Determination of protein-ligand binding affinities by thermal shift assay

Mohammad F. Khan, Mohammad M. Rahman, Yue Xin, Abdur Mustafa, Brian J. Smith, Karen M. Ottemann, Anna Roujeinikova

Research output: Contribution to journalArticlepeer-review

Abstract

Quantification of protein-ligand interactions is crucial for understanding the protein’s biological function and for drug discovery. In this study, we employed three distinct approaches for determination of protein-ligand binding affinities by a thermal shift assay using a single ligand concentration. We present the results of the comparison of the performance of the conventional curve fitting (CF) method and two newly introduced methods - assuming zero heat capacity change across small temperature ranges (ZHC) and utilizing the unfolding equilibrium constant (UEC); the latter has the advantage of reducing calculations by obtaining the unfolding equilibrium constant directly from the experimental data. Our results highlight superior performance of the ZHC and UEC methods over the conventional CF method in estimating the binding affinity, irrespective of the ligand concentration. In addition, we evaluated how the new methods can be applied to high-throughput screening for potential binders, when the enthalpy (ΔHL) and molar heat capacity change (ΔCPL) of ligand binding are unknown. Our results suggest that, in this scenario, using the −300 cal K-1 mol-1 assumption for ΔCpL and either −5 kcal mol-1 or the average enthalpy efficiency-based estimation for ΔHL(T) can still provide reasonable estimates of the binding affinity. Incorporating the new methods into the workflow for screening of small drug-like molecules, typically conducted using single-concentration libraries, could greatly simplify and streamline the drug discovery process.

Original languageEnglish
Pages (from-to)3096-3107
Number of pages12
JournalACS Pharmacology and Translational Science
Volume7
Issue number10
Early online date17 Sept 2024
DOIs
Publication statusPublished - 11 Oct 2024

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