Un estere misto degli acidi ialuronico, butirrico e retinoico è in grado di agire come rimodellante inverso della matrice cellulare sui fibroblasti cardiaci

Le cardiomiopatie che insorgono a seguito di infarto miocardico sono causa di elevata morbilità e mortalità dalle importanti ricadute cliniche, dovute alle patologie insorgenti a seguito dell’ischemia e della cicatrice post-infatuale. Il ventricolo sinistro danneggiato va incontro a un rimodellamento progressivo, con perdita di cardiomiociti e proliferazione dei fibroblasti, risultante in un’architettura e in una funzionalità dell’organo distorta. I fibroblasti cardiaci sono i principali responsabili della fibrosi, il processo di cicatrizzazione caratterizzato da un’eccessiva deposizione di matrice extracellulare (ECM). Negli ultimi anni gli sforzi del nostro laboratorio sono stati volti a cercare di risolvere questo problema, attraverso l’uso di una molecola da noi sintetizzata, un estere misto degli acidi butirrico, retinoico e ialuronico, HBR, capace di commissionare le cellule staminali in senso cardio-vascolare. Studi in vivo mostrano come l’iniezione diretta di HBR in cuori di animali sottoposti a infarto sperimentale, sia in grado, tra le atre cose, di diminuire la fibrosi cardiaca. Sulla base di questa evidenza abbiamo cercato di capire come e se HBR agisse direttamente sui fibroblasti, indagando i meccanismi coinvolti nella riduzione della fibrosi in vivo.. In questa tesi abbiamo dimostrato come HBR abbia un’azione diretta su fibroblasti, inibendone la proliferazione, senza effetti citotossici. Inoltre HBR induce una significativa riduzione della deposizione di collagene.. HBR agisce sull’espressione genica e sulla sintesi proteica, sopprimendo la trascrizione dei geni del collagene, così come dell’a-sma, inibendo la trasizione fibroblasti-miofibroblasti, e promuovendo la vasculogenesi (attraverso VEGF), la chemoattrazione di cellule staminali (attraverso SDF) e un’attività antifibrotica (inibendo CTGF). HBR sembra modulare l’espressione genica agendo direttamente sulle HDAC, probabilmente grazie alla subunità BU. L’abilità di HBR di ridurre la fibrosi post-infartuale, come dimostrato dai nostri studi in vivo ed in vitro, apre la strada a importanti prospettive terapeutiche.

Synthesis of new bioactive β-lactam compounds

New biologically active β-lactams were designed and synthesized, developing novel antibiotics and enzymatic inhibitors directed toward specific targets. Within a work directed to the synthesis of mimetics for RGD (Arg-Gly-Asp) sequence able to interact with αvβ3 and α5β1-type integrins, new activators were developed and their Structure-Activity Relationships (SAR) analysis deepened, enhancing their activity range towards the α4β1 isoform. Moreover, to synthesize novel compounds active both against bacterial infections and pulmonary conditions of cystic fibrosis patients, new β-lactam candidates were studied. Among the abundant library of β-lactams prepared, mainly with antioxidant and antibacterial double activities, it was identified a single lead to be pharmacologically tested in vivo. Its synthesis was optimized up to the gram-scale, and pretreatment method and HPLC-MS/MS analytical protocol for sub-nanomolar quantifications were developed. Furthermore, replacement of acetoxy group in 4-acetoxy-azetidinone derivatives was studied with different nucleophiles and in aqueous media. A phosphate group was introduced and the reactivity exploited using different hydroxyapatites, obtaining biomaterials with multiple biological activities. Following the same kind of reactivity, a small series of molecules with a β-lactam and retinoic hybrid structure was synthesized as epigenetic regulators. Interacting with HDACs, two compounds were respectively identified as an inhibitor of cell proliferation and a differentiating agent on steam cells. Additionally, in collaboration with Professor L. De Cola at ISIS, University of Strasbourg, some new photochemically active β-lactam Pt (II) complexes were designed and synthesized to be used as bioprobes or theranostics. Finally, it was set up and optimized the preparation of new chiral proline-derived α-aminonitriles through an enantioselective Strecker reaction, and it was developed a chemo-enzymatic oxidative method for converting alcohols to aldehydes or acid in a selective manner, and amines to relative aldehydes, amides or imines. Moreover, enzymes and other green chemistry methodologies were used to prepare Active Pharmaceutical Ingredients (APIs).

Atropisomeric xanthines: Synthesis, stereodynamics and absolute configuration

During the thesis period a new class of atropisomeric xanthine derivatives has been studied. We decided to focus our attention on these purine bases because of their various biological activities, that could play an important role in the discovery of new bioactive atropisomers. The synthesized compounds bear an Aryl-N chiral axis in position 1 of the xanthine scaffold, around which the rotation is prevented by the presence of bulky ortho substituents. Through a retro synthetic analysis we synthesized three atropisomeric structures bearing in position 1 of the purine scaffold respectively an o-tolyl, o-nitrophenyl and a 1-naphthyl group. The conformational studies by DFT simulations showed that the interconversion energy barrier between the two available skewed conformations is higher enough to obtain thermally stable atropisomers. After the separation of the atropisomers, the experimental energy of interconversion was investigated by means of kinetic studies following the thermal racemization process using an enantioselective HPLC column. The absolute configuration of each atropisomer was assigned by experimental ECD analysis and TD-DFT simulations of the ECD spectra.

Nuovi coniugati fra acido azelaico ed azaeterocicli: sintesi e valutazione dell'attività biologica

The well-known antiproliferative properties of the 9-hydroxystearic acid (9-HSA) on human colon cancer cells (HT-29 cell line) have inspired this thesis work in order to obtain new derivatives maintaining the C1-C8 chain of the HSA linked to an heterocyclic moiety at the C-9 carbon atom and to investigate their biological activity. First, thiazoles, thiadiazoles and benzothiazoles, that are compounds of interest in many fields for their biological activities, have been introduced through an amide bond starting from their 2-amino precursors. The products have been obtained by treatment with methyl 9-chloro-9-oxononanoate according to a Schotten-Baumann type reaction. The acylation reaction occurred at the endocyclic nitrogen atom of the heterocycle, as ascertained through NOESY-1D experiment. After, methyl 9-chloro-9-oxononanoate was reacted with indole, N-methylindole, and triptamine giving a serie of new indole derivatives. Finally, the biological activity of some compounds has been tested through assays on HT-29 cancer cells and bacterial and fungal microorganisms; docking calculations have also been performed to evaluate the possible interactions with the active site of histone deacetylase, which are molecular targets of the 9-HSA.

Plant volatiles: production, function and pharmacology

Plant volatiles typically occur as a complex mixture of low-molecular weight lipophilic compounds derived from different biosynthetic pathways, and are seemingly produced as part of a defense strategy against biotic and abiotic stress, as well as contributing to various physiological functions of the producer organism. The biochemistry and molecular biology of plant volatiles is complex, and involves the interplay of several biochemical pathways and hundreds of genes. All plants are able to store and emit volatile organic compounds (VOCs), but the process shows remarkable genotypic variation and phenotypic plasticity. From a physiological standpoint, plant volatiles are involved in three critical processes, namely plant–plant interaction, the signaling between symbiotic organisms, and the attraction of pollinating insects. Their role in these ‘‘housekeeping’’ activities underlies agricultural applications that range from the search for sustainable methods for pest control to the production of flavors and fragrances. On the other hand, there is also growing evidence that VOCs are endowed with a range of biological activities in mammals, and that they represent a substantially under-exploited and still largely untapped source of novel drugs and drug leads. This review summarizes recent major developments in the study of biosynthesis, ecological functions and medicinal applications of plant VOCs.

Characterization of Molecular Determinants of the Conformational Stability of Macrophage Migration Inhibitory Factor: Leucine 46 Hydrophobic Pocket

Macrophage Migration Inhibitory Factor (MIF) is a key mediator of inflammatory responses and innate immunity and has been implicated in the pathogenesis of several inflammatory and autoimmune diseases. The oligomerization of MIF, more specifically trimer formation, is essential for its keto-enol tautomerase activity and probably mediates several of its interactions and biological activities, including its binding to its receptor CD74 and activation of certain signaling pathways. Therefore, understanding the molecular factors governing the oligomerization of MIF and the role of quaternary structure in modulating its structural stability and multifunctional properties is crucial for understanding the function of MIF in health and disease. Herein, we describe highly conserved intersubunit interactions involving the hydrophobic packing of the side chain of Leu46 onto the β-strand β3 of one monomer within a hydrophobic pocket from the adjacent monomer constituted by residues Arg11, Val14, Phe18, Leu19, Val39, His40, Val41, Val42, and Pro43. To elucidate the structural significance of these intersubunit interactions and their relative contribution to MIF’s trimerization, structural stability and catalytic activity, we generated three point mutations where Leu46 was replaced by glycine (L46G), alanine (L46A) and phenylalanine (L46F), and their structural properties, stability, oligomerization state, and catalytic activity were characterized using a battery of biophysical methods and X-ray crystallography. Our findings provide new insights into the role of the Leu46 hydrophobic pocket in stabilizing the conformational state of MIF in solution. Disrupting the Leu46 hydrophobic interaction perturbs the secondary and tertiary structure of the protein but has no effect on its oligomerization state.

QSAR Studies on Thiazolidines: A Biologically Privileged Scaffold

A large number of drugs and biologically relevant molecules contain heterocyclic systems. Often the presence of hetero atoms or groupings imparts preferential specificities in their biological responses. Amongst the heterocyclic systems, thiazolidine is a biologically important scaffold known to be associated with several biological activities. Some of the prominent biological responses attributed to this skeleton are antiviral, antibacterial, antifungal, antihistaminic, hypoglycemic, anti-inflammatory activities. This diversity in the biological response profiles of thiazolidine has attracted the attention of many researchers to explore this skeleton to its multiple potential against several activities. Many of these synthetic and biological explorations have been subsequently analyzed in detailed quantitative structure-activity relationship (QSAR) studies to correlate the respective structural features and physicochemical properties with the activities to identify the important structural components in deciding their activity behavior. In this, drugs or any biologically active molecules may be viewed as structural frames consisting of strategically positioned functional groups that will interact effectively with the complementary groups/sites of the receptor. With this in focus, the present article reviews the QSAR studies of diverse biological activities of the thiazolidines published during the past decade.

Screeningof Indian Plants for BiologicalActivity-Part XI

Alcoholic extracts of two hundred and ninety two botanically identified plant species have been tested for a wide variety of biological activities including chemotherapeutic and pharmacological activities. Biological activities have been confirmed in fractions of 46 of these extracts. Follow-up studies have been carried out in some plants with confirmed activity. The active principles and results of these studies have also been reported.

MQN-Mapplet: Visualization of Chemical Space with Interactive Maps of DrugBank, ChEMBL, PubChem, GDB-11, and GDB-13

The MQN-mapplet is a Java application giving access to the structure of small molecules in large databases via color-coded maps of their chemical space. These maps are projections from a 42-dimensional property space defined by 42 integer value descriptors called molecular quantum numbers (MQN), which count different categories of atoms, bonds, polar groups, and topological features and categorize molecules by size, rigidity, and polarity. Despite its simplicity, MQN-space is relevant to biological activities. The MQN-mapplet allows localization of any molecule on the color-coded images, visualization of the molecules, and identification of analogs as neighbors on the MQN-map or in the original 42-dimensional MQN-space. No query molecule is necessary to start the exploration, which may be particularly attractive for nonchemists. To our knowledge, this type of interactive exploration tool is unprecedented for very large databases such as PubChem and GDB-13 (almost one billion molecules). The application is freely available for download at www.gdb.unibe.ch.

Expression of Foxi3 is regulated by ectodysplasin in skin appendage placodes

BACKGROUND Foxi3 is a member of the large forkhead box family of transcriptional regulators, which have a wide range of biological activities including manifold developmental processes. Heterozygous mutation in Foxi3 was identified in several hairless dog breeds characterized by sparse fur coat and missing teeth. A related phenotype called hypohidrotic ectodermal dysplasia (HED) is caused by mutations in the ectodysplasin (Eda) pathway genes. RESULTS Expression of Foxi3 was strictly confined to the epithelium in developing ectodermal appendages in mouse embryos, but no expression was detected in the epidermis. Foxi3 was expressed in teeth and hair follicles throughout embryogenesis, but in mammary glands only during the earliest stages of development. Foxi3 expression was decreased and increased in Eda loss- and gain-of-function embryos, respectively, and was highly induced by Eda protein in embryonic skin explants. Also activin A treatment up-regulated Foxi3 mRNA levels in vitro. CONCLUSIONS Eda and activin A were identified as upstream regulators of Foxi3. Foxi3 is a likely transcriptional target of Eda in ectodermal appendage placodes suggesting that HED phenotype may in part be produced by compromised Foxi3 activity. In addition to hair and teeth, Foxi3 may have a role in nail, eye, and mammary, sweat, and salivary gland development.

THE ESTROGENIC PROPERTIES OF O,P'-DDT

o,p'-DDT is a major component of the pesticide DDT (dichlorodiphenyltrichloro ethane, technical grade). Although possessing little insecticidal ability, the o,p'- isomer has two major biological activities which affect mammalian reproductive systems: it is estrogenic, and it induces hepatic mixed function oxidase enzymes. The focus of this work is the characterization of the estrogenic properties of o,p'-DDT in rodents.^ Initial studies examined the ability of o,p'-DDT to bind to and interact with elements of the estrogen receptor system. In an in vitro assay, DDT was shown to compete with 17(beta)-estradiol (E(,2)) for binding to cytoplasmic estrogen receptors (R(,c)) from normal and neoplastic tissues in two rodent species. The following phenomena were studied by measuring receptor levels from uteri (whole uteri and/or uterine cell types) taken from immature ovariectomized rats given one acute injection of o,p'-DDT or E(,2): the translocation of the R(,c) to the nucleus, nuclear receptor (R(,n)) retention patterns, and the subsequent reappearance of R(,c) in the cytoplasm.^ The magnitude and temporal patterns of the biological responses of uteri from similar immature rats were compared following o,p'-DDT and E(,2) exposure. The responses examined included increased "Induced Protein" synthesis (in vitro); and uterine wet weight, DNA synthesis and mitosis (in vivo).^ From dose-response data, correlations were made between R(,n) levels and levels of subsequent biological responses. The aim was to lend support to the premise that biological responses to o,p'-DDT exposure occur as a result of its interaction with the classical estrogen receptor system. Correlation coefficients of 0.95 to 0.98 were obtained between R(,n) levels and levels of responses examined, strongly supporting this hypothesis.^ Finally, o,p'-DDT was shown to be as effective as E(,2) in supporting the growth of a transplantable estrogen-responsive mammary tumor in adult rats (although it was unable to support the growth of a transplantable estrogen-dependent renal tumor in hamsters). While the positive result cannot be directly extrapolated to human or animal exposure to environmental estrogens, it suggests that hyperplastic responses of estrogen sensitive tissues should be considered as a possible toxicity of o,p'-DDT, related compounds having estrogenic properties, and other environmental estrogens. ^

Heme oxygenase 1 is required for mammalian iron reutilization

The majority of iron for essential mammalian biological activities such as erythropoiesis is thought to be reutilized from cellular hemoproteins. Here, we generated mice lacking functional heme oxygenase 1 (Hmox1; EC 1.14.99.3), which catabolizes heme to biliverdin, carbon monoxide, and free iron, to assess its participation in iron homeostasis. Hmox1-deficient adult mice developed an anemia associated with abnormally low serum iron levels, yet accumulated hepatic and renal iron that contributed to macromolecular oxidative damage, tissue injury, and chronic inflammation. Our results indicate that Hmox1 has an important recycling role by facilitating the release of iron from hepatic and renal cells, and describe a mouse model of human iron metabolic disorders.

The crystal structure of human interferon β at 2.2-Å resolution

Type I interferons (IFNs) are helical cytokines that have diverse biological activities despite the fact that they appear to interact with the same receptor system. To achieve a better understanding of the structural basis for the different activities of α and β IFNs, we have determined the crystal structure of glycosylated human IFN-β at 2.2-Å resolution by molecular replacement. The molecule adopts a fold similar to that of the previously determined structures of murine IFN-β and human IFN-α2b but displays several distinct structural features. Like human IFN-α2b, human IFN-β contains a zinc-binding site at the interface of the two molecules in the asymmetric unit, raising the question of functional relevance for IFN-β dimers. However, unlike the human IFN-α2b dimer, in which homologous surfaces form the interface, human IFN-β dimerizes with contact surfaces from opposite sides of the molecule. The relevance of the structure to the effects of point mutations in IFN-β at specific exposed residues is discussed. A potential role of ligand–ligand interactions in the conformational assembly of IFN receptor components is discussed.

Glial cell line-derived neurotrophic factor promotes the development of adrenergic neurons in mouse neural crest cultures

Growth of mouse neural crest cultures in the presence of glial cell line-derived neurotrophic factor (GDNF) resulted in a dramatic dose-dependent increase in the number of tyrosine hydroxylase (TH)-positive cells that developed when 5% chicken embryo extract was present in the medium. In contrast, growth in the presence of bone morphogenetic protein (BMP)-2, BMP-4, BMP-6, transforming growth factor (TGF) β1, TGF-β2, and TGF-β3 elicited no increase in the number of TH-positive cells. The TH-positive cells that developed in the presence of GDNF had neuronal morphology and contained the middle and low molecular weight neurofilament proteins. Numerous TH-negative cells with the morphology of neurons also were observed in GDNF-treated cultures. Analysis revealed that the period from 6 to 12 days in vitro was the critical time for exposure to GDNF to generate the increase in TH-positive cell number. The growth factors neurotrophin-3 and fibroblast growth factor-2 elicited increases in the number of TH-positive cells similar to that seen in response to GDNF. In contrast, nerve growth factor was unable to substitute for GDNF. These findings extend the previously reported biological activities of GDNF by showing that it can act on mouse neural crest cultures to promote the development of neurons.

Soluble complexes of regulated upon activation, normal T cells expressed and secreted (RANTES) and glycosaminoglycans suppress HIV-1 infection but do not induce Ca2+ signaling

Chemokines comprise a family of low-molecular-weight proteins that elicit a variety of biological responses including chemotaxis, intracellular Ca2+ mobilization, and activation of tyrosine kinase signaling cascades. A subset of chemokines, including regulated upon activation, normal T cell expressed and secreted (RANTES), macrophage inflammatory protein-1α (MIP-1α), and MIP-1β, also suppress infection by HIV-1. All of these activities are contingent on interactions between chemokines and cognate seven-transmembrane spanning, G protein-coupled receptors. However, these activities are strongly inhibited by glycanase treatment of receptor-expressing cells, indicating an additional dependence on surface glycosaminoglycans (GAG). To further investigate this dependence, we examined whether soluble GAG could reconstitute the biological activities of RANTES on glycanase-treated cells. Complexes formed between RANTES and a number of soluble GAG failed to induce intracellular Ca2+ mobilization on either glycanase-treated or untreated peripheral blood mononuclear cells and were unable to stimulate chemotaxis. In contrast, the same complexes demonstrated suppressive activity against macrophage tropic HIV-1. Complexes composed of 125I-labeled RANTES demonstrated saturable binding to glycanase-treated peripheral blood mononuclear cells, and such binding could be reversed partially by an anti-CCR5 antibody. These results suggest that soluble chemokine–GAG complexes represent seven-transmembrane ligands that do not activate receptors yet suppress HIV infection. Such complexes may be considered as therapeutic formulations for the treatment of HIV-1 infection.