Two isothermal titration calorimetry (ITC) techniques for measuring the kinetics of enzyme inhibition have been developed. According to researchers at McGill University, these complementary methods will allow for the measurement of inhibitor association and dissociates rates, which are important in assessing a drug candidate’s efficacy.
The new methods are detailed in a paper published today in Nature Communications. The McGill team, led by chemistry professors Nicolas Moitessier and Anthony Mittermaier, demonstrate the use of ITC to measure the heat generated by enzyme activity and thereby the rates at which inhibitor substances blocked that activity.
"One key difference between ITC and other methods is that ITC measures the rate of reaction directly," Mittermaier explains. Existing methods for measuring enzyme activity look at that activity indirectly, by measuring changes in concentration caused by enzymatic catalysis as a function of time. These measurements often depend on special reagents that change color or fluorescence when acted on by the enzyme, and require a unique test to be developed for each enzyme being studied.
In addition to its generality, the ITC method gives a direct read-out of enzyme activity because it detects heat flow in real time. By providing a direct window on the reaction, ITC offers researchers a better insight into the mechanisms by which enzyme inhibition proceeds. It is usually very challenging, and sometimes next to impossible, to obtain this information from conventional assays.
The real-time nature of ITC is particularly promising for researchers investigating covalent inhibitors. These strongly binding molecules have potential as long-acting drugs but had previously fallen out of favor in drug development due to toxicity concerns. The insight ITC offers into the relationship between an inhibitor's molecular structure and how it reacts with its target will support renewed interest in covalent inhibitors and facilitate the work of developing them into drugs that are both highly effective and safe.