Establishing a screening platform for the biological evaluation and modulation of complement-related integrin receptors

The family of β2-integrin receptors consists of conformationally flexible heterodimeric transmembrane receptors, which are critically involved in host defense and homeostasis by mediating immune cell adhesion and migration. Two members of the family, complement receptors 3 (CR3) and 4 (CR4), are furthermore involved in complement-dependent phagocytosis. Together with their molecular siblings leukocyte function-associated antigen-1 and CD11d/CD18 they exert key roles in immune surveillance and inflammation, and inappropriate engagement of these receptors may contribute to several clinical conditions. While a therapeutic modulation of β2-integrin signaling has been recognized as an attractive option for drug development, the poor druggability of integrins, the shortage of validated assay systems, and gaps in our understanding of disease-related molecular mechanisms have hampered drug development efforts. While the β2-integrin family is generally recognized for its highly complex mode of function,featuring different ligands and distinct conformational states, CR3 in particular is known for its profound number of reported ligands. This ligand binding spectrum largely defines CR3’s involvement in a broad variety of physiological and pathophysiological processes. To better understand the role of CR3 in health and disease, an in view of the many gaps and inconsistencies in current literature, We carefully catalogued the different interaction partners of CR3, with a focus on describing the binding mechanism and its functional implications. For facilitating molecular and functional studies on the β2-integrin family, and enabling the screening of receptor-selective modulators, we aimed to provide a consistent and validated assay platform. For this purpose, the αI domains of all four β2-integrins, which present the major ligand-binding areas, were recombinantly expressed in low- and high affinity states. In direct binding studies based on surface plasmon resonance, the expected activity and selectivity profiles of the recombinant αI domains for their endogenous ligands could be demonstrated. In addition, direct binding studies were used to develop competitive assays to demonstrate the inhibitory activity of the reported β2-integrin inhibitor simvastatin. Moreover, bead- and cell-based adhesion assays were developed to validate and extend the results of the direct binding assays. The combined assay panel could thereby be established as suitable platform for the functional characterization of β2-integrins and the screening of receptor modulators. After its successful validation, we employed the assay plaform to drive the discovery and characterization of αI-binding cyclic peptides. The goal was to select macrocyclic peptides by phage display library screening, which could be used as tool compounds to determine the role of ligand binding to the β2 integrin family. Peptides binding to one or several αI domains were synthesized and characterized using our in vitro assay platform. Several peptide sequences were enriched in multiple rounds of phage display, of which two peptides showed the strongest binding and were therefore selected for further characterization. Both lead peptides showed binding to the αI domains of all four β2 integrins, thereby indicating a contact site at a conserved area. In a competitive assay, both peptides showed dose-dependent inhibition of αI domain binding to their major ligands iC3b and ICAM-1, which was confirmed in bead- and cell-based adhesion assays. While additional studies are needed to fully explore the structure-activity relationship profile of the newly discovered peptide ligands, the direct competition of the lead compounds with key β2 integrin interactions raises the expectation that our peptides can be used as tool compounds to provide valuable insights into the (patho)physiology of CR3 and the other members of the β2 integrin family. Both the functional insight and the idenfitication of β2 integrin modulators driven by our assay platform could guide the development of potential therapeutic approaches for autoimmune, inflammatory, and age-related diseases.