Rapid measurement of inhibitor binding kinetics by isothermal titration calorimetry

Trani, Justin M. Di; Cesco, Stéphane De; O’Leary, Rebecca A.; Plescia, Jessica; Nascimento, Cláudia Jorge do; Moitessier, Nicolas; Mittermaier, Anthony

doi:10.1038/s41467-018-03263-3

Cited by 47 publications

(49 citation statements)

References 32 publications

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“…In addition to simple two-component (one-to-one) binding processes, ITC may also be used to study more complex processes such as competitive binding [ 1 , 6 ], binding cooperativity [ 7 ], and binding events coupled to changes in the protonation state [ 8 , 9 ] or tautomeric state [ 10 ] of one or more components. Provided reaction rates are slower than cell mixing times, ITC can even be used to study the kinetics of association [ 11 – 15 ].…”

Section: Introductionmentioning

confidence: 99%

Bayesian analysis of isothermal titration calorimetry for binding thermodynamics

Nguyen¹,

Rustenburg²,

Krimmer³

et al. 2018

PLoS ONE

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Isothermal titration calorimetry (ITC) is the only technique able to determine both the enthalpy and entropy of noncovalent association in a single experiment. The standard data analysis method based on nonlinear regression, however, provides unrealistically small uncertainty estimates due to its neglect of dominant sources of error. Here, we present a Bayesian framework for sampling from the posterior distribution of all thermodynamic parameters and other quantities of interest from one or more ITC experiments, allowing uncertainties and correlations to be quantitatively assessed. For a series of ITC measurements on metal:chelator and protein:ligand systems, the Bayesian approach yields uncertainties which represent the variability from experiment to experiment more accurately than the standard data analysis. In some datasets, the median enthalpy of binding is shifted by as much as 1.5 kcal/mol. A Python implementation suitable for analysis of data generated by MicroCal instruments (and adaptable to other calorimeters) is freely available online.

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Section: Introductionmentioning

confidence: 99%

Bayesian analysis of isothermal titration calorimetry for binding thermodynamics

Nguyen¹,

Rustenburg²,

Krimmer³

et al. 2018

PLoS ONE

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“…Our lab has recently designed a pair of experiments which build on the multiple injection ITC experiment to give additional information on inhibitor association and dissociation rates (Di Trani et al, 2018a). In the association experiment (Figure 6C), the syringe contains the inhibitor and the sample cell contains dilute enzyme and sufficient substrate to maintain an essentially constant concentration throughout the experiment.…”

Section: Enzyme Inhibitorsmentioning

confidence: 99%

“…This method exploits the fact that ITC measures enzyme velocity directly. A traditional concentration-based enzyme assay would detect the gradual decreases and increases in enzyme velocity vividly illustrated in Figures 6D,F as slight curvature in the product buildup curve, making quantitative analysis far more difficult (Di Trani et al, 2018a).…”

Section: Enzyme Inhibitorsmentioning

confidence: 99%

Enzyme Kinetics by Isothermal Titration Calorimetry: Allostery, Inhibition, and Dynamics

Wang

Moitessier

et al. 2020

Front. Mol. Biosci.

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Isothermal titration calorimetry (ITC) involves accurately measuring the heat that is released or absorbed in real time when one solution is titrated into another. This technique is usually used to measure the thermodynamics of binding reactions. However, there is mounting interest in using it to measure reaction kinetics, particularly enzymatic catalysis. This application of ITC has been steadily growing for the past two decades, and the method is proving to be sensitive, generally applicable, and capable of providing information on enzyme activity that is difficult to obtain using traditional biochemical assays. This review aims to give a broad overview of the use of ITC to measure enzyme kinetics. It describes several different classes of ITC experiment, their strengths and weaknesses, and recent methodological advancements. A summary of applications in the literature is given and several examples where ITC has been used to investigate challenging aspects of enzyme behavior are presented in more detail. These include examples of allostery, where small-molecule binding outside the active site modulates activity. We describe the use of ITC to measure the strength, mode (i.e., competitive, uncompetitive, or mixed), and association and dissociation kinetics of enzyme inhibitors. Further, we provide examples of ITC applied to complex, heterogeneous mixtures, such as insoluble substrates and live cells. These studies exemplify the wide range of problems where ITC can provide answers, and illustrate the versatility of the technique and potential for future development and applications.

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“…The development of non-covalent inhibitors in a drug-design study is usually guided by the optimization of the affinity or dissociation constants (i.e., K i , K d , IC 50 ). However, dealing with covalent inhibition is even more complex, and in order to address the full potential of a covalent-inhibitor we need not only to measure their affinities but also kinetic binding parameters (e.g., residence time t r , the average time that a ligand remains bound in the binding site) ( De Cesco et al, 2017 ; Trani et al, 2018 ). The development of docking methodologies to predict poses and binding affinities of ligands that bind covalently to the receptor is a challenging task.…”

Section: Challenging Topics and Promising Strategiesmentioning

confidence: 99%

Empirical Scoring Functions for Structure-Based Virtual Screening: Applications, Critical Aspects, and Challenges

2018

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Structure-based virtual screening (VS) is a widely used approach that employs the knowledge of the three-dimensional structure of the target of interest in the design of new lead compounds from large-scale molecular docking experiments. Through the prediction of the binding mode and affinity of a small molecule within the binding site of the target of interest, it is possible to understand important properties related to the binding process. Empirical scoring functions are widely used for pose and affinity prediction. Although pose prediction is performed with satisfactory accuracy, the correct prediction of binding affinity is still a challenging task and crucial for the success of structure-based VS experiments. There are several efforts in distinct fronts to develop even more sophisticated and accurate models for filtering and ranking large libraries of compounds. This paper will cover some recent successful applications and methodological advances, including strategies to explore the ligand entropy and solvent effects, training with sophisticated machine-learning techniques, and the use of quantum mechanics. Particular emphasis will be given to the discussion of critical aspects and further directions for the development of more accurate empirical scoring functions.

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Rapid measurement of inhibitor binding kinetics by isothermal titration calorimetry

Cited by 47 publications

References 32 publications

Bayesian analysis of isothermal titration calorimetry for binding thermodynamics

Bayesian analysis of isothermal titration calorimetry for binding thermodynamics

Enzyme Kinetics by Isothermal Titration Calorimetry: Allostery, Inhibition, and Dynamics

Empirical Scoring Functions for Structure-Based Virtual Screening: Applications, Critical Aspects, and Challenges

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