Design and evaluation of 'Linkerless' hydroxamic acids as selective HDAC8 inhibitors

University of California, San Francisco, San Francisco, California, United States
Bioorganic & Medicinal Chemistry Letters (Impact Factor: 2.42). 05/2007; 17(10):2874-8. DOI: 10.1016/j.bmcl.2007.02.064
Source: PubMed


In this report, we describe new HDAC inhibitors designed to exploit a unique sub-pocket in the HDAC8 active site. These compounds were based on inspection of the available HDAC8 crystal structures bound to various inhibitors, which collectively show that the HDAC8 active site is unusually malleable and can accommodate inhibitor structures that are distinct from the canonical 'zinc binding group-linker-cap group' structures of SAHA, TSA, and similar HDAC inhibitors. Some inhibitors based on this new scaffold are >100-fold selective for HDAC8 over other class I and class II HDACs with IC(50) values <1microM against HDAC8. Furthermore, treatment of human cells with the inhibitors described here shows a unique pattern of hyperacetylated proteins compared with the broad-spectrum HDAC inhibitor TSA.

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    • "High potassium solution (KPSS) with composition as per PSS with an isosmotic substitution of NaCl) with KCl (60 mmol/L final concentration ). Phenylephrine (PE) hydrochloride and acetylcholine chloride stocks were made up daily in distilled water; TSA stock was made up with ethanol and compound 2 (manufactured as described previously Krennhrubec et al. 2008) and plumbagin in dimethylsulfoxide (DMSO). "
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    ABSTRACT: There is increasing interest in establishing the roles that lysine acetylation of non nuclear proteins may exert in modulating cell function. Lysine deacetylase 8 (KDAC8), for example, has been suggested to interact with α-actin and control the differentiation of smooth muscle cells. However, a direct role of smooth muscle non nuclear protein acetylation in regulating tone is unresolved. We sought to define the actions of two separate KDAC inhibitors on arterial tone and identify filament-interacting protein targets of acetylation and association with KDAC8. Compound 2 (a specific KDAC8 inhibitor) or Trichostatin A (TSA, a broad-spectrum KDAC inhibitor) inhibited rat arterial contractions induced by phenylephrine (PE) or high potassium solution. In contrast to the predominantly nuclear localization of KDAC1 and KDAC2, KDAC8 was positioned in extranuclear areas of native vascular smooth muscle cells. Several filament-associated proteins identified as putative acetylation targets colocalized with KDAC8 by immunoprecipitation (IP): cortactin, α-actin, tropomyosin, HSPB1 (Hsp27) and HSPB6 (Hsp20). Use of anti-acetylated lysine antibodies showed that KDAC inhibition increased acetylation of each protein. A custom-made antibody targeting the C-terminal acetylated lysine of human HSPB6 identified this as a novel target of acetylation that was increased by KDAC inhibition. HSPB6 phosphorylation, a known vasodilatory modification, was concomitantly increased. Interrogation of publicly available mass spectrometry data identified 50 other proteins with an acetylated C-terminal lysine. These novel data, in alliance with other recent studies, alert us to the importance of elucidating the mechanistic links between changes in myofilament-associated protein acetylation, in conjunction with other posttranslational modifications, and the regulation of arterial tone.
    11/2013; 1(6):e00127. DOI:10.1002/phy2.127
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    • "Similarly to pan-HDACi induction of miR-183, induction was stronger in the MYCN-amplified cells. In contrast, the HDAC6-selective inhibitor, Tubacin, and the HDAC8-selective inhibitor, Compound 2 (36), did not induce miR-183 (Figure 4A and Supplementary Table S1). These results suggest only single HDACs play a role in miR-183 regulation. "
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    ABSTRACT: MYCN is a master regulator controlling many processes necessary for tumor cell survival. Here, we unravel a microRNA network that causes tumor suppressive effects in MYCN-amplified neuroblastoma cells. In profiling studies, histone deacetylase (HDAC) inhibitor treatment most strongly induced miR-183. Enforced miR-183 expression triggered apoptosis, and inhibited anchorage-independent colony formation in vitro and xenograft growth in mice. Furthermore, the mechanism of miR-183 induction was found to contribute to the cell death phenotype induced by HDAC inhibitors. Experiments to identify the HDAC(s) involved in miR-183 transcriptional regulation showed that HDAC2 depletion induced miR-183. HDAC2 overexpression reduced miR-183 levels and counteracted the induction caused by HDAC2 depletion or HDAC inhibitor treatment. MYCN was found to recruit HDAC2 in the same complexes to the miR-183 promoter, and HDAC2 depletion enhanced promoter-associated histone H4 pan-acetylation, suggesting epigenetic changes preceded transcriptional activation. These data reveal miR-183 tumor suppressive properties in neuroblastoma that are jointly repressed by MYCN and HDAC2, and suggest a novel way to bypass MYCN function.
    Nucleic Acids Research 04/2013; 41(12). DOI:10.1093/nar/gkt346 · 9.11 Impact Factor
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    • "It is therefore clear that HDAC8 fulfils critical functions in cancer cells and that these are associated with its overexpression. This has stimulated interest in the development of HDAC8-specific inhibitors and treatment of neuroblastoma cells with a " linkerless " hydroxamic acid, selective HDAC8 inhibitor [100], inhibited cell proliferation and induced differentiation, reproducing the siRNAinduced phenotype [98]. Interestingly, knockdown of HDAC8 failed to affect global histone H4 acetylation, suggesting that the principal targets of this enzyme are either non-histone proteins or specific gene promoters. "
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    ABSTRACT: The histone modifying enzymes (HME) represent particularly promising targets for the development of alternatives to praziquantel, the only currently available drug to combat schistosomiasis. The inhibition of these enzymes frequently arrests the cell cycle or induces apoptosis in cancer cells, but not in normal cells and numerous HME inhibitors are under investigation as potential anticancer agents. The recent resolution of the genome sequences of Schistosoma mansoni and Schistosoma japonicum has allowed us to identify all the schistosome genes encoding histone acetyltransferases, deacetylases, methyltransferases and demethylases. We have chosen a strategy using phylogenetic screening with inhibitors of HME classes, screening of individual HME targets by both high-throughput and reasoned (in silico docking using resolved crystal structures) approaches in a project funded by the European Community, named SEtTReND (Schistosome Epigenetics: Targets, Regulation, New Drugs). The initial focus is on the class I histone deacetylase (HDAC) 8 since the comparison of the catalytic site of the schistosome and human enzymes shows crucial differences, rendering possible the development of inhibitors specific for SmHDAC8. However, phenotypic screening shows that inhibitors of all HME classes tested were able to induce apoptosis and death in parasites in vitro, indicating that other enzymes may prove to be viable targets.
    Current pharmaceutical design 05/2012; 18(24):3567-78. · 3.45 Impact Factor
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