Moderate to high throughput in vitro binding kinetics for drug discovery

Area of Science:

• Pharmacology
• Biochemistry
• Analytical Chemistry

Background:

• Drug-target binding kinetics are crucial for understanding drug efficacy and safety.
• Traditional methods for kinetic analysis can be low-throughput and time-consuming.
• Advancements in technology are enabling faster and more comprehensive kinetic profiling.

Purpose of the Study:

• To provide a concise summary of state-of-the-art, moderate to high-throughput in vitro technologies for drug-target binding kinetics.
• To cover a wide kinetic timescale from milliseconds to days.
• To highlight best practices and applications in drug discovery.

Main Methods:

• Automated stopped-flow for millisecond to second kinetics.
• Surface plasmon resonance (SPR) biosensors for second to hour kinetics.
• Filtration plate-based assays and jump dilution for slower kinetics (hours to days).
• Liquid chromatography coupled tandem mass spectrometry (LC/MS/MS) as a label-free technology.

Main Results:

• Technologies reviewed cover kinetic timescales spanning nine orders of magnitude.
• Methods discussed include automated stopped flow, SPR, global progress curve analysis, radioligand/fluorescent binding assays, and LC/MS/MS.
• Best practices and simulated data are provided for each technology.

Conclusions:

• High-throughput in vitro technologies are essential for routine kinetic analysis in drug discovery.
• Binding kinetics are integral to mechanism of action elucidation and quantitative pharmacology.
• These advanced methods facilitate a new era in translational drug development.