Protocol for quantifying cellular drug-target engagement across a temperature series using the MICRO-TAG enzyme...

Area of Science:

• Biochemistry
• Molecular Biology
• Pharmacology

Background:

• Quantifying cellular target engagement is crucial for drug discovery and development.
• Existing methods may have limitations in dynamic range or throughput.
• Developing robust assays for target engagement under various conditions is essential.

Purpose of the Study:

• To present a novel protocol for quantifying cellular target engagement.
• To enable target engagement assessment across a programmable temperature series.
• To validate the protocol using a split-RNase S complementation assay.

Main Methods:

• Expressing target proteins as S-tag fusions in mammalian cells.
• Applying thermal challenge and compound treatment to cells.
• Measuring RNase-mediated cleavage of an RNA fluorescence resonance energy transfer (FRET) substrate using real-time quantitative PCR.
• Analyzing fluorescence signals and performing four-parameter logistic fitting to derive EC50 values.

Main Results:

• The protocol successfully quantifies cellular target engagement.
• The assay is adaptable to a range of temperatures and compound concentrations.
• EC50 values can be reliably derived from the fluorescence data.

Conclusions:

• The presented protocol offers a robust method for assessing cellular target engagement.
• This assay is valuable for early-stage drug discovery and compound screening.
• The method provides quantitative insights into drug-target interactions under thermal stress.