Rapid Countermeasure Discovery against Francisella tularensis Based on a Metabolic Network Reconstruction Sidhartha Chaudhury 1 , Mohamed Diwan M. Abdulhameed 1 , Narender Singh 1 , Gregory J. Tawa 1 , Patrik M. D’haeseleer 2 , Adam T. Zemla 2 , Ali Navid 2 , Carol E. Zhou 2 , Matthew C. Franklin 3 , Jonah Cheung 3 , Michael J. Rudolph 3 , James Love 3 , John F. Graf 4 , David A. Rozak 5 , Jennifer L. Dankmeyer 5 , Kei Amemiya 5 , Simon Daefler 6 , Anders Wallqvist 1 * 1 Department of Defense Biotechnology High Performance Computing Software Applications Institute, Telemedicine and Advanced Technology Research Center, U.S. Army Medical Research and Materiel Command, Fort Detrick, Maryland, United States of America, 2 Pathogen Bioinformatics, Lawrence Livermore National Laboratory, Livermore, California, United States of America, 3 New York Structural Biology Center, New York, New York, United States of America, 4 Computational Biology and Biostatistics Laboratory, Diagnostics and Biomedical Technologies, GE Global Research, General Electric Company, Niskayuna, New York, United States of America, 5 Bacteriology Division, U.S. Army Medical Research Institute for Infectious Diseases, Fort Detrick, Maryland, United States of America, 6 Mount Sinai School of Medicine, New York, New York, United States of America 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 mM, 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. Citation: Chaudhury S, Abdulhameed MDM, Singh N, Tawa GJ, D’haeseleer PM, et al. (2013) Rapid Countermeasure Discovery against Francisella tularensis Based on a Metabolic Network Reconstruction. PLoS ONE 8(5): e63369. doi:10.1371/journal.pone.0063369 Editor: Peter Csermely, Semmelweis University, Hungary Received February 12, 2013; Accepted March 30, 2013; Published May 21, 2013 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: 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. 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. * E-mail: awallqvist@bhsai.org Introduction The threat of emergent, highly infectious, drug-resistant pathogens, whether through genetic engineering of bioterrorism agents or evolution and adaptation of naturally occurring strains, demands capabilities to rapidly screen and identify potential novel antimicrobials for a given pathogen. Francisella tularensis, the causative agent of tularemia, is classified by the Centers for Disease Control and Prevention (CDC) as a category A select agent, and its role as a biological weapon is known to have been investigated in the United States, the former Soviet Union, and Japan [1]. Strains of F. tularensis [2] are among the most virulent known, requiring only 10 organisms to infect a human intrave- nously and ,10–50 organisms through inhalation [3]. Currently, there is no Food and Drug Administration (FDA)-approved small- molecule inhibitor that specifically targets this pathogen, and treatment of tularemia is typically limited to a few antibiotics, such as fluoroquinolones [1]. Although several experimental live- attenuated vaccines [4–6] are under development, no vaccine for F. tularensis is currently available. Traditional antibiotic development is characterized by long development times and high costs. For example, a recent review [7] outlining antibacterial discovery efforts at the pharmaceutical company GlaxoSmithKline revealed that 67 high-throughput screening campaigns over the course of six years, at a cost of PLOS ONE | www.plosone.org 1 May 2013 | Volume 8 | Issue 5 | e63369