Histone deacetylases (HDACs) have roles beyond histone deacetylation and epigenetic regulation with non-histone and even non-peptide substrates. Class IIB member HDAC10 is known to be an effective N8-acetylspermidine deacetylase with poor activity on protein substrates and may be involved in chemotherapy resistance of neuroblastoma via autophagy. HDAC10 deletion in mice was shown to increase the suppressive function of T-regulatory cells, therefore playing an important role in transplant rejection and inflammatory disorders. Selective inhibition of HDAC10 has potential for therapeutic applications, but has been challenging so far, especially within the same HDAC class. We developed the first selective chemical probes for HDAC10 with unprecedented selectivity over other HDAC isozymes. Taking inspiration from the distinct polyamine substrate specificity of HDAC10, 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 selective HDAC10 inhibitor. Optimization of the aza-SAHA structure yielded DKFZ-748, which has a 22 nM IC50 against HDAC10 in cells and &gt;500-fold selectivity over the closest relative HDAC6, as well as the Class I enzymes (HDAC1, 2, 3, 8). Potency and selectivity of our aza-SAHA derivatives was rationalized with HDAC10 co-crystal structures and HDAC6 docking models. Furthermore, we demonstrated cellular target-engagement in a cell-based bioluminescence resonance energy transfer (BRET) assay and confirmed that our inhibitors do not lead to hyperacetylation of tubulin nor histones. Treatment of neuroblastoma cells with DKFZ-748, followed by quantification of acetylated polyamines, confirmed for the first time in cells the suspected physiological function of HDAC10 as a polyamine deacetylase. Selective HDAC10 chemical probes provide a valuable pharmacological tool for target validation and will enable further studies on the enigmatic biology of HDAC10 and acetylated polyamines. HDAC10-selective aza-SAHA derivatives are not cytotoxic, which opens the doors to novel therapeutic applications as immunomodulators or in combination cancer therapy.

From pan-inhibitor to selective HDAC10 inhibitor by substitution of a single methylene in vorinostat (SAHA)

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ACS Fall 2022

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