Santi Spampinato

Antiangiogenic Effect of Dual/Selective α5β1/αvβ3 Integrin Antagonists Designed on Partially Modified Retro-Inverso Cyclotetrapeptide Mimetics

Recent evidence highlighted the role of alpha(5)beta(1) integrin in angiogenesis and in regulating alpha(v)beta(3) integrin function. As a consequence, selective alpha(5)beta(1) integrin inhibitors or dual alpha(5)beta(1)/alpha(v)beta(3) integrin inhibitors are considered promising candidates for the development of cancer therapeutic agents. In this paper, we describe the synthesis and pharmacological characterization of a minilibrary of cyclotetrapeptide mimetics containing a PMRI Arg-Gly-Asp sequence. In particular, c[(R)-betaPhepsi(NHCO)Asppsi(NHCO)Gly-Arg] (3) displayed a good activity in inhibiting the alpha(v)beta(3) integrin-mediated cell adhesion of fibronectin or vitronectin, as well as the adhesion of fibronectin to the alpha(5)beta(1) integrin. Interestingly, the diastereomeric compound c[(S)-betaPhepsi(NHCO)Asppsi(NHCO)Gly-Arg] (2) maintained a good efficacy in inhibiting alpha(5)beta(1) integrin while gaining a certain selectivity over alpha(v)beta(3) integrin. These two integrin antagonists significantly inhibited bFGF-induced human endothelial cell tube formation at submicromolar concentrations. Conformational analysis and Molecular Docking calculations suggest that the different alpha(5)beta(1) versus alpha(v)beta(3) selectivity of 2 and 3 can be rationalized on the basis of the alternative display of the aromatic side chain adjacent to Asp.

The Inverse Type II β‐Turn on D‐Trp‐Phe, a Pharmacophoric Motif for MOR Agonists

Herein we propose the D‐Trp‐Phe sequence within an inverse typeu2005II β‐turn as a new kind of pharmacophoric motif for μ‐opioid receptor (MOR) cyclopeptide agonists. Initially, we observed that c[Tyr‐D‐Pro‐D‐Trp‐Phe‐Gly] (4), an analogue of endomorphin‐1 (H‐Tyr‐Pro‐Trp‐Phe‐NH2) lacking the crucial protonatable amino group of Tyru20091, is a MOR agonist with 10−8u2009M affinity. Molecular docking analysis suggested that the relevant interactions with the receptor involve D‐Trp‐Phe. The bioactive conformation of this region was investigated by selected derivatives of 4 designed to adopt an inverse typeu2005II β‐turn. These efforts led to c[Tyr‐Gly‐D‐Trp‐Phe‐Gly] (14) and to the cyclotetrapeptide c[D‐Asp‐1‐amide‐β‐Ala‐D‐Trp‐Phe] (15), showing improved nanomolar affinity. Both 14 and 15 selectively bind MOR, as they have negligible affinity for the κ‐ and δ‐opioid receptors. Both 14 and 15 behave as partial MOR agonists in functional assays. Conformational and docking analyses confirm the role of the inverse β‐turn in binding. These results indicate that the D‐Trp‐Phe inverse β‐turn structure can be used for designing non‐endomorphin‐like peptidomimetic opioid agonists in general, characterized by an atypical mechanism of interaction between ligand and receptor.

Ligand based approach to L-type calcium channel by imidazo[2,1-b]thiazole-1,4-dihydropyridines: from heart activity to brain affinity.

The synthesis, characterization, and functional in vitro assay in cardiac and smooth muscle (vascular and nonvascular) of a series of 4-imidazo[2,1-b]thiazole-1,4-dihydropyridines are reported. To define the calcium blocker nature of the imidazo[2,1-b]thiazole-1,4-DHPs and their selectivity on Cav1.2 and Cav1.3 isoforms, we performed binding studies on guinea pig atrial and ventricular membranes on intact cells expressing the cloned Cav1.2a subunit and on rat brain cortex. To get major insights into the reasons for the affinity for Cav1.2 and/or Cav1.3, molecular modeling studies were also undertaken. Some physicochemical and pharmacokinetic properties of selected compounds were calculated and compared. All the biological data collected and reported herein allowed us to rationalize the structure-activity relationship of the 4-imidazo[2,1-b]thiazole-1,4-DHPs and to identify which of these enhanced the activity at the central level.

Opioid Activity Profiles of Oversimplified Peptides Lacking in the Protonable N-Terminus

Recently, we described cyclopeptide opioid agonists containing the d-Trp-Phe sequence. To expand the scope of this atypical pharmacophore, we tested the activity profiles of the linear peptides Ac-Xaa-Phe-Yaa (Xaa = l/d-Trp, d-His/Lys/Arg; Yaa = H, GlyNH(2)). Ac-d-Trp-PheNH(2) appeared to be the minimal binding sequence, while Ac-d-Trp-Phe-GlyNH(2) emerged as the first noncationizable short peptide (partial) agonist with high μ-opioid receptor affinity and selectivity. Conformational analysis suggested that 5 adopts in solution a β-turn conformation.

Protective and worsening peripheral nociceptin/orphanin FQ receptor-mediated effect in a rat model of experimental colitis.

Nociceptin/orphanin FQ (N/OFQ) and nociceptin orphanin peptide (NOP) receptors represent an endogenous system modulating gastrointestinal functions and inflammation. We investigated the peripheral effect of N/OFQ and of UFP-101, the NOP antagonist, in a model of colitis induced by TNBS (2,4,6 trinitrobenzenesulphonic acid; 60mg/kg). Male rats received two intraperitoneal injections per day of N/OFQ, UFP-101 or saline for 3 days after colitis induction. Four days after TNBS, animals were sacrificed and colonic histological damage, myeloperoxidase (MPO) activity and cytokine (IL-1β and IL-10) levels were evaluated. N/OFQ plasmatic levels were assessed by radioimmunoassay. TNBS increased all the inflammatory variables considered. In colitic rats, N/OFQ (0.02 and 0.2nmol/kg) improved microscopic damage, MPO activity and decreased IL-1β levels in comparison with TNBS group, whereas at the highest dose (20nmol/kg) the peptide worsened colitis. UFP-101 at the dose of 1nmol/kg, without pharmacological activity, antagonised the protective effect of N/OFQ (0.2nmol/kg) on colitis, but at a dose level of 3 and 10nmol/kg worsened inflammation, revealing the endogenous N/OFQergic system protective role. N/OFQ plasmatic levels were not modified in TNBS-treated rats compared with controls, whereas they were reduced in rats treated with the doses of UFP-101 aggravating colitis. In conclusion, peripheral low doses of N/OFQ have a beneficial effect on colonic inflammation in rats. In contrast, N/OFQ at a dose 100-1000-fold higher than those that protect worsens colitis, probably through different mechanisms. The peripheral N/OFQergic system can represent a new field of investigation in some intestinal inflammatory conditions.

Therapeutic Targeting of Eosinophil Adhesion and Accumulation in Allergic Conjunctivitis

Considerable evidence indicates that eosinophils are important effectors of ocular allergy. Increased worldwide prevalence of allergic eye pathologies has stimulated the identification of novel drug targets, including eosinophils and adhesion molecules. Accumulation of eosinophils in the eye is a key event in the onset and maintenance of allergic inflammation and is mediated by different adhesion molecules. Antihistamines with multiple mechanisms of action can be effective during the early and late phases of allergic conjunctivitis by blocking the interaction between β1 integrins and vascular cell adhesion molecule (VCAM)-1. Small molecule antagonists that target key elements in the process of eosinophil recruitment have been identified and reinforce the validity of α4β1 integrin as a therapeutic target. Glucocorticoids are among the most effective drugs for ocular allergy, but their use is limited by adverse effects. Novel dissociated glucocorticoids can prevent eosinophil accumulation and induce apoptosis of eosinophils, making them promising candidates for ophthalmic drugs. This article reviews recent understanding of the role of adhesion molecules in eosinophil recruitment in the inflamed conjunctiva along with effective treatments for allergic conjunctivitis.

β-Arrestin 2-mediated heterologous desensitization of IGF-IR by prolonged exposure of SH-SY5Y neuroblastoma cells to a mu opioid agonist

Prolonged (12 h) exposure of SH‐SY5Y neuroblastoma cells to the mu‐opioid receptor (MOPr) agonist [D‐Ala2,N‐Me‐Phe4,Gly5‐ol]‐enkephalin (DAMGO) causes homologous desensitization as well as heterologous desensitization of the extracellular signal‐regulated kinase 1/2 (ERK 1/2) phosphorylation induced by insulin‐like growth factor (IGF)‐I. Brief (15 min) but not prolonged exposure to DAMGO transregulates the insulin‐like growth factor‐I (IGF‐I) receptor, as evidenced by its phosphorylation in the absence of IGF‐I. Silencing of β‐arrestin 2 uncouples the crosstalk between the two receptors, thus maintaining IGF‐I‐mediated receptor phosphorylation and ERK 1/2 activation even after prolonged DAMGO exposure. Furthermore, MOPr‐induced activation of IGF‐I receptor requires the tyrosine kinase c‐Src.

Design and synthesis of opioid peptide analogues and mimetics

Background and Aim: Native opioid peptides possess enormous promise for the treatment of pain. However, despite this potential, such peptides have seen no use as clinically viable drugs for their low stability against proteolysis, resulting in a short duration of in vivo activity, and for their poor ability to cross the blood-brain barrier. One way to overcome these disadvantages is the use of modified peptides, the so called peptidomimetics [Gentilucci et al., 2010]. Methods: Starting from the structure of Endomorphin-1 (Tyr-Pro-Trp-PheNH2), the endogenous ligand of μ-opioid receptor (MOR), we prepared modified peptides and peptidomimetics by introduction of unnatural amino acids, halogenation of the Trp, introduction of Pro-analogues, lipidization, and size reduction. The syntheses of (S)or (R)-halo-tryphtophans and other Trp-analogues have been performed by tandem 1,4-addition and stereoselective enolate protonation of DHA-containing peptides. Otherwise, substituted tryphtophans have been prepared in optically pure form by enzymatic resolution. Oxazolidin-2one4-carboxylate has been introduced in position 2 of endomorphin-1 as a proline mimetic, to give a trans conformation of the Tyr1-Xaa2 peptide bond. Lipidization and a rationale search for focused structure modifications lead to the endomorphin mimetic N-Ac-DTrp-Phe-Gly-NH2 and halo-substituted derivatives. Results: The modified peptides and mimetics have been bio-assayed in vitro by binding experiments, to determine the affinity for MORs, and in vivo, to verify the analgesic activity; the bioactivity has been rationalized by molecular docking computations. Conclusion: The new endomorphin analogues and mimetics described here constitute promising leads for the development of potentially useful, lipophilic analgesics.

REST is up-regulated by epidermal growth factor in HeLa cells and inhibits apoptosis by influencing histone H3 acetylation.

REST (repressor element 1-silencing transcription factor) is a transcription factor that recruits histone deacetylases to silence gene transcription. REST appears to play a paradoxical role in cancer cells: it exhibits tumor suppressor activity or promotes tumorigenesis, depending upon the setting. The extracellular signaling molecules that control REST gene expression in cancer cells remain poorly understood. In this study, we report that REST expression in HeLa cells is elevated in cells exposed to epidermal growth factor or serum, whereas the rate of cell apoptosis is low. Apoptosis induced by serum withdrawal is significantly increased in HeLa cells treated with an antisense phosphorothioate oligodeoxynucleotide (AS ODN) capable of down-regulating REST expression, whereas in HeLa cells transfected with a REST expressing plasmid, REST overexpression reduces the marked apoptosis caused, in absence of serum, by exposure to an anti-Fas receptor antibody imitating the Fas ligand activity plus PD 98059, a blocker of extracellular signal-regulated kinase 1/2 activation. REST knockdown also reduces mRNA levels of the antiapoptotic protein Bcl-X(L) whereas in HeLa cells overexpressing REST, the reduction of Bcl-X(L) mRNA caused by the anti-Fas receptor antibody plus PD 98059 is significantly decreased. Finally, we report that acetylation of histone H3 is increased in HeLa cells exposed to AS ODN or anti-Fas receptor antibody, whereas it is reduced in cells transfected with the REST expressing plasmid. Our findings indicate that REST is a novel gene regulated by EGF in HeLa cells that potentially contributes to the modulation of apoptosis via epigenetic mechanisms.

Pain and Child: A Translational Hypothesis on the Pathophysiology of a Mild Type-2 Diabetes Model

Pediatric pain management underwent many changes since the undertreatment of pain in children was reported in the literature in 1980. Increasing data also suggest that long-term behavioural effects can be observed in children, following pain episodes as early as in the neonatal period. Therefore, the knowledge about safe and effective management of pain in children should be applied with greater effectiveness into clinical practice. Other advances in the field include the findings of long-term residual behavioural and metabolic effects induced by pain experienced during the critical periods of development in laboratory animals. Recent data in laboratory animals and clinical data in children suggest that early repeated and/or severe pain and other stressful procedures applied in the perinatal periods may produce not only behavioral, but also important hormonal, immune and metabolic long-term effects. In this paper we shall report data on some metabolic conditions described in adult humans following disruption of hormonal-metabolic programming produced in the peri-natal period. Quite similar signs can be found between animal models and human conditions, most of them being connected with hypothalamus-pituitary-adrenal hormones (HPA) dysfunction. In addition, some signs in animal models, such as overweight and abdominal overweight are prevented by treatment with the μ- and δ-opioid receptor antagonist naloxone during the lactating period. This indicates that some long-term consequences following stress received during the early phases of life in mammals may be bound to the HPA system dysregulation, whereas others are bound to different (e,g., opioid) endogenous brain receptors and/or neuromediators alteration.