Rino Ragno

Effects of Mentha suaveolens Essential Oil Alone or in Combination with Other Drugs in Candida albicans

Candidosis is the most important cause of fungal infections in humans. The yeast Candida albicans can form biofilms, and it is known that microbial biofilms play an important role in human diseases and are very difficult to treat. The prolonged treatment with drugs has often resulted in failure and resistance. Due to the emergence of multidrug resistance, alternatives to conventional antimicrobial therapy are needed. This study aims to analyse the effects induced by essential oil of Mentha suaveolens Ehrh (EOMS) on Candida albicans and its potential synergism when used in combination with conventional drugs. Morphological differences between control and EOMS treated yeast cells or biofilms were observed by scanning electron microscopy and transmission electron microscopy (SEM and TEM resp.,). In order to reveal the presence of cell cycle alterations, flow cytometry analysis was carried out as well. The synergic action of EOMS was studied with the checkerboard method, and the cellular damage induced by different treatments was analysed by TEM. The results obtained have demonstrated both the effects of EOMS on C. albicans yeast cells and biofilms and the synergism of EOMS when used in combination with conventional antifungal drugs as fluconazole (FLC) and micafungin (MCFG), and therefore we can hypothesize on its potential use in therapy. Further studies are necessary to know its mechanism of action.

3-(4-Aroyl-1-methyl-1H-2-pyrrolyl)-N-hydroxy-2-propenamides as a New Class of Synthetic Histone Deacetylase Inhibitors. 2. Effect of Pyrrole-C2 and/or -C4 Substitutions on Biological Activity

Previous SAR studies (Part 1: Mai, A.; et al. J. Med. Chem. 2003, 46, 512-524) performed on some portions (pyrrole-C4, pyrrole-N1, and hydroxamate group) of 3-(4-benzoyl-1-methyl-1H-pyrrol-2-yl)-N-hydroxy-2-propenamide (1a) highlighted its 4-phenylacetyl (1b) and 4-cynnamoyl (1c) analogues as more potent compounds in inhibiting maize HD2 activity in vitro. In the present paper, we investigated the effect on anti-HD2 activity of chemical substitutions performed on the pyrrole-C2 ethene chains of 1a-c, which were replaced with methylene, ethylene, substituted ethene, and 1,3-butadiene chains (compounds 2). Biological results clearly indicated the unsubstituted ethene chain as the best structural motif to get the highest HDAC inhibitory activity, the sole exception to this rule being the introduction of the 1,3-butadienyl moiety into the 1a chemical structure (IC50(2f) = 0.77 microM; IC50(1a) = 3.8 microM). IC50 values of compounds 3, prepared as 1b homologues, revealed that between benzene and carbonyl groups at the pyrrole-C(4) position a hydrocarbon spacer length ranging from two to five methylenes is well accepted by the APHA template, being that 3a (two methylenes) and 3d (five methylenes) are more potent (2.3- and 1.4-fold, respectively) than 1b, while the introduction of a higher number of methylene units (see 3e,f) decreased the inhibitory activities of the derivatives. Particularly, 3a (IC50 = 0.043 microM) showed the same potency as SAHA in inhibiting HD2 in vitro, and it was 3000- and 2.6-fold more potent than sodium valproate and HC-toxin and was 4.3- and 6-fold less potent than trapoxin and TSA, respectively. Finally, conformationally constrained forms of 1b,c (compounds 4), prepared with the aim to obtain some information potentially useful for a future 3D-QSAR study, showed the same (4a,b) or higher (4c,d) HD2 inhibiting activities in comparison with those of the reference drugs. Molecular modeling and docking calculations on the designed compounds performed in parallel with the chemistry work fully supported the synthetic effort and gave insights into the binding mode of the more flexible APHA derivatives (i.e., 3a). Despite the difference of potency between 1b and 3a in the enzyme assay, the two APHA derivatives showed similar antiproliferative and cytodifferentiating activities in vivo on Friends MEL cells, being that 3a is more potent than 1b in the differentiation assay only at the highest tested dose (48 microM).

5-alkyl-6-benzyl-2-(2-oxo-2-phenylethylsulfanyl)pyrimidin-4(3H)-ones, a series of anti-HIV-1 agents of the dihydro-alkoxy-benzyl-oxopyrimidine family with peculiar structure - Activity relationship profile

A series of dihydro-alkylthio-benzyl-oxopyrimidines (S-DABOs) bearing a 2-aryl-2-oxoethylsulfanyl chain at pyrimidine C2, an alkyl group at C5, and a 2,6-dichloro-, 2-chloro-6-fluoro-, and 2,6-difluoro-benzyl substitution at C6 (oxophenethyl- S-DABOs, 6-8) is here described. The new compounds showed low micromolar to low nanomolar (in one case subnanomolar) inhibitory activity against wt HIV-1. Against clinically relevant HIV-1 mutants (K103N, Y181C, and Y188L) as well as in enzyme (wt and K103N, Y181I, and L100I mutated RTs) assays, compounds carrying an ethyl/ iso-propyl group at C5 and a 2,6-dichloro-/2-chloro-6-fluoro-benzyl moiety at C6 were the most potent derivatives, also characterized by low fold resistance ratio. Interestingly, the structure-activity relationship (SAR) data drawn from this DABO series are more related to HEPT than to DABO derivatives. These findings were at least in part rationalized by the description of a fair superimposition between the 6-8 and TNK-651 (a HEPT analogue) binding modes in both WT and Y181C RTs.

Combining 3-D Quantitative Structure−Activity Relationship with Ligand Based and Structure Based Alignment Procedures for in Silico Screening of New Hepatitis C Virus NS5B Polymerase Inhibitors

The viral NS5B RNA-dependent RNA-polymerase (RdRp) is one of the best-studied and promising targets for the development of novel therapeutics against hepatitis C virus (HCV). Allosteric inhibition of this enzyme has emerged as a viable strategy toward blocking replication of viral RNA in cell based systems. Herein, we describe how the combination of a complete computational procedure together with biological studies led to the identification of novel molecular scaffolds, hitherto untested toward NS5B polymerase. Structure based 3-D quantitative structure-activity relationship (QSAR) models were generated employing NS5B non-nucleoside inhibitors (NNIs), whose bound conformations were readily available from the protein database (PDB). These were grouped into two training sets of structurally diverse NS5B NNIs, based on their binding to the enzyme thumb (15 NNIs) or palm (10 NNIs) domains. Ligand based (LB) and structure based (SB) alignments were rigorously investigated to assess the reliability on the correct molecular alignment for unknown binding mode modeled compounds. Both Surflex and Autodock programs were able to reproduce with minimal errors the experimental binding conformations of 24 experimental NS5B allosteric inhibitors. Eighty-one (thumb) and 223 (palm) modeled compounds taken from literature were LB and SB aligned and used as external validation sets for the development of 3-D QSAR models. Low error of prediction proved the 3-D QSARs to be useful scoring functions for the in silico screening procedure. Finally, the virtual screening of the NCI Diversity Set led to the selection for enzymatic assays of 20 top-scoring molecules for each final model. Among the 40 selected molecules, preliminary data yielded four derivatives exhibiting IC(50) values ranging between 45 and 75 microM. Binding mode analysis of hit compounds within the NS5B polymerase thumb domain showed that one of them, NSC 123526, exhibited a docked conformation which was in good agreement with the thumb training set most active compound (6).

Effects ofMentha suaveolensEssential Oil onChlamydia trachomatis

Chlamydia trachomatis, the most common cause of sexually transmitted bacterial infection worldwide, has a unique biphasic developmental cycle alternating between the infectious elementary body and the replicative reticulate body. C. trachomatis is responsible for severe reproductive complications including pelvic inflammatory disease, ectopic pregnancy, and obstructive infertility. The aim of our study was to evaluate whether Mentha suaveolens essential oil (EOMS) can be considered as a promising candidate for preventing C. trachomatis infection. Specifically, we investigated the in vitro effects of EOMS towards C. trachomatis analysing the different phases of chlamydial developmental cycle. Our results demonstrated that EOMS was effective towards C. trachomatis, whereby it not only inactivated infectious elementary bodies but also inhibited chlamydial replication. Our study also revealed the effectiveness of EOMS, in combination with erythromycin, towards C. trachomatis with a substantial reduction in the minimum effect dose of antibiotic. In conclusion, EOMS treatment may represent a preventative strategy since it may reduce C. trachomatis transmission in the population and, thereby, reduce the number of new chlamydial infections and risk of developing of severe sequelae.

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 QSAR studies on N-aryl heteroarylisopropanolamines, a new class of non-peptidic HIV-1 protease inhibitors.

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

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

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

Molecular modeling of azole antifungal agents active against Candida albicans. 1. A comparative molecular field analysis study.

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.

Identification of glutathione-methacrylates adducts in gingival fibroblasts and erythrocytes by HPLC-MS and capillary electrophoresis

A series of N-aryl heteroarylisopropanolamines in which an indole or a 3-arylpyrrole moiety was linked to an aryl group through an isopropanolamine linker, were designed and synthesized as potential anti-HIV-1-PR agents. Series was tested for their ability in blocking PR activity. As a rule, indole derivatives of class 1 exhibited more potency than pyrrole analogues of class 2 while tert-butylamide substituents increased anti-PR potency. In fact, bis tert-butylamide 1e showed the highest activity with IC(50)=25 microM. Even if not very potent, a simple class of anti-PR agents, with a facile synthetic pathway was discovered. QSAR studies on isopropanolamines 1 and 2 were performed in comparison with diarylbutanols, a new class of non peptidic anti-PR agents, recently discovered by Agouron Pharmaceuticals. QSAR and CoMFA models based on 30 diarylbutanols used as a training set were developed. The obtained models were used to investigate the binding mode of the newly synthesized derivatives 1 and 2. The results of this study suggest that N-aryl heteroarylisopropanolamines bind to the PR active site similarly to the diarylbutanols of Agouron.