Primed-site Probing of Papain-like Cysteine Proteases

A general concept is presented for the development and enhancement of novel molecular probes for papain-like cysteine proteases. To achieve an increase in affinity and selectivity of such probes for the target enzymes, the specificity of the primed binding subsites of the active-site clefts were investigated by the use of partially randomized dipeptide libraries containing Ep-460, a trans-epoxysuccinyl thiol-reactive unit. Once covalently grafted to the catalytic cysteine residue, the anchor ensured proper alignment of the peptide moiety to the primed sites of the enzymes. After determining the inhibition potencies of these libraries, the best candidates were deconvoluted in a second cycle of synthesis and kinetic measurements. Proof of concept was demonstrated by application of this strategy to cathepsins B and L as well as to μ-calpain. By this approach, compounds of markedly enhanced inhibitory potency were obtained for cathepsin B and L.