Electrophilic inhibitors have, until recently, been underutilised in drug design. But there has been a growing recognition for their utility as powerful tools for exploring biological mechanisms. The irreversible covalent interactions formed make identifying targets of compounds in cells more straight-forward than for non-covalent inhibitors, as chemical proteomics approaches can be utilised to isolate and identify interacting proteins. This is particularly attractive for phenotypic screening approaches, where the mode of action and protein targets are unknown.
Generation of these electrophilic screening libraries typically uses a limited synthetic toolkit, resulting in libraries with poor diversity that do not efficiently explore biologically relevant chemical space. Therefore, we aimed to develop a workflow that moves from the creation of a library of electrophilic compounds through to inhibitor discovery and profiling biological mechanisms in cells using chemical proteomics. Building a structurally diverse compound library using an iridium-catalysed dehydrogenative coupling based around sulfonyl fluoride warheads, to exploit their reactivity and stability under physiological conditions, and their reactive promiscuity to several protein amino acid side chains, aiming to reveal novel protein targets not accessible to traditional synthetic design.
Working in collaboration with the Smith group at the University of St. Andrews, the sulfonyl fluoride-based library was then screened in phenotypic assays against the human African trypanosomiasis parasite T. brucei, obtaining several active compounds with sub-micromolar potency with significant selectivity over human HeLa cells. We then designed chemical probes, analogous compounds containing an alkyne tag to directly explore the protein targets and mode of action of the active compounds using chemical proteomics methods. Initial work was undertaken to visualise these protein-probe interactions by SDS-PAGE from in-gel fluorescence. From here, future work should focus on the identification of these reactive proteins by mass spectrometry.
