Teresa Riccioni

Identification of Placenta Growth Factor Determinants for Binding and Activation of Flt-1 Receptor

Placenta growth factor (PlGF) belongs to the vascular endothelial growth factor (VEGF) family and represents a key regulator of angiogenic events in pathological conditions. PlGF exerts its biological function through the binding and activation of the seven immunoglobulin-like domain receptor Flt-1, also known as VEGFR-1. Here, we report the first detailed mutagenesis studies that provide a basis for understanding molecular recognition between PlGF-1 and Flt-1, highlighting some of the residues that are critical for receptor recognition. Mutagenesis analysis, performed on the basis of a structural model of interaction between PlGF and the minimal binding domain of Flt-1, has led to the identification of several PlGF-1 residues involved in Flt-1 recognition. The two negatively charged residues, Asp-72 and Glu-73, located in the β3-β4 loop, are critical for Flt-1 binding. Other mutations, which bring about a significant decrease in PlGF binding activity, are Gln-27, located in the N-terminal α-helix, and Pro-98 and Tyr-100 on the β6 strand. The mutation of one of the two glycosylated residues of PlGF, Asn-84, generates a PlGF variant with reduced binding activity. This indicates that, unlike in VEGF, glycosylation plays an important role in Flt-1 binding. The double mutation of residues Asp-72 and Glu-73 generates a PlGF variant unable to bind and activate the receptor molecules on the cell surface. This variant failed to induce in vitro capillary-like tube formation of primary endothelial cells or neo-angiogenesis in an in vivo chorioallantoic membrane assay.

Propionyl-l-Carnitine Improves Postischemic Blood Flow Recovery and Arteriogenetic Revascularization and Reduces Endothelial NADPH-Oxidase 4–Mediated Superoxide Production

Objective—The beneficial effect of the natural compound propionyl-l-carnitine (PLC) on intermittent claudication in patients with peripheral arterial disease is attributed to its anaplerotic function in ischemic tissues, but inadequate information is available concerning action on the vasculature. Methods and Results—We investigated the effects of PLC in rabbit hind limb collateral vessels after femoral artery excision, mouse dorsal air pouch, chicken chorioallantoic membrane, and vascular cells by angiographic, Doppler flow, and histomorphometrical and biomolecular analyses. PLC injection accelerated hind limb blood flow recovery after 4 days (P<0.05) and increased angiographic quadriceps collateral vascularization after 7 days (P<0.001) Histomorphometry confirmed the increased vascular area (P<0.05), with unchanged intramuscular capillary density. PLC-induced dilatative adaptation, and growth was found associated with increased inducible nitric oxide synthase and reduced arterial vascular endothelial growth factor and intracellular adhesion molecule-1 expression. PLC also increased vascularization in air pouch and chorioallantoic membrane (P<0.05), particularly in large vessels. PLC increased endothelial and human umbilical vascular endothelial cell proliferation and rapidly reduced inducible nitric oxide synthase and NADPH-oxidase 4–mediated reactive oxygen species production in human umbilical vascular endothelial cells; NADPH-oxidase 4 also regulated NF-&kgr;B–independent intracellular adhesion molecule-1 expression. Conclusion—Our results provided strong evidence that PLC improves postischemic flow recovery and revascularization and reduces endothelial NADPH-oxidase–related superoxide production. We recommend that PLC should be included among therapeutic interventions that target endothelial function.

Synthesis and Biological Activity of Fluorinated Combretastatin Analogues

With the aim of understanding the influence of fluorine on the double bond of the cis-stilbene moiety of combretastatin derivatives and encouraged by a preliminary molecular modeling study showing a different biological environment on the interaction site with tubulin, we prepared, through various synthetic approaches, a small library of compounds in which one or both of the olefinic hydrogens were replaced with fluorine. X-ray analysis on the difluoro-CA-4 analogue demonstrated that the spatial arrangement of the molecule was not modified, compared to its nonfluorinated counterpart. SAR analysis confirmed the importance of the cis-stereochemistry of the stilbene scaffold. Nevertheless, some unpredicted results were observed on a few trans-fluorinated derivatives. The position of a fluorine atom on the double bond may affect the inhibition of tubulin polymerization and cytotoxic activity of these compounds.

Biological and molecular properties of a new αvβ3/αvβ5 integrin antagonist

The aim of the present study was to identify specific αvβ3/αvβ5 integrin antagonists active on tumor-induced angiogenesis. To this purpose, in vitro integrin-binding assays were used to screen a library of conformationally constrained bicyclic lactam Arg-Gly-Asp–containing pseudopeptides. The results identified ST1646 as a high-affinity specific ligand for αvβ3 and αvβ5 integrins with negligible interacting with α5β1 integrin. In all the assays, ST1646 was equipotent to or more potent than the well-characterized integrin antagonists c(RGDfV) and cyclo(Arg-Gly-Asp-d-Phe-[NMe]Val) (EMD121974). In the chorioallantoic membrane assay, topical administration of ST1646 was able to prevent the angiogenic responses elicited by recombinant fibroblast growth factor-2 or vascular endothelial growth factor. In addition, systemic administration of ST1646 in mice exerted a significant antiangiogenic activity on neovascularization triggered by mammary carcinoma MDA-MB435 cells implanted s.c. in a dorsal air sac via a (Millipore Filter Corporation, Bedford, MA) chamber. Moreover, ST1646 delivery via an osmotic pump inhibited the growth and vascularization of tumor xenografts originating from the injection of αvβ3/αvβ5-expressing human ovarian carcinoma cells in nude mice. In agreement with the biochemical and pharmacologic studies, Monte Carlo/Stochastic Dynamics simulation showed that the bicyclic scaffold in ST1646 forced the compound to assume a preferred conformation superimposable to the X-ray conformation of αvβ3-bound EMD121974. Accordingly, computer-docking studies indicated that the ST1646-αvβ3 integrin complex maintains the ligand-receptor distances and interactions observed in the crystalline EMD121974-αvβ3 integrin complex. Taken together, these observations indicate that ST1646 represents a dual αvβ3/αvβ5 integrin antagonist with interesting biochemical and biological features to be tested in cancer therapy.

Biological and molecular properties of a new alpha(v)beta3/alpha(v)beta5 integrin antagonist.

The aim of the present study was to identify specific αvβ3/αvβ5 integrin antagonists active on tumor-induced angiogenesis. To this purpose, in vitro integrin-binding assays were used to screen a library of conformationally constrained bicyclic lactam Arg-Gly-Asp–containing pseudopeptides. The results identified ST1646 as a high-affinity specific ligand for αvβ3 and αvβ5 integrins with negligible interacting with α5β1 integrin. In all the assays, ST1646 was equipotent to or more potent than the well-characterized integrin antagonists c(RGDfV) and cyclo(Arg-Gly-Asp-d-Phe-[NMe]Val) (EMD121974). In the chorioallantoic membrane assay, topical administration of ST1646 was able to prevent the angiogenic responses elicited by recombinant fibroblast growth factor-2 or vascular endothelial growth factor. In addition, systemic administration of ST1646 in mice exerted a significant antiangiogenic activity on neovascularization triggered by mammary carcinoma MDA-MB435 cells implanted s.c. in a dorsal air sac via a (Millipore Filter Corporation, Bedford, MA) chamber. Moreover, ST1646 delivery via an osmotic pump inhibited the growth and vascularization of tumor xenografts originating from the injection of αvβ3/αvβ5-expressing human ovarian carcinoma cells in nude mice. In agreement with the biochemical and pharmacologic studies, Monte Carlo/Stochastic Dynamics simulation showed that the bicyclic scaffold in ST1646 forced the compound to assume a preferred conformation superimposable to the X-ray conformation of αvβ3-bound EMD121974. Accordingly, computer-docking studies indicated that the ST1646-αvβ3 integrin complex maintains the ligand-receptor distances and interactions observed in the crystalline EMD121974-αvβ3 integrin complex. Taken together, these observations indicate that ST1646 represents a dual αvβ3/αvβ5 integrin antagonist with interesting biochemical and biological features to be tested in cancer therapy.

Synthesis of (E)-8-(3-Chlorostyryl)caffeine Analogues Leading to 9-Deazaxanthine Derivatives as Dual A2A Antagonists/MAO-B Inhibitors

A systematic modification of the caffeinyl core and substituents of the reference compound (E)-8-(3-chlorostyryl)caffeine led to the 9-deazaxanthine derivative (E)-6-(4-chlorostyryl)-1,3,5,-trimethyl-1H-pyrrolo[3,2-d]pyrimidine-2,4-(3H,5H)-dione (17f), which acts as a dual human A(2a) antagonist/MAO-B inhibitor (K(i)(A(2A)) = 260 nM; IC(50)(MAO-B) = 200 nM; IC(50)(MAO-A) = 10 μM) and dose dependently counteracts haloperidol-induced catalepsy in mice from 30 mg/kg by the oral route. The compound is the best balanced A(2A) antagonist/MAO-B inhibitor reported to date, and it could be considered as a new lead in the field of anti-Parkinsons agents. A number of analogues of 17f were synthesized and qualitative SARs are discussed. Two analogues of 17f, namely 18b and 19a, inhibit MAO-B with IC(50) of 68 and 48 nM, respectively, being 5-7-fold more potent than the prototypical MAO-B inhibitor deprenyl (IC(50) = 334 nM).

ST1936 stimulates cAMP, Ca2+, ERK1/2 and Fyn kinase through a full activation of cloned human 5-HT6 receptors.

5-HT(6) receptor is one of the most recently cloned serotonin receptors, and it might play important roles in Alzheimers disease, depression, and learning and memory disorders. Availability of only very few 5-HT(6) receptor agonists, however, does not allow examining their contribution in psychopharmacological processes. Therefore, a new 5-HT(6) receptor agonist, ST1936, was synthesized. ST1936 binds to human 5-HT(6) receptors with good affinity (K(i)=28.8 nM). ST1936 also exhibited some moderate binding affinity for 5HT(2B), 5HT(1A), 5HT(7) receptors and adrenergic α receptors. ST1936 behaved as a full 5-HT(6) agonist on cloned cells and was able to increase Ca(2+) concentration, phosphorylation of Fyn kinase, and regulate the activation of ERK1/2 that is a downstream target of Fyn kinase. These effects were completely antagonized by two 5-HT(6) receptor antagonists, SB271046 and SB258585. The other 5-HT(6) receptor agonist, WAY181187 also increased Fyn kinase activity. These results suggest that both ST1936 and WAY181187 mediate 5-HT(6) receptor-dependent signal pathways, such as cAMP, Fyn and ERK1/2 kinase, as specific agonists.

Biological and molecular properties of a new v 3/ v 5 integrin antagonist

The aim of the present study was to identify specific αvβ3/αvβ5 integrin antagonists active on tumor-induced angiogenesis. To this purpose, in vitro integrin-binding assays were used to screen a library of conformationally constrained bicyclic lactam Arg-Gly-Asp–containing pseudopeptides. The results identified ST1646 as a high-affinity specific ligand for αvβ3 and αvβ5 integrins with negligible interacting with α5β1 integrin. In all the assays, ST1646 was equipotent to or more potent than the well-characterized integrin antagonists c(RGDfV) and cyclo(Arg-Gly-Asp-d-Phe-[NMe]Val) (EMD121974). In the chorioallantoic membrane assay, topical administration of ST1646 was able to prevent the angiogenic responses elicited by recombinant fibroblast growth factor-2 or vascular endothelial growth factor. In addition, systemic administration of ST1646 in mice exerted a significant antiangiogenic activity on neovascularization triggered by mammary carcinoma MDA-MB435 cells implanted s.c. in a dorsal air sac via a (Millipore Filter Corporation, Bedford, MA) chamber. Moreover, ST1646 delivery via an osmotic pump inhibited the growth and vascularization of tumor xenografts originating from the injection of αvβ3/αvβ5-expressing human ovarian carcinoma cells in nude mice. In agreement with the biochemical and pharmacologic studies, Monte Carlo/Stochastic Dynamics simulation showed that the bicyclic scaffold in ST1646 forced the compound to assume a preferred conformation superimposable to the X-ray conformation of αvβ3-bound EMD121974. Accordingly, computer-docking studies indicated that the ST1646-αvβ3 integrin complex maintains the ligand-receptor distances and interactions observed in the crystalline EMD121974-αvβ3 integrin complex. Taken together, these observations indicate that ST1646 represents a dual αvβ3/αvβ5 integrin antagonist with interesting biochemical and biological features to be tested in cancer therapy.

5-HT6 pharmacology inconsistencies.

5-HT(6) receptors are relatively recently-discovered receptors. After an uncertain beginning, where results were ambiguous, findings are now apparently more congruent. Nevertheless, discrepancies still exist. The aim of the present manuscript is to point out some of these discrepancies, in order to reflect on the current status of the field of the 5-HT(6) receptor neuropharmacology, and where the field should move next. Examples of 5-HT(6) receptor ligand-induced changes in behavior, neurochemistry and binding highlight areas where discrepancies remain and further experimental attention is needed. Possible methodological as well as conceptual issues underlying the inconsistencies are considered in an effort to increase awareness of the need for more thorough consideration of these aspects in future research.

Synthesis and Biological Evaluation of Metabolites of 2-n-Butyl-9-methyl-8-[1,2,3]triazol-2-yl-9H-purin-6-ylamine (ST1535), A Potent Antagonist of the A2A Adenosine Receptor for the Treatment of Parkinson’s Disease

The synthesis and preliminary in vitro evaluation of five metabolites of the A2A antagonist ST1535 (1) are reported. The metabolites, originating in vivo from enzymatic oxidation of the 2-butyl group of the parent compound, were synthesized from 6-chloro-2-iodo-9-methyl-9H-purine (2) by selective C-C bond formation via halogen/magnesium exchange reaction and/or palladium-catalyzed reactions. The metabolites behaved in vitro as antagonist ligands of cloned human A2A receptor with affinities (Ki 7.5-53 nM) comparable to that of compound 1 (Ki 10.7 nM), thus showing that the long duration of action of 1 could be in part due to its metabolites. General behavior after oral administration in mice was also analyzed.