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

## TL;DR

This paper introduces a validated assay to identify inhibitors of isoprenoid biosynthesis, revealing new insights into enzyme inhibition and isoprenoid patterns in cells.

## Contribution

A novel whole-cell screening assay was developed to distinguish free and pyrophosphorylated isoprenoids and identify enzyme-specific inhibitors.

## Key findings

- The assay successfully characterized 11 isoprenoid biosynthesis inhibitors, including 6-fluoromevalonate as a new isomerase inhibitor.
- Dephosphorylated isoprenoids were the predominant form detected in both cellular and extracellular matrices.
- Eight of the 11 tested inhibitors influenced isoprenoid patterns in HL60 cells.

## Abstract

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.

A fully validated assay is reported that can be used to identify inhibitors of isoprenoid biosynthesis by gas chromatography‐mass spectrometry (GC‐MS). The free isoprenoids can be distinguished from their pyrophosphorylated species. From 11 literature‐described isoprenoid biosynthesis inhibitors, eight influenced the isoprenoid patterns of HL60 cells. The assay can be applied to cellular and extracellular matrices, making it an ideal approach for isoprenoid trafficking.