PEPTIDYL SUBSTRATES CONTAINING UNNATURAL AMINO-ACID AT THE P'1 POSITION ARE POTENT INHIBITORS OF PROHORMONE CONVERTASES

In order to study further the importance of the P'1 residue upon the a ctivity of human PCl and human furin, two important members of subtili sin/kexin family of enzymes, we have prepared by solid-phase Fmoc or r ecently introduced FastMoc chemistry a series of 10 peptidyl substrate analogs, The structures of these analogs are based upon the core sequ ence of pro-mPC 1(83-93) namely, D-Tyr-Lys-Glu-Arg-Ser Lys-Arg-Xaa-Val -Gln-Lys-Asp, where D-Tyr replaces the native L-Tyr residue and Xaa, r epresenting the P'1 position, corresponds to L-Ser or to nonproteinaco us amino acids such as Tle, Sarc, MLeu, Aib, D-Tic or L-Tic, Two more analogs with L-Tic at P'1 position but with one amino acid less, namel y P5 Glu or P'3 Gin, and one with a Cit residue in place of Arg at P1 site of the dodecapeptide were also obtained. These peptides were all fully characterized by a combination of MS, H-1-NMR and amino acid ana lysis. In contrast to the Ser analog, which is an excellent substrate for both hPCl and hfurin, these analogs displayed moderate to strong i nhibition of both hPCl and hfurin activity in a reversible competitive manner. They all exhibited higher potency for hfurin than for hPCl, w ith an inhibition constant (Ki) ranging from 0.8 to 10 mu M and from 1 .0 to 170 mu M, respectively. Incorporation of L-Tic yielded an analog with a two to four-fold increased inhibition of either enzymes when c ompared to its D-Tic counterpart, the effect being more pronounced for hPCl than for hfurin. Comparison of these data with those for the cor responding N-terminal Fmoc protected peptides revealed that the highly hydrophobic N-terminal Fmoc function occupying the P8 position can co ntribute positively or negatively towards proteinase inhibition depend ing on the nature of the unnatural amino acid at P'1 and the enzyme us ed. Finally, none of the analogs was significantly cleaved by either e nzyme. FTIR data on these analogs revealed some important structural d ifferences between the substrate and inhibitor analogs, as there appea rs to be a conformational shift from a more beta-sheet-like structure for the substrates to a more a-helical-like structure for the inhibito rs. (C) Munksgaard 1995.