Detecting drug-target binding in cells using fluorescence-activated cell sorting coupled with mass spectrometry...

Area of Science:

• Biochemistry
• Pharmacology
• Drug Discovery

Background:

• Assessing drug-target engagement within cells is crucial but challenging for difficult protein targets.
• Current methods are often limited to soluble proteins and struggle with targets exhibiting poor in vitro properties.
• Difficult targets include those with low solubility, stability, or purification challenges.

Purpose of the Study:

• To introduce a novel technique, the Toxicity-Affinity-Permeability-Selectivity (TAPS) assay, for measuring intracellular drug-target interactions.
• To evaluate the TAPS assay's capability in assessing compound permeability, specificity, and cytotoxicity.
• To demonstrate the TAPS assay's utility with a challenging intracellular membrane protein, human kynurenine 3-monooxygenase (KMO).

Main Methods:

• Development and application of the TAPS assay for intracellular drug-target engagement.
• Utilizing the TAPS assay to analyze the human kynurenine 3-monooxygenase (KMO) target.
• Assessing compound permeability, specificity, and cytotoxicity alongside target binding.

Main Results:

• The TAPS assay successfully measured intracellular compound-target complex formation.
• The assay demonstrated its ability to assess cellular permeability, specificity, and cytotoxicity.
• TAPS confirmed the binding of known KMO inhibitors to the target protein within cells.

Conclusions:

• The TAPS assay provides a robust method for assessing drug-target engagement for challenging intracellular proteins.
• This technique facilitates intracellular hit validation across a broad range of drug targets.
• TAPS enhances the efficiency and reliability of early-stage drug discovery.