Is thermodynamic data useful for drug discovery?

Just over a decade ago Ernesto Freire suggested that small molecules whose binding energy is dominated by the enthalpic – rather than the entropic – term make superior drugs. He also suggested that such molecules may be more selective for their target. But the backlash came quickly, and a couple years ago we wrote that focusing on thermodynamics probably isn’t particularly practical. A new perspective in Drug Disc. Today by Gerhard Klebe (Philipps-University Marburg) revisits this topic.

Klebe suggests that enthalpy was initially embraced “because readily accessible and easily recordable parameters are much sought after for the support of the nontrivial decision over which molecules to take to the next level of development.” (I would be interested to know whether sales of isothermal titration calorimetry (ITC) instruments spiked around 2010.) Unfortunately, both theoretical and practical reasons make thermodynamic measurements less useful than hoped.

First, and as we noted previously, “in an ITC experiment… the balance sheet of the entire process is measured.” In particular, water molecules – which make up the bulk of the solution – can affect both enthalpic and entropic terms. Klebe describes an example in which the most flexible of a series of ligands binds with the most favorable entropy to the target protein; this is counterintuitive because the ligand adopts a more ordered state once bound to the protein. It turned out that in solution the ligand traps a water molecule that is released when the ligand binds to the protein, thus accounting for the favorable entropy.

Indeed, water turns out to be a major confounding factor. We’ve previously written about “high-energy” water; Klebe notes that an individual water molecule can easily contribute more than 2 kcal/mol to the overall thermodynamic signature. And of course, proteins in solution are literally bathed in water. The structure of this water network, which may change upon ligand binding, is rarely known experimentally, but optimizing for it can improve affinity of a ligand by as much as 50-fold. Conversely, attaching a polar substituent to a solvent-exposed portion of a molecule to improve solubility sometimes causes a loss in affinity, and Klebe suggests this can be due to disruption of the water sheath.

Beyond these theoretical considerations, experimental problems abound. We’ve previously discussed how spurious results can be obtained when testing mixtures of ligands in an ITC experiment, but even with single protein-ligand complexes things can get complicated. Klebe shows examples where the relative enthalpic and entropic components to free energy change dramatically simply because of changes in buffer or temperature. This means that the growing body of published thermodynamic data needs to be treated cautiously.

So what is to be done? First, thermodynamic data should always be treated relatively: “we should avoid classifying ligands as enthalpy- or entropy-driven binders; in fact, we can only differentiate them as enthalpically or entropically more favored binders relative to one another.”

Klebe argues that collecting data on a variety of ligands for a given target under carefully controlled conditions will be useful for developing computational binding models. This is important, but not the kind of work for which people usually win grants, let alone venture funding.

He also suggests that, by collecting thermodynamic data across a series of ligands, unexpected changes in thermodynamic profiles might reveal “changes in binding modes, protonation states, or water-mediated interactions.” Maybe. But it takes serious effort to collect high-quality ITC data. Are there examples where you’ve found it to be worthwhile?