The UDP-2,3-diacylglucosamine pyrophosphatase, LpxH, is a well-conserved, essential enzyme in the biosynthesis of the crucial saccharolipid, lipid A, in Gram-negative bacteria. LpxH is vital to outer membrane biogenesis and cell viability in the majority of Gram-negative bacteria and is an excellent novel antibiotic target. In 2015, AstraZeneca identified a LpxH-targeting sulfonyl piperazine compound (referred to as AZ1). Previous work by our lab has investigated LpxH’s viability as an antibiotic target through developing a library of AZ1-based derivatives, and conjointly elucidating the apo- and drug bound crystal structures of Klebsiella pneumoniae LpxH (KpLpxH) in complex with AZ1 and other derivatives. These structures provided insights into the molecular details of AZ1 inhibition by showing that both AZ1 and its first generation of inhibitors all fit snugly into the L-shaped acyl chain-binding chamber of LpxH with their indoline rings situating adjacent to the active site, their sulfonyl groups adopting a sharp kink, and their N-CF₃-phenyl substituted piperazine groups reaching out to the far side of the LpxH acyl chain-binding chamber. The lead derivative, JH-LPH-33 displayed striking improvement in antibiotic activity over AZ1 against wild-type K. pneumoniae. Surprisingly, none of our first generation of sulfonyl piperazine compounds occupied the active site of LpxH, including JH-LPH-33. We next prepared and evaluated a new series of sulfonyl piperazine analogs with modifications in the phenyl and N-acetyl group. From this series, we discovered and solved the structures of two extended N-acyl chain analogs in complex with KpLpxH, revealing both binding and chelation of the catalytically essential dimanganese cluster within the LpxH active site. Due to this chelation, both leads showed significantly improved activity over JH-LPH-33. We expect that this work will provide guiding design principles for new LpxH inhibitors and establish important frameworks for the future development of antibiotics against multi-drug resistant Gram-negative pathogens.

Colleen Cochrane

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