Target profiling of PARP inhibitors and necroptosis inhibitors using photoaffinity labelling

Abstract

Target profiling of a small molecule therapeutic is essential to fully understand how that compound works in the clinic. Photoaffinity labelling (PAL) has become a widely utilised strategy for in-cell target identification campaigns for reversible, small molecule drugs. After an overview of target profiling and PAL, this Thesis discusses the application of PAL to two classes of molecules with incomplete target profiles. The Thesis focusses initially on the generation of the first photo-activatable probe for inhibitors of the PARP family of enzymes, PARPYnD, based on a novel anti-cancer PARP1/2/6 inhibitor AZ0108 with unexplained off-target toxicity. The design, synthesis and validation of the probe is discussed, along with the application of PARPYnD to PAL studies. Herein, simultaneous live-cell target engagement of PARP1/2 is shown for the first time by a photo-activatable probe, and this labelling is used to quantify live-cell engagement of these PARPs by known PARP inhibitors in competitive PAL experiments. For AZ0108 and clinical PARP inhibitor olaparib, novel off-targets are identified, demonstrating the power of PAL to capture weaker, secondary binders. Finally, PARPYnD fails to label PARP6 in live cells, but is able to label recombinant PARP6, highlighting a biomolecular disparity that raises questions about the proposed mechanism of action of AZ0108. PAL is then applied to a novel series of inhibitors of necroptosis, an inflammatory form of cell death, with an unknown mechanism of action. Design and synthesis of cell-active photo-activatable probe 7PQYnD1 is presented, along with the development of a bespoke live-cell necroptosis assay to evaluate necroptosis inhibitors in-house. 7PQYnD1 is then applied to the PAL workflow and five bona fide target proteins are identified through proteomics. Preliminary functional analysis of these hits is then undertaken to begin to identify the target interaction(s) responsible for the anti-necroptosis phenotype of these compounds.