Please use this identifier to cite or link to this item: https://hdl.handle.net/10356/81171
Title: A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening
Authors: Yu, Yong
New, Siu Yee
Lin, Jiaxian
Su, Xiaodi
Tan, Yen Nee
Keywords: Fluorescence
Binding constant
Stability
Issue 104
Gold nanoclusters
Synthesis
Human serum albumin
Drug screening
Protein unfolding
Bioengineering
Issue Date: 2015
Source: Yu, Y., New, S. Y., Lin, J., Su, X., & Tan, Y. N. (2015). A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening. Journal of Visualized Experiments, 104, e53261-.
Series/Report no.: Journal of Visualized Experiments
Abstract: We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold nanoclusters (Au NCs) within the drug-loaded protein, based on the differential fluorescence signal emitted by the Au NCs. Albumin proteins such as human serum albumin (HSA) and bovine serum albumin (BSA) are selected as the model proteins. Four small molecular drugs (e.g., ibuprofen, warfarin, phenytoin, and sulfanilamide) of different binding affinities to the albumin proteins are tested. It was found that the formation rate of fluorescent Au NCs inside the drug loaded albumin protein under denaturing conditions (i.e., 60 °C or in the presence of urea) is slower than that formed in the pristine protein (without drugs). Moreover, the fluorescent intensity of the as-formed NCs is found to be inversely correlated to the binding affinities of these drugs to the albumin proteins. Particularly, the higher the drug-protein binding affinity, the slower the rate of Au NCs formation, and thus a lower fluorescence intensity of the resultant Au NCs is observed. The fluorescence intensity of the resultant Au NCs therefore provides a simple measure of the relative binding strength of different drugs tested. This method is also extendable to measure the specific drug-protein binding constant (KD) by simply varying the drug content preloaded in the protein at a fixed protein concentration The measured results match well with the values obtained using other prestige but more complicated methods.
URI: https://hdl.handle.net/10356/81171
http://hdl.handle.net/10220/39185
ISSN: 1940-087X
DOI: 10.3791/53261
Rights: © 2015 Journal of Visualized Experiments. This paper was published in Journal of Visualized Experiments and is made available as an electronic reprint (preprint) with permission of Journal of Visualized Experiments. The published version is available at: [http://dx.doi.org/10.3791/53261]. One print or electronic copy may be made for personal use only. Systematic or multiple reproduction, distribution to multiple locations via electronic or other means, duplication of any material in this paper for a fee or for commercial purposes, or modification of the content of the paper is prohibited and is subject to penalties under law.
Fulltext Permission: open
Fulltext Availability: With Fulltext
Appears in Collections:SCBE Journal Articles

SCOPUSTM   
Citations 20

1
checked on Sep 5, 2020

WEB OF SCIENCETM
Citations 50

1
checked on Oct 24, 2020

Page view(s) 50

130
checked on Oct 26, 2020

Download(s) 50

91
checked on Oct 26, 2020

Google ScholarTM

Check

Altmetric


Plumx

Items in DR-NTU are protected by copyright, with all rights reserved, unless otherwise indicated.