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 activity of human PC1 and human furin, two important members of subtilisin/kexin family of enzymes, we have prepared by solid-phase Fmoc or recently introduced FastMoc chemistry a series of 10 peptidyl substrate analogs. The structures of these analogs are based upon the core sequence of pro-mPC1(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, representing the P'1 position, corresponds to L-Ser or to nonproteinacous 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, namely P5 Glu or P'3 Gln, 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, 1H-NMR and amino acid analysis. In contrast to the Ser analog, which is an excellent substrate for both hPC1 and hfurin, these analogs displayed moderate to strong inhibition of both hPC1 and hfurin activity in a reversible competitive manner. They all exhibited higher potency for hfurin than for hPC1, with an inhibition constant (Ki) ranging from 0.8 to 10 microM and from 1.0 to 170 microM, respectively. Incorporation of L-Tic yielded an analog with a two to four-fold increased inhibition of either enzymes when compared to its D-Tic counterpart, the effect being more pronounced for hPC1 than for hfurin. Comparison of these data with those for the corresponding N-terminal Fmoc protected peptides revealed that the highly hydrophobic N-terminal Fmoc function occupying the P8 position can contribute positively or negatively towards proteinase inhibition depending on the nature of the unnatural amino acid at P'1 and the enzyme used. Finally, none of the analogs was significantly cleaved by either enzyme. FTIR data on these analogs revealed some important structural differences between the substrate and inhibitor analogs, as there appears to be a conformational shift from a more beta-sheet-like structure for the substrates to a more alpha-helical-like structure for the inhibitors.