Mechanism of Action

The mechanism of action (MoA) for this series, highlighted by the lead compound OSM-S-106, is currently being studied. The Plasmodium falciparum asparagine tRNA synthetase (PfAsnRs) is a key target under consideration.

The newest paper (Reaction hijacking inhibition of Plasmodium falciparum asparagine tRNA synthetase) presents comprehensive biological studies focused on this novel target and its MoA. The mechanism of action opens a novel approach to targeting a class of enzymes considered viable drug targets in Plasmodium and other pathogens.

The ongoing work on the series would improve the OSM-S-106 based on the understanding of reaction hijacking. In addition to PfAsnRs, we are also exploring other potential mechanisms, such as PfCA, and will provide updates as new findings emerge.

Here are the molecules (106 and 106 derivatives) from the latest SAR: (Note: New analogues' latest results will be updated on the Issues page.)

Pf AsnRs

All the results related to biological experiments are provided in the paper here.

About the new target:

Stanley from the University of Melbourne investigated Pf AsnRS as a potential target by examining OSM-S-106's ability to inhibit protein translation. This inhibition led to uncharged tRNA accumulation and eIF2α phosphorylation, supporting the targeting of Pf AsnRS by OSM-S-106.
In LCMS analyses, a strong signal for the Asn-OSM-S-106 adduct (m/z 421.0746) was detected when OSM-S-106 was treated in Pf cell culture.
Further characterisation of OSM-S-106’s targets showed: (1) PfAsnRS^R487S mutation line had 2.3-fold lower sensitivity to OSM-S-106, implying PfAsnRS as a key target. (2) Knockdown of PfNT4, PfGDH3, and PfCA (another potential 106 target) did not significantly impact parasite growth, consistent with previous findings. (3) No differential sensitivity to OSM-S-106 was observed following knockdown of PfAlaRS, PfGlyRS, PfGDH3, or PfCA, suggesting these proteins are not major targets.

106 selectivity of Pf AsnRs but not Hs AsnRs:

Recombinant HsAsnRS showed limited capacity to form Asn-OSM-S-106 adducts compared to PfAsnRS (m/z 421.0735), with 18-fold lower intensity.
OSM-S-106 reduces ATP usage by PfAsnRS but not HsAsnRS. In the presence of tRNA, 106 increases ATP consumption, indicating productive aminoacylation. It effectively inhibits PfAsnRS, PfAsnRS^R487S, and partially HsAsnRS. Susceptibility to reaction hijacking appears to depend more on the ability to generate the Asn-OSM-S-106 adduct than on binding the preformed conjugate.
The structural difference between PfAsnRS and HsAsnRS lies in a unique Plasmodium-specific insertion near the flipping loop. This loop secures the activated Asn-AMP intermediate and shifts to accommodate the tRNA acceptor stem. In PfAsnRS, a large insert may prolong Asn-tRNA binding, allowing more time for OSM-S-106 reaction. In HsAsnRS, quicker Asn-tRNA release reduces reaction hijacking chances.

Below are previous studies history Some results were inconsistent with our findings that OSM-S-106 inhibits Pf AsnRs due to the absence of tRNA involvement.

There is experimental evidence from resistant mutant generation by the MalDA consortium that OSM-S-106 inhibits Pfal asparagine tRNA ligase. This is an interesting, unique target, e.g. here. The tRNA synthetases are of interest as drug targets generally and specifically for infectious disease. Several experiments were scheduled to validate this finding:

a) Marcus Lee (Sanger) is doing CRISPR-Cas9 validation (this supported the synthetase being the target of OSM-S-106) and Jacquin Niles (MIT) is doing conditional knock-downs.

b) Stuart Ralph (Uni Melbourne) is happy to investigate by using radiolabelled amino acids +/- OSM-S-106 to see if protein synthesis is affected (as predicted), and to see if the IC50 alters in the presence of excess asparagine. The former experiment is something that Sabine Ottilie in Elizabeth's lab is also happy to do.

c) Ho Leung has built a homology model of the target, and has started docking of ligand and target. He is happy to express the protein in his lab if helpful towards a biophysics or co-crystal structure validation. Protein expression was also been volunteered by the SGC (Al Edwards, offline suggestion to Mat Todd) leading to the biochemical assay mentioned below. Shozeb Haider also developed a preliminary homology model for this target.

d) Valentina Garcia in Joe DeRisi's lab at UCSF have conducted cell-free translation inhibition experiments that appeared to show OSM-S-106 does not inhibit translation, but this is not inconsistent with inhibition of Arg-tRNA ligase.

e) Metabolomics experiments on OSM-S-106 by Darren Creek et al. had been inconclusive, with a "general death" profile observed.

f) A direct biochemical assay on recombinant protein performed at SGC-Unicamp by Rafa Counago suggested that NRS is not the target, given that no inhibition was seen, though the assay lacked a positive control (because there wasn't one available). Subsequent work by Leann Tilley's group has elucidated why this biochemical assay gave no inhibition - to be published shortly.

Leann Tilley has also published her group's work on the related target, Pfal tyrosine tRNA synthetase.

There is significant interest in these kinds of targets for other pathogens too, e.g. bacteria, giving us a strong rationale for cross-screening.

Another possibility 1: Carbonic Anhydrase

Carbonic anhydrase (CA) has also been raised as a potential target worthy of further investigation multiple times also here. Claudiu T. Supuran and his team from Uni of Florence ran an inhibition assay of OSM-S-106 and OSM-S-123 against PfηCA. OSM-S-106 looks like a promising inhibitor of PfηCA.

Sally-Ann Poulsen published an important study examining 31 primary sulfonamide compounds sourced from the GlaxoSmithKline (GSK) Tres Cantos antimalarial set (TCAMS) for their ability selectively to inhibit the in vitro growth of Pfal asexual stage malaria parasites. The aromatic sulfonamide moiety is known to inhibit CA from many organisms so the compounds were assessed for recombinant Pfal CA (PfCA) mediated inhibition of CO2 hydration. The PfCA inhibition activity did not correlate with antiplasmodial potency. OSM-S-106 was (we think) not included in this paper, though it was in the TCAMS set.

Sally-Ann Poulsen and her student Sarah Mueller have now obtained a crystal structure of human CA with OSM-S-106 bound.

Earlier, Chris Swain had conducted a study on a homology model of Pfη Carbonic Anhydrase in Issue 21 which can be used for further study if necessary - note the crystallography above is of the human enzyme, which is easier to crystallise than the Pf enzyme.

Benzenesulfonamides bearing various substituted (hetero)aryl rings in the para-position were evaluated as human carbonic anhydrase (hCA, EC 4.2.1.1) inhibitors against isoforms hCA I, II, IX, and XII. Most of the prepared sulfonamides showed low inhibition against hCA I isoform, whereas the other cytosolic isoenzyme, hCA II, was strongly affected. (DOI: 10.1021/acs.jmedchem.5b01771) So it matters which CA is under study.

Another possibility 2: Kinases

Clues to the MoA may be gleaned from the surrounding chemical space, but a possible MoA, based on the general similarity of OSM-S-106 to other known active compounds, is the inhibition of one or more kinases. Early predictive modeling suggested several targets (CLK1/CLK3, PKA-R, thymidylate kinase, PfPK5) over others (Sir2a, PI3K, PI4K-3B). Additional modelling further supported PKA as a promising target. It was concluded that the compounds may have a promiscuous role vs. several kinase targets, but the docking studies were consistent with the experimental observations that small structural changes, of the kinds that had been experimentally investigated, would negatively impact binding (explaining the low number of active analogs discovered to date). (A control compound, OSM-S-123, lacking the sulfonamide and which is inactive vs. the parasite, has been used to investigate other potential kinases and can be used as a check for any binding hypotheses).

Subsequent production of two relevant proteins (Pf CLK1 and PKA-R) was successful (see also discussion here). The below molecules (high, medium, low potency vs. Pfal) were sent to the relevant laboratory in Kansas State University for evaluation against these proteins (alongside a known, relevant kinase inhibitor, sunitinib). A known, structurally related compound caboxamycin was suggested for these experiments, and given the expense, the synthesis has been started.

The same molecules were sent to the University of North Carolina for screening against a broader range of kinases (shipment; experiments courtesy of Carrow Wells in David Drewry's lab; data). The preliminary results suggested several possible targets for OSM-S-106 (2 non-mutant human kinases TYK2 (1, 2) and MAP3K19; and PfCDPK1), OSM-S-137 (WEE1 but may be a false positive) and OSM-S-126 (relatively narrow spectrum human FLT3 inhibitor). Kd determinations are ongoing. A SwissTarget prediction report was also carried out and showed kinases as the likely target. Chris Swain verified this with other prediction packages

Another possibility 3: A combination of mechanisms

It is possible that the carbonic anhydrase binding sequesters the drug into erythrocytes, essentially acting as a drug delivery mechanism that enhances its concentration around blood stage plasmodium parasites. i.e. the binding is real, but the CA inhibition is of little consequence to the molecular MoA. This is discussed in more detail in Issue 14.