ABSTRACT
Title
Novel antagonists of αvβ3, α5β1 and α4β1 integrins: attractive targets for cancer and anti-inflammatory therapeutics
Authors
S. D. Dattoli1, M. Baiula1, A. Viola2, R. De Marco2, S. Spampinato1
1Dept. of Pharmacology, University of Bologna, Bologna, Italy
2 Dept. Of Chemistry, University of Bologna, Bologna, Italy
1Dept. of Pharmacology, University of Bologna, Bologna, Italy
2 Dept. Of Chemistry, University of Bologna, Bologna, Italy
Abstract
Integrins are noncovalently associated heterodimeric cell surface adhesion molecules. In vertebrates, 18 α subunits and 8 β subunits form 24 known αβ pairs. Both the integrin α and β subunits are type I transmembrane glycoproteins with large extracellular domains, single-spanning transmembrane domains, and, with the exception of β4, short cytoplasmic domains. Integrins are expressed in a cell specific manner and their ligand binding is characterized by both specificity and redundancy. More specifically, integrins may exhibit different specificities when expressed on different cell types; furthermore, the same integrin can bind different ligands or different integrins can bind to the same ligand. Integrins such as αvβ3 and α5β1 recognize the RGD sequence (Arg-Gly-Asp) in their ligands, whereas others recognize alternative short peptide sequences, for example α4β1 recognizes the LDV motif.
In particular, we focused on integrin αvβ3, α5β1 because they are considered a privileged target for anti-angiogenic therapy, and on integrin α4β1 for its involvement in inflammatory diseases (Kummer et al. 2006). It is established that tumor associated vessels express integrin αvβ3. Interestingly, the α5β1 integrin has been shown to affect αvβ3-mediated endothelial cell migration and angiogenesis via regulation of αvβ3 integrin function.
As the crystal structure of the extracellular segment of the αvβ3 integrin is known, it is possible to design novel integrin antagonists based on the receptor-bound conformation of the RGD tripeptide. Therefore, small RGD mimetic molecules that are able to interfere with the α5β1 and αvβ3 integrins are currently considered of interest because of their possible anti-angiogenic and anti-tumoral role (Gentilucci et al. 2010).
We tested some compounds that constitute a library of peptidomimetics capable of interact with αvβ3 and α5β1 integrins through functional groups that mimicking the guanidine and carboxylic moieties present in the side chain of arginine and aspartic acid. Their efficacy and potency are established with adhesion assays, using different cell lines expressing integrins. The ability of compounds synthesized to inhibit the adhesion of K562 (human erythroleukemic cells, expressing α5β1 integrin) or SK-MEL-24 cells, expressing αvβ3 integrin) to immobilized fibronectin was evaluated.
Compound EM 391 exhibits the highest potency as an inhibitor of cell adhesion mediated by αvβ3 and α5β1 integrin (IC50 values:8,79x10-9 M vs αvβ3 and 1,05x10-9 M vs α5β1). Instead, compound EM490, STA177 and STA204 can be considered selective antagonists: in fact, they were more effective toward α5β1 integrin (IC50 values: STA177 1,9x10-8 M and STA204 1x10-9 M), while compound EM490 was a more selective antagonist toward αvβ3 integrin (IC50 value:3x10-7 M)
In order to develop α4β1 integrin antagonists, a library of compounds was synthesized and tested in cell adhesion assay on Jurkat E6.1 cells. Some compounds showed very interesting IC50 values; for example, compounds DS23, DS70 and RDM355 displayed the following IC50 :3,51x10-10 M, 3,098x10-9 M and 2,589x10-9 M respectively. Moreover, a SPA (scintillation proximity assay) assay was built up and employed in order to define the affinity of compounds toward α4β1 integrin.
Gentilucci et al (2010), J.Med.Chem. 53:106-118
Kummer et al (2006), Biochem. Pharm. 72:1460-1468
In particular, we focused on integrin αvβ3, α5β1 because they are considered a privileged target for anti-angiogenic therapy, and on integrin α4β1 for its involvement in inflammatory diseases (Kummer et al. 2006). It is established that tumor associated vessels express integrin αvβ3. Interestingly, the α5β1 integrin has been shown to affect αvβ3-mediated endothelial cell migration and angiogenesis via regulation of αvβ3 integrin function.
As the crystal structure of the extracellular segment of the αvβ3 integrin is known, it is possible to design novel integrin antagonists based on the receptor-bound conformation of the RGD tripeptide. Therefore, small RGD mimetic molecules that are able to interfere with the α5β1 and αvβ3 integrins are currently considered of interest because of their possible anti-angiogenic and anti-tumoral role (Gentilucci et al. 2010).
We tested some compounds that constitute a library of peptidomimetics capable of interact with αvβ3 and α5β1 integrins through functional groups that mimicking the guanidine and carboxylic moieties present in the side chain of arginine and aspartic acid. Their efficacy and potency are established with adhesion assays, using different cell lines expressing integrins. The ability of compounds synthesized to inhibit the adhesion of K562 (human erythroleukemic cells, expressing α5β1 integrin) or SK-MEL-24 cells, expressing αvβ3 integrin) to immobilized fibronectin was evaluated.
Compound EM 391 exhibits the highest potency as an inhibitor of cell adhesion mediated by αvβ3 and α5β1 integrin (IC50 values:8,79x10-9 M vs αvβ3 and 1,05x10-9 M vs α5β1). Instead, compound EM490, STA177 and STA204 can be considered selective antagonists: in fact, they were more effective toward α5β1 integrin (IC50 values: STA177 1,9x10-8 M and STA204 1x10-9 M), while compound EM490 was a more selective antagonist toward αvβ3 integrin (IC50 value:3x10-7 M)
In order to develop α4β1 integrin antagonists, a library of compounds was synthesized and tested in cell adhesion assay on Jurkat E6.1 cells. Some compounds showed very interesting IC50 values; for example, compounds DS23, DS70 and RDM355 displayed the following IC50 :3,51x10-10 M, 3,098x10-9 M and 2,589x10-9 M respectively. Moreover, a SPA (scintillation proximity assay) assay was built up and employed in order to define the affinity of compounds toward α4β1 integrin.
Gentilucci et al (2010), J.Med.Chem. 53:106-118
Kummer et al (2006), Biochem. Pharm. 72:1460-1468