Phenotypic Screening of Histone Deacetylase (HDAC) Inhibitors against Schistosoma mansoni.

Schistosomiasis is a prevalent yet neglected tropical parasitic disease caused by the Schistosoma genus of blood flukes. Praziquantel is the only currently available treatment, hence drug resistance poses a major threat. Recently, histone deacetylase 8 (HDAC8) selective inhibitors have been proposed as a viable treatment for schistosomiasis. Herein, we report the phenotypic screening of a focused library of small molecules of varying HDAC isozyme-inhibition profiles, including eight HDAC8 inhibitors with >10-fold selectivity in comparable functional inhibition assays and IC50 values against HDAC8<100 nM. HDAC8-selective inhibitors showed the lowest potency against Schistosoma mansoni newly transformed schistosomula (NTS). Pan-HDAC inhibitors MMH258, MMH259, and MMH373, as assessed by functional inhibition assays, with minimal or no-observed hHDAC8 and SmHDAC8 activities, were active against both NTS (MMH258, IC50 =1.5 µM; MMH259, IC50 =2.3 µM) and adult S. mansoni (MMH258, IC50 =2.1 µM; MMH373, IC50 =3.4 µM). Our results indicate that neither hHDAC8 nor SmHDAC8 activity were directly correlated to their NTS and adult S. mansoni activities.

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

Histone deacetylases (HDACs) are important epigenetic regulators involved in many diseases, especially cancer. Five HDAC inhibitors have been approved for anticancer therapy and many are in clinical trials. Among the 11 zinc-dependent HDACs, HDAC10 has received relatively little attention by drug discovery campaigns, despite its involvement, e. g., in the pathogenesis of neuroblastoma. This is due in part to a lack of robust enzymatic conversion assays. In contrast to the protein lysine deacetylase and deacylase activity of most other HDAC subtypes, it has recently been shown that HDAC10 has strong preferences for deacetylation of oligoamine substrates like acetyl-putrescine or -spermidine. Hence, it is also termed a polyamine deacetylase (PDAC). Here, we present the first fluorescent enzymatic conversion assay for HDAC10 using an aminocoumarin-labelled acetyl-spermidine derivative to measure its PDAC activity, which is suitable for high-throughput screening. Using this assay, we identified potent inhibitors of HDAC10-mediated spermidine deacetylation in vitro. Based on the oligoamine preference of HDAC10, we also designed inhibitors with a basic moiety in appropriate distance to the zinc binding hydroxamate that showed potent inhibition of HDAC10 with high selectivity, and we solved a HDAC10-inhibitor structure using X-ray crystallography. We could demonstrate selective cellular target engagement for HDAC10 but a lysosomal phenotype in neuroblastoma cells that was previously associated with HDAC10 inhibition was not observed. Thus, we have developed new chemical probes for HDAC10 that allow further clarification of the biological role of this enzyme.

Species-selective targeting of pathogens revealed by the atypical structure and active site of Trypanosoma cruzi histone deacetylase DAC2.

Writing and erasing of posttranslational modifications are crucial to phenotypic plasticity and antigenic variation of eukaryotic pathogens. Targeting pathogens' modification machineries, thus, represents a valid approach to fighting parasitic diseases. However, identification of parasitic targets and the development of selective anti-parasitic drugs still represent major bottlenecks. Here, we show that the zinc-dependent histone deacetylases (HDACs) of the protozoan parasite Trypanosoma cruzi are key regulators that have significantly diverged from their human counterparts. Depletion of T. cruzi class I HDACs tcDAC1 and tcDAC2 compromises cell-cycle progression and division, leading to cell death. Notably, tcDAC2 displays a deacetylase activity essential to the parasite and shows major structural differences with human HDACs. Specifically, tcDAC2 harbors a modular active site with a unique subpocket targeted by inhibitors showing substantial anti-parasitic effects in cellulo and in vivo. Thus, the targeting of the many atypical HDACs in pathogens can enable anti-parasitic selective chemical impairment.