SBIR-STTR Award

Mice lacking a functional c-src gene develop osteopetrosis due to a defect in bone resorption by osteoclasts (1). This evidence implicates the role of Src in osteoclast resorption activity. We aim to develop a drug that blocks the action of Src protein kinase as a potential treatment of osteoporosis. To do this, we are targeting a domain of the Src protein kinase termed SH2 (src homology 2). SH2 domains are found on a number of intracellular proteins and function as receptors for tyrosine-phosphorylated polypeptides. Our Phase I goal is to identify peptides that tightly bind to the Src SH2 domain using a technique termed "phage display". A library of pentapeptldes is expressed on the surface of M13 phage fused to the gene III protein, a minor M13 coat protein. Each phage displays a single peptide sequence on its surface. The phage library is mixed with immobilized SH2 domain and non-binding phage are removed by washing. Bound phage are eluted and propagated through E. coli to produce a population of phage enriched for sequences that recognize the SH2 domain. Since SH2 domains exhibit a higher affinity for tyrosine-phosphorylated peptides versus nonphosphorylated peptides, we will use several novel approaches to phosphorylate the phage-displayed peptides. The selection cycle is repeated 5-10 times then individual phage are cloned, and the encoded peptides are sequenced. Peptides derived from the selected clones will be synthesized and tested for binding to Src SH2 domain.Awardee's statement of the potential commercial applications of the research: Peptides that inhibit Src will serve as lead compounds in the design of small molecule drugs to treat osteoporosis.National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)

The growth and maintenance of the skeleton is a dynamic process in which osteoblast cells form new bone and osteoclasts degrade bone. Src gene disruption experiments in mice demonstrated that Src, an intracellular protein tyrosine kinase, is essential for resorption activity of osteoclasts. Osteoporosis is a serious disease of essential bone degradation. The properties of Src deficient osteoclasts suggest that inhibitors of Src function would be useful drugs for the prevention or treatment of osteoporosis, by slowing down the rate of osteoclast mediated bone resorption. The investigators aim to exploit their knowledge of Src structure and function to develop orally active small molecule drugs that block Src function in osteoclasts. Src becomes activated in response to a variety of extracellular signals. The protein is composed of multiple structural domains, and among these, the SH2 and SH3 domains are critical for Src function. These non-catalytic domains promote protein-protein interactions between Src and key cellular proteins Our aims are to 1) identify peptide ligands for Src SH2 and SH3 domains that bind with high affinity and specificity; 2) evaluate the peptides m vitro and in cultured cells for their ability to inhibit known Src functions, and 3) evaluate the peptides in cultured osteoclasts for their effect on Src-mediated signal transduction and resorption, and 4) determine the structures of domain-peptide complexes. These studies test the concept that a Src inhibitor will block osteoclast function and provide the foundation for structure-based drug development.Awardee's statement of the potential commercial applieations of the research:The protein tyrosine kinase, Src, is required for osteoclast mediated bone degradation. The inhibitors of Src that will be developed from the high affinity Src-llgands identified in this proposal could be used for the treatment of osteoporosis, a disease involving loss of bone mass.National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)