Histone Deacetylase 6 (HDAC6) is an enzyme involved in cellular motility and epigenetic modifications and has been found to be upregulated in multiple cancers. As such, the development and (Phase II) clinical testing of HDAC6-selective inhibitors, ACY-241 (Citarinostat) and ACY-1215 (Ricolinostat), has shown substantial therapeutic promise, as well as overcoming some of the challenges associated with conventional pan-HDAC isoform inhibitors. Herein, we describe the design, synthesis, and biological evaluation of novel HDAC6-selective inhibitors. We have expanded on the current paradigm of HDAC inhibition to design and explore novel binding modes and conformations within the inhibitor-catalytic binding pocket. Three lead small molecules have been identified with potencies ranging from 0.7-3.0 nM against HDAC6 in an EMSA-based functional assay. Moreover, in the same assay, tested against the 10 other HDAC isoforms, the lead compounds demonstrated in vitro selectivity ranging from 30-250-fold. The selectivity profile compares favourably to HDAC6-selctive inhibitors, ACY-241 and ACY-1215, which exhibit 5-6 fold selectivity in the same assay conditions. The impressive selectivity of the lead candidates is recapitulated in cellulo, with induced accumulation of acetylated-tubulin over acetylation of histone H3 and H4, at sub-100 nM concentrations of inhibitor in refractory medulloblastoma, multiple myeloma, and acute myeloid leukemia cells. In HELA cells, using NanoBRETTM technology, lead compounds showed up to 5-fold greater residence time with the HDAC6 target, as well as selectivity relative to HDAC1. In addition, lead compounds showed potent biological activity in the same cell lines, and at the same time, showed limited toxicity against a range of healthy cells, included pooled human fibroblasts and hematopoietic stem cells, with large therapeutic indexes of &gt;100. Notably, lead HDAC6 inhibitors presented exhibit excellent pharmacokinetic properties with greater Cmax, t1/2, AUC, and MRT than ACY-241.

Design and synthesis of selective HDAC-isoform-targeting small molecule inhibitors

ACS Fall 2020 Virtual Meeting

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