CYP51A1 is a heme-thiolate monoxygenase which 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 anti-cancer 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 re-purposing 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.

The options for cholesterol detection included a simple kit-based enzyme assay or a cumbersome method using gas chromatography (GC). While the kit method did not have the necessary sensitivity, the GC method was time- consuming, lacked acceptable throughput, and was not cost-effective. To overcome these limitations, we devised a method using the RapidFire 365 HTMS system to develop the assay. RapidFire is a label-free technology which uses mass spectrometry for monitoring multiple analytes in complex sample mixtures. The short cycle time of 7 s per sample enables a high throughput analysis of required analytes and this platform is thus ideal for performing rapid screening of large compound libraries.

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