Small-molecule inhibitor: E64

Name

History: E64 was described by Hanada et al. (1978).
Peptidases inhibited: The majority of peptidases in family C1 are inactivated, although rates vary widely. Half-times (s) for inhibition at 10 micromolar inhibitor (Salvesen & Nagase, 2001) are: 0.1 (papain), 0.8 (cathepsin B), 17 (cathepsin H), 0.7 (cathepsin L), 9.2 (calpain). Falcipain 1 may not be inhibited (Goh et al., 2005). Staphopain is inhibited (Hofmann et al., 1993) as is the vaccinia virus I7L peptidase: Byrd & Hruby, 2005). E64 is very definitely not a general inhibitor of cysteine peptidases. A subset of the families in clan CA contain peptidases with a preference for glycyl bonds that resist inhibition by E64, and peptidases in clan CE have similar characteristics (Golubtsov et al., 2006). E64 shows no irreversible inactivation of peptidases in clan CD such as the caspases, for example.
There is reversible inhibition of some peptidases not in clan CA including clostripain (Barrett et al., 1982), trypsin (Sreedharan et al., 1996) and gingipain R (Rangarajan et al., 1997); these are arginine-specific enzymes, and the (4-guanidino)butane moiety of the inhibitor play a role.
Mechanism: Inhibition is irreversible, by S-alkylation of the active site cysteine with opening of the epoxide ring (Matsumoto et al., 1989; Varughese et al., 1989). Crystal structures show that the inhibitor occupies S rather than S" subsites.
DrugBank: DB04276

ChEBI: 30270
Structure
Chemical/biochemical name: 4-[(2S,3S)-3-carboxyoxiran-2-ylcarbonyl-L-leucylamido(4-guanidino)butane; L->trans-epoxysuccinyl-leucylamido(4-guanidino)butane
Formula weight: 357

Inhibitor class: This compound is of the epoxysuccinate class of inhibitors, which affect primarily cysteine peptidases in clan CA. The compounds inhibit irreversibly by S-alkylation of the catalytic cysteine, which results in opening of the epoxide ring. A thioester bond is formed by nucleophilic attack at C2 or C3 of the epoxide ring. Powers et al. (2002) (pp. 4664-4675) provide an authoritative review of epoxysuccinyl peptides as peptidase inhibitors.
Comment: An early study comparing reaction rate constants of epoxides, and showing the usefulness of E64 in the active-site-titration of some cysteine peptidases, was that of Barrett et al. (1982).