Dansyl-conjugated beta-lactam antibiotic as a fluorescent drug-based sensor for beta-lactamase detection and in vitro drug screening

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Dansyl-conjugated beta-lactam antibiotic as a fluorescent drug-based sensor for beta-lactamase detection and in vitro drug screening

 

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
Degree: M.Phil.
Year: 2015
Subject: Beta lactam antibiotics
Beta lactamases.
Hong Kong Polytechnic University -- Dissertations
Department: Dept. of Applied Biology and Chemical Technology
Pages: vii, 107 pages : illustrations
Language: English
InnoPac Record: http://library.polyu.edu.hk/record=b2813594
URI: http://theses.lib.polyu.edu.hk/handle/200/8023
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.

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