Bacterial Cell Growth Inhibitors Targeting Undecaprenyl Diphosphate Synthase and Undecaprenyl Diphosphate Phosphatase.

We synthesized a series of benzoic acids and phenylphosphonic acids and investigated their effects on the growth of Staphylococcus aureus and Bacillus subtilis. One of the most active compounds, 5-fluoro-2-(3-(octyloxy)benzamido)benzoic acid (7, ED50 ∼0.15 µg mL-1 ) acted synergistically with seven antibiotics known to target bacterial cell-wall biosynthesis (a fractional inhibitory concentration index (FICI) of ∼0.35, on average) but had indifferent effects in combinations with six non-cell-wall biosynthesis inhibitors (average FICI∼1.45). The most active compounds were found to inhibit two enzymes involved in isoprenoid/bacterial cell-wall biosynthesis: undecaprenyl diphosphate synthase (UPPS) and undecaprenyl diphosphate phosphatase (UPPP), but not farnesyl diphosphate synthase, and there were good correlations between bacterial cell growth inhibition, UPPS inhibition, and UPPP inhibition.

Isoprenoid Biosynthesis Inhibitors Targeting Bacterial Cell Growth.

We synthesized potential inhibitors of farnesyl diphosphate synthase (FPPS), undecaprenyl diphosphate synthase (UPPS), or undecaprenyl diphosphate phosphatase (UPPP), and tested them in bacterial cell growth and enzyme inhibition assays. The most active compounds were found to be bisphosphonates with electron-withdrawing aryl-alkyl side chains which inhibited the growth of Gram-negative bacteria (Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa) at ∼1-4 µg mL-1 levels. They were found to be potent inhibitors of FPPS; cell growth was partially "rescued" by the addition of farnesol or overexpression of FPPS, and there was synergistic activity with known isoprenoid biosynthesis pathway inhibitors. Lipophilic hydroxyalkyl phosphonic acids inhibited UPPS and UPPP at micromolar levels; they were active (∼2-6 µg mL-1 ) against Gram-positive but not Gram-negative organisms, and again exhibited synergistic activity with cell wall biosynthesis inhibitors, but only indifferent effects with other inhibitors. The results are of interest because they describe novel inhibitors of FPPS, UPPS, and UPPP with cell growth inhibitory activities as low as ∼1-2 µg mL-1 .

Structure, Function, and Inhibition of Staphylococcus aureus Heptaprenyl Diphosphate Synthase.

We report the first structure of heptaprenyl diphosphate synthase from Staphylococcus aureus (SaHepPPS), together with an investigation of its mechanism of action and inhibition. The protein is involved in the formation of menaquinone, a key electron transporter in many bacteria, including pathogens. SaHepPPS consists of a "catalytic " subunit (SaHepPPS-2) having two "DDXXD" motifs and a "regulatory" subunit (SaHepPPS-1) that lacks these motifs. High concentrations of the substrates, isopentenyl diphosphate and farnesyl diphosphate, inhibit the enzyme, which is also potently inhibited by bisphosphonates. The most active inhibitors (Ki ∼200 nm) were N-alkyl analogues of zoledronate containing ∼C6 alkyl side chains. They were modestly active against S. aureus cell growth, and growth inhibition was partially "rescued" by the addition of menaquinone-7. Because SaHepPPS is essential for S. aureus cell growth, its structure is of interest in the context of the development of menaquinone biosynthesis inhibitors as potential antibiotic leads.

Characterization of potential drug targets farnesyl diphosphate synthase and geranylgeranyl diphosphate synthase in Schistosoma mansoni.

Schistosomiasis affects over 200 million people worldwide, with over 200,000 deaths annually. Currently, praziquantel is the only drug available against schistosomiasis. We report here that Schistosoma mansoni farnesyl diphosphate synthase (SmFPPS) and geranylgeranyl diphosphate synthase (SmGGPPS) are potential drug targets for the treatment of schistosomiasis. We expressed active, recombinant SmFPPS and SmGGPPS for subsequent kinetic characterization and testing against a variety of bisphosphonate inhibitors. Recombinant SmFPPS was found to be a soluble 44.2-kDa protein, while SmGGPPS was a soluble 38.3-kDa protein. Characterization of the substrate utilization of the two enzymes indicates that they have overlapping substrate specificities. Against SmFPPS, several bisphosphonates had 50% inhibitory concentrations (IC50s) in the low micromolar to nanomolar range; these inhibitors had significantly less activity against SmGGPPS. Several lipophilic bisphosphonates were active against ex vivo adult worms, with worm death occurring over 4 to 6 days. These results indicate that FPPS and GGPPS could be of interest in the context of the emerging resistance to praziquantel in schistosomiasis therapy.

Structural characterization of the monolayer-multilayer transition in a pulmonary surfactant model: IR studies of films transferred at continuously varying surface pressures.

The four-component system acyl chain perdeuterated 1,2-dipalmitoylphosphatidylcholine (DPPC)/1,2-dipalmitoylphosphatidylglycerol/ (DPPG)/pulmonary surfactant protein SP-C/cholesterol provides a useful model for in vitro biophysical studies of the reversible monolayer to multilayer transition that occurs during compression <--> expansion cycles in the lung. Monolayer films of this mixture (with chain perdeuterated DPPC-d62) at the air/water interface have been transferred to solid substrates under conditions of continuously varying surface pressure, an approach termed COVASP (continuously varying surface pressures) (Langmuir 2007, 23, 4958). The thermodynamic properties of the Langmuir films have been examined with pressure-area isotherms, while the molecular properties of the film constituents in the transferred films in the monolayer and multilayer phases have been examined with IR spectroscopy. Quantitative intensity measurements of the DPPC-d62, DPPG, and SP-C components in each phase reveal that the DPPG and SP-C constituents are relatively enriched in the multilayer compared with the DPPC-d62, although all three species are present in both phases. Some molecular structure information is available from the surface-pressure-induced variation in IR parameters. The DPPC-d62 exhibits slightly increased conformational order in the multilayer phase as detected from decreases in the CD2 stretching frequencies upon compression, while the lipid phosphate residues become dehydrated, as deduced from increases in the 1245 cm-1 symmetric PO2- stretching frequency. A small increase is observed in the protein amide I frequency; possible interpretations of these changes are presented. The current observations are compared with ideas contained in the "squeeze-out hypothesis" (Handbook of Physiology, The Respiratory System; American Physiological Society Press: Bethesda, MD, 1986; Vol. III, p 247) and in the "liquid crystalline collapse" model (Biophys. J. 2003, 84, 3792). Within the limitation of the current procedures, the data contain elements from both these descriptions of the monolayer transformation. Extensions and possible limitations of the COVASP-IR method are discussed.