of chemotherapy
Integrins are non-covalent heterodimeric transmembrane glycopro- teins existing as 24 combinations of 18α and 8β subunits (each of them is made up several domains linked through flexible linkers), able to mediate cell-cell and cell-extracellular matrix (ECM) interactions: they can be divided in five classes in which the same β subunit is combined with dif- ferent α ones, producing series of proteins with different ligand-binding specificities [118]. The half of them binds extracellular matrix proteins like fibronectin, vitronectin, VCAM-1 and collagen through a specific recognition sequence, and are fundamental for adhesion, migration, sig- naling and survival of cells. Integrins exist in different conformational states that include (i) the bent inactive, in which the inactivators are bound, (ii) the extended and (iii) the fully activated forms in which inte- grins are linked to actin and ECM protein together (Figure 5.2). According to the inside-out activation, a specific protein of the intracellular space links the integrin in the cytoplasmatic domain: talin links the cytoplas- matic β-domain of integrin in its inactive form, breaking the bridges be- tween the two subunits in the extracellular domain inducing changes in the tilt angle of the protein; kindlins binds the same cytoplasmatic do- main enhancing the tail-induced activation. For the outside-in activa- tion, the ECM ligand induces the transition between the "‘closed"’ to the "‘open"’ conformation of the β-subunit domain, without affecting the intracellular domain; when the talin in the intracellular space links the protein, integrin is completely activated [119]. Integrins are widely involved in cancer progression and several sub-types are highly over- expressed on cancer cells. Among them it is possible to mention α4β1,
Figure 5.2: Integrin activation.
α4β7, αvβ3and α5β1. Integrin-ligand combinations generate four main classes, based on the nature of the molecular interaction.
α4β1and α4β7integrins
α4β1, α4β7, α9β1, the four members of the β2subfamily, and αEβ7 recognize related sequences in their ligands. In particular, the α4-subclasses are expressed on the surface of leukocytes [120] and therefore involved
in inflammatory processes whose disregulation leads to pathogenesis of chronic inflammations and autoimmune disease like rheumatoid arthritis, multiple sclerosis and Crohn’s disease [121]. α4β1, known as "VLA-4", plays a central role in the trafficking of leukocytes and in their activa- tion and migration through the blood-endothelial barrier while inflam- matory response is working. It binds its natural ligand, Vascular Cell Adhesion Molecule-1 (VCAM-1, CD106), and a portion of the type III connecting segment of fibronectin (FN) [118]; its pivotal role regards the tumor angiogenesis associated with chronic inflammation, but it is involved also in the recruitment of progenitor cells in the formation of new blood vessels. As consequence of their overexpression in melanoma cells, these integrins are used as markers for prediction of metastatic risk [115][122] [123] [124] [125]. α4β7 is involved in lymphocyte hom- ing to mucosal tissue by adhesion to Mucosal Addressed Cell Adhesion Molecule (MAdCAM), but it links also FN and VCAM-1 [126][127]. It partecipates to (i) the infiltration of leukocytes in the islets of Langer- hans in type I diabetes, (ii) the demyelination in multiple sclerosis, (iii) the migration of T lymphocyte in the gut in inflammatory bowel dis- ease (IBD) subtypes of Crohn’s disease [128]. When FN and VCAM-1 are bound, α4β1 forms cluster on the cell surface named focal adhe- sions: this is the cross point between the ECM, the actine cytoskele- ton and the focal adhesion kinase, that activate a signaling cascade that involves extracellular regulated kinase (ERK), promoting proliferation and migration [129]. So, molecules able to interfere with these pro- teins can be employed for the treatment of inflammations, autoimmune diseases and cancer therapy. Actually, Natalizumab and Vedolizumab, anti-α4 and anti-α4β7 antibody respectively, are used as chemothera- peutics: Natalizumab, for Crohn’s disease and multiple sclerosis even if in same cases it induces progressive multifocal encephalopathy (PML) [130][131][132]; Vedolizumab, for treatment of ulcerative colitis and
Crohn’s disease [133][134]. However, the research of small molecules as protein ligands rapidly grew up in the last years, leading to the develop- ment of selective or dual ligands of the two types of integrins. They can be cyclic or linear peptides, mimicking the LDV, IDS or LDT recognition sequences: the first is part of fibronectin, the second of VCAM-1 and the tirth of MAdCAM (Figure 5.3). With the aim to develop analogues of
Figure 5.3: LDV, IDS and LDT recognition sequences.
these sequences, several examples of tyrosine and phenylalanine deriva- tives have been reported, in particular regarding the chemistry of pep- tidomimetic molecules [135][136]. To understand the allowed changes in the development of new ligands, studies on the binding poses of known compounds have been done by studying the crystal structure of the α4β1 binding fragment of VCAM-1 [137], together to 3D models [138], in sil- ico screening [139] and 3D QSAR studies [140]. The fundamental fea- tures for a good binding are the presence of a carboxylate group, a donor of H-bond (amide, for example) in the central part of the molecule and a lipophilic chain mimicking the Leu side chain in LDV [135]. More- over, PUPA (4[(N-2-methylphenyl)ureido]-phenylacetyl motif) gives en- hancement of activity, like observed in BIO1211 (Figure 5.4) [141].
αvβ3and α5β1integrins
αVβ3 and α5β1 support cell adhesion and proliferation of cancer cells, differentiation of mesenchymal stem cells (MSCs) and, in particu-
Figure 5.4: Structure of the bioactive α4β1ligand BIO1211.
lar for αvβ3, critical regulation of physiological and pathological angio- genesis, that is a critical step in the progression of a tumor and its metas- tasis. In tumor at early stages, the hypoxia can induce the "‘angiogenic switch"’ in so called dormient cells inducing the production of growth factors and the upregulation of integrins. By interaction with ECM lig- ands, this integrin partecipates in the formation of blood vassels, needed for the trafficking of oxygen and nutrients towards cancer cells. While αvβ3 can bind several ECM ligands like vitronectin (VN), fibronectin (FN), osteopontin and bone sialoprotein, α5β1recognize a specific se- quence of FN due to the presence on the cell attachment site of the protein of the synergistic amino acid sequence PHSRN. Them both recognized the RGD sequence as minimal adhesive binding motif [142]. Among the drugs active on these integrins reproducing the above-mentioned se- quence, surely Cilengitide is the most studied antagonist that reached the clinical phase III in trials for the treatment of glioblastoma and the phase II for other types of cancers [143] (Figure 5.5). By studying the X-ray crystallographic results of the complex αvβ3-cilengitide, it was found that the main interaction is of electrostatic nature in regions of the protein with opposite charges: the Arg interacts with two Asp residues on the α domain, while the carboxylate group of the ligand interacts
Figure 5.5: Structure of RGD sequence and its synthetic analogue Cilengitide.
with the MIDAS centre, the metal-ion-dependent adhesion site [144]. The research toward peptidomimetic analogues of the RGD sequence is tipically voted to the incorporation of rigid heterocyclic scaffolds with acidic and basic moieties properly oriented and distanced or enantiopure non-proteinogenic amino acids. We recently reported a small library of peptidomimetic isoxazoline-containing molecules designed as αvβ3and α5β1 integrin ligands: the isoxazoline core was functionalized in order to mimic the appendages of arginine and aspartate side chains; the result- ing structures showed good affinity results, with IC50in the nanomolar range for them both the integrins (Figure 5.6) [145].