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1J

16. Johanna Vappiani

GlaxoSmithKline
Photoaffinity probes and quantitative proteomics enable assessment of target engagement and compound potency in live cel
Heidelberg, Germany
Late stage failure in drug development is a critical challenge for the pharmaceutical industry and is mainly caused by a lack of efficacy or safety considerations. The comprehensive preclinical assessment of target and off-target engagement in relevant biological systems could contribute to a reduction of attrition in clinical trials. Photoaffinity labeling (PAL) combined with proteomics enables detecting interactions between probe molecules and their corresponding protein targets in live cells, but PAL approaches described so far predominantly provide qualitative information. We have further developed this technology into a quantitative target engagement assay that enables the determination of apparent dissociation constants (Kdapp) for drug-target interactions in live cells. Trifunctional PAL probes consist of a small molecule ligand for target binding, a photo reactive moiety for covalent labeling, and the bioorthogonal handle trans-cyclooctene (TCO) for enrichment. The inverse electron-demand Diels-Alder reaction using TCO and tetrazine provides high reaction rates in complex biological systems, and thus enables a robust and simple enrichment workflow. We synthesized PAL probes through a modular approach in which building blocks consisting of different photo reactive groups and TCO can be easily reacted to any amine functionalized small molecule. Experimental conditions for quantitative assays were established using a PAL-probe derived from the histone deacetylase (HDAC) inhibitor Vorinostat. In a first step, the affinity of a photoreactive probe to its targets was assessed using a saturation binding model. In a second step, IC50s were measured for each target in cell-based competition binding experiments between the drug molecule and the PAL-probe. Using the Cheng-Prusoff relationship, Kdapps were determined from IC50s using correction factors determined in step 1. With this approach, we determined apparent interaction constants between Vorinostat and endogenous HDACs in lysate and in live cells. Target affinities were in good agreement with published reports. In addition, we identified Tetratricopeptide repeat protein 38 (TTC38) as a novel Vorinostat target in cell-based experiments. Next, we applied photo-affinity labeling to probe the interaction of cyanopindolol with the G protein coupled receptor ADRB2. With PAL-modified desisopropyl propranolol, ADRB2 was captured from an over-expressing cell line, as well as from a cell line with endogenous expression levels. Out of more than 1000 proteins identified in a proteomics competition experiment with the adenoreceptor antagonist cyanopindolol, the cognate target ADRB2 was the only protein showing dose-dependent competition of probe binding (Kdapp = 1.0 nM). In summary, we present a quantitative target engagement assay, that enables assessment of drug-target interactions in living cells. The method is applicable to multipass membrane proteins, which is challenging with many other chemoproteomics methods.