title: Development of a one-pot assay for screening and identification of Mur pathway inhibitors in Mycobacterium tuberculosis.
abstract: The cell wall of Mycobacterium tuberculosis (Mtb) consists of peptidoglycan, arabinogalactan and mycolic acids. The cytoplasmic steps in the peptidoglycan biosynthetic pathway, catalyzed by the Mur (A-F) enzymes, involve the synthesis of UDP-n-acetylmuramyl pentapeptide, a key precursor molecule required for the formation of the peptidoglycan monomeric building blocks. Mur enzymes are indispensable for cell integrity and their lack of counterparts in eukaryotes suggests them to be promising Mtb drug targets. However, the caveat is that most of the current assays utilize a single Mur enzyme, thereby identifying inhibitors against only one of the enzymes. Here, we report development of a one-pot assay that reconstructs the entire Mtb Mur pathway in vitro and has the advantage of eliminating the requirement for nucleotide intermediates in the pathway as substrates. The MurA-MurF enzymes were purified and a one-pot assay was developed through optimization of successive coupled enzyme assays using UDP-n-acetylglucosamine as the initial sugar substrate. The assay is biochemically characterized and optimized for high-throughput screening of molecules that could disrupt multiple targets within the pathway. Furthermore, we have validated the assay by performing it to identify D-Cycloserine and furan-based benzene-derived compounds with known Mur ligase inhibition as inhibitors of Mtb MurE and MurF.
authors.affiliation: Department of Biomedical Science, Acharya Narendra Dev College, University of Delhi, New Delhi 110019, India.
authors.forename: Kandasamy
authors.initials: K
authors.lastname: Eniyan
authors.affiliation: Infectious Disease Research Institute, Seattle WA 98102, USA.
authors.forename: Anuradha
authors.initials: A
authors.lastname: Kumar
authors.affiliation: CSIR-IGIB, South Campus, Mathura road, New Delhi 110025, India.
authors.forename: Geetha Vani
authors.initials: GV
authors.lastname: Rayasam
authors.affiliation: National Institute of Chemistry, Hajdrihova 19, 1001 Ljubljana, Slovenia.
authors.forename: Andrej
authors.initials: A
authors.lastname: Perdih
authors.affiliation: Department of Biomedical Science, Acharya Narendra Dev College, University of Delhi, New Delhi 110019, India.
authors.forename: Urmi
authors.initials: U
authors.lastname: Bajpai
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D001554:Benzene / Q000494:pharmacology; D001681:Biological Assay / Q000379:methods*; D002473:Cell Wall / Q000187:drug effects / Q000378:metabolism; D003523:Cycloserine / Q000737:chemistry / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology*; D005663:Furans / Q000737:chemistry / Q000494:pharmacology; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000378:metabolism*; D010453:Peptide Synthases / Q000378:metabolism; D010457:Peptidoglycan / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D014538:Uridine Diphosphate N-Acetylmuramic Acid / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D005663:Furans; D010457:Peptidoglycan; D014538:Uridine Diphosphate N-Acetylmuramic Acid; C002225:UDP-N-acetylmuramic acid pentapeptide; D003523:Cycloserine; D019883:Alkyl and Aryl Transferases; C022566:UDP-N-acetylglucosamine 1-carboxyvinyltransferase; D010453:Peptide Synthases; D001554:Benzene; C039281:furan