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.