Dynamic undocking for better predictions

Computational screening continues to improve, due in part to a better understanding of the energetics of protein-ligand interactions. But for low affinity fragments, differentiating binders from nonbinders is still challenging. In a recent paper in Nature Chemistry, Xavier Barril and collaborators at the Universitat de Barcelona, Discngine, Vernalis, and the University of York describe a new approach that sidesteps thermodynamics.

The researchers started with the notion that, in many cases, a single hydrogen bond is critical for the stability of a protein-ligand complex. Rather than trying to calculate the binding energy of the complex, they instead ran “dynamic undocking” (DUck) experiments. This involved “steered molecular dynamics” simulations in which the researchers calculated how much work (W_QB) is required to move the ligand from the bound state to a quasi-bound state in which the key hydrogen bond is broken. The calculations do not consider what happens under equilibrium conditions (ie, unbinding and rebinding), so W_QB should not necessarily correlate with binding affinity. Still, one might expect ligands that require a particularly high energy to dissociate (for example, W_QB > 6 kcal/mol) to have higher affinities. This turned out to be the case for ligands targeting several different proteins: the kinase CDK2, the GPCR adenosine A2A receptor, and the protease trypsin. Indeed, receiver operating characteristic curves (an analysis comparing known binders and decoys) showed a significant enrichment of true binders.

Next, the researchers compared DUck with several commonly used computational docking approaches. Again, and not surprisingly, there was essentially no correlation. However, the researchers argue that this is a feature, not a bug, since the very orthogonality of the approaches should provide better predictions: a molecule that docks favorably and has a high W_QB is more likely to be a real hit.

This is a nice idea, but does it work in practice? To find out, the researchers turned to the old work-horse protein HSP90 and performed docking experiments on 280,000 fragments. Of the top 450 hits, 139 diverse molecules were chosen for DUck. Several dozen of these were then tested for binding using three different ligand-observed NMR experiments.

Of 21 molecules with W_QB > 6 kcal/mol, 8 confirmed as binders by all three NMR methods – an impressive hit rate of 38%. SPR confirmed binding for four of these (with dissociation constants between 0.077 and 0.73 mM), while three yielded crystal structures. In contrast, only one out of 15 molecules with W_QB between 3 and 6 kcal/mol confirmed, while none of 11 molecules with W_QB < 3 kcal/mol were clear hits. In other words, not only is DUck able to improve identification of true binders, it appears to have a fairly low false negative rate.

In a sense, this approach addresses the question of kinetics. Molecules that dissociate slowly from their target are becoming increasingly fashionable; perhaps DUck can be used to identify them. Although the researchers do not make this claim, several of the authors described an experimental “off-rate screening” approach a few years ago. It will be fun to see further developments, particularly as the method is extended to incorporate information beyond a single hydrogen bond.