HMG-CoA Reductase Inhibition Assays: Updated Protocols for Drug Discovery and Enzyme Kinetics

Hypercholesterolemia contributes to the global burden of disease as a major risk factor for cardiovascular disease, and cholesterol-lowering therapies are key to global health. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase, EC 1.1.1.34) is the rate-limiting enzyme of cholesterol biosynthesis and an important target for drug development. Statins, the most commonly used HMG-CoA reductase inhibitors, have been shown to successfully reduce low-density lipoprotein cholesterol (LDL-C) and minimize coronary events, but also long-term use is associated with side effects, including myopathy, hepatotoxicity, and increased risk of diabetes. Consequently, researchers are turning their attention to examining alternate HMG-CoA reductase inhibitors, such as ATP-citrate lyase inhibitors (e.g., bempedoic acid), PCSK9 inhibitors, and bioactive compounds from plants and microbes. Here, we update the classical detailed method for measuring HMG-CoA reductase activity and inhibition based on recent advances in enzyme kinetics, high-throughput screening (HTS) techniques, and computational modeling. The spectrophotometric assay using NADPH oxidation at 340 nm is popular for measuring enzyme activity. Improvements in assay conditions have been made to increase accuracy and reproducibility. Furthermore, this chapter describes an IC₅₀ four-parameter logistic model to accurately evaluate the inhibition spectrum. This procedure simplifies the pursuit of more effective cholesterol-lowering therapeutics by restructuring the screening of novel compounds with greater efficacy and safety.