SELECTION OF A HISTIDINE-CONTAINING INHIBITOR OF GELATINASES THROUGH DECONVOLUTION OF COMBINATORIAL TETRAPEPTIDE LIBRARIES

A fully automated peptide synthesizer was used to generate tetrapeptid e sublibraries from 24 natural and nonnatural amino acids, from which new inhibitors of gelatinases (matrix metalloproteinases MMP-2 and MMP -9) were selected as potential anticancer drugs. MMP-2 and MMP-9 from mouse Balbc/3T3 fibroblasts conditioned media were assayed in their li near range response by zymography to quantify inhibition at each step of the tetrapeptide library deconvolution. The histidine-e-amino capro ic acid-beta-alanine-histidine (His-epsilon Ahx-beta Ala-His) sequence was found to yield optimal inhibition of both MMP-2 and MMP-9. Inhibi tion by selected tetrapeptides was also evaluated with two other techn iques, a native type IV collagen degradation assay and a fluorogenic e nzymatic assay, confirming the tetrapeptide potency. The His-epsilon A hx-beta Ala-His tetrapeptide also inhibited purified human MMP-2 and M MP-9 and the corresponding enzymes present in conditioned media from h uman tumour cells. Finally, the length of the spacer between the two t erminal histidines was found to be crucial to the inhibitory potential . This approach may thus be considered as a successful strategy to yie ld specific peptide or pseudopeptide inhibitors, although their potenc y remains moderate, since it was measured before any chemical optimiza tion was undertaken.