Development of screening assays to test novel integrin antagonists in allergic inflammation

Aim of the research: to develop a prototype of homogeneous high-throughput screening (HTS) for identification of novel integrin antagonists for the treatment of ocular allergy and to better understand the mechanisms of action of integrin-mediated levocabastine antiallergic action. Results: This thesis provides evidence that adopting scintillation proximity assay (SPA) levocabastine (IC50=406 mM), but not the first-generation antihistamine chlorpheniramine, displaces [125I]fibronectin (FN) binding to human a4b1 integrin. This result is supported by flow cytometry analysis, where levocabastine antagonizes the binding of a primary antibody to integrin a4 expressed in Jurkat E6.1 cells. Levocabastine, but not chlorpheniramine, binds to a4b1 integrin and prevents eosinophil adhesion to VCAM-1, FN or human umbilical vein endothelial cells (HUVEC) cultured in vitro. Similarly, levocabastine affects aLb2/ICAM-1-mediated adhesion of Jurkat E6.1 cells. Analyzing the supernatant of TNF-a-treated (24h) eosinophilic cells (EoL-1), we report that levocabastine reduces the TNF-a-induced release of the cytokines IL-12p40, IL-8 and VEGF. Finally, in a model of allergic conjunctivitis, levocastine eye drops (0.05%) reduced the clinical aspects of the early and late phase reactions and the conjunctival expression of a4b1 integrin by reducing infiltrated eosinophils. Conclusions: SPA is a highly efficient, amenable to automation and robust binding assay to screen novel integrin antagonists in a HTS setting. We propose that blockade of integrinmediated cell adhesion might be a target of the anti-allergic action of levocabastine and may play a role in preventing eosinophil adhesion and infiltration in allergic conjunctivitis.

Role of αvβ3 – Integrin and TLR2 in the innate response to Herpes Simplex Virus infection and Delivery of retargeted oncolytic Herpes Simplex via carrier cells

In the first part of my thesis I studied the mechanism of initiation of the innate response to HSV-1. Innate immune response is the first line of defense set up by the cell to counteract pathogens infection and it is elicited by the activation of a number of membrane or intracellular receptors and sensors, collectively indicated as PRRs, Patter Recognition Receptors. We reported that the HSV pathogen-associated molecular patterns (PAMP) that activate Toll-like receptor 2 (TLR2) and lead to the initiation of innate response are the virion glycoproteins gH/gL and gB, which constitute the conserved fusion core apparatus across the Herpesvirus. Specifically gH/gL is sufficient to initiate a signaling cascade which leads to NF-κB activation. Then, by gain and loss-of-function approaches, we found that αvβ3-integrin is a sensor of and plays a crucial role in the innate defense against HSV-1. We showed that αvβ3-integrin signals through a pathway that concurs with TLR2, affects activation/induction of interferons type 1, NF-κB, and a polarized set of cytokines and receptors. Thus, we demonstrated that gH/gL is sufficient to induce IFN1 and NF-κB via this pathway. From these data, we proposed that αvβ3-integrin is considered a class of non-TLR pattern recognition receptors. In the second part of my thesis I studied the capacity of human mesenchymal stromal cells isolated by fetal membranes (FM-hMSCs) to be used as carrier cells for the delivery of retargeted R-LM249 virus. The use of systemically administrated carrier cells to deliver oncolytic viruses to tumoral targets is a promising strategy in oncolytic virotherapy. We observed that FM-hMSCs can be infected by R-LM249 and we optimized the infection condition; then we demonstrate that stromal cells sustain the replication of retargeted R-LM249 and spread it to target tumoral cells. From these preliminary data FM-hMSCs resulted suitable to be used as carrier cells

Discovery and Pharmacological Characterization of a Novel Small Molecule Antagonist of Integrin α4β1: Focus on Antiallergic Activity

Allergy is a common hypersensitivity disorder that affects 15% to 20% of the population and its prevalence is increasing worldwide. Its severity correlates with the degree of eosinophil infiltration into the conjunctiva, which is mediated by chemokines that stimulate the production of adhesion molecules like intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) on the endothelial cell surface. The α4β1 and α4β7 integrins are expressed in eosinophils and contribute to their activation and infiltration in AC through the binding to VCAM-1 or fibronectin, expressed on vascular endothelial cells. Blockade of α4 integrins might be a therapeutical achievement in allergic eye diseases. DS 70, that show an IC50 in the nanomolar range against α4β1 integrin in Jurkat cells and in the eosinophilic cell line EOL-1. This compound was able to prevent cell adhesion to VCAM-1 and FN in vitro. In a scintillation proximity assay DS70 displaced 125I-FN binding to human α4β1 integrin and, in flow cytometry analysis, it antagonized the binding of a primary antibody to α4β1 integrin expressed on the Jurkat cells surface as well. Furthermore, we analysed also its effects on integrin α4β1 signalling. In an vivo model of allergic conjunctivitis, topical DS70 reduced the clinical aspects of EPR (early phase reaction) and LPR (late phase reaction), by reducing clinical score, eosinophil accumulation, mRNA levels of cytochines and chemochines pro-inflammatory and the conjunctival expression of α4 integrin. In conclusion, DS70 seems a novel antiallergic ocular agent that has significant effects on both early and late phases of ocular allergy.

Peptidomimetic ligands for tuning integrin-mediated signalling: rational design, synthesis and theranostic applications

Integrins are α/β-heterodimeric transmembrane adhesion receptors that mediate cell-cell and cell-ECM interactions. Integrins are bidirectional signalling receptors that respond to external signals (“outside-in” signalling) and in parallel, transduce internal signals to the matrix (“inside-out” signalling), to regulate vital cellular functions including migration, survival, growth and differentiation. Therefore, dysregulation of these tightly regulated processes often results in uncontrolled integrin activation and abnormal tissue expression that is responsible for many diseases. Because of their important roles in physiological and pathological events, they represent a validated target for therapeutic and diagnostic purposes. The aim of the present Thesis was focused on the development of peptidic ligands for α4β1 and αvβ3 integrin subtypes, involved in inflammatory responses (leukocytes recruitment and extravasation) and cancer progression (angiogenesis, tumor growth, metastasis), respectively. Following the peptidomimetic strategy, we designed and synthesized a small library of linear and cyclic hybrid α/β-peptidomimetics based on the phenylureido-LDV scaffolds for the treatment of chronic inflammatory autoimmune diseases. In order to implement a fast and non-invasive diagnostic method for monitoring the course of the inflammatory processes, a flat glass-surface of dye-loaded Zeolite L-crystal nanoparticles was coated with bioactive α4β1-peptidomimetics to detect specific integrin-expressing cells as biomarkers of inflammatory diseases. Targeted drug delivery has been considered a promising alternative to overcome the pharmacokinetic limitations of conventional anticancer drugs. Thus, a novel Small-Molecule Drug Conjugate was synthesized by connecting the highly cytotoxic Cryptophycin to the tumor-targeting RGDfK-peptide through a protease-cleavable linker. Finally, in view to making the peptide synthesis more sustainable and greener, we developed an alternative method for peptide bonds formation employing solvent-free mechanochemistry and ultra-mild minimal solvent-grinding conditions in common, inexpensive laboratory equipment. To this purpose, standard amino acids, coupling agents and organic-green solvents were used in the presence of nanocrystalline hydroxyapatite as a reusable, bio-compatible inorganic basic catalyst.

Stereoselective synthesis of heterocycles as intermediates of biologically active compounds

The aim of this thesis was to investigate the synthesis of enantiomerically enriched heterocycles and dehydro-β-amino acid derivatives which can be used as scaffolds or intermediates of biologically active compounds, in particular as novel αvβ3 and α5β1 integrin ligands. The starting materials of all the compounds here synthesized are alkylideneacetoacetates. Alkylidene derivates are very usefull compounds, they are usually used as unsaturated electrophiles and they have the advantage of introducing different kind of functionality that may be further elaborated. In chapter 1, regio- and stereoselective allylic amination of pure carbonates is presented. The reaction proceeds via uncatalyzed or palladium-catalyzed conditions and affords enantiopure dehydro-β-amino esters that are useful precursor of biologically active compounds. Chapter 2 illustrates the synthesis of substituted isoxazolidines and isoxazolines via Michael addition followed by intramolecular hemiketalisation. The investigation on the effect of the Lewis acid catalysis on the regioselectivity of the addition it also reported. Isoxazolidines and isoxazolines are interesting heterocyclic compounds that may be regarded as unusual constrained -amino acids or as furanose mimetics. The synthesis of unusual cyclic amino acids precursors, that may be envisaged as proline analogues, as scaffolds for the design of bioactive peptidomimetics is presented in chapter 3. The synthesis of 2-substituted-3,4-dehydropyrrole derivatives starting from allylic carbonates via a two step allylic amination/ring closing metathesis (RCM) protocol is carried out. The reaction was optimized by testing different Grubbs’ catalysts and carbamate nitrogen protecting groups. Moreover, in view of a future application of these dehydro-β-amino acids as central core of peptidomimetics , the malonate chain was also used to protect nitrogen prior to RCM. Finally, chapter 4 presents the synthesis of two novel different classes of integrin antagonists, one derived from dehydro-β-amino acid prepared as described in chapter 1 and the other one has isoxazolidines synthesized in chapter 2 as rigid constrained core. Since that these compounds are promising RGD mimetics for αvβ3 and α5β1 integrins, they have been submitted to biological assay. and to interpret on a molecular basis their different affinities for the αvβ3 receptor, docking studies were performed using Glide program.

Synthesis of constrained peptidomimetics for therapeutic, diagnostic and theranostic applications

This thesis work deals, principally, with the development of different chemical protocols ranging from environmental sustainability peptide synthesis to asymmetric synthesis of modified tryptophans to a series of straightforward procedures for constraining peptide backbones without the need for a pre-formed scaffold. Much efforts have been dedicated to the structural analysis in a biomimetic environment, fundamental for predicting the in vivo conformation of compounds, as well as for giving a rationale to the experimentally determined bioactivity. The conformational analyses in solution has been done mostly by NMR (2D gCosy, Roesy, VT, titration experiments, molecular dynamics, etc.), FT-IR and ECD spectroscopy. As a practical application, 3D rigid scaffolds have been employed for the synthesis of biological active compounds based on peptidomimetic and retro-mimetic structures. These mimics have been investigated for their potential as antiflammatory agents and actually the results obtained are very promising. Moreover, the synthesis of Amo ring permitted the development of an alternative high effective synthetic pathway for obtaining Linezolid antibiotic. The final section is, instead, dedicated to the construction of a new biosensor based on zeolite L SAMs functionalized with the integrin ligand c[RGDfK], that has showed high efficiency for the selective detection of tumor cells. Such kind of sensor could, in fact, enable the convenient, non-invasive detection and diagnosis of cancer in early stages, from a few drops of a patient's blood or other biological fluids. In conclusion, the researches described herein demonstrate that the peptidomimetic approach to 3D definite structures, allows unambiguous investigation of the structure-activity relationships, giving an access to a wide range bioactive compounds of pharmaceutical interest to use not only as potential drugs but also for diagnostic and theranostic applications.

Innovative therapeutic approaches for the atrophic Age-related Macular Degeneration (“dry” AMD)

Age related macular degeneration (AMD) is a major concern regarding blindness in the world. In western countries, where visual alterations due to minor pathologies as cataract and uncorrected refractive errors are easily resolved, AMD represent the main cause of blindness. Of the two existing forms of the disease, while the neovascular is more aggressive and progress quickly, geographic atrophy is the one still lacking an appropriate therapy. My PhD program was focused on investigating AMD features, trying to understand if some approaches I tested could be able to provide some suggestion about potential future therapies on “dry” AMD. In my research I developed three main projects. The most important part of the work regards the study of integrins and their fundamental role in cell adhesion in a context of interaction between retinal pigmented epithelium (RPE) and immune cells. I investigated how co-culture of these different cell lines can lead to simulate an inflammatory state inducing cell signaling, cytokine production and cell death. The use of integrin antagonists developed in our laboratory, showed how these effects can be reverted. A secondary approach regards the use of antioxidants and their role in epigenetic modifications in ARPE-19 cells to investigate how these compounds might exert their well-known protective role on AMD. Commonly used antioxidants as Lutein and Quercetin do not induce clear epigenetic modifications through histone H3 acetylation indicating only a limited involvement.

Harnessing the power of Drosophila model to obtain new insights on MYC and ABCC1 cellular functions: from neuroblastoma to autism spectrum disorder

MYCN amplification is a genetic hallmark of the childhood tumour neuroblastoma. MYCN-MAX dimers activate the expression of genes promoting cell proliferation. Moreover, MYCN seems to transcriptionally repress cell differentiation even in absence of MAX. We adopted the Drosophila eye as model to investigate the effect of high MYC to MAX expression ratio on cells. We found that dMyc overexpression in eye cell precursors inhibits cell differentiation and induces the ectopic expression of Antennapedia (the wing Hox gene). The further increase of MYC/MAX ratio results in an eye-to-wing homeotic transformation. Notably, dMyc overexpression phenotype is suppressed by low levels of transcriptional co-repressors and MYCN associates to the promoter of Deformed (the eye Hox gene) in proximity to repressive sites. Hence, we envisage that, in presence of high MYC/MAX ratio, the “free MYC” might inhibit Deformed expression, leading in turn to the ectopic expression of Antennapedia. This suggests that MYCN might reinforce its oncogenic role by affecting the physiological homeotic program. Furthermore, poor neuroblastoma outcome associates with a high level of the MRP1 protein, encoded by the ABCC1 gene and known to promote drug efflux in cancer cells. Intriguingly, this correlation persists regardless of chemotherapy and ABCC1 overexpression enhances neuroblastoma cell motility. We found that Drosophila dMRP contributes to the adhesion between the dorsal and ventral epithelia of the wing by inhibiting the function of integrin receptors, well known regulators of cell adhesion and migration. Besides, integrins play a crucial role during synaptogenesis and ABCC1 locus is included in a copy number variable region of the human genome (16p13.11) involved in neuropsychiatric diseases. Interestingly, we found that the altered dMRP/MRP1 level affects nervous system development in Drosophila embryos. These preliminary findings point out novel ABCC1 functions possibly defining ABCC1 contribution to neuroblastoma and to the pathogenicity of 16p13.11 deletion/duplication

Multifarious tumor-oriented small molecules: development of synthetic methodologies and application for diagnostic platforms

Cancer represents one of the most relevant and widespread diseases in the modern age. In this context, integrin receptors are important for the interactions of cells with extracellular matrix and for the development of both inflammation and carcinogenic phenomena. There are many tricks to improve the bioactivity and receptor selectivity of exogenous ligands; one of these is to integrate the amino acid sequence into a cyclic peptide to restrict its conformational space. Another approach is to develop small peptidomimetic molecules in order to enhance the molecular stability and open the way to versatile synthetic strategies. Starting from isoxazoline-based peptidomimetic molecules we recently reported, in this thesis we are going to present the synthesis of new integrin ligands obtained by modifying or introducing appendages on already reported structures. Initially, we are going to introduce the synthesis of linear and cyclic α-dehydro-β-amino acids as scaffolds for the preparation of bioactive peptidomimetics. Subsequently, we are going to present the construction of small molecule ligands (SMLs) based delivery systems performed starting from a polyfunctionalised isoxazoline scaffold, whose potency towards αVβ3 and α5β1 integrins has already been established by our research group. In the light of these results and due to the necessity to understand the behaviour of a single enantiomer of the isoxazoline-based compounds, the research group decided to synthesise the enantiopure heterocycle using a 1,3-dipolar cycloaddiction approach. Subsequently, we are going to introduce the synthesis of a Reporting Drug Delivery System composed by a carrier, a first spacer, a linker, a self-immolative system, a second spacer and a latent fluorophore. The last part of this work will describe the results obtained during the internship abroad in Prof. Aggarwal’s laboratory at the University of Bristol. The project was focused on the Mycapolyol A synthesis.

β-lactams from their structural features to their biological applications

β-lactam compounds represent an important class of four-membered cyclic amides (azetidin-2-ones) thanks to their valuable and varied biological activities. The presence of a β-lactam ring in a series of bioactive molecules targeting different proteins, allows us to consider the azetidin-2-one a privileged structure. The constrained four-membered cyclic amide could easily undergo ring-opening reactions by nucleophilic residues in the active sites of enzymes and this is the mechanism suggested for antibacterial activity; moreover, the rigid core structure could favour and actually enhance directional noncovalent bonding for an effective ligand−receptor recognition. Nowadays monocyclic β-lactams are known as anticancer, antidiabetic, anti-tubercular, anti-inflammatory agents and as ligands of integrin receptors. In order to consider different facets of 4-azetidin-2-ones, this theis will be divided into two sections: the first one will be dedicated to the design, synthesis and characterization of biological active β-lactams (new β-lactam based integrin ligands and their different applications and novel N-thio-alkyl substituted azetidinones for the treatment of Tuberculosis); the second one instead, will be based on two projects which consider two different proprieties of β-lactams: stereochemistry, evaluated by biocatalytic methods and reactivity at C-4 position. In the first case we want to obtain enantiomerically pure 4-acetoxy-2-azetidinone, useful for synthesis of stereo-chemically defined bioactive β-lactams, while in the second case we want to study in which conditions the nucleophilic substitutions occur. A final section will be instead dedicated to the research project conducted in Philochem AG, Zurich, under the supervision of Prof. Dario Neri and Dr. Samuele Cazzamalli, based on the study of new cleavable disulfide linkers for small molecule drug conjugates targeting Fibroblast activation protein (FAP).