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Related Experiment Video

Updated: May 21, 2026

A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

Published on: March 10, 2017

Fully functionalized small-molecule probes for integrated phenotypic screening and target identification.

Justin S Cisar¹, Benjamin F Cravatt

¹The Skaggs Institute for Chemical Biology and Department of Chemical Physiology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.

Journal of the American Chemical Society

|June 7, 2012

Summary

This study developed a functionalized small-molecule library for identifying drug targets. A novel compound selectively inhibited cancer cell proliferation by targeting NADH:ubiquinone oxidoreductase (complex 1).

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Area of Science:

Chemical Biology
Drug Discovery
Proteomics

Background:

Phenotypic screening identifies bioactive small molecules but target identification is challenging due to off-target effects.
Characterizing small molecule interactions in living systems remains difficult, hindering mechanistic understanding of pharmacological activity.

Purpose of the Study:

To develop a novel, fully functionalized small-molecule library for accelerated target identification.
To create a tool for visualizing and identifying protein targets of bioactive compounds in situ.

Main Methods:

A small-molecule library was designed with diversity elements, a photoreactive group for cross-linking, and an alkyne handle for reporter conjugation.
Quantitative chemoproteomics and in vitro assays were employed to identify and validate the target of an active compound.

Main Results:

A library member selectively inhibited cancer cell proliferation under low-glucose conditions.
Quantitative chemoproteomics identified NADH:ubiquinone oxidoreductase (complex 1) as the specific target.
The compound inhibited complex 1 activity in vitro, consistent with observed cell death under nutrient limitation.

Conclusions:

Fully functionalized compound libraries coupled with chemoproteomics enable rapid target identification and mechanistic characterization.
This approach accelerates the discovery of bioactive probes for complex biological processes.