A Small-Molecule Antagonist to Integrin LFA-1 Reveals a Crucial Inter-Domain Communication as a Novel Therapeutic Target.

The integrin LFA-1 (αLβ2) is an αβ heterodimeric adhesion molecule critical in the effective trafficking of leukocytes and in facilitating subsequent antigen-specific inter-action. Participation of αLβ2 in multiple steps critical for T-cell-mediated immunity in vivo makes αLβ2 a valid therapeutic target for anti-inflammation therapy. Many small-molecule antagonists to αLβ2 have been developed as anti-inflammatory agents, out of which polysubstituted (S)-2-benzoylamino propionic acids, represented by XVA143 (XVA), have emerged as the most potent antagonists. αLβ2 is a large glycoprotein with a complex multi-domain organization, where a conserved von Willebrand factor-type A domain is contained in each subunit, the inserted (I) domain in the α-subunit and the I-like domain in the β subunit. The α-subunit I domain directly binds ligand, whereas the β-subunit I-like domain is thought to play a regulatory role by interacting with a part of the I domain. Thus far, it remains to be elucidated which domain the antagonists bind to and how they inhibit αLβ2 function. Here we investigate a mechanistic basis of XVA activity. XVA blocked the αLβ2-ICAM-1 interaction with EC50 of < 1 nM and suppressed mixed lymphocyte reaction as potently as cyclosporin A. XVA did not block ligand binding by αLβ2 directly, as it did not block αLβ2 containing a mutant I domain that is stabilized in the high-affinity conformation. Rather, XVA interfered with conformational changes that convert the I domain to the high-affinity state. Surface plasmon resonance analysis using an isolated I domain showed that XVA did not target the I domain. Interestingly, XVA stabilized non-covalent αβ association sufficiently to make it resistant to denaturation with SDS. Stabilization of mutant αβ complexes was utilized to test compound binding to αLβ2 mutants and locate the inhibitor-binding site. As binding of XVA was found to be metal-dependent, alanine-scanning of the metal binding sites indicated that this compound binds to the metal ion-dependent adhesion site in the I-like domain, where it disrupts the interaction of the I-like domain with the I domain. XVA inhibits αLβ2 allosterically by perturbing the inter-domain communication that is critical to relay conformational signals which induce the active I domain conformation. Furthermore, XVA stabilized a global conformation of αLβ2 in the active extended form, whereas the ligand binding I domain was left in the inactive conformation, as demonstrated by exposure of activation-dependent epitopes in αLβ2 on the cell surface and electron microscopic images of the soluble recombinant αLβ2. The results strongly suggest that XVA would serve as a mimetic for the intrinsic ligand that is involved in receptor-ligand like interaction between the I domain and I-like domain. This inhibitor revealed a crucial intersection for relaying conformational signals within the integrin αLβ2. While blocking signals in one direction (to the I domain), the antagonists induce the active conformation of the I-like domain as well as the rest of domains, and thus transmit conformational signals in the opposite direction toward the transmembrane domains.