Identification of inhibitors of prohormone convertases 1 and 2 using a peptide combinatorial library

A positional scanning synthetic peptide combinatorial library containing approximately 52 million hexapeptides was used to identify potential inhibitory peptides for recombinant mouse prohormone convertase 1 (PC1) and PC2 and to provide information on the specificity of these enzymes. 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 Arg were most inhibitory against PC2. Using information derived from the library surveys, hexapeptide sets were synthesized and screened for inhibition of PC1 and PC2. The data obtained revealed the preference of both enzymes for a P3 Val. At P5, many substitutions were well tolerated. 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 Ki value for peptide Ac-Leu-Leu-Arg-Val-Lys-Arg-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 Ile at P6, as evidenced by the small (1.5-fold) difference in Ki values for these two peptides. Several hexapeptides synthesized as a result of the screen were found to represent potent inhibitors of PC2 (with Ki values in the submicromolar range) and, particularly, of PC1 (with Ki 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 enzymes and inhibited PC1 and PC2 in a competitive, fast-binding manner with Ki values of 3.2 and 360 nM, respectively. The four most potent peptide inhibitors of PC1 and PC2 were also tested against soluble human furin and found to exhibit a different rank order of inhibition; for example, Ac-Leu-Leu-Arg-Val-Lys-Arg-NH2 was 440-fold less potent against furin than against PC1, with a Ki of 1400 nM.