A Colorimetric Assay to Identify and Characterize Bacterial Primase Inhibitors

Allan H. Pang; Oleg V. Tsodikov

doi:10.1007/978-1-0716-2855-3_15

A Colorimetric Assay to Identify and Characterize Bacterial Primase Inhibitors

Research output: Chapter in Book/Report/Conference proceeding › Chapter › peer-review

Abstract

Bacterial DNA primase DnaG is an attractive target for antibiotic discovery since it plays an essential role in DNA replication. Over the last 10 years, we have developed and optimized a robust colorimetric assay that enabled us to identify and validate inhibitors of bacterial primases. Here, we provide a detailed protocol for this colorimetric assay for DnaG from three different pathogenic bacteria (Mycobacterium tuberculosis, Bacillus anthracis, and Staphylococcus aureus), which can be performed in high throughput. We also describe secondary assays to characterize hits from this high-throughput screening assay. These assays are designed to identify inhibitors of the coupled enzyme inorganic pyrophosphatase, DNA binding agents, and elucidate the mode of inhibition of primase inhibitors.

Original language	English
Title of host publication	Methods in Molecular Biology
Pages	283-301
Number of pages	19
DOIs	https://doi.org/10.1007/978-1-0716-2855-3_15
State	Published - 2023

Publication series

Name	Methods in Molecular Biology
Volume	2601
ISSN (Print)	1064-3745
ISSN (Electronic)	1940-6029

Bibliographical note

Publisher Copyright:
© 2023, The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.

Keywords

DNA intercalation
DNA replication
Drug discovery
High-throughput assay
Inorganic pyrophosphatase
Mode of inhibition

ASJC Scopus subject areas

Molecular Biology
Genetics

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

Access to Document

10.1007/978-1-0716-2855-3_15

Cite this

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abstract = "Bacterial DNA primase DnaG is an attractive target for antibiotic discovery since it plays an essential role in DNA replication. Over the last 10 years, we have developed and optimized a robust colorimetric assay that enabled us to identify and validate inhibitors of bacterial primases. Here, we provide a detailed protocol for this colorimetric assay for DnaG from three different pathogenic bacteria (Mycobacterium tuberculosis, Bacillus anthracis, and Staphylococcus aureus), which can be performed in high throughput. We also describe secondary assays to characterize hits from this high-throughput screening assay. These assays are designed to identify inhibitors of the coupled enzyme inorganic pyrophosphatase, DNA binding agents, and elucidate the mode of inhibition of primase inhibitors.",

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AU - Tsodikov, Oleg V.

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KW - DNA replication

KW - Drug discovery

KW - High-throughput assay

KW - Inorganic pyrophosphatase

KW - Mode of inhibition

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A Colorimetric Assay to Identify and Characterize Bacterial Primase Inhibitors

Abstract

Publication series

Bibliographical note

Keywords

ASJC Scopus subject areas

UN SDGs

Access to Document

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