Characterization and Inhibition of 1-Deoxy-d-Xylulose 5-Phosphate Reductoisomerase: A Promising Drug Target in Acinetobacter baumannii and Klebsiella pneumoniae.

The ESKAPE pathogens comprise a group of multidrug-resistant bacteria that are the leading cause of nosocomial infections worldwide. The prevalence of antibiotic resistant strains and the relative ease by which bacteria acquire resistance genes highlight the continual need for the development of novel antibiotics against new drug targets. The methylerythritol phosphate (MEP) pathway is an attractive target for the development of new antibiotics. The MEP pathway governs the synthesis of isoprenoids, which are key lipid precursors for vital cell components such as ubiquinone and bacterial hopanoids. Additionally, the MEP pathway is entirely distinct from the corresponding mammalian pathway, the mevalonic acid (MVA) pathway, making the first committed enzyme of the MEP pathway, 1-deoxy-d-xylulose 5-phosphate reductoisomerase (IspC), an attractive target for antibiotic development. To facilitate drug development against two of the ESKAPE pathogens, Acinetobacter baumannii and Klebsiella pneumoniae, we cloned, expressed, purified, and characterized IspC from these two Gram-negative bacteria. Enzyme inhibition assays using IspC from these two pathogens, and compounds fosmidomycin and FR900098, indicate IC50 values ranging from 19.5-45.5 nM. Antimicrobial susceptibility tests with these inhibitors reveal that A. baumannii is susceptible to FR900098, whereas K. pneumoniae is susceptible to both compounds. Finally, to facilitate structure-based drug design of inhibitors targeting A. baumannii IspC, we determined the 2.5 Å crystal structure of IspC from A. baumannii in complex with inhibitor FR900098, and cofactors NADPH and magnesium.

Inhibition of the Yersinia pestis Methylerythritol Phosphate Pathway of Isoprenoid Biosynthesis by α-Phenyl-Substituted Reverse Fosmidomycin Analogues.

Fosmidomycin inhibits IspC (1-deoxy-d-xylulose 5-phosphate reductoisomerase), the first committed enzyme in the methylerythritol phosphate (MEP) pathway of isoprenoid biosynthesis. The MEP pathway of isoprenoid biosynthesis is essential to the causative agent of the plague, Yersinia pestis, and is entirely distinct from the corresponding mammalian pathway. To further drug development, we established structure-activity relationships of fosmidomycin analogues by assessing a suite of 17 α-phenyl-substituted reverse derivatives of fosmidomycin against Y. pestis IspC. Several of these compounds showed increased potency over fosmidomycin with IC50 values in the nanomolar range. Additionally, we performed antimicrobial susceptibility testing with Y. pestis A1122 (YpA1122). The bacteria were susceptible to several compounds with minimal inhibitory concentration (MIC) values ranging from 128 to 512 µg/mL; a correlation between the IC50 and MIC values was observed.

Discovery of novel inhibitors of multidrug-resistant Acinetobacter baumannii.

The recent emergence of multidrug-resistant Acinetobacter baumannii strains and the non-efficacy of currently available antibiotics against such infections have led to an urgent need for the development of novel antibacterials. In an effort to address this problem, we have identified three novel inhibitors, namely, D5, D12 and D6 using in silico screening with a homology model of the outer membrane protein W2 (OmpW2) from A. baumannii, as the proposed new drug target. OmpW is an eight-stranded ß-barrel protein involved in the transport of hydrophobic molecules across the outer membrane and maintenance of homeostasis under cellular stress. The antimicrobial activities of compounds D5, D12 and D6 were evaluated against a panel of clinical isolates of A. baumannii strains. These compounds inhibited the growth of the strains with minimum inhibitory concentration (MIC) ranges of 1-32µg/mL. Time-kill kinetic studies with the highly virulent and multidrug-resistant strain, A. baumannii 5075, indicated that D6 exhibited the highest bactericidal activity asa≥3log10 CFU/mL (99.9%) reduction in colony count from the initial inoculum was observed after 30min incubation. D5 and D12 reduced at least 1log10 CFU/mL (90%) of the initial inoculum after 24h. In conclusion, these three lead inhibitors have provided two distinct chemical scaffolds for further analog design and optimizations, using chemical synthesis, to develop more potent inhibitors of the pathogen.

A conformational change in the peripheral anionic site of Torpedo californica acetylcholinesterase induced by a bis-imidazolium oxime.

As part of ongoing efforts to design improved nerve agent antidotes, two X-ray crystal structures of Torpedo californica acetylcholinesterase (TcAChE) bound to the bis-pyridinium oxime, Ortho-7, or its experimental bis-imidazolium analogue, 2BIM-7, were determined. Bis-oximes contain two oxime groups connected by a hydrophobic linker. One oxime group of Ortho-7 binds at the entrance to the active-site gorge near Trp279, and the second binds at the bottom near Trp84 and Phe330. In the Ortho-7-TcAChE complex the oxime at the bottom of the gorge was directed towards the nucleophilic Ser200. In contrast, the oxime group of 2BIM-7 was rotated away from Ser200 and the oxime at the entrance induced a significant conformational change in the peripheral anionic site (PAS) residue Trp279. The conformational change alters the surface of the PAS and positions the imidazolium oxime of 2BIM-7 further from Ser200. The relatively weaker binding and poorer reactivation of VX-inhibited, tabun-inhibited or sarin-inhibited human acetylcholinesterase by 2BIM-7 compared with Ortho-7 may in part be owing to the unproductively bound states caught in crystallo. Overall, the reactivation efficiency of 2BIM-7 was comparable to that of 2-pyridine aldoxime methyl chloride (2-PAM), but unlike 2-PAM the bis-imidazolium oxime lacks a fixed charge, which may affect its membrane permeability.

Evaluation and computational characterization of the facilitated transport of Glc carbon C-1 oxime reactivators across a blood brain barrier model.

We are evaluating a facilitative transport strategy to move oximes across the blood brain barrier (BBB) to reactivate inhibited brain acetylcholinesterase (AChE). We selected glucose (Glc) transporters (GLUT) for this purpose as these transporters are highly represented in the BBB. Glc conjugates have successfully moved drugs across the BBB and previous work has shown that Glc-oximes (sugar-oximes, SOxs) can reduce the organophosphonate induced hypothermia response. We previously evaluated the reactivation potential of Glc carbon C-1 SOxs. Here we report the reactivation parameters for VX- and GB-inhibited human (Hu) AChE of the best SOx (13c) and our findings that the kinetics are similar to those of the parent oxime. Although crystals of Torpedo californica AChE were produced, neither soaked or co-crystallized experiments were successful at concentrations below 20mM 13c, and higher concentrations cracked the crystals. 13c was non-toxic to neuroblastoma and kidney cell lines at 12-18 mM, allowing high concentrations to be used in a BBB kidney cell model. The transfer of 13c from the donor side was asymmetric with the greatest loss of 13c from the apical- or luminal-treated side. There was no apparent transfer from the basolateral side. The 13cP(app) results indicate a 'low' transport efficiency; however, mass accounting revealed only a 20% recovery from the apical dose in which high concentrations were found in the cell lysate fraction. Molecular modeling of 13c through the GLUT-1 channel demonstrated that transport of 13c was more restricted than Glc. Selected sites were compared and the 13c binding energies were greater than two times those of Glc.

Aerosolized delivery of oxime MMB-4 in combination with atropine sulfate protects against soman exposure in guinea pigs.

We evaluated the efficacy of aerosolized acetylcholinesterase (AChE) reactivator oxime MMB-4 in combination with the anticholinergic atropine sulfate for protection against respiratory toxicity and lung injury following microinstillation inhalation exposure to nerve agent soman (GD) in guinea pigs. Anesthetized animals were exposed to GD (841 mg/m(3), 1.2 LCt(50)) and treated with endotracheally aerosolized MMB-4 (50 µmol/kg) plus atropine sulfate (0.25 mg/kg) at 30 sec post-exposure. Treatment with MMB-4 plus atropine increased survival to 100% compared to 38% in animals exposed to GD. Decreases in the pulse rate and blood O(2) saturation following exposure to GD returned to normal levels in the treatment group. The body-weight loss and lung edema was significantly reduced in the treatment group. Similarly, bronchoalveolar cell death was significantly reduced in the treatment group while GD-induced increase in total cell count was decreased consistently but was not significant. GD-induced increase in bronchoalveolar protein was diminished after treatment with MMB-4 plus atropine. Bronchoalveolar lavage AChE and BChE activity were significantly increased in animals treated with MMB-4 plus atropine at 24 h. Lung and diaphragm tissue also showed a significant increase in AChE activity in the treatment group. Treatment with MMB-4 plus atropine sulfate normalized various respiratory dynamics parameters including respiratory frequency, tidal volume, peak inspiratory and expiratory flow, time of inspiration and expiration, enhanced pause and pause post-exposure to GD. Collectively, these results suggest that aerosolization of MMB-4 plus atropine increased survival, decreased respiratory toxicity and lung injury following GD inhalation exposure.

Hydrolysis potential of recombinant human skin and kidney prolidase against diisopropylfluorophosphate and sarin by in vitro analysis.

Human prolidase (PROL), which has structural homology to bacterial organophosphate acid anhydrolase that hydrolyze organophosphates and nerve agents has been proposed recently as a potential catalytic bioscavenger. To develop PROL as a catalytic bioscavenger, we evaluated the in vitro hydrolysis efficiency of purified recombinant human PROL against organophosphates and nerve agents. Human liver PROL was purified by chromatographic procedures, whereas recombinant human skin and kidney PROL was expressed in Trichoplusia ni larvae, affinity purified and analyzed by gel electrophoresis. The catalytic efficiency of PROL against diisopropylfluorophosphate (DFP) and nerve agents was evaluated by acetylcholinesterase back-titration assay. Partially purified human liver PROL hydrolyzed DFP and various nerve agents, which was abolished by specific PROL inhibitor showing the specificity of hydrolysis. Both the recombinant human skin and kidney PROL expressed in T. ni larvae showed ∼99% purity and efficiently hydrolyzed DFP and sarin. In contrast to human liver PROL, both skin and kidney PROL showed significantly low hydrolyzing potential against nerve agents soman, tabun and VX. In conclusion, compared to human liver PROL, recombinant human skin and kidney PROL hydrolyze only DFP and sarin showing the substrate specificity of PROL from various tissue sources.

In vitro efficacy of paraoxonase 1 from multiple sources against various organophosphates.

Paraoxonase 1 (PON1) has been described as a potential catalytic bioscavenger due to its ability to hydrolyze organophosphate (OP) insecticides and nerve agents. In vitro catalytic efficiency of purified human and rabbit serum PON1 against different OP substrates was compared to human recombinant PON1, expressed in Trichoplusia ni larvae. Highly purified human and rabbit serum PON1s were prepared by multiple chromatography methods. Purified enzymes showed higher catalytic activity with the substrate p-nitrophenyl acetate compared to diethyl paraoxon. The hydrolyzing potential of PON1s against multiple OPs was evaluated by using an in vitro acetylcholinesterase back-titration assay. Significant differences in the catalytic efficiency of all the three PON1s with regard to various OP substrates were observed. Purified PON1s showed higher catalytic activity towards diisopropylfluorophosphate followed by diethylparaoxon compared to dimethyl paraoxon. Heat inactivation or incubation of PON1 with specific inhibitor resulted in complete loss of the enzyme catalytic activity indicating that OP hydrolysis was intrinsic to PON1. In conclusion, purified PON1s from multiple sources show significant differences in the catalytic activity against several OP substrates. These results underscore the importance of systematic analysis of candidate PON1 molecules for developing as an effective catalytic bioscavenger against toxic OPs and chemical warfare nerve agents.

Efficient hydrolysis of the chemical warfare nerve agent tabun by recombinant and purified human and rabbit serum paraoxonase 1.

Paraoxonase 1 (PON1) has been described as an efficient catalytic bioscavenger due to its ability to hydrolyze organophosphates (OPs) and chemical warfare nerve agents (CWNAs). It is the future most promising candidate as prophylactic medical countermeasure against highly toxic OPs and CWNAs. Most of the studies conducted so far have been focused on the hydrolyzing potential of PON1 against nerve agents, sarin, soman, and VX. Here, we investigated the hydrolysis of tabun by PON1 with the objective of comparing the hydrolysis potential of human and rabbit serum purified and recombinant human PON1. The hydrolysis potential of PON1 against tabun, sarin, and soman was evaluated by using an acetylcholinesterase (AChE) back-titration Ellman method. Efficient hydrolysis of tabun (100 nM) was observed with ∼25-40 mU of PON1, while higher concentration (80-250 mU) of the enzyme was required for the complete hydrolysis of sarin (11 nM) and soman (3 nM). Our data indicate that tabun hydrolysis with PON1 was ∼30-60 times and ∼200-260 times more efficient than that with sarin and soman, respectively. Moreover, the catalytic activity of PON1 varies from source to source, which also reflects their efficiency of hydrolyzing different types of nerve agents. Thus, efficient hydrolysis of tabun by PON1 suggests its promising potential as a prophylactic treatment against tabun exposure.