Mechanism of Action
The molecular mechanism of action of OSA_000822 will be investigated using two proteomic techniques:
- Multiplexed inhibitor beads with mass spectroscopic detection (MIB-MS), a protein-capture technique performed with and without compound present.
- Cellular thermal shift assay (CETSA), which detects a shift in melting point when compound is bound.
Both the MIB-MS and CETSA experiments will be conducted in collaboration with the UNC Proteomics Core.
The image below represents the basic experimental design. MRSA cells are incubated with either an active compound (OSA_822), inactive compound (OSA_820), or DMSO control. Cells are lysed and passed over kinase inhibitors bound to immobilized beads. The bound proteins are then analyzed and identified by MS. Decreased binding in the presence of active compound compared to DMSO indicates that the compound is competing for the binding site of the immobilized kinase inhibitor, and thereby preventing protein binding to the bead.
Adapted from: doi.org/10.1074/jbc.RA119.011083
The inhibitors used and their kinase inhibition profiles are shown below. The inhibitor bead cocktail consists of reversible, promiscuous mammalian ATP-site binders in order to capture as much of the kinome as possible. However, beads oftentimes bind other proteins that are not kinases. The results provided by the UNC team contain ALL proteins identified, not just kinases. The targets in our dataset were identified by mass spec using a MRSA-specific database and are known MRSA proteins.
From: doi.org/10.18632/oncotarget.24337
Raw data from the first MIB experiment may be found here, reported as a log2 fold change (FC) of each sample compared to DMSO. A log2 FC ≤ -0.5 can be considered a kinase with decreased MIB binding/abundance. A log2 FC ≥ 0.5 can be considered a kinase with increased MIB binding/abundance. A more stringent cutoff is log2 FC -1 or lower/1 or higher. With a competition assay, the most compelling data will often be the protein groups that respond in a dose-dependent manner in either direction.
Kinases most meriting follow-up are those that show significant dose-dependent changes in MIB binding when treated with OSA_822, but not OSA_820. Filtering the results in this order
- Those showing -0.5 < log2 FC < 0.5 at 50 uM OSA_822 (28)
- Those that did not show a dose-dependent change when treated with OSA_822
- Those that showed dose-dependent changes when treated with both OSA_822 and OSA_820
led to the identification of the proteins below as being of interest as possible targets:
Discussion of these results and next steps may be found at #15 and #53
The data from the proteomics work done at UNC may be found as follows:
ProteomeXchange title: Kinase Selectivity Profiling of anti-MRSA Diarylimidazoles in HEK293 Cells ProteomeXchange accession: PXD040208 PubMed ID: 37991879 Publication DOI: Not applicable Project Webpage: http://www.ebi.ac.uk/pride/archive/projects/PXD040208 FTP Download: https://ftp.pride.ebi.ac.uk/pride/data/archive/2024/01/PXD040208