Targeting an Essential GTPase Obg for the Development of Broad-Spectrum Antibiotics

• Published in: PloS one 2016-02, Vol.11 (2), p.e0148222-e0148222
• Main Authors: Bonventre, Josephine A, Zielke, Ryszard A, Korotkov, Konstantin V, Sikora, Aleksandra E
• Format: Article
• Language: English
• Subjects: Analogs; Anti-Bacterial Agents - pharmacology; Antibiotics; Assaying; Bacteria; Bacterial infections; Bacterial Proteins - antagonists & inhibitors; Biochemistry; Biology and Life Sciences; Biosynthesis; Chelating Agents - pharmacology; Colorimetry; Drug Evaluation, Preclinical; Drug resistance; Drug Resistance, Multiple - drug effects; E coli; Enzyme Inhibitors - pharmacology; Escherichia coli; Fluorescence; Fourier transforms; Genetic aspects; Gonorrhea; GTP; GTP-Binding Proteins - antagonists & inhibitors; GTPases; Guanine; Guanosine triphosphatases; Guanosine triphosphate; Guanosinetriphosphatase; High-throughput screening; Inhibitors; Klebsiella; Lead compounds; Medical screening; Medicine and Health Sciences; Methicillin; Molecular Targeted Therapy; Multidrug resistance; Neisseria gonorrhoeae - drug effects; Neisseria gonorrhoeae - enzymology; Neonates; Nucleotide analogs; Pharmaceutical sciences; Pharmacy; Physical Sciences; Physiological aspects; Pneumonia; Proteins; Research and Analysis Methods; Sexually transmitted diseases; Solvents - pharmacology; Spectrum analysis; Staphylococcus aureus; Staphylococcus infections; STD; Vibrio harveyi
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0148222

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.