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A high-throughput effector screen identifies a novel small molecule scaffold for inhibition of ten-eleven translocation dioxygenase 2.

ABSTRACT: Ten-eleven translocation dioxygenases (TETs) are the erasers of 5-methylcytosine (mC), the central epigenetic regulator of mammalian DNA. TETs convert mC to three oxidized derivatives with unique physicochemical properties and inherent regulatory potential, and it initializes active demethylation by the base excision repair pathway. Potent small molecule inhibitors would be useful tools to study TET functions by conditional control. To facilitate the discovery of such tools, we here report a high-throughput screening pipeline and its application to screen and validate 31.5k compounds for inhibition of TET2. Using a homogenous fluorescence assay, we discover a novel quinoline-based scaffold that we further validate with an orthogonal semi-high throughput MALDI-MS assay for direct monitoring of substrate turnover. Structure-activity relationship (SAR) studies involving >20 derivatives of this scaffold led to the identification of optimized inhibitors, and together with computational studies suggested a plausible model for its mode of action.

SUBMITTER: Palei S

PROVIDER: S-EPMC9749932 | biostudies-literature | 2022 Dec

REPOSITORIES: biostudies-literature

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A high-throughput effector screen identifies a novel small molecule scaffold for inhibition of ten-eleven translocation dioxygenase 2.

Palei Shubhendu S Weisner Jörn J Vogt Melina M Gontla Rajesh R Buchmuller Benjamin B Ehrt Christiane C Grabe Tobias T Kleinbölting Silke S Müller Matthias M Clever Guido H GH Rauh Daniel D Summerer Daniel D

RSC medicinal chemistry 20220902 12

Ten-eleven translocation dioxygenases (TETs) are the erasers of 5-methylcytosine (mC), the central epigenetic regulator of mammalian DNA. TETs convert mC to three oxidized derivatives with unique physicochemical properties and inherent regulatory potential, and it initializes active demethylation by the base excision repair pathway. Potent small molecule inhibitors would be useful tools to study TET functions by conditional control. To facilitate the discovery of such tools, we here report a hig ...[more]

PMID: 36545435

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Project description:Liver-specific ten-eleven translocation (Tet) methylcytosine dioxygenases 2 and 3 (Tet2 plus Tet3)-deficient hepatitis B virus (HBV) transgenic mice fail to support viral biosynthesis. The levels of viral transcription and replication intermediates are dramatically reduced. Hepatitis B core antigen is only observed in a very limited number of pericentral hepatocytes in a pattern that is similar to glutamate-ammonia ligase (Glul), a β-catenin target gene. HBV transcript abundance in adult Tet-deficient mice resembles that observed in wild-type neonatal mice. Furthermore, the RNA levels of several β-catenin target genes including Glul, Lhpp, Notun, Oat, Slc1a2, and Tbx3 in Tet-deficient mice were also similar to that observed in wild-type neonatal mice. As HBV transcription is regulated by β-catenin, these findings support the suggestion that neonatal Tet deficiency might limit β-catenin target gene expression, limiting viral biosynthesis. Additionally, HBV transgene DNA displays increased 5-methylcytosine (5mC) frequency at CpG sequences consistent with neonatal Tet deficiency being responsible for decreased developmental viral DNA demethylation mediated by 5mC oxidation to 5-hydroxymethylcytosine, a process that might be responsible for the reduction in cellular β-catenin target gene expression and viral transcription and replication.IMPORTANCEChronic hepatitis B virus (HBV) infection causes significant worldwide morbidity and mortality. There are no curative therapies available to resolve chronic HBV infections, and the small viral genome limits molecular targets for drug development. An alternative approach to drug development is to target cellular genes essential for HBV biosynthesis. In the liver, ten-eleven translocation (Tet) genes encode cellular enzymes that are not essential for postnatal mouse development but represent essential activities for viral DNA demethylation and transcription. Consequently, Tet inhibitors may potentially be developed into therapeutic agents capable of inducing and/or maintaining HBV covalently closed circular DNA methylation, resulting in transcriptional silencing and the resolution of chronic viral infection.

Project description:BackgroundStroke is a leading cause of morbidity and mortality worldwide. Neuroinflammation plays a key role in acute brain injury of ischemic stroke. MicroRNA-210 (miR210) is the master hypoxamir and regulates microglial activation and inflammation in a variety of diseases. In this study, we uncovered the mechanism of miR210 in orchestrating ischemic stroke-induced neuroinflammation through repression of TET2 (ten-eleven translocation methylcytosine dioxygenase 2) in the adult mouse brain.MethodsIschemic stroke was induced in adult WT (wild type) or miR210 KO (miR210 deficient) mice by transient intraluminal middle cerebral artery occlusion. Injection of TET2 silencing RNA or miR210 complementary locked nucleic acid oligonucleotides, or miR210 KO mice were used to validate miR210-TET2 axis and its role in ischemic brain injury. Furthermore, the effect of TET2 overexpression on miR210-stimulated proinflammatory cytokines was examined in BV2 microglia. Post assays included magnetic resonance imaging scan for brain infarct size; neurobehavioral tests, reverse transcription-quantitative polymerase chain reaction, and Western blot for miR210; and TET2 levels, flow cytometry, and ELISA for neuroinflammation in the brain after stroke or microglia in vitro.ResultsmiR210 injection significantly reduced TET2 protein abundance in the brain, while miR210 complementary locked nucleic acid oligonucleotides or miR210 KO preserved TET2 regardless of ischemic brain injury. TET2 knockdown reversed the protective effects of miR210 inhibition or miR210 KO on ischemic stroke-induced brain infarct size and neurobehavioral deficits. Moreover, flow cytometry and ELISA assays showed that TET2 knockdown also significantly dampened the anti-inflammatory effect of miR210 inhibition on microglial activation and IL (interleukin)-6 release after stroke. In addition, overexpression of TET2 in BV2 microglia counteracted miR210-induced increase in cytokines.ConclusionsmiR210 inhibition reduced ischemic stroke-induced neuroinflammatory response via repression of TET2 in the adult mouse brain, suggesting that miR210 is a potential treatment target for acute brain injury after ischemic stroke.

Dataset Information

A high-throughput effector screen identifies a novel small molecule scaffold for inhibition of ten-eleven translocation dioxygenase 2.

Publications

A high-throughput effector screen identifies a novel small molecule scaffold for inhibition of ten-eleven translocation dioxygenase 2.

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