Fragment linking: too strained, too flexible, or too positive?

One of the most rewarding fragment-based outcomes is to link two fragments together and get a pop in activity, as predicted by William Jencks almost three decades ago. Unfortunately, all too frequently linking two fragments gives a less than additive boost in binding energies, and often it doesn’t work at all. A new paper from James Stivers and colleagues published in Nature Chemical Biology investigates why.

The authors had previously discovered bipartite inhibitors of the DNA repair enzyme uracil DNA glycosylase (UNG): one piece of the inhibitor was fixed as the substrate uracil, and the other piece was chosen empirically from a library of fragments; the two fragments were each attached by rigid oxime connectors to a flexible linker. A crystal structure of one of these inhibitors bound to UNG showed that the uracil fragment does indeed bind in the uracil-binding pocket, while the other fragment binds in a nearby phosphodiester-binding pocket. However, the linker exhibited an unusual kink, suggesting it might be strained. The researchers have now followed up on this observation to explore the effects of varying the linker.

Four of the molecules made and tested are shown below; these vary only in how fragments connect to the linker. Importantly, the authors were able to determine co-crystal structures of all of these molecules bound to UNG. The most potent molecule, MA1, has a (flexible) secondary amine connection to the uracil fragment and a (rigid) oxime connection to the benzoate fragment; the linker appears unstrained in the crystal structure. In contrast, although the two fragments of DO bind in the same manner as the two fragments of MA1, the linker assumes the apparently strained “kinked” conformation previously observed, and the molecule binds with a thirty-fold lower affinity. In the case of the other two molecules in this set, the uracil fragment still binds as expected, but the benzoate is either not visible in the electron density (MA2) or binds to a nearby molecule of UNG in the crystal (DA). Both of these molecules are weak inhibitors, with IC50s comparable to that of uracil connected to the linkers alone, without the benzoate fragment (700-750 micromolar).



The authors argue that the greater linker flexibility in MA1 compared to DO reduces linker strain when the two fragments assume their optimal positions. They also suggest that the effect of the linker on the tighter-interacting uracil fragment will be less pronounced than on the more “loosely interacting” benzoate fragment; uracil’s specific binding interactions are able to overcome linker strain, while the weaker benzoate requires more precise positioning. In other words, the flexible connector to the uracil and the rigid connector to the benzoate give a Goldilocks-type situation for MA1.

This is reasonable. But there may be more than strain and flexibility at work here. In particular, introducing a (positively charged) secondary amine near the benzoate may have a considerable electrostatic effect on binding. The authors consider this possibility unlikely, and provide some evidence against it, but given that this enzyme binds to negatively charged DNA, I can’t dismiss it. Although the linkers are solvent-exposed, the electrostatic surface of UNG is quite positive, and in fact the benzoate linkage is not far from a histidine residue. It would be interesting to see whether replacement of the oxime linkages with similarly uncharged ethers or methylenes has the same effects as the amines.

Still, this type of systematic analysis is a valuable experimental addition to the field of fragments. I hope someone will do high-level computational modeling on these complexes to try to further dissect the origins of the differences in affinities.