Deconstructing an HTS hit for GyrB inhibitors

COVID-19 is deservedly engaging most of our collective mindspace when it comes to infectious diseases. Unfortunately, plenty other threats are out there, including antibiotic-resistant bacteria. A paper recently published in ACS Omega by Fumihito Ushiyama and colleagues at Taisho reports progress in this area.

The researchers were specifically interested in the protein DNA Gyrase B (GyrB), which is essential for bacterial replication (see here for previous work on the same target). A high-throughput screen against the E. coli protein led to a few dozen hits that were validated using a variety of biophysical methods including SPR, isothermal titration calorimetry (ITC), and crystallography. Compound 1 binds in the ATP-binding site, which is also where the natural product inhibitor novobiocin binds. The latter molecule makes an interaction with an arginine residue in the protein, but introducing a carboxylic acid moiety onto compound 1 to make a similar interaction was not successful (compound 8e).

Taking a step back, the researchers stripped compound 1 down to the core fragment 2a, which makes both polar and hydrophobic interactions with GyrB. Unfortunately, this fragment was too weak to show any affinity by ITC, as were 120 related fragments.

Looking closer at the structure of compound 1 bound to the protein revealed a small unfilled hydrophobic pocket near the 2-quinolinone fragment. Making appropriately substituted fragments was “relatively complicated,” and most of them were inactive. However, compound 2d showed binding by ITC as well as excellent ligand efficiency. Growing from this fragment ultimately led to compound 13e, with low nanomolar affinity. In addition to binding, compound 13e is a potent inhibitor of GyrB and is selective against a panel of 96 human kinases. Unfortunately though, it displays only modest antibacterial activity, likely due to efflux.

Nonetheless, this is a nice example of thoughtful structure-based design. In particular, the dramatic boost in potency gained by filling a small pocket (nearly 400-fold from compound 8e to 13e) validates the willingness to explore difficult chemistry rather than sticking with available analogs. The paper ends by noting that optimization is continuing, and I wish them well. By my count only a single fragment-derived antibacterial agent has entered clinical development, and that program is no longer active. We could use more.