Isoform-specific screening assay for 5-alpha reductase inhibitors using dual-substrate mass spectrometry

Overview

This offering is a cell-based screening assay designed to evaluate and differentiate inhibition of the two isoforms of steroid 5-alpha reductase (SRD5A1 and SRD5A2) in a single experimental well. The platform addresses a key challenge in androgen-related drug discovery: determining whether a candidate compound selectively inhibits one isoform over the other. By exploiting the substrate selectivity of the two enzymes—particularly the fact that the 11-oxygenated androgen 11-ketotestosterone is metabolised by SRD5A2 but not SRD5A1—the assay reveals isoform-specific activity through distinct product profiles (DHT versus 11-keto-DHT) measured via mass spectrometry.

The technology is relevant to pharmaceutical and biotechnology partners developing treatments for conditions such as benign prostatic hyperplasia, androgenetic alopecia, and androgen-sensitive cancers, where isoform selectivity influences efficacy and side-effect profiles.

Technical specifications

Key features:

• Co-expression of SRD5A1 and SRD5A2 in non-steroidogenic HEK293 cells, enabling dual-isoform screening in a single well
• Use of isoform-selective substrates (testosterone for SRD5A1; 11-ketotestosterone for SRD5A2) to distinguish inhibition profiles
• Multi-steroid high-throughput mass spectrometry readout with run times of approximately five minutes per sample
• Liquid-liquid extraction workflow with deuterated internal standards for quantitative accuracy
• Inclusion of dutasteride and finasteride as reference controls for assay benchmarking
• Flexible compound delivery in DMSO, ethanol, or water
• 48- or 96-well plate format suitable for screening-scale throughput
• Option to follow up hits with single-isoformat assays for confirmation

How it works:

HEK293 cells co-transfected with SRD5A1 and SRD5A2 are treated with a substrate mixture in the presence or absence of test compounds. After a four-to-six-hour incubation targeting 40–60% substrate conversion, media are collected and steroids are extracted and quantified by mass spectrometry. A reduction in DHT production without a corresponding reduction in 11-keto-DHT indicates SRD5A1 inhibition, while reduced 11-keto-DHT production signals SRD5A2 inhibition. Compounds that reduce both products can be re-evaluated against each isoform individually.

Technology readiness level

The assay is built on validated laboratory methods, including established HEK293 co-expression protocols for SRD5A1 and SRD5A2 and published multi-steroid mass spectrometry screening workflows. The underlying substrate selectivity of the isoforms has been demonstrated in prior peer-reviewed work. The platform is positioned for immediate collaborative use, with a clear validation roadmap that includes stable transfection via CRISPR-Cas9 if additional funding is secured. The technology is ready for sponsored research collaborations, co-development partnerships, and pilot screening engagements with industry partners seeking isoform-resolved inhibitor profiling.