Determination of Protein-Ligand Binding Affinities by Thermal Shift Assay.

Autor: Khan MF; Department of Microbiology, Biomedicine Discovery Institute, Monash University, Melbourne, Victoria 3800, Australia., Rahman MM; Department of Microbiology, Biomedicine Discovery Institute, Monash University, Melbourne, Victoria 3800, Australia., Xin Y; Department of Microbiology, Biomedicine Discovery Institute, Monash University, Melbourne, Victoria 3800, Australia., Mustafa A; School of Computing, Mathematics and Engineering, Charles Sturt University, Albury, New South Wales 2678, Australia., Smith BJ; La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria 3086, Australia., Ottemann KM; Department of Microbiology and Environmental Toxicology, University of California Santa Cruz, Santa Cruz, California 95064, United States., Roujeinikova A; Department of Microbiology, Biomedicine Discovery Institute, Monash University, Melbourne, Victoria 3800, Australia.; Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Victoria 3800, Australia.
Jazyk: angličtina
Zdroj: ACS pharmacology & translational science [ACS Pharmacol Transl Sci] 2024 Sep 17; Vol. 7 (10), pp. 3096-3107. Date of Electronic Publication: 2024 Sep 17 (Print Publication: 2024).
DOI: 10.1021/acsptsci.4c00293
Abstrakt: 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 (Δ H L ) and molar heat capacity change (Δ C PL ) of ligand binding are unknown. Our results suggest that, in this scenario, using the -300 cal K -1 mol -1 assumption for Δ C pL and either -5 kcal mol -1 or the average enthalpy efficiency-based estimation for Δ H L ( 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.
Competing Interests: The authors declare no competing financial interest.
(© 2024 The Authors. Published by American Chemical Society.)
Databáze: MEDLINE
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