Molecular View on the iRGD Peptide Binding Mechanism: Implications for Integrin Activity and Selectivity Profiles.

Receptor-selective peptides are widely used as smart carriers for specific tumor-targeted delivery. A remarkable example is the cyclic nonapeptide iRGD (CRGDKPGDC, 1) that couples intrinsic cytotoxic effects with striking tumor-homing properties. These peculiar features are based on a rather complex multistep mechanism of action, where the primary event is the recognition of RGD integrins. Despite the high number of preclinical studies and the recent success of a phase I trial for the treatment of pancreatic ductal adenocarcinoma (PDAC), there is little information available about the iRGD three-dimensional (3D) structure and integrin binding properties. Here, we re-evaluate the peptide's affinity for cancer-related integrins including not only the previously known targets αvß3 and αvß5 but also the αvß6 isoform, which is known to drive cell growth, migration, and invasion in many malignancies including PDAC. Furthermore, we use parallel tempering in the well-tempered ensemble (PT-WTE) metadynamics simulations to characterize the in-solution conformation of iRGD and extensive molecular dynamics calculations to fully investigate its binding mechanism to integrin partners. Finally, we provide clues for fine-tuning the peptide's potency and selectivity profile, which, in turn, may further improve its tumor-homing properties.

Design, Pharmacological Characterization, and Molecular Docking of Minimalist Peptidomimetic Antagonists of α4ß1 Integrin.

Integrin receptors mediate cell-cell interactions via the recognition of cell-adhesion glycoproteins, as well as via the interactions of cells with proteins of the extracellular matrix, and upon activation they transduce signals bi-directionally across the cell membrane. In the case of injury, infection, or inflammation, integrins of ß2 and α4 families participate in the recruitment of leukocytes, a multi-step process initiated by the capturing of rolling leukocytes and terminated by their extravasation. In particular, α4ß1 integrin is deeply involved in leukocyte firm adhesion preceding extravasation. Besides its well-known role in inflammatory diseases, α4ß1 integrin is also involved in cancer, being expressed in various tumors and showing an important role in cancer formation and spreading. Hence, targeting this integrin represents an opportunity for the treatment of inflammatory disorders, some autoimmune diseases, and cancer. In this context, taking inspiration from the recognition motives of α4ß1 integrin with its natural ligands FN and VCAM-1, we designed minimalist α/ß hybrid peptide ligands, with our approach being associated with a retro strategy. These modifications are expected to improve the compounds' stability and bioavailability. As it turned out, some of the ligands were found to be antagonists, being able to inhibit the adhesion of integrin-expressing cells to plates coated with the natural ligands without inducing any conformational switch and any activation of intracellular signaling pathways. An original model structure of the receptor was generated using protein-protein docking to evaluate the bioactive conformations of the antagonists via molecular docking. Since the experimental structure of α4ß1 integrin is still unknown, the simulations might also shed light on the interactions between the receptor and its native protein ligands.

Quantitative Systems Pharmacology and Biased Agonism at Opioid Receptors: A Potential Avenue for Improved Analgesics.

Chronic pain is debilitating and represents a significant burden in terms of personal and socio-economic costs. Although opioid analgesics are widely used in chronic pain treatment, many patients report inadequate pain relief or relevant adverse effects, highlighting the need to develop analgesics with improved efficacy/safety. Multiple evidence suggests that G protein-dependent signaling triggers opioid-induced antinociception, whereas arrestin-mediated pathways are credited with modulating different opioid adverse effects, thus spurring extensive research for G protein-biased opioid agonists as analgesic candidates with improved pharmacology. Despite the increasing expectations of functional selectivity, translating G protein-biased opioid agonists into improved therapeutics is far from being fully achieved, due to the complex, multidimensional pharmacology of opioid receptors. The multifaceted network of signaling events and molecular processes underlying therapeutic and adverse effects induced by opioids is more complex than the mere dichotomy between G protein and arrestin and requires more comprehensive, integrated, network-centric approaches to be fully dissected. Quantitative Systems Pharmacology (QSP) models employing multidimensional assays associated with computational tools able to analyze large datasets may provide an intriguing approach to go beyond the greater complexity of opioid receptor pharmacology and the current limitations entailing the development of biased opioid agonists as improved analgesics.

New 4-Aminoproline-Based Small Molecule Cyclopeptidomimetics as Potential Modulators of α4ß1 Integrin.

Integrin α4ß1 belongs to the leukocyte integrin family and represents a therapeutic target of relevant interest given its primary role in mediating inflammation, autoimmune pathologies and cancer-related diseases. The focus of the present work is the design, synthesis and characterization of new peptidomimetic compounds that are potentially able to recognize α4ß1 integrin and interfere with its function. To this aim, a collection of seven new cyclic peptidomimetics possessing both a 4-aminoproline (Amp) core scaffold grafted onto key α4ß1-recognizing sequences and the (2-methylphenyl)ureido-phenylacetyl (MPUPA) appendage, was designed, with the support of molecular modeling studies. The new compounds were synthesized through SPPS procedures followed by in-solution cyclization maneuvers. The biological evaluation of the new cyclic ligands in cell adhesion assays on Jurkat cells revealed promising submicromolar agonist activity in one compound, namely, the c[Amp(MPUPA)Val-Asp-Leu] cyclopeptide. Further investigations will be necessary to complete the characterization of this class of compounds.

The Bacterial Toxin CNF1 Protects Human Neuroblastoma SH-SY5Y Cells against 6-Hydroxydopamine-Induced Cell Damage: The Hypothesis of CNF1-Promoted Autophagy as an Antioxidant Strategy.

Several chronic neuroinflammatory diseases, including Parkinson's disease (PD), have the so-called 'redox imbalance' in common, a dynamic system modulated by various factors. Among them, alteration of the mitochondrial functionality can cause overproduction of reactive oxygen species (ROS) with the consequent induction of oxidative DNA damage and apoptosis. Considering the failure of clinical trials with drugs that eliminate ROS directly, research currently focuses on approaches that counteract redox imbalance, thus restoring normal physiology in a neuroinflammatory condition. Herein, we used SH-SY5Y cells treated with 6-hydroxydopamine (6-OHDA), a neurotoxin broadly employed to generate experimental models of PD. Cells were pre-treated with the Rho-modulating Escherichia coli cytotoxic necrotizing factor 1 (CNF1), before the addition of 6-OHDA. Then, cell viability, mitochondrial morphology and dynamics, redox profile as well as autophagic markers expression were assessed. We found that CNF1 preserves cell viability and counteracts oxidative stress induced by 6-OHDA. These effects are accompanied by modulation of the mitochondrial network and an increase in macroautophagic markers. Our results confirm the Rho GTPases as suitable pharmacological targets to counteract neuroinflammatory diseases and evidence the potentiality of CNF1, whose beneficial effects on pathological animal models have been already proven to act against oxidative stress through an autophagic strategy.

Chiral ß-lactam-based integrin ligands through Lipase-catalysed kinetic resolution and their enantioselective receptor response.

Obtainment and testing of pure enantiomers are of great importance for bioactive compounds, because of the assessed implications of enantioselectivity in receptor-mediated responses. Herein we evaluated the use of biocatalysis to obtain enantiomerically pure ß-lactam intermediates further exploited in the synthesis of novel integrin ligands as single enantiomers. From a preliminary screening on a set of commercially available hydrolases, Burkholderia Cepacia Lipase (BCL) emerged as a suitable and highly performing enzyme for the kinetic resolution of a racemic azetidinone, key intermediate for the synthesis of novel agonists of integrins. Upon optimization of the biocatalytic protocol in terms of enzymes, acylating agents and procedures, the two ß-lactam enantiomers were obtained in excellent enantiomeric excesses (94% and 98% ee). Synthetic elaborations on the separated enantiomers allowed the synthesis of four chiral ß-lactams which were evaluated in cell adhesion assays on Jurkat cell line expressing α4ß1 integrin, and K562 cell line expressing α5ß1 integrin. Biological tests revealed that only (S)-enantiomers maintained the agonist activity of racemates with a nanomolar potency, and a specific enantio-recognition by integrin receptors was demonstrated.

Selective detection of α4ß1 integrin (VLA-4)-expressing cells using peptide-functionalized nanostructured materials mimicking endothelial surfaces adjacent to inflammatory sites.

Persistent accumulation of immune cells mediated by α4ß1 integrin (VLA-4) is a hallmark of the inflammatory diseases and of chronic inflammation observed in the affected tissues of autoimmune diseases. Aiming at exploring new methods for monitoring the course of the inflammatory processes, we designed the first peptide-functionalized nanostructured devices capable to mimic the high-density multivalency binding between the α4ß1 integrin-expressing cells and the ligands overexpressed on the endothelial surfaces, in the proximity of the sites of inflammation. Specifically, we describe the first examples of monolayers constituted by dye-loaded zeolite L crystals, coated with α4ß1 integrin peptide ligands, and we analyze the adhesion of model Jurkat cells in comparison to non-α4ß1 integrin-expressing cells. In particular, the peptidomimetic diphenylurea-Leu-Asp-Val-diamine allows significant and selective detection of α4ß1 integrin-expressing Jurkat cells, after very rapid incubation time, supporting the possible implementation in a diagnostic device capable to detect the desired cells from biological fluids, obtainable from patients in a noninvasive way.

5-aminomethyloxazolidine-2,4-dione hybrid α/ß-dipeptide scaffolds as inductors of constrained conformations: Applications to the synthesis of integrin antagonists.

Peptidomimetics represent an attractive starting point for drug discovery programs; in particular, peptidomimetics that result from the incorporation of a heterocycle may take advantage of increased enzymatic stability and higher ability to reproduce the bioactive conformations of the parent peptides, resulting in enhanced therapeutic potential. Herein, we present mimetics of the α4ß1 integrin antagonist BIO1211 (MPUPA-Leu-Asp-Val-Pro-OH), containing a aminomethyloxazolidine-2,4-dione scaffold (Amo). Interestingly, the retro-sequences PhCOAsp(OH)-Amo-APUMP including either (S)- or (R)-configured Amo displayed significant ability to inhibit the adhesion of α4ß1 integrin expressing cells, and remarkable stability in mouse serum. Possibly, the conformational bias exerted by the Amo scaffold determined the affinity for the receptors. These peptidomimetics could be of interest for the development of small-molecule agents effective against inflammatory processes and correlated autoimmune diseases.

Conjecturing about Small-Molecule Agonists and Antagonists of α4ß1 Integrin: From Mechanistic Insight to Potential Therapeutic Applications.

Integrins are heterodimeric cell-surface receptors that regulate cell-cell adhesion and cellular functions through bidirectional signaling. On the other hand, anomalous trafficking of integrins is also implicated in severe pathologies as cancer, thrombosis, inflammation, allergies, and multiple sclerosis. For this reason, they are attractive candidates as drug targets. However, despite promising preclinical data, several anti-integrin drugs failed in late-stage clinical trials for chronic indications, with paradoxical side effects. One possible reason is that, at low concentration, ligands proposed as antagonists may also act as partial agonists. Hence, the comprehension of the specific structural features for ligands' agonism or antagonism is currently of the utmost interest. For α4ß1 integrin, the situation is particularly obscure because neither the crystallographic nor the cryo-EM structures are known. In addition, very few potent and selective agonists are available for investigating the mechanism at the basis of the receptor activation. In this account, we discuss the physiological role of α4ß1 integrin and the related pathologies, and review the few agonists. Finally, we speculate on plausible models to explain agonism vs. antagonism by comparison with RGD-binding integrins and by analysis of computational simulations performed with homology or hybrid receptor structures.

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.

Design and Pharmacological Characterization of α4ß1 Integrin Cyclopeptide Agonists: Computational Investigation of Ligand Determinants for Agonism versus Antagonism.

α4ß1 integrin is a cell adhesion receptor deeply involved in the migration and accumulation of leukocytes. Therefore, integrin antagonists that inhibit leukocytes recruitment are currently regarded as a therapeutic opportunity for the treatment of inflammatory disorder, including leukocyte-related autoimmune diseases. Recently, it has been suggested that integrin agonists capable to prevent the release of adherent leukocytes might serve as therapeutic agents as well. However, very few α4ß1 integrin agonists have been discovered so far, thus precluding the investigation of their potential therapeutic efficacy. In this perspective, we synthesized cyclopeptides containing the LDV recognition motif found in the native ligand fibronectin. This approach led to the discovery of potent agonists capable to increase the adhesion of α4 integrin-expressing cells. Conformational and quantum mechanics computations predicted distinct ligand-receptor interactions for antagonists or agonists, plausibly referable to receptor inhibition or activation.

Eosinophil as a cellular target of the ocular anti-allergic action of mapracorat, a novel selective glucocorticoid receptor agonist.

PURPOSE: Glucocorticoids can either suppress gene transcription (transrepression) or activate it (transactivation). This latter process may contribute to certain side effects caused by these agents. Mapracorat (also known as BOL-303242-X or ZK 245186) is a novel selective glucocorticoid receptor agonist that maintains a beneficial anti-inflammatory activity but seems to be less effective in transactivation, resulting in a lower potential for side effects; it has been proposed for the topical treatment of inflammatory skin disorders. This study assessed the anti-allergic activity of mapracorat at the ocular level and whether eosinophils and mast cells are targets of its action. METHODS: With in vitro studies apoptosis was evaluated in human eosinophils by flow cytometry and western blot of caspase-3 fragments. Eosinophil migration toward platelet-activating factor was evaluated by transwell assays. Interleukin (IL)-6, IL-8, tumor necrosis factor-α (TNF-α), and the chemokine (C-C motif) ligand 5 (CCL5)/regulated upon activation normal T cell expressed, and presumably secreted (RANTES) were measured using a high-throughput multiplex luminex technology. Annexin I and the chemochine receptor C-X-C chemokine receptor 4 (CXCR4) were detected by flow cytometry. With in vivo studies, allergic conjunctivitis was induced in guinea pigs sensitized to ovalbumin by an ocular allergen challenge and evaluated by a clinical score. Conjunctival eosinophils were determined by microscopy or eosinophil peroxidase assay. RESULTS: In cultured human eosinophils, mapracorat showed the same potency as dexamethasone but displayed higher efficacy in increasing spontaneous apoptosis and in counteracting cytokine-sustained eosinophil survival. These effects were prevented by the glucocorticoid receptor antagonist mifepristone. Mapracorat inhibited eosinophil migration and IL-8 release from eosinophils or the release of IL-6, IL-8, CCL5/RANTES, and TNF-α from a human mast cell line with equal potency as dexamethasone, whereas it was clearly less potent than this glucocorticoid in inducing annexin I and CXCR4 expression on the human eosinophil surface; this was taken as a possible sign of glucocorticoid-dependent transactivation. In the guinea pig, mapracorat or dexamethasone eye drops induced an analogous reduction in clinical symptoms of allergic conjunctivitis and conjunctival eosinophil accumulation. CONCLUSIONS: Mapracorat appears to be a promising candidate for the topical treatment of allergic eye disorders. It maintains an anti-allergic profile similar to that of dexamethasone but seems to have fewer transactivation effects in comparison to this classical glucocorticoid. Some of its cellular targets may contribute to eosinophil apoptosis and/or to preventing their recruitment and activation and to inhibiting the release of cytokines and chemokines.

Monitoring Opioid Receptor Interaction in Living Cells by Bioluminescence Resonance Energy Transfer (BRET).

Bioluminescence resonance energy transfer (BRET ) is a natural phenomenon that has been successfully applied for the study of protein-protein interactions, including opioid receptor oligomers. The discovery of opioid receptor homomers and heteromers has brought to the discovery of new functions and new way of signaling and trafficking; therefore, opioid receptor oligomers may be considered as novel drug targets. Fusing receptors of interest with Renilla luciferase and with a fluorescent protein (such as EYFP ) it is possible to study opioid receptor dimerization using BRET .

An in Vitro Assay to Study the Role of Opioids in Modulating Immune Cell Adhesion.

Opioids play a pivotal role in pain transmission but are also able to modulate immune cell functions. In the last decades a connection between opioids and integrins-adhesion molecules involved, among many other processes, in leukocyte recruitment at inflamed site-has been established. To study immune cell integrin-mediated adhesion, cell adhesion assay is a simple, reproducible, and valuable tool capable of unraveling concentration-dependent effects of a test candidate on integrin-mediated cell adhesion.

Experimental Pharmacotherapy for Dry Eye Disease: A Review.

Dry eye disease (DED) is a complex multifactorial disease showing heterogenous symptoms, including dryness, photophobia, ocular discomfort, irritation and burning but also pain. These symptoms can affect visual function leading to restrictions in daily life activities and reduction in work productivity with a consequently high impact on quality of life. Several pathological mechanisms contribute to the disease: evaporative water loss leads to impairment and loss of tear homeostasis inducing either directly or indirectly to inflammation, in a self-perpetuating vicious cycle. Dysregulated ocular immune responses result in ocular surface damage, which further contributes to DED pathogenesis. Currently, DED treatment is based on a flexible stepwise approach to identify the most beneficial intervention. Although most of the available treatments may control to a certain extent some signs and symptoms of DED, they show significant limitations and do not completely address the needs of patients suffering from DED. This review provides an overview of the emerging experimental therapies for DED. Several promising therapeutic strategies are under development with the aim of dampening inflammation and restoring the homeostasis of the ocular surface microenvironment. Results from early phase clinical trials, testing the effects of EnaC blockers, TRPM8 agonist or mesenchymal stem cells in DED patients, are especially awaited to demonstrate their therapeutic value for the treatment of DED. Moreover, the most advanced experimental strategies in the pipeline for DED, tivanisiran, IL-1R antagonist EBI-005 and SkQ1, are being tested in Phase III clinical trials, still ongoing. Nevertheless, although promising results, further studies are still needed to confirm efficacy and safety of the new emerging therapies for DED.

Design of α/ß-Hybrid Peptide Ligands of α4ß1 Integrin Equipped with a Linkable Side Chain for Chemoselective Biofunctionalization of Microstructured Materials.

Arg-Gly-Asp (RGD)-binding integrins, e.g., αvß3, αvß1, αvß5 integrins, are currently regarded as privileged targets for the delivery of diagnostic and theranostic agents, especially in cancer treatment. In contrast, scarce attention has been paid so far to the diagnostic opportunities promised by integrins that recognize other peptide motifs. In particular, α4ß1 integrin is involved in inflammatory, allergic, and autoimmune diseases, therefore, it represents an interesting therapeutic target. Aiming at obtaining simple, highly stable ligands of α4ß1 integrin, we designed hybrid α/ß peptidomimetics carrying linkable side chains for the expedient functionalization of biomaterials, nano- and microparticles. We identified the prototypic ligands MPUPA-(R)-isoAsp(NHPr)-Gly-OH (12) and MPUPA-Dap(Ac)-Gly-OH (13) (MPUPA, methylphenylureaphenylacetic acid; Dap, 2,3-diamino propionic acid). Modification of 12 and 13 by introduction of flexible linkers at isoAsp or Dap gave 49 and 50, respectively, which allowed for coating with monolayers (ML) of flat zeolite crystals. The resulting peptide-zeolite MLs were able to capture selectively α4ß1 integrin-expressing cells. In perspective, the α4ß1 integrin ligands identified in this study can find applications for preparing biofunctionalized surfaces and diagnostic devices to control the progression of α4ß1 integrin-correlated diseases.

Selective Integrin Ligands Promote Cell Internalization of the Antineoplastic Agent Fluorouracil.

Drug conjugates consisting of an antineoplastic drug and a targeting receptor ligand could be effective to overcome the heavy side effects of unselective anticancer agents. To address this need, we report here the results of a project aimed to study agonist and antagonist integrin ligands as targeting head of molecular cargoes for the selective delivery of 5-fluorouracil (5-FU) to cancer or noncancer cells. Initially, two fluorescent ß-lactam-based integrin ligands were synthesized and tested for an effective and selective internalization mediated by α4ß1 or α5ß1 integrins in Jurkat and K562 cells, respectively. No cellular uptake was observed for both fluorescent compounds in HEK293 noncancerous control cells. Afterward, three conjugates composed of the ß-lactam-based integrin ligand, suitable linkers, and 5-FU were realized. The best compound E, acting as α5ß1 integrin agonist, is able to selectively deliver 5-FU into tumor cells, successfully leading to cancer cell death.

Integrin-mediated adhesive properties of neutrophils are reduced by hyperbaric oxygen therapy in patients with chronic non-healing wound.

In recent years, several studies suggested that the ability of hyperbaric oxygen therapy (HBOT) to promote healing in patients with diabetic ulcers and chronic wounds is due to the reduction of inflammatory cytokines and to a significant decrease in neutrophils recruitment to the damaged area. α4 and ß2 integrins are receptors mediating the neutrophil adhesion to the endothelium and the comprehension of the effects of hyperbaric oxygenation on their expression and functions in neutrophils could be of great importance for the design of novel therapeutic protocols focused on anti-inflammatory agents. In this study, the α4 and ß2 integrins' expression and functions have been evaluated in human primary neutrophils obtained from patients with chronic non-healing wounds and undergoing a prolonged HBOT (150 kPa per 90 minutes). The effect of a peptidomimetic α4ß1 integrin antagonist has been also analyzed under these conditions. A statistically significant decrease (68%) in ß2 integrin expression on neutrophils was observed during the treatment with HBO and maintained one month after the last treatment, while α4 integrin levels remained unchanged. However, cell adhesion function of both neutrophilic integrins α4ß1 and ß2 was significantly reduced 70 and 67%, respectively), but α4ß1 integrin was still sensitive to antagonist inhibition in the presence of fibronectin, suggesting that a combined therapy between HBOT and integrin antagonists could have greater antinflammatory efficacy.

Leukocyte Integrin Antagonists as a Novel Option to Treat Dry Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is a complex multifactorial degenerative disease that leads to irreversible blindness. AMD affects the macula, the central part of the retina responsible for sharp central vision. Retinal pigment epithelium (RPE) is the main cellular type affected in dry AMD. RPE cells form a monolayer between the choroid and the neuroretina and are in close functional relationship with photoreceptors; moreover, RPE cells are part of the blood retina barrier that is disrupted in ocular diseases such as AMD. During ocular inflammation lymphocytes and macrophages are recruited, contact RPE and produce pro-inflammatory cytokines, which play an important role in AMD pathogenesis. The interaction between RPE and immune cells is mediated by leukocyte integrins, heterodimeric transmembrane receptors, and adhesion molecules, including VCAM-1 and ICAM-1. Within this frame, this study aimed to characterize RPE-leukocytes interaction and to investigate any potentially beneficial effects induced by integrin antagonists (DS-70, MN27 and SR714), developed in previous studies. ARPE-19 cells were co-cultured for different incubation times with Jurkat cells and apoptosis and necrosis levels were analyzed by flow cytometry. Moreover, we measured the mRNA levels of the pro-inflammatory cytokine IL-1ß and the expression of adhesion molecules VCAM-1 and ICAM-1. We found that RPE-lymphocyte interaction increased apoptosis and necrosis levels in RPE cells and the expression of IL-1ß. This interaction was mediated by the binding of α4ß1 and αLß2 integrins to VCAM-1 and ICAM-1, respectively. The blockade of RPE-lymphocyte interaction with blocking antibodies highlighted the pivotal role played by integrins. Therefore, α4ß1 and αLß2 integrin antagonists were employed to disrupt RPE-lymphocyte crosstalk. Small molecule integrin antagonists proved to be effective in reducing RPE cell death and expression of IL-1ß, demonstrating that integrin antagonists could protect RPE cells from detrimental effects induced by the interaction with immune cells recruited to the retina. Overall, the leukocyte integrin antagonists employed in the present study may represent a novel opportunity to develop new drugs to fight dry AMD.

Side chain effect in the modulation of αvß3/α5ß1 integrin activity via clickable isoxazoline-RGD-mimetics: development of molecular delivery systems.

Construction of small molecule ligand (SML) based delivery systems has been performed starting from a polyfunctionalized isoxazoline scaffold, whose αvß3 and α5ß1 integrins' potency has been already established. The synthesis of this novel class of ligands was obtained by conjugation of linkers to the heterocyclic core via Huisgen-click reaction, with the aim to use them as "shuttles" for selective delivery of diagnostic agents to cancer cells, exploring the effects of the side chains in the interaction with the target. Compounds 17b and 24 showed excellent potency towards α5ß1 integrin acting as selective antagonist and agonist respectively. Further investigations confirmed their effects on target receptor through the analysis of fibronectin-induced ERK1/2 phosphorylation. In addition, confocal microscopy analysis allowed us to follow the fate of EGFP conjugated α5ß1 integrin and 17b FITC-conjugated (compound 31) inside the cells. Moreover, the stability in water solution at different values of pH and in bovine serum confirmed the possible exploitation of these peptidomimetic molecules for pharmaceutical application.