21 March 2016

Fragments vs bacterial GyrB

Anti-bacterial targets are not common among fragment-based lead discovery efforts. We’ve written previously about AstraZeneca’s work on DNA gyrase, which led to a clinical candidate. In a recent paper in Bioorg. Med. Chem. Lett., Michael Mesleh and collaborators at Cubist and Evotec describe their efforts on this protein.

Bacterial DNA gyrase has two subunits, GyrA and GyrB, and is essential during DNA replication. It is also well-validated, being the target of decades-old antibiotics such as the fluoroquinolones. The researchers started by screening a library of 5643 fragments against Staphylococcus aureus GyrB using STD NMR, yielding 304 hits. These were winnowed down to 46 based on intensity of the STD signal, novelty, and ease of follow-up chemistry. These were then tested using chemical shift perturbations and crystallography. Although several crystal structures of fragments bound to GyrB were obtained, these did not suggest clear ways to advance the hits.

On the other hand, compound 5, which showed only weak binding by NMR and did not result in a crystal structure, was appealing because of its novelty and polarity. The researchers knew that most ligands that interact with GyrB make a pair of hydrogen-bond donor-acceptor interactions, and they used that knowledge to surmise a binding model. This suggested that growing the fragment towards a pair of arginine residues could improve affinity and led to the synthesis of compound 8, with low micromolar activity.

A crystal structure of a related molecule confirmed the model and also suggested that removing the methyl group would stabilize a more planar conformation better matched to the binding site. Doing so (compound 9) yielded a ten-fold boost in potency. (This methyl is also a nice example of Teddy’s “Sauron Atom”). In a separate paper published last year, the researchers further optimized this molecule to compound 2, with low nanomolar potency and activity in animal models.

Several things stand out about this paper. First, the researchers were willing to pursue a fragment with an affinity lower than other hits. Second, careful modeling and conformational analyses were critical in advancing the molecules. Finally, crystallography was not used in the initial fragment growing. Of course, it helped that the researchers were working with a well-characterized protein amenable to modeling. Still, it is nice to see another example of advancing fragments in the absence of experimentally-determined structures.