Determining the True Selectivity Profile of αv Integrin Ligands Using Radioligand Binding: Applying an Old Solution to a New Problem.

The arginyl-glycinyl-aspartic acid (RGD) integrin subfamily contains five members that partner with the αv subunit: αvß1, αvß3, αvß5, αvß6, and αvß8. Within the αv integrins, the epithelially restricted αvß6 has been identified as playing a key role in the activation of transforming growth factor ß that is hypothesized to be pivotal in the development of idiopathic pulmonary fibrosis (IPF). As part of a drug discovery program to identify a selective αvß6 RGD mimetic for IPF, cell adhesion and radioligand binding assays were investigated to screen compounds to determine affinity and αv integrin selectivity. In this study, a pan-αv radioligand was characterized against all the αv integrins and used to determine accurate selectivity profiles for literature and novel RGD ligands, as well as enable an early readout on αvß6 dissociation kinetics. It has been shown that while cell adhesion offers a high throughput and reliable format for ranking compounds, there are downsides to this format when comparing selectivity across αv integrins. By accurately defining the relationship between these assay formats, a medicinal chemistry effort has identified novel, high-affinity, and selective αvß6 RGD mimetics with slow dissociation kinetics, with the potential to be developed into clinical candidates for IPF.

Pharmacological Characterization of the αvß6 Integrin Binding and Internalization Kinetics of the Foot-and-Mouth Disease Virus Derived Peptide A20FMDV2.

A20FMDV2 is a peptide derived from the foot-and-mouth disease virus with a high affinity and selectivity for the alpha-v beta-6 (αvß6) arginyl-glycinyl-aspartic acid (RGD)-binding integrin. It has been shown to be an informative tool ligand in pre-clinical imaging studies for selective labelling of the αvß6 integrin in a number of disease models. In a radioligand binding assay using a radiolabelled form of the peptide ([3H]A20FMDV2), its high affinity (K(D): 0.22 nmol/l) and selectivity (at least 85-fold) for αvß6 over the other members of the RGD integrin family was confirmed. [3H]A20FMDV2 αvß6 binding could be fully reversed only in the presence of EDTA, whereas a partial reversal was observed in the presence of excess concentrations of an RGD-mimetic small molecule (SC-68448) or unlabelled A20FMDV2. Using flow cytometry on bronchial epithelial cells, the ligand-induced internalization of αvß6 by A20FMDV2 and latency-associated peptide-1 was shown to be fast (t(1/2): 1.5 and 3.1 min, respectively), concentration-dependent (EC50: values 1.1 and 3.6 nmol/l, respectively) and was followed by a moderately slow return of integrin to the surface. The results of the radioligand binding studies suggest that the binding of A20FMDV2 to the RGD-binding site on αvß6 is required to maintain its engagement with the hypothesised A20FMDV2 synergy site on the integrin. In addition, there is evidence from flow cytometric studies that the RGD-ligand engagement of αvß6 post-internalization plays a role in delaying recycling of the integrin to the cell surface. This mechanism may act as a homeostatic control of membrane αvß6 following RGD ligand engagement.

Cyclic glycopeptidomimetics through a versatile sugar-based scaffold.

Cyclic peptidomimetics are attracting structures to obtain a distinct, bioactive conformation. Even more attractive are sugar-containing cyclic peptidomimetics which present turn structures induced by the pyranose ring when incorporated in cyclic peptides. The use of a new and versatile saccharidic scaffold to achieve sugar-based peptidomimetics is here reported together with the successful synthesis of diastereomerically pure cyclic SAA peptidomimetics 15 and 16.

SB-267268, a nonpeptidic antagonist of alpha(v)beta3 and alpha(v)beta5 integrins, reduces angiogenesis and VEGF expression in a mouse model of retinopathy of prematurity.

PURPOSE: To determine whether SB-267268, a nonpeptidic antagonist of the alpha(v)beta3 and alpha(v)beta5 integrins, attenuates angiogenesis in a murine model of retinopathy of prematurity (ROP) and alters the expression of vascular endothelial growth factor (VEGF) and its second receptor (VEGF-R2). METHODS: In receptor binding, SB-267268 exhibited nanomolar potency for human, monkey, and murine alpha(v)beta3 and alpha(v)beta5. SB-267268 inhibited the attachment of alpha(v)beta3-transfected HEK293 cells to microtiter plate wells precoated with RGD-containing matrix proteins, and vitronectin-mediated human and rat aortic smooth-muscle-cell migration. At postnatal day (P)12, C57BL/6 mice were exposed to 80% oxygen for 7 days followed by 7 days in room air (angiogenic period). Between P12 and P17, ROP mice were administered sterile saline (vehicle intraperitoneal [i.p.]) or SB-267268 (60 mg/kg bi-daily, i.p.). Shams were exposed to room air from P0 and administered either vehicle or SB-267268 during P12 to 17. In at least 3 randomly chosen paraffin sections from each eye, the number of blood vessel profiles in the inner retina were counted. In situ hybridization for VEGF and VEGFR-2 was performed on at least 8 randomly chosen paraffin sections from each eye. RESULTS: SB-267268 reduced pathologic angiogenesis in ROP mice by approximately 50% and had no effect on developmental retinal angiogenesis in shams. Both VEGF and VEGFR-2 mRNA were upregulated in the inner retina of ROP mice and reduced with SB-267268. CONCLUSIONS: Nonpeptidic inhibition of alpha(v)beta3 and alpha(v)beta5 integrins is effective in ROP and may be a suitable anti-angiogenic therapy for other ischemic retinal pathologies.