Highly ligand efficient and selective N-2-(Thioethyl)picolinamide histone deacetylase inhibitors inspired by the natural product psammaplin†A.

Novel picolinamide-based histone deacetylase (HDAC) inhibitors were developed, drawing inspiration from the natural product psammaplin A. We found that the HDAC potency and isoform selectivity provided by the oxime unit of psammaplin A could be reproduced by using carefully chosen heterocyclic frameworks. The resulting (hetero)aromatic amide based compounds displayed very high potency and isoform selectivity among the HDAC family, in addition to excellent ligand efficiency relative to previously reported HDAC inhibitors. In particular, the high HDAC1 isoform selectivity provided by the chloropyridine motif represents a valuable design criterion for the development of new lead compounds and chemical probes that target HDAC1.

Defining the mechanism of action and enzymatic selectivity of psammaplin A against its epigenetic targets.

Psammaplin A (11c) is a marine metabolite previously reported to be a potent inhibitor of two classes of epigenetic enzymes: histone deacetylases and DNA methyltransferases. The design and synthesis of a focused library based on the psammaplin A core has been carried out to probe the molecular features of this molecule responsible for its activity. By direct in vitro assay of the free thiol generated upon reduction of the dimeric psammaplin scaffold, we have unambiguously demonstrated that 11c functions as a natural prodrug, with the reduced form being highly potent against HDAC1 in vitro (IC(50) 0.9 nM). Furthermore, we have shown it to have high isoform selectivity, being 360-fold selective for HDAC1 over HDAC6 and more than 1000-fold less potent against HDAC7 and HDAC8. SAR around our focused library revealed a number of features, most notably the oxime functionality to be important to this selectivity. Many of the compounds show significant cytotoxicity in A549, MCF7, and W138 cells, with the SAR of cytotoxicity correlating to HDAC inhibition. Furthermore, compound treatment causes upregulation of histone acetylation but little effect on tubulin acetylation. Finally, we have found no evidence for 11c functioning as a DNMT inhibitor.

Synthesis and biochemical analysis of 2,2,3,3,4,4,5,5,6,6,7,7-dodecafluoro-N-hydroxy-octanediamides as inhibitors of human histone deacetylases.

Inhibition of human histone deacetylases (HDACs) has emerged as a novel concept in the chemotherapeutic treatment of cancer. Two chemical entities, SAHA (ZOLINZA, Merck) and romidepsin (Istodax, Celgene) have been recently approved by the FDA as first-in-class drugs against cutaneous T-cell lymphoma. Clinical use of these drugs revealed several side effects including gastro-intestinal symptoms, fatigue, thrombocytopenia, thrombosis. Romidepsin is associated with an yet unresolved cardiotoxicity issue. A general hypothesis for the diminishment of unwanted adverse effects and an improved therapeutical window suggests the development of more isotype selective inhibitors. In this study the first time HDAC inhibitors with perfluorinated spacers between the zinc chelating moiety and the aromatic capping group were synthesized and tested against representatives of HDAC classes I, IIa and IIb. Competitive binding assays and a combined approach by using blind docking and molecular dynamics support binding of the perfluorinated analogs of SAHA to the active site of the HDAC-like amidohydrolase from Bordetella/Alcaligenes and presumably also to human HDACs. In contrast to the alkyl spacer of SAHA and derivatives, the perfluorinated alkyl spacer seems to contribute to or facilitate the induction of selectivity for class II, particularly class IIa, HDACs even though the overall potency of the perfluorinated SAHA analogs in this study against human HDACs remained still rather moderate in the micromolar range.

Identification of selective class II histone deacetylase inhibitors using a novel dual-parameter binding assay based on fluorescence anisotropy and lifetime.

Histone deacetylases (HDACs) are important epigenetic factors regulating a variety of vital cellular functions such as cell cycle progression, differentiation, cell migration, and apoptosis. Consequently, HDACs have emerged as promising targets for cancer therapy. The drugability of HDACs has been shown by the discovery of several structural classes of inhibitors (HDACis), particularly by the recent approval of two HDACis, vorinostat (ZOLINZA) and romidepsin (Istodax), for the treatment of cutaneous T-cell lymphoma by the US Food and Drug Administration. The outstanding potential of HDACis, with a defined isoform selectivity profile as drugs against a plurality of diseases, vindicates increased effort in developing high-throughput capable assays for screening campaigns. In this study, a dual-competition assay exploiting changes in fluorescence anisotropy and lifetime was used to screen the LOPAC (Sigma-Aldrich, St Louis, MO) library against the bacterial histone deacetylase homologue HDAH from Bordetella, which shares 35% identity with the second deacetylase domain of HDAC6. The binding assay proved to be highly suitable for high-throughput screening campaigns. Several LOPAC compounds have been identified to inhibit HDAH in the lower micromolar range. Most interestingly, some of the hit compounds turned out to be weak but selective inhibitors of human class IIa and IIb HDACs.

Non-isotopic dual parameter competition assay suitable for high-throughput screening of histone deacetylases.

Histone deacetylases reside among the most important and novel target classes in oncology. Selective lead structures are intensively developed to improve efficacy and reduce adverse effects. The common assays used so far to identify new lead structures suffer from many false positive hits due to auto-fluorescence of compounds or triggering undesired signal transduction pathways. These drawbacks are eliminated by the dual parameter competition assay reported in this study. The assay involves a new fluorescent inhibitor probe that shows an increase in both, fluorescence anisotropy and fluorescence lifetime upon binding to the enzyme. The assay is well suited for high-throughput screening.