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Methods for the expression, purification, and crystallization of histone deacetylase 6-inhibitor complexes
Jeremy D Osko1, David W Christianson1
1Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania, Philadelphia, PA, United States.
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.
Insights
Selective inhibition of Histone deacetylase 6 (HDAC6) is crucial for cancer therapy. Zebrafish HDAC6 CD2 serves as a reliable surrogate for structural studies, aiding in the design of novel HDAC6 inhibitors.
Area of Science:
- Biochemistry
- Structural Biology
- Drug Discovery
Background:
- Histone deacetylase (HDAC) isozymes regulate cellular pathways, making them key drug targets.
- HDAC6, a cytosolic tubulin deacetylase, is implicated in cancer cell cycle arrest and apoptosis upon inhibition.
- Selective HDAC6 inhibitors are needed to minimize off-target effects and improve therapeutic outcomes.
Purpose of the Study:
- To present methods for preparing, purifying, and crystallizing zebrafish HDAC6 CD2 (zCD2)-inhibitor complexes.
- To highlight the utility of zCD2 as a surrogate for human HDAC6 CD2 in structure-based drug design.
- To facilitate the discovery of novel and selective HDAC6 inhibitors through structural analysis.
Main Methods:
- Heterologous expression of zCD2 in Escherichia coli.
- Purification of zCD2 and subsequent complex formation with potential inhibitors.
- X-ray crystallography of zCD2-inhibitor complexes for structural determination.
Main Results:
- The crystal structure of zCD2 is highly similar to human HDAC6 CD2, validating its use as a surrogate.
- Established protocols for preparing and crystallizing zCD2-inhibitor complexes.
- Structural insights into the interaction of inhibitors with the zCD2 active site.
Conclusions:
- Zebrafish HDAC6 CD2 is a practical and effective surrogate for structural studies of human HDAC6.
- The presented methods enable rapid structural data acquisition for designing selective HDAC6 inhibitors.
- This approach accelerates the discovery of new cancer therapeutics targeting HDAC6.
