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 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; cell growth was partially "rescued" by addition of farnesol or overexpression of FPPS, and there was synergistic activity with known isoprenoid biosynthesis pathway inhibitors. Lipophilic hydroxy-alkyl phosphonic acids inhibited UPPS and UPPP at μM levels; they were active (~2-6 μg/ml) 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 since they describe novel FPPS, UPPS as well as UPPP inhibitors with cell growth inhibition activities as low as ~1-2 μg/ml.

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 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. Since 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.

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

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.

Terpenes and isoprenoids are the most diverse small organic molecules on Earth. Here, we obtained the structures of sesquisabinene and santalene synthases from Santalum album L. in the presence or absence of substrate, substrate-like, or product-like molecules for a total of eight structures. All proteins crystallized in an "open", noncatalytic conformation and, in the case of the FPP structure, three Mg 2+ (MgABC) were bound in the active site region. We found that one of these Mg 2+ (MgB), involved in pocket closure to the catalytically active conformation, interacted with a single innersphere amino acid, a bidentate diphosphate, and a facial arrangement of three water molecules, H 2 O-a,b,c: fac-[Mg(H 2 O) 3 XPP(Asn/Asp)]. Paradoxically, the same arrangement of ligands is found in closed-form, catalytically active "head-to-tail" prenyltransferases, such as farnesyl diphosphate synthase (FPPS), leading to the proposal that in these proteins, pocket closure is due to interaction with outer-sphere ligands (typically, two Asps) with H 2 O-a,b. We also discovered an extensive H-bond network that is present in related cyclases and prenyltransferases: the D-motif. This motif is centered around the totally conserved outer-sphere Asp that is bonded via H 2 O-c to MgB and comprises five amino acids, including a very highly conserved residue in the cyclases, an Arg, and a highly conserved residue in the αHT proteins, a Gln, and we propose that this motif may be involved in catalysis per se. Finally, using bioinformatics and mutagenesis, we show that many of the most highly conserved residues in these and other cyclases are outside the active site region and are in the noncatalytic β-domain, reflecting their evolutionary origin from βγ domain proteins, while a highly conserved α-domain Arg forms a salt bridge with a Glu that serves to stabilize the catalytically active αβ-domain structure.