of Research Plan: This project will determine the high resolution three dimensional structure (3DS) of OP-1 to atomic resolution and use this structure to design small molecules or peptidomimetic agents for treating osteoporosis and other metabolic bone disease. The synergistic relationship between the most advanced techniques of 3DS analysis and biotechnology will be utilized to work toward effective drug development. Large quantities of recombinant OP-1 expressed in Chinese Hamster Ovary cells have been purified, renatured and examined by gel electropohoresis and HPLC to ensure homogeneity. Biologically active OP-1 has been used to grow crystals for single crystal x-ray diffraction studies and the 3DS solved to 2.8 A resolution. Parts of this structure remain poorly defined and the accuracy of the present model limits our ability to use sophisticated structure guided drug design techniques. Nonetheless, some inferences about the 3DS and potential receptor binding sites have been made, and some potential peptides of interest have been identified. In the proposed project, the high resolution structure studies would be pursued while these initial peptides are synthesized and tested for receptor binding and biological activity in OP-1 assays. In phase I, the OP-1 crystals will be used to determine the 3DS of OP-1 to atomic resolution at reduced temperatures. The atomic model of OP-1 determined the at 2.8 A will be used to extend the 2.9 A x-ray structure to a resolution of 2.0A. Low temperature x-ray data will be collected to define the portions of the structure that were poorly defined in the 2.8A structure and to complete the refinement at 2.0A. Analysis of the x-ray structure with computational programs and detailed comparison with other growth factors will be used to select and synthesize potential active peptides. These peptides will be tested for receptor binding. Those peptides that show significant receptor binding will then be tested in bioassays for agonist and antagonist activity. The information gained from the peptides will be correlated with the 3DS to design lead compounds for the development of potential non-protein based therapeutic agents.
