Four crystal structures are presented of histone deacetylase 6 (HDAC6) complexes with para-substituted phenylhydromaxamate inhibitors, including bulky peptoids. These structures provide insight regarding the design of capping groups that confer selectivity for binding to HDAC6, specifically with regard to interactions in a pocket formed by the L1 loop. Capping group interactions may also influence hydroxamate-Zn coordination with monodentate or bidentate geometry.

Dual- or multi-target drugs have emerged as a promising alternative to combination therapies. Proteasome inhibitors (PIs) possess synergistic activity with histone deacetylase (HDAC) inhibitors due to the simultaneous blockage of the ubiquitin-degradation and aggresome pathways. Here, we present the design, synthesis, binding modes and anticancer properties of RTS-V5 as the first-in-class dual HDAC-proteasome ligand. The inhibition of both targets was confirmed by biochemical and cellular assays as well as X-ray crystal structures of the 20S proteasome and HDAC6 complexed with RTS-V5. Cytotoxicity assays with leukemia and multiple myeloma cell lines as well as therapy-refractory primary patient-derived leukemia cells demonstrated that RTSV5 possesses potent and selective anticancer activity. Our results will thus guide the structure-based optimization of dual HDAC-proteasome inhibitors for the treatment of hematological malignancies.

Histone deacetylase 6 (HDAC6) is emerging as a target for inhibition in therapeutic strategies aimed at treating cancer, neurodegenerative disease, and other disorders. Among the metal-dependent HDAC isozymes, HDAC6 is unique in that it contains two catalytic domains, CD1 and CD2. CD2 is a tubulin deacetylase and a tau deacetylase, and the development of HDAC6-selective inhibitors has focused exclusively on this domain. In contrast, there is a dearth of structural and functional information regarding CD1, which exhibits much narrower substrate specificity in comparison with CD2. As the first step in addressing the CD1 information gap, we now present X-ray crystal structures of seven inhibitor complexes with wild-type, Y363F, and K330L HDAC6 CD1. These structures broaden our understanding of molecular features that are important for catalysis and inhibitor binding. The active site of HDAC6 CD1 is wider than that of CD2, which is unexpected in view of the narrow substrate specificity of CD1. Amino acid substitutions between HDAC6 CD1 and CD2, as well as conformational differences in conserved residues, define striking differences in active site contours. Catalytic activity measurements with HDAC6 CD1 confirm the preference for peptide substrates containing C-terminal acetyllysine residues. However, these measurements also show that CD1 exhibits weak activity for peptide substrates bearing certain small amino acids on the carboxyl side of the scissile acetyllysine residue. Taken together, these results establish a foundation for understanding the structural basis of HDAC6 CD1 catalysis and inhibition, pointing to possible avenues for the development of HDAC6 CD1-selective inhibitors.

Inhibition of histone deacetylase 6 (HDAC6) has emerged as a promising therapeutic strategy for the treatment of cancer, chemotherapy-induced peripheral neuropathy, and neurodegenerative disease. The recent X-ray crystal structure determination of HDAC6 enables an understanding of structural features directing affinity and selectivity in the active site. Here, we present the X-ray crystal structures of five HDAC6-inhibitor complexes that illuminate key molecular features of inhibitor linker and capping groups that facilitate and differentiate binding to HDAC6. In particular, aromatic and heteroaromatic linkers nestle within an aromatic cleft defined by F583 and F643, and different aromatic linkers direct the capping group toward shallow pockets defined by the L1 loop, the L2 loop, or somewhere in between these pockets. These results expand our understanding of factors contributing to the selective inhibition of HDAC6, particularly regarding interactions that can be targeted in the region of the L2 pocket.

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