Fragments vs LpxC revisited

Back in 2020 we described fragment-derived inhibitors of the highly conserved bacterial enzyme LpxC, which is essential for biosynthesis of the outer membrane in Gram-negative bacteria. In a recent (open access) paper in J. Med. Chem., a different group consisting of Ralph Holl and collaborators at Universität Hamburg and several other academic centers describe a new series.

 

The researchers started with compound 9, a molecule they had previously discovered. The substrate for LpxC is a rather large small molecule called (UDP)-3-O-[(R)-3-hydroxymyristoyl]-N-acetylglucosamine. Compound 9 does not occupy the UDP-binding site, so the researchers initially tried building towards it with a series of simple linkers connected to a phenyl group. The (S) enantiomers tended to be more active than the (R)-enantiomers, and the most potent was compound (S)-13a, which showed sub-micromolar activity against LpxC from E. coli as well as P. aeruginosa in an enzymatic assay. (For simplicity only the E. coli data are shown here.)

 

Seeking to improve affinity, the researchers screened 650 fragments in pools of five against LpxC in the presence of compound 9 using STD NMR and WaterLOGSY. After deconvolution, this led to 97 hits. STD-based epitope mapping, which we wrote about here, was used to prioritize fragments likely to have a single, well-defined binding mode, culling the number to 19. Finally, NMR-ILOE experiments (see here) suggested that nine of this set bound in close proximity to compound 9, while the other ten did not. Four of these fragments, including the simple indole F3, were then linked to compound 9 at various positions. This is akin to SAR by NMR, but with less information about the relative binding modes so more trial and error is necessary.

 

Among the roughly two dozen molecules made, compound (S)-13j was the most potent against LpxC, with low nanomolar activity. This compound (and several others) also showed antibacterial activity against E. coli and several other strains of Gram-negative bacteria. In vitro stability studies of compound (S)-13j were promising, though the researchers noted the need for improvement. And, since the molecule contains a hydroxamic acid moiety potentially capable of binding to multiple metalloproteins, it was tested against a handful of mammalian zinc-dependent enzymes and shown to be nearly inactive.

 

Compound (S)-13j is 15-fold more potent than the simple phenyl analog (S)-13a, and molecular modeling suggested this may be due to a hydrogen bond from the protein to the indole NH. Although one could argue that it would have been possible to arrive at compound (S)-13j using standard medicinal chemistry starting from (S)-13a, this may have taken longer without knowledge of the indole fragment. Whether or not the molecules advance further, this is a nice example of using fragment screening to find a second-site binder to improve affinity of an existing lead.