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Affinity Selection-Mass Spectrometry Identifies a Novel Antibacterial RNA Polymerase Inhibitor.

ABSTRACT: The growing prevalence of drug resistant bacteria is a significant global threat to human health. The antibacterial drug rifampin, which functions by inhibiting bacterial RNA polymerase (RNAP), is an important part of the antibacterial armamentarium. Here, in order to identify novel inhibitors of bacterial RNAP, we used affinity-selection mass spectrometry to screen a chemical library for compounds that bind to Escherichia coli RNAP. We identified a novel small molecule, MRL-436, that binds to RNAP, inhibits RNAP, and exhibits antibacterial activity. MRL-436 binds to RNAP through a binding site that differs from the rifampin binding site, inhibits rifampin-resistant RNAP derivatives, and exhibits antibacterial activity against rifampin-resistant strains. Isolation of mutants resistant to the antibacterial activity of MRL-436 yields a missense mutation in codon 622 of the rpoC gene encoding the RNAP ?' subunit or a null mutation in the rpoZ gene encoding the RNAP ? subunit, confirming that RNAP is the functional cellular target for the antibacterial activity of MRL-436, and indicating that RNAP ?' subunit residue 622 and the RNAP ? subunit are required for the antibacterial activity of MRL-436. Similarity between the resistance determinant for MRL-436 and the resistance determinant for the cellular alarmone ppGpp suggests a possible similarity in binding site and/or induced conformational state for MRL-436 and ppGpp.

SUBMITTER: Walker SS

PROVIDER: S-EPMC5800494 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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Affinity Selection-Mass Spectrometry Identifies a Novel Antibacterial RNA Polymerase Inhibitor.

Walker Scott S SS Degen David D Nickbarg Elliott E Carr Donna D Soriano Aileen A Mandal Mihir M Painter Ronald E RE Sheth Payal P Xiao Li L Sher Xinwei X Murgolo Nicholas N Su Jing J Olsen David B DB Ebright Richard H RH Young Katherine K

ACS chemical biology 20170331 5

The growing prevalence of drug resistant bacteria is a significant global threat to human health. The antibacterial drug rifampin, which functions by inhibiting bacterial RNA polymerase (RNAP), is an important part of the antibacterial armamentarium. Here, in order to identify novel inhibitors of bacterial RNAP, we used affinity-selection mass spectrometry to screen a chemical library for compounds that bind to Escherichia coli RNAP. We identified a novel small molecule, MRL-436, that binds to R ...[more]

PMID: 28323406

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Project description:The primary objective of early drug development is to identify hits and leads for a target of interest. To achieve this aim, rapid, and reliable screening techniques for a huge number of compounds are needed. Mass spectrometry based binding assays (MS Binding Assays) represent a well-established technique for library screening based on competitive binding experiments revealing active sublibraries due to reduced binding of a reporter ligand and following hit identification for active libraries by deconvolution in further competitive binding experiments. In the present study, we combined the concepts of MS Binding Assays and affinity selection mass spectrometry (ASMS) to improve the efficiency of the hit identification step. In that case, only a single competitive binding experiment is performed that is in the first step analyzed for reduced binding of the reporter ligand and-only if a sublibrary is active-additionally for specific binding of individual library components. Subsequently, affinities of identified hits as well as activities of reduced sublibraries (i.e., all sublibrary components without hit) are assessed in additional competitive binding experiments. We exemplified this screening concept for the identification of ligands addressing the most widespread GABA transporter subtype in the brain (GAT1) studying in the beginning a library composed of 128 and further on a library of 1,280 well-characterized GAT1 inhibitors, drug substances, and pharmacological tool compounds. Determination of sublibraries' activities was done by quantification of bound NO711 as reporter ligand and hit identification for the active ones achieved in a further LC-ESI-MS/MS run in the multiple reaction monitoring mode enabling detection of all sublibrary components followed by hit verification and investigation of reduced sublibraries in further competitive binding experiments. In this way, we could demonstrate that all GAT1 inhibitors reducing reporter ligand binding below 50% at a concentration of 1 μM are detected reliably without generation of false positive or false negative hits. As the described strategy is apart from its reliability also highly efficient, it can be assumed to become a valuable tool in early drug research, especially for membrane integrated drug targets that are often posing problems in established screening techniques.

Dataset Information

Affinity Selection-Mass Spectrometry Identifies a Novel Antibacterial RNA Polymerase Inhibitor.

Publications

Affinity Selection-Mass Spectrometry Identifies a Novel Antibacterial RNA Polymerase Inhibitor.

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