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In Vitro Assays for the Discovery of PCSK9 Autoprocessing Inhibitors

Research Highlights Life Science

HTRF, a way to identify specific conformation protease inhibitors

Expert opinion

Do you want to identify specific conformation protease inhibitors, but you’re not sure which assay to use? You’ll find the answer in this interesting article by Scott P. Salowe from Merck.PCSK9 is a hot therapeutic target today, with two drugs recently approved by the FDA and over 20 clinical trials on the way. The quest for the next generation hypercholestolemia treatment has begun!Searching for new chemicals that inhibit serine proteases seems reasonable. On the other hand, it is much trickier to identify potent inhibitors that target a specific conformation change obtained from self-cleavage.You can start by checking auto-processing of recombinant proPCSK9 using conventional Western Blot techniques, then run quantitative antibody-based assays to determine potencies and kinetic features of your inhibitory compounds.Let your curiosity guide you! See which assays Scott P. Salowe developed to identify a new PCSK9 autoprocessing inhibitor. Or you can keep looking for a needle in a haystack.

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.

Details

Journal of Biomolecular Screening, 2016 Dec; 21(10):1034-1041.

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