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Updated: Jan 13, 2026
Assessing Cellular Target Engagement by SHP2 PTPN11 Phosphatase Inhibitors
Published on: July 17, 2020
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.
Abstract:
Human loss-of-function variants in 17βHSD13 have been associated with reduced risk and progression of metabolic and alcohol-related liver disease. Although in vitro experiments implicate 17βHSD13 in the processing of steroidal and lipogenic substrates, establishing an unambiguous mechanistic link to disease causation remains challenging, and the absence of robust pharmacodynamic biomarkers complicates clinical dose selection based on tissue target occupancy. To address this, we implemented a medium-throughput cellular thermal shift assay (CETSA) in humanized mouse liver homogenates to quantify the binding of BI-3231, an uncompetitive 17βHSD13 inhibitor, and compared results with functional cell assays. We systematically profiled isothermal dose-response fingerprints (ITDRFCETSA) across 58-70.5 °C to account for temperature-induced relaxation of binding equilibria at elevated temperatures, which produced pronounced right-shifts in the apparent potency and confirmed NAD+-dependent binding. Complementary surface plasmon resonance (SPR) measurements across 5-42 °C defined the temperature dependence of NAD+ and BI-3231 binding. Extrapolating SPR affinities to the CETSA temperature range showed convergence with ITDRFCETSA fingerprints, supporting the use of the SPR-derived KD for BI-3231 (2.5 nM at 37 °C; pKD = 8.60 ± 0.07, 95% CI) to estimate the occupancy also in liver homogenates. This work provides a generalizable approach to quantify target engagement for CETSA-responsive drug targets, while underscoring that occupancy estimates for uncompetitive inhibitors must incorporate cofactor saturation.
Insights
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.
