Fragments vs LpxC, two ways

Gram negative bacteria such as Pseudomonas aeruginosa are a continuing threat, and antibacterial drug discovery is not keeping pace. The enzyme UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) is critical for the synthesis of the bacterial cell wall lipopolysaccharide. In a new J. Med. Chem. paper, Yousuke Yamada, Rod Hubbard, and collaborators at Taisho and Vernalis describe progress against this target.

 

LpxC is a zinc hydrolase, and although previous potent inhibitors have been reported against the metalloenzyme, these contained hydroxamate moieties. Unfortunately, hydroxamic acids are rather nonspecific zinc binders, and many of them hit human enzymes such as HDACs and MMPs. Thus, the researchers turned to fragments to find new metallophilic starting points.

 

The 1152 members of the Vernalis fragment library were screened against LpxC using three NMR experiments: STD, WaterLOGSY, and CPMG in pools of six. This yielded a remarkable 252 hits in at least one assay. These were retested individually and for competition with a substrate pocket-binding small molecule, resulting in 28 hits, two of which were advanced.

 

A crystal structure of compound 6 bound to LpxC suggested that adding a hydroxyl group could make additional interactions with the protein, and this was confirmed in the form of compound 10. Further fiddling in this region of the molecule was not successful, and the phenyl ring did not provide good vectors to a hydrophobic tunnel. However, replacing the phenyl with a more shapely piperidine yielded compound 17. Although this molecule had slightly lower affinity, it did provide a better starting point for further optimization, ultimately leading to compound 21, with low nanomolar potency against LpxC. Unfortunately, this and other members of the series showed only weak antibacterial activity.

 

Compound 9 was weaker than the other fragment starting point, but making and testing related compounds led to improved binders such as compound 27. This was the first molecule in this series to be structurally characterized, and crystallography revealed that the imidazole was making a single interaction with the zinc at the heart of the LpxC active site. Adding a hydroxyl led to bidentate chelator 29 (i.e. two interactions with the zinc) that had better activity, and further structure-based design ultimately led to low nanomolar inhibitors such as compound 43. In contrast to the other series, this one did show antibacterial activity, and the researchers eventually discovered molecules with in vivo efficacy. Both series were also selective against a small panel of human metalloproteases.

 

 

This is a nice fragment to lead story (expect it to be included in the next compilation). As the researchers note, it provides two important lessons. First, fragments can provide multiple different starting points for a target. Second, because fragment libraries tend to be small, it can be valuable to take some time to refine a fragment before launching into fragment growing or merging. Indeed, compound 38 (itself fragment-sized) contains only four more atoms than the initial fragment hit, yet has more than a thousand-fold higher affinity. During lead optimization you often need to add molecular weight, lipophilicity, and possibly polar atoms, so it is crucial to get the core binding elements as good as possible.