Quantification of In Vivo Target Engagement Using Microfluidic Activity-Based Protein Profiling

Area of Science:

• Biochemistry
• Pharmacology
• Analytical Chemistry

Background:

• Accurate measurement of in vivo drug-target interactions is crucial for drug development.
• Current assays often rely on indirect biomarkers, limiting direct assessment of target engagement.
• Activity-based protein profiling (ABPP) offers a direct method to quantify enzyme activity using covalent probes.

Purpose of the Study:

• To adapt and validate microfluidic electrophoresis for ABPP analysis of probe-labeled proteins.
• To compare the efficiency, quantification, and sample requirements of microfluidic ABPP versus conventional gel electrophoresis.
• To enable earlier integration of ABPP into drug discovery workflows for assessing target engagement.

Main Methods:

• Adapted protocols for a commercial LabChip GXII microfluidic instrument for ABPP.
• Performed electrophoretic separation of probe-labeled proteins in tissue lysates and plasma on microfluidic chips.
• Quantified fluorescence labeling and compared results with conventional polyacrylamide gel electrophoresis.

Main Results:

• Microfluidic chip electrophoresis provided rapid (40 s/sample) separation and automatic quantification of labeled proteins.
• Overall time savings of 3-5 hours per 96-well plate were achieved compared to conventional gels.
• Microfluidic ABPP yielded comparable percent inhibition values to gels with significantly lower sample volume requirements.
• Consistent performance was observed across chips and sample replicates.

Conclusions:

• Microfluidic electrophoresis offers a time-efficient and miniaturized approach for ABPP.
• This method facilitates early-stage drug discovery by enabling routine assessment of in vivo target engagement.
• The reduced sample consumption is advantageous for analyzing scarce biological samples.