Molecular Targeted Engagement of DPP9 in Rat Tissue Using CETSA, SP3 Processing, and Absolute Quantitation Mass...

Area of Science:

• Biochemistry
• Pharmacology
• Analytical Chemistry

Background:

• Cellular thermal shift assay (CETSA) is crucial for assessing small molecule ligand binding to protein targets.
• Improving quantitation and specificity in CETSA is vital for accurate target engagement studies.
• Mass spectrometry offers enhanced analytical capabilities for precise quantification in biological assays.

Purpose of the Study:

• To evaluate target engagement of dipeptidyl peptidase 9 (DPP9) in rat tissue using CETSA.
• To implement advanced analytical methods for absolute quantitation of DPP9 engagement.
• To demonstrate the utility of CETSA coupled with mass spectrometry for in vivo drug discovery.

Main Methods:

• Cellular thermal shift assay (CETSA) was performed on rat tissue samples.
• Single-pot, solid-phase-enhanced sample preparation (SP3) was utilized for sample processing.
• High-resolution mass spectrometry was employed for absolute quantitation of DPP9.

Main Results:

• CETSA demonstrated a temperature-dependent melting curve for DPP9 upon ex vivo compound incubation.
• In vivo studies showed dose-dependent engagement of DPP9 in response to treatment.
• Absolute quantitation by mass spectrometry enabled precise measurement of DPP9 engagement in dosed animals.

Conclusions:

• The study successfully extended CETSA to in vivo dosed-animal samples.
• Absolute quantitation of DPP9 by mass spectrometry provides a robust method for target engagement assessment.
• This integrated approach offers a viable strategy for interrogating therapeutic molecules in drug discovery.