NMR Studies On The Low-Affinity Binding Of Drugs To Protein

Nuclear magnetic resonance (NMR) spectroscopy is a powerful approach in investigating the low-affinity binding of drugs to protein. Several NMR parameters have been used to achieve the binding constants, screening active drugs, and determining the orientation of ligand on the binding site. In this dissertation, we proposed a chemical model CLAB for studying the competitive low-affinity binding of two drugs to protein, and a new epitope mapping method for characterizing the orientation of ligand on the binding site. We also studied the structure and interaction between matrine and human serum albumin.Firstly, a competitive low-affinity binding model, CLAB, was proposed for determining the number of mutual (overlapped) and specific binding sites of two ligands (A, B) on a protein (P). A titration experiment can be carried out by adding either A or B into the three-component system (A-B-P). Then the mutual and specific binding sites of the two ligands on the protein can be obtained by fitting the titration curve to the proposed model (CLAB). The model was examined by using human serum albumin (HSA) as a receptor and tolmetin (TOL) and salicylic acid (SAL) as ligands. Proton longitudinal relaxation rates (R₁) were measured on a 500-MHz NMR spectrometer during the titration and used to derive the mutual binding sites. It was found that among the binding sites of 32±4 for SAL and 28±2 for TOL on HSA, there were 17±5 mutual sites for the two ligands. This result indicates that, although HSA has large binding capacities to most ligands, there are still a reasonable amount of the low-affinity binding sites that are structural selective.Secondly, the transferred nuclear Overhauser effect (trNOE) during diffusion NMR experiment was used and a new epitope mapping method was proposed. During diffusion...