A Small Molecule Agonist of an Integrin, αLβ2

Abstract

Integrins are a large family of ␣/␤ heterodimeric cell surface receptors that mediate cell-cell and cell-extracellular matrix adhesion and transduce signals bidirectionally across the plasma membrane. Integrin ␣ L ␤ 2 (lymphocyte function associated antigen-1 (LFA-1)) 5 belongs to the ␤ 2 integrin subfamily and is constitutively expressed on all leukocytes. ␣ L ␤ 2 remains in a low affinity state in resting lymphocytes and undergoes dramatic conformational change during lymphocyte activation, which greatly increases its binding affinity for its ligands intercellular adhesion molecule -1, -2, and -3 (ICAM-1, -2, and -3). Regulation of ␣ L ␤ 2 activation is pivotal for controlling leukocyte trafficking and immune responses in health and diseases (1-3).␣ L ␤ 2 is an important pharmaceutical target for treating autoimmune and inflammatory diseases (4 -8). A humanized antibody to ␣ L ␤ 2 that blocks its binding to the ligand ICAM-1 has been approved by the FDA for treatment of psoriasis, a T cellmediated autoimmune disease of the skin (9, 10). Furthermore, small molecule antagonists of ␣ L ␤ 2 have been discovered and are in development (11)(12)(13)(14)(15)(16)(17).␣ L ␤ 2 contains two von Willebrand factor-type A domains, the inserted (I) domains in the ␣ L and the ␤ 2 subunits (18 -20). Both ␣ L I and ␤ 2 I domains have a Rossman fold (i.e. a central ␤-sheet surrounded by ␣-helices) with a metal ion-dependent adhesion site (MIDAS) formed by ␤-␣ loops at the "top" face of the domain (20 -23). In ligand binding the Mg 2ϩ ion in the MIDAS of the ␣ L I domain coordinates directly to a Glu residue that is in the center of the ligand binding sites in domain 1 of 24). The affinity of the ␣ L I domain for ICAMs is regulated by downward axial displacement of its C-terminal ␣7 helix, which is conformationally linked to reshaping of MIDAS loops and increases affinity for ligand by up to 10,000-fold (25, 26). During activation, the ␤ I domain undergoes similar ␣7 helix downward axial movement, which is induced by the swing out of the hybrid domain (27)(28)(29)(30). 6 Previous data suggested that when activated, the ␤ 2 I domain binds (through the Mg 2ϩ in its MIDAS) to the Glu residue (Glu-310) in the C-terminal linker of the ␣ L I domain, exerts a downward pull on its ␣7 helix, and thereby activates the ␣ L I domain (Fig. 1A) (32, 33).Two distinct classes of small molecule antagonists of ␣ L ␤ 2 have been developed as anti-inflammatory agents. One group of antagonists binds the hydrophobic pocket underneath the ␣7 helix of the ␣ L I domain (e.g. LFA703 or BIRT377), blocks the downward axial movement of the ␣7 helix, and inhibits ligand binding of ␣ L ␤ 2 allosterically by stabilizing the ␣ L I domain in the low affinity conformation (11)(12)(13)(14)34). These antagonists are called ␣ I allosteric inhibitors. The other group of antagonists appears to bind to the ␤ 2 I domain MIDAS near a key regulatory interface with the ␣ L I domain, blocking communication of conformational change to the ␣ L I domain while at the same time ac...

Aberrant activation of integrin α4β7 suppresses lymphocyte migration to the gut

“…This idea is further supported by the recent finding that a smallmolecule agonist to α L β 2 suppressed transendothelial migration by preventing uropod detachment (48). Because mutational activation of α L β 2 and α 4 β 7 reduced the accumulation of leukocytes in acutely inflamed tissues (14) as well as chronically inflamed tissues as reported in the present study, perturbing de-adhesion of integrins might represent a novel approach for interfering with leukocyte migration to sites of inflammation.…”

“…After washing with L-15 medium (Cambrex Bioscience), cells were added to the chambers, and differential interference contrast images were acquired using an Axiovert S200 epifluorescence microscope (Zeiss) equipped with a ×63 oil objective coupled to an Orca charge-coupled device camera (Hamamatsu) at 10-second intervals over the course of 25 minutes. Cell migration was analyzed by manually tracing the outline of each cell in 180-second intervals as previously described (48).…”

Small Molecule Agonist of Very Late Antigen-4 (VLA-4) Integrin Induces Progenitor Cell Adhesion

“…More recently, a small molecule antagonist of ␣L␤2 was found to be a partial agonist of cell adhesion under low affinity conditions (23). These findings led us to hypothesize that we can convert a known integrin antagonist to a full agonist.…”

Distinct roles for LFA-1 affinity regulation during T-cell adhesion, diapedesis, and interstitial migration in lymph nodes

“…Jerky migratory behaviors have also been seen in the disturbed TEM of human lymphocytes aberrantly activated by a small-molecule agonist for LFA-1. 46 Alternatively, aberrantly regulated signaling through constitutively, or irreversibly, active ␣ L -I306A LFA-1 might perturb appropriate LFA-1 redistribution on the membrane, thereby suppressing efficient cell migration, as regulated redistribution of IA LFA-1 at the leading edge and HA in the midbody have been proposed to be important for facilitating T-cell migration. 47 Since its first implementation, MP-IVM has revealed the unexpectedly motile nature of naive T cells in the LN interstitial compartment.…”