Fragment linking: flexible rules

Linking two fragments together to achieve a boost in potency has been done a number of times (see here, here, here, and here), though it often doesn’t work as well as might be hoped (see here). To better understand the energetics of fragment-linking, Marc Nazaré, Hans Matter, and colleagues at Sanofi-Aventis Deutschland have analyzed ligands for the blood coagulation enzyme factor Xa (fXa) and published their results in a recent issue of Angew. Chem. Int. Ed.

The researchers “deconstructed” potent fXa inhibitors into component fragments, measured their inhibition constants (and thereby inferred their binding energies), and compared these binding energies with those of the original linked molecules. One of the first observations was that many of the component fragments bound so weakly as to show no measurable activity, a phenomenon that has been observed previously.

In an exemplary case, cleaving a single bond connecting the two component fragments of a 2 nM ligand (1a, below) yielded one fragment (1g) with 58 micromolar activity and another (1d) whose activity was worse than 10 millimolar. Because the second fragment has such low affinity, the binding energy of linking is really just a lower estimate, but it seems to be at least 3.3 kcal/mol, which is greater than the binding energy of fragment 1d itself. In other words, the affinity brought about by linking is greater than the affinity of the weakly binding fragment. The superadditivity provided by the linker in this case is about 300-fold, a similar value to that observed in the unrelated MMP-12 system. This is perhaps all the more remarkable given the fact that the fragments are connected by a linker containing several rotatable bonds, the entropy of which should partially counter the advantages of linking.

In fact, a common strategy to improve the potency of two linked fragments is to rigidify the linker. Often this doesn’t work: in a second case, the Sanofi-Aventis researchers cleaved one bond of a 3 nM ligand (2a, below) to yield two fragments with roughly equal potency. However, even though the linker is more rigid than in the previous example, the binding energy due to linking is less – just 2.0 kcal/mol, representing a boost of about 30-fold.

As the authors note:

The introduction of rigid aromatic moieties as a common approach to increase affinity does not necessarily maximize the benefit from the linker effect as detrimental affinity contributions might originate from suboptimal orientation and accommodation of specific binding elements.

There are many more examples in this paper than can be covered in a blog post; the authors dissect compounds 1a and 2a at a number of different points, and while the component fragments typically bind less tightly than simple additivity would suggest, there are lots of interesting details.

Finally, it is interesting to note that ligands 1a and 2a consist of a relatively hydrophobic fragment (1g or 2g) connected to a more polar fragment (1d or 2h). The fact that these show superadditivity is consistent with Mark Whittaker and colleagues' proposal last year that linking such fragments is likely to maximize additivity, although given the precise interactions made by both parts of the molecules the details get a bit messy. We’re not yet at the point where the universe of molecular interactions can be distilled to rigid rules.