title: Rapid countermeasure discovery against Francisella tularensis based on a metabolic network reconstruction.
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.
authors.affiliation: 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.
authors.forename: Sidhartha
authors.initials: S
authors.lastname: Chaudhury
authors.forename: Mohamed Diwan M
authors.initials: MD
authors.lastname: Abdulhameed
authors.forename: Narender
authors.initials: N
authors.lastname: Singh
authors.forename: Gregory J
authors.initials: GJ
authors.lastname: Tawa
authors.forename: Patrik M
authors.initials: PM
authors.lastname: D'haeseleer
authors.forename: Adam T
authors.initials: AT
authors.lastname: Zemla
authors.forename: Ali
authors.initials: A
authors.lastname: Navid
authors.forename: Carol E
authors.initials: CE
authors.lastname: Zhou
authors.forename: Matthew C
authors.initials: MC
authors.lastname: Franklin
authors.forename: Jonah
authors.initials: J
authors.lastname: Cheung
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Rudolph
authors.forename: James
authors.initials: J
authors.lastname: Love
authors.forename: John F
authors.initials: JF
authors.lastname: Graf
authors.forename: David A
authors.initials: DA
authors.lastname: Rozak
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Dankmeyer
authors.forename: Kei
authors.initials: K
authors.lastname: Amemiya
authors.forename: Simon
authors.initials: S
authors.lastname: Daefler
authors.forename: Anders
authors.initials: A
authors.lastname: Wallqvist
mesh_terms: D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000737:chemistry; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D055808:Drug Discovery; D004353:Drug Evaluation, Preclinical; D005604:Francisella tularensis / Q000187:drug effects* / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D053858:Metabolic Networks and Pathways / Q000187:drug effects*; D008826:Microbial Sensitivity Tests; D050296:Microbial Viability / Q000187:drug effects
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins