Histone deacetylases are promising molecular targets for the development of antitumor agents. A novel series of histone deacetylase inhibitors of the hydroxamic acid type were synthesized for structure-activity studies. Thirteen tricyclic dibenzodiazepine, -oxazepine, and -thiazepine analogues were studied and shown to induce variable degrees of histone H3/H4 and tubulin acetylation in a cellular model of myeloid leukemia sensitive to all-trans retinoic acid (ATRA). Multiparametric correlations between acetylation of the three substrates, tumor cell growth inhibition, and ATRAdependent cytodifferentiation were performed, providing information on the chemical functionalities governing these activities. For two analogues, antitumor activity in the animal was demonstrated.KEYWORDS HDAC inhibitors, hydroxamic acid, retinoic acid, antiproliferative effect, differentiation H istone deacetylase (HDAC) proteins are classified in four groups (class I-IV) based on function and sequence similarity. A common observation in neoplastic cells is high level expression of class I and II HDACs with corresponding hypoacetylation of histones.1 Increased HDAC activity may play a critical role in the pathogenesis of leukemia.2 HDACs of significance for cancer cell biology reside not only in the nucleus but also in the cytoplasm, where they act on substrates other than histones. Within class I, HDACs 1, 2, and 8 are primarily found in the nucleus. Class II HDACs (HDAC 4, 5, 6, 7, 9, and 10) are able to shuttle in and out of the nucleus depending on different signals.3 HDAC 6 is primarily a cytoplasmic enzyme, deacetylating proteins like tubulin, Hsp90, and cortactin. 4 Transformed cells are generally more sensitive to HDAC inhibitor-induced growth inhibition and apoptosis than their normal counterparts.5 Hence, anticancer therapeutic strategies based on HDAC inhibitors have raised significant interest. Some of the most powerful natural and synthetic HDAC inhibitors 6 are derivatives of hydroxamic acid. Here, we describe the chemical synthesis and the pharmacologic characterization of a novel series of hydroxamic acid derivatives characterized by tricyclic dibenzo-diazepine, -oxazepine, and -thiazepine rings. As the catalytic domain of all HDAC isoforms is highly conserved, 7 our approach was to design novel molecules targeting the variable external part of the channel leading to the catalytic center. We studied the effects of a limited number of systematic modifications to the tricyclic core, keeping the hydroxamic group and the linker chain of our molecules constant. The tricyclic core was selected, as structural modifications affecting hydrogen bonding, electronic features, and angles between the two aromatic rings can be easily obtained by accessible chemical modifications. Test compounds (0.01, 0.1, 1.0, 10, and 50 μM) were initially screened using an enzymatic assay measuring the total HDAC activity in HeLa cell extracts, which resulted in the selection of 13 molecules. Scheme 1 illustrates the synthesis and structures o...

Retinoid-related molecules (RRM) are novel agents with tumor-selective cytotoxic/antiproliferative activity, a different mechanism of action from classic retinoids and no cross-resistance with other chemotherapeutics. ST1926 and CD437 are prototypic RRMs, with the former currently undergoing phase I clinical trials. We show here that ST1926, CD437, and active congeners cause DNA damage. Cellular and subcellular COMET assays, H2AX phosphorylation (;-H2AX), and scoring of chromosome aberrations indicate that active RRMs produce DNA double-strand breaks (DSB) and chromosomal lesions in NB4, an acute myeloid leukemia (AML) cell line characterized by high sensitivity to RRMs. There is a direct quantitative correlation between the levels of DSBs and the cytotoxic/antiproliferative effects induced by RRMs. NB4.437r blasts, which are selectively resistant to RRMs, do not show any sign of DNA damage after treatment with ST1926, CD437, and analogues. DNA damage is the major mechanism underlying the antileukemic activity of RRMs in NB4 and other AML cell lines. In accordance with the S-phase specificity of the cytotoxic and antiproliferative responses of AML cells to RRMs, increases in DSBs are maximal during the S phase of the cell cycle. Induction of DSBs precedes inhibition of DNA replication and is associated with rapid activation of ataxia telangectasia mutated, ataxia telangectasia RAD3-related, and DNAdependent protein kinases with subsequent stimulation of the p38 mitogen-activated protein kinase. Inhibition of ataxia telangectasia mutated and DNA-dependent protein kinases reduces phosphorylation of H2AX. Cells defective for homologous recombination are particularly sensitive to ST1926, indicating that this process is important for the protection of cells from the RRM-dependent DNA damage and cytotoxicity. [Mol Cancer Ther 2008;7(9):2941 -54]

The peptidyl-prolyl-isomerase Pin1 interacts with phosphorylated proteins, altering their conformation. The retinoic acid receptor RARA and the acute-promyelocytic-leukemia-specific counterpart PML-RARA directly interact with Pin1. Overexpression of Pin1 inhibits ligand-dependent activation of RARA and PML-RARA. Inhibition is relieved by Pin1-targeted short interfering RNAs and by pharmacologic inhibition of the catalytic activity of the protein. Mutants of Pin1 catalytically inactive or defective for client-protein-binding activity are incapable of inhibiting ligand-dependent RARA transcriptional activity. Functional inhibition of RARA and PML-RARA by Pin1 correlates with degradation of the nuclear receptors via the proteasome-dependent pathway. In the acute myelogenous leukemia cell lines HL-60 and NB4, Pin1 interacts with RARA in a constitutive fashion. Suppression of Pin1 by a specific short hairpin RNA in HL-60 or NB4 cells stabilizes RARA and PML-RARA, resulting in increased sensitivity to the cytodifferentiating and antiproliferative activities of all-trans retinoic acid. Treatment of the two cell lines and freshly isolated acute myelogenous leukemia blasts (M1 to M4) with ATRA and a pharmacologic inhibitor of Pin1 causes similar effects. Our results add a further layer of complexity to the regulation of nuclear retinoic acid receptors and suggest that Pin1 represents an important target for strategies aimed at increasing the therapeutic index of retinoids. [Cancer Res 2009;69(3):1016-26]

Aldehyde oxidases are molybdo-flavoenzymes structurally related to xanthine oxidoreductase. They catalyze the oxidation of aldehydes or N-heterocycles of physiological, pharmacological, and toxicological relevance. Rodents are characterized by four aldehyde oxidases as follows: AOX1 and aldehyde oxidase homologs 1-3 (AOH1, AOH2, and AOH3). Humans synthesize a single functional aldehyde oxidase, AOX1. Here we define the structure and the characteristics of the aldehyde oxidase genes and proteins in chicken and dog. The avian genome contains two aldehyde oxidase genes, AOX1 and AOH, mapping to chromosome 7. AOX1 and AOH are structurally very similar and code for proteins whose sequence was deduced from the corresponding cDNAs. AOX1 is the ortholog of the same gene in mammals, whereas AOH represents the likely ancestor of rodent AOH1, AOH2, and AOH3. The dog genome is endowed with two structurally conserved and active aldehyde oxidases clustering on chromosome 37. Cloning of the corresponding cDNAs and tissue distribution studies demonstrate that they are the orthologs of rodent AOH2 and AOH3. The vestiges of dog AOX1 and AOH1 are recognizable upstream of AOH2 and AOH3 on the same chromosome. Comparison of the complement and the structure of the aldehyde oxidase and xanthine oxidoreductase genes in vertebrates and other animal species indicates that they evolved through a series of duplication and inactivation events. Purification of the chicken AOX1 protein to homogeneity from kidney demonstrates that the enzyme possesses retinaldehyde oxidase activity. Unlike humans and most other mammals, dog and chicken are devoid of liver aldehyde oxidase activity. Molybdo-flavoenzymes (MOFEs)4 constitute a small family of homodimeric oxidoreductases characterized by conserved structures (1). Until a few years ago, it was believed that the family of mammalian MOFEs consisted of only two members, i.e. xanthine oxidoreductase (XOR) (2-4) and the aldehyde oxidase AOX1 5 (5-8). XOR has been extensively studied and is the key enzyme in the catabolism of purines, oxidizing hypoxanthine to xanthine and xanthine to uric acid (9 -14). This function is conserved throughout evolution, as the enzyme is present from bacteria to man (1). In mammals, the protein also plays an important role in milk secretion (15-17) and kidney development (18). The function of AOX1 is ill-defined, and the enzyme lacks a recognized physiological substrate. AOX1 metabolizes N-heterocyclic compounds and aldehydes of pharmacological and toxicological relevance (19 -22). XOR and AOX1 are the products of two genes mapping on distinct chromosomes in rodents and different arms of chromosome 2 in humans (4,7,23,24).Recently, we demonstrated that the family of mammalian MOFEs is larger than originally anticipated (25-28). Mice and rats are endowed with three extra MOFEs structurally and biochemically more similar to AOX1 than to XOR. We named these proteins aldehyde oxidase homologs 1-3 (AOH1, AOH2, and AOH3). In rodents, AOH1 is synthesized predominantly in liver and...

Supplementary Figures 6-9 from Inhibition of the Peptidyl-Prolyl-Isomerase Pin1 Enhances the Responses of Acute Myeloid Leukemia Cells to Retinoic Acid via Stabilization of RARα and PML-RARα