The design, synthesis and structure-activity relationships of novel isoindoline-based histone deacetylase inhibitors

The design, synthesis and biological evaluation of a novel series of isoindoline-based hydroxamates is described. Several analogs were shown to inhibit HDAC1 with IC₅₀ values in the low nanomolar range and inhibit cellular proliferation of HCT116 human colon cancer cells in the sub-micromolar range. The cellular potency of compound 17e was found to have greater in vitro anti-proliferative activity than several compounds in late stage clinical trials for the treatment of cancer. The in vitro safety profiles of selected compounds were assessed and shown to be suitable for further lead optimization.     

Synthesis and biological evaluation of 2-quinolineacrylamides

A series of C6-substituted N-hydroxy-2-quinolineacrylamides (3–15), with four types of bridging groups have been synthesized. Most of these compounds exhibit antiproliferative activity against A549 and HCT116 cells and Western blot analysis revealed that they are able to inhibit HDAC. Measurement of the HDAC isoform activity of ether-containing compounds showed that compound 9 has distinct HDAC6 selectivity, more than 300-fold over other isoforms. This paper describes the development of 6-aryloxy-N-hydroxy-2-quinolineacrylamides as potential HDAC6 inhibitors.     

Design,synthesis and biological evaluation of 4-piperazinyl-containing Chidamide derivatives as HDACs inhibitors

The synthesis and biological evaluation of a variety of 4-piperazinyl-containing Chidamide derivatives is described. Some of these compounds were shown to inhibit HDAC1 with IC₅₀ values below micromolar range, and inhibited proliferation of several human cancer cells, not possessing toxicity to human normal cells and hERG K⁺ ion channels. Compound 9g, proved to be the most potent and efficacious derivative in this series, was orally active in an HCT116 xenograft model in vivo.     

Identification of Highly Selective and Potent Histone Deacetylase 3 Inhibitors Using Click Chemistry-Based Combinatorial Fragment Assembly

To find histone deacetylase 3 (HDAC3)-selective inhibitors, a series of 504 candidates was assembled using “click chemistry”, by reacting nine alkynes bearing a zinc-binding group with 56 azide building blocks in the presence of Cu(I) catalyst. Screening of the 504-member triazole library against HDAC3 and other HDAC isozymes led to the identification of potent and selective HDAC3 inhibitors T247 and T326. These compounds showed potent HDAC3 inhibition with submicromolar IC₅₀s, whereas they did not strongly inhibit other isozymes. Compounds T247 and T326 also induced a dose-dependent selective increase of NF-κB acetylation in human colon cancer HCT116 cells, indicating selective inhibition of HDAC3 in the cells. In addition, these HDAC3-selective inhibitors induced growth inhibition of cancer cells, and activated HIV gene expression in latent HIV-infected cells. These findings indicate that HDAC3-selective inhibitors are promising candidates for anticancer drugs and antiviral agents. This work also suggests the usefulness of the click chemistry approach to find isozyme-selective HDAC inhibitors.     

Requirement of Krüppel-like factor 4 in preventing entry into mitosis following DNA damage

Previous studies indicate that Krüppel-like factor 4 (KLF4 or GKLF) controls the G1/S cell cycle checkpoint upon DNA damage. We present evidence for an equally important role of KLF4 in maintaining the integrity of the G2/M checkpoint following DNA damage. HCT116, a colon cancer cell line with wild type p53 alleles, underwent sustained G2 arrest up to 4 days after gamma-irradiation. In contrast, HCT116 cells null for p53 were able to enter mitosis following irradiation. Western blot analyses of irradiated HCT116 cells showed increased levels of p53, KLF4, and p21WAF1/CIP1 and decreased levels of cyclin B1 when compared with unirradiated controls. In contrast, the levels of cyclin B1 increased in irradiated HCT116 p53-/- cells, in which KLF4 failed to increase due to the absence of p53. When KLF4 was inhibited by small interfering RNA, irradiated HCT116 cells exhibited increased mitotic indices and a rise in cyclin B1 levels. Conversely, irradiated HCT116 p53-/- cells that were infected with KLF4-expressing adenoviruses demonstrated a concurrent reduction in mitotic indices and cyclin B1 levels. In each case, Cdc2 kinase measurements showed an inverse correlation between Cdc2 kinase activities and KLF4 levels. Co-transfection experiments showed that KLF4 repressed the cyclin B1 promoter through a specific GC-rich element. Moreover, chromatin immunoprecipitation experiments demonstrated that both KLF4 and HDAC were associated with the cyclin B1 promoter in irradiated HCT116 cells. We conclude that KLF4 is essential in preventing mitotic entry following gamma-irradiation and does so by inhibiting cyclin B1 expression.     

HDAC inhibitors show differential epigenetic regulation and cell survival strategies on p53 mutant colon cancer cells

Besides inactivating tumour suppressor activity in cells, mutations in p53 confer significant oncogenic functions and promote metastasis and resistance to anticancer therapy. A variety of therapies involving genetic and epigenetic signalling events regulate tumorogenesis and progression in such cases. Pharmacological interventions with HDAC inhibitors have shown promise in therapy. This work explores the changes in efficacy of the four HDAC inhibitors SAHA, MS-275, valproic acid and sodium butyrate on a panel of colon cancer cell lines – HCT116 (p53 wt), HCT116 p53-/-, HT29 and SW480 (with mutations in p53). Clonogenic assays, gene profiling and epigenetic expression done on these cells point to p53 dependent differential activity of the 4 HDAC inhibitors which also elevate methylation levels in p53 mutant cell lines. In silico modelling establishes the alterations in interactions that lead to such differential activity of valproic acid, one of the inhibitors considered for the work. Molecular Dynamic simulations carried out on the valproic acid complex ensure stability of the complex. This work establishes a p53 dependent epigenetic signalling mechanism triggered by HDAC inhibition expanding the scope of HDAC inhibitors in adjuvant therapy for p53 mutant tumours.     

Discovery of adamantane based highly potent HDAC inhibitors

Herein, we report the development of highly potent HDAC inhibitors for the treatment of cancer. A series of adamantane and nor-adamantane based HDAC inhibitors were designed, synthesized and screened for the inhibitory activity of HDAC. A number of compounds exhibited GI₅₀ of 10-100 nM in human HCT116, NCI-H460 and U251 cancer cells, in vitro. Compound 32 displays efficacy in human tumour animal xenograft model.     

Anticancer Activity of MPT0E028, a Novel Potent Histone Deacetylase Inhibitor, in Human Colorectal Cancer HCT116 Cells In Vitro and In Vivo

Recently, histone deacetylase (HDAC) inhibitors have emerged as a promising class of drugs for treatment of cancers, especially subcutaneous T-cell lymphoma. In this study, we demonstrated that MPT0E028, a novel N-hydroxyacrylamide-derived HDAC inhibitor, inhibited human colorectal cancer HCT116 cell growth in vitro and in vivo. The results of NCI-60 screening showed that MPT0E028 inhibited proliferation in both solid and hematological tumor cell lines at micromolar concentrations, and was especially potent in HCT116 cells. MPT0E028 had a stronger apoptotic activity and inhibited HDACs activity more potently than SAHA, the first therapeutic HDAC inhibitor proved by FDA. In vivo murine model, the growth of HCT116 tumor xenograft was delayed and inhibited after treatment with MPT0E028 in a dose-dependent manner. Based on in vivo study, MPT0E028 showed stronger anti-cancer efficacy than SAHA. No significant body weight difference or other adverse effects were observed in both MPT0E028-and SAHA-treated groups. Taken together, our results demonstrate that MPT0E028 has several properties and is potential as a promising anti-cancer therapeutic drug.     

Phenylglycine and phenylalanine derivatives as potent and selective HDAC1 inhibitors (SHI-1)

An HTS screening campaign identified a series of low molecular weight phenols that showed excellent selectivity (>100-fold) for HDAC1/HDAC2 over other Class I and Class II HDACs. Evolution and optimization of this HTS hit series provided HDAC1-selective (SHI-1) compounds with excellent anti-proliferative activity and improved physical properties. Dose-dependent efficacy in a mouse HCT116 xenograft model was demonstrated with a phenylglycine SHI-1 analog.     

Synthesis, biological evaluation and molecular docking studies of 3-(1,3-diphenyl-1H-pyrazol-4-yl)-N-phenylacrylamide derivatives as inhibitors of HDAC activity

In present study, a series of 3-(1,3-diphenyl-1H-pyrazol-4-yl)-N-phenylacrylamide derivatives (5a-8d) were designed, synthesized, and evaluated for HDAC inhibition and tumor cell antiproliferation. All of these compounds are reported for the first time, the chemical structures of these compounds were confirmed by means of (1)H NMR, ESI-MS and elemental analyzes. Among the compounds, compound 8c showed the most potent biological activity against HCT116 cancer cell line (IC(50) of 0.42 ± 0.02 μM for HDAC-1 and IC(50)=0.62 ± 0.02 for HCT116). Docking simulation was performed to position compound 8c into the HDAC active site to determine the probable binding model. The results of antiproliferative assay and western-blot demonstrated that compound 8c with potent inhibitory activity in tumor growth inhibition may be a potential anticancer agent against HCT116 cancer cell.     

HDAC2 promotes the EMT of colorectal cancer cells and via the modular scaffold function of ENSG00000274093.1

Histone deacetylase 2 (HDAC2), a member of the Histone deacetylase family, plays a vital role in various carcinomas. In this study, we identified that HDAC2 expression levels are associated with liver metastasis, higher T stages and poor prognosis in colorectal cancer. HDAC2 down-regulation via lentivirus-mediated expression of HDAC2-targeting shRNA reduced the in vitro migration and invasion ability of HCT116 cell as well as their liver metastasis in nude mouse xenografts. Mechanistically, HDAC2 promotes epithelial-mesenchymal transition (EMT) in colorectal cancer cells by combining HDAC1 with EZH2 (a key histone methyltransferase), possibly through the modular scaffold function of a new lncRNA, ENSG00000274093.1. HDAC2 thus appears to promote CRC cell migration and invasion through binding HDAC1 and EZH2 via ENSG00000274093.1.     

Differential protein acetylation induced by novel histone deacetylase inhibitors

Histone deacetylase (HDAC) inhibitors induce the hyperacetylation of nucleosomal histones in carcinoma cells resulting in the expression of repressed genes that cause growth arrest, terminal differentiation, and/or apoptosis. In vitro selectivity of several novel hydroxamate HDAC inhibitors including succinimide macrocyclic hydroxamates and the non-hydroxamate alpha-ketoamide inhibitors was investigated using isolated enzyme preparations and cellular assays. In vitro selectivity for the HDAC isozymes (HDAC1/2, 3, 4/3, and 6) was not observed for these HDAC inhibitors or the reference HDAC inhibitors, MS-275 and SAHA. In T24 and HCT116 cells these compounds caused the accumulation of acetylated histones H3 and H4; however, the succinimide macrocyclic hydroxamates and the alpha-ketoamides did not cause the accumulation of acetylated alpha-tubulin. These data suggest "selectivity" can be observed at the cellular level with HDAC inhibitors and that the nature of the zinc-chelating moiety is an important determinant of activity against tubulin deacetylase.     

Pim-2 phosphorylation of p21Cip1/WAF1 enhances its stability and inhibits cell proliferation in HCT116 cells

Pim-2 kinase is one of the three highly conserved Pim family members which are known to be involved in cell survival and cell proliferation. Here we demonstrate that like Pim-1, Pim-2 also phosphorylates the cell cycle inhibitor p21^Cip1/WAF1 (p21) on Thr145 in vitro and in vivo. Overexpression of Pim-2 in HCT116 cells leads to the increased stability of p21 and results in enhanced levels of both exogenous and endogenous p21 proteins. Knockdown of Pim-2 expression via siRNA results in reduced level of endogenous p21, indicating that like Pim-1, Pim-2 is another legitimate p21 kinase. However, Pim-2 has no influence on the nuclear localization of p21 in HCT116 cells. In addition, Pim-2 is able to arrest the cell cycle at G1/S phase and inhibit cell proliferation through phosphorylation of p21 in HCT116 cells. These data suggest that Pim-2 phosphorylation of p21 enhances p21's stability and inhibits cell proliferation in HCT116 cells.     

The role of p53 deacetylation in p21Waf1 regulation by laminar flow

Laminar flow arrests vascular endothelial cells at the G0/G1 phase with concurrent increase in p53 and p21Waf1. We investigated the molecular mechanism by which laminar flow activates p53 and p21Waf1 in endothelial cells. The application of a laminar flow (12 dyn/cm2) increased the deacetylation at Lys-320 and Lys-373 of p53 and the acetylation at Lys-382 in human umbilical vein endothelial cells. Laminar flow increased the activity of histone deacetylase (HDAC) and the association of p53 with HDAC1. Treating human umbilical vein endothelial cells with trichostatin A (TSA), an HDAC inhibitor, abolished the flow-induced p53 deacetylation at Lys-320 and Lys-373. To investigate the role of the HDAC-deacetylated p53 in the flow activation of p21Waf1, we found that TSA inhibited the activation at both the mRNA and protein levels. Deletion and mutation analyses of the p21Waf1 promoter revealed that flow activated p21Waf1 through p53 and TSA abrogated this p53-dependent activation. The expression plasmid encoding the p53 mutant, with Lys-320 and Lys-373 replaced by Arg, increased the activity of the co-transfected p21Waf1 promoter, which demonstrates that HDAC-deacetylated p53 can transactivate the p21Waf1 gene. The regulation of the p53-p21Waf1 pathway by laminar flow was further supported by observations that flow caused an increase of p21Waf1 level in the wild-type HCT116 (p53+/+) cells but not in the p53-null HCT116 cells.