Fragment linking, enthalpy, and entropy: not quite so simple

The strategy of fragment linking dates to the origins of fragment-based lead discovery. The idea that two low affinity binders can be linked to produce a more potent molecule is based on the theory that the binding energies of linked fragments will at least be additive. Indeed, sometimes superadditivity can be observed; in those cases, the binding energy of the linked molecule is considerably better than the sum of the binding energies of the separate fragments. The most common explanation for this is that linking two fragments “pre-pays” the entropic cost of binding to the protein; rather than two fragments locking into fixed binding modes, only a single linked ligand pays this entropic penalty. This makes sense intuitively, but is it correct?

An early example of fragment linking was reported by Abbott researchers in 1997: two fragments that bound to the matrix metalloproteinase stromelysin were linked together to give a molecule that bound about 14-fold more tightly than the product of the affinities of the two fragments. Thermodynamic analyses were conducted to explore the roles of entropy and enthalpy, but these were complicated by the fact that one of the fragments contained an acidic phenol that was removed in the course of linking. In a new paper published in Bioorg. Med. Chem. Lett., Eric Toone and colleagues at Duke University have re-examined this system.

The researchers dissected several of the originally reported linked molecules into component fragments and examined their thermodynamics of binding using isothermal titration calorimetry. All of the experiments produced similar results; a particularly illustrative example is shown in the figure, in which a single bond in compound 1 was conceptually broken to yield component fragments 5 and 8.

As the researchers note, weirdly, the “favorable additivity in ligand binding – that is a free energy of binding greater than the sum of those for the constituent ligand fragments – is enthalpic in origin,” not entropic. It is not clear why this is the case, but what is clear is that the results are completely different from those obtained by Claudio Luchinat and colleagues on another matrix metalloproteinase. In that report, the enhanced affinity of the linked molecule was entirely entropic in origin, as might be expected. So what’s going on here?

One clue is provided by Fesik and colleagues in their original analysis of their stromelysin inhibitors. They noted that, when fragment 8 (acetohydroxamic acid) was added to the protein, biphenyl ligands similar to fragment 5 bound considerably more tightly than when fragment 8 was not present. In other words, the ligands displayed cooperative binding even when they were not covalently linked, probably due to non-covalent interactions between the two bound ligands or possibly to changes in protein structure and dynamics.

It is easy to assume that two ligands bind independently to two sites on a rigid protein, when in fact proteins are anything but rigid, and the addition of one ligand to a protein can dramatically change its properties. Thermodynamics measures changes in the entire system, not just the ligands, and if the protein changes upon ligand binding things can quickly get complicated. As Fesik and coworkers noted:

The observed cooperativity between the two ligands is a factor that should be considered when optimizing compounds for binding to nearby sites, since a portion of the binding energy is due to the cooperativity rather than interactions between the ligands and the protein.

All of which is to say that we remain woefully ignorant of the forces driving ligand binding, let alone fragment linking. But assessing how much better (or worse) a linked molecule binds than its component fragments can still be a useful exercise to guide optimization, even if the thermodynamic origins of the effects are unclear.