IN-VITRO CLEAVAGE OF INTERNALLY QUENCHED FLUOROGENIC HUMAN PROPARATHYROID HORMONE AND PROPARATHYROID-RELATED PEPTIDE-SUBSTRATES BY FURIN - GENERATION OF A POTENT INHIBITOR

The cleavage of parathyroid hormone (PTH) from its precursor proparath yroid hormone (pro-PTH) is accomplished efficiently by the proprotein convertase furin (Hendy, G. N., Bennett, H. P. J., Gibbs, B. F., Lazur e, C., Day, R., and Seidah, N. G. (1995) J. Biol. Chem. 270, 9517-9525 ). We also showed that a synthetic peptide comprising the -6 to +7 seq uence of human pro-PTH is appropriately cleaved by purified furin in v itro. The human pro-PTH processing site Lys-Ser-Val-Lys-Lys-Arg differ s from the consensus furin site Arg-Xaa-(Lys/Arg)-Arg that is represen ted by Arg-Arg-Leu-Lys-Arg in the cleavage site of pro-PTH-related pep tide (pro-PTHrP). An earlier study demonstrated that an internally que nched fluorogenic substrate bearing an O-aminobenzoyl fluorescent dono r at the NH2 terminus and an acceptor 3-nitrotyrosine near the COOH te rminus was appropriately cleaved by the convertases furin and PC1 (Jea n, F., Basak, A., DiMaio, J., Seidah, N. G., and Lazure, C. (1995) Bio chem. J. 307, 689-695). Here, we have synthesized a series of internal ly quenched fluorogenic substrates based upon the pro-PTH and pro-PTHr P sequences to determine which residues are important for furin cleava ge. Purified recombinant furin and PC1 cleaved the human pro-PTH inter nally quenched substrate at the appropriate site in an identical manne r to that observed with the nonfluorescent peptide. Several substituti ons in the P-6-P-3 sequence were well tolerated; however, replacement of the Lys at the P-6 position with Gly and replacement of the P-3 Lys by an acidic residue led to markedly compromised cleavage by furin. F urin activity was very sensitive to substitution in P' positions. Repl acement of Ser at P-1' with Gly and Val at P-2' with Ala generated sub strates that were less well cleaved. Substitution at the P-1' position of Val for Ser in conjunction with Ala for Val at P-2', as well as a single substitution of Lys for Val at P-2', generated specific inhibit ors of furin cleavage. The findings of this study open the way to the rational design of inhibitors of furin with therapeutic potential.