Wednesday , 2 December 2020

Fluorescence resonance energy transfer (FRET) based high throughput drug screening for the determination of antineoplastic activity of natural compounds from herbs

Sharmistha Gupta1 and Mithun Mukherjee2
1West Bengal State Council of Science and Technology, West Bengal-India 
2Ramakrishna Vivekananda Mission Institute of Advanced Studies, West Bengal-India

ABSTRACT
Objectives:  In the present experiment a very useful cell-based high throughput screening method that uses a novel bio-sensor which specifically identifies apoptosis based on fluorescence resonance energy transfer technology.
Methodology: A stable HeLa cell line expressing a FRET-based bio-sensor protein was produced. Cells undergoing apoptosis have an activated enzyme ‘caspase-3’ which is a protease this activated enzyme breaks sensor protein and causes the fluorescence emission spectra to shift from 590 nm to 512 nm changing color from yellowish green to blue. A reduction in the green/blue emission ratio indicated apoptosis. After growing the sensor cells in 96-well plates different chemical compounds are added to the wells wherein the fluorescent profile was measured using fluorescent plate reader.
Results: It is a novel cell based HTS method and was proved to be highly effective and sensitive process for detecting anti-neoplastic compounds which induce apoptosis. In this experiment we screened compounds obtained from two plants used in Indian medicines.
Keywords: Drug discovery, HTS, FRET, fluorescent protein, apoptosis, caspase, anticancer drugs; herbal medicine, tannins, quercetins.

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