Phenylthiomethyl Ketone-Based Fragments Show Selective and Irreversible Inhibition of Enteroviral 3C Proteases

Lead
structure discovery mainly focuses on the identification of
noncovalently binding ligands. Covalent linkage, however, is an essential
binding mechanism for a multitude of successfully marketed drugs,
although discovered by serendipity in most cases. We present a concept
for the design of fragments covalently binding to
proteases. Covalent linkage enables fragment binding unrelated to
affinity to shallow protein binding sites and at the same time allows
differentiated targeted hit verification and binding location verification
through mass spectrometry. We describe a systematic and rational computational
approach for the identification of covalently binding fragments from
compound collections inhibiting enteroviral 3C protease, a target
with high therapeutic potential. By implementing reactive groups potentially
forming covalent bonds as a chemical feature in our 3D pharmacophore
methodology, covalent binders were discovered by high-throughput virtual
screening. We present careful experimental validation of the virtual
hits using enzymatic assays and mass spectrometry unraveling a novel,
previously unknown irreversible inhibition of the 3C protease by phenylthiomethyl
ketone-based fragments. Subsequent synthetic optimization through
fragment growing and reactivity analysis against catalytic and noncatalytic
cysteines revealed specific irreversible 3C protease inhibition.