Development of Calpain-specific Inactivators by Screening of Positional Scanning Epoxide Libraries

Abstract

Calpains are calcium-dependent proteases that are required for numerous intracellular processes but also play an important role in the development of pathologies such as ischemic injury and neurodegeneration. Many current small molecule calpain inhibitors also inhibit other cysteine proteases, including cathepsins, and need improved selectivity. The specificity of inhibition of several calpains and papain was profiled using synthetic positional scanning libraries of epoxide-based compounds that target the active-site cysteine. These peptidomimetic libraries probe the P4, P3, and P2 positions, display (S,S)-or (R,R)-epoxide stereochemistries, and incorporate both natural and nonnatural amino acids. To facilitate library screening, an SDS-PAGE assay that measures the extent of hydrolysis of an inactive recombinant m-calpain was developed. Individual epoxide inhibitors were synthesized guided by calpain-specific preferences observed from the profiles and tested for inhibition against calpain. The most potent compounds were assayed for specificity against cathepsins B, L, and K. Several compounds demonstrated high inhibition specificity for calpains over cathepsins. The best of these inhibitors, WRH(R,R), irreversibly inactivates m-and -calpain rapidly (k 2 /K i ‫؍‬ 131,000 and 16,500 M ؊1 s ؊1 , respectively) but behaves exclusively as a reversible and less potent inhibitor toward the cathepsins. X-ray crystallography of the proteolytic core of rat -calpain inactivated by the epoxide compounds WR ␥-cyano-␣-aminobutyric acid (S,S) and WR allylglycine (R,R) reveals that the stereochemistry of the epoxide influences positioning and orientation of the P2 residue, facilitating alternate interactions within the S2 pocket. Moreover, the WR ␥-cyano-␣-aminobutyric acid (S,S)-complexed structure defines a novel hydrogen-bonding site within the S2 pocket of calpains.

Probing of primed and unprimed sites of calpains: Design, synthesis and evaluation of epoxysuccinyl-peptide derivatives as selective inhibitors

“…Guided by the results of our earlier study with a group of azaglycine substituted substrate peptides exhibiting inhibitory properties [14], we have prepared a limited set of the analogues for the present investigations. Our results confirmed the findings of Courrier et al [18] that at position P2 the Leu residue was preferred. Although its substitution to Val (5) resulted in a moderate calpain 2 inhibitory activity, without inhibiting the other enzymes.…”

“…Our observation about the preference of Trp residue at this position is also in harmony with findings in the literature [18]. Trp was the most preferred amino acid next to the Arg at this position, and Pro was one of the most unfavorable..…”

“…Using L-or D-epoxysuccinyl analogues the effect of changes in positions P 4 -P 2 was studied on calpain 1 and 2 inhibition [18]. As a result of this systematic study a peptide analogue, WRH(D-Eps)-OEt was identified as a selective and irreversible calpain inhibitor.…”

“…Epoxysuccinyl inhibitors have been co-crystallized with calpain 1 (2nqg [18], 2nqi, 2nqg [18], 1tlo [27]), papain (1cvz [28]) and cathepsin B (1ito [29] Figure 1): residues P 2 -P 4 are linked to Lys61, Gly103, Glu193, Ser196 of domain II of the enzyme, the epoxysuccinyl part is bound by the amide nitrogen of Gly198, P 1 ' Ser by the carbonyl oxygen of Gly261 of domain II and the P 5 ' Ser by Asp425 and Met426 of domain III -it is thus bound in a conformation where not the P 2 Leu, but the P 1 ' Ser is immersed in the S 2 pocket. This is not so surprising, since the S 2 pocket is lined by polar residues (Thr200, Thr201, Ser241, Glu339), whereby it is far from being an ideal docking site for Leu, and although the P 1 ' Ser is too short to reach these residues, it benefits from the polarized environment created by them.…”

“…Gly198 (and the corresponding Gly208 of calpain 1, Gly66 of papain and Gly74 of cathepsin B) forms a similar H-bond with the carbonyl oxygen atom of the Leu at position P 2 of calpastatin (3df0 [30], ltl9 [27]), or the epoxysuccinyl carbonyl oxygen of E64 (1tlo [27]), E64C (1ito [29]), WR18 (2nqg [18]), WR13 (2nqi [18]) or also of CLIK148 [28].…”