Mariarosaria Conte

Selective class II HDAC inhibitors impair myogenesis by modulating the stability and activity of HDAC–MEF2 complexes

Histone deacetylase (HDAC) inhibitors are promising new epi‐drugs, but the presence of both class I and class II enzymes in HDAC complexes precludes a detailed elucidation of the individual HDAC functions. By using the class II‐specific HDAC inhibitor MC1568, we separated class I‐ and class II‐dependent effects and defined the roles of class II enzymes in muscle differentiation in cultured cells and in vivo. MC1568 arrests myogenesis by (i) decreasing myocyte enhancer factor 2D (MEF2D) expression, (ii) by stabilizing the HDAC4–HDAC3–MEF2D complex, and (iii) paradoxically, by inhibiting differentiation‐induced MEF2D acetylation. In vivo MC1568 shows an apparent tissue‐selective HDAC inhibition. In skeletal muscle and heart, MC1568 inhibits the activity of HDAC4 and HDAC5 without affecting HDAC3 activity, thereby leaving MEF2–HDAC complexes in a repressed state. Our results suggest that HDAC class II‐selective inhibitors might have a therapeutic potential for the treatment of muscle and heart diseases.

Synthesis of Benzamides Related to Anacardic Acid and Their Histone Acetyltransferase (HAT) Inhibitory Activities

A group of benzamides related to anacardic acid amide CTPB with alkyl chains of defined length were prepared by a five‐step sequence starting from 2,6‐dihydroxybenzoic acid, and their activities were compared with those reported for the HAT inhibitor anacardic acid (AA). The subset of 4‐cyano‐3‐trifluoromethylphenylbenzamides with shorter chains exhibited activities similar to that of AA, as they behaved as human p300 inhibitors, induced a decrease in histone acetylation levels in immortalized HEK cells, and counteracted the action of the HDAC inhibitor SAHA in MCF7 breast cancer cells. Moreover, an analogue with the shortest alkyl chain induced significant apoptosis at 50 μM in U937 leukemia cells.

Synthesis and biological validation of novel synthetic histone/protein methyltransferase inhibitors.

In eukaryotic cells, genes are complexed with core histones and other chromosomal proteins to form the chromatin. The basic unit of chromatin is the nucleosome, a nucleoprotein particle that consists of 147 base pairs of DNA wrapped around a core of histones (H2A, H2B, H3, and H4). The histone lysineand arginine-rich N-terminal tails protrude out of the histone core and are the sites of many types of post-translational modifications such as acetylation, methylation, and phosphorylation. The post-translational modification of histone tails regulates the level of chromatin condensation, and is in turn important for gene transcription. Histone acetylation is one of the best understood histone modifications. The highly regulated activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs) are responsible for the control of specific acetylation levels. Indeed, actively transcribed regions of chromatin (euchromatin) are hyperacetylated in comparison with condensed regions (heterochromatin), which are not accessible to transcription factors. In this scenario, small molecule inhibitors of HDACs can affect the heritable changes in gene expression of specific genes, and are used as drugs for cancer therapy. Histone methylation has also been shown to be important in establishing stable gene-expression patterns. Histone methylation does not alter the overall charge of the histone tails, but has an influence on basicity, hydrophobicity, and on the affinity for anionic molecules such as DNA. Histone tails can be mono-, di-, and trimethylated on the e-amino group of lysine residues, and either monoor dimethylated on arginine residues. Depending on the context, lysine methylation provides either activating or repressing modification. Thus, trimethylation of Lys9 in histone H3 is associated primarily with transcriptional silencing, whereas Lys4 methylation correlates with transcriptional activation. Moreover, aberrant histone methylation has been linked to a number of human diseases such as cancer. Protein arginine methyltransferases (PRMTs) are grouped into two major classes, type I enzymes catalyzing the formation of asymmetric w-N,N-dimethylarginine tails, and type II enzymes catalyzing the formation of symmetric w-N,N-dimethylarginine tails. To date, no mutations have been identified in PRMTs in tumour cells. However, the coactivator-associated arginine methyltransferase (CARM1/PRMT4) is over-expressed in both grade-3 breast tumours and in hormone-dependent prostate tumours. In addition to their role in histone modification, PRMTs target several proteins involved in cell proliferation, signal transduction, mRNA splicing, RNA transport, and protein–protein interactions. PRMT1 regulates the nuclear cytoplasmic shuttling of the heterogeneous nuclear ribonuleoprotein (hnRNP) Npl3p, and methylates Arg3 in H4 facilitating acetylation of H4 by the HAT p300, which leads to transcriptional activation. CARM1 binds the p160 family of nuclear hormone receptor coactivators, and enhances the nuclear receptor-mediated transcription activation through methylation of H3. Whereas studies on PRMTs are in their infancy, it is likely that they hold crucial roles in chromatin remodelling with regulation of gene expression and cellular processes. As such, PRMTs are likely to provide useful targets in the design of new anticancer agents. In 2004, a series of dyes and dye-like compounds were evaluated as small molecule modulators of PRMT and histone lysine methyltransferase (HKMT) activity. In this screen, AMI-1 was described as the first specific PRMT inhibitor, and AMI-5 was one of the most potent, though less selective, compounds (Figure 1). Recently, the fungal metabolite chaetocin was identified and characterized as the first specific inhibitor of the HKMT SU ACHTUNGTRENNUNG(VAR)3-9 (Figure 1). As a part of our medicinal chemistry project aimed at discovering new entities as small molecule modulators of epigenetic targets, we chose the AMI-5 chemical structure as a template and designed a new series of simplified analogues starting from a pharmacophore hypothesis. In this hypothesis, we identified the presence of two o-bromoor o,o-dibromophenol moieties as crucial for having antimethyltransferase activity, and inserted a hydrophobic spacer between the above fragments. In particular, we prepared a series of substituted 1,5-diphenyl-1,4-pentadien-3-ones 1–12 (Figure 2), in which some of them share two or more bro[a] Prof. A. Mai, Dr. S. Valente, Dr. A. Perrone, Dr. R. Ragno, Dr. S. Simeoni Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Studi Farmaceutici Universit

Targeting Histone Deacetylases in Diseases: Where Are We?

degli Studi di Roma “La Sapienza”, P.le A. Moro 5, 00185 Roma (Italy) Fax: (+39)06-491491 E-mail : antonello.mai@uniroma1.it [b] Dr. D. Cheng, Prof. M. T. Bedford University of Texas M.D. Anderson Cancer Center, Science Park-Research Division, Smithville, Texas 78957 (USA) Fax: (+1)512-237-2475 E-mail : mtbedford@mdanderson.org [c] Prof. G. Sbardella Dipartimento di Scienze Farmaceutiche, Universit

Indole-Derived Psammaplin A Analogues as Epigenetic Modulators with Multiple Inhibitory Activities

degli Studi di Salerno, via Ponte Don Melillo, 84084 Fisciano (SA) (Italy) [d] Prof. G. Brosch Division of Molecular Biology, Biocenter, Innsbruck Medical University, FritzPreglstrasse 3, 6020 Innsbruck (Austria) [e] Dr. A. Nebbioso, Dr. M. Conte, Prof. L. Altucci Dipartimento di Patologia Generale, Seconda Universit

HDAC Inhibitors Repress BARD1 Isoform Expression in Acute Myeloid Leukemia Cells via Activation of miR-19a and/or b

degli Studi di Napoli, vico L. De Crecchio 7, 80138 Napoli (Italy) Fax: (+39)081-450-169 E-mail : lucia.altucci@unina2.it Supporting information for this article is available on the WWW under http://www.chemmedchem.org or from the author. Supporting information includes experimental procedures, characterization data for compounds 1–14, molecular modelling investigation, and further biological data on U937 cell line.

TNF-related apoptosis-inducing ligand: Signalling of a ‘smart’ molecule

SIGNIFICANCE Epigenetic inactivation of pivotal genes involved in cell growth is a hallmark of human pathologies, in particular cancer. Histone acetylation balance obtained through opposing actions of histone deacetylases (HDACs) and histone acetyltransferases is one epigenetic mechanism controlling gene expression and is, thus, associated with disease etiology and progression. Interfering pharmacologically with HDAC activity can correct abnormalities in cell proliferation, migration, vascularization, and death. RECENT ADVANCES Histone deacetylase inhibitors (HDACi) represent a new class of cytostatic agents that interfere with the function of HDACs and are able to increase gene expression by indirectly inducing histone acetylation. Several HDACi, alone or in combination with DNA-demethylating agents, chemopreventive, or classical chemotherapeutic drugs, are currently being used in clinical trials for solid and hematological malignancies, and are, thus, promising candidates for cancer therapy. CRITICAL ISSUES (i) Non-specific (off-target) HDACi effects due to activities unassociated with HDAC inhibition. (ii) Advantages/disadvantages of non-selective or isoform-directed HDACi. (iii) Limited number of response-predictive biomarkers. (iv) Toxicity leading to dysfunction of critical biological processes. FUTURE DIRECTIONS Selective HDACi could achieve enhanced clinical utility by reducing or eliminating the serious side effects associated with current first-generation non-selective HDACi. Isoform-selective and pan-HDACi candidates might benefit from the identification of biomarkers, enabling better patient stratification and prediction of response to treatment.

Context-Selective Death of Acute Myeloid Leukemia Cells Triggered by the Novel Hybrid Retinoid-HDAC Inhibitor MC2392

A SAR study has been carried out around a modified scaffold of the natural product psammaplin A obtained by replacing the o-bromophenol unit by an indole ring. A series of indole psammaplin A constructs were generated in a short synthetic sequence that starts with the functionalization of the C3 indole position with in situ generated nitrosoacrylate, and this is followed by protection of the β-indole-α-oximinoesters, saponification, condensation with symmetrical diamines, and deprotection. Biochemical and cellular characterization using U937 and MCF-7 cells confirmed that many of these analogues displayed more potent actitivies than the parent natural product. Moreover, in addition to the reported HDAC and DNMT dual epigenetic inhibitory profile of the parent compound, some analogues, notably 4a (UVI5008), also inhibited the NAD(+)-dependent SIRT deacetylase enzymes. The SAR study provides structural insights into the mechanism of action of these multiple epigenetic ligands and paves the way for additional structural exploration to optimize their pharmacological profiles. Because of their multi(epi)target features and their action in ex vivo samples, the indole-based psammaplin A derivatives are attractive molecules for the modulation of epigenetic disorders.

Molecular pathways: the complexity of the epigenome in cancer and recent clinical advances.

Over the past years BARD1 (BRCA1-associated RING domain 1) has been considered as both a BRCA1 (BReast Cancer susceptibility gene 1, early onset) interactor and tumor suppressor gene mutated in breast and ovarian cancers. Despite its role as a stable heterodimer with BRCA1, increasing evidence indicates that BARD1 also has BRCA1-independent oncogenic functions. Here, we investigate BARD1 expression and function in human acute myeloid leukemias and its modulation by epigenetic mechanism(s) and microRNAs. We show that the HDACi (histone deacetylase inhibitor) Vorinostat reduces BARD1 mRNA levels by increasing miR-19a and miR-19b expression levels. Moreover, we identify a specific BARD1 isoform, which might act as tumor diagnostic and prognostic markers.

Molecular analysis of the apoptotic effects of BPA in acute myeloid leukemia cells

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor super-family and signals via two death receptors, TRAIL-R1 and TRAIL-R2, and two decoy receptors, TRAIL-R3 and TRAIL-R4, differently expressed in normal and cancer cells. TRAIL is mainly studied for its capacity to induce apoptosis preferentially in cancer cells. TRAIL is expressed in a variety of human tissues, in particular in the lymphoid system, suggesting a strong physiological role in the innate immunity. This review will focus on TRAIL gene structure and regulation, protein folding, tissue expression and molecular signalling. Finally, the potential use of TRAIL as anticancer treatment alone or in combination therapy as well as the use of drugs which signal via TRAIL and its receptors will be analyzed.