Goulart Stollmaier Juana, Herbst-Gervasoni Corey J, Christianson David W
Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania, Philadelphia, PA, United States.
Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania, Philadelphia, PA, United States.
Methods Enzymol. 2025;715:19-40. doi: 10.1016/bs.mie.2025.01.074. Epub 2025 Feb 11.
The class IIb histone deacetylase HDAC10 is responsible for the deacetylation of intracellular polyamines, in particular N-acetylspermidine. HDAC10 is emerging as an attractive target for drug design owing to its role as an inducer of autophagy, and high-resolution crystal structures enable structure-based drug design efforts. The only crystal structure available to date is that of HDAC10 from Danio rerio (zebrafish), but a construct containing the A24E and D94A substitutions yields an active site contour that more closely resembles that of human HDAC10. The use of this "humanized" construct has advanced our understanding of HDAC10-inhibitor structure-activity relationships. Here, we outline the preparation, purification, assay, and crystallization of humanized zebrafish HDAC10-inhibitor complexes. The plasmid containing the humanized zebrafish HDAC10 construct for heterologous expression in Escherichia coli is available through Addgene (#225542).
IIb类组蛋白去乙酰化酶HDAC10负责细胞内多胺的去乙酰化，尤其是N - 乙酰亚精胺。由于其作为自噬诱导剂的作用，HDAC10正成为药物设计的一个有吸引力的靶点，并且高分辨率晶体结构有助于基于结构的药物设计研究。迄今为止唯一可用的晶体结构是来自斑马鱼的HDAC10，但含有A24E和D94A替换的构建体产生的活性位点轮廓与人类HDAC10的活性位点轮廓更相似。这种“人源化”构建体的使用推进了我们对HDAC10 - 抑制剂构效关系的理解。在这里，我们概述了人源化斑马鱼HDAC10 - 抑制剂复合物的制备、纯化、测定和结晶。含有用于在大肠杆菌中异源表达的人源化斑马鱼HDAC10构建体的质粒可通过Addgene（#225542）获得。
