Publications by authors named "Giuseppe Fracchiolla"

33 Publications

Virucidal and antiviral effects of Thymus vulgaris essential oil on feline coronavirus.

Res Vet Sci 2021 Apr 22;137:44-47. Epub 2021 Apr 22.

Department of Veterinary Medicine, University of Aldo Moro of Bari, Valenzano, Italy. Electronic address:

Feline infectious peritonitis (FIP) is a fatal systemic disease of felids caused by a Coronavirus (CoV) (FIPV). In spite of its clinical relevance and impact on feline health, currently the therapeutic possibilities for treatment of FIP in cats are limited. The emergence of the pandemic Severe Respiratory Syndrome (SARS) coronavirus (CoV) type 2 (SARS-CoV-2), etiological agent of the 2019 Coronavirus Disease (COVID-19), able to infect a broad spectrum of animal species including cats, triggered the interest for the development of novel molecules with antiviral activity for treatment of CoV infections in humans and animals. Essential oils (EOs) have raised significant attention for their antiviral properties integrating and, in some cases, replacing conventional drugs. Thymus vulgaris EO (TEO) has been previously shown to be effective against several RNA viruses including CoVs. In the present study the antiviral efficacy of TEO against FIPV was evaluated in vitro. TEO at 27 μg/ml was able to inhibit virus replication with a significant reduction of 2 log10 TCID/50 μl. Moreover, virucidal activity was tested using TEO at 27 and 270 μg/ml, over the cytotoxic threshold, determining a reduction of viral titre as high as 3.25 log10 TCID/50 μl up to 1 h of time contact. These results open several perspectives in terms of future applications and therapeutic possibilities for coronaviruses considering that FIPV infection in cats could be a potential model for the study of antivirals against CoVs.
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http://dx.doi.org/10.1016/j.rvsc.2021.04.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8061179PMC
April 2021

Anti-Biofilm Inhibitory Synergistic Effects of Combinations of Essential Oils and Antibiotics.

Antibiotics (Basel) 2020 Sep 24;9(10). Epub 2020 Sep 24.

Department of Pharmacy-Drug Sciences, University of Bari "Aldo Moro", via E. Orabona, 4-70125 Bari, Italy.

In recent years, the increase of bacteria antibiotic- resistance has been a severe problem for public health. A useful solution could be to join some phytochemicals naturally present in essential oils (EOs) to the existing antibiotics, with the aim to increase their efficacy in therapies. According to in vitro studies, EOs and their components could show such effects. Among them, we studied the activity of , , , and EOs on bacterial biofilm and their synergism when used in association with some common antibiotics such as norfloxacin, oxacillin, and gentamicin. The chemical composition of EOs was determined using gas chromatography (GC) coupled with mass spectrometry (MS) techniques. The EOs drug efficacy was evaluated on four different strains of Gram-positive bacteria forming biofilms. The synergistic effects were tested through the chequerboard microdilution method. The association EOs-antibiotics showed a strong destruction of the biofilm growth of the four bacterial species considered. The interaction of norfloxacin with EOs was the most effective in all the tested combinations against the strains object of this study. These preliminary results suggest the formulation of a new generation of antimicrobial agents based on a combination of antimicrobial compounds with different origin.
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http://dx.doi.org/10.3390/antibiotics9100637DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7598638PMC
September 2020

Virucidal activity of ginger essential oil against caprine alphaherpesvirus-1.

Vet Microbiol 2019 Mar 5;230:150-155. Epub 2019 Feb 5.

Department of Veterinary Medicine, University of Aldo Moro of Bari, Valenzano, Italy.

The emergence of alphaherpesvirus strains resistant to commonly used antiviral drugs has prompted the research for alternative, biologically active anti-herpetic agents. Essential oils (EOs) have shown anti-infective properties against human herpes simplex viruses (HSV-1 and -2). Caprine alphaherpesvirus 1 (CpHV-1) induces genital lesions in its natural host and it is regarded as a useful homologous animal model for the study of HSV-2 infection, chiefly for the assessment of antiviral drugs in in vivo studies. In the present study we evaluated the activity in vitro of ginger EO (GEO) against CpHV-1. GEO was found to be effective as virucide on cell-free virus, inactivating CpHV-1 up to 100%. The virucidal activity of GEO is likely accounted for by disruption of herpesvirus envelope and its associated structures which are necessary for virus adsorption and entry into host cells. On the opposite, GEO was not able to inhibit virus adsorption and/or replication, as treatment of cells before and after infection did not abolish virus infectivity. GEO could be suggested for topical applications in in vivo experiments using CpHV-1/goat model, since the lipophilic nature of EOs favours their adsorption through the cutaneous/mucosal barrier, either alone or in conjunction with other molecules. These findings open several perspectives in terms of therapeutic possibilities for a number of human and animal alphaherpesviruses.
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http://dx.doi.org/10.1016/j.vetmic.2019.02.001DOI Listing
March 2019

Elucidation of the synergistic action of Mentha Piperita essential oil with common antimicrobials.

PLoS One 2018 1;13(8):e0200902. Epub 2018 Aug 1.

Department of Pharmacy-Drug Sciences, University of Bari "A. Moro", Bari, Italy.

Mentha piperita L. essential oil (EO) is employed for external use as antipruritic, astringent, rubefacient and antiseptic. Several studies demonstrated its significant antiviral, antifungal and antibacterial properties. The aim of this work is the study of the synergistic effects of M. piperita EO with antibacterials and antifungals that are widely available and currently prescribed in therapies against infections. The observed strong synergy may constitute a potential new approach to counter the increasing phenomenon of multidrug resistant bacteria and fungi. In vitro efficacy of the association M. piperita EO/drugs was evaluated against a large panel of Gram-positive and Gram-negative bacteria and yeast strains. The antimicrobial effects were studied by checkerboard microdilution method. The synergistic effect of M. piperita EO with gentamicin resulted in a strong growth inhibition for all the bacterial species under study. The synergistic effect observed for M. piperita EO and antifungals was less pronounced.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0200902PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6070247PMC
January 2019

Repositioning of Endonuclear Receptors Binders as Potential Antibacterial and Antifungal Agents. Eptyloxìm: A Potential and Novel Gyrase B and Cytochrome Cyp51 Inhibitor.

Mol Inform 2016 09 12;35(8-9):326-32. Epub 2016 May 12.

Department of Pharmacy - Drug Sciences, University of Bari "Aldo Moro", Via E. Orabona, 4, I-70125, Bari, Italy.

A novel class of antibacterial and antifungal agents is here identified by means of dockings and virtual screening techniques. Biological data proved the initial effort, formulated on the structure similarity of nuclear receptors binders with known quinolones or thiazole derivatives, to reposition PPARs agonists as likely bacterial type II topoisomerases inhibitors.
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http://dx.doi.org/10.1002/minf.201600021DOI Listing
September 2016

Kidney CLC-K chloride channels inhibitors: structure-based studies and efficacy in hypertension and associated CLC-K polymorphisms.

J Hypertens 2016 May;34(5):981-92

aDipartimento di Farmacia - Scienze del Farmaco, Università degli Studi di Bari, Bari bIstituto di Biofisica, CNR, Genova, Italy *Antonella Liantonio and Paola Imbrici contributed equally to the writing of this article.

Objective: Alterations in the handling of renal salt reabsorption may contribute to interindividual differences in blood pressure regulation and susceptibility to hypertension. CLC-K chloride channels and their accessory subunit barttin play a pivotal role in kidney by controlling chloride and water absorption. Compounds selective for CLC-Ks, such as the benzofuran derivative MT-189, may have a significant therapeutic potential. Here, we assessed the feasibility of using CLC-K blockers in hypertension and aimed at enhancing drug inhibitory affinity.

Methods And Results: We demonstrated that acute in-vivo administration of MT-189 to spontaneously hypertensive rats (SHR) caused a reduction of blood pressure and defined the CLC-K/barttin gene expression pattern in kidney of SHR in comparison with normotensive Wistar-Kyoto rats. Based on MT-189, we designed and tested a new series of benzofuran derivatives on CLC-K chloride channels heterologously expressed in HEK293 cells. These studies enabled us to elucidate the causative molecular relationship for obtaining the most potent and selective inhibitor (SRA-36) described so far, with an IC50 of 6.6 ± 1 μmol/l. The biophysical and pharmacological characterization of A447T CLC-Ka and Y315F CLC-Ka, both polymorphisms associated with hypertension, showed that SRA-36 is an efficacious inhibitor of the chloride currents sustained by these polymorphisms. Molecular docking studies allowed hypothesizing an inhibition mechanism for the considered ligands, laying the foundations for the rational design of new and more effective CLC-K inhibitors.

Conclusion: The SRA-36 molecule represents a new potential therapeutic option for hypertension.
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http://dx.doi.org/10.1097/HJH.0000000000000876DOI Listing
May 2016

Synthesis, in vitro evaluation, and molecular modeling investigation of benzenesulfonimide peroxisome proliferator-activated receptors α antagonists.

Eur J Med Chem 2016 May 27;114:191-200. Epub 2016 Feb 27.

Unità di Chimica Farmaceutica, Dipartimento di Farmacia, Università"G. d'Annunzio", Chieti, Italy. Electronic address:

Recent evidences suggest a moderate activation of Peroxisome Proliferator-Activated Receptors (PPARs) could be favorable in metabolic diseases, reducing side effects given from full agonists. PPAR partial agonists and antagonists represent, to date, interesting tools to better elucidate biological processes modulated by these receptors. In this work are reported new benzenesulfonimide compounds able to block PPARα, synthesized and tested by transactivation assays and gene expression analysis. Some of these compounds showed a dose-dependent antagonistic behavior on PPARα, submicromolar potency, different profiles of selectivity versus PPARγ, and a repressive effect on CPT1A expression. Dockings and molecular dynamics on properly selected benzenesulfonimide derivatives furnished fresh insights into the molecular determinant most likely responsible for PPARα antagonism.
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http://dx.doi.org/10.1016/j.ejmech.2016.02.064DOI Listing
May 2016

Design, synthesis and biological evaluation of a class of bioisosteric oximes of the novel dual peroxisome proliferator-activated receptor α/γ ligand LT175.

Eur J Med Chem 2015 Jan 24;90:583-94. Epub 2014 Nov 24.

Dipartimento di Farmacia-Scienze del Farmaco, Università degli Studi di Bari 'Aldo Moro', Via E. Orabona 4, 70125 Bari, Italy. Electronic address:

The effects resulting from the introduction of an oxime group in place of the distal aromatic ring of the diphenyl moiety of LT175, previously reported as a PPARα/γ dual agonist, have been investigated. This modification allowed the identification of new bioisosteric ligands with fairly good activity on PPARα and fine-tuned moderate activity on PPARγ. For the most interesting compound (S)-3, docking studies in PPARα and PPARγ provided a molecular explanation for its different behavior as full and partial agonist of the two receptor isotypes, respectively. A further investigation of this compound was carried out performing gene expression studies on HepaRG cells. The results obtained allowed to hypothesize a possible mechanism through which this ligand could be useful in the treatment of metabolic disorders. The higher induction of the expression of some genes, compared to selective agonists, seems to confirm the importance of a dual PPARα/γ activity which probably involves a synergistic effect on both receptor subtypes.
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http://dx.doi.org/10.1016/j.ejmech.2014.11.044DOI Listing
January 2015

Molecular determinants for nuclear receptors selectivity: chemometric analysis, dockings and site-directed mutagenesis of dual peroxisome proliferator-activated receptors α/γ agonists.

Eur J Med Chem 2013 May 24;63:321-32. Epub 2013 Feb 24.

Dipartimento di Farmacia-Scienze del Farmaco, Università degli studi di Bari 'Aldo Moro', via E. Orabona 4, 70125 Bari, Italy.

A series of previously synthesized chiral derivatives of clofibric and phenylacetic acids, acting as dual agonists towards the peroxisome proliferator-activated receptors (PPARs) α and γ, was taken into account, and the efficacy of these compounds was analyzed by means of 2D-, 3D-QSAR and docking studies with the goal to gain deeper insights into the three-dimensional determinants governing ligands selectivity for PPARs. By multiregressional analysis a correlation between the lipophilicity and PPARα activity was found, whereas for PPARγ the correlation was achieved once efficacy was related to the presence of polar groups on agonists scaffold. Docking of these compounds further corroborated this hypothesis, and then provided a valid support for subsequent chemometric analysis and pharmacophore models development for both receptors subtypes. Computational results suggested site directed mutagenesis experiments which confirmed the importance of amino acid residues in PPAR activity, allowing the identification of critical hotspots most likely taking over PPARs selectivity.
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http://dx.doi.org/10.1016/j.ejmech.2013.02.015DOI Listing
May 2013

Synthesis, biological evaluation and molecular investigation of fluorinated peroxisome proliferator-activated receptors α/γ dual agonists.

Bioorg Med Chem 2012 Mar 28;20(6):2141-51. Epub 2012 Jan 28.

Dipartimento Farmaco-Chimico, Università degli Studi di Bari 'Aldo Moro', Via Orabona 4, 70126 Bari, Italy.

PPARs are transcription factors that govern lipid and glucose homeostasis and play a central role in cardiovascular disease, obesity, and diabetes. Thus, there is significant interest in developing new agonists for these receptors. Given that the introduction of fluorine generally has a profound effect on the physical and/or biological properties of the target molecule, we synthesized a series of fluorinated analogs of the previously reported compound 2, some of which turned out to be remarkable PPARα and PPARγ dual agonists. Docking experiments were also carried out to gain insight into the interactions of the most active derivatives with both receptors.
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http://dx.doi.org/10.1016/j.bmc.2012.01.025DOI Listing
March 2012

Might the observed α(2A)-adrenoreceptor agonism or antagonism of allyphenyline analogues be ascribed to different molecular conformations?

Bioorg Med Chem 2012 Mar 31;20(6):2082-90. Epub 2012 Jan 31.

Scuola di Scienze del Farmaco e dei Prodotti della Salute, Università degli Studi di Camerino, via S. Agostino 1, 62032 Camerino, Italy.

We recently reported that the α(2)-adrenoreceptor (AR) ligand allyphenyline (9) significantly enhanced morphine analgesia (due to its α(2C)-AR agonism), was devoid of sedative side effects (due to its α(2A)-AR antagonism), prevented and reversed morphine tolerance and dependence. To highlight the molecular characteristics compatible with this behaviour and to obtain novel agents potentially useful in chronic pain and opioid addiction management, the allyl group of 9 was replaced by substituents of moderate steric bulk (MR) and positive or negative lipophilic (π) and electronic (σ) contributions in all the possible combinations. Effective novel α(2C)-agonists/α(2A)-antagonists (2, 3, 10, 12, and 17) were obtained. This study also demonstrated that contradictory combinations of the physicochemical parameters were similarly able to induce the α(2A)-activation. Since we had previously observed that the absolute configuration affected only the potency, but not the functional profile of the ligands, we hypothesized that the α(2A)-activation was governed by a ligand preferred conformation. From a structural overlay investigation it emerged that an extended conformation appeared to be associated with dual α(2C)-agonism/α(2A)-antagonism, whereas a folded conformation associated with α(2C)-/α(2A)-agonism.
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http://dx.doi.org/10.1016/j.bmc.2012.01.035DOI Listing
March 2012

In-vivo administration of CLC-K kidney chloride channels inhibitors increases water diuresis in rats: a new drug target for hypertension?

J Hypertens 2012 Jan;30(1):153-67

Section of Pharmacology, Department of Pharmacobiology, Faculty of Pharmacy, University of Bari, Bari, Italy.

Objective: The human kidney-specific chloride channels ClC-Ka (rodent ClC-K1) and ClC-Kb (rodent ClC-K2) are important determinants of renal function, participating to urine concentration and blood pressure regulation mechanisms. Here we tested the hypothesis that these chloride channels could represent new drug targets for inducing diuretic and antihypertensive effects.

Methods: To this purpose, the CLC-K blockers benzofuran derivatives MT-189 and RT-93 (10, 50, 100 mg/kg), were acutely administered by gavage in Wistar rats, and pharmacodynamic and pharmacokinetic parameters determined by functional, bioanalytical, biochemical and molecular biology assays.

Results: Plasma concentration values for MT-189 and RT-93 were indicative of good bioavailability. Both MT-189 and RT-93 dose-dependently increased urine volume without affecting electrolyte balance. A comparable reduction of SBP was observed in rats after MT-189, RT-93 or furosemide administration. Benzofuran derivatives treatment did not affect kidney CLC-K mRNA level or inner medulla osmolality, whereas a significant vasopressin-independent down-regulation of aquaporin water channel type 2 was observed at protein and transcriptional levels. In rats treated with benzofuran derivatives, the observed polyuria was mainly water diuresis; this finding indirectly supports a cross-talk between chloride and water transport in nephron. Moreover, preliminary in-vitro evaluation of the drugs capability to cross the blood-inner ear barrier suggests that these compounds have a limited ability to induce potential auditory side effects.

Conclusion: CLC-K blockers may represent a new class of drugs for the treatment of conditions associated with expanded extracellular volume, with a hopeful high therapeutic potential for hypertensive patients carrying ClC-K gain-of-function polymorphisms.
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http://dx.doi.org/10.1097/HJH.0b013e32834d9eb9DOI Listing
January 2012

Structural nucleotide analogs are potent activators/inhibitors of pancreatic β cell KATP channels: an emerging mechanism supporting their use as antidiabetic drugs.

J Pharmacol Exp Ther 2012 Feb 25;340(2):266-76. Epub 2011 Oct 25.

Department of Pharmacobiology, Faculty of Pharmacy, University of Bari, Via Orabona No. 4, I-70126 Bari, Italy.

The 2H-1,4-benzoxazine derivatives are novel drugs structurally similar to nucleotides; however, their actions on the pancreatic β cell ATP-sensitive K+ (KATP) channel and on glucose disposal are unknown. Therefore, the effects of the linear/branched alkyl substituents and the aliphatic/aromatic rings at position 2 of the 2H-1,4-benzoxazine nucleus on the activity of these molecules against the pancreatic β cell KATP channel and the Kir6.2ΔC36 subunit were investigated using a patch-clamp technique. The effects of these compounds on glucose disposal that followed glucose loading by intraperitoneal glucose tolerance test and on fasting glycemia were investigated in normal mice. The 2-n-hexyl analog blocked the KATP (IC₅₀ = 10.1 × 10⁻⁹ M) and Kir6.2ΔC36 (IC₅₀ = 9.6 × 10⁻⁹ M) channels, which induced depolarization. In contrast, the 2-phenyl analog was a potent opener (drug concentration needed to enhance the current by 50% = 0.04 × 10⁻⁹ M), which induced hyperpolarization. The ranked order of the potency/efficacy of the analog openers was 2-phenyl > 2-benzyl > 2-cyclohexylmethyl. The 2-phenylethyl and 2-isopropyl analogs were not effective as blockers/openers. The 2-n-hexyl (2-10 mg/kg) and 2-phenyl analogs (2-30 mg/kg) reduced and enhanced the glucose areas under the curves, respectively, after glucose loading in mice. These compounds did not affect the fasting glycemia as is observed with glibenclamide. The linear alkyl chain and the aromatic ring at position 2 of the 1,4-benzoxazine nucleus are the determinants, which confer the KATP channel blocking action with glucose-lowering effects and the opening action with increased glucose levels, respectively. The opening/blocking actions of these compounds mimic those that were observed with ATP and ADP. The results support the use of these compounds as novel antidiabetic drugs.
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http://dx.doi.org/10.1124/jpet.111.185835DOI Listing
February 2012

Structural insight into peroxisome proliferator-activated receptor gamma binding of two ureidofibrate-like enantiomers by molecular dynamics, cofactor interaction analysis, and site-directed mutagenesis.

J Med Chem 2010 Jun;53(11):4354-66

Istituto di Cristallografia, Consiglio Nazionale delle Ricerche, Montelibretti, 00015 Monterotondo Stazione, Roma, Italia.

Molecular dynamics simulations were performed on two ureidofibrate-like enantiomers to gain insight into their different potency and efficacy against PPARgamma. The partial agonism of the S enantiomer seems to be due to its capability to stabilize different regions of the receptor allowing the interaction with both coactivators and corepressors as shown by fluorescence resonance energy transfer (FRET) assays. The recruitment of the corepressor N-CoR1 by the S enantiomer on two different responsive elements of PPARgamma regulated promoters was confirmed by chromatin immunoprecipitation assays. Cell-based transcription assays show that PPARgamma coactivator 1alpha (PGC-1alpha) and cAMP response element binding protein-binding protein (CBP) enhance the basal and ligand-stimulated receptor activity acting as coactivators of PPARgamma, whereas the receptor interacting protein 140 (RIP140) and the nuclear corepressor 1 (N-CoR1) repress the transcriptional activity of PPARgamma. We also tested the importance of the residue Q286 on the transcriptional activity of the receptor by site-directed mutagenesis and confirmed its key role in the stabilization of helix 12. Molecular modeling studies were performed to provide a molecular explanation for the different behavior of the mutants.
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http://dx.doi.org/10.1021/jm9013899DOI Listing
June 2010

New 2-aryloxy-3-phenyl-propanoic acids as peroxisome proliferator-activated receptors alpha/gamma dual agonists with improved potency and reduced adverse effects on skeletal muscle function.

J Med Chem 2009 Oct;52(20):6382-93

Dipartimento Farmaco-Chimico, Università degli Studi di Bari, via Orabona 4, 70126 Bari, Italia.

The preparation of a new series of 2-aryloxy-3-phenyl-propanoic acids, resulting from the introduction of a linker into the diphenyl system of the previously reported PPARalpha/gamma dual agonist 1, allowed the identification of new ligands with improved potency on PPARalpha and unchanged activity on PPARgamma. For the most interesting stereoisomers S-2 and S-4, X-ray studies in PPARgamma and docking experiments in PPARalpha provided a molecular explanation for their different behavior as full and partial agonists of PPARalpha and PPARgamma, respectively. Due to the adverse effects provoked by hypolipidemic drugs on skeletal muscle function, we also investigated the blocking activity of S-2 and S-4 on skeletal muscle membrane chloride channel conductance and found that these ligands have a pharmacological profile more beneficial compared to fibrates currently used in therapy.
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http://dx.doi.org/10.1021/jm900941bDOI Listing
October 2009

Synthesis, SAR, and biological evaluation of alpha-sulfonylphosphonic acids as selective matrix metalloproteinase inhibitors.

ChemMedChem 2009 Mar;4(3):352-62

Dipartimento Farmaco-Chimico, Università degli Studi di Bari, Via Orabona 4, 70126 Bari, Italy.

Eleven simple alpha-sulfonylphosphonates, new analogues of previously reported alpha-sulfonylaminophosphonates, were prepared and tested as MMP inhibitors. The IC(50) values of most of these compounds are in the nanomolar range against MMP-2, -8, -13, and -14. Compound 11 proved to be the most effective inhibitor of MMP-2 (IC(50) = 60 nM), with interesting selectivity versus the antitarget enzymes MMP-3 and MMP-9. The mode of binding of the new phosphonates in the active site of MMP-2 was studied, and variations in inhibition was explained by means of molecular modeling.
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http://dx.doi.org/10.1002/cmdc.200800324DOI Listing
March 2009

Crystal structure of the peroxisome proliferator-activated receptor gamma (PPARgamma) ligand binding domain complexed with a novel partial agonist: a new region of the hydrophobic pocket could be exploited for drug design.

J Med Chem 2008 Dec;51(24):7768-76

Consiglio Nazionale delle Ricerche, Roma 00016, Italy.

The peroxisome proliferator-activated receptors (PPARs) are ligand-dependent transcription factors regulating glucose and lipid metabolism. The search for new PPAR ligands with reduced adverse effects with respect to the marketed antidiabetic agents thiazolidinediones (TZDs) and the dual-agonists glitazars is highly desired. We report the crystal structure and activity of the two enantiomeric forms of a clofibric acid analogue, respectively complexed with the ligand-binding domain (LBD) of PPARgamma, and provide an explanation on a molecular basis for their different potency and efficacy against PPARgamma. The more potent S-enantiomer is a dual PPARalpha/PPARgamma agonist which presents a partial agonism profile against PPARgamma. Docking of the S-enantiomer in the PPARalpha-LBD has been performed to explain its different subtype pharmacological profile. The hypothesis that partial agonists show differential stabilization of helix 3, when compared to full agonists, is also discussed. Moreover, the structure of the complex with the S-enantiomer reveals a new region of the PPARgamma-LBD never sampled before by other ligands.
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http://dx.doi.org/10.1021/jm800733hDOI Listing
December 2008

Synthesis, biological evaluation, and molecular modeling investigation of chiral 2-(4-chloro-phenoxy)-3-phenyl-propanoic acid derivatives with PPARalpha and PPARgamma agonist activity.

Bioorg Med Chem 2008 Nov 19;16(21):9498-510. Epub 2008 Sep 19.

Dipartimento Farmaco-Chimico, Università degli Studi di Bari, via Orabona 4, 70126 Bari, Italy.

PPARs are ligand-activated transcription factors that govern lipid and glucose homeostasis and play a central role in cardiovascular disease, obesity, and diabetes. Herein, we present screening results for a series of chiral 2-(4-chloro-phenoxy)-3-phenyl-propanoic acid derivatives, some of which are potent PPARgamma agonists as well as PPARalpha agonists. To investigate the binding modes of the most interesting derivatives into the PPARalpha and PPARgamma binding clefts and evaluate their agonist activity, docking experiments, molecular dynamics simulations, and MM-PBSA analysis were performed.
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http://dx.doi.org/10.1016/j.bmc.2008.09.045DOI Listing
November 2008

Molecular determinants for the activating/blocking actions of the 2H-1,4-benzoxazine derivatives, a class of potassium channel modulators targeting the skeletal muscle KATP channels.

Mol Pharmacol 2008 Jul 10;74(1):50-8. Epub 2008 Apr 10.

Department of Pharmacobiology, Faculty of Pharmacy, via Orabona no. 4, Bari, Italy.

The 2H-1,4-benzoxazine derivatives are modulators of the skeletal muscle ATP-sensitive-K(+) channels (K(ATP)), activating it in the presence of ATP but inhibiting it in the absence of nucleotide. To investigate the molecular determinants for the activating/blocking actions of these compounds, novel molecules with different alkyl or aryl-alkyl substitutes at position 2 of the 1,4-benzoxazine ring were prepared. The effects of the lengthening of the alkyl chain and of branched substitutes, as well as of the introduction of aliphatic/aromatic rings on the activity of the molecules, were investigated on the skeletal muscle K(ATP) channels of the rat, in excised-patch experiments, in the presence or absence of internal ATP (10(-4) M). In the presence of ATP, the 2-n-hexyl analog was the most potent activator (DE(50) = 1.08 x 10(-10) M), whereas the 2-phenylethyl was not effective. The rank order of efficacy of the openers was 2-n-hexyl > or =2-cyclohexylmethyl >2-isopropyl = 2-n-butyl > or = 2-phenyl > or = 2-benzyl = 2-isobutyl analogs. In the absence of ATP, the 2-phenyl analog was the most potent inhibitor (IC(50) = 2.5 x 10(-11) M); the rank order of efficacy of the blockers was 2-phenyl > or = 2-n-hexyl > 2-n-butyl > 2-cyclohexylmethyl, whereas the 2-phenylethyl, 2-benzyl, and 2-isobutyl 1,4-benzoxazine analogs were not effective; the 2-isopropyl analog activated the K(ATP) channel even in the absence of nucleotide. Therefore, distinct molecular determinants for the activating or blocking actions for these compounds can be found. For example, the replacement of the linear with the branched alkyl substitutes at the position 2 of the 1,4-benzoxazine nucleus determines the molecular switch from blockers to openers. These compounds were 100-fold more potent and effective as openers than other KCO against the muscle K(ATP) channels.
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http://dx.doi.org/10.1124/mol.108.046615DOI Listing
July 2008

Molecular switch for CLC-K Cl- channel block/activation: optimal pharmacophoric requirements towards high-affinity ligands.

Proc Natl Acad Sci U S A 2008 Jan 23;105(4):1369-73. Epub 2008 Jan 23.

Unità di Farmacologia and Dipartimento Farmacochimico, Dipartimento Farmacobiologico, Facoltà di Farmacia, Università di Bari, Bari 70125, Italy.

ClC-Ka and ClC-Kb Cl(-) channels are pivotal for renal salt reabsorption and water balance. There is growing interest in identifying ligands that allow pharmacological interventions aimed to modulate their activity. Starting from available ligands, we followed a rational chemical strategy, accompanied by computational modeling and electrophysiological techniques, to identify the molecular requisites for binding to a blocking or to an activating binding site on ClC-Ka. The major molecular determinant that distinguishes activators from blockers is the level of planarity of the aromatic portions of the molecules: only molecules with perfectly coplanar aromatic groups display potentiating activity. Combining several molecular features of various CLC-K ligands, we discovered that phenyl-benzofuran carboxylic acid derivatives yield the most potent ClC-Ka inhibitors so far described (affinity <10 microM). The increase in affinity compared with 3-phenyl-2-p-chlorophenoxy-propionic acid (3-phenyl-CPP) stems primarily from the conformational constraint provided by the phenyl-benzofuran ring. Several other key structural elements for high blocking potency were identified through a detailed structure-activity relationship study. Surprisingly, some benzofuran-based drugs inhibit ClC-Kb with a similar affinity of <10 microM, thus representing the first inhibitors for this CLC-K isoform identified so far. Based on our data, we established a pharmacophore model that will be useful for the development of drugs targeting CLC-K channels.
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http://dx.doi.org/10.1073/pnas.0708977105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2234145PMC
January 2008

Enantiomeric separation of 2-aryloxyalkyl- and 2-arylalkyl-2-aryloxyacetic acids on a Penicillin G Acylase-based chiral stationary phase: influence of the chemical structure on retention and enantioselectivity.

J Pharm Biomed Anal 2007 Oct 12;45(2):211-8. Epub 2007 Jun 12.

Dipartimento di Chimica Farmaceutica, Università di Pavia, Via Taramelli 12, I-27100 Pavia, Italy.

The chiral recognition mechanism of Penicillin G Acylase (PGA) was investigated with a set of 18 new chiral acidic compounds. A series of 2-aryloxyalkyl- and 2-arylalkyl-2-aryloxyacetic acids in which the absolute configuration has been reported to exert a strong influence on pharmacological activity, were synthesized and analysed on PGA-based chiral stationary phase (CSP) and 11 racemates were completely resolved with a mobile phase composed of 50 mM phosphate buffer (pH 7.0). The influence of structural variations of analytes on retention and enantioselectivity was investigated by application of molecular modelling studies. Docking experiments were also carried out to rationalize the observed enantioselective behaviour. The computation approach revealed to be helpful in elucidating the molecular basis of the enantioselectivity observed on PGA-CSP.
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http://dx.doi.org/10.1016/j.jpba.2007.06.005DOI Listing
October 2007

Insights into the mechanism of partial agonism: crystal structures of the peroxisome proliferator-activated receptor gamma ligand-binding domain in the complex with two enantiomeric ligands.

J Biol Chem 2007 Jun 2;282(23):17314-24. Epub 2007 Apr 2.

Istituto di Cristallografia, Consiglio Nazionale delle Ricerche, Montelibretti, 00016 Monterotondo Stazione, Roma, Italia.

The peroxisome proliferator-activated receptors (PPARs) are transcriptional regulators of glucose and lipid metabolism. They are activated by natural ligands, such as fatty acids, and are also targets of synthetic antidiabetic and hypolipidemic drugs. By using cell-based reporter assays, we studied the transactivation activity of two enantiomeric ureidofibrate-like derivatives. In particular, we show that the R-enantiomer, (R)-1, is a full agonist of PPARgamma, whereas the S-enantiomer, (S)-1, is a less potent partial agonist. Most importantly, we report the x-ray crystal structures of the PPARgamma ligand binding domain complexed with the R- and the S-enantiomer, respectively. The analysis of the two crystal structures shows that the different degree of stabilization of the helix 12 induced by the ligand determines its behavior as full or partial agonist. Another crystal structure of the PPARgamma.(S)-1 complex, only differing in the soaking time of the ligand, is also presented. The comparison of the two structures of the complexes with the partial agonist reveals significant differences and is suggestive of the possible coexistence in solution of transcriptionally active and inactive forms of helix 12 in the presence of a partial agonist. Mutation analysis confirms the importance of Leu(465), Leu(469), and Ile(472) in the activation by (R)-1 and underscores the key role of Gln(286) in the PPARgamma activity.
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http://dx.doi.org/10.1074/jbc.M702316200DOI Listing
June 2007

Synthesis, biological evaluation, and molecular modeling investigation of chiral phenoxyacetic acid analogues with PPARalpha and PPARgamma agonist activity.

ChemMedChem 2007 May;2(5):641-54

Dipartimento Farmaco-Chimico, Università degli Studi di Bari via Orabona 4, 70126 Bari, Italy.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that govern lipid and glucose homeostasis, and play a central role in cardiovascular disease, obesity, and diabetes. Thus, there is significant interest in developing new and specific agonists for these receptors. Herein we present screening results for a series of chiral phenoxyacetic acid analogues, some of which are potent PPARalpha agonists as well as PPARgamma agonists. The stereochemistry of these compounds plays an important role in determining their activity; the S isomers were observed to be more active than the corresponding R isomers. Interestingly, for one of these analogues, the stereoselectivity toward PPARalpha was reversed, and for this reason docking experiments were performed to rationalize this peculiar behavior.
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http://dx.doi.org/10.1002/cmdc.200600307DOI Listing
May 2007

Exploring the molecular basis of the enantioselective binding of penicillin G acylase towards a series of 2-aryloxyalkanoic acids: a docking and molecular dynamics study.

J Mol Graph Model 2007 Mar 8;25(6):773-83. Epub 2006 Aug 8.

Dipartimento di Chimica Farmaceutica e Tossicologica, Università di Napoli Federico II, Via D. Montesano, 49, I-80131 Napoli, Italy.

In the present paper, molecular modeling studies were undertaken in order to shed light on the molecular basis of the observed enantioselectivity of penicillin G acylase (PGA), a well known enzyme for its industrial applications, towards 16 racemic 2-aryloxyalkanoic acids, which have been reported to affect several biological systems. With this intention docking calculations and MD simulations were performed. Docking results indicated that the (S)-enantiomers establish several electrostatic interactions with SerB1, SerB386 and ArgB263 of PGA. Conversely, the absence of specific polar interactions between the (R)-enantiomers and ArgB263 seems to be the main reason for the different binding affinities observed between the two enantiomers. Results of molecular dynamics simulations demonstrated that polar interactions are responsible for both the ligand affinity and PGA enantiospecificity. Modeling calculations provided possible explanations for the observed enantioselectivity of the enzyme that rationalize available experimental data and could be the basis for future protein engineering efforts.
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http://dx.doi.org/10.1016/j.jmgm.2006.07.001DOI Listing
March 2007

Elucidation of the enantioselective recognition mechanism of a penicillin G acylase-based chiral stationary phase towards a series of 2-aryloxy-2-arylacetic acids.

Chirality 2006 Aug;18(8):633-43

Dipartimento di Chimica Farmaceutica, Università di Pavia, Pavia, Italy.

A series of structurally related 2-aryloxy-2-arylacetic acids (1-3, 5-16) together with a thioisostere derivative (4) have been synthesized and characterized by GC-MS and 1H NMR. The designed compounds were analyzed on a Penicillin G Acylase chiral stationary phase (PGA-CSP) and the influence of the structure variations on retention and enantioselectivity was investigated. The chromatographic study includes the direct separation of the enantiomers of the synthesized compounds and the determination of the elution order of selected racemic mixtures. 10 out of 16 racemates were separated; high chromatographic enantioseparation factors (alpha > 2) were achieved for some compounds. For the enantiomers of four compounds whose absolute configuration was known (1, 3, 12, 16), the elution order was R:S with the exception of 2-(4-chloro-phenoxy)phenylacetic acid (1), for which the elution order was reversed. Preliminary molecular modeling studies suggest that both polar and charge-transfer interactions as well as steric effects play an important role in determining the retention factors and the enantioselectivities observed.
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http://dx.doi.org/10.1002/chir.20300DOI Listing
August 2006

Activation and inhibition of kidney CLC-K chloride channels by fenamates.

Mol Pharmacol 2006 Jan 21;69(1):165-73. Epub 2005 Oct 21.

Unità di Farmacologia, Dipartimento Farmacobiologico, Facoltà di Farmacia, Università di Bari, Bari, Italy.

CLC-K Cl(-) channels are selectively expressed in kidney and ear, where they are pivotal for salt homeostasis, and loss-of-function mutations of CLC-Kb produce Bartter's syndrome type III. The only ligand known for CLC-K channels is a derivative of the 2-p-chlorophenoxypropionic acid (CPP), 3-phenyl-CPP, which blocks CLC-Ka, but not CLC-Kb. Here we show that in addition to this blocking site, CLC-K channels bear an activating binding site that controls channel opening. Using the voltage-clamp technique on channels expressed in Xenopus laevis oocytes, we found that niflumic acid (NFA) increases CLC-Ka and CLC-Kb currents in the 10 to 1000 microM range. Flufenamic acid (FFA) derivatives or high doses of NFA produced instead an inhibitory effect on CLC-Ka, but not on CLC-Kb, and on blocker-insensitive CLC-Ka mutants, indicating that the activating binding site is distinct from the blocker site. Evaluation of the sensitivity of CLC-Ka to derivatives of NFA and FFA together with a modeling study of these ligands allow us to conclude that one major characteristic of activating compounds is the coplanarity of the two rings of the molecules, whereas block requires a noncoplanar configuration. These molecules provide a starting point for identification of diuretics or drugs useful in the treatment of Bartter's syndrome.
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http://dx.doi.org/10.1124/mol.105.017384DOI Listing
January 2006

Synthesis, biological evaluation, and molecular modeling investigation of new chiral fibrates with PPARalpha and PPARgamma agonist activity.

J Med Chem 2005 Aug;48(17):5509-19

Dipartimento di Scienze Farmacologiche, Università degli Studi di Milano, via Balzaretti 9, 20133 Milano, Italia.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that govern lipid and glucose homeostasis playing a central role in cardiovascular diseases, obesity, and diabetes. Medications targeted to PPARs have been established to treat hyperlipidemia (fibrates) and insulin resistance (glitazones). Thus, there is significant interest in developing new and specific ligands for these receptors. Here, we present the results of the screening of new ligands of PPARalpha and PPARgamma. Optical isomers of new chiral fibrates were synthesized and tested in cell-based assays. Compound (S)-7 showed a dual PPARalpha/gamma activity, and its stereochemistry was crucial in receptor activation. Protease protection experiments suggested that this compound binds directly to PPAR. Moreover, computational studies showed that it properly docks to PPARalpha and gamma ligand binding pockets. Interestingly, (S)-7 exhibited only a modest capacity to induce the differentiation of murine fibroblasts 3T3-L1 into adipocytes compared to rosiglitazone, a well-known PPARgamma agonist.
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http://dx.doi.org/10.1021/jm0502844DOI Listing
August 2005

Stereospecific synthesis of "para-hydroxymexiletine" and sodium channel blocking activity evaluation.

Chirality 2004 Feb;16(2):72-8

Dipartimento Farmaco-Chimico, Facoltà di Farmacia, Università degli Studi di Bari, Bari, Italy.

Both enantiomers of "para-hydroxymexiletine" (PHM), one of the main metabolites of mexiletine, were synthesized and fully characterized. Properties of (R)- and (S)-PHM, in terms of blocking potency and stereoselectivity on frog skeletal muscle Na(+) channels, were evaluated. The presence of a hydroxy group on the aryloxy moiety in the 4-position, as in PHM, reduced potency with respect to mexiletine in reducing I(Na max). However, PHM showed clear use-dependent behavior similar to that of mexiletine and, in contrast with what is observed with the parent compound, maintained its stereoselectivity during the use-dependent block. Chirality 16:72-78, 2004.
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http://dx.doi.org/10.1002/chir.10307DOI Listing
February 2004

Investigations of pharmacologic properties of the renal CLC-K1 chloride channel co-expressed with barttin by the use of 2-(p-Chlorophenoxy)propionic acid derivatives and other structurally unrelated chloride channels blockers.

J Am Soc Nephrol 2004 Jan;15(1):13-20

Unità di Farmacologia, Dipartimento Farmacobiologico, Facoltà di Farmacia, Università di Bari, Bari, Italy.

CLC-K chloride channels are expressed in the kidney, where they play a pivotal role in the mechanisms of urine concentration and Na(+) reabsorption. The identification of barttin as an essential beta-subunit of CLC-K channels allowed performance of a pharmacologic characterization of wild-type CLC-K1 expressed in Xenopus oocytes. To this end, a series of 2-(p-chlorophenoxy)propionic acid (CPP) derivatives were screened using the two-microelectrode voltage-clamp technique. Several chemical modifications regarding the phenoxy group of the side chain (elimination of the oxygen atom or of methylenic groups, substitutions of the chlorine atom) did not alter the drug blocking activity, with five different derivatives showing a similar potency. Among these, a derivative of CPP carrying a benzyl group on the chiral center in the place of the methyl group represented the minimal structure for blocking CLC-K1. It inhibited the channel from the extracellular side with an affinity in the 150 micro M range. The blocking potency of this compound is fourfold increased by lowering the extracellular chloride concentration, suggesting that the drug interacts with the channel pore. Concomitantly, the effect of some "classical" Cl(-) channel blockers (9-anthracenecarboxylic acid, 2-(phenylamino)benzoic acid, iminodibenzoic acid, niflumic acid, 5-nitro-2-(3-phenylpropylamino)benzoic acid, 4,4'-diisothiocyanato-2,2'-stilbenedisulfonic acid disodium salt, and 4-acetamido-4'-isothiocyanato-2,2'-stilbenedisulfonic acid disodium salt) was screened. 4,4'-Diisothiocyanato-2,2'-stilbenedisulfonic acid disodium salt was the only one capable of blocking CLC-K1 with a potency similar to the CPP derivative, although in an irreversible manner. The newly identified substances provide a useful tool to investigate the biophysical and physiologic role of these renal channels and a starting point for the development of therapeutic drugs with diuretic action.
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http://dx.doi.org/10.1097/01.asn.0000103226.28798.eaDOI Listing
January 2004

Structural requisites of 2-(p-chlorophenoxy)propionic acid analogues for activity on native rat skeletal muscle chloride conductance and on heterologously expressed CLC-1.

Br J Pharmacol 2003 Aug;139(7):1255-64

Unità di Farmacologia, Dipartimento Farmacobiologico, Facoltà di Farmacia, Università di Bari, Italy.

(1) The 2-(p-chlorophenoxy)propionic acid (CPP) modulates in a stereoselective manner the macroscopic chloride conductance (gCl), the electrical parameter sustained by the CLC-1 channel, of skeletal muscle. In order to determine the structural requirements for modulating native gCl and to identify high-affinity ligands, the effects of newly synthesised CPP analogues have been evaluated on gCl of rat EDL muscle fibres by means of the two-microelectrode current-clamp technique. (2) Each type of the following independent modification of CPP structure led to a three- to 10-fold decrease or to a complete lack of gCl-blocking activity: replacement of the electron-attractive chlorine atom of the aromatic ring, substitution of the oxygen atom of the phenoxy group, modification at the chiral centre and substitution of the carboxylic function with a phosphonate one. (3) The analogues bearing a second chlorophenoxy group on the asymmetric carbon atom showed a significant gCl-blocking activity. Similar to racemate CPP, the analogue with this group, spaced by an alkyl chain formed by three methylenic groups, blocked gCl by 45% at 100 micro M. (4) These latter derivatives were tested on heterelogously expressed CLC-1 performing inside-out patch-clamp recordings to further define how interaction between drug and channel protein could take place. Depending on the exact chemical nature of modification, these derivatives strongly blocked CLC-1 with K(D) values at -140 mV ranging from about 4 to 180 micro M. (5) In conclusion, we identified four molecular determinants pivotal for the interaction with the binding site on muscle CLC-1 channels: (a) the carboxylic group that confers the optimal acidity and the negative charge; (b) the chlorophenoxy moiety that might interact with a hydrophobic pocket; (c) the chiral centre that allows the proper spatial disposition of the molecule; (d) an additional phenoxy group that remarkably stabilises the binding by interacting with a second hydrophobic pocket.
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http://dx.doi.org/10.1038/sj.bjp.0705364DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1573959PMC
August 2003