Biology of novel LpxC inhibitors for Neisseria gonorrhoeae informs strategy for therapeutic innovation

ABSTRACT Historically, LpxC inhibitors, which target the UDP-3- O-(R- 3-hydroxymyristoyl)- N- acetylglucosamine deacetylase catalyzing the rate-limiting step in lipid A biosynthesis, have a hydroxamate zinc-chelating head group that targets the active site and can participate in non-specific metal chelation. We synthesized novel molecules with a 2-(1 S -hydroxymethyl)-imidazole head group to target the Neisseria gonorrhoeae LpxC enzyme. The gonococcal LpxC structure was generated with AlphaFold, and relative affinities of PF-04753299, CHIR-090, and our novel inhibitors were compared in docking simulations. The MIC of the most potent novel inhibitor for human challenge and multidrug-resistant N. gonorrhoeae was less than 1 μg/mL, intermediate to PF-04753299 and CHIR-090 MICs. Potency in bactericidal assays, which were designed to reflect in vivo conditions, and the ability to inhibit induction of TNF-α in THP-1 monocytes infected with gonococci, reflected relative MIC values. Cytotoxicity of the inhibitors was minimal in hemolysis and lactate dehydrogenase release assays. We showed that treating gonococci with the most potent novel inhibitor reduced lipooligosaccharide expression. Alternatives to differences in affinity for LpxC that could affect potency, including iron binding via the hydroxamate moieties and variable permeability, were explored utilizing synergism experiments and mutational analyses. We isolated spontaneous mutants resistant to the inhibitors and demonstrated that this resistance could be transferred by DNA-mediated transformation. DNA sequence analysis indicated that resistance was not due to lpxC alterations. Overall, the non-hydroxamate inhibitors exhibited high efficacy and low cytotoxicity. Furthermore, exploration of multiple antibacterial mechanisms led us to develop a promising dual therapy approach that could inform strategies to combat multidrug resistance. IMPORTANCE Neisseria gonorrhoeae is a major pathogen worldwide and the second most commonly reported cause of sexually transmitted infection in the USA. A total of 601,319 cases of gonorrhea were reported to the Centers for Disease Control and Prevention (CDC) in 2023. The bacteria typically infect the urogenital tract, but infections also occur in the eye, throat, and rectum. The CDC has classified gonorrhea as an urgent public health threat, as there is no vaccine and the bacteria have developed resistance to all but one class of antibiotic. We developed novel inhibitors of an enzyme that plays a critical role in the synthesis of lipooligosaccharide, an important bacterial cell surface toxin, that have high efficacy and low cytotoxicity. Importantly, we found that the inhibitors may have relatively low propensity to engender resistance and, furthermore, we identified a potential dual therapy approach utilizing the novel inhibitors that could have clinical applicability.