Complete Kinetic Characterization of Enzyme Inhibition in a Single Isothermal Titration Calorimetric Experiment

Abstract

Techniques for rapidly measuring both the strength and mode of enzyme inhibitors are crucial to lead generation and optimization in drug development. Isothermal titration calorimetry (ITC) is emerging as a powerful tool for measuring enzyme kinetics with distinct advantages over traditional techniques. ITC measures heat flow, a feature of nearly all chemical reactions, and gives an instantaneous readout of enzyme velocity, eliminating the need for artificial substrates or postreaction processing. In principle, ITC is an ideal method for characterizing enzyme inhibition. However, existing ITC experiments are not well-suited to rapid throughput and few studies to date have employed this approach. We have developed a new ITC experiment, in which substrate and inhibitor are premixed in the injection syringe, that yields complete kinetic characterization of an enzyme inhibitor in an hour or less. This corresponds to savings in time and material of 5-fold or greater compared to previous ITC methods. We validated the approach using the trypsin inhibitor benzamidine as a model system, recapitulating both its competitive inhibition mode and binding constant. Our approach combines the rapid throughput of optimized spectroscopic assays with the universality and precision of ITC-based methods, providing substantially improved inhibitor characterization for biochemistry and drug development applications.

Evaluation of the In Vitro Stability of Stimuli-Sensitive Fatty Acid-Based Microparticles for the Treatment of Lung Cancer

“… 39 , 40 Additional information can be extracted from the evolution of the rate of the heat flow following a single injection as a function of time. 41 Figures S1 and S2 (see the Supporting information) show examples of energy flow following a single injection of 10 mg/mL albumin solution or 0.5 mg/mL unilamellar lipid vesicles formed from DOPC to microparticles formed from fatty acids alone or from fatty acids mixed with NP and PAX. In addition to the differences mentioned before regarding the direction of energy flux, there are additional, less obvious differences.…”

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

“…Error bars correspond to the standard deviations of three repeat experiments. (C) Single injection assay with trypsin in the sample cell and one of its substrates, benzoyl-L-arginine ethyl ester, in the syringe (Di Trani et al, 2018b). Data collected during (after) the 30 s injection are plotted in orange (blue).…”

“…In a single injection ITC kinetic assay, the amount of enzyme is typically chosen to be large enough so that the injected substrate can be fully converted to product on the timescale of minutes or tens of minutes. The concentration of substrate is chosen so that the injection appreciably saturates the enzyme, i.e., the concentration of substrate in the sample cell immediately after the injection is several-fold higher than the K m (Transtrum et al, 2015;Di Trani et al, 2018b). Single injection assays can be initiated either by injecting substrate (syringe) into enzyme (cell) or enzyme (syringe) into substrate (cell).…”

“…It should be noted that single injection assays are usually performed with substantially more dilute enzyme, leading to peaks that are much broader, on the order of 20-60 min, in contrast to the 100 s shown here. Our group has been developing ways to collect and analyze data for rapid single injection experiments, such as those shown in Figure 3C, offering over 10-fold reductions in measurement time and opening the door to new types of experiment (Di Trani et al, 2018b;Wang et al, 2019). Single-injection ITC data can be fitted directly by numerically integrating Equation 2 to give [S](t) and ν 0 (t), and calculating dQ/dt as a function of time according to Equation 6, or using non-linear least squares optimization to find the values of K m and k cat that best reproduce the experimental values.…”

“…The defining feature of this approach is that a full enzyme kinetic characterization is achieved in a single injection. Thus, with substrate in the syringe, it is straightforward to perform many single injection measurements within the same ITC experiment, simply by programming several injections (as many as 10 or 20) spaced at appropriate intervals (Cai et al, 2001;Ertan et al, 2012;Pedroso et al, 2014;Siddiqui et al, 2014;Maximova and Trylska, 2015;Di Trani et al, 2018b;Mason et al, 2018;Abis et al, 2019;Maximova et al, 2019;Wang et al, 2019). For the sake of clarity, we will refer to these as recurrent single injection experiments, to distinguish them from the very different approach termed multiple injection experiments (see section "Multiple Injection Assays, " above).…”