Our last post covered SPR. While we’re on the topic of biophysical methods, we should touch on isothermal titration calorimetry (ITC). A Perspective in last month’s issue of Nature Reviews Drug Discovery gives a very readable and concise summary of the technique, along with its applications for fragment-based drug discovery.
In ITC, two samples are mixed together, and the change in heat is precisely measured. If one solution contains a protein and the other a small molecule, one can determine the enthalpy (deltaH) as well as the overall free energy (deltaG) of binding (and thus the affinity), entropy (deltaS), and stoichiometry. In their article, John Ladbury, Gerhard Klebe, and Ernesto Freire, all long-time proponents of the technique, describe the importance of enthalpically-driven versus entropically-driven protein-ligand interactions.
It turns out that compounds derived from medicinal chemistry efforts have a greater entropic component to their affinities than do natural ligands, which rely more heavily on enthalpy. This is because it is easier to improve entropy than enthalpy: enthalpy is dependent on the number and strength of non-covalent bonds between a protein and its ligand, and as anyone who has tried to engineer a specific hydrogen bond can attest, this is easier said than done. Entropy, on the other hand, can often be increased just by making a compound more hydrophobic. However, increasing hydrophobicity too much decreases solubility and can cause other problems. The authors suggest that, while it may be easier to improve entropy than enthalpy, focusing on the later parameter will lead to better drugs. In fact, for statins and HIV protease inhibitors, first-in-class compounds were largely entropically-driven, while best-in-class compounds have their affinities dominated by enthalpy. Just as natural ligands have evolved to rely more on enthalpy than entropy, drug developers are also selecting for enthalpically driven binders as they optimize other parameters. But this selection has been indirect, and the authors suggest that researchers should deliberately select for enthalpic binders.
The authors acknowledge that commercially available ITC instruments are not sufficiently high-throughput for primary screening, and also that fragment interactions are sometimes so weak that dissociation constants may not be measurable with the technology. Nevertheless, it is possible to measure enthalpy of binding even for fragments, and, as we noted last year, this can lead to superior molecules.
Despite its power, ITC does not seem to be used often in fragment campaigns: at a roundtable discussion at the recent Tri-Conference, not one of the dozen or so participants had direct experience with the method. I suspect this has to do both with the availability of instruments as well as perceived difficulties with the experiments. Hopefully this will change, but whether the technique will become as popular as SPR remains to be seen.