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. Co-crystal structures of our aza-SAHA derivatives with HDAC10 provide a structural rationale for potency, and chemoproteomic profiling con-firmed 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, confirmed for the first time the suspected cellular function of HDAC10 as a polyamine deacetylase. Finally, in a polyamine-limited 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.

Histone deacetylase-10 liberates spermidine to support polyamine homeostasis and tumor cell growth

“…No significant J o u r n a l P r e -p r o o f differences were detected in spermidine concentrations with HDAC10 knockout in either cell line. These results are consistent with those observed in a neuroblastoma cell line with HDAC10-targeting siRNA (20) and confirm an association of N 8 -AcSpd with HDAC10 in cell lines derived from multiple tissues.…”

“…S1 ) followed by Sanger sequencing to confirm biallelic generation of indels. HeLa cell clones lacking HDAC10 were previously reported ( 20 ). Mass spectrometry analyses verified significantly increased levels of N 8 -AcSpd in both HDAC10 -KO cell lines ( Fig.…”

“…N 8 -AcSpd is a metabolite present in feces and the intestinal lumen ( 19 ). We therefore extended our recent studies using HDAC10-selective inhibitors ( 20 ) to determine if inhibiting deacetylase activity was sufficient to prevent polyamine restoration and growth rescue by N 8 -AcSpd in HCT116 colon cancer cells. DKFZ-728 and DKFZ-748 ( Fig.…”

First Fluorescent Acetylspermidine Deacetylation Assay for HDAC10 Identifies Selective Inhibitors with Cellular Target Engagement**

“…In a parallel, yet independent study, it has been shown very recently that a benzoyl instead of a benzyl-group at the "outer" nitrogen also leads to piperidine-hydroxamate HDAC10 ChemBioChem inhibitors but with decreased potency (93 nM in FRET assay) and selectivity as compared to our new inhibitors. [57] In this amide series, open chain analogues (aza-vorinostats) showed higher potency and selectivity than the piperidines. We do not have the matched pairs for the amines in the open chain series, but judging from the comparison in the piperidine series and the specific additional interactions of our second basic center with E24 we would argue that this second amine function is beneficial for potency and selectivity.…”

“…The same was observed for other new selective HDAC10 inhibitors mentioned above. [57] Thus, the exact HDAC subtype selectivity profile for the induction of the lysosomal phenotype remains elusive so far.…”