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Biochemical characterization and novel inhibitor identification of Mycobacterium tuberculosis Endonuclease VIII 2 (Rv3297).

ABSTRACT: Nei2 (Rv3297) is a DNA Base Excision Repair (BER) glycosylase that is essential for survival of Mycobacterium tuberculosis in primates. We show that MtbNei2 is a bifunctional glycosylase that specifically acts on oxidized pyrimidine-containing single-stranded, double-stranded, 5'/3' fork and bubble DNA substrates. MtbNei2 possesses Uracil DNA glycosylase activity unlike E. coli Nei. Mutational studies demonstrate that Pro2 and Glu3 located in the active site are essential for glycosylase activity of MtbNei2. Mutational analysis demonstrated that an unstructured C-terminal zinc finger domain that was important for activity in E. coli Nei and Fpg, was not required for the glycosylase activity of MtbNei2. Lastly, we screened the NCI natural product compound database and identified three natural product inhibitors with IC50 values ranging between 41.8 ?M-92.7 ?M against MtbNei2 in in vitro inhibition assays. Surface Plasmon Resonance (SPR) experiments showed that the binding affinity of the best inhibitor, NSC31867, was 74 nM. The present results set the stage for exploiting this important target in developing new therapeutic strategies that target Mycobacterial BER.

SUBMITTER: Lata K

PROVIDER: S-EPMC5613217 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Biochemical characterization and novel inhibitor identification of Mycobacterium tuberculosis Endonuclease VIII 2 (Rv3297).

Lata Kiran K Afsar Mohammad M Ramachandran Ravishankar R

Biochemistry and biophysics reports 20170731

Nei2 (Rv3297) is a DNA Base Excision Repair (BER) glycosylase that is essential for survival of Mycobacterium tuberculosis in primates. We show that MtbNei2 is a bifunctional glycosylase that specifically acts on oxidized pyrimidine-containing single-stranded, double-stranded, 5'/3' fork and bubble DNA substrates. MtbNei2 possesses Uracil DNA glycosylase activity unlike E. coli Nei. Mutational studies demonstrate that Pro2 and Glu3 located in the active site are essential for glyco ...[more]

PMID: 28955788

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Project description:Tuberculosis (TB) continues to be an important public health threat worldwide, due in part to drug resistant Mycobacterium tuberculosis (Mtb) strains. The United States recently reported a shortage of isoniazid (INH), which could drive higher INH resistance rates. Changes in the Mtb proteome before and after acquisition of INH resistance in a clean genetic background remain understudied and may elucidate alternate drug targets. Here, we focused on Mtb clonal strains to characterize the consequences of INH resistance on mycobacterial metabolism. Proteomic analysis was conducted by liquid-chromatography tandem mass spectrometry (LC-MS/MS) of cellular and secreted fractions, followed by a normalized spectral counting (NSAF) analysis (data are available via ProteomeXchange with identifier PXD009549). Two different Mtb clonal pairs representing a specific genetic lineage (one clinical and one generated in the laboratory) but sharing a katG mutation associated with INH resistance, were used in our analysis. Overall, we found 26 Mtb proteins with altered abundances after acquisition of INH resistance across both Mtb genetic lineages studied. These proteins were involved in ATP synthesis, lipid metabolism, regulatory events, and virulence, detoxification, and adaptation processes. Proteomic findings were validated by Western blotting analyses whenever possible. Mycolic acid (MA) analysis through LC/MS in the clonal Mtb pairs did not reveal a common trend in the alteration of these fatty acids across both INHr strains but revealed a significant reduction in levels of the two more abundant ?-MA features in the clinical INHr strain. Interestingly, the clinical clonal pair demonstrated more variation in the abundance of the proteins involved in the FAS II pathway. Together, the proteomic and lipidomic data highlight the identification of potential drug targets such as alternative lipid biosynthetic pathways that may be exploited to combat clinically relevant Mtb INHr strains.

Dataset Information

Biochemical characterization and novel inhibitor identification of Mycobacterium tuberculosis Endonuclease VIII 2 (Rv3297).

Publications

Biochemical characterization and novel inhibitor identification of <i>Mycobacterium tuberculosis</i> Endonuclease VIII 2 (Rv3297).

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