Expedited SARS-CoV-2 Main Protease Inhibitor Discovery through Modular 'Direct-to-Biology' Screening.

Reactive fragment (RF) screening has emerged as an efficient method for ligand discovery across the proteome, irrespective of a target's perceived tractability. To date, however, the efficiency of subsequent optimisation campaigns has largely been low-throughput, constrained by the need for synthesis and purification of target compounds. We report an efficient platform for 'direct-to-biology' (D2B) screening of cysteine-targeting chloroacetamide RFs, wherein synthesis is performed in 384-well plates allowing direct assessment in downstream biological assays without purification.

One-Pot Oxidative Amidation of Aldehydes via the Generation of Nitrile Imine Intermediates.

A one-pot procedure for the oxidative amidation of aldehydes via the generation of reactive nitrile imine (NI) intermediates has been developed. Distinct from our progenitor processes, mechanistic and control experiments revealed that the NI undergoes rapid oxidation to an acyl diazene species, which then facilitates -acylation of an amine. A range of substrates have been explored, including application in the synthesis of pharmaceutically relevant compounds.

Novel WRN Helicase Inhibitors Selectively Target Microsatellite-Unstable Cancer Cells.

Microsatellite-unstable (MSI) cancers require WRN helicase to resolve replication stress due to expanded DNA (TA)n dinucleotide repeats. WRN is a promising synthetic lethal target for MSI tumors, and WRN inhibitors are in development. In this study, we used CRISPR-Cas9 base editing to map WRN residues critical for MSI cells, validating the helicase domain as the primary drug target.

Kinetic Optimization of Lysine-Targeting Covalent Inhibitors of HSP72.

The covalent inhibition mechanism of action, which overcomes competition with high-affinity, high-abundance substrates of challenging protein targets, can deliver effective chemical probes and drugs. The success of this strategy has centered on exposed cysteine residues as nucleophiles but the low abundance of cysteine in the proteome has limited its application. We have recently reported our discovery that lysine-56 in the difficult-to-drug target HSP72 could form a covalent bond with a small-molecule inhibitor.

Lysine-Targeting Covalent Inhibitors.

Targeted covalent inhibitors have gained widespread attention in drug discovery as a validated method to circumvent acquired resistance in oncology. This strategy exploits small-molecule/protein crystal structures to design tightly binding ligands with appropriately positioned electrophilic warheads. Whilst most focus has been on targeting binding-site cysteine residues, targeting nucleophilic lysine residues can also represent a viable approach to irreversible inhibition.

An Irreversible Inhibitor of HSP72 that Unexpectedly Targets Lysine-56.

The stress-inducible molecular chaperone, HSP72, is an important therapeutic target in oncology, but inhibiting this protein with small molecules has proven particularly challenging. Validating HSP72 inhibitors in cells is difficult owing to competition with the high affinity and abundance of its endogenous nucleotide substrates. We hypothesized this could be overcome using a cysteine-targeted irreversible inhibitor.