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      Isothermal Analysis of ThermoFluor Data can readily provide Quantitative Binding Affinities

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          Abstract

          Differential scanning fluorimetry (DSF), also known as ThermoFluor or Thermal Shift Assay, has become a commonly-used approach for detecting protein-ligand interactions, particularly in the context of fragment screening. Upon binding to a folded protein, most ligands stabilize the protein; thus, observing an increase in the temperature at which the protein unfolds as a function of ligand concentration can serve as evidence of a direct interaction. While experimental protocols for this assay are well-developed, it is not straightforward to extract binding constants from the resulting data. Because of this, DSF is often used to probe for an interaction, but not to quantify the corresponding binding constant (K d). Here, we propose a new approach for analyzing DSF data. Using unfolding curves at varying ligand concentrations, our “isothermal” approach collects from these the fraction of protein that is folded at a single temperature (chosen to be temperature near the unfolding transition). This greatly simplifies the subsequent analysis, because it circumvents the complicating temperature dependence of the binding constant; the resulting constant-temperature system can then be described as a pair of coupled equilibria (protein folding/unfolding and ligand binding/unbinding). The temperature at which the binding constants are determined can also be tuned, by adding chemical denaturants that shift the protein unfolding temperature. We demonstrate the application of this isothermal analysis using experimental data for maltose binding protein binding to maltose, and for two carbonic anhydrase isoforms binding to each of four inhibitors. To facilitate adoption of this new approach, we provide a free and easy-to-use Python program that analyzes thermal unfolding data and implements the isothermal approach described herein ( https://sourceforge.net/projects/dsf-fitting).

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          The role of ligand efficiency metrics in drug discovery.

          The judicious application of ligand or binding efficiency metrics, which quantify the molecular properties required to obtain binding affinity for a drug target, is gaining traction in the selection and optimization of fragments, hits and leads. Retrospective analysis of recently marketed oral drugs shows that they frequently have highly optimized ligand efficiency values for their targets. Optimizing ligand efficiency metrics based on both molecular mass and lipophilicity, when set in the context of the specific target, has the potential to ameliorate the inflation of these properties that has been observed in current medicinal chemistry practice, and to increase the quality of drug candidates.
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            Evaluation of fluorescence-based thermal shift assays for hit identification in drug discovery.

            The fluorescence-based thermal shift assay is a general method for identification of inhibitors of target proteins from compound libraries. Using an environmentally sensitive fluorescent dye to monitor protein thermal unfolding, the ligand-binding affinity can be assessed from the shift of the unfolding temperature (Delta Tm) obtained in the presence of ligands relative to that obtained in the absence of ligands. In this article, we report that the thermal shift assay can be conducted in an inexpensive, commercially available device for temperature control and fluorescence detection. The binding affinities obtained from thermal shift assays are compared with the binding affinities measured by isothermal titration calorimetry and with the IC(50) values from enzymatic assays. The potential pitfalls in the data analysis of thermal shift assays are also discussed.
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              Using circular dichroism collected as a function of temperature to determine the thermodynamics of protein unfolding and binding interactions.

              Circular dichroism (CD) is an excellent spectroscopic technique for following the unfolding and folding of proteins as a function of temperature. One of its principal applications is to determine the effects of mutations and ligands on protein and polypeptide stability. If the change in CD as a function of temperature is reversible, analysis of the data may be used to determined the van't Hoff enthalpy and entropy of unfolding, the midpoint of the unfolding transition and the free energy of unfolding. Binding constants of protein-protein and protein-ligand interactions may also be estimated from the unfolding curves. Analysis of CD spectra obtained as a function of temperature is also useful to determine whether a protein has unfolding intermediates. Measurement of the spectra of five folded proteins and their unfolding curves at a single wavelength requires approximately 8 h.
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                Author and article information

                Contributors
                john.karanicolas@fccc.edu
                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group UK (London )
                2045-2322
                25 February 2019
                25 February 2019
                2019
                : 9
                : 2650
                Affiliations
                [1 ]ISNI 0000 0004 0456 6466, GRID grid.412530.1, Program in Molecular Therapeutics, , Fox Chase Cancer Center, ; Philadelphia, PA 19111 USA
                [2 ]ISNI 0000 0001 2106 0692, GRID grid.266515.3, Department of Molecular Biosciences, , University of Kansas, ; Lawrence, KS 66045 USA
                [3 ]ISNI 0000 0001 2150 1785, GRID grid.17088.36, Department of Biochemistry & Molecular Biology and Department of Computational Mathematics, Science and Engineering, , Michigan State University, ; East Lansing, MI 48824 USA
                Author information
                http://orcid.org/0000-0003-0300-726X
                Article
                37072
                10.1038/s41598-018-37072-x
                6389909
                30804351
                fbb3fa58-c257-49f1-aa8f-0b23e0726611
                © The Author(s) 2019

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 23 July 2018
                : 30 November 2018
                Funding
                Funded by: FundRef https://doi.org/10.13039/501100000854, Human Frontier Science Program (HFSP);
                Award ID: RGP0022/2017
                Award Recipient :
                Funded by: FundRef https://doi.org/10.13039/100000057, U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS);
                Award ID: R01GM112736
                Award Recipient :
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