title: In Vitro Assays for the Discovery of PCSK9 Autoprocessing Inhibitors.
abstract: PCSK9 plays a significant role in regulating low-density lipoprotein (LDL) cholesterol levels and has become an important drug target for treating hypercholesterolemia. Although a member of the serine protease family, PCSK9 only catalyzes a single reaction, the autocleavage of its prodomain. The maturation of the proprotein is an essential prerequisite for the secretion of PCSK9 to the extracellular space where it binds the LDL receptor and targets it for degradation. We have found that a construct of proPCSK9 where the C-terminal domain has been truncated has sufficient stability to be expressed and purified from Escherichia coli for the in vitro study of autoprocessing. Using automated Western analysis, we demonstrate that autoprocessing exhibits the anticipated first-order kinetics. A high-throughput time-resolved fluorescence resonance energy transfer assay for autocleavage has been developed using a PCSK9 monoclonal antibody that is sensitive to the conformational changes that occur upon maturation of the proprotein. Kinetic theory has been developed that describes the behavior of both reversible and irreversible inhibitors of autocleavage. The analysis of an irreversible lactone inhibitor validates the expected relationship between potency and the reaction end point. An orthogonal liquid chromatography-mass spectrometry assay has also been implemented for the confirmation of hits from the antibody-based assays.
authors.affiliation: 1 Department of In Vitro Pharmacology, Merck Research Laboratories, Kenilworth, NJ, USA.
authors.forename: Scott P
authors.initials: SP
authors.lastname: Salowe
authors.affiliation: 1 Department of In Vitro Pharmacology, Merck Research Laboratories, Kenilworth, NJ, USA.
authors.forename: Lei
authors.initials: L
authors.lastname: Zhang
authors.affiliation: 1 Department of In Vitro Pharmacology, Merck Research Laboratories, Kenilworth, NJ, USA.
authors.forename: Hratch J
authors.initials: HJ
authors.lastname: Zokian
authors.affiliation: 1 Department of In Vitro Pharmacology, Merck Research Laboratories, Kenilworth, NJ, USA.|2 Current address: Janssen Research & Development LLC, Spring House, PA, USA.
authors.forename: Jennifer J
authors.initials: JJ
authors.lastname: Gesell
authors.affiliation: 1 Department of In Vitro Pharmacology, Merck Research Laboratories, Kenilworth, NJ, USA.
authors.forename: Deborah L
authors.initials: DL
authors.lastname: Zink
authors.affiliation: 1 Department of In Vitro Pharmacology, Merck Research Laboratories, Kenilworth, NJ, USA.
authors.forename: Judyann
authors.initials: J
authors.lastname: Wiltsie
authors.affiliation: 1 Department of In Vitro Pharmacology, Merck Research Laboratories, Kenilworth, NJ, USA.
authors.forename: Xi
authors.initials: X
authors.lastname: Ai
authors.affiliation: 1 Department of In Vitro Pharmacology, Merck Research Laboratories, Kenilworth, NJ, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Kavana
authors.affiliation: 3 Department of Cardiometabolic Diseases, Merck Research Laboratories, Kenilworth, NJ, USA.
authors.forename: Shirly
authors.initials: S
authors.lastname: Pinto
mesh_terms: D016503:Drug Delivery Systems / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D056945:Hep G2 Cells; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D006937:Hypercholesterolemia / Q000188:drug therapy* / Q000235:genetics; D007700:Kinetics; D007783:Lactones / Q000037:antagonists & inhibitors; D013058:Mass Spectrometry / Q000379:methods; D000091362:PCSK9 Inhibitors; D000071449:Proprotein Convertase 9 / Q000737:chemistry* / Q000235:genetics; D011487:Protein Conformation / Q000187:drug effects; D011973:Receptors, LDL / Q000235:genetics
chemical_list: D007783:Lactones; D000091362:PCSK9 Inhibitors; D011973:Receptors, LDL; C472125:PCSK9 protein, human; D000071449:Proprotein Convertase 9
keywords: PCSK9; autocleavage; high-throughput screening; kinetics