Comparative Assessment of the Ligand and Metal Ion Binding Properties of Integrins α9β1 and α4β1

Abstract

Integrins alpha9beta1 and alpha4beta1 form a distinct structural class, but while alpha4beta1 has been subjected to extensive study, alpha9beta1 remains poorly characterized. We have used the small molecule N-(benzenesulfonyl)-(L)-prolyl-(L)-O-(1-pyrrolidinylcarbonyl)tyrosine (3) to investigate the biochemical properties of alpha9beta1 and directly compare these properties with those of alpha4beta1. Compound 3 has a high affinity for both integrins with K(D) values of < or =3 and 180 pM for alpha9beta1 in 1 mM Mn2+ (activating) and 1 mM Ca2+ and 1 mM Mg2+ (nonactivating) conditions and < or =5 and 730 pM for alpha4beta1 under the corresponding conditions. Ca2+ treatment promoted the binding of 3 to both integrins (EC50 = 30 microM Ca2+ in both cases). Compound 3 binding to both integrins was also stimulated by the addition of the activating monoclonal antibody TS2/16. These findings indicate that the mechanisms by which metal ions and TS2/16 regulate ligand binding to alpha9beta1 and alpha4beta1 are similar. The binding of 3 to both integrins induced the mAb 9EG7 LIBS epitope, a property consistent with occupancy of the receptor's ligand binding site by 3. But whereas EGTA treatment inhibited the binding of 9EG7 to alpha4beta1, it stimulated the binding of 9EG7 to alpha9beta1. The 9EG7 and TS2/16 effects point to contributions of the beta1-chains on binding. Cross-linking data revealed that the integrin alpha-chains are also involved in binding the small molecule, as stable linkages were observed on both the alpha9 chain of alpha9beta1 and the alpha4 chain of alpha4beta1. Extensive structure-activity analyses with natural and synthetic ligands indicate distinct features of the ligand binding pockets. Most notable was the estimated >1000-fold difference in the affinity of the integrins for VCAM-1, which binds alpha4beta1with an apparent K(D) of 10 nM and alpha9beta1 with an apparent K(D) of >10 microM. Differences were also seen in the binding of alpha9beta1 and alpha4beta1 to osteopontin. Compound 3 competed effectively for the binding of VCAM-1 and osteopontin to both integrins. While these studies show many similarities in the biochemical properties of alpha9beta1 and alpha4beta1, they identify important differences in their structure and function that can be exploited in the design of selective alpha9beta1 and alpha4beta1 inhibitors.

Therapeutic targeting and rapid mobilization of endosteal HSC using a small molecule integrin antagonist

“…In this study, we demonstrate targeting of a 9 b 1 /a 4 b 1 using a single dose of BOP 22 , induces rapid mobilization of long-term repopulating HSC through inhibiting integrin-dependent binding, most likely to trOpn 15,16 and VCAM-1 (ref. 17).…”

“…Bone has a very high mineral content and is the primary storage site of the inorganic salts of calcium and magnesium as well as trace metals such as manganese 56,57 : all of which are known to play a role in a 9 b 1 and a 4 b 1 adopting the conformational changes required for ligand binding 21,22,58 . Using XFM and quantitative ICP-MS, we demonstrate that endosteal BM harbours a significantly greater concentration of elemental calcium, magnesium and manganese relative to the central medullary region.…”

“…21) based on the small molecule N-(benzenesulfonyl)-L-prolyl-L-O-(1-pyrrolidinylcarbonyl)tyrosine 22 (BOP), which we previously demonstrated to bind effectively to a 9 b 1 and a 4 b 1 integrins in the presence of divalent metal cations 21 . Based on the interaction between a 9 b 1 /a 4 b 1 and trOpn being restricted to endosteal BM, we postulated that this family of compounds would target potent endosteal HSC for mobilization.…”

“…2f). The reduced in vivo efficacy of R-BC154 relative to BOP is most likely the result of the former having lower binding affinities as determined by association and dissociation kinetics studies in vitro 21,22 and thus reduced inhibitory potency towards integrin-dependent adhesive interactions. Fig.…”

Integrin α9 on lymphatic endothelial cells regulates lymphocyte egress

“…The avidity of binding of integrins to their ligand is regulated, at least in part, by the integrin activation state (29). We noted an increased activation state of the β1 integrin subunit, the partner of α9, as defined by staining with the activation-specific antibody 9EG7 (30) in lymphatic sinuses of inflamed LNs (Fig. 4A).…”