title: Mycobacterial dihydrofolate reductase inhibitors identified using chemogenomic methods and in vitro validation.
abstract: The lack of success in target-based screening approaches to the discovery of antibacterial agents has led to reemergence of phenotypic screening as a successful approach of identifying bioactive, antibacterial compounds. A challenge though with this route is then to identify the molecular target(s) and mechanism of action of the hits. This target identification, or deorphanization step, is often essential in further optimization and validation studies. Direct experimental identification of the molecular target of a screening hit is often complex, precisely because the properties and specificity of the hit are not yet optimized against that target, and so many false positives are often obtained. An alternative is to use computational, predictive, approaches to hypothesize a mechanism of action, which can then be validated in a more directed and efficient manner. Specifically here we present experimental validation of an in silico prediction from a large-scale screen performed against Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. The two potent anti-tubercular compounds studied in this case, belonging to the tetrahydro-1,3,5-triazin-2-amine (THT) family, were predicted and confirmed to be an inhibitor of dihydrofolate reductase (DHFR), a known essential Mtb gene, and already clinically validated as a drug target. Given the large number of similar screening data sets shared amongst the community, this in vitro validation of these target predictions gives weight to computational approaches to establish the mechanism of action (MoA) of novel screening hit.
authors: European Molecular Biology Laboratory, European Bioinformatics Institute (EMBL-EBI), Wellcome Trust Genome Campus, Hinxton, Cambridge, United Kingdom.
authors: Grace
authors: G
authors: Mugumbate
authors: Institute of Microbiology and Infection (IMI), School of Biosciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom.
authors: Katherine A
authors: KA
authors: Abrahams
authors: Institute of Microbiology and Infection (IMI), School of Biosciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom.
authors: Jonathan A G
authors: JA
authors: Cox
authors: European Molecular Biology Laboratory, European Bioinformatics Institute (EMBL-EBI), Wellcome Trust Genome Campus, Hinxton, Cambridge, United Kingdom.
authors: George
authors: G
authors: Papadatos
authors: European Molecular Biology Laboratory, European Bioinformatics Institute (EMBL-EBI), Wellcome Trust Genome Campus, Hinxton, Cambridge, United Kingdom.
authors: Gerard
authors: G
authors: van Westen
authors: Diseases of the Developing World, GlaxoSmithKline, Severo Ochoa 2, 28760 Tres Cantos, Madrid, Spain.
authors: Joël
authors: J
authors: Lelièvre
authors: Institute of Microbiology and Infection (IMI), School of Biosciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom.
authors: Szymon T
authors: ST
authors: Calus
authors: Institute of Microbiology and Infection (IMI), School of Biosciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom.
authors: Nicholas J
authors: NJ
authors: Loman
authors: Diseases of the Developing World, GlaxoSmithKline, Severo Ochoa 2, 28760 Tres Cantos, Madrid, Spain.
authors: Lluis
authors: L
authors: Ballell
authors: Diseases of the Developing World, GlaxoSmithKline, Severo Ochoa 2, 28760 Tres Cantos, Madrid, Spain.
authors: David
authors: D
authors: Barros
authors: European Molecular Biology Laboratory, European Bioinformatics Institute (EMBL-EBI), Wellcome Trust Genome Campus, Hinxton, Cambridge, United Kingdom.
authors: John P
authors: JP
authors: Overington
authors: Institute of Microbiology and Infection (IMI), School of Biosciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom.
authors: Gurdyal S
authors: GS
authors: Besra
mesh_terms: D003198:Computer Simulation; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005493:Folic Acid Antagonists / Q000032:analysis* / Q000378:metabolism / Q000494:pharmacology*; D023281:Genomics; D006801:Humans; D008024:Ligands; D062105:Molecular Docking Simulation; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D010641:Phenotype; D011487:Protein Conformation; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000378:metabolism*
chemical_list: D005493:Folic Acid Antagonists; D008024:Ligands; D013762:Tetrahydrofolate Dehydrogenase