An internally quenched fluorogenic substrate of prohormone convertase 1 and furin leads to a potent prohormone convertase inhibitor.
Based upon the observed cleavage of various peptidyl substrates by the recombinant prohormone convertases PC1 and furin, an intramolecularly quenched fluorogenic peptidyl substrate, (o-aminobenzoyl)-Lys-Glu-Arg-Ser-Lys-Arg-Ser-Ala-Leu-Arg-Asp-(3-nitro)Ty r-Ala, was synthesized. In spite of the distance (approx. 33 A) separating the fluorescent donor/acceptor pair, the highly fluorescent o-aminobenzoyl group is efficiently quenched by long-range resonance energy transfer to the (3-nitro)Tyr moiety. Both recombinant human PC1 and human furin recognize and cleave specifically this substrate at the expected Arg-Ser site in a sensitive manner. The Km values for human PC1 and human furin were 17 microM and 30 microM respectively, with Vmax. values of 6.4 microM/h and 18 microM/h. These values differ significantly from those obtained when using a 7-amino-4-methylcoumarin-containing pentapeptidyl substrate where, for similar Km values, the Vmax. values were much lower. The peptide sequence was used to synthesize another peptide incorporating a ketomethylene arginyl pseudopeptide bond. This compound proved to be a potent competitive inhibitor of both human PC1 and human furin, displaying Ki values of 7.2 microM and 2.4 microM respectively.
Jean
F
F
Neuropeptides Structure and Metabolism Laboratory, Clinical Research Institute of Montréal, Québec, Canada.
Basak
A
A
DiMaio
J
J
Seidah
N G
NG
Lazure
C
C
D000595:Amino Acid Sequence; D001120:Arginine / Q000378:metabolism; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D001665:Binding Sites; D002863:Chromogenic Compounds / Q000378:metabolism / Q000494:pharmacology; D004792:Enzyme Precursors / Q000037:antagonists & inhibitors / Q000378:metabolism; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000378:metabolism* / Q000494:pharmacology*; D045683:Furin; D006801:Humans; D007536:Isomerism; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism* / Q000494:pharmacology*; D043484:Proprotein Convertases; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology; D012694:Serine / Q000378:metabolism; D013381:Subtilisins / Q000378:metabolism*
D002863:Chromogenic Compounds; D004792:Enzyme Precursors; D005456:Fluorescent Dyes; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012694:Serine; D001120:Arginine; D043484:Proprotein Convertases; D013381:Subtilisins; D045683:Furin; D016282:Aspartic Acid Endopeptidases