[3H]A20FMDV2, a radiolabelled peptide derived from the foot-and-mouth disease virus, demonstrates a complex binding interaction with the α vβ 6 integrin

Rob Slack1,2, Rebecca Rogers1, Jane Denyer1, David Flint2, Susan Pyne2. 1GlaxoSmithKline, Stevenage, Hertfordshire, UK, 2University of Strathclyde, Glasgow, UK

A20FMDV2 is a peptide derived from the foot-and-mouth disease virus (FMDV) that has been shown to be >1,000 fold selective for the αvβ6 integrin over related integrins (Hausner et al, 2007). A20FMDV2 was radiolabelled with [3H] to allow pharmacological characterisation of the binding profile of this RGD peptide with human αvβ6 integrin.

Radioligand filtration binding experiments were performed with [3H]A20FMDV2 and purified human recombinant αvβ6 in binding buffer (25mM HEPES, 100mM NaCl, 2mM MgCl2 and 1mM CHAPS at pH 7.4) at 37ºC, with non-specific binding (NSB) determined by addition of 10μM SC-68448 (a peptidomimetic antagonist of αvβ3) or 1μM A20FMDV2. For divalent cation dependency experiments, 2mM MgCl2 was replaced with either 2mM CaCl2 or MnCl2. Saturation, association, dissociation and competition binding studies were performed to determine receptor binding kinetics at αvβ6 (equilibrium dissociation constant (K D), total number of receptors (B max), association rate (k on), dissociation rate (k off), and dissociation half-life (t1/2 )). Dissociation was initiated by addition of either 10μM SC-68448, 1μM A20FMDV2, 10μM SC-68448 and 1μM A20FMDV2 or 10mM EDTA following a 1 h pre-incubation of [3H]A20FMDV2 and αvβ6.

Binding of [3H]A20FMDV2 to αvβ6 required the presence of divalent cations with 2mM Ca2+, Mg2+ or Mn2+ having a comparable propensity to support binding. [3H]A20FMDV2 saturation binding studies (equilibrated for 6 h) resulted in a profile suggestive of two site binding, which was independent of unlabelled ligand used to define NSB. Scatchard transformations for these data were non-linear. The high affinity binding site was further characterised with the saturation parameters shown in Table 1 (all data shown are mean ± SEM, n=4).

Table 1. The receptor binding kinetic parameters for [3H]A20FMDV2 at the human α vβ 6 integrin.

Association of [3H]A20FMDV2 to αvβ6 was measured at ~KD and 5 x KD concentrations of radioligand. Global fitting of the association kinetic model to specific binding resulted in a kon of 5.7 ± 0.2 x 107 M-1/min-1. The dissociation profiles of [3H]A20FMDV2 initiated by 10 µM SC-68448, 1 µM A20FMDV2 or 10 µM SC-68448 and 1 µM A20FMDV2 in combination, were all comparable with only a partial dissociation observed. At 24 h, under all these conditions, ~67 % of the radioligand remained bound. 10mM EDTA resulted in a single phase dissociation with complete dissociation achieved at 24 h with a t1/2 of 5.9 ± 0.6 min. In contrast, full competition was observed when radioligand and unlabelled ligands were exposed to αvβ6 simultaneously in competition binding studies (equilibrated for 6 h).

In conclusion, the data generated in this study suggests there are two binding sites on the αvβ6 integrin for [3H]A20FMDV2, the high affinity RGD-binding site and a low affinity non-RGD-binding site that is also EDTA sensitive. This provides further evidence for a synergy site (DiCara et al, 2008) on the αvβ6 integrin for the FMDV.

DiCara et al, J Virol 82:1537, 2008

Hausner et al, Cancer Res 67:7833, 2007