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Herein, we report arylazopyrazole ureas and sulfones as a novel class of photoswitchable serine hydrolase inhibitors and present a chemoproteomic platform for rapid discovery of optically controlled serine hydrolase targets in complex proteomes. Specifically, we identify highly potent and selective photoswitchable inhibitors of the drug-metabolizing enzymes carboxylesterases 1 and 2 and demonstrate their pharmacological application by optically controlling the metabolism of the immunosuppressant drug mycophenolate mofetil. Collectively, this proof-of-concept study provides a first example of photopharmacological tools to optically control drug metabolism by modulating the activity of a metabolizing enzyme. Our arylazopyrazole ureas and sulfones offer synthetically accessible scaffolds that can be expanded to identify specific photoswitchable inhibitors for other serine hydrolases, including lipases, peptidases, and proteases. Our chemoproteomic platform can be applied to other photoswitches and scaffolds to achieve optical control over diverse protein classes.

For the discovery of novel chemical matter generally endowed with bioactivity, strategies may be particularly efficient that combine previous insight about biological relevance, e.g., natural product (NP) structure, with methods that enable efficient coverage of chemical space, such as fragment-based design. We describe the de novo combination of different 5-membered NP-derived N-heteroatom fragments to structurally unprecedented “pseudo-natural products” in an efficient complexity-generating and enantioselective one-pot synthesis sequence. The pseudo-NPs inherit characteristic elements of NP structure but occupy areas of chemical space not covered by NP-derived chemotypes, and may have novel biological targets. Investigation of the pseudo-NPs in unbiased phenotypic assays and target identification led to the discovery of the first small-molecule ligand of the RHO GDP-dissociation inhibitor 1 (RHOGDI1), termed Rhonin. Rhonin inhibits the binding of the RHOGDI1 chaperone to GDP-bound RHO GTPases and alters the subcellular localization of RHO GTPases.

We present a novel chemical scaffold for cysteine-reactive covalent inhibitors. Chloromethyl triazoles (CMTs) are readily accessed in only two chemical steps, thus enabling the rapid optimization of the pharmacological properties of these inhibitors. We demonstrate the tunability of the CMTs towards a specific biological target by synthesizing AA-CW236 as the first potent non-pseudosubstrate inhibitor of the O(6) -alkylguanine DNA methyltransferase (MGMT), a protein of major clinical significance for the treatment of several severe cancer forms. Using quantitative proteomics profiling techniques, we show that AA-CW236 exhibits a high degree of selectivity towards MGMT. Finally, we validate the effectiveness of our MGMT inhibitor in combination with the DNA alkylating drug temozolomide in breast and colon cancer cells by fluorescence imaging and a cell-viability assay. Our results may open a new avenue towards the development of a clinically approved MGMT inhibitor.