Targeting the isoprenoid pathway in choleste biosynthesis: An approach to identify isoprenoid biosynthesis inhibitors

The development of novel Cholesterol biosynthesis inhibitors is a task of major concern due to the diverse roles of Cholesterol and its precursors in physiological processes. Therefore, appropriate screening assays are required, which can be used to identify and quantify specific inhibitors targeting the desired Enzyme. Here, we developed a whole-cell screening assay based on a HL60 cell line, which can be used to characterize inhibitors interacting with Enzymes of the isoprenoid part of Cholesterol biosynthesis. Due to the change of the isoprenoid pattern under Enzyme inhibition, an identification of the targeted Enzyme is possible. With the described assay, we can distinguish between free and pyrophosphorylated isoprenoids after enzymatic cleavage in cellular and extracellular matrices. The approach was validated in line with the European Medicines Agency guideline on bioanalytical method validation. As proof of concept, literature-described inhibitors of the isoprenoid pathway were tested. We characterized the effect of 11 isoprenoid biosynthesis inhibitors, and we identified 6-fluoromevalonate as an isopentenyl pyrophosphate isomerase inhibitor, a biological activity that was previously unknown. Furthermore, isoprenoid patterns revealed that, independent of the analyzed matrix, the predominant form of the detected isoprenoids were dephosphorylated isoprenoids and only small amounts were present as pyrophosphates.

6‐fluoromevalonate; GC‐MS; isoprenoid trafficking; pre‐squalene pathway; pyrophosphate.