|Author:||Yip, Chun Yin Karl|
|Title:||Dansyl-conjugated beta-lactam antibiotic as a fluorescent drug-based sensor for beta-lactamase detection and in vitro drug screening|
|Subject:||Beta lactam antibiotics|
Hong Kong Polytechnic University -- Dissertations
|Department:||Department of Applied Biology and Chemical Technology|
|Pages:||vii, 107 pages : illustrations|
|Abstract:||Beta-lactam antibiotics have been widely used as antibacterial agents in the clinical treatment of bacterial infections. These drugs can irreversibly bind to the active site of penicillin-binding proteins in bacteria, thus inhibiting these proteins from synthesizing cell walls and leading to cell death. The overuse of beta-lactam antibiotics has, however, led to the increasing emergence of various beta-lactamases, which are enzymes produced by bacterial to inactivate beta-lactam antibiotics. These enzymes can efficiently catalyze the hydrolysis of the beta-lactam ring, thus rendering the antibiotic clinically inactive. The TEM family is a large group of beta-lactamase which has more than 100 variants derived from the ancestor TEM-1 through one or more amino acid mutation(s). Many TEM-type beta-lactamases, including TEM-1, are clinically relevant, and therefore the detection of such enzymes and the development of new beta-lactam antibiotics/inhibitors against TEM-type beta-lactamases are important in combating antibiotic-resistant bacteria capable of producing TEM-type beta-lactamases. Nitrocefin, which is a colorimetric beta-lactam antibiotic, has been routinely used as a probe for detecting beta-lactamase activity. This colorimetric antibiotic is, however, very expensive and unstable in aqueous solution. As such, it is highly desirable to develop a convenient tool that can provide both beta-lactamase sensing and in vitro drug screening functions.|
In this project, we have successfully developed a versatile dansyl-conjugated beta-lactam antibiotic as a 'turn-on' fluorescent sensor which can detect the activity of the TEM-1 beta-lactamase and perform in vitro drug screening. Mass spectrometric studies have shown that this fluorescent antibiotic can bind to the active site of TEM-1 through the formation of covalent enzyme-substrate complex. Upon binding to TEM-1, the fluorescent antibiotic exhibits a characteristic blue shift in emission wavelength (555 to 513 nm) and stronger fluorescence, presumably due to experiencing a hydrophobic environment upon binding to the active site. Time-course fluorescence measurements indicated that this 'turn-on' fluorescent antibiotic can detect TEM-1 at sub-nanomolar level (0.1 nM). Our fluorescence studies on the fluorescent antibiotic with different proteins have shown that this sensor can specifically recognize TEM-1. The ability of the fluorescent antibiotic to perform in vitro drug screening was also studied by time-course fluorescence measurements. In the presence of inhibitors that can inactivate TEM-1, the binding of the fluorescent antibiotic to TEM-1 is largely suppressed, thus making the fluorescent antibiotic to fluoresce weakly at a longer wavelength. In contrast, in the presence of drug candidates that are unable to bind and hence inactivate TEM-1, the binding of the fluorescent antibiotic to TEM-1 becomes favourable and therefore makes the fluorescent antibiotic to fluoresce stronger at a shorter wavelength. These characteristic fluorescence profiles highlight the useful in vitro drug screening function of the fluorescent antibiotic. The characteristic fluorescence responses of the dansyl-conjugated beta-lactam antibiotic to beta-lactamase binding and other drug candidates in drug screening highlight its versatile functions in beta-lactamase detection and in vitro drug screening.
Files in This Item:
|b28135945.pdf||For PolyU Staff & Students||3.98 MB||Adobe PDF||View/Open|
|27583.pdf||For All Users (Non-printable)||4.01 MB||Adobe PDF||View/Open|
As a bona fide Library user, I declare that:
- I will abide by the rules and legal ordinances governing copyright regarding the use of the Database.
- I will use the Database for the purpose of my research or private study only and not for circulation or further reproduction or any other purpose.
- I agree to indemnify and hold the University harmless from and against any loss, damage, cost, liability or expenses arising from copyright infringement or unauthorized usage.
By downloading any item(s) listed above, you acknowledge that you have read and understood the copyright undertaking as stated above, and agree to be bound by all of its terms.
Please use this identifier to cite or link to this item: