PDGF-AA-DIMER STRUCTURE AND ATHEROSCLEROSIS DRUG DESIGN

THIS PROJECT WILL DEVELOP THE BASIS FOR STRUCTURE ANALYSIS OF PDGF-AA IN PHASE I, LEADING TO ITS 3-D STRUCTURE DETERMINATION IN PHASE II. THE ULTIMATE GOAL IS TO USE THE 3-D STRUCTURE OF PDGF TO DEVELOP DRUGS THAT INHIBIT THE ACTIONS OF PDGF-AA IN ATHEROSCLEROSIS. THIS APPROACH WILL TAKE ADVANTAGE OF THE MOST CONTEMPORARY TECHNIQUES IN BIOTECHNOLOGY AND STRUCTURE ANALYSIS. MILLIGRAM QUANTITIES OF PDGF-AA-HOMODIMER WILL BE PRODUCED BY RECOMBINANT DNA METHODOLOGY; THE CLONED PROTEIN WILL BE EXPRESSED IN ESCHERICHIA COLI, FOLLOWED BY ITS RENATURATION AND FINAL PURIFICATION. BIOLOGICALLY ACTIVE PDGF-AA WILL THEN BE USED TO GROW HOMODIMER CRYSTALS SUITABLE FOR SINGLE-CRYSTAL X-RAY DIFFRACTION STUDIES. NUCLEAR MAGNETIC RESONANCE (NMR) SPECTROSCOPY WILL BE PURSUED CONCURRENTLY ON THE PROTEIN IN SOLUTION IN ORDER TO ESTABLISH THE BASIS FOR 2-D NMR ANALYSIS OF ITS STRUCTURE. CRYSTALS SUITABLE FOR X-RAY DIFFRACTION STUDIES TO HIGH-RESOLUTION AND/OR NMR SPECTRAL DATA SUITABLE FOR PLANNING A DETAILED 3-D STRUCTURE ANALYSIS WILL FORM THE BASIS OF A PHASE II STRATEGY. THE FEASIBILITY OF USING COMPUTATIONAL ALGORITHMS TO EXPEDITE MODELING WILL ALSO BE ASSESSED. A DETAILED 3-D STRUCTURE OF PDGF-AA-HOMODIMER WILL BE DETERMINED IN THE PHASE II PROGRAM, LEADING TO A MOLECULAR BASIS FOR RATIONAL DESIGN OF DRUGS THAT BLOCK PDGF ACTION IN THE DEVELOPMENT OF ATHEROSCLEROTIC PLAQUE.