AZIRIDINE ANALOGS OF -(EPOXYSUCCINYL)-L-LEUCYL]AMINO]-4-GUANIDINOBUTANE (E-64) AS INHIBITORS OF CYSTEINE PROTEASES

Aziridine derivatives of E-64 have been synthesized, and their charact erization against the cysteine proteases cathepsin B, cathepsin L, and papain is reported. The inhibition was found to be strongly pH-depend ent, with maximum activity observed at pH 4, indicating that the proto nated aziridinium ion form of the inhibitor is the more reactive form. At low pH, the peptide aziridine HO-(L)Az-Leu-NH-iAm inactivated papa in with a second-order rate constant, k(inac)/K-i, of 7.0 x 10(4) M(-1 ) s(-1), a value very close to that observed with E-64 or with the cor responding epoxysuccinyl analog HO-(L)Eps-Leu-NH-iAm. This demonstrate s that with the correct peptide sequence, aziridine analogs of E-64 ca n be good irreversible inhibitors of cysteine proteases. Substitution of the epoxysuccinyl moiety by an aziridine does not affect the specif icity of inhibition against the three proteases used in this study. Th e D-diastereomer is the preferred (by 10-fold) diastereomer for the in hibition of cysteine proteases. The reactivity of both diastereomers o f iBuNH-Az-LeuPro-OH against cathepsin B was also found to be much low er than that of iBuNH-(L)Eps-LeuPro-OH, which is a potent selective in hibitor of cathepsin B. These differences are attributed mainly to the presence of the protonated aziridine ring, which can modify the bindi ng mode of aziridine analogs at the active site of cysteine proteases.