Anna Bottoncetti

Click-chemistry-derived triazole ligands of arginine-glycine-aspartate (RGD) integrins with a broad capacity to inhibit adhesion of melanoma cells and both in vitro and in vivo angiogenesis.

A click chemistry approach was applied for the discovery of triazole-based arginine-glycine-aspartate (RGD) mimetics by Cu(I)-catalyzed 1,3-dipolar alkyne-azide coupling reaction, which showed binding affinity properties toward α(v)β(3)/α(v)β(5) integrins. Biological assays showed compound 18 capable of binding α(v)β(3) integrin with nanomolar affinity according to a two-sites model, and molecular modeling studies revealed a peculiar π-stacking interaction between the triazole ring and Tyr178 side chain. Accordingly, compound 18 inhibited the adhesion of integrin-expressing human melanoma cells to RGD-containing proteins of the extracellular matrix, such as vitronectin, fibronectin, and osteopontin, and also angiogenesis in in vitro and in vivo experimental models. The relevant biological effects exerted by compound 18 suggest its potential application as an antiangiogenic agent in the diagnosis and therapy of tumors where α(v)β(3) integrin expression is up-regulated.

Discovery of Subnanomolar Arginine-Glycine-Aspartate-Based αVβ3/αVβ5 Integrin Binders Embedding 4-Aminoproline Residues

The embodiment of 4-aminoproline residues (Amp) into the arginine-glycine-aspartate (RGD) sequence led to the discovery of a novel class of high-affinity alpha Vbeta 3/alpha Vbeta 5 integrin binders [IC 50 h (alpha Vbeta 3) 0.03-5.12 nM; IC 50 h (alpha Vbeta 5) 0.88-154 nM]. A total of eight cyclopeptides of type cyclo-[-Arg-Gly-Asp-Amp-], 5- 12, were assembled by a standard solid-phase peptide synthesis protocol that involved the C2-carboxyl and C4-amino functionalities of the proline scaffolds, leaving the N (alpha)-nuclear site untouched. Functionalization of this vacant proline site with either alkyl or acyl substituents proved feasible, with significant benefit to the integrin binding capabilities of the ligands. Notably, six out of eight cyclopeptide inhibitors, 5- 7 and 9- 11, showed moderate yet significant selectivity toward the alpha Vbeta 3 receptor. The three-dimensional structure in water was determined by NMR techniques and molecular dynamics calculations. Docking studies to the X-ray crystal structure of the extracellular segment of integrin alpha Vbeta 3 complexed with reference compound 1 were also performed on selected analogues to highlight the structural features required for potent ligand binding affinity.

125I-Radiolabeled Morpholine-Containing Arginine–Glycine–Aspartate (RGD) Ligand of αvβ3 Integrin As a Molecular Imaging Probe for Angiogenesis

In this paper, using a hybrid small-animal Micro SPECT/CT imaging system, we report that a new (125)I-Cilengitide-like RGD-cyclopentapeptide, containing d-morpholine-3-carboxylic acid, interacts in vivo with α(v)β(3) integrin expressed by melanoma cells. Images clearly show that the (125)I-compound has the capacity to monitor the growth of a melanoma xenograft. Indeed, retention of the labeled ligand in the tumor mass has a good tumor/background ratio, and a significant reduction of its uptake was observed after injection of unlabeled ligand. These results suggest that the use of (125)I-labeled morpholine-based RGD-cyclopentapeptides targeting α(v)β(3) positive tumors may play a role in future therapeutic strategies.

Morpholine-based RGD-cyclopentapeptides as αvβ3/αvβ5 integrin ligands: Role of configuration towards receptor binding affinity

Two c[RGDfX] cyclopeptides, having either L- or D-morpholine-3-COOH (Mor) as the X amino acid were developed as ligands for alpha(v)beta(3)/alpha(v)beta(5) integrins. Biological assays showed only d-Mor-containing cyclopentapeptide capable to bind alpha(v)beta(3) integrin with a low nanomolar affinity according to a two-site model, thus revealing a connection between the configuration of Mor and the preferred binding to alpha(v)beta(3) integrin. Conformational analysis showed different structural preferences for the two peptides induced by the two enantiomeric cyclic amino acids, suggesting a role of the stereochemistry of Mor on the overall peptide conformation and on the presentation of the pharmacophoric Arg and Asp side chains.

Synthesis, SAR and in vitro evaluation of new cyclic Arg-Gly-Asp pseudopentapeptides containing a s-cis peptide bond as integrin αvβ3 and αvβ5 ligands

The solid-phase synthesis of two diastereomeric cyclic pseudopeptides containing the Arg-Gly-Asp sequence and the dipeptide isostere 2-amino-3-oxotetrahydro-1H-pyrrolizine-7a(5H)-carboxylic acid (GPTM) is described. Competition binding assays to purified alphavbeta3 and alphavbeta5 integrins with respect to [125I]echistatin showed a high inhibitory activity for the (2S,7aS)-GPTM derivative. Effects of the structural constraint induced by the two enantiomeric scaffolds (2R,7aR)-GPTM and (2S,7aS)-GPTM on the conformation of Arg-Gly-Asp sequence have been computationally investigated using as a reference the recently solved X-ray structure of cyclo(Arg-Gly-Asp-d-Phe-[N-Me]Val) in complex with the extracellular fragment of the alphavbeta3 receptor. The computational method disclosed the key role played by a bridging water molecule on differentiating the two ligands by a diverse stabilization of the ligand-protein complex.

A PPAR-gamma agonist attenuates pulmonary injury induced by irradiation in a murine model.

PURPOSE/OBJECTIVE(S) Due to its anti-inflammatory, antifibrotic and antineoplastic properties, the PPAR-γ agonist rosiglitazone is of interest in the prevention and therapy of radiation-induced pulmonary injury. We evaluated the radioprotective effects of rosiglitazone in a murine model of pulmonary damage to determine whether radioprotection was selective for normal and tumor tissues. METHODS Lungs in C57BL/6J mice were irradiated (19 Gy) with or without rosiglitazone (RGZ, 5mg/kg/day for 16 weeks, oral gavage). Computed tomography (CT) was performed and Hounsfield Units (HU) were determined during the observation period. Histological analysis and evaluation of fibrosis/inflammatory markers by western blot were performed at 16 weeks. A549 tumor-bearing CD1 mice were irradiated (16 Gy) with or without RGZ, and tumor volumes were measured at 35 days. RESULTS Rosiglitazone reduced radiologic and histologic signs of fibrosis, inflammatory infiltrate, alterations to alveolar structures, and HU lung density that was increased due to irradiation. RGZ treatment also significantly decreased Col1, NF-kB and TGF-β expression and increased Bcl-2 protein expression compared to the irradiation group and reduced A549 clonogenic survival and xenograft tumor growth. CONCLUSIONS Rosiglitazone exerted a protective effect on normal tissues in radiation-induced pulmonary injury, while irradiated lung cancer cells were not protected in vivo and in vitro. Thus, rosiglitazone could be proposed as a radioprotective agent in the treatment of lung cancer.

Radiosynthesis and micro-SPECT analysis of triazole-based RGD integrin ligands as non-peptide molecular imaging probes for angiogenesis

Taking advantage of click chemistry, we synthesized triazole-containing RGD peptidomimetics capable of binding to αvβ3 integrin with diverse potency, and selected (125)I-labeled compounds proved to interact in vitro and in vivo with αvβ3 integrin expressed by melanoma cells. Two (125)I-compounds containing either 2-aminobenzimidazole or 2-aminopyridine groups as the arginine bioisostere with the capacity to selectively bind cells of highly expressing αvβ3 melanoma xenografts were found using micro-SPECT imaging studies.

Cyclic DGR-peptidomimetic containing a bicyclic reverse turn inducer as a selective αvβ5 integrin ligand

Abstract3-Aza-6,8-dioxabicyclo[3.2.1]octane-based amino acids as reverse turn inducers have been introduced into cyclic peptidomimetics containing the RGD or DGR retro-sequence, in order to achieve a stereochemical scanning of the binding capability of the resulting molecules towards αvβ3 and αvβ5 integrins, resulting in retro-inverso DGR peptides as micromolar ligands. A comparative analysis between the conformational preferences of 4 and of its isomer 3, having the opposite RGD sequence, was reported with respect to the binding activity, giving insight into the factors affecting the preferential binding of 4 to the αvβ5 integrin.

Synthesis and preliminary biological characterization of a new potential 125I-Radioligand for dopamine and serotonin receptors

The synthesis and a preliminary biological characterization of a new class of N-benzyl-aminoalcohols which have serotonin (5-HT(2)) and dopamine (D(2)) receptor affinity is described. In vitro competition binding studies were conducted with the new molecules and (3)H-spiperone on crude membrane preparation from rat striatum and frontal cortex. One of these compounds, 3-benzylamino-1-(4-fluoro-2-iodophenyl)-propan-1-ol (6f), whose IC(50) values are in the micromolar range for both the D(2) and 5-HT(2) receptors, was prepared in iodine-125 labelled form (6i) by nucleophilic substitution of the bromine atom of 3-benzylamino-1-(2-bromo-4-fluorophenyl)-propan-1-ol (6d). In the in vivo studies, conducted on rats, the radiolabelled molecule 6i shows a good capacity to cross the blood-brain barrier (BBB) with a mean value of first pass cerebral extraction (E) of ca. 50% when the regional cerebral blood flow, measured with microsphere technique, is in the experimental animals physiologic range (0.8-1 mL/min/g). A preliminary in vitro autoradiographic distribution on coronal rat brain slices of the radioiodinated molecule showed that it was preferentially localized in the striatum and in the cerebral regions rich in dopamine- and serotonin receptors, even if a high non-specific binding was observed.

Prevention of physostigmine-induced lethality by the opioid analgesic meptazinol in the mouse.

1 The prophylactic action of meptazinol against physostigmine‐and neostigmine‐induced lethality was evaluated in mice. Meptazinol proved to be effective against physostigmine (1 mg kg−1 i.p.), but not against neostigmine (0.5 mg kg−1 i.p.). 2 The antagonism by meptazinol of physostigmine‐induced poisoning was maximal when the drug was administered 15 min before physostigmine. Under these conditions the ED50 (95% confidence limits) of meptazinol was 24 (22.0–26.1) mg kg−1 s.c. A 30 mg kg−1 dose of the drug prevented lethality in 89% of the animals. 3 The action of meptazinol was not antagonized by naloxone hydrochloride (2 mg kg−1 i.p.), injected 10 min before meptazinol. 4 Pretreatment of mice with 30 mg kg−1 meptazinol 15 min before physostigmine (1 mg kg−1) poisoning increased brain acetylcholinesterase (AChE) activity on average, from 8 to 31% of control values. 5 The protection of cholinesterases against physostigmine‐and neostigmine‐induced inactivation was demonstrated in vitro directly on purified preparations of the enzymes using a dilution method. The ED50 values (95% confidence limits) for the protective effect of meptazinol of electric eel AChE against 1 and 3 μm physostigmine and 1 μm neostigmine were 2.6 (1.4‐4.9), 9.5 (5–18) and 3 (1.6‐5.7) μm, respectively, while for protection of horse serum butyrylcholinesterase (BuChE) against the same inhibitors, the ED50 values were 12 (5.4–26.4), 42 (27–65.1) and 8 (3.6–17.6) μm, respectively. 6 It is suggested that prevention of physostigmine‐induced lethality by meptazinol is a consequence of its protective action on AChE in the central nervous system.