Aza-SAHA Derivatives Are Selective Histone Deacetylase 10 Chemical Probes That Inhibit Polyamine Deacetylation and Phenocopy HDAC10 Knockout

Abstract

We report the first well-characterized selective chemical probe for histone deacetylase 10 (HDAC10) with unprecedented selectivity over other HDAC isozymes. HDAC10 deacetylates polyamines and has a distinct substrate specificity, making it unique among the 11 zinc-dependent HDAC hydrolases. Taking inspiration from HDAC10 polyamine substrates, we systematically inserted an amino group (“aza-scan”) into the hexyl linker moiety of the approved drug Vorinostat (SAHA). This one-atom replacement (C→N) transformed SAHA from an unselective pan-HDAC inhibitor into a specific HDAC10 inhibitor. Optimization of the aza-SAHA structure yielded the HDAC10 chemical probe DKFZ‑748, with potency and selectivity demonstrated by cellular and biochemical target engagement, as well as thermal shift assays. Cocrystal structures of our aza-SAHA derivatives with HDAC10 provide a structural rationale for potency, and chemoproteomic profiling confirmed exquisite cellular HDAC10-selectivity of DKFZ‑748 across the target landscape of HDAC drugs. Treatment of cells with DKFZ‑748, followed by quantification of selected polyamines, validated for the first time the suspected cellular function of HDAC10 as a polyamine deacetylase. Finally, in a polyamine-limiting in vitro tumor model, DKFZ‑748 showed dose-dependent growth inhibition of HeLa cells. We expect DKFZ‑748 and related probes to enable further studies on the enigmatic biology of HDAC10 and acetylated polyamines in both physiological and pathological settings.

Chemical Versatility in Catalysis and Inhibition of the Class IIb Histone Deacetylases

“…(e) Dose-dependent increases in N 8 -acetylspermidine levels resulting from HDAC10 inhibition are observed in the neuroblastoma cell line BE(2)-C following 24 h of incubation with increasing concentrations of DKFZ-748 (compound 49 is the corresponding ester, which served as a negative control). Plates (b)–(e) are reproduced with permission from ref . Copyright 2022 American Chemical Society.…”

“…Optimization of the DKFZ-728 capping group yielded DKFZ-748, in which a naphthyl group was substituted for the phenyl group of DKFZ-728. DKFZ-748 exhibited superior affinity and selectivity for HDAC10 binding as well as cellular engagement (Figure d).…”

“…Using the structure of the clinically approved pan-HDAC inhibitor SAHA as a starting point, Miller and colleagues systematically substituted a methylamino group in place of individual methylene groups in the aliphatic linker, thereby determining the optimal separation of the hydroxamate zinc-binding group and a tertiary ammonium cation targeting interaction with E274 (Figure a) . The design rationale underlying this “aza scan” was confirmed in the crystal structure determination of the HDAC10 complex with DKFZ-711, the tertiary ammonium group of which forms a water-mediated hydrogen bond with E274 (Figure b) similar to that observed for the substrate N 8 -acetylspermidine (Figure c).…”

“…Although histone lysine deacetylase activity was initially discovered in the mammalian cell nucleus, where class I HDACs can serve epigenetic functions, subsequent studies showed that HDACs exhibit remarkable diversity in subcellular localization, substrate specificity, and catalytic function, thereby regulating biological processes far beyond gene expression. For example, the class IIb HDACs are localized predominantly (but not exclusively) in the cytosol, where HDAC6 CD2 serves as a tubulin deacetylase or a tau deacetylase, and HDAC10 serves as a highly specific N 8 -acetylspermidine deacetylase in polyamine homeostasis. , HDAC6 additionally serves as a lamin A lysine decrotonylase in the nucleus . The class IV deacetylase HDAC11 is localized in the nucleus, cytosol, and/or mitochondrion depending on tissue and cell type, where it serves as a lysine-fatty acid deacylase. − Thus, the HDAC substrate repertoire is vast and includes thousands of proteins and even small molecules throughout the cell and indeed throughout all domains of life. − …”

Near‐Infrared Light‐Driven Nanoparticles for Cancer Photoimmunotherapy by Synergizing Immune Cell Death and Epigenetic Regulation

“…[14][15][16][17][18][19] Nevertheless, a single HDACi is ineffective for the majority of solid or advanced cancers. [20][21][22] Hence, it would be a good option to combine epigenetic modification with other anticancer treatments to increase antitumor effectiveness. Immunogenic cell death (ICD) regulates the transition between "cold" and "hot" tumors by affecting the function and phenotype of immune cells in the tumor microenvironment (TME), with important implications for epigenetic regulation.…”