Silvia Simeoni

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

Design, Synthesis and Biological Evaluation of Carboxy Analogues of Arginine Methyltransferase Inhibitor 1 (AMI-1)

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

3-D QSAR studies on histone deacetylase inhibitors. A GOLPE/GRID approach on different series of compounds

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

Small-molecule interferon inducers. Toward the comprehension of the molecular determinants through ligand-based approaches.

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.

New pyrrole-based histone deacetylase inhibitors: binding mode, enzyme- and cell-based investigations.

Here we report the synthesis of a number of compounds structurally related to arginine methyltransferase inhibitor 1 (AMI‐1). The structural alterations that we made included: 1) the substitution of the sulfonic groups with the bioisosteric carboxylic groups; 2) the replacement of the ureidic function with a bis‐amidic moiety; 3) the introduction of a N‐containing basic moiety; and 4) the positional isomerization of the aminohydroxynaphthoic moiety. We have assessed the biological activity of these compounds against a panel of arginine methyltransferases (fungal RmtA, hPRMT1, hCARM1, hPRMT3, hPRMT6) and a lysine methyltransferase (SET7/9) using histone and nonhistone proteins as substrates. Molecular modeling studies for a deep binding‐mode analysis of test compounds were also performed. The bis‐carboxylic acid derivatives 1 b and 7 b emerged as the most effective PRMT inhibitors, both in vitro and in vivo, being comparable or even better than the reference compound (AMI‐1) and practically inactive against the lysine methyltransferase SET7/9.

Investigation of a potential mechanism for the inhibition of SmTGR by Auranofin and its implications for Plasmodium falciparum inhibition

Docking simulation and three-dimensional quantitative structure-activity relationships (3D-QSARs) analyses were conducted on four series of HDAC inhibitors. The studies were performed using the GRID/GOLPE combination using structure-based alignment. Twelve 3-D QSAR models were derived and discussed. Compared to previous studies on similar inhibitors, the present 3-D QSAR investigation proved to be of higher statistical value, displaying for the best global model r2, q2, and cross-validated SDEP values of 0.94, 0.83, and 0.41, respectively. A comparison of the 3-D QSAR maps with the structural features of the binding site showed good correlation. The results of 3D-QSAR and docking studies validated each other and provided insight into the structural requirements for anti-HDAC activity. To our knowledge this is the first 3-D QSAR application on a broad molecular diversity training set of HDACIs.

Aroyl-Pyrrolyl Hydroxyamides: Influence of Pyrrole C4-Phenylacetyl Substitution on Histone Deacetylase Inhibition†

Hepatitis C is becoming an increasingly common cause of mortality especially in the HIV-coinfected group. Due to the efficacy of interferon (IFN) based therapy in the treatment of hepatitis C, various compounds possessing IFN-inducing activity have been hitherto reported. In the present study, we describe how steric, electrostatic, hydrophobic, and hydrogen-bonding interactions might influence the biological activity of a published set of IFN inducers, using a three-dimensional quantitative structure-activity relationship (3-D QSAR) approach. Analyses were conducted evaluating different series of compounds structurally related to 8-hydroxyadenines and 1H-imidazo[4,5-c]quinolines. A ligand-based alignment protocol in combination with the GRID/GOLPE approach was applied: 62 3-D QSAR models were derived using different GRID probes and several training sets. Performed 3-D QSAR investigations proved to be of good statistical value displaying r2, q2CV-LOO, and cross-validated SDEP values of 0.73, 0.61, 0.61 and 0.89, 0.64, 0.58 using the OH or the DRY probe, respectively. Additionally, the predictive performance was evaluated using an external test set of 20 compounds. Analyses of the resulting models led to the definition of a pharmacophore model that can be of interest to explain the observed affinities of known compounds as well as to design novel low molecular weight IFN inducers (IFNIs). To the best of our knowledge, this is the first 3-D QSAR application on IFN-inducing agents.

Identification of the Schistosoma mansoni molecular target for the antimalarial drug artemether.

Aroyl-pyrrolyl-hydroxy-amides (APHAs) are a class of synthetic HDAC inhibitors described by us since 2001. Through structure-based drug design, two isomers of the APHA lead compound 1, the 3-(2-benzoyl-1-methyl-1H-pyrrol-4-yl)-N-hydroxy-2-propenamide 2 and the 3-(2-benzoyl-1-methyl-1H-pyrrol-5-yl)-N-hydroxy-2-propenamide 3 (iso-APHAs) were designed, synthesized and tested in murine leukemia cells as antiproliferative and cytodifferentiating agents. To improve their HDAC activity and selectivity, chemical modifications at the benzoyl moieties were investigated and evaluated using three maize histone deacetylases: HD2, HD1-B (class I human HDAC homologue), and HD1-A (class II human HDAC homologue). Docking experiments on HD1-A and HD1-B homology models revealed that the different compounds selectivity profiles could be addressed to different binding modes as observed for the reference compound SAHA. Smaller hydrophobic cap groups improved class II HDAC selectivity through the interaction with HD1-A Asn89-Ser90-Ile91, while bulkier aromatic substituents increased class I HDAC selectivity. Taking into account the whole enzyme data and the functional test results, the described iso-APHAs showed a behaviour of class I/IIb HDACi, with 4b and 4i preferentially inhibiting class IIb and class I HDACs, respectively. When tested in the human leukaemia U937 cell line, 4i showed altered cell cycle (S phase arrest), joined to high (51%) apoptosis induction and significant (21%) differentiation activity.

Novel Cinnamyl Hydroxyamides and 2-Aminoanilides as Histone Deacetylase Inhibitors: Apoptotic Induction and Cytodifferentiation Activity

Schistosoma mansoni and Plasmodium falciparum are pathogen parasites that spend part of their lives in the blood stream of the human host and are therefore heavily exposed to fluxes of toxic reactive oxygen species (ROS). SmTGR, an essential enzyme of the S. mansoni ROS detoxification machinery, is known to be inhibited by Auranofin although the inhibition mechanism has not been completely clarified. Auranofin also kills P. falciparum, even if its molecular targets are unknown. Here, we used computational and docking techniques to investigate the molecular mechanism of interaction between SmTGR and Auranofin. Furthermore, we took advantage of the homology relationship and of docking studies to assess if PfTR, the SmTGR malaria parasite homologue, can be a putative target for Auranofin. Our findings support a recently hypothesized molecular mechanism of inhibition for SmTGR and suggest that PfTR is indeed a possible and attractive drug target in P. falciparum.

Histone deacetylation in epigenetics: an attractive target for anticancer therapy.

The novel aroyl‐pyrrolyl hydroxyamides 4 a–a′ are analogues of the lead compound 3‐(1‐methyl‐4‐phenylacetyl‐1H‐pyrrol‐2‐yl)‐N‐hydroxy‐2‐propenamide (2) and are active as HDAC inhibitors. The benzene ring of 2 was substituted with a wide range of electron‐donating and electron‐withdrawing groups, and the effect was evaluated on three HDACs from maize, namely HD2, HD1‐B (a class I HDAC), and HD1‐A (a class II HDAC). Inhibition studies show that the benzene 3′ and, to a lesser extent, 4′ positions of 2 were the most suitable for the introduction of substituents, with the 3′‐chloro (in 4 b) and the 3′‐methyl (in 4 k) derivatives being the most potent compounds, reaching the same activity as SAHA. Inhibition data for 4 b,k against mouse HDAC1 were consistent with those observed in the maize enzyme. The substituent insertion on the benzene ring of 2 (compounds 4 a–a′) abated the slight (3‐fold) selectivity for class II HDACs displayed by 2. Compound 4 b showed interesting, dose‐dependent antiproliferative and cytodifferentiation properties against human acute promyelocytic leukemia HL‐60 cells.