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.

Document Type

Article

Publication Date

2-5-2016

Notes/Citation Information

Published in PLOS ONE, v. 11, no. 2, e0148222, p. 1-18.

© 2016 Bonventre et al.

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Digital Object Identifier (DOI)

https://doi.org/10.1371/journal.pone.0148222

Funding Information

Funding for this work was provided to AES by grant R01-AI117235 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health and by a pilot grant provided by the College of Pharmacy (OSU). Research reported in this publication was partially supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103486 to KVK.

journal.pone.0148222.s001.DOCX (7607 kB)
S1 Fig. Schematic outline of ObgGC architecture with introduced mutations.

journal.pone.0148222.s002.DOCX (30 kB)
S2 Fig. Evaluation of inhibitory potential of GTPase EngA inhibitor, Garcinol, and potential lead compounds identified in a pilot ObgGC screen, A & B, using the Biomol® Green assay.

journal.pone.0148222.s003.DOCX (55 kB)
S3 Fig. Kinetic characterization of ObgGC.

journal.pone.0148222.s004.DOCX (17 kB)
S1 Table. Timeline of the ObgGC HTS procedures.