Targeting isoprenoid and quinone biosynthesis for antimicrobial discovery

Enzymes in isoprenoid biosynthesis pathway play important roles in all living organisms. Here we report the first structure of heptaprenyl diphosphate synthase from Staphylococcus aureus (SaHepPPS) together with an investigation of its mechanism of action, and inhibition. HepPPS is involved in menaquinone biosynthesis, a key electron transporter in many pathogens. It is a heterodimer consisting of a catalytic subunit and a regulatory subunit. High concentrations of substrates (FPP and IPP) inhibit the enzyme, which is also inhibited by bisphosphonates. The most active inhibitors (Ki ~ 200 nM) were N-alkyl analogs of zoledronate containing ~C6 alkyl side-chains. They were modestly active against S. aureus cell growth, and growth inhibition was partially “rescued” by addition of menaquinone-7. Helicobacter pylori utilizes a completely different menaquinone biosynthesis pathway. Orthologs of Men genes (MenA-MenG) involved in menaquinone biosynthesis are absent in H. pylori. Recently, an alternate biosynthesis, called futalosine pathway was discovered in H. pylori, which uses a novel substrate, 6-carboxy-1,4-naphthoquinone as an intermediate in the menaquinone biosynthesis. We report the first expression and characterization of H. pylori UbiD and UbiX. We demonstrate that UbiD is the menaquinone decarboxylase that is dependent on a prenyltransferase enzyme, UbiX. UbiD family of enzymes are reported in literature as phenolic acid decarboxylase and ubiquinone decarboxylase. We report the first UbiD functioning as menaquinone decarboxylase in futalosine pathway, active with 6-carboxy-1,4-naphthoquinone substrate. We successfully expressed active, recombinant farnesyl diphosphate synthase (FPPS) and geranylgeranyl diphosphate synthase (GGPPS) for subsequent kinetic characterization and testing against a variety of bisphosphonates. Characterization of the substrate utilization of FPPS and GGPPS indicates that they have overlapping substrate specificities. Against FPPS, several bisphosphonates had IC50s in the low micromolar to nanomolar range; these inhibitors had significantly less activity against GGPPS. Several lipophilic bisphosphonates were active against ex vivo adult S. mansoni worms with worm death occurring over 4-6 days. These results indicate that FPPS and GGPPS are important for development of new drug against schistosomiasis. FPPS is also a suitable target for development of new antibiotics against gram-negative bacteria. We report bisphosphonates with electron-withdrawing aryl-alkyl sidechains, which inhibited the growth of gram-negative bacteria (Acinetobacter baumannii, Klebsiella pneumoniae, E. coli and Pseudomonas aeruginosa) at ~1-4 µg/mL levels. They were potent inhibitors of FPPS; bacterial cell growth was partially "rescued" by addition of farnesol or overexpression of FPPS; and there was synergistic activity with known isoprenoid biosynthesis pathway inhibitors. In bacteria, farnesyl diphosphate (FPP) is further utilized in downstream isoprenoid biosynthesis pathway to synthesize undecaprenyl diphosphate (UPP, catalyzed by UPP synthase) and undecaprenyl monophosphate (UP, catalyzed by UPP phosphatase). Undecaprenyl monophosphate is an essential lipid carrier involved in cell wall biosynthesis in bacteria, which is not present in humans; thus it’s a suitable drug target. We report a series of lipophilic hydroxy-monophosphonates, dihydroxy acids, and benzoic acids that inhibit UPPS and UPPP at µM levels. Lipophilic hydroxy-phosphonates were active (~2-6 µg/mL) against gram-positive organisms (S. aureus and B. subtilis) and exhibited synergistic activity with known isoprenoid/cell wall biosynthesis inhibitors, but only indifferent effects with other inhibitors. One of the most active benzoic acid (IC50 ~ 0.15 μg/mL) acted synergistically with seven antibiotics known to target bacterial cell wall biosynthesis (fractional inhibitory concentration index, FICI~0.35, on average). Benzoic acids were active against UPPS and UPPP and there were good correlations between S. aureus cell growth inhibition and SaUPPS inhibition (r = 0.85)