Inhibition of aspartic proteases by pepstatin and 3-methylstatine derivatives of pepstatin. Evidence for collected-substrate enzyme inhibition.

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)