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. 2013 May 21;8(5):e63369.
doi: 10.1371/journal.pone.0063369. Print 2013.

Rapid Countermeasure Discovery Against Francisella Tularensis Based on a Metabolic Network Reconstruction

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Free PMC article

Rapid Countermeasure Discovery Against Francisella Tularensis Based on a Metabolic Network Reconstruction

Sidhartha Chaudhury et al. PLoS One. .
Free PMC article

Abstract

In the future, we may be faced with the need to provide treatment for an emergent biological threat against which existing vaccines and drugs have limited efficacy or availability. To prepare for this eventuality, our objective was to use a metabolic network-based approach to rapidly identify potential drug targets and prospectively screen and validate novel small-molecule antimicrobials. Our target organism was the fully virulent Francisella tularensis subspecies tularensis Schu S4 strain, a highly infectious intracellular pathogen that is the causative agent of tularemia and is classified as a category A biological agent by the Centers for Disease Control and Prevention. We proceeded with a staggered computational and experimental workflow that used a strain-specific metabolic network model, homology modeling and X-ray crystallography of protein targets, and ligand- and structure-based drug design. Selected compounds were subsequently filtered based on physiological-based pharmacokinetic modeling, and we selected a final set of 40 compounds for experimental validation of antimicrobial activity. We began screening these compounds in whole bacterial cell-based assays in biosafety level 3 facilities in the 20th week of the study and completed the screens within 12 weeks. Six compounds showed significant growth inhibition of F. tularensis, and we determined their respective minimum inhibitory concentrations and mammalian cell cytotoxicities. The most promising compound had a low molecular weight, was non-toxic, and abolished bacterial growth at 13 µM, with putative activity against pantetheine-phosphate adenylyltransferase, an enzyme involved in the biosynthesis of coenzyme A, encoded by gene coaD. The novel antimicrobial compounds identified in this study serve as starting points for lead optimization, animal testing, and drug development against tularemia. Our integrated in silico/in vitro approach had an overall 15% success rate in terms of active versus tested compounds over an elapsed time period of 32 weeks, from pathogen strain identification to selection and validation of novel antimicrobial compounds.

Conflict of interest statement

Competing Interests: The authors would like to disclose the following interest. JFG is employed by the General Electric Company. There are no patents, products in development or marketed products to declare. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

Figures

Figure 1
Figure 1. Summary of workflow, timelines, and outcomes.
An overview of the major phases of the drug discovery pipelines from target identification, structure determination, in silico screening, in silico pharmacological characterization and experimental validation. The overall timeline and the number of targets and inhibitors at each phase are shown as well.
Figure 2
Figure 2. Crystal structures determined in this work.
Crystal structures for aroG (A), nadC (B), and lpcA (C) color coded according to their secondary structure elements, with the active site region used in structure-based virtual screening shown as a gray surface.
Figure 3
Figure 3. Distribution of calculated pharmacokinetic parameters.
Predicted PK parameters for each of the set of 190 candidate compounds are shown. Each column represents one compound, and compounds are grouped according to their target. PK parameter values are represented by a color spectrum ranging from “undesirable” (red) to “desirable” limits (green), defined by the respective 10 and 90 percentile values among the set of Federal Drug Administration-approved drugs for each parameter. For volume of distribution (Vd), the limits were <0.25 l/kg (red) and >3.30 l/kg (green). For clearance (CL), the limits were >4.5 ml·min−1·kg−1 (red) and <1.30 ml·min−1·kg−1 (green). For mean residence time (MRT), the limits were <1.0 h (red) and >14.0 h (green). Finally, for half-life (T ½), the limits were <1.5 h (red) and >13.0 h (green). The 40 compounds selected for experimental testing are highlighted (∗). The putative protein targets are shown in Table 2.
Figure 4
Figure 4. Potency and toxicity of identified antimicrobials against F. tularensis.
Compound ID, chemical structure, putative target, minimum inhibitory concentration (MIC), and toxicity for each of the active antimicrobials identified in this study.

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Grant support

This work was supported by the Defense Threat Reduction Agency through projects TMTI0004.09.BH.T, HDTRA1-08-C-0052, TMTI10049.09.RD.T, and W911SR-11-C-0014. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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