Synthesis and structure-activity relationship of histone deacetylase (HDAC) inhibitors with triazole-linked cap group

Histone deacetylase (HDAC) inhibition is a recent, clinically validated therapeutic strategy for cancer treatment. Small molecule HDAC inhibitors identified so far fall in to three distinct structural motifs: the zinc-binding group (ZBG), a hydrophobic linker, and a recognition cap group. Here we report the suitability of a 1,2,3-triazole ring as a surface recognition cap group-linking moiety in suberoylanilide hydroxamic acid-like (SAHA-like) HDAC inhibitors. Using “click” chemistry (Huisgen cycloaddition reaction), several triazole-linked SAHA-like hydroxamates were synthesized. Structure–activity relationship revealed that the position of the triazole moiety as well as the identity of the cap group markedly affected the in vitro HDAC inhibition and cell growth inhibitory activities of this class of compounds.     

The new low-toxic histone deacetylase inhibitor S-(2) induces apoptosis in various acute myeloid leukaemia cells

Histone deacetylase inhibitors (HDACi) induce tumour cell cycle arrest and/or apoptosis, and some of them are currently used in cancer therapy. Recently, we described a series of powerful HDACi characterized by a 1,4-benzodiazepine (BDZ) ring hybridized with a linear alkyl chain bearing a hydroxamate function as Zn(++)--chelating group. Here, we explored the anti-leukaemic properties of three novel hybrids, namely the chiral compounds (S)-2 and (R)-2, and their non-chiral analogue 4, which were first comparatively tested in promyelocytic NB4 cells. (S)-2 and partially 4--but not (R)-2--caused G0/G1 cell-cycle arrest by up-regulating cyclin G2 and p21 expression and down-regulating cyclin D2 expression, and also apoptosis as assessed by cell morphology and cytofluorimetric assay, histone H2AX phosphorylation and PARP cleavage. Notably, these events were partly prevented by an anti-oxidant. Moreover, novel HDACi prompted p53 and α-tubulin acetylation and, consistently, inhibited HDAC1 and 6 activity. The rank order of potency was (S)-2 > 4 > (R)-2, reflecting that of other biological assays and addressing (S)-2 as the most effective compound capable of triggering apoptosis in various acute myeloid leukaemia (AML) cell lines and blasts from patients with different AML subtypes. Importantly, (S)-2 was safe in mice (up to 150 mg/kg/week) as determined by liver, spleen, kidney and bone marrow histopathology; and displayed negligible affinity for peripheral/central BDZ-receptors. Overall, the BDZ-hydroxamate (S)-2 showed to be a low-toxic HDACi with powerful anti-proliferative and pro-apototic activities towards different cultured and primary AML cells, and therefore of clinical interest to support conventional anti-leukaemic therapy.     

Acetylation of retinal histones in diabetes increases inflammatory proteins: effects of minocycline and manipulation of histone acetyltransferase (HAT) and histone deacetylase (HDAC)

Histone acetylation was significantly increased in retinas from diabetic rats, and this acetylation was inhibited in diabetics treated with minocycline, a drug known to inhibit early diabetic retinopathy in animals. Histone acetylation and expression of inflammatory proteins that have been implicated in the pathogenesis of diabetic retinopathy were increased likewise in cultured retinal Müller glia grown in a diabetes-like concentration of glucose. Both the acetylation and induction of the inflammatory proteins in elevated glucose levels were significantly inhibited by inhibitors of histone acetyltransferase (garcinol and antisense against the histone acetylase, p300) or activators of histone deacetylase (theophylline and resveratrol) and were increased by the histone deacetylase inhibitor, suberolylanilide hydroxamic acid. We conclude that hyperglycemia causes acetylation of retinal histones (and probably other proteins) and that the acetylation contributes to the hyperglycemia-induced up-regulation of proinflammatory proteins and thereby to the development of diabetic retinopathy.     

The role of porcine thyroid peroxidase and FAD-containing monooxygenase in the metabolism of 1-methyl-2-thioimidazole (methimazole)

The peroxidase and FAD-containing monooxygenase activities of porcine thyroid subcellular preparations were measured and it was observed that FAD-containing monooxygenase activity was considerably lower than that of peroxidase. The end product of 1-methyl-2[14C]thioimidazole oxidation catalysed by thyroid peroxidase was confirmed to be 1-methylimidazole by mass spectrometry. In the presence of thyroid peroxidase 1-methyl-2-thioimidazole would appear initially to be oxidised to bis(1-methylimidazole)-2,2'-disulphide. The extent of oxidation was dependent on the iodide concentration in the reaction mixture.     

The role of MutY homolog (Myh1) in controlling the histone deacetylase Hst4 in the fission yeast Schizosaccharomyces pombe

The DNA glycosylase MutY homolog (Myh1) excises adenines misincorporated opposite guanines or 7,8-dihydro-8-oxo-guanines on DNA by base excision repair thereby preventing G:C to T:A mutations. Schizosaccharomyces pombe (Sp) Hst4 is an NAD⁺-dependent histone/protein deacetylase involved in gene silencing and maintaining genomic integrity. Hst4 regulates deacetylation of histone 3 Lys56 at the entry and exit points of the nucleosome core particle. Here, we demonstrate that the hst4 mutant is more sensitive to H₂O₂ than wild-type cells. H₂O₂ treatment results in an SpMyh1-dependent decrease in SpHst4 protein level and hyperacetylation of histone 3 Lys56. Furthermore, SpHst4 interacts with SpMyh1 and the cell cycle checkpoint Rad9-Rad1-Hus1 (9-1-1) complex. SpHst4, SpMyh1, and SpHus1 are physically bound to telomeres. Following oxidative stress, there is an increase in the telomeric association of SpMyh1. Conversely, the telomeric association of spHst4 is decreased. Deletion of SpMyh1 strongly abrogated telomeric association of SpHst4 and SpHus1. However, telomeric association of SpMyh1 is enhanced in hst4Δ cells in the presence of chronic DNA damage. These results suggest that SpMyh1 repair regulates the functions of SpHst4 and the 9-1-1 complex in maintaining genomic stability.     

Replacing dietary glucose with fructose increases ChREBP activity and SREBP-1 protein in rat liver nucleus

Diets high in fructose cause hypertriglyceridemia and insulin resistance in part due to simultaneous induction of gluconeogenic and lipogenic genes in liver. We investigated the mechanism underlying the unique pattern of gene induction by dietary fructose. Male Sprague-Dawley rats (n = 6 per group) were meal-fed (4 h/d) either 63% (w/w) glucose or 63% fructose diet. After two weeks, animals were killed at the end of the last meal. Nuclear SREBP-1 was 2.2 times higher in fructose-fed rats than glucose-fed rats. Nuclear FoxO1 was elevated 1.7 times in fructose group, but did not reach significance (P = 0.08). Unexpectedly, no difference was observed in nuclear ChREBP between two groups. However, ChREBP DNA binding was 3.9× higher in fructose-fed animals without an increase in xylulose-5-phospate, a proposed ChREBP activator. In conclusion, the gene induction by dietary fructose is likely to be mediated in part by simultaneously increased ChREBP activity, SREBP-1 and possibly FoxO1 protein in nucleus.     

Dual regulation of macrophage migration inhibitory factor (MIF) expression in hypoxia by CREB and HIF-1

Macrophage migration inhibitory factor (MIF) is a well-described pro-inflammatory mediator that has also been implicated in the process of oncogenic transformation and tumor progression. However, despite the compelling evidence that MIF is overexpressed in, and contributes to, the pathology of inflammatory and malignant diseases the mechanisms that contribute to exaggerated expression of MIF have been poorly described. Here we show that hypoxia, and specifically HIF-1alpha, is a potent and rapid inducer of MIF expression. In addition, we demonstrate that hypoxia-induced MIF expression is dependent upon a HRE in the 5'UTR of the MIF gene but is further modulated by CREB expression. We propose a model where hypoxia-induced MIF expression is driven by HIF-1 but amplified by hypoxia-induced degradation of CREB. Given the importance of MIF in inflammatory and malignant diseases these data reveal a HIF-1-mediated pathway as a potential therapeutic target for suppression of MIF expression in hypoxic tissues.     

15-Deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) mediates repression of TNF-alpha by decreasing levels of acetylated histone H3 and H4 at its promoter

Thirty-nine missense mutations, which had been identified in rod monochromacy or related disorders, in the CNGA3 subunit of cone photoreceptor cGMP-gated channels were analyzed. HEK293 cells were transfected with cDNA of the human CNGA3 subunit harboring each of these mutations in an expression vector. Patch-clamp recordings demonstrated that 32 of the 39 mutants did not show cGMP-activated current, suggesting that these 32 mutations cause a loss of function of the channels. From the remaining 7 mutants that showed cGMP-activated current, two mutations in the cyclic nucleotide-binding domain, T565M or E593K, were further studied. The half-maximal activating concentration (K(1/2)) for cGMP in the homomeric CNGA3-T565M channels (160microM) was 17.8-fold higher than that of the homomeric wild-type CNGA3 channels (9.0microM). Conversely, the K(1/2) for cGMP in the homomeric CNGA3-E593K channels (3.0microM) was 3-fold lower than that of the homomeric wild-type CNGA3 channels. These results suggest that the T565M and E593K mutations alter the apparent affinity for cGMP of the channels to cause cone dysfunction, resulting in rod monochromacy.     

Expression and function of fibroblast growth factor (FGF) 9 in hepatic stellate cells and its role in toxic liver injury

Hepatic injury and regeneration of the liver are associated with activation of hepatic stellate cells (HSC). Fibroblast growth factors (FGFs) and their receptors are important regulators of repair in various tissues. HSC express FGFR3IIIc as well as FGFGR4 and different spliced FGFR1IIIc and FGFR2IIIc isoforms which differ in the presence or absence of the acid box and of the first Ig-like domain. Expression of FGF9, known to be capable to activate the HSC FGFR2/3-isoforms, was increased in HSC in liver slice cultures after exposition to carbon tetrachloride, as an acute liver injury model. FGF9 significantly stimulated 3-H thymidine incorporation of hepatocytes, but failed to induce DNA synthesis in HSC despite the fact that FGF9 induced a sustained activation of extracellular signal-related kinases (ERK) 1/2. FGF9 induced an increased phosphorylation of Tyr436 of the fibroblast growth factor receptor substrate (FRS) 2, while phosphorylation of Tyr196 which is required for efficient Grb2 recruitment remained unchanged. Our findings suggest that HSC FGF9 provide a paracrine mitogenic signal to hepatocytes during acute liver injury, while the autocrine FGF9 signaling appears to be not sufficient to induce cell proliferation.     

突变型人HGF(tvNK1)对CCl_4诱导的大鼠肝纤维化的影响

目的:探讨突变型人肝细胞生长因子(HGF~(K132E,R134E),tvNK1)对四氯化碳(CCl_4)诱导的SD大鼠肝纤维化的影响。方法:生物发酵大量制备tvNK1,并经腹腔注射于CCl_4诱导的纤维化SD大鼠体内,6周后取肝脏组织,通过real-time PCR和Western blot检测tvNK1对纤维化SD大鼠肝脏中I型胶原蛋白(Collagen type I,Col1A1)、IV型胶原蛋白(Collagen type IV,Col4A1)和α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)在mRNA和蛋白水平表达的影响,并进一步通过HE和Masson染色观察tvNK1对纤维化SD大鼠肝脏形态和肝组织胶原纤维的影响。结果:SDSPAGE检测结果显示,获得纯度≥95%的tvNK1。Real-time PCR和Western blot结果显示,tvNK1降低纤维化大鼠肝脏中Col1A1、Col4A1和α-SMA在mRNA和蛋白水平上的表达。HE和Masson染色结果显示,tvNK1减缓纤维化大鼠肝结构的病理性改变,并降低肝脏中胶原纤维含量。结论:tvNK1能抑制CCl_4诱导的大鼠肝纤维化,为预防肝纤维化和其他器官纤维化疾病提供实验依据。