Kinetic Drug Screening Based On Small Molecule Microarray

The receptor theory of drug action posits that the drug exhibitsfunctions only when it interacts with its target protein, which can be quantified by the binding kinetic analysis. Small molecule microarray is miniaturized assembles of molecules organized on a planar surface, which enlarges the speed of drug discovery. This thesis contributes to a small molecule microarray and its kinetic screening analysis with protein targets by developments of surface chemistry and chip structure.A universal label-free method for small molecules immobilization was carried on a three-dimensional(3D) surface using a carbene-based photocrosslinking reaction by atom transfer radical polymerization(ATRP) polymer assembled on gold surface, which wasoptimized from 2D self-assembled monolayer and 3D surface initiated polymerization surface chemistry. The surface realizedsimultaneous display of a large number of photoaffinity sites coupled on a single PEG polymer chain which significantly increased the probability of the attachment of the label to a random position of a small molecule. Based on the kinetic screening of targeting proteins, the method provided an opportunity to calculate the association and disassociation kinetic constants from the real-time binding between thousands of small molecules and targeting proteins. A competition analysis method was designed to verify the binding site of targeting protein among the inhibitors. The chip was aimed at a powerful tool to explore the kinetic information of the interactions for the drug discovery, repositioning, function evaluation and personal medicine.To achieve further sensitivity and throughput improvement for kinetic analysis, Tri-layered chip and silver chip were developed, which addressed the inherent instability of silver by a gold buffer layer. A comparable stabilitywith gold chip wasverified. Meanwhile, the chip substantiated 53% and 101% sensitivity improvement respectively, which fully exhibited the intrinsic characteristic of silver. A protein microarray with multipleanalytesproved the applicability of the new high-throughput kinetic screening platforms.