INTEGRIN ALPHAVBETA3 AS THERAPEUTIC TARGET AND IMAGING BIOMARKER FOR VASCULAR ENDOTHELIAL CELLS AND CANCER EPITHELIAL CELLS

Integrins are heterodimeric transmembrane proteins belonging to a family of cell adhesion receptors evolutionary old and that play pivotal roles in physiological and pathological processes, such as neo-angiogenesis and cancer spreading. Integrins are composed by an extracellular domain, a single transmembrane region and a short cytoplasmic tail. In particular, the cytoplasmic domain is crucial for the regulation of integrin activity and function. It controls the integrin affinity state and its ECM ligand-binding activity, but it also promotes cellular responses upon extracellular ligand binding. Integrins are in fact activated upon binding with their extracellular ligands (inside-out signalling) and may change different conformations according to individual variations. This leads to the so-called outside-in signalling, which activates complex and cell-specific signalling events depending on the other molecular partners involved. Given that integrins lack of intrinsic kinase activity, ligated integrins cluster with few signalling and adaptor proteins from the cytoplasm, including growth factor receptors, cytokine receptors and trafficking molecules. This clustering gives rise to dynamic macromolecular protein structures globally termed adhesion complexes. Integrin ligation finally promotes signalling events leading to cell spreading, migration, survival and proliferation. The concomitant engagement of integrins and growth factor receptors optimizes the global yield of the activation through the integration of different signalling patways. Again, this cooperative signalling plays a pivotal role in the regulation of tumor cell adhesion, migration, invasion, survival and in the homeostasis of the angiogenic endothelium. The cross-talk between integrins and growth factors or cytokines receptors plays an important role also in the biology of several host cell types, with a particular focus on endothelial cell migration, proliferation and survival. Therefore, integrins act as molecular bridges between the extracellular matrix and the cytoskeleton, displaying mechanical functions and affecting cell biology by means of complex intracellular signalling pathways. Integrins are able to mediate cell adhesion to immune cells, depending upon specific short peptide sequences recognized by integrins on their ligands. To this regard, it is noteworthy that the same ligand may be bound by different integrins because of their redundancy. As previously underlined, integrins play pivotal roles in traction for cell motility and invasion, in remodelling of the ECM by means of localization of proteases, in cell growth through adhesion-dependent control of proliferation, in cell signalling thanks to the cross-talk with growth factors and cytokine receptors. Few data confirm that some endothelial integrins, such as the RGD sequence recognizing integrins \u3b1v\u3b23, \u3b1v\uf0625 and \u3b15\u3b21, are very important in the regulation of cell growth, survival and migration during angiogenesis and lymphangiogenesis. In particular, the \u3b1v integrins have key roles in embryonic development of blood vessels in placenta and brain, whereas fibronectin-binding integrins are essential for developmental angiogenesis. In addition, it has been demonstrated that a number of integrins is highly expressed in human metastatic cancers. Integrins are able to promote migration, survival and invasion because of their ability to degrade basement membrane by interacting with proteolytic enzymes (e.g. metalloproteases). Integrins may either enhance cell survival or initiate apoptosis depending on environmental cues. Integrin \u3b1v\u3b23 drive cancer cell invasion, proliferation, survival and also the EMT. Integrin \u3b1v\u3b23 cross-talks with VEGFR in melanoma, glioblastoma and angiogenic endothelial cells. Moreover, it mediates the adhesion of malignant cells to platelets or to vascular endothelial cells. Integrin \u3b1v\u3b23, together with integrin \u3b15\u3b21, is highly up-regulated in some tumors but is expressed in low amounts in adult epithelia. Integrins \u3b1v\u3b23 and \u3b1v\u3b25 play important roles in promoting cancer cell biology as well. Moreover, the fibronectin-binding integrin \u3b15\u3b21 may promote solid tumor invasiveness. Integrin \u3b1v\u3b25 is normally expressed by epithelial cells but may contribute to tumor migration, proliferation and survival. Integrin \u3b1v\u3b25 is generally over-expressed in malignant tumor cells, angiogenic endothelial cells within the tumor and metastatic breast or pancreatic cancer cells. Integrins on tumor cells are able to enhance metastasis, facilitate invasion and movements across blood vessels. As previously seen integrins are widely expressed in various cell types. Together with their ability to cross-talk with growth factors, cytokines and their receptors, this points out that integrins are appealing therapeutic targets. Integrin targeting agents might then be able to inhibit both tumor cells and tumor-associated host cells, and several integrin targeting methods have been extensively studied in order to treat integrins-related diseases. Apart from function blocking antibodies, integrin antagonists comprise synthetic peptides and peptidomimetics. Synthetic peptides mimic the structure of natural integrin binding ligands. The largest class of synthetic peptides so far developed comprises RGD peptides, given that the RGD sequence is commonly found in several glycoproteins of the extracellular matrix. Conformational constrained sequences and their chemical modifications are able to enhance the binding affinity and the bioavailability of the peptides. On the other hand, peptidomimetics are chemically synthesized products mimicking the functions and structures of the biological integrin antagonists. The peptidomimetic compounds generally display higher bioavailability and are able to avoid enzymatic degradation. In 2000 Belvisi et al. studied peptide secondary structure mimics and reported the synthesis and conformational analysis of a series of 1-aza-2-oxobicycloalkane amino acids. Such azabicycloalkane scaffolds showing different reverse-turn mimetic properties may constrain the RGD sequence into different conformations, possibly providing the required activity and selectivity for integrin antagonism and enhancing ligand binding. A small library of cyclic RGD peptidomimetics was then synthesized. In addition, in 2009 Manzoni et al. reported the functionalization of the previously cited azabicycloalkanes with heteroalkyl side chains ending with a hydroxyl group. The hydroxyl group can easily be converted into other suitable functional groups or directly used for the conjugation of various chemical entities (applications in medical diagnosis and therapy). Thus, the functionalization allowed to generate high affinity ligands potentially active per se but able to behave as intelligent vectors as well. Finally, gold nanoparticles conjugated on their surface with the cyclic-RGD integrin ligand were recently generated. Gold nanoparticles are in fact the ideal candidate for optical imaging because they are not susceptible to photobleaching and may be functionalized in several ways. During this work the small library of cyclic RGD peptidomimetics alone or conjugated with imaging probes or nanoparticles was screened. It was determined that the main target of such compounds was integrin \u3b1v\u3b23, by means of receptor binding assays. The expression of integrin \u3b1v\u3b23 was further evaluated in a panel of human vascular endothelial and epithelial cancer cells, in order to chose the most suitable cell models to deepen the biological role of the peptidomimetics. In particular, one compound, namely Compound 31, was selected because of its high affinity for integrin \u3b1v\u3b23 receptor. Compound 31 displayed a strong anti-adhesive activity in both vascular endothelial and cancer cells, thus pointing to its putative employment in further in vivo studies. On the other hand, the heteroalkyl substituent of Compound 31 allowed its conjugation with fluorescein in order to give rise to imaging agents (namely Compound 13 and 15). Both of them were able to positively stain endothelial and cancer cells expressing the integrin \u3b1v\u3b23 receptor on their surface, without displaying cytotoxicity. This evidence strongly suggested the possible role of fluorochrome-conjugated cyclic RGD compounds in cancer diagnosis. Finally, the functionalization of gold nanoparticles with the cyclic RGD scaffold and fluorescein was performed. These gold nanoparticles were demonstrated to positively stain in a very specific way cancer cells expressing integrin \u3b1v\u3b23. In fact, the confocal analysis of cells stained with gold nanoparticles revealed that the signal given by fluorescein and the reflection spectrum given by gold were perfectly merged and were localized near the cell adhesive contacts. This ultimately suggested that the internalization took place in the membrane zone where integrin \u3b1v\u3b23 is mainly expressed, that is focal contacts. In conclusion, the results obtained in this work by means of basically cell biology tests could pave the way for a further evaluation of cyclic RGD peptidomimetics and their functional derivatives in vascular biology, cancer treatment (in association with known chemotherapeutic drugs) and diagnosis

STD and trNOESY NMR Study of Receptor-Ligand Interactions in Living Cancer Cells

Integrin antagonists: Saturation transfer difference (STD) and transferred NOE provided useful information on membrane-bound receptor-ligand interactions in living cells in which the integrin \u3b1v\u3b23 is highly expressed. The epitopes of two cyclic RGD mimics were identified by STD. The different activities of these compounds were interpreted in terms of different binding modes. The experimental evidence was confirmed by docking calculations

New potent alpha(v)beta(3) integrin ligands based on azabicycloalkane (gamma,alpha)-dipeptide mimics

We have designed a new synthetic strategy for the preparation of a new class of cyclic RGD integrin ligands in which the azabicycloalkane scaffold can be envisaged as a (\u3b3,\u3b1) dipeptide mimic. The synthesis and in vitro biological evaluation of these RGD derivatives, as well as the computational study of their conformational properties and binding modes to \u3b1V\u3b23 integrin are described. Compound has shown to be a promising candidate as \u3b1V\u3b23 integrin antagonist able to interfere with both cell adhesion and movement on vitronectin with no evidence of cytotoxic effects

Cyclic RGD-containing functionalized azabicycloalkane peptides as potent integrin antagonists for tumor targeting

Cyclic RGD-containing functionalized azabicycloalkane peptides were synthesized with the aim of developing high-affinity selective integrin ligands as carriers for therapeutic and diagnostic purposes. Herein we describe the synthesis and in vitro screening of these RGD derivatives, as well as the determination of their conformational properties in solution by spectroscopic and computational methods. Docking studies with the X-ray crystal structure of the extracellular domain of integrin alpha(v)beta(3) were also performed to elucidate the structural binding requirements and to rationalize the biological results. One compound in particular was found to be the best alpha(v)beta(3) integrin binder (IC(50)=53.7 nM) among the new functionalized RGD cyclic peptides, thus emerging as a promising candidate for covalent bonding and selective homing of useful functional units