Carboxyl-modified amino acids and peptides as protease inhibitors

Several types of carboxyl-modified amino acids and peptides were prepared in forms having N-terminal modifications (carrier fragments) suitable for one of several representative protease enzymes, and their inhibitory action toward those enzymes were evaluated. The carboxyl modifications (inhibitory units) included (b) CONH2, (c) CSNH2, (d) CN, (e) trans-CH = CHCO2Me, and (f) trans-CH = CHSO2Me. The carrier fragments included NH2(PhCH2)CHX (1), AcNH(PhCH2)CHX (2), H2NCH2CONH(PhCH2)CHX (3), and AcNH(PhCH2)CHCONHCH2X (4). Compounds 1b, 1d, 1e, and 1f were competitive inhibitors of both microsomal and cytosolic leucine aminopeptidase (Ki = 14.8, 67, 61, and 3.7 mM with the former and 14.1, 26.4, 27.3, and 8.8 mM with the latter, respectively). Neither compound 1c nor leucine thioamide had any detectable effect on either enzyme. Compounds 2b-f were also competitive inhibitors toward chymotrypsin (Ki = 13.9, 23.0, 5.3, 30.8, and 29.4 mM, respectively). While 4b, 4c, and 4d were competitive inhibitors of papain (Ki = 4.7, 0.095, and 0.0011 mM, respectively), 4e proved to be an irreversible affinity label (Ki = 0.026 mM and k2 = 0.0018 s-1). Inactivation of papain by 4e was retarded in the presence of 4d and could not be reversed by dialysis. Similarly 3b and 3d were competitive inhibitors of dipeptidyl aminopeptidase I (DPP-I, EC 3.4.14.1) (Ki = 6.2 and 0.0027 mM, respectively), while 3e and 3f were irreversible affinity labels (Ki = 0.22 and 0.18 mM, and k2 = 0.015 and 0.010 s-1, respectively). Inhibition of DPP-I by 3d provides only the second example of a cysteine protease which is strongly inhibited by a nitrile analogue of a specific substrate. Further studies are needed to determine the generality and potential utility of this finding. Compounds 3e, 3f, and 4e exemplify a new class of specific affinity labels for cysteine proteases whose activity probably derives from irreversible Michael addition of the catalytic cysteine to the activated double bond.