Pim Kinase Inhibitors Evaluated with a Single-Molecule Engineered Nanopore Sensor

Area of Science:

• Biochemistry
• Nanotechnology
• Pharmacology

Background:

• Protein kinases, particularly Pim kinases, are crucial targets in leukemia and cancer therapy.
• Existing assays for kinase inhibitors often suffer from bias and false-positives.
• Developing robust and sensitive methods for evaluating kinase inhibitors is essential.

Purpose of the Study:

• To develop a novel protein nanopore sensor for detecting Pim kinase activity.
• To establish a label-free method for determining the inhibition constants of Pim kinase inhibitors.
• To demonstrate the superiority of this method over existing techniques like differential scanning fluorimetry.

Main Methods:

• Engineering a protein nanopore sensor with a pseudosubstrate peptide specific for Pim kinases.
• Measuring analyte binding via modulation of ionic current through a single nanopore.
• Observing synergistic binding of MgATP and kinase to the sensor.

Main Results:

• Demonstrated a novel method for label-free determination of Pim kinase inhibitor constants.
• Identified a potent inhibitor missed by differential scanning fluorimetry, highlighting assay advantages.
• The developed sensor showed synergistic binding of MgATP and kinase, enhancing detection sensitivity.
• The approach is adaptable for high-throughput screening applications.

Conclusions:

• The protein nanopore sensor provides a superior, bias-free method for evaluating Pim kinase inhibitors.
• This technique offers a sensitive and reliable platform for drug discovery in oncology.
• The method's amenability to high-throughput chips facilitates accelerated identification of novel cancer therapeutics.