Selective Identification of Allosteric Inhibitors and Co-Drug Combinations Targeting Kinases by Using a Nanopore...

Area of Science:

• Biophysics
• Pharmacology
• Biotechnology

Background:

• Allosteric inhibitors offer potential for selective drugs and overcoming resistance in kinase therapies.
• Current screening methods for allosteric inhibitors are limited by low throughput and dependence on known binding sites.

Purpose of the Study:

• To develop a label-free, high-throughput screening method for identifying allosteric kinase inhibitors.
• To demonstrate the utility of nanopore tweezers for detecting allosteric binders to Abl kinase.

Main Methods:

• A cytolysin A (ClyA) nanopore was used to trap Abl kinase with a blocked active site.
• Ligand-induced ionic current fluctuations were monitored to detect co-binding of ATP-competitive and allosteric inhibitors at the single-molecule level.
• This method does not require prior structural information of allosteric binding sites.

Main Results:

• The nanopore tweezer approach successfully detected allosteric binders to Abl kinase.
• Real-time, single-molecule observation of inhibitor co-binding was achieved.
• The method demonstrated adaptability for high-throughput screening across various kinases.

Conclusions:

• Nanopore tweezers provide a structure-based platform for identifying allosteric modulators and co-drug combinations.
• This approach can accelerate the discovery of highly selective therapeutic agents.
• The method is adaptable for screening other enzyme targets beyond kinases.