title: Targeting an Essential GTPase Obg for the Development of Broad-Spectrum Antibiotics.
abstract: A promising new drug target for the development of novel broad-spectrum antibiotics is the highly conserved small GTPase Obg (YhbZ, CgtA), a protein essential for the survival of all bacteria including Neisseria gonorrhoeae (GC). GC is the agent of gonorrhea, a prevalent sexually transmitted disease resulting in serious consequences on reproductive and neonatal health. A preventive anti-gonorrhea vaccine does not exist, and options for effective antibiotic treatments are increasingly limited. To address the dire need for alternative antimicrobial strategies, we have designed and optimized a 384-well GTPase assay to identify inhibitors of Obg using as a model Obg protein from GC, ObgGC. The assay was validated with a pilot screen of 40,000 compounds and achieved an average Z' value of 0.58 ± 0.02, which suggests a robust assay amenable to high-throughput screening. We developed secondary assessments for identified lead compounds that utilize the interaction between ObgGC and fluorescent guanine nucleotide analogs, mant-GTP and mant-GDP, and an ObgGC variant with multiple alterations in the G-domains that prevent nucleotide binding. To evaluate the broad-spectrum potential of ObgGC inhibitors, Obg proteins of Klebsiella pneumoniae and methicillin-resistant Staphylococcus aureus were assessed using the colorimetric and fluorescence-based activity assays. These approaches can be useful in identifying broad-spectrum Obg inhibitors and advancing the therapeutic battle against multidrug resistant bacteria.
authors: Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, OR, 97330, United States of America.
authors: Josephine A
authors: JA
authors: Bonventre
authors: Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, OR, 97330, United States of America.
authors: Ryszard A
authors: RA
authors: Zielke
authors: Department of Molecular and Cellular Biochemistry, and Center for Structural Biology, University of Kentucky, Lexington, KY, 40536, United States of America.
authors: Konstantin V
authors: KV
authors: Korotkov
authors: Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, OR, 97330, United States of America.
authors: Aleksandra E
authors: AE
authors: Sikora
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors*; D002614:Chelating Agents / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical; D018432:Drug Resistance, Multiple / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D019204:GTP-Binding Proteins / Q000037:antagonists & inhibitors*; D058990:Molecular Targeted Therapy; D009344:Neisseria gonorrhoeae / Q000187:drug effects / Q000201:enzymology; D012997:Solvents / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D002614:Chelating Agents; D004791:Enzyme Inhibitors; C090542:Obg GTP-binding protein, Bacteria; D012997:Solvents; D019204:GTP-Binding Proteins