Cellular LC-MS based assay for 5-alpha reductase inhibition screening
A cell-based enzymatic assay using LC-MS coupled with a flow scintillation counter to screen and evaluate inhibitors of 5-alpha reductase, the enzyme responsible for converting testosterone to dihydrotestosterone (DHT). The platform supports drug discovery for androgen-related conditions and enables profiling of small molecules, plant extracts, and synthetic compounds against multiple 5-alpha reductase isozymes.
Overview
This offering provides a cellular LC-MS based assay designed to screen and quantify 5-alpha reductase inhibition. 5-alpha reductase is an enzyme that converts testosterone to dihydrotestosterone (DHT), a potent androgen linked to conditions such as benign prostatic hyperplasia, androgenetic alopecia, and certain hormone-dependent cancers. The assay uses cells that naturally express 5-alpha reductase (LnCap cells) incubated with labeled testosterone and test compounds. After incubation, samples are analyzed via LC-MS linked to a flow scintillation counter, enabling precise quantification of both substrate and product levels. The approach delivers a relatively low-cost, high-throughput method for identifying and characterizing enzyme inhibitors, making it valuable for pharmaceutical, nutraceutical, and cosmeceutical research programs.
Technical specifications
Key features:
- Cell-based enzymatic assay using LnCap cells that naturally express 5-alpha reductase
- Tritiated testosterone as substrate, with Finasteride serving as a standard positive control
- Triplicate treatment format with 4-hour incubation for reproducible results
- LC-MS coupled to a flow scintillation counter for simultaneous detection of labeled testosterone and its metabolic products, including DHT
- Quantitative measurement of enzyme inhibition, with demonstrated ability to identify fractions reducing conversion to DHT by over 50% (p<0.01)
- Validated through screening of 64 isolated fractions of plant extracts with tentative 5-alpha reductase activity
- Planned expansion to include stably transfected cell lines expressing SRD5A-1, SRD5A-2, and SRD5A-3, enabling isozyme-specific inhibition profiling
Technology readiness level
The assay has been validated and applied in a working laboratory setting, having successfully screened 64 plant extract fractions and identified multiple fractions with statistically significant inhibitory activity. The current platform uses cells naturally expressing 5-alpha reductase and is operational for contract research engagements. The next phase of development involves creating stably transfected cell lines for each of the three human 5-alpha reductase isozymes (SRD5A-1, SRD5A-2, and SRD5A-3), with cell line creation and validation expected within 6 to 12 months. This expansion will enable isozyme-selective inhibitor screening, adding a layer of specificity valuable for targeted drug development.
About Phytor Ltd.
Phytor Ltd. is a biotechnology research company based in Jerusalem, Israel, that specializes in providing drug development, regulatory, and consulting services for the life sciences industry. The company operates a laboratory focused on biologically led assays and the synthesis of novel chemical entities derived from plant molecules. Their R&D activities include the development of therapeutics for oncology and neurodegenerative diseases, alongside the creation of diagnostic tools. By utilizing expertise in medicinal chemistry, pharmacology, toxicology, and regulatory compliance, Phytor assists partners in translating research from the bench to commercial applications in fields such as pharmaceuticals, dietary supplements, and food products.
The company serves as a contract research organization (CRO) for a diverse range of clients, offering outsourcing R&D and strategic regulatory support to ensure products meet market and safety standards. Phytor’s scientific team leverages state-of-the-art instrumentation to perform specialized analyses, including flow cytometry, chemical characterization, and cell-based assays. Their work has been applied to various collaborative projects, including the development of plant-based dietary supplements and novel antimicrobial agents, with their scientific contributions and research findings documented in peer-reviewed publications.
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