</xref>.</p></list-item></list></p></sec></sec><sec id="s4-2"><title>Ligand ensemble preparation with near-‘native’ perturbation</title><p>Each molecule in our two compound libraries was prepared for the docking simulations. Hydrogen atoms of each molecule were added for the compounds lacking modeled hydrogens using the program Omega (v. 2.3.2, OpenEye) (<xref ref-type="bibr" rid="bib4">Bostrom et al., 2003</xref>). Ligand atoms were represented by the most similar Rosetta atom type, their coordinates were re-centered to the origin, and their partial charges were assigned by OpenEye’s AM1-BCC implementation. We then generated the ligand perturbation ensemble near the crystal conformation (CSD set) or starting conformation (FC set) of each molecule. For each rotatable bond of the ligand, a small degree torsion angle deviation (±5°) was applied. K-mean clustering method was used to generate the ligand perturbation ensemble and similar/redundant conformations (rmsd to the selected conformation is less than 0.5 Å) were omitted. Finally, up to 100 conformations for each ligand were generated and made available for Rosetta LigandDock.</p></sec><sec id="s4-3"><title>Rosetta LigandDock with additional near ‘native’ perturbation sampling</title><p>We adopted the docking algorithm based on the method previously described in the RosettaLigand docking paper (<xref ref-type="bibr" rid="bib43">Meiler and Baker, 2006</xref>; <xref ref-type="bibr" rid="bib12">Davis and Baker, 2009</xref>). In general, the algorithm includes three stages: coarse-grained stage, Monte Carlo minimization (MCM) stage and gradient-based minimization stage. Whereas the original RosettaLigand method performed a full sampling of torsional degrees of freedom in the internal ligands and protein side-chains, we made modifications to enable the fast run time required by the screening method. Specially, we sampled the ligand and protein side-chain torsion angles in near-‘native’ perturbation fashion, where only the near-‘native’ conformation of side-chain and ligand rotamers were allowed and any conformation far away from the starting conformation was omitted. For each protein side-chain, the deviations (±0.33, 0.67, 1 SD) around each input torsion were applied based on the standard deviation value of the same torsion bin from the backbone-dependent Dunbrack rotamer library. For each internal torsional angle of the ligand, the deviations (±5°) around the input torsion were applied as described above.</p><p>To optimize possible interactions (H-bonding or packing) between compound and fiber, we carried out random perturbations to the TS rigid-body degrees of freedom (5 Å for translational degrees of freedom; 360° for full rotational degrees of freedom) to explore different rigid body arrangements. For each rigid-body perturbation, different conformations of fiber sidechains, and compounds were explored to maximize the binding interactions. We next carried out simultaneous quasi-Newton optimization of the compound rigid body orientation and the sidechain torsion angles, and in some cases, the torsion angles of the compound and the backbone torsion angles in the binding site, using the complete Rosetta energy function.</p></sec><sec id="s4-4"><title>Docking of molecules to KLVFFA and Aβ fibrillar structure</title><p>The structure of KLVFFA fiber was taken from the co-crystal structure of KLVFFA with orange G (pdb entry: 3OVJ) (<xref ref-type="bibr" rid="bib36">Landau et al., 2011</xref>). After removing orange G, the sidechain torsion of KLVFFA was optimized to correct any conformational bias from the presence of orange G, and then the optimized structure were inspected to ensure that sidechain torsions are still within the original conformation of the co-crystal structure. The Aβ fibrillar structure was from ssNMR fiber structure of full-length Aβ (pdb entry: 2LMO) <sup>40</sup>. The same optimization step was applied before docking. The comparison of docking onto both KLVFFA and Aβ fibrillar structure are discussed in <xref ref-type="fig" rid="fig13">Figure 13</xref>.<fig-group><fig id="fig13" position="float"><object-id pub-id-type="doi">10.7554/eLife.00857.035</object-id><label>Figure 13.</label><caption><title>BAFs bind to in-register β-sheets and are compatible to both parallel and antiparallel amyloid β-sheets.</title><p>A subtlety of our procedure for compound discovery is that it involves both parallel (<bold>A</bold>) and antiparallel (<bold>B</bold>) amyloid β-sheets. In the X-ray structure of orange G bound to the segment Aβ<sub>16–21</sub>(KLVFFA) of Aβ, the sheets are antiparallel (<bold>B</bold>). The library of compounds is initially selected based on docking to the antiparallel β-sheet of Aβ<sub>16–21</sub>. In the next step of our procedure, each compound is further screened against the solid-state-NMR-derived model of full-length Aβ fiber, which is a parallel sheet (<bold>A</bold>). The structure models of orange G docked onto Aβ<sub>16–21</sub> structure and full-length Aβ model are shown in <xref ref-type="fig" rid="fig13s1">Figure 13—figure supplement 1</xref>. As simplified here in (<bold>A</bold> and <bold>B</bold>), sulfate ions (red) of orange G are respectively hydrogen bonded to two lysine residues (light brown), which keep nearly identical geometry (the same ∼9.6 Å distance between the two lysine residues) in either parallel or antiparallel sheet. Evidence that orange G, as well as BAF compounds identified by our procedure, all bind to both antiparallel and parallel sheets is given by the NMR experiments summarized in <xref ref-type="fig" rid="fig5">Figure 5</xref>, where orange G and BAFs are shown to bind to both Aβ<sub>16–21</sub> and full-length Aβ fibers. Apparently both parallel and antiparallel amyloid β-sheets are effective in binding to the same compounds.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00857.035">http://dx.doi.org/10.7554/eLife.00857.035</ext-link></p></caption><graphic xlink:href="elife00857f013"/></fig><fig id="fig13s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00857.036</object-id><label>Figure 13—figure supplement 1.</label><caption><title>Structural models of orange G docked onto the antiparallel Aβ<sub>16–21</sub> (A) and parallel full-length Aβ (B) fiber.</title><p>(<bold>A</bold>). The side view of orange G (in an orange color) docked on the Aβ<sub>16–21</sub> fiber (in a grey color) with a predicted binding energy of—8 kcal/mol. (<bold>B</bold>) side view of orange G (in an orange color) docked on the Aβ full fiber (in a light pink color) with a predicted binding energy of—9 kcal/mol. The charge interactions between the orange G and Lysine 16 are highlighted by dark grey lines.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00857.036">http://dx.doi.org/10.7554/eLife.00857.036</ext-link></p></caption><graphic xlink:href="elife00857fs014"/></fig></fig-group></p></sec><sec id="s4-5"><title>Post-docking analysis to rank the compounds</title><p>The docked compounds were filtered based on the following criteria: (1) The docking models with a compound-fiber van der Waals attractive energy > −7.0 kcal/mol were removed; (2) The docking models with a compound-fiber hydrogen-binding energy >−0.2 kcal/mol were eliminated. The remaining docked compounds were then ranked according to the energy of binding of compound to fiber. We used not only the total binding energy but also on each of the energy components separately (Lennard-Jones interactions, solvation, hydrogen bonding, and electrostatics) (<xref ref-type="bibr" rid="bib38">Lazaridis and Karplus, 1999</xref>; <xref ref-type="bibr" rid="bib32">Kuhlman and Baker, 2000</xref>; <xref ref-type="bibr" rid="bib29">Kortemme et al., 2003</xref>) for ranking. The compounds ranked in the top 40% according to all of these measures were selected. Finally, the compounds were ranked by tightest binding energy (<xref ref-type="bibr" rid="bib43">Meiler and Baker, 2006</xref>) and best shape complementarity (<xref ref-type="bibr" rid="bib37">Lawrence and Colman, 1993</xref>).</p></sec><sec id="s4-6"><title>Description of geometrical parameters of the interactions between BAFs and Aβ fiber defined based on structure-based screening of Aβ toxicity inhibitor</title><p>Based on the rounds of computing search and experimental test, general rules of the essential interactions of BAF binding to Aβ fibers are summarized here. As illustrated in <xref ref-type="fig" rid="fig10">Figure 10</xref>, the geometrical parameters of those key interactions are specified as followings:<list list-type="order"><list-item><p>H-bond acceptor (or negative charge) of the inhibitor makes either hydrogen bond or salt bridge to sidechain nitrogen atoms (NZ) of at least two Lysine residues from adjacent Aβ strands along the fiber axis. Our data suggest that the BAFs need to have good contacts across 2 to 4 adjacent Aβ strands, in order to effectively bind to Aβ fiber and reduce Aβ toxicity.</p></list-item><list-item><p>The hydrogen bond or salt bridge described in 1) follows the general rule of H-bond geometry, which are:</p><list list-type="alpha-upper"><list-item><p>Distance (d<sub>1</sub>, as shown in the figure) between the NZ atom of Lys16 and H-bond acceptor atoms of BAFs: 2.8∼3.5 angstrom;</p></list-item><list-item><p>Angle (Θ<sub>1</sub>) at BAF H-bond acceptor atoms:100∼150°;</p></list-item><list-item><p>Angle (Θ<sub>2</sub>) at the NZ atom of Lys16: 130∼180°.</p></list-item></list></list-item><list-item><p>Hydrophobic interactions between the apolar residues (phenylalanine18 and valine 20) and the planar aromatic portion of the compounds. The aromatic portion of compounds should be planar or semi-planar to pack against the flat surface of Aβ which spans across at least two adjacent Aβ strands.</p></list-item><list-item><p>The hydrophobic interactions described in 3) follow the pi-pi stacking geometry, which are:</p><list list-type="alpha-upper"><list-item><p>Distance (d<sub>2</sub>) between the center of the apolar sidechains and the center of BAF aromatic rings: 4.0∼5.0 angstrom;</p></list-item><list-item><p>Dihedral angle (Φ) between the surface plane defined by Phe18 and Val20 and the aromatic ring of the BAFs: 0∼40°.</p></list-item></list></list-item></list></p></sec><sec id="s4-7"><title>Experimental procedures</title><sec id="s4-7-1"><title>Chemicals and reagents</title><p>Chemicals were obtained from a variety of companies (<xref ref-type="table" rid="tbl1">Table 1</xref>) and were of the highest purity available.</p></sec><sec id="s4-7-2"><title>Source of KLVFFA(Aβ<sub>16–21</sub>) and Aβ<sub>1–42</sub> peptide</title><p>N-terminal acetylated and C-terminal amidated KLVFFA(Aβ<sub>16–21</sub>) peptide was synthesized by Celtek Bioscience Peptides (Nashville, TN). Aβ<sub>1–42</sub> peptide was overexpressed through <italic>Escherichia coli</italic> recombinant expression system and was purified as reported previously (<xref ref-type="bibr" rid="bib19">Finder et al., 2010</xref>). The fusion construct for Aβ<sub>1–42</sub> expression contains an N-terminal His tags, followed by 19 repeats of Asn-Ala-Asn-Pro, TEV protease site and the human Aβ<sub>1–42</sub> sequence. Briefly, the fusion construct was expressed into inclusion bodies in <italic>E.coli</italic> BL21(DE3) cells. 8 M urea was used to solubilize the inclusion bodies. Fusion proteins were purified through HisTrap HP Columns, followed by Reversed-phase high-performance liquid chromatography (RP-HPLC). After TEV cleavage, Aβ<sub>1–42</sub> peptide was purified from the cleavage solution by RP–HPLC followed by lyophilization. To disrupt preformed aggregation, lyophilized Aβ<sub>1–42</sub> was resuspended in 100% Hexafluoroisopropanol (HFIP) which was finally removed by evaporation.</p></sec><sec id="s4-7-3"><title>Preparation of KLVFFA (Aβ<sub>16–21</sub>) and Aβ<sub>1–42</sub> fiber samples for 1D <sup>1</sup>H NMR titration measurement</title><p>KLVFFA (Aβ<sub>16–21</sub>) peptide was dissolved in PBS buffer, pH 7.4 at the concentration of 1 mM and incubated at 37° with continuing shaking for 3 months. Pre-disaggregated Aβ<sub>1–42</sub> was dissolved in PBS buffer, pH 7.4 at the concentration of 200 μM and incubated at 37° with continuing shaking for 2 months. For NMR titration samples preparation, KLVFFA (Aβ<sub>16–21</sub>) or Aβ<sub>1–42</sub> fiber stocks were diluted in the PBS buffer solution at the indicated concentrations, followed by adding the small molecules from 100 mM stock solutions in DMSO into fibrillar solution. The final concentration of the small molecule was 50uM or 100 μM. The final volume of NMR samples was 500 μL containing 5% D<sub>2</sub>O. Prior to NMR spectra collection, samples were incubated at room temperature for 0.5 hr. 500 MHz <sup>1</sup>H NMR spectra were collected on a Bruker DRX500 at 283 K with either 256 or 1024 scans collected depending on the intensity of the small molecule signal. H<sub>2</sub>O resonance was suppressed via excitation sculpting (<xref ref-type="bibr" rid="bib24">Hwang and Shaka, 1995</xref>); DMSO resonance was suppressed via a frequency shifted presaturation of the DMSO peak. Spectra were processed with XWINNMR 3.6.</p></sec><sec id="s4-7-4"><title>Dissociation constant (Kd) of small molecules to fibers calculated from NMR data</title><p>NMR data were analyzed to estimate the binding constant for the interaction between the BAF compounds and KLVFFA fibers. We monitored the decrease in the <sup>1</sup>H aromatic resonance of the compounds as a function of increasing concentrations of KLVFFA fibers. The general equation for deriving the apparent dissociation constant (Kd) is as follows:</p><p>For a general reaction of a ligand binding to fibers (containing N monomers):<disp-formula id="equ1"><mml:math id="m1"><mml:mrow><mml:mi>F</mml:mi><mml:msub><mml:mrow><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mi mathvariant="italic">ibril</mml:mi></mml:mrow><mml:mo>)</mml:mo></mml:mrow></mml:mrow><mml:mrow><mml:mi>N</mml:mi></mml:mrow></mml:msub><mml:mo>+</mml:mo><mml:mi>L</mml:mi><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mi mathvariant="italic">igand</mml:mi></mml:mrow><mml:mo>)</mml:mo></mml:mrow><mml:mo>↔</mml:mo><mml:msub><mml:mi>F</mml:mi><mml:mi>N</mml:mi></mml:msub><mml:mi>L</mml:mi></mml:mrow></mml:math></disp-formula>.</p><p>We estimated the concentration of fibers at any given monomer concentration as:<disp-formula id="equ2"><mml:math id="m2"><mml:mrow><mml:mrow><mml:mo>[</mml:mo><mml:mrow><mml:mtext>F</mml:mtext><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mtext>iber</mml:mtext></mml:mrow><mml:mo>)</mml:mo></mml:mrow><mml:mtext>N</mml:mtext></mml:mrow><mml:mo>]</mml:mo></mml:mrow><mml:mo>=</mml:mo><mml:mrow><mml:mo>[</mml:mo><mml:mrow><mml:mtext>Fmomomer</mml:mtext></mml:mrow><mml:mo>]</mml:mo></mml:mrow><mml:mo>∗</mml:mo><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mrow><mml:mrow><mml:mn>1</mml:mn><mml:mo> </mml:mo><mml:mtext>fiber</mml:mtext></mml:mrow><mml:mo>/</mml:mo><mml:mrow><mml:mtext>N monomers</mml:mtext></mml:mrow></mml:mrow></mml:mrow><mml:mo>),</mml:mo></mml:mrow></mml:mrow></mml:math></disp-formula>and then we could get:</p><p><inline-formula><mml:math id="inf1"><mml:mrow><mml:mrow><mml:mo>[</mml:mo><mml:mrow><mml:msub><mml:mi>F</mml:mi><mml:mi>N</mml:mi></mml:msub></mml:mrow><mml:mo>]</mml:mo></mml:mrow><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:msub><mml:mrow><mml:mrow><mml:mo>[</mml:mo><mml:mi>F</mml:mi><mml:mo> ]</mml:mo></mml:mrow></mml:mrow><mml:mi>T</mml:mi></mml:msub></mml:mrow><mml:mi>N</mml:mi></mml:mfrac><mml:mo>−</mml:mo><mml:mrow><mml:mo>[</mml:mo><mml:mrow><mml:msub><mml:mi>F</mml:mi><mml:mi>N</mml:mi></mml:msub><mml:mi>L</mml:mi></mml:mrow><mml:mo>]</mml:mo></mml:mrow></mml:mrow></mml:math></inline-formula>, <inline-formula><mml:math id="inf2"><mml:mrow><mml:mrow><mml:mo>[</mml:mo><mml:mi>L</mml:mi><mml:mo>]</mml:mo></mml:mrow><mml:mo>=</mml:mo><mml:msub><mml:mrow><mml:mo>[</mml:mo><mml:mi>L</mml:mi><mml:mo>]</mml:mo></mml:mrow><mml:mi>T</mml:mi></mml:msub><mml:mo>−</mml:mo><mml:mrow><mml:mo>[</mml:mo><mml:msub><mml:mi>F</mml:mi><mml:mi>N</mml:mi></mml:msub><mml:mi>L</mml:mi><mml:mo>]</mml:mo></mml:mrow></mml:mrow></mml:math></inline-formula>, where [F]<sub>T</sub> is the total monomer concentration, [L]<sub>T</sub> is the total ligand concentration and [F<sub>N</sub>L] is the concentration of bound fiber;<disp-formula id="equ3"><mml:math id="m3"><mml:mrow><mml:msub><mml:mi>K</mml:mi><mml:mi>d</mml:mi></mml:msub><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:mrow><mml:mo>[</mml:mo><mml:mrow><mml:msub><mml:mi>F</mml:mi><mml:mi>N</mml:mi></mml:msub></mml:mrow><mml:mo>]</mml:mo></mml:mrow><mml:mrow><mml:mo>[</mml:mo><mml:mi>L</mml:mi><mml:mo>]</mml:mo></mml:mrow></mml:mrow><mml:mrow><mml:mo>[</mml:mo><mml:msub><mml:mi>F</mml:mi><mml:mi>N</mml:mi></mml:msub><mml:mi>L</mml:mi><mml:mo>]</mml:mo></mml:mrow></mml:mfrac><mml:mo> </mml:mo><mml:mtext>and</mml:mtext><mml:mo> </mml:mo><mml:msub><mml:mi>K</mml:mi><mml:mi>d</mml:mi></mml:msub><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mfrac><mml:mrow><mml:msub><mml:mrow><mml:mrow><mml:mo>[</mml:mo><mml:mi>F</mml:mi><mml:mo> ]</mml:mo></mml:mrow></mml:mrow><mml:mi>T</mml:mi></mml:msub></mml:mrow><mml:mi>N</mml:mi></mml:mfrac><mml:mo>−</mml:mo><mml:mrow><mml:mo>[</mml:mo><mml:mrow><mml:msub><mml:mi>F</mml:mi><mml:mi>N</mml:mi></mml:msub><mml:mi>L</mml:mi></mml:mrow><mml:mo>]</mml:mo></mml:mrow></mml:mrow><mml:mo>)</mml:mo></mml:mrow><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:msub><mml:mrow><mml:mrow><mml:mo>[</mml:mo><mml:mi>L</mml:mi><mml:mo>]</mml:mo></mml:mrow></mml:mrow><mml:mi>T</mml:mi></mml:msub><mml:mo>−</mml:mo><mml:mrow><mml:mo>[</mml:mo><mml:mrow><mml:msub><mml:mi>F</mml:mi><mml:mi>N</mml:mi></mml:msub><mml:mi>L</mml:mi></mml:mrow><mml:mo>]</mml:mo></mml:mrow></mml:mrow><mml:mo>)</mml:mo></mml:mrow></mml:mrow><mml:mrow><mml:mrow><mml:mo>[</mml:mo><mml:mrow><mml:msub><mml:mi>F</mml:mi><mml:mi>N</mml:mi></mml:msub><mml:mi>L</mml:mi></mml:mrow><mml:mo>]</mml:mo></mml:mrow></mml:mrow></mml:mfrac><mml:mo>,</mml:mo></mml:mrow></mml:math></disp-formula>and thus<disp-formula id="equ4"><mml:math id="m4"><mml:mrow><mml:msup><mml:mrow><mml:mrow><mml:mo>[</mml:mo><mml:mrow><mml:msub><mml:mi>F</mml:mi><mml:mi>N</mml:mi></mml:msub><mml:mi>L</mml:mi></mml:mrow><mml:mo>]</mml:mo></mml:mrow></mml:mrow><mml:mn>2</mml:mn></mml:msup><mml:mo>−</mml:mo><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mfrac><mml:mrow><mml:msub><mml:mrow><mml:mrow><mml:mo>[</mml:mo><mml:mi>F</mml:mi><mml:mo> ]</mml:mo></mml:mrow></mml:mrow><mml:mi>T</mml:mi></mml:msub></mml:mrow><mml:mi>N</mml:mi></mml:mfrac><mml:mo>+</mml:mo><mml:msub><mml:mrow><mml:mrow><mml:mo>[</mml:mo><mml:mi>L</mml:mi><mml:mo>]</mml:mo></mml:mrow></mml:mrow><mml:mi>T</mml:mi></mml:msub><mml:mo>+</mml:mo><mml:msub><mml:mi>K</mml:mi><mml:mi>d</mml:mi></mml:msub></mml:mrow><mml:mo>)</mml:mo></mml:mrow><mml:mrow><mml:mo>[</mml:mo><mml:mrow><mml:msub><mml:mi>F</mml:mi><mml:mi>N</mml:mi></mml:msub><mml:mi>L</mml:mi></mml:mrow><mml:mo>]</mml:mo></mml:mrow><mml:mo>+</mml:mo><mml:mfrac><mml:mrow><mml:msub><mml:mrow><mml:mrow><mml:mo>[</mml:mo><mml:mi>F</mml:mi><mml:mo> ]</mml:mo></mml:mrow></mml:mrow><mml:mi>T</mml:mi></mml:msub><mml:msub><mml:mrow><mml:mrow><mml:mo>[</mml:mo><mml:mi>L</mml:mi><mml:mo>]</mml:mo></mml:mrow></mml:mrow><mml:mi>T</mml:mi></mml:msub></mml:mrow><mml:mi>N</mml:mi></mml:mfrac><mml:mo>=</mml:mo><mml:mn>0</mml:mn><mml:mo>.</mml:mo></mml:mrow></mml:math></disp-formula></p><p>Finally, we could get the concentration of bound complex [F<sub>N</sub>L]:<disp-formula id="equ5"><label>(1)</label><mml:math id="m5"><mml:mrow><mml:mrow><mml:mo>[</mml:mo><mml:mrow><mml:msub><mml:mi>F</mml:mi><mml:mi>N</mml:mi></mml:msub><mml:mi>L</mml:mi></mml:mrow><mml:mo>]</mml:mo></mml:mrow><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mfrac><mml:mrow><mml:msub><mml:mrow><mml:mrow><mml:mo>[</mml:mo><mml:mi>F</mml:mi><mml:mo> ]</mml:mo></mml:mrow></mml:mrow><mml:mi>T</mml:mi></mml:msub></mml:mrow><mml:mi>N</mml:mi></mml:mfrac><mml:mo>+</mml:mo><mml:msub><mml:mrow><mml:mrow><mml:mo>[</mml:mo><mml:mi>L</mml:mi><mml:mo>]</mml:mo></mml:mrow></mml:mrow><mml:mi>T</mml:mi></mml:msub><mml:mo>+</mml:mo><mml:msub><mml:mi>K</mml:mi><mml:mi>d</mml:mi></mml:msub></mml:mrow><mml:mo>)</mml:mo></mml:mrow><mml:mo>−</mml:mo><mml:msqrt><mml:mrow><mml:msup><mml:mrow><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mfrac><mml:mrow><mml:msub><mml:mrow><mml:mrow><mml:mo>[</mml:mo><mml:mi>F</mml:mi><mml:mo> ]</mml:mo></mml:mrow></mml:mrow><mml:mi>T</mml:mi></mml:msub></mml:mrow><mml:mi>N</mml:mi></mml:mfrac><mml:mo>+</mml:mo><mml:msub><mml:mrow><mml:mrow><mml:mo>[</mml:mo><mml:mi>L</mml:mi><mml:mo>]</mml:mo></mml:mrow></mml:mrow><mml:mi>T</mml:mi></mml:msub><mml:mo>+</mml:mo><mml:msub><mml:mi>K</mml:mi><mml:mi>d</mml:mi></mml:msub></mml:mrow><mml:mo>)</mml:mo></mml:mrow></mml:mrow><mml:mn>2</mml:mn></mml:msup><mml:mo>−</mml:mo><mml:mfrac><mml:mrow><mml:mn>4</mml:mn><mml:msub><mml:mrow><mml:mrow><mml:mo>[</mml:mo><mml:mi>F</mml:mi><mml:mo> ]</mml:mo></mml:mrow></mml:mrow><mml:mi>T</mml:mi></mml:msub><mml:msub><mml:mrow><mml:mrow><mml:mo>[</mml:mo><mml:mi>L</mml:mi><mml:mo>]</mml:mo></mml:mrow></mml:mrow><mml:mi>T</mml:mi></mml:msub></mml:mrow><mml:mi>N</mml:mi></mml:mfrac></mml:mrow></mml:msqrt></mml:mrow><mml:mn>2</mml:mn></mml:mfrac></mml:mrow></mml:math></disp-formula></p><p>We then applied this <xref ref-type="disp-formula" rid="equ5">equation (1)</xref> to our NMR experiments, where we monitored the integrated area of each NMR peak (<bold><italic>A</italic></bold>) of the compounds over a range of KLVFFA fiber concentrations. Assuming the complex of the BAF compound with fiber is in fast exchange, the peak area is the average of the peak signals for free and bound states, weighted by the fraction of the observed molecule in each state:<disp-formula id="equ6"><mml:math id="m6"><mml:mrow><mml:mi>A</mml:mi><mml:mo>=</mml:mo><mml:msub><mml:mi>f</mml:mi><mml:mi>L</mml:mi></mml:msub><mml:msub><mml:mi>A</mml:mi><mml:mi>L</mml:mi></mml:msub><mml:mo>+</mml:mo><mml:msub><mml:mi>f</mml:mi><mml:mrow><mml:msub><mml:mi>F</mml:mi><mml:mi>N</mml:mi></mml:msub><mml:mi>L</mml:mi></mml:mrow></mml:msub><mml:msub><mml:mi>A</mml:mi><mml:mrow><mml:msub><mml:mi>F</mml:mi><mml:mi>N</mml:mi></mml:msub><mml:mi>L</mml:mi></mml:mrow></mml:msub><mml:mo>.</mml:mo></mml:mrow></mml:math></disp-formula></p><p>And the change in NMR peak area (<bold><italic>Δ</italic><italic>A</italic></bold>),<disp-formula id="equ7"><mml:math id="m7"><mml:mrow><mml:mi>Δ</mml:mi><mml:mi>A</mml:mi><mml:mo>=</mml:mo><mml:msub><mml:mi>A</mml:mi><mml:mi>L</mml:mi></mml:msub><mml:mo>−</mml:mo><mml:mi>A</mml:mi><mml:mo>=</mml:mo><mml:msub><mml:mi>f</mml:mi><mml:mrow><mml:msub><mml:mi>F</mml:mi><mml:mi>N</mml:mi></mml:msub><mml:mi>L</mml:mi></mml:mrow></mml:msub><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:msub><mml:mi>A</mml:mi><mml:mi>L</mml:mi></mml:msub><mml:mo>−</mml:mo><mml:msub><mml:mi>A</mml:mi><mml:mrow><mml:msub><mml:mi>F</mml:mi><mml:mi>N</mml:mi></mml:msub><mml:mi>L</mml:mi></mml:mrow></mml:msub></mml:mrow><mml:mo>)</mml:mo></mml:mrow></mml:mrow></mml:math></disp-formula><disp-formula id="equ8"><mml:math id="m8"><mml:mrow><mml:mfrac><mml:mrow><mml:mi>Δ</mml:mi><mml:mi>A</mml:mi></mml:mrow><mml:mrow><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:msub><mml:mi>A</mml:mi><mml:mi>L</mml:mi></mml:msub><mml:mo>−</mml:mo><mml:msub><mml:mi>A</mml:mi><mml:mrow><mml:msub><mml:mi>F</mml:mi><mml:mi>N</mml:mi></mml:msub><mml:mi>L</mml:mi></mml:mrow></mml:msub></mml:mrow><mml:mo>)</mml:mo></mml:mrow></mml:mrow></mml:mfrac><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:mi>Δ</mml:mi><mml:mi>A</mml:mi></mml:mrow><mml:mrow><mml:mi>Δ</mml:mi><mml:msub><mml:mi>A</mml:mi><mml:mrow><mml:mi mathvariant="italic">max</mml:mi></mml:mrow></mml:msub></mml:mrow></mml:mfrac><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:mrow><mml:mrow><mml:mi>Δ</mml:mi><mml:mi>A</mml:mi></mml:mrow><mml:mo>/</mml:mo><mml:mrow><mml:msub><mml:mi>A</mml:mi><mml:mi>L</mml:mi></mml:msub></mml:mrow></mml:mrow></mml:mrow><mml:mrow><mml:mrow><mml:mrow><mml:mi>Δ</mml:mi><mml:msub><mml:mi>A</mml:mi><mml:mrow><mml:mi mathvariant="italic">max</mml:mi></mml:mrow></mml:msub></mml:mrow><mml:mo>/</mml:mo><mml:mrow><mml:msub><mml:mi>A</mml:mi><mml:mi>L</mml:mi></mml:msub></mml:mrow></mml:mrow></mml:mrow></mml:mfrac><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:msub><mml:mo>%</mml:mo><mml:mrow><mml:mi>Δ</mml:mi><mml:mi>A</mml:mi></mml:mrow></mml:msub></mml:mrow><mml:mrow><mml:msub><mml:mo>%</mml:mo><mml:mrow><mml:mi>Δ</mml:mi><mml:msub><mml:mi>A</mml:mi><mml:mrow><mml:mi mathvariant="italic">max</mml:mi></mml:mrow></mml:msub></mml:mrow></mml:msub></mml:mrow></mml:mfrac><mml:mo>=</mml:mo><mml:msub><mml:mi>f</mml:mi><mml:mrow><mml:msub><mml:mi>F</mml:mi><mml:mi>N</mml:mi></mml:msub><mml:mi>L</mml:mi></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:mrow><mml:mo>[</mml:mo><mml:mrow><mml:msub><mml:mi>F</mml:mi><mml:mi>N</mml:mi></mml:msub><mml:mi>L</mml:mi></mml:mrow><mml:mo>]</mml:mo></mml:mrow></mml:mrow><mml:mrow><mml:msub><mml:mrow><mml:mrow><mml:mo>[</mml:mo><mml:mi>L</mml:mi><mml:mo>]</mml:mo></mml:mrow></mml:mrow><mml:mi>T</mml:mi></mml:msub></mml:mrow></mml:mfrac></mml:mrow></mml:math></disp-formula><disp-formula id="equ9"><mml:math id="m9"><mml:mrow><mml:mrow><mml:mrow><mml:mi>Δ</mml:mi><mml:mi>A</mml:mi></mml:mrow><mml:mo>/</mml:mo><mml:mrow><mml:msub><mml:mi>A</mml:mi><mml:mi>L</mml:mi></mml:msub></mml:mrow></mml:mrow><mml:mo>=</mml:mo><mml:mrow><mml:mrow><mml:mi>Δ</mml:mi><mml:msub><mml:mi>A</mml:mi><mml:mrow><mml:mi mathvariant="italic">max</mml:mi></mml:mrow></mml:msub></mml:mrow><mml:mo>/</mml:mo><mml:mrow><mml:msub><mml:mi>A</mml:mi><mml:mi>L</mml:mi></mml:msub></mml:mrow></mml:mrow><mml:mfrac><mml:mrow><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mfrac><mml:mrow><mml:msub><mml:mrow><mml:mrow><mml:mo>[</mml:mo><mml:mi>F</mml:mi><mml:mo> ]</mml:mo></mml:mrow></mml:mrow><mml:mi>T</mml:mi></mml:msub></mml:mrow><mml:mi>N</mml:mi></mml:mfrac><mml:mo>+</mml:mo><mml:msub><mml:mrow><mml:mrow><mml:mo>[</mml:mo><mml:mi>L</mml:mi><mml:mo>]</mml:mo></mml:mrow></mml:mrow><mml:mi>T</mml:mi></mml:msub><mml:mo>+</mml:mo><mml:msub><mml:mi>K</mml:mi><mml:mi>d</mml:mi></mml:msub></mml:mrow><mml:mo>)</mml:mo></mml:mrow><mml:mo>−</mml:mo><mml:msqrt><mml:mrow><mml:msup><mml:mrow><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mfrac><mml:mrow><mml:msub><mml:mrow><mml:mrow><mml:mo>[</mml:mo><mml:mi>F</mml:mi><mml:mo> ]</mml:mo></mml:mrow></mml:mrow><mml:mi>T</mml:mi></mml:msub></mml:mrow><mml:mi>N</mml:mi></mml:mfrac><mml:mo>+</mml:mo><mml:msub><mml:mrow><mml:mrow><mml:mo>[</mml:mo><mml:mi>L</mml:mi><mml:mo>]</mml:mo></mml:mrow></mml:mrow><mml:mi>T</mml:mi></mml:msub><mml:mo>+</mml:mo><mml:msub><mml:mi>K</mml:mi><mml:mi>d</mml:mi></mml:msub></mml:mrow><mml:mo>)</mml:mo></mml:mrow></mml:mrow><mml:mn>2</mml:mn></mml:msup><mml:mo>−</mml:mo><mml:mfrac><mml:mrow><mml:mn>4</mml:mn><mml:msub><mml:mrow><mml:mrow><mml:mo>[</mml:mo><mml:mi>F</mml:mi><mml:mo> ]</mml:mo></mml:mrow></mml:mrow><mml:mi>T</mml:mi></mml:msub><mml:msub><mml:mrow><mml:mrow><mml:mo>[</mml:mo><mml:mi>L</mml:mi><mml:mo>]</mml:mo></mml:mrow></mml:mrow><mml:mi>T</mml:mi></mml:msub></mml:mrow><mml:mi>N</mml:mi></mml:mfrac></mml:mrow></mml:msqrt></mml:mrow><mml:mrow><mml:mn>2</mml:mn><mml:msub><mml:mrow><mml:mo>[</mml:mo><mml:mi>L</mml:mi><mml:mo>]</mml:mo></mml:mrow><mml:mi>T</mml:mi></mml:msub></mml:mrow></mml:mfrac><mml:mo>.</mml:mo></mml:mrow></mml:math></disp-formula></p><p>Hence, the observed fraction of peak area change during the titration of increasing fiber concentration against fixed small compound,<disp-formula id="equ10"><mml:math id="m10"><mml:mrow><mml:msub><mml:mi>f</mml:mi><mml:mrow><mml:mi mathvariant="italic">obs</mml:mi></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:msub><mml:mi>f</mml:mi><mml:mrow><mml:mi mathvariant="italic">max</mml:mi></mml:mrow></mml:msub><mml:mfrac><mml:mrow><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mfrac><mml:mrow><mml:msub><mml:mrow><mml:mrow><mml:mo>[</mml:mo><mml:mi>F</mml:mi><mml:mo> ]</mml:mo></mml:mrow></mml:mrow><mml:mi>T</mml:mi></mml:msub></mml:mrow><mml:mi>N</mml:mi></mml:mfrac><mml:mo>+</mml:mo><mml:msub><mml:mrow><mml:mrow><mml:mo>[</mml:mo><mml:mi>L</mml:mi><mml:mo>]</mml:mo></mml:mrow></mml:mrow><mml:mi>T</mml:mi></mml:msub><mml:mo>+</mml:mo><mml:msub><mml:mi>K</mml:mi><mml:mi>d</mml:mi></mml:msub></mml:mrow><mml:mo>)</mml:mo></mml:mrow><mml:mo>−</mml:mo><mml:msqrt><mml:mrow><mml:msup><mml:mrow><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mfrac><mml:mrow><mml:msub><mml:mrow><mml:mrow><mml:mo>[</mml:mo><mml:mi>F</mml:mi><mml:mo> ]</mml:mo></mml:mrow></mml:mrow><mml:mi>T</mml:mi></mml:msub></mml:mrow><mml:mi>N</mml:mi></mml:mfrac><mml:mo>+</mml:mo><mml:msub><mml:mrow><mml:mrow><mml:mo>[</mml:mo><mml:mi>L</mml:mi><mml:mo>]</mml:mo></mml:mrow></mml:mrow><mml:mi>T</mml:mi></mml:msub><mml:mo>+</mml:mo><mml:msub><mml:mi>K</mml:mi><mml:mi>d</mml:mi></mml:msub></mml:mrow><mml:mo>)</mml:mo></mml:mrow></mml:mrow><mml:mn>2</mml:mn></mml:msup><mml:mo>−</mml:mo><mml:mfrac><mml:mrow><mml:mn>4</mml:mn><mml:msub><mml:mrow><mml:mrow><mml:mo>[</mml:mo><mml:mi>F</mml:mi><mml:mo> ]</mml:mo></mml:mrow></mml:mrow><mml:mi>T</mml:mi></mml:msub><mml:msub><mml:mrow><mml:mrow><mml:mo>[</mml:mo><mml:mi>L</mml:mi><mml:mo>]</mml:mo></mml:mrow></mml:mrow><mml:mi>T</mml:mi></mml:msub></mml:mrow><mml:mi>N</mml:mi></mml:mfrac></mml:mrow></mml:msqrt></mml:mrow><mml:mrow><mml:mn>2</mml:mn><mml:msub><mml:mrow><mml:mo>[</mml:mo><mml:mi>L</mml:mi><mml:mo>]</mml:mo></mml:mrow><mml:mi>T</mml:mi></mml:msub></mml:mrow></mml:mfrac><mml:mo>.</mml:mo></mml:mrow></mml:math></disp-formula></p><p>Our structural model suggests that one BAF compound binds three fiber monomers. To obtain the Kd, we fit the equation for 1:3 (small molecule:fiber) binding to the NMR titration curve (N = 3), with <inline-formula><mml:math id="inf3"><mml:mrow><mml:msub><mml:mi>f</mml:mi><mml:mrow><mml:mi mathvariant="italic">obs</mml:mi></mml:mrow></mml:msub></mml:mrow></mml:math></inline-formula> defined as the fraction of peak area decrease <inline-formula><mml:math id="inf4"><mml:mrow><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mfrac><mml:mrow><mml:mi>Δ</mml:mi><mml:mi>A</mml:mi></mml:mrow><mml:mrow><mml:msub><mml:mi>A</mml:mi><mml:mi>L</mml:mi></mml:msub></mml:mrow></mml:mfrac></mml:mrow><mml:mo>)</mml:mo></mml:mrow></mml:mrow></mml:math></inline-formula> for each titration experiment, and <inline-formula><mml:math id="inf5"><mml:mrow><mml:msub><mml:mi>f</mml:mi><mml:mrow><mml:mi mathvariant="italic">max</mml:mi></mml:mrow></mml:msub></mml:mrow></mml:math></inline-formula> defined as the fraction maximum of peak area decrease <inline-formula><mml:math id="inf6"><mml:mrow><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mfrac><mml:mrow><mml:msub><mml:mi>A</mml:mi><mml:mrow><mml:mi mathvariant="italic">max</mml:mi></mml:mrow></mml:msub></mml:mrow><mml:mrow><mml:msub><mml:mi>A</mml:mi><mml:mi>L</mml:mi></mml:msub></mml:mrow></mml:mfrac></mml:mrow><mml:mo>)</mml:mo></mml:mrow></mml:mrow></mml:math></inline-formula> for the saturated complex.</p></sec><sec id="s4-7-5"><title>MTT cell viability assay</title><p>We performed MTT-based cell viability assay to assess the cytotoxicity of Aβ<sub>1–42</sub> with or without the addition of BAFs and orange G. A CellTiter 96 aqueous non-radioactive cell proliferation assay kit (MTT) (Promega cat. #G4100, Madison, WI) was used. HeLa and PC-12 (ATCC; cat. # CRL-1721, Manassas, VA) cell lines were used for measuring the toxicity of Aβ<sub>1–42</sub>. Prior to toxicity test, both HeLa and PC-12 cell lines were plated at 10,000 cells per well in 96-well plates (Costar cat. # 3596, Washington, DC). HeLa cells were cultured in DMEM medium with 10% fetal bovine serum, PC-12 cells were cultured in ATCC-formulated RPMI 1640 medium (ATCC; cat.# 30–2001) with 10% heat-inactivated horse serum and 5% fetal bovine serum. Cells were cultured in 96-well plates for 20 hr at 37°C in 5% CO<sub>2</sub>. For Aβ<sub>1–42</sub> and BAFs samples preparation, purified Aβ<sub>1–42</sub> was dissolved in PBS at the final concentration of 5 μM, followed by the addition of BAFs at indicated concentrations. The mixtures were filtered with a 0.2-μm filter and further incubated for 16 hr at 37°C without shaking for fiber formation. To start the MTT assay, 10 μl of pre-incubated mixture was added to each well containing 90 μl medium. After 24 hr incubation at 37°C in 5% CO<sub>2</sub>, 15 μl Dye solution (Promega cat. #G4102) was added into each well. After incubation for 4 hr at 37°C, 100 μl solubilization Solution/Stop Mix (Promega cat. #G4101) was added to each well. After 12 hr incubation at room temperature, the absorbance was measured at 570 nm with background absorbance recorded at 700 nm. Four replicates were measured for each of the samples. The MTT cell viability assay measured the percentage of survival cell upon the treatment of the mixture of Aβ<sub>1–42</sub> and BAFs. The toxicity inhibition (%) or rescuing percentage (%) of each BAF compound was calculated by normalizing the cell survival rate using the PBS buffer-treated cells as 100% and 0.5 μM (final concentration) Aβ<sub>1–42</sub> fiber alone-treated cell as 0% viability<bold>.</bold></p></sec><sec id="s4-7-6"><title>Transmission electron microscopy (TEM)</title><p>TEM was performed to visualize the fibrillation of Aβ<sub>1–42</sub> in presence of BAFs. The samples of Aβ<sub>1–42</sub> and BAFs mixture for TEM measurement were the same as those for MTT assay. For specimen preparation, 5 μl solution was spotted onto freshly glow-discharged carbon-coated electron microscopy grids (Ted Pella, Redding, CA). Grids were rinsed twice with 5 μl distilled water after 3 min incubation, followed by staining with 1% uranyl acetate for 1 min. A CM120 electron microscope at an accelerating voltage of 120 kV was used to examine the specimens. Images were recorded digitally by TIETZ F224HD CCD camera.</p></sec><sec id="s4-7-7"><title>ThT fibrillation assay</title><p>Purified Aβ<sub>1–42</sub> was dissolved in 10 mM NaOH at the concentration of 200 μM, followed by sonication for further solubilizing Aβ<sub>1–42</sub>. Aβ<sub>1–42</sub> was diluted into PBS buffer at the final concentration of 20 μM, and was mixed with 20 μM Thioflavin T (ThT) and different concentrations of BAFs. The reaction mixture was filtered with a 0.2 μm filter, split into four replicates and placed in a 96-well plate (black with flat optic bottom). The ThT fluorescence signal was measured every 5 min using the Varioskan plate reader (Thermo Fisher Scientific, Inc) with excitation and emission wavelengths of 444 and 484 nm, respectively, at 37°C.</p></sec></sec></sec></body><back><ack id="ack"><title>Acknowledgements</title><p>We thank N Wu for help with initial computational work, R Peterson and E Hartman for help with NMR experiments, A Soragni, MR Sawaya, H Chang, D Li, D Anderson, P Bajaj, J Bowie, T Yeates, and F Guo for discussion, and HHMI, NIH, NSF, and DOE for support.</p></ack><sec sec-type="additional-information"><title>Additional information</title><fn-group content-type="competing-interest"><title>Competing interests</title><fn fn-type="conflict" id="conf1"><p>The authors declare that no competing interests exist.</p></fn></fn-group><fn-group content-type="author-contribution"><title>Author contributions</title><fn fn-type="con" id="con1"><p>LJ, Conceived and designed the projects; Created structure-based screening protocols; Identified the compounds and analyzed the data; Contributed to the crystallographic efforts; Wrote the manuscript coordinating contributions from other authors</p></fn><fn fn-type="con" id="con2"><p>CL, Conceived and designed the projects; Performed the EM and cell viability assays; Contributed to the crystallographic efforts</p></fn><fn fn-type="con" id="con3"><p>DL, Performed NMR studies</p></fn><fn fn-type="con" id="con4"><p>ML, Contributed to the crystallographic efforts</p></fn><fn fn-type="con" id="con5"><p>MZ, Contributed to the crystallographic efforts</p></fn><fn fn-type="con" id="con6"><p>MPH, Contributed to the crystallographic efforts</p></fn><fn fn-type="con" id="con7"><p>DSE, Conceived and designed the projects; Wrote the manuscript coordinating contributions from other authors</p></fn></fn-group></sec><sec sec-type="supplementary-material"><title>Additional files</title><supplementary-material id="SD1-data"><object-id pub-id-type="doi">10.7554/eLife.00857.037</object-id><label>Supplementary file 1.</label><caption><title>Compound Library Set 1: Cambridge Structure Database (CSD) set.</title><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00857.037">http://dx.doi.org/10.7554/eLife.00857.037</ext-link></p></caption><media mime-subtype="xlsx" mimetype="application" xlink:href="elife00857s001.xlsx"/></supplementary-material><supplementary-material id="SD2-data"><object-id pub-id-type="doi">10.7554/eLife.00857.038</object-id><label>Supplementary file 2.</label><caption><title>Compound Library Set 2: Flat Compound (FC) set.</title><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00857.038">http://dx.doi.org/10.7554/eLife.00857.038</ext-link></p></caption><media mime-subtype="xlsx" mimetype="application" xlink:href="elife00857s002.xlsx"/></supplementary-material></sec><ref-list><title>References</title><ref id="bib1"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Alavez</surname><given-names>S</given-names></name><name><surname>Vantipalli</surname><given-names>MC</given-names></name><name><surname>Zucker</surname><given-names>DJ</given-names></name><name><surname>Klang</surname><given-names>IM</given-names></name><name><surname>Lithgow</surname><given-names>GJ</given-names></name></person-group><year>2011</year><article-title>Amyloid-binding compounds maintain protein homeostasis during ageing and extend lifespan</article-title><source>Nature</source><volume>472</volume><fpage>226</fpage><lpage>9</lpage><pub-id pub-id-type="doi">10.1038/nature09873</pub-id></element-citation></ref><ref id="bib2"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bartolini</surname><given-names>M</given-names></name><name><surname>Andrisano</surname><given-names>V</given-names></name></person-group><year>2010</year><article-title>Strategies for the inhibition of protein aggregation in human diseases</article-title><source>Chembiochem</source><volume>11</volume><fpage>1018</fpage><lpage>35</lpage><pub-id pub-id-type="doi">10.1002/cbic.200900666</pub-id></element-citation></ref><ref id="bib3"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bieschke</surname><given-names>J</given-names></name><name><surname>Herbst</surname><given-names>M</given-names></name><name><surname>Wiglenda</surname><given-names>T</given-names></name><name><surname>Friedrich</surname><given-names>RP</given-names></name><name><surname>Boeddrich</surname><given-names>A</given-names></name><name><surname>Schiele</surname><given-names>F</given-names></name><etal/></person-group><year>2011</year><article-title>Small-molecule conversion of toxic oligomers to nontoxic beta-sheet-rich amyloid fibrils</article-title><source>Nat Chem Biol</source><volume>8</volume><fpage>93</fpage><lpage>101</lpage><pub-id pub-id-type="doi">10.1038/nchembio.719</pub-id></element-citation></ref><ref id="bib4"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bostrom</surname><given-names>J</given-names></name><name><surname>Greenwood</surname><given-names>JR</given-names></name><name><surname>Gottfries</surname><given-names>J</given-names></name></person-group><year>2003</year><article-title>Assessing the performance of OMEGA with respect to retrieving bioactive conformations</article-title><source>J Mol Graph Model</source><volume>21</volume><fpage>449</fpage><lpage>62</lpage><pub-id pub-id-type="doi">10.1016/S1093-3263(02)00204-8</pub-id></element-citation></ref><ref id="bib5"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Chen</surname><given-names>J</given-names></name><name><surname>Armstrong</surname><given-names>AH</given-names></name><name><surname>Koehler</surname><given-names>AN</given-names></name><name><surname>Hecht</surname><given-names>MH</given-names></name></person-group><year>2010</year><article-title>Small molecule microarrays enable the discovery of compounds that bind the alzheimer’s abeta peptide and reduce its cytotoxicity</article-title><source>J Am Chem Soc</source><volume>132</volume><fpage>17015</fpage><lpage>22</lpage><pub-id pub-id-type="doi">10.1021/ja107552s</pub-id></element-citation></ref><ref id="bib6"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Cheng</surname><given-names>PN</given-names></name><name><surname>Liu</surname><given-names>C</given-names></name><name><surname>Zhao</surname><given-names>M</given-names></name><name><surname>Eisenberg</surname><given-names>D</given-names></name><name><surname>Nowick</surname><given-names>JS</given-names></name></person-group><year>2012</year><article-title>Amyloid beta-sheet mimics that antagonize protein aggregation and reduce amyloid toxicity</article-title><source>Nat Chem</source><volume>4</volume><fpage>927</fpage><lpage>33</lpage><pub-id pub-id-type="doi">10.1038/nchem.1433</pub-id></element-citation></ref><ref id="bib7"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Chiti</surname><given-names>F</given-names></name><name><surname>Dobson</surname><given-names>CM</given-names></name></person-group><year>2006</year><article-title>Protein misfolding, functional amyloid, and human disease</article-title><source>Annu Rev Biochem</source><volume>75</volume><fpage>333</fpage><lpage>66</lpage><pub-id pub-id-type="doi">10.1146/annurev.biochem.75.101304.123901</pub-id></element-citation></ref><ref id="bib8"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Cleary</surname><given-names>JP</given-names></name><name><surname>Walsh</surname><given-names>DM</given-names></name><name><surname>Hofmeister</surname><given-names>JJ</given-names></name><name><surname>Shankar</surname><given-names>GM</given-names></name><name><surname>Kuskowski</surname><given-names>MA</given-names></name><name><surname>Selkoe</surname><given-names>DJ</given-names></name><etal/></person-group><year>2005</year><article-title>Natural oligomers of the amyloid-beta protein specifically disrupt cognitive function</article-title><source>Nat Neurosci</source><volume>8</volume><fpage>79</fpage><lpage>84</lpage><pub-id pub-id-type="doi">10.1038/nn1372</pub-id></element-citation></ref><ref id="bib9"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Colletier</surname><given-names>JP</given-names></name><name><surname>Laganowsky</surname><given-names>A</given-names></name><name><surname>Landau</surname><given-names>M</given-names></name><name><surname>Zhao</surname><given-names>M</given-names></name><name><surname>Soriaga</surname><given-names>AB</given-names></name><name><surname>Goldschmidt</surname><given-names>L</given-names></name><etal/></person-group><year>2011</year><article-title>Molecular basis for amyloid-beta polymorphism</article-title><source>Proc Natl Acad Sci USA</source><volume>108</volume><fpage>16938</fpage><lpage>43</lpage><pub-id pub-id-type="doi">10.1073/pnas.1112600108</pub-id></element-citation></ref><ref id="bib10"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Cremades</surname><given-names>N</given-names></name><name><surname>Cohen</surname><given-names>SI</given-names></name><name><surname>Deas</surname><given-names>E</given-names></name><name><surname>Abramov</surname><given-names>AY</given-names></name><name><surname>Chen</surname><given-names>AY</given-names></name><name><surname>Orte</surname><given-names>A</given-names></name><etal/></person-group><year>2012</year><article-title>Direct observation of the interconversion of normal and toxic forms of alpha-synuclein</article-title><source>Cell</source><volume>149</volume><fpage>1048</fpage><lpage>59</lpage><pub-id pub-id-type="doi">10.1016/j.cell.2012.03.037</pub-id></element-citation></ref><ref id="bib11"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Dasilva</surname><given-names>KA</given-names></name><name><surname>Shaw</surname><given-names>JE</given-names></name><name><surname>McLaurin</surname><given-names>J</given-names></name></person-group><year>2010</year><article-title>Amyloid-beta fibrillogenesis: structural insight and therapeutic intervention</article-title><source>Exp Neurol</source><volume>223</volume><fpage>311</fpage><lpage>21</lpage><pub-id pub-id-type="doi">10.1016/j.expneurol.2009.08.032</pub-id></element-citation></ref><ref id="bib12"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Davis</surname><given-names>IW</given-names></name><name><surname>Baker</surname><given-names>D</given-names></name></person-group><year>2009</year><article-title>RosettaLigand docking with full ligand and receptor flexibility</article-title><source>J Mol Biol</source><volume>385</volume><fpage>381</fpage><lpage>92</lpage><pub-id pub-id-type="doi">10.1016/j.jmb.2008.11.010</pub-id></element-citation></ref><ref id="bib13"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>De Felice</surname><given-names>FG</given-names></name><name><surname>Houzel</surname><given-names>JC</given-names></name><name><surname>Garcia-Abreu</surname><given-names>J</given-names></name><name><surname>Louzada</surname><given-names>PR</given-names><suffix>Jnr.</suffix></name><name><surname>Afonso</surname><given-names>RC</given-names></name><etal/></person-group><year>2001</year><article-title>Inhibition of alzheimer’s disease beta-amyloid aggregation, neurotoxicity, and in vivo deposition by nitrophenols: implications for alzheimer’s therapy</article-title><source>FASEB J</source><volume>15</volume><fpage>1297</fpage><lpage>9</lpage></element-citation></ref><ref id="bib14"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ehrnhoefer</surname><given-names>DE</given-names></name><name><surname>Bieschke</surname><given-names>J</given-names></name><name><surname>Boeddrich</surname><given-names>A</given-names></name><name><surname>Herbst</surname><given-names>M</given-names></name><name><surname>Masino</surname><given-names>L</given-names></name><name><surname>Lurz</surname><given-names>R</given-names></name><etal/></person-group><year>2008</year><article-title>EGCG redirects amyloidogenic polypeptides into unstructured, off-pathway oligomers</article-title><source>Nat Struct Mol Biol</source><volume>15</volume><fpage>558</fpage><lpage>66</lpage><pub-id pub-id-type="doi">10.1038/nsmb.1437</pub-id></element-citation></ref><ref id="bib15"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Eisenberg</surname><given-names>D</given-names></name><name><surname>Jucker</surname><given-names>M</given-names></name></person-group><year>2012</year><article-title>The amyloid state of proteins in human diseases</article-title><source>Cell</source><volume>148</volume><fpage>1188</fpage><lpage>203</lpage><pub-id pub-id-type="doi">10.1016/j.cell.2012.02.022</pub-id></element-citation></ref><ref id="bib16"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Evans</surname><given-names>CG</given-names></name><name><surname>Wisen</surname><given-names>S</given-names></name><name><surname>Gestwicki</surname><given-names>JE</given-names></name></person-group><year>2006</year><article-title>Heat shock proteins 70 and 90 inhibit early stages of amyloid beta-(1-42) aggregation in vitro</article-title><source>J Biol Chem</source><volume>281</volume><fpage>33182</fpage><lpage>91</lpage><pub-id pub-id-type="doi">10.1074/jbc.M606192200</pub-id></element-citation></ref><ref id="bib17"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ewing</surname><given-names>TJ</given-names></name><name><surname>Makino</surname><given-names>S</given-names></name><name><surname>Skillman</surname><given-names>AG</given-names></name><name><surname>Kuntz</surname><given-names>ID</given-names></name></person-group><year>2001</year><article-title>DOCK 4.0: search strategies for automated molecular docking of flexible molecule databases</article-title><source>J Comput Aided Mol Des</source><volume>15</volume><fpage>411</fpage><lpage>28</lpage><pub-id pub-id-type="doi">10.1023/A:1011115820450</pub-id></element-citation></ref><ref id="bib18"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Findeis</surname><given-names>MA</given-names></name></person-group><year>2002</year><article-title>Peptide inhibitors of beta amyloid aggregation</article-title><source>Curr Top Med Chem</source><volume>2</volume><fpage>417</fpage><lpage>23</lpage><pub-id pub-id-type="doi">10.2174/1568026024607508</pub-id></element-citation></ref><ref id="bib19"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Finder</surname><given-names>VH</given-names></name><name><surname>Vodopivec</surname><given-names>I</given-names></name><name><surname>Nitsch</surname><given-names>RM</given-names></name><name><surname>Glockshuber</surname><given-names>R</given-names></name></person-group><year>2010</year><article-title>The recombinant amyloid-beta peptide Abeta1-42 aggregates faster and is more neurotoxic than synthetic Abeta1-42</article-title><source>J Mol Biol</source><volume>396</volume><fpage>9</fpage><lpage>18</lpage><pub-id pub-id-type="doi">10.1016/j.jmb.2009.12.016</pub-id></element-citation></ref><ref id="bib20"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Frid</surname><given-names>P</given-names></name><name><surname>Anisimov</surname><given-names>SV</given-names></name><name><surname>Popovic</surname><given-names>N</given-names></name></person-group><year>2007</year><article-title>Congo red and protein aggregation in neurodegenerative diseases</article-title><source>Brain Res Rev</source><volume>53</volume><fpage>135</fpage><lpage>60</lpage><pub-id pub-id-type="doi">10.1016/j.brainresrev.2006.08.001</pub-id></element-citation></ref><ref id="bib21"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Guha</surname><given-names>R</given-names></name><name><surname>Howard</surname><given-names>MT</given-names></name><name><surname>Hutchison</surname><given-names>GR</given-names></name><name><surname>Murray-Rust</surname><given-names>P</given-names></name><name><surname>Rzepa</surname><given-names>H</given-names></name><name><surname>Steinbeck</surname><given-names>C</given-names></name><etal/></person-group><year>2006</year><article-title>The blue obelisk-interoperability in chemical informatics</article-title><source>J Chem Inf Model</source><volume>46</volume><fpage>991</fpage><lpage>8</lpage><pub-id pub-id-type="doi">10.1021/ci050400b</pub-id></element-citation></ref><ref id="bib22"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hard</surname><given-names>T</given-names></name><name><surname>Lendel</surname><given-names>C</given-names></name></person-group><year>2012</year><article-title>Inhibition of amyloid formation</article-title><source>J Mol Biol</source><volume>421</volume><fpage>441</fpage><lpage>65</lpage><pub-id pub-id-type="doi">10.1016/j.jmb.2011.12.062</pub-id></element-citation></ref><ref id="bib23"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hartley</surname><given-names>DM</given-names></name><name><surname>Walsh</surname><given-names>DM</given-names></name><name><surname>Ye</surname><given-names>CP</given-names></name><name><surname>Diehl</surname><given-names>T</given-names></name><name><surname>Vasquez</surname><given-names>S</given-names></name><name><surname>Vassilev</surname><given-names>PM</given-names></name><etal/></person-group><year>1999</year><article-title>Protofibrillar intermediates of amyloid beta-protein induce acute electrophysiological changes and progressive neurotoxicity in cortical neurons</article-title><source>J Neurosci</source><volume>19</volume><fpage>8876</fpage><lpage>84</lpage></element-citation></ref><ref id="bib24"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hwang</surname><given-names>TL</given-names></name><name><surname>Shaka</surname><given-names>AJ</given-names></name></person-group><year>1995</year><article-title>Water suppression that works. excitation sculpting using arbitrary waveforms and pulsed field gradients</article-title><source>J Magn Reson</source><volume>112</volume><fpage>175</fpage><lpage>279</lpage><pub-id pub-id-type="doi">10.1016/j.jmr.2006.10.014</pub-id></element-citation></ref><ref id="bib25"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Irwin</surname><given-names>JJ</given-names></name><name><surname>Shoichet</surname><given-names>BK</given-names></name></person-group><year>2005</year><article-title>ZINC–a free database of commercially available compounds for virtual screening</article-title><source>J Chem Inf Model</source><volume>45</volume><fpage>177</fpage><lpage>82</lpage><pub-id pub-id-type="doi">10.1021/ci049714+</pub-id></element-citation></ref><ref id="bib26"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kayed</surname><given-names>R</given-names></name><name><surname>Head</surname><given-names>E</given-names></name><name><surname>Thompson</surname><given-names>JL</given-names></name><name><surname>McIntire</surname><given-names>TM</given-names></name><name><surname>Milton</surname><given-names>SC</given-names></name><name><surname>Cotman</surname><given-names>CW</given-names></name><etal/></person-group><year>2003</year><article-title>Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis</article-title><source>Science</source><volume>300</volume><fpage>486</fpage><lpage>9</lpage><pub-id pub-id-type="doi">10.1126/science.1079469</pub-id></element-citation></ref><ref id="bib27"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Klunk</surname><given-names>WE</given-names></name><name><surname>Engler</surname><given-names>H</given-names></name><name><surname>Nordberg</surname><given-names>A</given-names></name><name><surname>Wang</surname><given-names>Y</given-names></name><name><surname>Blomqvist</surname><given-names>G</given-names></name><name><surname>Holt</surname><given-names>DP</given-names></name><etal/></person-group><year>2004</year><article-title>Imaging brain amyloid in alzheimer’s disease with pittsburgh compound-B</article-title><source>Ann Neurol</source><volume>55</volume><fpage>306</fpage><lpage>19</lpage><pub-id pub-id-type="doi">10.1002/ana.20009</pub-id></element-citation></ref><ref id="bib28"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kokkoni</surname><given-names>N</given-names></name><name><surname>Stott</surname><given-names>K</given-names></name><name><surname>Amijee</surname><given-names>H</given-names></name><name><surname>Mason</surname><given-names>JM</given-names></name><name><surname>Doig</surname><given-names>AJ</given-names></name></person-group><year>2006</year><article-title>N-Methylated peptide inhibitors of beta-amyloid aggregation and toxicity. Optimization of the inhibitor structure</article-title><source>Biochemistry</source><volume>45</volume><fpage>9906</fpage><lpage>18</lpage><pub-id pub-id-type="doi">10.1021/bi060837s</pub-id></element-citation></ref><ref id="bib29"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kortemme</surname><given-names>T</given-names></name><name><surname>Morozov</surname><given-names>AV</given-names></name><name><surname>Baker</surname><given-names>D</given-names></name></person-group><year>2003</year><article-title>An orientation-dependent hydrogen bonding potential improves prediction of specificity and structure for proteins and protein-protein complexes</article-title><source>J Mol Biol</source><volume>326</volume><fpage>1239</fpage><lpage>59</lpage><pub-id pub-id-type="doi">10.1016/S0022-2836(03)00021-4</pub-id></element-citation></ref><ref id="bib30"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Krishnan</surname><given-names>R</given-names></name><name><surname>Goodman</surname><given-names>JL</given-names></name><name><surname>Mukhopadhyay</surname><given-names>S</given-names></name><name><surname>Pacheco</surname><given-names>CD</given-names></name><name><surname>Lemke</surname><given-names>EA</given-names></name><name><surname>Deniz</surname><given-names>AA</given-names></name><etal/></person-group><year>2012</year><article-title>Conserved features of intermediates in amyloid assembly determine their benign or toxic states</article-title><source>Proc Natl Acad Sci USA</source><volume>109</volume><fpage>11172</fpage><lpage>7</lpage><pub-id pub-id-type="doi">10.1073/pnas.1209527109</pub-id></element-citation></ref><ref id="bib31"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kroth</surname><given-names>H</given-names></name><name><surname>Ansaloni</surname><given-names>A</given-names></name><name><surname>Varisco</surname><given-names>Y</given-names></name><name><surname>Jan</surname><given-names>A</given-names></name><name><surname>Sreenivasachary</surname><given-names>N</given-names></name><name><surname>Rezaei-Ghaleh</surname><given-names>N</given-names></name><etal/></person-group><year>2012</year><article-title>Discovery and structure activity relationship of small molecule inhibitors of toxic beta-amyloid-42 fibril formation</article-title><source>J Biol Chem</source><volume>287</volume><fpage>34786</fpage><lpage>800</lpage><pub-id pub-id-type="doi">10.1074/jbc.M112.357665</pub-id></element-citation></ref><ref id="bib32"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kuhlman</surname><given-names>B</given-names></name><name><surname>Baker</surname><given-names>D</given-names></name></person-group><year>2000</year><article-title>Native protein sequences are close to optimal for their structures</article-title><source>Proc Natl Acad Sci USA</source><volume>97</volume><fpage>10383</fpage><lpage>8</lpage><pub-id pub-id-type="doi">10.1073/pnas.97.19.10383</pub-id></element-citation></ref><ref id="bib33"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ladiwala</surname><given-names>AR</given-names></name><name><surname>Bhattacharya</surname><given-names>M</given-names></name><name><surname>Perchiacca</surname><given-names>JM</given-names></name><name><surname>Cao</surname><given-names>P</given-names></name><name><surname>Raleigh</surname><given-names>DP</given-names></name><name><surname>Abedini</surname><given-names>A</given-names></name><etal/></person-group><year>2012</year><article-title>Rational design of potent domain antibody inhibitors of amyloid fibril assembly</article-title><source>Proc Natl Acad Sci USA</source><volume>109</volume><fpage>19965</fpage><lpage>70</lpage><pub-id pub-id-type="doi">10.1073/pnas.1208797109</pub-id></element-citation></ref><ref id="bib34"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ladiwala</surname><given-names>AR</given-names></name><name><surname>Dordick</surname><given-names>JS</given-names></name><name><surname>Tessier</surname><given-names>PM</given-names></name></person-group><year>2011</year><article-title>Aromatic small molecules remodel toxic soluble oligomers of amyloid beta through three independent pathways</article-title><source>J Biol Chem</source><volume>286</volume><fpage>3209</fpage><lpage>18</lpage><pub-id pub-id-type="doi">10.1074/jbc.M110.173856</pub-id></element-citation></ref><ref id="bib35"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Laganowsky</surname><given-names>A</given-names></name><name><surname>Liu</surname><given-names>C</given-names></name><name><surname>Sawaya</surname><given-names>MR</given-names></name><name><surname>Whitelegge</surname><given-names>JP</given-names></name><name><surname>Park</surname><given-names>J</given-names></name><name><surname>Zhao</surname><given-names>M</given-names></name><etal/></person-group><year>2012</year><article-title>Atomic view of a toxic amyloid small oligomer</article-title><source>Science</source><volume>335</volume><fpage>1228</fpage><lpage>31</lpage><pub-id pub-id-type="doi">10.1126/science.1213151</pub-id></element-citation></ref><ref id="bib36"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Landau</surname><given-names>M</given-names></name><name><surname>Sawaya</surname><given-names>MR</given-names></name><name><surname>Faull</surname><given-names>KF</given-names></name><name><surname>Laganowsky</surname><given-names>A</given-names></name><name><surname>Jiang</surname><given-names>L</given-names></name><name><surname>Sievers</surname><given-names>SA</given-names></name><etal/></person-group><year>2011</year><article-title>Towards a pharmacophore for amyloid</article-title><source>PLOS BIOL</source><volume>9</volume><fpage>e1001080</fpage><pub-id pub-id-type="doi">10.1371/journal.pbio.1001080</pub-id></element-citation></ref><ref id="bib37"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lawrence</surname><given-names>MC</given-names></name><name><surname>Colman</surname><given-names>PM</given-names></name></person-group><year>1993</year><article-title>Shape complementarity at protein/protein interfaces</article-title><source>J Mol Biol</source><volume>234</volume><fpage>946</fpage><lpage>50</lpage><pub-id pub-id-type="doi">10.1006/jmbi.1993.1648</pub-id></element-citation></ref><ref id="bib38"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lazaridis</surname><given-names>T</given-names></name><name><surname>Karplus</surname><given-names>M</given-names></name></person-group><year>1999</year><article-title>Effective energy function for proteins in solution</article-title><source>Proteins</source><volume>35</volume><fpage>133</fpage><lpage>52</lpage><pub-id pub-id-type="doi">10.1002/(SICI)1097-0134(19990501)35:23.0.CO;2-N</pub-id></element-citation></ref><ref id="bib39"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Leaver-Fay</surname><given-names>A</given-names></name><name><surname>Tyka</surname><given-names>M</given-names></name><name><surname>Lewis</surname><given-names>SM</given-names></name><name><surname>Lange</surname><given-names>OF</given-names></name><name><surname>Thompson</surname><given-names>J</given-names></name><name><surname>Jacak</surname><given-names>R</given-names></name><etal/></person-group><year>2011</year><article-title>ROSETTA3: an object-oriented software suite for the simulation and design of macromolecules</article-title><source>Methods Enzymol</source><volume>487</volume><fpage>545</fpage><lpage>74</lpage><pub-id pub-id-type="doi">10.1016/B978-0-12-381270-4.00019-6</pub-id></element-citation></ref><ref id="bib40"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Liu</surname><given-names>C</given-names></name><name><surname>Zhao</surname><given-names>M</given-names></name><name><surname>Jiang</surname><given-names>L</given-names></name><name><surname>Cheng</surname><given-names>PN</given-names></name><name><surname>Park</surname><given-names>J</given-names></name><name><surname>Sawaya</surname><given-names>MR</given-names></name><etal/></person-group><year>2012</year><article-title>Out-of-register beta-sheets suggest a pathway to toxic amyloid aggregates</article-title><source>Proc Natl Acad Sci USA</source><volume>109</volume><fpage>20913</fpage><lpage>8</lpage><pub-id pub-id-type="doi">10.1073/pnas.1218792109</pub-id></element-citation></ref><ref id="bib41"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Luhrs</surname><given-names>T</given-names></name><name><surname>Ritter</surname><given-names>C</given-names></name><name><surname>Adrian</surname><given-names>M</given-names></name><name><surname>Riek-Loher</surname><given-names>D</given-names></name><name><surname>Bohrmann</surname><given-names>B</given-names></name><name><surname>Dobeli</surname><given-names>H</given-names></name><etal/></person-group><year>2005</year><article-title>3D structure of Alzheimer’s amyloid-beta(1-42) fibrils</article-title><source>Proc Natl Acad Sci USA</source><volume>102</volume><fpage>17342</fpage><lpage>7</lpage><pub-id pub-id-type="doi">10.1073/pnas.0506723102</pub-id></element-citation></ref><ref id="bib42"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>McLaurin</surname><given-names>J</given-names></name><name><surname>Kierstead</surname><given-names>ME</given-names></name><name><surname>Brown</surname><given-names>ME</given-names></name><name><surname>Hawkes</surname><given-names>CA</given-names></name><name><surname>Lambermon</surname><given-names>MH</given-names></name><name><surname>Phinney</surname><given-names>AL</given-names></name><etal/></person-group><year>2006</year><article-title>Cyclohexanehexol inhibitors of Abeta aggregation prevent and reverse alzheimer phenotype in a mouse model</article-title><source>Nat Med</source><volume>12</volume><fpage>801</fpage><lpage>8</lpage><pub-id pub-id-type="doi">10.1038/nm1423</pub-id></element-citation></ref><ref id="bib43"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Meiler</surname><given-names>J</given-names></name><name><surname>Baker</surname><given-names>D</given-names></name></person-group><year>2006</year><article-title>ROSETTALIGAND: protein-small molecule docking with full side-chain flexibility</article-title><source>Proteins</source><volume>65</volume><fpage>538</fpage><lpage>48</lpage><pub-id pub-id-type="doi">10.1002/prot.21086</pub-id></element-citation></ref><ref id="bib44"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Morris</surname><given-names>GM</given-names></name><name><surname>Huey</surname><given-names>R</given-names></name><name><surname>Lindstrom</surname><given-names>W</given-names></name><name><surname>Sanner</surname><given-names>MF</given-names></name><name><surname>Belew</surname><given-names>RK</given-names></name><name><surname>Goodsell</surname><given-names>DS</given-names></name><etal/></person-group><year>2009</year><article-title>AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility</article-title><source>J Comput Chem</source><volume>30</volume><fpage>2785</fpage><lpage>91</lpage><pub-id pub-id-type="doi">10.1002/jcc.21256</pub-id></element-citation></ref><ref id="bib45"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Necula</surname><given-names>M</given-names></name><name><surname>Kayed</surname><given-names>R</given-names></name><name><surname>Milton</surname><given-names>S</given-names></name><name><surname>Glabe</surname><given-names>CG</given-names></name></person-group><year>2007</year><article-title>Small molecule inhibitors of aggregation indicate that amyloid beta oligomerization and fibrillization pathways are independent and distinct</article-title><source>J Biol Chem</source><volume>282</volume><fpage>10311</fpage><lpage>24</lpage><pub-id pub-id-type="doi">10.1074/jbc.M608207200</pub-id></element-citation></ref><ref id="bib46"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Nelson</surname><given-names>R</given-names></name><name><surname>Sawaya</surname><given-names>MR</given-names></name><name><surname>Balbirnie</surname><given-names>M</given-names></name><name><surname>Madsen</surname><given-names>AO</given-names></name><name><surname>Riekel</surname><given-names>C</given-names></name><name><surname>Grothe</surname><given-names>R</given-names></name><etal/></person-group><year>2005</year><article-title>Structure of the cross-beta spine of amyloid-like fibrils</article-title><source>Nature</source><volume>435</volume><fpage>773</fpage><lpage>8</lpage><pub-id pub-id-type="doi">10.1038/nature03680</pub-id></element-citation></ref><ref id="bib47"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ono</surname><given-names>K</given-names></name><name><surname>Li</surname><given-names>L</given-names></name><name><surname>Takamura</surname><given-names>Y</given-names></name><name><surname>Yoshiike</surname><given-names>Y</given-names></name><name><surname>Zhu</surname><given-names>L</given-names></name><name><surname>Han</surname><given-names>F</given-names></name><etal/></person-group><year>2012</year><article-title>Phenolic compounds prevent amyloid beta-protein oligomerization and synaptic dysfunction by site-specific binding</article-title><source>J Biol Chem</source><volume>287</volume><fpage>14631</fpage><lpage>43</lpage><pub-id pub-id-type="doi">10.1074/jbc.M111.325456</pub-id></element-citation></ref><ref id="bib48"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Petkova</surname><given-names>AT</given-names></name><name><surname>Leapman</surname><given-names>RD</given-names></name><name><surname>Guo</surname><given-names>Z</given-names></name><name><surname>Yau</surname><given-names>WM</given-names></name><name><surname>Mattson</surname><given-names>MP</given-names></name><name><surname>Tycko</surname><given-names>R</given-names></name></person-group><year>2005</year><article-title>Self-propagating, molecular-level polymorphism in Alzheimer’s beta-amyloid fibrils</article-title><source>Science</source><volume>307</volume><fpage>262</fpage><lpage>5</lpage><pub-id pub-id-type="doi">10.1126/science.1105850</pub-id></element-citation></ref><ref id="bib49"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Petkova</surname><given-names>AT</given-names></name><name><surname>Yau</surname><given-names>WM</given-names></name><name><surname>Tycko</surname><given-names>R</given-names></name></person-group><year>2006</year><article-title>Experimental constraints on quaternary structure in Alzheimer’s beta-amyloid fibrils</article-title><source>Biochemistry</source><volume>45</volume><fpage>498</fpage><lpage>512</lpage><pub-id pub-id-type="doi">10.1021/bi051952q</pub-id></element-citation></ref><ref id="bib50"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Porat</surname><given-names>Y</given-names></name><name><surname>Abramowitz</surname><given-names>A</given-names></name><name><surname>Gazit</surname><given-names>E</given-names></name></person-group><year>2006</year><article-title>Inhibition of amyloid fibril formation by polyphenols: structural similarity and aromatic interactions as a common inhibition mechanism</article-title><source>Chem Biol Drug Des</source><volume>67</volume><fpage>27</fpage><lpage>37</lpage><pub-id pub-id-type="doi">10.1111/j.1747-0285.2005.00318.x</pub-id></element-citation></ref><ref id="bib51"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Sacchettini</surname><given-names>JC</given-names></name><name><surname>Kelly</surname><given-names>JW</given-names></name></person-group><year>2002</year><article-title>Therapeutic strategies for human amyloid diseases</article-title><source>Nat Rev Drug Discov</source><volume>1</volume><fpage>267</fpage><lpage>75</lpage><pub-id pub-id-type="doi">10.1038/nrd769</pub-id></element-citation></ref><ref id="bib52"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Sawaya</surname><given-names>MR</given-names></name><name><surname>Sambashivan</surname><given-names>S</given-names></name><name><surname>Nelson</surname><given-names>R</given-names></name><name><surname>Ivanova</surname><given-names>MI</given-names></name><name><surname>Sievers</surname><given-names>SA</given-names></name><name><surname>Apostol</surname><given-names>MI</given-names></name><etal/></person-group><year>2007</year><article-title>Atomic structures of amyloid cross-beta spines reveal varied steric zippers</article-title><source>Nature</source><volume>447</volume><fpage>453</fpage><lpage>7</lpage><pub-id pub-id-type="doi">10.1038/nature05695</pub-id></element-citation></ref><ref id="bib53"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Schutz</surname><given-names>AK</given-names></name><name><surname>Soragni</surname><given-names>A</given-names></name><name><surname>Hornemann</surname><given-names>S</given-names></name><name><surname>Aguzzi</surname><given-names>A</given-names></name><name><surname>Ernst</surname><given-names>M</given-names></name><name><surname>Bockmann</surname><given-names>A</given-names></name><etal/></person-group><year>2011</year><article-title>The amyloid-Congo red interface at atomic resolution</article-title><source>Angew Chem Int Ed Engl</source><volume>50</volume><fpage>5956</fpage><lpage>60</lpage><pub-id pub-id-type="doi">10.1002/anie.201008276</pub-id></element-citation></ref><ref id="bib54"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Shahnawaz</surname><given-names>M</given-names></name><name><surname>Soto</surname><given-names>C</given-names></name></person-group><year>2012</year><article-title>Microcin amyloid fibrils A are reservoir of toxic oligomeric species</article-title><source>J Biol Chem</source><volume>287</volume><fpage>11665</fpage><lpage>76</lpage><pub-id pub-id-type="doi">10.1074/jbc.M111.282533</pub-id></element-citation></ref><ref id="bib55"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Silveira</surname><given-names>JR</given-names></name><name><surname>Raymond</surname><given-names>GJ</given-names></name><name><surname>Hughson</surname><given-names>AG</given-names></name><name><surname>Race</surname><given-names>RE</given-names></name><name><surname>Sim</surname><given-names>VL</given-names></name><name><surname>Hayes</surname><given-names>SF</given-names></name><etal/></person-group><year>2005</year><article-title>The most infectious prion protein particles</article-title><source>Nature</source><volume>437</volume><fpage>257</fpage><lpage>61</lpage><pub-id pub-id-type="doi">10.1038/nature03989</pub-id></element-citation></ref><ref id="bib56"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Takahashi</surname><given-names>T</given-names></name><name><surname>Mihara</surname><given-names>H</given-names></name></person-group><year>2008</year><article-title>Peptide and protein mimetics inhibiting amyloid beta-peptide aggregation</article-title><source>Acc Chem Res</source><volume>41</volume><fpage>1309</fpage><lpage>18</lpage><pub-id pub-id-type="doi">10.1021/ar8000475</pub-id></element-citation></ref><ref id="bib57"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Williams</surname><given-names>AD</given-names></name><name><surname>Sega</surname><given-names>M</given-names></name><name><surname>Chen</surname><given-names>M</given-names></name><name><surname>Kheterpal</surname><given-names>I</given-names></name><name><surname>Geva</surname><given-names>M</given-names></name><name><surname>Berthelier</surname><given-names>V</given-names></name><etal/></person-group><year>2005</year><article-title>Structural properties of Abeta protofibrils stabilized by a small molecule</article-title><source>Proc Natl Acad Sci USA</source><volume>102</volume><fpage>7115</fpage><lpage>20</lpage><pub-id pub-id-type="doi">10.1073/pnas.0408582102</pub-id></element-citation></ref><ref id="bib58"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Wood</surname><given-names>SJ</given-names></name><name><surname>MacKenzie</surname><given-names>L</given-names></name><name><surname>Maleeff</surname><given-names>B</given-names></name><name><surname>Hurle</surname><given-names>MR</given-names></name><name><surname>Wetzel</surname><given-names>R</given-names></name></person-group><year>1996</year><article-title>Selective inhibition of Abeta fibril formation</article-title><source>J Biol Chem</source><volume>271</volume><fpage>4086</fpage><lpage>92</lpage><pub-id pub-id-type="doi">10.1074/jbc.271.48.30392</pub-id></element-citation></ref><ref id="bib59"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Xue</surname><given-names>WF</given-names></name><name><surname>Hellewell</surname><given-names>AL</given-names></name><name><surname>Gosal</surname><given-names>WS</given-names></name><name><surname>Homans</surname><given-names>SW</given-names></name><name><surname>Hewitt</surname><given-names>EW</given-names></name><name><surname>Radford</surname><given-names>SE</given-names></name></person-group><year>2009</year><article-title>Fibril fragmentation enhances amyloid cytotoxicity</article-title><source>J Biol Chem</source><volume>284</volume><fpage>34272</fpage><lpage>82</lpage><pub-id pub-id-type="doi">10.1074/jbc.M109.049809</pub-id></element-citation></ref><ref id="bib60"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Yang</surname><given-names>F</given-names></name><name><surname>Lim</surname><given-names>GP</given-names></name><name><surname>Begum</surname><given-names>AN</given-names></name><name><surname>Ubeda</surname><given-names>OJ</given-names></name><name><surname>Simmons</surname><given-names>MR</given-names></name><name><surname>Ambegaokar</surname><given-names>SS</given-names></name><etal/></person-group><year>2005</year><article-title>Curcumin inhibits formation of amyloid beta oligomers and fibrils, binds plaques, and reduces amyloid in vivo</article-title><source>J Biol Chem</source><volume>280</volume><fpage>5892</fpage><lpage>901</lpage><pub-id pub-id-type="doi">10.1074/jbc.M404751200</pub-id></element-citation></ref></ref-list></back><sub-article article-type="article-commentary" id="SA1"><front-stub><article-id pub-id-type="doi">10.7554/eLife.00857.039</article-id><title-group><article-title>Decision letter</article-title></title-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Kuriyan</surname><given-names>John</given-names></name><role>Reviewing editor</role><aff><institution>Howard Hughes Medical Institute, University of California, Berkeley</institution>, <country>United States</country></aff></contrib></contrib-group></front-stub><body><boxed-text><p>eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see <ext-link ext-link-type="uri" xlink:href="http://elife.elifesciences.org/review-process">review process</ext-link>). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.</p></boxed-text><p>Thank you for sending your work entitled “Structure-based discovery of fiber-binding compounds that reduce the cytotoxicity of amyloid beta” for consideration at <italic>eLife</italic>. Your article has been favorably peer reviewed by two reviewers, one of whom is a Senior editor.</p><p>The Senior editor has assembled the following comments to help you prepare a revised submission in which you should respond to the issues raised by the reviewers.</p><p>In this paper, David Eisenberg and colleagues use computational docking to discover small molecules that bind to and stabilize a variety of peptide fibers formed by fragments of amylogenic proteins, and in this way suppress the toxicity of amyloid fibrils of the Alzheimer’s Abeta peptide. They validate the binding of the peptides by NMR, followed up by assays for fibril formation, and also show that the small molecules alleviate the toxicity of the amylogenic peptides to mammalian cells. This work builds on earlier results from the Eisenberg group that established high-resolution crystal structures for many different kinds of peptide fibrils and revealed how small molecules such as Orange G bind to them. The work also follows on earlier studies from others (for example, Pastore and Wanker) on the effects of small molecules on amylogenic peptides. What is new in the present work, however, is the use of computational docking to come up with novel pharmacophores. This is interesting because the binding sites on the fibrils are quite different from the deep hydrophobic cavities that are the targets of conventional docking efforts. Here, the binding sites are flat, and although hydrophobicity is still important, the small molecules lie along the flat surfaces of the fibrils and are anchored by polar residues in a way that stabilizes the fibrils.</p><p>The paper is generally clearly written and illustrated. The great interest in developing drugs against amyloid diseases as well as the creative use of computational methods towards that end makes this paper suitable for publication in <italic>eLife</italic>.</p><p>Major comment:</p><p>A stronger case would be made if the authors tested whether their small molecule compounds bind to and reduce toxicity of oligomers. This would be a good test of their hypothesis that their compounds target fibrils (not oligomers) and prevent toxicity by reducing the release of toxic oligomers from fibrils (i.e., shifting the equilibrium toward fibrils). Do the authors have data that can address this question, or can such data be obtained relatively easily?</p></body></sub-article><sub-article article-type="reply" id="SA2"><front-stub><article-id pub-id-type="doi">10.7554/eLife.00857.040</article-id><title-group><article-title>Author response</article-title></title-group></front-stub><body><p><italic>A stronger case would be made if the authors tested whether their small molecule compounds bind to and reduce toxicity of oligomers. This would be a good test of their hypothesis that their compounds target fibrils (not oligomers) and prevent toxicity by reducing the release of toxic oligomers from fibrils (i.e., shifting the equilibrium toward fibrils). Do the authors have data that can address this question, or can such data be obtained relatively easily</italic>?</p><p>We thank the reviewers for raising this important question. To explore the possibility raised by the reviewers that our small molecule compounds (termed BAFs) might target oligomers, we designed experiments to test whether BAFs are capable of inhibiting pre-formed toxic Abeta oligomers using the MTT cell viability assay. The results of these new experiments are given in new <xref ref-type="table" rid="tbl7">Table 7</xref> and <xref ref-type="fig" rid="fig11s1">Figure 11—figure supplement 1</xref>: four BAFs, which significantly reduce Abeta toxicity, show no protective effects on the toxicity of pre-formed Abeta oligomers. These data verify that BAFs detoxify Abeta42 <italic>not</italic> by targeting toxic oligomers, as requested by the reviewers.</p><p>Also there are theoretical reasons for expecting that compounds designed to bind fibers will not bind oligomers. These reasons are based on recent work (Laganowsky et al., Science 2012; Liu et al., PNAS 2012), which shows that the structural features of a toxic oligomer are distinct from those observed in fibrils. As we detail in the Discussion section of the present paper, we applied our structure-based approach to search for BAFs that bind to flat in-register β-sheets in fibril structures rather than out-of-register β-strands found in toxic oligomeric structures. As highlighted in <xref ref-type="fig" rid="fig11">Figure 11</xref>, these small molecules are predicted to bind along the hydrophobic in-register β-sheet surfaces of the fibrils and to be anchored by polar residues (i.e., Lysine 16 of Abeta fibril in this case). The distances between the Lys residues that interact with the small molecules have distinct differences between in-register β-sheets (left panel) and out-of-register β-strands (right panel). Since we search for the small molecules (in a green color) compatible with the geometry of Lys residues in in-register β-sheets, these small molecules are not expected to bind out-of-register oligomer. Together with our new experimental results, we speculate that the BAFs are unable to bind to Abeta oligomers, and this difference accounts for the diminished toxicity that accompanies specific binding of the small molecules.</p></body></sub-article></article> |