Fragments vs riboswitches

Most of fragment-based lead discovery – indeed, most of lead discovery – is directed against proteins. However, RNA is also an essential biomolecule, and in a new paper in Chem. Biol. Adrian R. Ferré-D’Amaré and colleagues at the National Heart, Lung, and Blood Institute, along with collaborators at the University of Cambridge and the University of North Carolina Chapel Hill, demonstrate that fragments can potentially make an impact here as well. This is the first example I know of where crystallography has been used to assess fragment hits against RNA molecules.

The story begins several years ago, when Chris Abell and colleagues became interested in the TPP riboswitch thiM. This is a bacterial stretch of RNA that binds to the essential cofactor thiamine pyrophosphate (TPP). This binding causes a change in conformation that regulates protein translation; small molecules that interfere with this process could lead to new antibiotics. In 2010 the researchers described a fragment screen using equilibrium dialysis, in which the RNA was added to one chamber along with radiolabeled thiamine, which binds with low micromolar affinity. This chamber was separated from another chamber containing fragments by a dialysis membrane permeable to small molecules and fragments but not to (larger) RNA. Fragments were screened in pools of five, and pools that caused displacement of radioligand were then deconvoluted to identify the active fragments. A total of 20 fragment binders were identified out of roughly 1300 tested.

WaterLOGSY NMR was used to confirm the binding of these 20 fragments to the riboswitch, and all of them were then tested using isothermal titration calorimetry, which yielded dissociation constants for 17 of them ranging between 22 and 670 micromolar. When tested against a different riboswitch, 10 of them appeared to be selective for thiM. The chemical structures of all of these were reported in 2011, along with some speculation as to how they might bind.

Of course, speculation is just that, and in fact fragment hits have been identified against RNA and DNA before. In the new paper the researchers use X-ray crystallography to actually determine the structures of several fragments bound to the riboswitch. This provides several interesting observations.

First, despite the different chemical structures of the fragment hits, all four of those whose structures were determined bind in the same region where the pyrimidine moiety of the natural ligand TPP binds. In fact, fragment 1 (magenta), which is essentially a fragment of TPP (green), almost perfectly superimposes on the corresponding moiety of TPP.

More strikingly, the co-crystal structures of each of the fragments bound to the riboswitch reveal that one of the guanosine residues (magenta stick in figure above) rearranges to fill the pocket that would otherwise be occupied by the pyrophosphate moiety of TPP (orange and red above). This occurs with fragment 1 as well as other fragments that do not resemble the natural ligand.

The researchers also took the useful step of solving the crystal structure of thiamine (cyan) bound to thiM. Since thiamine is intermediate in size between TPP and fragment 1, you might expect the structure to resemble one or the other, but as it turns out it binds in yet a third mode in which the pyrimidine ring no longer superimposes with the other two structures, nor does the guanosine residue rearrange to fill the pyrophosphate-binding pocket. This provides an interesting example of fragmenting natural products (TPP to thiamine to fragment 1). Although all of the molecules bind with high ligand efficiencies, it is unlikely that their binding modes could have been accurately predicted.

As the researchers note, the conformational shifts observed with these fragments could lead to antibiotics that selectively target an inactive form of the riboswitch. Although they’ve got a long way to go, it is fun to see folks applying FBLD to non-traditional targets.