The synthesis of 10 analogues of pepstatin modified so that statine is replaced by 4-amino-3-hydroxy-3,6-dimethylheptanoic acid (Me3Sta) or 4-amino-3-hydroxy-3-methyl-5-phenylpentanoic acid (Me3AHPPA) residues is reported. Both the 3S,4S and 3R,4S diastereomers of each analogue were tested as inhibitors of the aspartic proteases, porcine pepsin, cathepsin D, and penicillopepsin. In all cases the 3R,4S diastereomer (rather than the 3S,4S diastereomer) of the Me3Sta and Me3AHPPA derivatives was found to be the more potent inhibitor of the aspartic protease (Ki = 1.5-10 nM for the best inhibitors), in contrast to the results obtained with statine (Sta) or AHPPA derivatives, where the 3S,4S diastereomer is the more potent inhibitor for each diastereomeric pair of analogues. The Me3Sta- and Me3AHPPA-containing analogues are only about 10-fold less potent than the corresponding statine and AHPPA analogues and 100-1000-fold more potent than the corresponding inhibitors lacking the C-3 hydroxyl group. Difference NMR spectroscopy indicates that the (3R,4S)-Me3Sta derivative induces conformational changes in porcine pepsin comparable to those induced by the binding of pepstatin and that the (3S,4S)-Me3Sta derivatives do not induce the difference NMR spectrum. These results require that the C-3 methylated analogues of statine-containing peptides must inhibit enzymes by a different mechanism than the corresponding statine peptides. It is proposed that pepstatin and (3S)-statine-containing peptides inhibit aspartic proteases by a collected-substrate inhibition mechanism. The enzyme-inhibitor complex is stabilized, relative to pepstatin analogues lacking the C-3 hydroxyl groups, by the favorable entropy derived when enzyme-bound water is returned to bulk solvent.(ABSTRACT TRUNCATED AT 250 WORDS)

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Five new cathepsin D inhibitors were synthesized and tested as inhibitors of bovine cathepsin D. The compounds were derived by replacing a Phe-Phe dipeptidyl unit of a good cathepsin D substrate, Boc-Phe-Leu-Ala-Phe-Phe-Val-Leu-OR, with statine [3S,4S)-4-amino-3-hydroxy-6-methylheptanoic acid, Sta) or with Sta-Phe. The best inhibitor, Boc-Phe-Leu-Ala-(S,S)-Sta-Val-Leu-OMe, inhibited cathepsin D with a Ki value of 1.1 nM. In general, the more effective inhibitors were consistent with the proposal that statine functions as a replacement for a dipeptidyl unit. Thirty-five known pepstatin analogues also were evaluated as cathepsin D inhibitors. Substituents in the P4 and P3' positions are important for maximal inhibition of this aspartic proteinase, and the P4 substituent appears more important for inhibition of cathepsin D than for inhibition of other aspartic proteinases.

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The synthesis of two new analogues of statine are reported corresponding to analogues with the lysine side chain and the ornithine side chain. These analogues were designed on the basis of substrate specificity and molecular modeling of three-dimensional structures of the penicillopepsin: Iva-Val-Sta-OEt crystal structure. 4,8-Diamino-3-hydroxyoctanoic acid [LySta] and 4,7-diamino-3-hydroxyheptanoic acid [OrnSta] were synthesized respectively from Boc-Lys(Z)-al and Boc-Orn(Bzl,Z)-al by addition of lithio ethyl acetate to the aldehyde group. The [LySta] derivative was converted to the trichloroethoxycarbonyl derivative and separated into the corresponding 3S,4S and 3R,4R diastereomers. The [OrnSta] derivative was used as a mixture of 3-position diastereomers. These new amino acids were used to prepare the following inhibitors: Iva-Val-Val-[LySta]-OEt and Iva-Val-Val-[OrnSta]-OEt as well as the corresponding synthetic intermediates. Inhibition constants (Ki values) were measured for inhibition of porcine pepsin and penicillopepsin. Both compounds were potent inhibitors of penicillopepsin with Ki values 10-100 times smaller (2.1 and 1.1 nM, respectively) than the Ki of Iva-Val-Val-Sta-OEt (47 nM). In contrast both inhibitors are exceptionally weak inhibitors of porcine pepsin with Ki values greater than 1 microM. These results are correlated with the ability of the basic group in the new inhibitors to bind to aspartic acid-77 in penicillopepsin.