|標題:||[學研計畫] 分子生物電子平台系統整合與應用( I )|
Molecular Bio-Electronic Platform for High Throughput Screening of Drug Discovery( I )
HUANG GUEWHA STEVEN
|關鍵字:||分子生物電子;藥物開發;高通量篩選;單分子偵測;酵素動力學;Molecular bioelectronics;drug discovery;high throughput screening;single molecule detection;enzyme kinetics|
High throughput screening (HTS) is an integral part of drug discovery industry. Here we propose a pro-T-based enzyme assay (pro-Ten) platform that enables HTS with single molecule sensitivity and versatility thus will facilitate the process and enhance the sensitivity of drug discovery. Enzymes are the most attractive targets in human diseases. Inhibitory molecules may serve as the starting points for drug discovery. The leads obtained from HTS represent the starting points for medicinal chemistry. Assay development and HTS often determine the successful flow of drug development. An ultra-sensitive and universal assay independent of substrate concentration and target properties would be beneficial in these steps. A single-molecule and real-time enzyme activity measuring platform based on single molecule protein transistor is established in our lab. An antibody-based protein transistor (pro-T) is fabricated through the specific recognition between antibody and gold nanoparticles with gold nanoparticles attaching to electrodes. The structure-sensing ability of pro-T provides an opportunity to monitor enzymatic action at single molecular level. The pro-T can be transformed to an enzyme transistor by binding to an enzyme-conjugated secondary antibody. Binding of beta-galactosidase-conjugated secondary antibody enables pro-T to sense the enzymatic hydrolysis of lactose (pro-Tgal). Pto-Tgal is able to monitor the enzymatic hydrolysis of -gal includes substrate binding, structural change of enzyme, hydrolysis and release of the end sugar, release of galactose, and recover of native enzyme structure. Pro-Tgal instantaneously distinguishes non-substrate, substrate, and inhibitor by the shape and intensity of the conductivity. Incorporation of other enzymes, such as glucose oxidase, horse relish peroxidase, pepsin, also exhibited single molecule sensitivity and specificity. We propose to establish an HTS platform to accelerate the drug discovery efficiency. The specific aims of this proposal are: 1. To develop an HTS platform to facilitate drug discovery process. We will combine the single molecule detection of our system (detecting substrate binding and structural change of protein) with single molecule fluorescence detection (detection product conversion and distance between chromophores). Microfluidic and multichannel sampling system will be amended. 2. To investigate single molecule enzyme reaction. The advantage of pro-Ten is the nano-second timescale to observe enzymatic reaction in real time. The single molecule FRET system detects “snapshot” of reaction, thus is also called “single molecule ensemble experiment”. This will be the first opportunity to verify most enzymatic hypotheses based on Mechalis-Menten kinetics. Additionally, kinetics of protein folding will be investigated. 3. To explore possible applications in bioelectronics. Incorporating into HTS system is not the only option for application of pro-T. Single cell photovoltics and integrated bio-circuitry will be investigated. Additionally, sensors in non-conventional environment, such as air or vacuum, will be investigated. Pro-T is most suitable to apply for biological detection. X-ray crystallography will be performed to monoclonal antibody and gold nanoparticle conjugate. This is to explore the possibility of extracting extra information for the bio-nano interface from structural data. We expect to generate high quality patents attractive to pharmaceutical society. High impact publications in Single molecule enzyme kinetics, protein folding, structural biology will be generated.
|Appears in Collections:||Research Plans|