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Mass Spectrometry-Based Screening Reveals Inhibitors of Cholesterol 25-Hydroxylase
Atikur Rahman1,2, Elijah H Hayes1,2, Drew J Adams3,1,2
1Chemical Biology Program, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, United States.
ACS Chemical Biology
|June 12, 2026
Summary
Researchers developed new cell-based assays to study Cholesterol 25-hydroxylase (CH25H), an enzyme linked to diseases. They identified three small molecules, including U73343, as the first cell-active inhibitors of CH25H.
Area of Science:
- Biochemistry
- Enzymology
- Medicinal Chemistry
Background:
- Cholesterol 25-hydroxylase (CH25H) is an ER-resident enzyme catalyzing cholesterol to 25-hydroxycholesterol (25HC).
- CH25H plays a pathological role in diseases like osteoarthritis and Alzheimer's Disease.
- No small molecule inhibitors for CH25H are currently available, hindering research.
Purpose of the Study:
- To establish cell-based assays for monitoring CH25H activity.
- To identify and characterize first-in-class small molecule inhibitors of CH25H.
- To develop tools for studying CH25H in disease contexts.
Main Methods:
- Development of a mass spectrometry-based cellular assay to quantify 25HC production.
- Screening of a focused small molecule library against the CH25H assay.
- Establishment of a secondary transcriptional reporter assay for CH25H activity.
- Evaluation of inhibitor selectivity against related enzymes.
Main Results:
- Three potent inhibitors of cellular CH25H activity were identified: U73343, Ciclopirox, and phenanthroline.
- U73343 demonstrated strong selectivity for CH25H over related diiron lipid oxidases.
- The developed assays successfully monitored CH25H function in a cellular context.
Conclusions:
- This study presents novel cell-based assays for assessing CH25H activity.
- First-in-class cell-active inhibitors of CH25H, including U73343, have been identified.
- These findings provide crucial tools for further investigation of CH25H in disease pathogenesis.
