A real-time temperature series MICRO-TAG cell target engagement assay using Applied Biosystems QuantStudio system

Area of Science:

• Biochemistry
• Molecular Biology
• Pharmacology

Background:

• Direct measurement of drug-target engagement is crucial for early drug discovery.
• Existing methods often require extensive optimization, prolonged incubations, or lack real-time compatibility.
• There is a need for streamlined, quantitative assays for cellular target engagement.

Purpose of the Study:

• To develop and validate a real-time temperature-series assay for cellular drug-target engagement.
• To integrate fluorescence-based detection with programmable thermal cycling for enhanced efficiency.
• To demonstrate the assay's utility for rapid binding assessment and EC50 determination.

Main Methods:

• Development of the temperature-series MICRO-TAG assay using an RNase-based FRET substrate.
• Integration with the Applied Biosystems QuantStudio™ Real-Time PCR System for programmable thermal cycling.
• Utilizing a ramp-hold-detect temperature program for streamlined workflow and signal acquisition.

Main Results:

• Demonstrated reproducible real-time fluorescence signal separation between ligand-treated and control samples.
• Showcased the ability to perform single-concentration binding assessment within a single run.
• Successfully determined EC50 values in a single experimental run using MAPK1 as a target.

Conclusions:

• The real-time temperature-series MICRO-TAG assay offers a streamlined and quantitative approach to cellular target engagement.
• This method eliminates the need for melting-temperature scouting, accelerating early drug discovery.
• The assay enables efficient assessment of drug-target interactions in intact cells with real-time data acquisition.