DESIGN OF POTENT REVERSIBLE INHIBITORS FOR THERMOLYSIN - PEPTIDES CONTAINING ZINC COORDINATING LIGANDS AND THEIR USE IN AFFINITY CHROMATOGRAPHY

A series of potent inhibitors for the zinc metal-loprotease thermolysin have been developed. Most contain the same tripeptide backbone (-Phe-l-Ala-Gly-NH2) to interact with thermolysin's extended substrate-binding site and a ligand to coordinate to the zinc atom in the active site of the enzyme. The most effective inhibitors with K1 values in the 10-6—10 7 M range at pH 7.2 and 25 °C were those with a hydroxamic acid, thiol, phosphoramidate, or N-hydroxy peptide functional group to coordinate to the zinc atom. Most of the inhibitors act as monodentate ligands for the zinc, and their effectiveness is determined by a combination of their ligating ability and their resemblance to the tetrahedral transition state for peptide bond hydrolysis. The N-hydroxy peptide and hydroxamic acids are proposed to act as bidentate ligands with both oxygen atoms interacting with the zinc atom. Hydroxamic acids were shown to exhibit the expected side-chain specificity. The derivative with a leucyl side chain at P/ was a slightly better inhibitor (k1 = 0.48 μM) than that with a phenylalanyl side chain (k1 = 0.66 μM). The hydroxamic acid with no side chain was an extremely poor inhibitor (K1 =1100 μM). A model was proposed to account for the inhibition of several aminopeptidases (metalloexopeptidases) by the natural inhibitors bestatin and amastatin. Two oxygen atoms of the α-hydroxycarbonyl functional group of the inhibitors are proposed to act as a bidentate ligand for the active-site zinc in a manner similar to that proposed for the N-hydroxy peptides and hydroxamic acids. One of the best hydroxamic acid inhibitors was attached to agarose via an aminopropyl spacer group and was shown to be a very potent and stable affinity support. Thermolysin was retained by the column and could be eluted under a variety of conditions. As expected, chymotrypsin and carboxypeptidase A were not retained. Two neutral proteases from BaciIIus subtilis which have a substrate specificity similar to that of thermolysin could be separated on the column by gradient elution. The results reported should be useful for the design of specific inhibitors or affinity supports for other zinc metalloproteases. © 1979, American Chemical Society. All rights reserved.