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

CYP51A1 is a heme-thiolate monooxygenase essential to the cholesterol biosynthetic pathway in humans and the ergosterol pathway in fungi. While inhibitors of this enzyme have long been used as antifungal agents, recent research highlights its potential as a cancer target, given the role of elevated cholesterol synthesis in proliferating tumor cells. Statins, which act upstream in the pathway, have shown mixed clinical outcomes in oncology, prompting interest in repurposing CYP51A1 inhibitors for anticancer use.

Aragen partnered with a client seeking to evaluate an internal collection of antifungal inhibitors for their ability to modulate cholesterol synthesis in human cells. Traditional detection methods — enzyme-based kits or gas chromatography — proved either insufficiently sensitive or too slow and costly. To overcome these barriers, we developed a high-throughput assay using the RapidFire 365 HTMS platform, enabling rapid, label-free mass spectrometry–based detection of cholesterol, lanosterol, and dihydrolanosterol in complex cellular mixtures. This approach provided the sensitivity, speed, and throughput necessary to accelerate screening of large compound libraries and address key challenges in oncology drug discovery.

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