A Novel Cell-Based Screening Assay For Cholesterol Biosynthetic Pathway Inhibition Using The RapidFire HTMS Platform

CYP51A1 is a heme-thiolate monooxygenase that participates in an obligatory step in the cholesterol biosynthetic pathway and catalyzes the formation of critical intermediates in humans. This enzyme is also a critical component of the ergosterol biosynthetic pathway in fungi. Inhibitors targeting this enzyme have been successfully marketed as antifungal agents for decades, while statins targeting HMG-CoA reductase (upstream of lanosterol in the cholesterol synthetic pathway) have dominated the cholesterol-lowering drug market. It is known that increased cholesterol synthesis is an important feature of actively proliferating cancer cells, and clinical trials have tried to assess the efficacy of statins as anticancer agents. However, conflicting evidence links statin intake to higher incidences of cancer-related death. Researchers exploring alternatives to statins for inhibiting the cholesterol synthetic pathway thus turned to CYP51A1 as a potential anticancer target, and antifungal CYP51A1 inhibitor drugs are being tested for anticancer efficacy.

As a full-service drug discovery CRDO, GVK BIO was commissioned to enable one such project. The client was exploring the possibility of repurposing an internal collection of antifungal CYP51A1 inhibitors for targeting cancers. The first step was to understand whether the compounds would be able to inhibit the cholesterol synthetic pathway in human cells, which required the quantitation of cholesterol or other intermediates within the cell.