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Systematic Application of a Cellular Thermal Shift Assay for Inferring Inhibitor Binding Affinity to 17βHSD13 in a
Sandra Lunnerdal1, Fredrik Wågberg1, Göran Dahl2
1Early CVRM, Biopharmaceuticals R&D, Gothenburg SE-431 83 Mölndal, Sweden.
Biochemistry
|January 12, 2026
Summary
Human variants in 17βHSD13 protect against liver disease. This study developed a method using cellular thermal shift assay (CETSA) to measure inhibitor binding, aiding drug development for liver conditions.
Area of Science:
- Biochemistry
- Pharmacology
- Hepatology
Background:
- Loss-of-function variants in 17β-hydroxysteroid dehydrogenase type 13 (17βHSD13) are linked to reduced risk and progression of metabolic and alcohol-related liver disease.
- In vitro studies suggest 17βHSD13's role in steroidal and lipogenic substrate processing, but a clear mechanistic link to disease and effective biomarkers for target occupancy remain elusive.
- This hinders precise clinical dose selection for 17βHSD13 inhibitors.
Purpose of the Study:
- To implement and validate a medium-throughput cellular thermal shift assay (CETSA) for quantifying the binding of BI-3231, an uncompetitive 17βHSD13 inhibitor, in humanized mouse liver homogenates.
- To establish a robust method for estimating target occupancy and inform dose selection for 17βHSD13-targeted therapies.
- To investigate the temperature dependence of binding equilibria and cofactor saturation effects on inhibitor potency.
Main Methods:
- Utilized a medium-throughput cellular thermal shift assay (CETSA) in humanized mouse liver homogenates to assess BI-3231 binding to 17βHSD13.
- Systematically profiled isothermal dose-response fingerprints (ITDRFCETSA) across a temperature range (58-70.5 °C) to capture binding dynamics.
- Performed complementary surface plasmon resonance (SPR) measurements (5-42 °C) to determine the temperature dependence of NAD+ and BI-3231 binding affinities.
Main Results:
- CETSA profiling revealed temperature-induced shifts in apparent potency, confirming NAD+-dependent binding of BI-3231.
- SPR experiments elucidated the temperature dependency of NAD+ and BI-3231 interactions, yielding a KD for BI-3231 of 2.5 nM at 37 °C (pKD = 8.60 ± 0.07).
- Extrapolation of SPR data to CETSA temperatures demonstrated convergence, validating the SPR-derived KD for estimating liver homogenate occupancy.
Conclusions:
- The study presents a generalizable CETSA-based approach for quantifying target engagement of CETSA-responsive drug targets.
- The findings underscore the necessity of incorporating cofactor saturation into occupancy estimates for uncompetitive inhibitors like BI-3231.
- This methodology facilitates improved pharmacodynamic biomarker strategies for 17βHSD13 inhibitors, crucial for liver disease therapeutics.
