Small molecule agonists of integrin CD11b/CD18 do not induce global conformational changes and are significantly better than activating antibodies in reducing vascular injury

Abstract

BACKGROUND CD11b/CD18 is a key adhesion receptor that mediates leukocyte adhesion, migration and immune functions. We recently identified novel compounds, leukadherins, that allosterically enhance CD11b/CD18-dependent cell adhesion and reduce inflammation in vivo, suggesting integrin activation to be a novel mechanism of action for the development of anti-inflammatory therapeutics. Since a number of well-characterized anti-CD11b/CD18 activating antibodies are currently available, we wondered if such biological agonists could also become therapeutic leads following this mechanism of action. METHODS We compared the two types of agonists using in vitro cell adhesion and wound-healing assays and using animal model systems. We also studied effects of the two types of agonists on outside-in signaling in treated cells. RESULTS Both types of agonists similarly enhanced integrin-mediated cell adhesion and decreased cell migration. However, unlike leukadherins, the activating antibodies produced significant CD11b/CD18 macro clustering and induced phosphorylation of key proteins involved in outside-in signaling. Studies using conformation reporter antibodies showed that leukadherins did not induce global conformational changes in CD11b/CD18 explaining the reason behind their lack of ligand-mimetic outside-in signaling. In vivo, leukadherins reduced vascular injury in a dose-dependent fashion, but, surprisingly, the anti-CD11b activating antibody ED7 was ineffective. CONCLUSIONS Our results suggest that small molecule allosteric agonists of CD11b/CD18 have clear advantages over the biologic activating antibodies and provide a mechanistic basis for the difference. GENERAL SIGNIFICANCE CD11b/CD18 activation represents a novel strategy for reducing inflammatory injury. Our study establishes small molecule leukadherins as preferred agonists over activating antibodies for future development as novel anti-inflammatory therapeutics.

Antagonism of CD11b with Neutrophil Inhibitory Factor (NIF) Inhibits Vascular Lesions in Diabetic Retinopathy

“…In contrast to our results demonstrating a beneficial effect of inhibiting the interaction between leukocytes and endothelial cells, other investigators have reported that enhancing the binding of leukocytes to endothelial cells inhibited vascular disease such as neointimal thickening following denudation [46]. However this approach may be less efficacious in diabetes where adhesion of activated leukocytes may cause additional occlusion of capillaries and leukocyte-mediated endothelial damage.…”

A Lupus-Associated Mac-1 Variant Has Defects in Integrin Allostery and Interaction with Ligands under Force

“…However, α M R77H with β 2 V124A (Figure 2E) or β 2 L132A (data not shown) remained impaired in ligand binding under flow. The small molecule Leukadherin-1 (LA-1) binds and activates the αI-domain without inducing global conformational changes (Faridi et al, 2013). While LA-1 pretreatment enhanced adhesion of α M WT expressing cells (data not shown) as described (Faridi et al, 2013), it did not rescue the ability of α M R77H to bind iC3b under flow (Figure 2F).…”

“…The small molecule Leukadherin-1 (LA-1) binds and activates the αI-domain without inducing global conformational changes (Faridi et al, 2013). While LA-1 pretreatment enhanced adhesion of α M WT expressing cells (data not shown) as described (Faridi et al, 2013), it did not rescue the ability of α M R77H to bind iC3b under flow (Figure 2F). Thus, neither an open, permissive βI-domain nor the direct activation of the αI-domain rescues R77H.…”

CD11b activation suppresses TLR-dependent inflammation and autoimmunity in systemic lupus erythematosus

“…Given that their expression is modulated by nuclear levels of FOXO3, we also quantified the levels of FOXO3 in the nucleus versus cytoplasm and found that, under basal conditions, the cells carrying a minor allele showed significantly reduced levels of FOXO3 in the nucleus, as compared with the cells carrying the common allele ( Figure 7B). Recent reports show that (38). Such LA1-mediated integrin activation significantly blunted generation of TLR-stimulated proinflammatory cytokines and chemokines in leukocytes, suppressing TLR-dependent intracellular signaling pathways in vitro and in vivo, and further explaining the observed strong antiinflammatory effects of LA1 in vivo.…”

“…LA1 binds to the CD11bA/I domain (CD11bA) in an allosteric pocket (Supplemental Figure 1), enhancing ligand binding. Molecular dynamics and isothermal calorimetry studies with CD11bA and fluid shear stress studies with cell surface-expressed full-length integrin CD11b/CD18 (Supplemental Methods; Supplemental Tables, 2 and (37) and no large conformational changes in the integrin heterodimer (38), defining this as its preferred binding mode. Given that the SLE-associated CD11b mutations are located outside of the ligand-binding αA domain (Supplemental Figure 2, A and B), it is unclear whether they would affect LA1 binding to the αA domain, and thus affect LA1's activity.…”