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

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          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.

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          Author and article information

          Journal
          0212271
          5787
          Methods Enzymol
          Meth. Enzymol.
          Methods in enzymology
          0076-6879
          1557-7988
          18 December 2019
          18 July 2019
          2019
          18 July 2020
          : 626
          : 447-474
          Affiliations
          Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6323, United States
          Author notes
          [* ]Author to whom correspondence should be sent: chris@ 123456sas.upenn.edu
          Article
          PMC6941440 PMC6941440 6941440 nihpa1064072
          10.1016/bs.mie.2019.06.028
          6941440
          31606087
          Categories
          Article

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