Could Dissecting the Molecular Framework of ß-Lactam Integrin Ligands Enhance Selectivity?

By dissecting the structure of ß-lactam-based ligands, a new series of compounds was designed, synthesized, and evaluated toward integrins αvß3, α5ß1, and α4ß1. New selective ligands with antagonist or agonist activities of cell adhesion in the nanomolar range were obtained. The best agonist molecules induced significant adhesion of SK-MEL-24 cells and Saos-2 cells as a valuable model for osteoblast adhesion. These data could lead to the development of new agents to improve cellular osseointegration and bone regeneration. Molecular modeling studies on prototypic compounds and αvß3 or α5ß1 integrin supported the notion that ligand carboxylate fixing to the metal ion-dependent adhesion site in the ß-subunit can be sufficient for binding the receptors, while the aryl side chains play a role in determining the selectivity as well as agonism versus antagonism.

Small molecule agonist of very late antigen-4 (VLA-4) integrin induces progenitor cell adhesion.

Activation of the integrin family of cell adhesion receptors on progenitor cells may be a viable approach to enhance the effects of stem cell-based therapies by improving cell retention and engraftment. Here, we describe the synthesis and characterization of the first small molecule agonist identified for the integrin α4ß1 (also known as very late antigen-4 or VLA-4). The agonist, THI0019, was generated via two structural modifications to a previously identified α4ß1 antagonist. THI0019 greatly enhanced the adhesion of cultured cell lines and primary progenitor cells to α4ß1 ligands VCAM-1 and CS1 under both static and flow conditions. Furthermore, THI0019 facilitated the rolling and spreading of cells on VCAM-1 and the migration of cells toward SDF-1α. Molecular modeling predicted that the compound binds at the α/ß subunit interface overlapping the ligand-binding site thus indicating that the compound must be displaced upon ligand binding. In support of this model, an analog of THI0019 modified to contain a photoreactive group was used to demonstrate that when cross-linked to the integrin, the compound behaves as an antagonist instead of an agonist. In addition, THI0019 showed cross-reactivity with the related integrin α4ß7 as well as α5ß1 and αLß2. When cross-linked to αLß2, the photoreactive analog of THI0019 remained an agonist, consistent with it binding at the α/ß subunit interface and not at the ligand-binding site in the inserted ("I") domain of the αL subunit. Co-administering progenitor cells with a compound such as THI0019 may provide a mechanism for enhancing stem cell therapy.

Real-time analysis of the inside-out regulation of lymphocyte function-associated antigen-1 revealed similarities to and differences from very late antigen-4.

Ten years ago, we introduced a fluorescent probe that shed light on the inside-out regulation of one of the major leukocyte integrins, very late antigen-4 (VLA-4, CD49d/CD29). Here we describe the regulation of another leukocyte integrin, lymphocyte function-associated antigen-1 (LFA-1, CD11a/CD18) using a novel small fluorescent probe in real time on live cells. We found that multiple signaling mechanisms regulate LFA-1 conformation in a manner analogous to VLA-4. LFA-1 can be rapidly activated by Gα(i)-coupled G protein-coupled receptors (GPCRs) and deactivated by Gα(s)-coupled GPCRs. The effects of Gα(s)-coupled GPCR agonists can be reversed in real time by receptor-specific antagonists. The specificity of the fluorescent probe binding has been assessed in a competition assay using the natural LFA-1 ligand ICAM-1 and the LFA-1-specific α I allosteric antagonist BIRT0377. Similar to VLA-4 integrin, modulation of the ligand dissociation rate can be observed for different LFA-1 affinity states. However, we also found a striking difference in the binding of the small fluorescent ligand. In the absence of inside-out activation ligand, binding to LFA-1 is extremely slow, at least 10 times slower than expected for diffusion-limited binding. This implies that an additional structural mechanism prevents ligand binding to inactive LFA-1. We propose that such a mechanism explains the inability of LFA-1 to support cell rolling, where the absence of its rapid engagement by a counterstructure in the inactive state leads to a requirement for a selectin-mediated rolling step.

Effect of divalent cations on the affinity and selectivity of alpha4 integrins towards the integrin ligands vascular cell adhesion molecule-1 and mucosal addressin cell adhesion molecule-1: Ca2+ activation of integrin alpha4beta1 confers a distinct ligand specificity.

A microtiter plate assay measuring the binding of cells expressing integrins alpha4beta1 or alpha4beta7 to VCAM-1 and MAdCAM-1, expressed as Ig fusion proteins, was used to explore the interplay between the variables of integrin beta-chain, identity and density of ligand, and identity and concentration of activating cations. Both Mn2+ and Mg2+ supported binding of either integrin to either ligand. Ca2+ supported only the binding of alpha4beta1 to VCAM-Ig. Cation concentrations required for half-maximal binding (EC50) ranged from 0.8-280 microM for Mn2+ and 0.8-30 mM for Mg2+, being thus 2-3 logs lower for Mn2+ compared to Mg2+ independent of ligand. EC50 values for binding of alpha4beta1 to VCAM-Ig were 30-45-fold lower compared to MAdCAM-Ig, while alpha4beta7 showed an opposite 3-15-fold selectivity for MAdCAM-Ig over VCAM-Ig. The density of ligand required for adhesion via alpha4beta1 was markedly lower with Mn2+ versus Mg2+, and with VCAM-Ig versus MAdCAM-Ig. These results were interpreted in terms of a coupled equilibrium model, in which binding of activating metal ions and of integrin ligands each stabilizes activated integrin. We conclude that Mn2+ and Mg2+ bind to common regulatory sites with different affinities, producing similar activated states of the integrin. The resulting activated alpha4beta1 binds more strongly to VCAM-Ig versus MAdCAM-Ig by 30-45-fold, while similarly activated alpha4beta7 binds more strongly to MAdCAM-Ig versus VCAM-Ig by 3-15-fold. Inhibition studies showed that Ca2+ also binds to regulatory sites on both integrins. However, the Ca2+-activated state of alpha4beta1 is distinct from that achieved by Mn2+ and Mg2+, possessing increased selectivity for binding to VCAM-1 versus MAdCAM-1.