Obbiettivo integrine e non solo: sintesi e valutazione biologica di peptidomimetici multifunzionali nella terapia del tumore

The work described in this PhD thesis concerns the design and synthesis of small molecular ligands targeting some subtypes of the integrin receptor family, in particular αVβ3 and α4β1 integrins. One major effort, which is described in Chapter 1a, is focused on the synthesis of dual small molecule conjugates as potential tools to impair tumor-associated angiogenesis. These constructs are formed by the covalent conjugation of a RGD unit, which is able to selectively bind to the extracellular segment of the αVβ3 integrin receptor, with a sunitinib-like moiety, a proven tyrosine kinase inhibitor (TKI) able to interact with the cytoplasmic domain of VEGFR2. Given the strict crosstalk between the VEGFR2 and αVβ3 receptors in both activated endothelial cells (ECs) and some cancer cell types, the blockage of the αVβ3/VEGFR2 couple may assume high anti-angiogenic and anti-tumor potential. On these bases, three dual conjugates were designed, synthesized and fully characterized, and subsequently tested in vitro to assess their properties as αVβ3 binders and VEGFR2 kinase inhibitors. All the conjugated compounds showed interesting anti-angiogenesis properties in vitro and one of them demonstrated a significant angiogenesis impairment in vivo, which proved superior to the action of the single RGD or sunitinib modules and their simple combinations. In Chapter 1b the work done during my period abroad is described. The coordination of platinum to DNA is an area of intense research that allowed the development of valuable platinum-based chemotherapeutics such as cisplatin and carboplatin. Besides DNA, platinum(II) is able to coordinate other biomolecules such as proteins, and this binding ability was exploited in bioconjugation reactions. In particular, extensive research in this area showed that some N-heteroaryl and S-donor groups in the side chains of suitable amino acids are preferential coordination sites for platinum in proteins. My work focused on the exploitation of the binding properties of Pt(II) versus suitable amino acids in order to synthesize cyclic peptides. In particular, four new cyclopeptides were prepared, where the central Pt(II) metal ion coordinates to different bidentate amine ligands and diverse RGD-based peptide sequences embedding suitable N-heteroaryl and S-donor groups. This coordination-based approach testifies that the platinum(II) ion may exert a double function: i) to favour peptide cyclization by exploiting its coordinative ability; and ii) to furnish peptide-Pt(II) complexes, which could be valuable tools for anti-cancer and anti-angiogenic interventions. As a further therapeutic target of interest, another integrin receptor, α4β1, was considered, given its primary role in mediating chronic inflammation, autoimmune diseases and cancer-related inflammation. In Chapter 2, our efforts in this field are described. In particular, the design and synthesis of seven new cyclic peptidomimetics are reported, all of which containing an aminoproline core scaffold grafted into key α4β1-recognizing peptide sequences. This work constitutes a first step towards further studies aiming at the evaluation of these small molecules as effective and selective α4β1 binders, with the final goal of gaining insights about the structural and functional aspects of this important biological target

Novel cycloAmpRGD-Sunitinib Dual Conjugates as Potent Targeted Anti-angiogenic Tools

In recent years, targeted therapies that selectively address receptors and pathways involved in tumor genesis and progression have attracted growing interest. Some integrin subfamilies (e.g. αVβ3, αVβ5 and α5β1) have shown to be involved, even by close cooperation with other cell receptors (e.g. vascular endothelial growth factor receptors, VEGFRs) in tumor angiogenesis, which plays crucial role in tumor development and dissemination. Recent studies clearly demonstrated a complex in vivo regulation of tumor angiogenesis events; in particular, the αVβ3 integrin receptor is physically and functionally correlated with the VEGFR2 receptor within endothelial cells (ECs), suggesting that dual specific agents capable of inhibiting them would have a great anti-angiogenesis potential. Our research group has recently introduced a new class of cyclic semipeptide ligands, cycloAmpRGD, containing the Arg-Gly-Asp (RGD) sequence and 4-aminoproline scaffolds. These ligands demonstrated to efficiently and selectively bind to the αVβ3 integrin and their binding capability is preserved even in the presence of covalently conjugated “bulky loads” (cytotoxic and chelating agents). Here we report the synthesis, characterization and biological evaluation of a series of dual conjugates of type I, wherein the ligand cycloAmpRGD is covalently associated to a Sunitinib-derived moiety, a clinically approved anti-angiogenic multikinase inhibitor. The binding competence of these candidates toward the αVβ3 integrin in EC lines, their kinase inhibitory activity toward VEGFR2, and their ability to block endothelial cell capillary formation in vitro are described, in comparison with the single agents and related combinations. Encouraging results point to the notion that the covalent conjugation of cycloAmpRGD and Sunitinib may be of high therapeutic potential for tumor angiogenesis inhibition

Synthesis of Novel c(AmpRGD)−Sunitinib Dual Conjugates as Molecular Tools Targeting the αvβ3 Integrin/VEGFR2 Couple and Impairing Tumor-Associated Angiogenesis

On the basis of a previously discovered anti-αVβ3 integrin peptidomimetic (c(AmpRGD)) and the clinically approved antiangiogenic kinase inhibitor sunitinib, three novel dual conjugates were synthesized (compounds 1–3), featuring the covalent and robust linkage between these two active modules. In all conjugates, the ligand binding competence toward αVβ3 (using both isolated receptors and αVβ3-overexpressing endothelial progenitor EP cells) and the kinase inhibitory activity (toward both isolated kinases and EPCs) remained almost untouched and comparable to the activity of the single active units. Compounds 1–3 showed interesting antiangiogenesis properties in an in vitro tubulogenic assay; furthermore, dimeric-RGD conjugate 3 strongly inhibited in vivo angiogenesis in Matrigel plug assays in FVB mice. These results offer proof-of-concept of how the covalent conjugation of two angiogenesis-related small modules may result in novel and stable molecules, which impair tumor-related angiogenesis with equal or even superior ability as compared to the single modules or their simple combinations

Gold Nanoparticles Functionalized with RGD-Semipeptides: A Simple yet Highly Effective Targeting System for αVβ3 Integrins

Effective and selective targeting of the αVβ3integrin subtype is of high relevance in cancer research for the development of therapeutic systems with improved efficacy and of diagnostic imaging probes. We report here a new class of highly selective, αVβ3-targeted gold nanoparticles (AuNPs), which carry cyclic 4-aminoproline-RGD semipeptides (cAmpRGD) as the targeting moiety immobilized at low surface density on the poly(ethylene glycol) (PEG)-based nanoparticle coating. We show that these nanoparticles are potent inhibitors of the integrin-mediated melanoma tumor cell adhesion to vitronectin and are selectively internalized via receptor-mediated endocytosis. Furthermore, we have developed bifunctional cAmpRGD-functionalized AuNPs by conjugation of a fluorophore (FAM or TAMRA) to a separate set of reactive groups on the PEG-based coating. These bifunctional AuNPs not only recapitulate the binding properties of cAmpRGD-AuNPs but also can be visualized via confocal laser microscopy, allowing direct observation of nanoparticle internalization. The peculiar molecular design of these nanoparticles and their precisely defined architecture at the molecular level accounts for their selective integrin binding with very low nonspecific background

Integrin-targeted AmpRGD sunitinib liposomes as integrated antiangiogenic tools

We report here the preparation, physico-chemical characterization, and biological evaluation of a new liposome formulation as a tool for tumor angiogenesis inhibition. Liposomes are loaded with sunitinib, a tyrosine kinase inhibitor, and decorated with cyclo-aminoprolineRGD units (cAmpRGD), efficient and selective ligands for integrin αVβ3. The RGD units play multiple roles since they target the nanovehicles at the integrin αVβ3-overexpressing cells (e.g. activated endothelial cells), favor their active cell internalization, providing drug accumulation in the cytoplasm, and likely take part in the angiogenesis inhibition by interfering in the αVβ3-VEGFR2 cross-talk. Both in vitro and in vivo studies show a better efficacy of this integrated antiangiogenic tool with respect to the free sunitinib and untargeted sunitinib-loaded liposomes. This system could allow a lower administration of the drug and, by increasing the vector specificity, reduce side-effects in a prolonged antiangiogenic therapy

Cell-targeted c(AmpRGD)-sunitinib molecular conjugates impair tumor growth of melanoma

Drug resistance and off-organ toxicity remain unsolved issues in chemotherapy of advanced-stage melanoma patients. Thus, the creation of new molecular conjugates able to combine a selective accumulation, high ability of internalization and signaling pathway inhibition, are highly requested. Recently, we reported a new class of molecular conjugates, compounds 1–3, where the anti-αVβ3 integrin peptidomimetic c(AmpRGD), which is a selective ligand for αVβ3 integrin, was covalently bound to the tyrosine kinase inhibitor sunitinib. Here, we report that these c(AmpRGD)-sunitinib conjugates and, in particular, compound 3, are selectively internalized by human melanoma cells through αVβ3 receptor-mediated endocytosis. Compound 3 is more effective than sunitinib in reducing in vitro melanoma cells proliferation, cloning efficiency, migration, and invasion. More interestingly, compound 3 is able to significantly reduce the growth of xenografted melanoma tumor developed in immune-compromised mice, more efficiently than an equimolar dose of sunitinib. Indeed, its targeting ability was demonstrated by the selective localization at the tumor level with respect to healthy tissues. Thus, c(AmpRGD)-sunitinib conjugates such as compound 3 could serve as intriguing multiple-target agents to selectively reach melanoma cells and interfere with the progression of the disease