Osteoporosis is present in 24 million Americans and contributes to more than 1.3 million fractures/year. Serious morbidity and mortality result from these fractures. Current therapies for osteoporosis are few, efficacy is limited, and side-effects problematic. This research proposal is focused on elucidating the role of the osteoclast integrin in bone resorption, the nature of ligand--integrin interactions, and development of antagonists for cell surface adhesion molecules, particularly the av2B3 vitronectin integrin receptor present on the surface of osteoclasts. Based on previous findings from the applicant's laboratory, peptides containing the """"""""RGD"""""""" motif are able to inhibit osteoclast-mediated bone resorption in vivo. They now propose to design more potent and selective inhibitors of bone resorption as a potential new therapeutic approach to osteoporosis based on a novel mechanism. Although extensive structure--activity studies have been conducted for RGD-containing ligands of integrin receptors in other systems, such as the fibrinogen receptor and platelet aggregation, there is little published work regarding structure--activity relations for ligands of osteoclast integrins. Hence, they intend to make a focused commitment to this new area. They will employ several independent approaches to design potent specific antagonists of the human osteoclast avB3 integrin. First, in an effort to rapidly identify the highest affinity ligands for the human avB3 integrin, they will generate combinatorial peptide libraries containing enormous structural diversity. For instance, the applicant's recently synthesized and chemically analyzed a library of 360,000 peptides, all of which contain RGD, and based on all the possible sequence combinations of extracellular matrix proteins known to bind avB3. Human avB3 receptors are now available from a cell line that expresses high levels of cloned receptor. The applicants will produce an affinity column of immobilized purified functional human avB3. The library of peptides will be screened by """"""""affinity-selection"""""""". The highest binding affinity peptide(s) will be eluted from the column and microsequenced. Receptor-favored sequences will be synthesized and evaluated in a battery in vitro bioassays. Promising analogs then will be tested in vivo for inhibition of bone resorption. avB3-selective ligands will be identified using a human platelet-based fibrinogen (GP IIb/IIa) receptor counter-screen. In addition, analogs which bind to integrins, but fail to stimulate intracellular signaling, will be sought. Information obtained from """"""""affinity-selection"""""""" of combinatorial libraries will be integrated and used to further the design of integrin antagonists. Through these investigations, insight will be gained into the role of integrins in bone biology and pathophysiology, and new directions will be developed for the design of potent human avB3-selective antagonists for the treatment of osteoporosis.
