IDENTIFICATION OF INHIBITORS OF PROHORMONE CONVERTASE-1 AND CONVERTASE-2 USING A PEPTIDE COMBINATORIAL LIBRARY

A positional scanning synthetic peptide combinatorial library containi ng approximately 52 million hexapeptides was used to identify potentia l inhibitory peptides for recombinant mouse prohormone convertase 1 (P C1) and PC2 and to provide information on the specificity of these enz ymes. The library surveys revealed that a P6 Leu, a P4 Arg, a P2 Lys, and a P1 Arg were most inhibitory against PC1, and a P6 Ile and a P4 A rg were most inhibitory against PC2, Using information derived from th e library surveys, hexapeptide sets were synthesized and screened for inhibition of PC1 and PC2, The data obtained revealed the preference o f both enzymes for a P3 Val, At P5, many substitutions were well toler ated. PC1 and PC2 proved to differ mainly in the selectivity of their S6 subsites, In PC1, this subsite displayed a strong preference toward occupation by Leu; the K-i value for peptide Ac-Leu-Leu-Arg-Val-Lys-A rg-NH2 was 28 times lower than that for peptide Ac-Ile-Ile-Arg-Val-Lys -Arg-NH2. In contrast, PC2 discriminated little between Leu and lie at P6, as evidenced by the small (1.5-fold) difference in K-i values for these two peptides, Several hexapeptides synthesized as a result of t he screen were found to represent potent inhibitors of PC2 (with K-i v alues in the submicromolar range) and, particularly, of PC1 (with K-i values in the low nanomolar range). The most potent inhibitor, Ac-Leu- Leu-Arg-Val-Lys-Arg-NH2, proved to be the same peptide for both enzyme s and inhibited PC1 and PC2 in a competitive, fast-binding manner with K-i values of 3.2 and 360 nm, respectively, The four most potent pept ide inhibitors of PC1 and PC2 were also tested against soluble human f urin and found to exhibit a different rank order of inhibition; for ex ample, Ac-Leu-Leu-Arg-Val-Lys-Arg-NH2, was 440-fold less potent agains t furin than against PC1, with a K-i of 1400 nM.