Structure-based design, synthesis and evaluation of conformationally constrained cysteine protease inhibitors

The inhibition of cysteine proteases is being studied as a strategy to comb at parasitic diseases such as Chagas' disease, leishmaniasis, and malaria. Cruzain is the major cysteine protease of Trypanosoma cruzi, the etiologic agent of Chagas' disease. A crystal structure of cruzain, covalently inacti vated by fluoromethyl ketone inhibitor 1 (Cbz-Phe-Ala-FMK), was used as a t emplate to design potential inhibitors. Conformationally constrained gamma- lactams containing electrophilic aldehyde (12, 17, 18, 25, 26, and 29) or v inyl sulfone (43, 44, and 46) units were synthesized. Constrained lactam 26 had IC50 values of ca. 20 nM against the Leishmania major protease and ca. 50 nM versus falcipain, an important cysteine protease isolated from Plasm odium falciparum. However, all of the conformationally constrained inhibito rs were weak inhibitors of cruzain, compared to unconstrained peptide aldeh yde (e.g. 5) and vinyl sulfone inhibitors (e.g. 48, which proved to be an e xcellent inhibitor of cruzain with an apparent second order inhibition rate constant (k(inact)/K-i) of 634,000 s(-1) M-1). A significant reduction in activity was also observed with acyclic inhibitors 30 and 51 containing alp ha-methyl phenylalanine residues at the P-2 position. These data indicate t hat the pyrrolidinone ring, especially the quarternary center at P-2, inter feres with the normal substrate binding mode with cruzain, but not with fal cipain or the leishmania protease. (C) 1998 Elsevier Science Ltd. All right s reserved.