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Updated: Jul 12, 2026
06:17
Covalent Fragment Screening Using the Quantitative Irreversible Tethering Assay
Published on: February 28, 2025
A FRET-Based Assay for Assessing Covalent Warhead Reactivity
Anna P Valaka1, Carlos Benitez-Martin1, Joakim Andréasson2
1Department of Chemistry and Molecular Biology, University of Gothenburg, 405 30 Gothenburg, Sweden.
ACS Omega
|July 10, 2026
Summary
This study introduces a novel fluorescent sensor to measure the reactivity of covalent drug warheads with biological molecules. The platform enables high-throughput screening, advancing drug discovery and chemical biology research.
Area of Science:
- Chemical Biology
- Drug Discovery
- Biochemistry
Background:
- Covalent modalities are crucial in drug discovery but evaluating electrophilic warhead reactivity is challenging.
- Förster resonance energy transfer (FRET)-based sensors are common in chemical biology but not used for warhead reactivity.
- Assessing covalent warhead reactivity requires reliable and high-throughput methods.
Purpose of the Study:
- To develop and validate a FRET-based fluorescent platform for assessing covalent warhead reactivity.
- To quantify the reactivity of a sulfone-based nucleophilic aromatic substitution (SNAr) warhead toward biological nucleophiles.
- To establish a high-throughput assay for screening nucleophiles and reaction conditions.
Main Methods:
- Designed a FRET dyad incorporating a coumarin donor, dinitrophenyl quencher, and SNAr warhead.
- Utilized the SNAr reaction to disrupt FRET, enabling fluorescence readout.
- Performed assays in a 96-well plate format for high-throughput screening.
Main Results:
- The FRET-based sensor effectively quantifies covalent warhead reactivity.
- Demonstrated strong selectivity for thiols as nucleophiles.
- The 96-well plate format facilitates high-throughput screening of nucleophiles and conditions.
Conclusions:
- Developed a versatile and robust FRET-based fluorescent platform for evaluating covalent warhead reactivity.
- This approach facilitates mechanistic studies in chemical biology and accelerates drug discovery.
- The sensor shows promise for assessing drug-target interactions involving covalent modifications.
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