Modeling magic methyls

Shamans have their magic mushrooms, we medicinal chemists have our magic methyls. The 'magic methyl effect' refers to the sometimes large and unexpected change in drug potency resulting from the addition of a single methyl group to a molecule (for laymen, a methyl group is a single carbon with three hydrogens, and you don't expect spectacular effects from the addition of such a small modification to a drug). This is part of a broader paradigm in chemistry in which small changes in molecular structure can bring about large changes in properties, especially biological activity.

In this study, researchers from Bill Jorgensen's group at Yale ask what exactly it is that a methyl group can do to a biologically active molecule. They look at more than 2000 cases of purported activity changes induced by methyl groups from two leading medicinal chemistry journals, and their work highlights some unexpected effects of methyls. The techniques used are free-energy perturbation, Monte-Carlo and molecular dynamics simulations to compare methylated and non-methylated versions of published inhibitors in an effort to gain insight into the factors dictating potency.

Firstly, they find that for all their reputation, methyls mostly confer a modest increase in potency. The greatest increase is about 3 kcal/mol in free energy, which considering the exponential relationship between free energy and binding constant, is actually quite substantial. But this happens in a negligible minority of cases; as they find, a 10 fold boost in potency with a methyl is seen in only 8% of the cases, while a 100 fold difference is seen in only 0.4%.

So what does a methyl do? For starters, a methyl is simply a nice, small, lipophilic group so you would expect it to give you some advantage simply by snugly fitting in in an otherwise unoccupied binding pocket. But the real advantage of a methyl is thought to come from kicking out 'unhappy' water molecules; often a small protein pocket is occupied by a highly constrained water molecule that is desperate to join its free brethren in the bulk. A methyl group is usually only too happy to oblige and kick the water out. Now, as the crystallographer Jack Dunitz demonstrated more than a decade back, the maximum free energy gain you could estimate from displacing a water molecule is about 2 kcal/mol. Considered in this light, you would expect a gain of at least that much from a hydrogen to methyl change, but the very rare 3 kcal/mol cases seen seem to call this belief into question. Clearly the common wisdom about methyls displacing waters is not telling us the entire story.

As the authors demonstrate, the common wisdom may indeed point to uncommon cases. They look at five cases where methyls give the greatest potency boost, and in no case do they find evidence for displacement of a water molecule. So where's the potency gain coming from? It turns out that it may be coming from a common but often underappreciated factor; conformational reorganization. When a ligand binds to a protein, it exists in several - often hundreds - of conformations in solution. How tightly the protein can bind the ligand depends on how much energy the protein can expend to twist and turn these conformations into the single bound conformation. You would expect that the more similar a molecule's unbound conformation in solution is to its protein-bound conformation, the easier it would be for the protein to latch on to it.

And indeed, that's what they find. Most of the cases they look at concern potency gains coming from putting methyls at the ortho position of a biaryl ring. Organic chemists are quite familiar with the steric, planarity-disrupting effects of ortho substituents on biaryl rings; in fact it's a tried and tested strategy to improve solubility by disrupting crystal packing effects. It turns out that the bound structures of the molecules present a twisted, non-planar conformation. In the absence of methyls, the rings would prefer to stay almost coplanar (or at least less non-planar) in the unbound conformation. But putting methyl groups on twists the rings in the unbound conformation into a form that's similar to the bound one; basically there's more overlap between the solution and bound conformations in case of the methylated versions compared to the non-methylated ones. Consequently, the protein has to expend less energy to turn an already similar conformation into its bound counterpart. This becomes clear simply by comparing the single dihedral angle bridging the two rings in the bound and unbound conformations of one particular molecule (as shown in the figure above).

The study seems to impart what is part of an important general lesson; when designing ligands or drugs to bind a protein, it is as important to take the solution conformations into account. It's not just how the protein interacts with the drug, but it's what the drug is doing before it meets the protein that's equally important.