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Identification of cellular targets of a series of boron heterocycles using TIPA II-A sensitive target identification platform.

ABSTRACT: One of the hurdles in the discovery of antibiotics is the difficulty of linking antibacterial compounds to their cellular targets. Our laboratory has employed a genome-wide approach of over-expressing essential genes in order to identify cellular targets of antibacterial inhibitors. Our objective in this project was to develop and validate a more sensitive disk diffusion based platform of target identification (Target Identification Platform for Antibacterials version 2; TIPA II) using a collection of cell clones in an Escherichia coli mutant (AS19) host with increased outer membrane permeability. Five known antibiotics/inhibitors and 28 boron heterocycles were tested by TIPA II assay, in conjunction with the original assay TIPA. The TIPA II was more sensitive than TIPA because eight boron heterocycles previously found to be inactive to AG1 cells in TIPA assays exhibited activity to AS19 cells. For 15 boron heterocycles, resistant colonies were observed within the zones of inhibition only on the inducing plates in TIPA II assays. DNA sequencing confirmed that resistant clones harbor plasmids with fabI gene as insert, indicating that these boron heterocycles all target enoyl ACP reductase. Additionally, cell-based assays and dose response curved obtained indicated that for two boron heterocycle inhibitors, the fabI cell clone in AG1 (wild-type) host cells exhibited at least 11 fold more resistance under induced conditions than under non-induced conditions. Moreover, TIPA II also identified cellular targets of known antibacterial inhibitors triclosan, phosphomycin, trimethoprim, diazaborine and thiolactomycin, further validating the utility of the new system.

SUBMITTER: Ward MS

PROVIDER: S-EPMC4992540 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

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Identification of cellular targets of a series of boron heterocycles using TIPA II-A sensitive target identification platform.

Ward Matthew S MS Silva Isba I Martinez Walfre W Jefferson Jameka J Rahman Shakila S Garcia Jeanie M JM Kanichar Divya D Roppiyakuda Lance L Kosmowska Ewa E Faust Michelle A MA Tran Kim P KP Chow Felicia F Buglo Elena E Zhou Feimeng F Groziak Michael P MP Xu H Howard HH

Bioorganic & medicinal chemistry 20160604 15

One of the hurdles in the discovery of antibiotics is the difficulty of linking antibacterial compounds to their cellular targets. Our laboratory has employed a genome-wide approach of over-expressing essential genes in order to identify cellular targets of antibacterial inhibitors. Our objective in this project was to develop and validate a more sensitive disk diffusion based platform of target identification (Target Identification Platform for Antibacterials version 2; TIPA II) using a collect ...[more]

PMID: 27301675

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Project description:Bioorthogonal chemistry has enabled the development of bioconjugates in physiological environments while averting interference from endogenous biomolecules. Reactions between carbonyl-containing molecules and alkoxyamines or hydrazines have experienced a resurgence in popularity in bioorthogonal chemistry owing to advances that allow the reactions to occur under physiological conditions. In particular, ortho-carbonyl-substituted phenylboronic acids (CO-PBAs) exhibit greatly accelerated rates of hydrazone and oxime formation via intramolecular Lewis acid catalysis. Unfortunately, the rate of the reverse reaction is also increased, yielding a kinetically less stable bioconjugate. When the substrate is a hydrazine derivative, an intramolecular reaction between the boronic acid and the hydrazone can lead to the formation of a heterocycle containing a boron-nitrogen bond. We have shown previously that ?-amino hydrazides undergo rapid reaction with CO-PBAs to form highly stable, tricyclic products, and that this reaction is orthogonal to the popular azide-alkyne and tetrazine-alkene reactions. In this work, we explore a series of heteroatom-substituted hydrazides for their ability to form tricyclic products with two CO-PBAs, 2-formylphenylboronic acid (2fPBA), and 2-acetylphenylboronic acid (AcPBA). In particular, highly stable products were formed using ?-hydroxy hydrazides and 2fPBA. C-Terminal ?-hydroxy hydrazide proteins are available using conventional biochemical methods, which alleviates one of the difficulties with applications of bioorthogonal chemical reactions: site-specific incorporation of a reactive group into the biomolecular target. Using sortase-mediated ligation (SML), C-terminal threonine and serine hydrazides were appended to a model eGFP protein in high yield. Subsequent labeling with 2fPBA functionalized probes could be performed quickly and quantitatively at neutral pH using micromolar concentrations of reactants. The SML process was applied directly to an expressed protein in cellular extract, and the C-terminal modified target protein was selectively immobilized using 2fPBA-agarose. Elution from the agarose yielded a highly pure protein that retained the hydrazide functionality. This strategy should be generally applicable for rapid, efficient site-specific protein labeling, protein immobilization, and preparation of highly pure functionalized proteins.

Dataset Information

Identification of cellular targets of a series of boron heterocycles using TIPA II-A sensitive target identification platform.

Publications

Identification of cellular targets of a series of boron heterocycles using TIPA II-A sensitive target identification platform.

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