During the last decade fragment-based approaches have been established as a valuable alternative to conventional high throughput screening [1]. Increasing sensitivity of analytical methods allow for identification of weakly low affinity fragment probes. The design of non-covalently binding inhibitors for proteases has shown limited success until now [2], while the design of reversible, covalent inhibitors is considered a promising strategy for these enzymes. Therefore we wanted to identify covalently binding fragments as potential starting points for the development of non-peptidic inhibitors of viral 3C protease. This cysteine protease is a key factor in the replication cycle of the picornaviridae, which includes a variety of human pathogenic viruses like enteroviruses, human rhinovirus or coxsackievirus [3]. To identify covalently binding fragments, 3D pharmacophore-based virtual screening of our in-house fragment library was performed. Only reactive (warhead-containing) fragments showing key interactions with the 3C protease were selected for the subsequent biochemical evaluation using an FRET-substrate based enzyme assay. Identified hit fragments were further analyzed using mass spectrometry to verify covalent binding. Structure-based in-silico analysis in combination with experimentally determined mass spectra allows for rationalization of the fragment hit binding mode. This experimentally confirmed binding mode is used for an in silico fragment growing strategy to design novel potent protease inhibitors. Commercially available building blocks are combined using tailored reaction rules to ensure synthetic accessibility of the newly designed ligands. 1. Scott, D.E., et al., Fragment-based approaches in drug discovery and chemical biology. Biochemistry, 2012. 51(25): p. 4990-5003. 2. Turk, B., Targeting proteases: successes, failures and future prospects. Nat Rev Drug Discov, 2006. 5(9): p. 785-99. 3. Hughes, A.L., Phylogeny of the Picornaviridae and differential evolutionary divergence of picornavirus proteins. Infection Genetics and Evolution, 2004. 4(2): p. 143-152.

Reaction-driven design of viral protease inhibitors from covalently binding fragments

251st National Meeting (2016)

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