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Methods for the expression, purification, and crystallization of histone deacetylase 6-inhibitor complexes.

ABSTRACT: Histone deacetylase (HDAC) isozymes modulate numerous regulatory signals and pathways in biological systems, hence serving as targets for drug design. For example, HDAC6 is the cytosolic tubulin deacetylase and its inhibition compromises microtubule dynamics, leading to cancer cell cycle arrest and apoptosis. The design of inhibitors that selectively target HDAC6 is desirable to avoid side effects resulting from the inhibition of off-target HDACs. High resolution X-ray crystal structures of HDAC6 have accelerated structure-based approaches to drug design targeting HDAC6. Crystal structure analysis reveals that the tubulin deacetylase domain of human HDAC6 (catalytic domain 2, also known as CD2) is very similar to that of HDAC6 CD2 from Danio rerio (zebrafish, designated zCD2). Thus, zCD2 is a valid surrogate of human HDAC6 CD2, the actual drug target; moreover, zCD2 is much more easily prepared and crystallized. A plasmid containing the zCD2 construct for heterologous expression in Escherichia coli is available through Addgene (#122031). In this chapter, we review the preparation, purification, and crystallization of zCD2-inhibitor complexes. These methods enable the rapid acquisition of structural data regarding optimal zinc-binding groups, capping groups, and linkers in the discovery of new and selective HDAC6 inhibitors.

SUBMITTER: Osko JD

PROVIDER: S-EPMC6941440 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Methods for the expression, purification, and crystallization of histone deacetylase 6-inhibitor complexes.

Osko Jeremy D JD Christianson David W DW

Methods in enzymology 20190718

Histone deacetylase (HDAC) isozymes modulate numerous regulatory signals and pathways in biological systems, hence serving as targets for drug design. For example, HDAC6 is the cytosolic tubulin deacetylase and its inhibition compromises microtubule dynamics, leading to cancer cell cycle arrest and apoptosis. The design of inhibitors that selectively target HDAC6 is desirable to avoid side effects resulting from the inhibition of off-target HDACs. High resolution X-ray crystal structures of HDAC ...[more]

PMID: 31606087

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Project description:Background Epigenetic modulators have been proposed as promising new drug targets to treat adverse remodeling in heart failure. Here, we evaluated the potential of 4 epigenetic drugs, including the recently developed histone deacetylase 6 (HDAC6) inhibitor JS28, to prevent endothelin-1 induced pathological gene expression in cardiac myocytes and analyzed the chromatin binding profile of the respective inhibitor targets. Methods and Results Cardiac myocytes were differentiated and puromycin-selected from mouse embryonic stem cells and treated with endothelin-1 to induce pathological gene expression (938 differentially expressed genes, q<0.05). Dysregulation of gene expression was at least in part prevented by epigenetic inhibitors, including the pan-BRD (bromodomain-containing protein) inhibitor bromosporine (290/938 genes), the BET (bromodomain and extraterminal) inhibitor JQ1 (288/938), the broad-spectrum HDAC inhibitor suberoylanilide hydroxamic acid (227/938), and the HDAC6 inhibitor JS28 (210/938). Although the 4 compounds were similarly effective toward pathological gene expression, JS28 demonstrated the least adverse effects on physiological gene expression. Genome-wide chromatin binding profiles revealed that HDAC6 binding sites were preferentially associated with promoters of genes involved in RNA processing. In contrast, BRD4 binding was associated with genes involved in core cardiac myocyte functions, for example, myocyte contractility, and showed enrichment at enhancers and intronic regions. These distinct chromatin binding profiles of HDAC6 and BRD4 might explain the different effects of their inhibitors on pathological versus physiological gene expression. Conclusions In summary, we demonstrated, that the HDAC6 inhibitor JS28 effectively prevented the adverse effects of endothelin-1 on gene expression with minor impact on physiological gene expression in cardiac myocytes. Selective HDAC6 inhibition by JS28 appears to be a promising strategy for future evaluation in vivo and potential translation into clinical application.

Project description:Eukaryotic cells maintain an immense amount of genetic information by tightly wrapping their DNA around positively charged histones. While this strategy allows human cells to maintain more than 25,000 genes, histone binding can also block gene expression. Consequently, cells express histone acetyl transferases (HATs) to acetylate histone lysines and release DNA for transcription. Conversely, histone deacetylases (HDACs) are employed for restoring the positive charge on the histones, thereby silencing gene expression by increasing histone-DNA binding. It has previously been shown that histones bind and silence viral DNA, while hyperacetylation of histones via HDAC inhibition restores viral gene expression. In this study, we demonstrate that treatment with Entinostat, an HDAC inhibitor, enhances transgene (luciferase) expression by up to 25-fold in human prostate and murine bladder cancer cell lines when used with cationic polymers for plasmid DNA delivery. Entinostat treatment altered cell cycle progression, resulting in a significant increase in the fraction of cells present in the G0/G1 phase at low micromolar concentrations. While this moderate G0/G1 arrest disappeared at higher concentrations, a modest increase in the fraction of apoptotic cells and a decrease in cell proliferation were observed, consistent with the known anticancer effects of the drug. DNase accessibility studies revealed no significant change in plasmid transcriptional availability with Entinostat treatment. However, quantitative PCR studies indicated that Entinostat treatment, at the optimal dose for enhancing transgene expression, led to an increase in the amount of plasmid present in the nucleus in two cancer cell lines. Taken together, our results show that Entinostat enhances polymer- mediated transgene expression and can be useful in applications related to transient protein expression in mammalian cells. Biotechnol. Bioeng. 2016;113: 1345-1356. © 2015 Wiley Periodicals, Inc.

Dataset Information

Methods for the expression, purification, and crystallization of histone deacetylase 6-inhibitor complexes.

Publications

Methods for the expression, purification, and crystallization of histone deacetylase 6-inhibitor complexes.

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