Publications by authors named "Giorgio Pochetti"

25 Publications

  • Page 1 of 1

A Novel N-Substituted Valine Derivative with Unique Peroxisome Proliferator-Activated Receptor γ Binding Properties and Biological Activities.

J Med Chem 2020 11 3;63(21):13124-13139. Epub 2020 Nov 3.

Aix Marseille University, INSERM, SSA, MCT, 13385 Marseille, France.

A proprietary library of novel -aryl-substituted amino acid derivatives bearing a hydroxamate head group allowed the identification of compound that possesses weak proadipogenic and peroxisome proliferator-activated receptor γ (PPARγ) activating properties. The systematic optimization of , in order to improve its PPARγ agonist activity, led to the synthesis of compound (-aryl-substituted valine derivative) that possesses dual PPARγ/PPARα agonistic activity. Structural and kinetic analyses reveal that occupies the typical ligand binding domain of the PPARγ agonists with, however, a unique high-affinity binding mode. Furthermore, is highly effective in preventing cyclin-dependent kinase 5-mediated phosphorylation of PPARγ serine 273. Although less proadipogenic than rosiglitazone, significantly increases adipocyte insulin-stimulated glucose uptake and efficiently promotes white-to-brown adipocyte conversion. In addition, prevents oleic acid-induced lipid accumulation in hepatoma cells. The unique biochemical properties and biological activities of compound suggest that it would be a promising candidate for the development of compounds to reduce insulin resistance, obesity, and nonalcoholic fatty liver disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jmedchem.0c01555DOI Listing
November 2020

Bone-Seeking Matrix Metalloproteinase Inhibitors for the Treatment of Skeletal Malignancy.

Pharmaceuticals (Basel) 2020 Jun 1;13(6). Epub 2020 Jun 1.

Department of Pharmacy and Pharmaceutical Sciences, University of Bari "A. Moro", via E. Orabona 4, 70125 Bari, Italy.

Matrix metalloproteinases (MMPs) are a family of enzymes involved at different stages of cancer progression and metastasis. We previously identified a novel class of bisphosphonic inhibitors, selective for MMPs crucial for bone remodeling, such as MMP-2. Due to the increasing relevance of specific MMPs at various stages of tumor malignancy, we focused on improving potency towards certain isoforms. Here, we tackled MMP-9 because of its confirmed role in tumor invasion, metastasis, angiogenesis, and immuno-response, making it an ideal target for cancer therapy. Using a computational analysis, we designed and characterized potent MMP-2/MMP-9 inhibitors. This is a promising approach to develop and clinically translate inhibitors that could be used in combination with standard care therapy for the treatment of skeletal malignancies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ph13060113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7344628PMC
June 2020

Insights into PPARγ Phosphorylation and Its Inhibition Mechanism.

J Med Chem 2020 05 15;63(9):4811-4823. Epub 2020 Apr 15.

Istituto di Cristallografia, Consiglio Nazionale delle Ricerche, Via Salaria km. 29.300, 00015 Monterotondo Stazione, Rome, Italy.

PPARγ represents a key target for the treatment of type 2 diabetes and metabolic syndrome. Synthetic antidiabetic drugs activating PPARγ are accompanied by serious undesirable side effects related to their agonism. In the search for new PPARγ regulators, inhibitors of PPARγ phosphorylation on S245 mediated by CDK5 represent an opportunity for the development of an improved generation of antidiabetic drugs acting through this nuclear receptor. We have employed a multidisciplinary approach, including protein-protein docking, X-ray crystallography, NMR, HDX, MD simulations, and site-directed mutagenesis to investigate conformational changes in PPARγ that impair the ability of CDK5 to interact with PPARγ and hence inhibit PPARγ phosphorylation. Finally, we describe an alternative inhibition mechanism adopted by a ligand bound far from the phosphorylation site.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jmedchem.0c00048DOI Listing
May 2020

Surface Plasmon Resonance as a Tool for Ligand Binding Investigation of Engineered GPR17 Receptor, a G Protein Coupled Receptor Involved in Myelination.

Front Chem 2019 10;7:910. Epub 2020 Jan 10.

Department of Biotechnology, University of Verona, Verona, Italy.

The aim of this study was to investigate the potential of surface plasmon resonance (SPR) spectroscopy for the measurement of real-time ligand-binding affinities and kinetic parameters for GPR17, a G protein-coupled receptor (GPCR) of major interest in medicinal chemistry as potential target in demyelinating diseases. The receptor was directly captured, in a single-step, from solubilized membrane extracts on the sensor chip through a covalently bound anti-6x-His-antibody and retained its ligand binding activity for over 24 h. Furthermore, our experimental setup made possible, after a mild regeneration step, to remove the bound receptor without damaging the antibody, and thus to reuse many times the same chip. Two engineered variants of GPR17, designed for crystallographic studies, were expressed in insect cells, extracted from crude membranes and analyzed for their binding with two high affinity ligands: the antagonist Cangrelor and the agonist Asinex 1. The calculated kinetic parameters and binding constants of ligands were in good agreement with those reported from activity assays and highlighted a possible functional role of the N-terminal residues of the receptor in ligand recognition and binding. Validation of SPR results was obtained by docking and molecular dynamics of GPR17-ligands interactions and by functional studies. The latter allowed us to confirm that Asinex 1 behaves as GPR17 receptor agonist, inhibits forskolin-stimulated adenylyl cyclase pathway and promotes oligodendrocyte precursor cell maturation and myelinating ability.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fchem.2019.00910DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6966494PMC
January 2020

Identification of the First PPARα/γ Dual Agonist Able To Bind to Canonical and Alternative Sites of PPARγ and To Inhibit Its Cdk5-Mediated Phosphorylation.

J Med Chem 2018 09 18;61(18):8282-8298. Epub 2018 Sep 18.

Dipartimento Farmacia-Scienze del Farmaco , Università degli Studi di Bari "Aldo Moro" , Via Orabona 4 , 70125 Bari , Italy.

A new series of derivatives of the PPARα/γ dual agonist 1 allowed us to identify the ligand ( S)-6 as a potent partial agonist of both PPARα and γ subtypes. X-ray studies in PPARγ revealed two different binding modes of ( S)-6 to the canonical site. However, ( S)-6 was also able to bind an alternative site as demonstrated by transactivation assay in the presence of a canonical PPARγ antagonist and supported from docking experiments. This compound did not activate the PPARγ-dependent program of adipocyte differentiation inducing a very less severe lipid accumulation compared to rosiglitazone but increased the insulin-stimulated glucose uptake in 3T3-L1 adipocytes. Finally, ( S)-6 inhibited the Cdk5-mediated phosphorylation of PPARγ at serine 273 that is currently considered the mechanism by which some PPARγ partial agonists exert antidiabetic effects similar to thiazolidinediones, without showing their typical side effects. This is the first PPARα/γ dual agonist reported to show this inhibitory effect representing the potential lead of a new class of drugs for treatment of dyslipidemic type 2 diabetes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jmedchem.8b00835DOI Listing
September 2018

Affinity-based separation methods for the study of biological interactions: The case of peroxisome proliferator-activated receptors in drug discovery.

Methods 2018 08 17;146:12-25. Epub 2018 Feb 17.

Dipartimento di Scienze del Farmaco, Università degli Studi di Pavia, Via Taramelli 12, 27100 Pavia, Italy. Electronic address:

Affinity-based methods using immobilized proteins are important approaches for understanding the interactions between small molecules and biological targets. This review is intended to provide an overview of different affinity based separation methods that have been applied to the study of peroxisome proliferator activated receptors (PPARs). The screening of compound to increase screening rates for synthetic and natural ligands of PPAR are reported. Pros and cons of the approaches in ligand discovery initiatives are discussed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ymeth.2018.02.006DOI Listing
August 2018

Betulinic acid is a PPARγ antagonist that improves glucose uptake, promotes osteogenesis and inhibits adipogenesis.

Sci Rep 2017 07 18;7(1):5777. Epub 2017 Jul 18.

Istituto di Cristallografia, Consiglio Nazionale delle Ricerche, Via Salaria Km. 29, 300, 00015, Monterotondo Stazione, Roma, Italy.

PPAR antagonists are ligands that bind their receptor with high affinity without transactivation activity. Recently, they have been demonstrated to maintain insulin-sensitizing and antidiabetic properties, and they serve as an alternative treatment for metabolic diseases. In this work, an affinity-based bioassay was found to be effective for selecting PPAR ligands from the dried extract of an African plant (Diospyros bipindensis). Among the ligands, we identified betulinic acid (BA), a compound already known for its anti-inflammatory, anti-tumour and antidiabetic properties, as a PPARγ and PPARα antagonist. Cell differentiation assays showed that BA inhibits adipogenesis and promotes osteogenesis; either down-regulates or does not affect the expression of a series of adipogenic markers; and up-regulates the expression of osteogenic markers. Moreover, BA increases basal glucose uptake in 3T3-L1 adipocytes. The crystal structure of the complex of BA with PPARγ sheds light, at the molecular level, on the mechanism by which BA antagonizes PPARγ, and indicates a unique binding mode of this antagonist type. The results of this study show that the natural compound BA could be an interesting and safe candidate for the treatment of type 2 diabetes and bone diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-017-05666-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5516003PMC
July 2017

Structural basis for PPAR partial or full activation revealed by a novel ligand binding mode.

Sci Rep 2016 10 6;6:34792. Epub 2016 Oct 6.

Dipartimento di Farmacia, Università degli Studi di Napoli, Via Montesano 49, 80131 Napoli, Italy.

The peroxisome proliferator-activated receptors (PPARs) are nuclear receptors involved in the regulation of the metabolic homeostasis and therefore represent valuable therapeutic targets for the treatment of metabolic diseases. The development of more balanced drugs interacting with PPARs, devoid of the side-effects showed by the currently marketed PPARγ full agonists, is considered the major challenge for the pharmaceutical companies. Here we present a structure-based virtual screening approach that let us identify a novel PPAR pan-agonist with a very attractive activity profile and its crystal structure in the complex with PPARα and PPARγ, respectively. In PPARα this ligand occupies a new pocket whose filling is allowed by the ligand-induced switching of the F273 side chain from a closed to an open conformation. The comparison between this pocket and the corresponding cavity in PPARγ provides a rationale for the different activation of the ligand towards PPARα and PPARγ, suggesting a novel basis for ligand design.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/srep34792DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5052532PMC
October 2016

Catechol-based matrix metalloproteinase inhibitors with additional antioxidative activity.

J Enzyme Inhib Med Chem 2016 24;31(sup4):25-37. Epub 2016 Aug 24.

b Dipartimento di Farmacia-Scienze del Farmaco, Università degli Studi "A. Moro" di Bari , Bari , Italy.

New catechol-containing chemical entities have been investigated as matrix metalloproteinase inhibitors as well as antioxidant molecules. The combination of the two properties could represent a useful feature due to the potential application in all the pathological processes characterized by increased proteolytic activity and radical oxygen species (ROS) production, such as inflammation and photoaging. A series of catechol-based molecules were synthesized and tested for both proteolytic and oxidative inhibitory activity, and the detailed binding mode was assessed by crystal structure determination of the complex between a catechol derivative and the matrix metalloproteinase-8. Surprisingly, X-ray structure reveals that the catechol oxygens do not coordinates the zinc atom.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/14756366.2016.1217853DOI Listing
February 2017

Screening of saponins and sapogenins from Medicago species as potential PPARγ agonists and X-ray structure of the complex PPARγ/caulophyllogenin.

Sci Rep 2016 06 10;6:27658. Epub 2016 Jun 10.

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

A series of saponins and sapogenins from Medicago species were tested for their ability to bind and activate the nuclear receptor PPARγ by SPR experiments and transactivation assay, respectively. The SPR analysis proved to be a very powerful and fast technique for screening a large number of compounds for their affinity to PPARγ and selecting the better candidates for further studies. Based on the obtained results, the sapogenin caulophyllogenin was proved to be a partial agonist towards PPARγ and the X-ray structure of its complex with PPARγ was also solved, in order to investigate the binding mode in the ligand binding domain of the nuclear receptor. This is the first known crystal structure of a sapogenin directly interacting with PPARγ. Another compound of the series, the echinocistic acid, showed antagonist activity towards PPARγ, a property that could be useful to inhibit the adipocyte differentiation which is a typical adverse effect of PPARγ agonists. This study confirms the interest on saponins and sapogenins as a valuable natural resource exploitable in the medical and food industry for ameliorating the metabolic syndrome.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/srep27658DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4901321PMC
June 2016

On the metabolically active form of metaglidasen: improved synthesis and investigation of its peculiar activity on peroxisome proliferator-activated receptors and skeletal muscles.

ChemMedChem 2015 Mar 29;10(3):555-65. Epub 2015 Jan 29.

Dipartimento di Farmacia-Scienze del Farmaco, Università degli Studi di Bari "Aldo Moro", 70126 Bari (Italy).

Metaglidasen is a fibrate-like drug reported as a selective modulator of peroxisome proliferator-activated receptor γ (PPARγ), able to lower plasma glucose levels in the absence of the side effects typically observed with thiazolidinedione antidiabetic agents in current use. Herein we report an improved synthesis of metaglidasen's metabolically active form halofenic acid (R)-2 and that of its enantiomer (S)-2. The activity of the two stereoisomers was carefully examined on PPARα and PPARγ subtypes. As expected, both showed partial agonist activity toward PPARγ; the investigation of PPARα activity, however, led to unexpected results. In particular, (S)-2 was found to act as a partial agonist, whereas (R)-2 behaved as an antagonist. X-ray crystallographic studies with PPARγ were carried out to gain more insight on the molecular-level interactions and to propose a binding mode. Given the adverse effects provoked by fibrate drugs on skeletal muscle function, we also investigated the capacity of (R)-2 and (S)-2 to block conductance of the skeletal muscle membrane chloride channel. The results showed a more beneficial profile for (R)-2, the activity of which on skeletal muscle function, however, should not be overlooked in the ongoing clinical trials studying its long-term effects.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cmdc.201402462DOI Listing
March 2015

Structural basis of the transactivation deficiency of the human PPARγ F360L mutant associated with familial partial lipodystrophy.

Acta Crystallogr D Biol Crystallogr 2014 Jul 29;70(Pt 7):1965-76. Epub 2014 Jun 29.

Istituto di Cristallografia, CNR, Via Salaria, Km 29,300, Monterotondo Stazione, 00015 Roma, Italy.

The peroxisome proliferator-activated receptors (PPARs) are transcription factors that regulate glucose and lipid metabolism. The role of PPARs in several chronic diseases such as type 2 diabetes, obesity and atherosclerosis is well known and, for this reason, they are the targets of antidiabetic and hypolipidaemic drugs. In the last decade, some rare mutations in human PPARγ that might be associated with partial lipodystrophy, dyslipidaemia, insulin resistance and colon cancer have emerged. In particular, the F360L mutant of PPARγ (PPARγ2 residue 388), which is associated with familial partial lipodystrophy, significantly decreases basal transcriptional activity and impairs stimulation by synthetic ligands. To date, the structural reason for this defective behaviour is unclear. Therefore, the crystal structure of PPARγ F360L together with the partial agonist LT175 has been solved and the mutant has been characterized by circular-dichroism spectroscopy (CD) in order to compare its thermal stability with that of the wild-type receptor. The X-ray analysis showed that the mutation induces dramatic conformational changes in the C-terminal part of the receptor ligand-binding domain (LBD) owing to the loss of van der Waals interactions made by the Phe360 residue in the wild type and an important salt bridge made by Arg357, with consequent rearrangement of loop 11/12 and the activation function helix 12 (H12). The increased mobility of H12 makes the binding of co-activators in the hydrophobic cleft less efficient, thereby markedly lowering the transactivation activity. The spectroscopic analysis in solution and molecular-dynamics (MD) simulations provided results which were in agreement and consistent with the mutant conformational changes observed by X-ray analysis. Moreover, to evaluate the importance of the salt bridge made by Arg357, the crystal structure of the PPARγ R357A mutant in complex with the agonist rosiglitazone has been solved.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1107/S1399004714009638DOI Listing
July 2014

LT175 is a novel PPARα/γ ligand with potent insulin-sensitizing effects and reduced adipogenic properties.

J Biol Chem 2014 Mar 22;289(10):6908-6920. Epub 2014 Jan 22.

Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, 20133 Milan, Italy. Electronic address:

Peroxisome proliferator-activated receptors (PPARs) are ligand-dependent transcription factors regulating lipid and glucose metabolism. Ongoing drug discovery programs aim to develop dual PPARα/γ agonists devoid of the side effects of the marketed antidiabetic agents thiazolidinediones and the dual agonists glitazars. Recently, we described a new dual PPARα/γ ligand, LT175, with a partial agonist profile against PPARγ and interacting with a newly identified region of the PPARγ-ligand binding domain (1). Here we show that LT175 differentially activated PPARγ target genes involved in fatty acid esterification and storage in 3T3-L1-derived adipocytes. This resulted in a less severe lipid accumulation compared with that triggered by rosiglitazone, suggesting that LT175 may have a lower adipogenic activity. Consistent with this hypothesis, in vivo administration of LT175 to mice fed a high-fat diet decreased body weight, adipocyte size, and white adipose tissue mass, as assessed by magnetic resonance imaging. Furthermore, LT175 significantly reduced plasma glucose, insulin, non-esterified fatty acids, triglycerides, and cholesterol and increased circulating adiponectin and fibroblast growth factor 21 levels. Oral glucose and insulin tolerance tests showed that the compound improves glucose homeostasis and insulin sensitivity. Moreover, we demonstrate that the peculiar interaction of LT175 with PPARγ affected the recruitment of the coregulators cyclic-AMP response element-binding protein-binding protein and nuclear corepressor 1 (NCoR1), fundamentals for the PPARγ-mediated adipogenic program. In conclusion, our results describe a new PPAR ligand, modulating lipid and glucose metabolism with reduced adipogenic activity, that may be used as a model for a series of novel molecules with an improved pharmacological profile for the treatment of dyslipidemia and type 2 diabetes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M113.506394DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3945352PMC
March 2014

Design and biophysical characterization of atrazine-sensing peptides mimicking the Chlamydomonas reinhardtii plastoquinone binding niche.

Phys Chem Chem Phys 2013 Aug;15(31):13108-15

Institute of Crystallography, CNR, Rome, Italy.

The plastoquinone (Q(B)) binding niche of the Photosystem II (PSII) D1 protein is the subject of intense research due to its capability to bind also anthropogenic pollutants. In this work, the Chlamydomonas reinhardtii D1 primary structure was used as a template to computationally design novel peptides enabling the binding of the herbicide atrazine. Three biomimetic molecules, containing the Q(B)-binding site in a loop shaped by two α-helices, were reconstituted by automated protein synthesis, and their structural and functional features deeply analysed by biophysical techniques. Standing out among the others, the biomimetic mutant peptide, D1pepMut, showed high ability to mimic the D1 protein in binding both Q(B) and atrazine. Circular dichroism spectra suggested a typical properly-folded α-helical structure, while isothermal titration calorimetry (ITC) provided a complete thermodynamic characterization of the molecular interaction. Atrazine binds to the D1pepMut with a high affinity (Kd = 2.84 μM), and a favourable enthalpic contribution (ΔH = -11.9 kcal mol(-1)) driving the interaction. Fluorescence spectroscopy assays, in parallel to ITC data, provided hyperbolic titration curves indicating the occurrence of a single atrazine binding site. The binding resulted in structural stabilisation of the D1pepMut molecule, as suggested by atrazine-induced cooperative profiles for the fold-unfold transition. The interaction dynamics and the structural stability of the peptides in response to the ligand were particularly considered as mandatory parameters for biosensor/biochip development. These studies paved the way to the set-up of an array of synthetic mutant peptides with a wide range of affinity towards different classes of target analytes, for the development of optical nanosensing platforms for herbicide detection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/c3cp51955dDOI Listing
August 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bmc.2012.01.025DOI Listing
March 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/jm900941bDOI Listing
October 2009

Extra binding region induced by non-zinc chelating inhibitors into the S1' subsite of matrix metalloproteinase 8 (MMP-8).

J Med Chem 2009 Feb;52(4):1040-9

Istituto di Cristallografia-CNR, Area della Ricerca Roma 1, Via Salaria Km.29,300, I-00016 Monterotondo Stazione, Roma, Italy.

The mode of binding and the activity of the first two non-zinc chelating, potent, and selective inhibitors of human neutrophil collagenase are reported. The crystal structures of the catalytic domain of MMP-8, respectively complexed with each inhibitor, reveals that both ligands are deeply inserted into the primary specificity subsite S(1)', where they induce a similar conformational change of the surrounding loop that is endowed with the main specificity determinants of MMPs. Accord to this rearrangement, both inhibitors remove the floor of the pocket formed by the Y227 side-chain, rendering available an extra binding region never explored before. The present data show that potent and more selective inhibitors can be obtained by developing ligands able to interact with the selectivity regions of the enzyme rather than with the catalytic zinc ion, which is the common feature of all MMP members.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/jm801166jDOI Listing
February 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/jm800733hDOI Listing
December 2008

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M702316200DOI Listing
June 2007

Stereoselectivity by enantiomeric inhibitors of matrix metalloproteinase-8: new insights from molecular dynamics simulations.

J Med Chem 2007 Jan;50(2):211-8

Dipartimento di Chimica, Ingegneria Chimica e Materiali, Università di L'Aquila, Italia.

Molecular Dynamics simulations in aqueous solution were performed for the matrix metalloproteinase-8 (MMP-8) free catalytic domain and for its complexes with the (R)- and (S)-[1-(4'-methoxybiphenyl-4-sulfonylamino)-2-methylpropyl] phosphonate. The 144-155 loop of the enzyme undergoes a drastic decrease of mobility once complexed with both enantiomers. The two enantiomers induce a different decrease of conformational entropy upon complexation. The higher affinity of the R-enantiomer can be related to the lower loss of conformational entropy accompanying its binding. The differences in the dynamical behavior of the protein induced by the two enantiomers are discussed at molecular level and the mode of binding of the simulated complexes is compared with that previously determined by X-ray crystallography.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/jm0608457DOI Listing
January 2007

Structural insight into the stereoselective inhibition of MMP-8 by enantiomeric sulfonamide phosphonates.

J Med Chem 2006 Feb;49(3):923-31

Istituto di Cristallografia, C.N.R., Monterotondo Stazione, Rome, Italy.

Potent and selective inhibitors of matrix metalloproteinases (MMPs), a family of zinc proteases that can degrade all the components of the extracellular matrix, could be useful for treatment of diseases such as cancer and arthritis. The most potent MMP inhibitors are based on hydroxamate as zinc-binding group (ZBG). alpha-Arylsulfonylamino phosphonates incorporate a particularly favorable combination of phosphonate as ZBG and arylsulfonylamino backbone so that their affinity exceptionally attains the nanomolar strength frequently observed for hydroxamate analogues. The detailed mode of binding of [1-(4'-methoxybiphenyl-4-sulfonylamino)-2-methylpropyl]phosphonate has been clarified by the crystal structures of the complexes that the R- and S-enantiomers respectively form with MMP-8. The reasons for the preferential MMP-8 inhibition by the R-phosphonate are underlined and the differences in the mode of binding of analogous alpha-arylsulfonylamino hydroxamates and carboxylates are discussed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/jm050787+DOI Listing
February 2006

N-Hydroxyurea as zinc binding group in matrix metalloproteinase inhibition: mode of binding in a complex with MMP-8.

Bioorg Med Chem Lett 2006 Jan 18;16(1):20-4. Epub 2005 Oct 18.

Dipartimento di Scienze del Farmaco, Università degli Studi 'G. d'Annunzio', Chieti, Italy.

The first crystallographic structure of an N-hydroxyurea inhibitor bound into the active site of a matrix metalloproteinase is reported. The ligand and three other analogues were prepared and studied as inhibitors of MMP-2, MMP-3, and MMP-8. The crystal structure of the complex with MMP-8 shows that the N-hydroxyurea, contrary to the analogous hydroxamate, binds the catalytic zinc ion in a monodentate rather than bidentate mode and with high out-of-plane distortion of the amide bonds.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bmcl.2005.09.057DOI Listing
January 2006

Overexpression system and biochemical profile of CTX-M-3 extended-spectrum beta-lactamase expressed in Escherichia coli.

FEMS Microbiol Lett 2004 Dec;241(2):229-32

Dipartimento di Scienze e Tecnologie Biomediche, Università di L'Aquila, Cattedra di Biochimica Clinica e Biologia Molecolare Clinica, Loc. Coppito, I-67100 L'Aquila, Italy.

An efficient over-expression system was developed for CTX-M-3 extended-spectrum-beta-lactamase. The recombinant enzyme was purified from 1 l of culture to yield 22 mg of pure enzyme. The N-terminal amino acid sequence was determined to be NH2-QTADVQ... Determination of kinetic parameters with the purified CTX-M-3 revealed efficient hydrolysis of penicillins and cephalosporins, while ceftazidime and aztreonam were very poor substrates. Clavulanic acid, sulbactam and especially tazobactam inhibited the CTX-M-3 enzyme.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.femsle.2004.10.031DOI Listing
December 2004

Computational study of the catalytic domain of human neutrophil collagenase. specific role of the S3 and S'3 subsites in the interaction with a phosphonate inhibitor.

J Comput Aided Mol Des 2002 Mar;16(3):213-25

Dipartimento di Chimica, Ingegneria Chimica e Materiali, Università degli Studi, L'Aquila, Italy.

Human neutrophil collagenase (HNC, MMP-8) is one of the target enzymes for drug treatment of pathologic extracellular matrix degradation. Peptidomimetic inhibitors bind in the S'-side of the enzyme active site occupying the S'1 primary specificity pocket by their large hydrophobic side-chains. The crystal structure of the complex between the catalytic domain of MMP-8 and Pro-Leu-L-TrpP(OH)2 (PLTP) showed that this phosphonate inhibitor binds in the S side of the active site. This finding was unexpected since it represents the first example of accommodation of the bulky Trp indolyl chain in the S1 rather than in the S'1 subsite. Dynamical and structural factors favouring this uncommon mode of binding were therefore investigated. MD simulations performed on the uncomplexed enzyme show that its structure in aqueous solution is only slightly different from the crystal structure found in the complex with PLTP. ED analysis of the MD simulations, performed on PLTP alternatively interacting with the S- or S'-side of the active site, shows that the enzyme fluctuation increases in both cases. The main contribution to the overall enzyme fluctuation is given by the loop 164-173. The fluctuation of this loop is spread over more degrees of freedom when PLTP interacts with the S-side. This dynamical factor can enhance the preference of PLTP for the S subsites of MMP-8. MD simulations also show that ligation of PLTP in the S subsites is further favoured by better zinc chelation, a cation-pi interaction at the S3 subsite and unstrained binding conformations. The role of the S3, S'3 and S'1 subsites in determining the inhibitor binding is discussed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1023/a:1020178822319DOI Listing
March 2002