Invited
Covalent inhibitors on demand - computational and empirical covalent ligand discovery

Covalent probes can display unmatched potency, selectivity and duration of action; however, their discovery is challenging. We developed two complimentary technologies for covalent ligand discovery: covalent virtual screening and empirical covalent fragment screening. To demonstrate the utility of covalent docking we report its use for the discovery of the first covalent MKK7 inhibitors. We optimized these compounds to low-micromolar inhibitors of JNK phosphorylation in cells. The crystal structure of a lead compound bound to MKK7 demonstrated that the binding mode was correctly predicted by docking. We asserted the selectivity of our inhibitors on a proteomic level and against a panel of 76 kinases, and validated an on-target effect using knockout cell lines. For the empirical covalent screening, we constructed a library of 993 mildly electrophilic fragments, characterized it by a new high-throughput thiol-reactivity assay and screened them against ten cysteine-containing proteins. Highly reactive and promiscuous fragments were rare and could be easily eliminated. By contrast, we found selective hits for most targets. Combination with high-throughput crystallography allowed rapid progression to potent and selective probes for two enzymes, the deubiquitinase OTUB2, and the pyrophosphatase NUDT7. No inhibitors were previously known for either. Combined, these two approaches allow the discovery of a new generation of covalent probes for many various protein targets.