Automation of Full-Kinome Profiling Using a Continuous Kinase Activity Platform

Abstract

Automated high throughput assessment of the kinase activity of lead compounds for optimization and selectivity assessment is essential for developing successful drug candidates. The typical high-throughput screen involves a primary assay of a huge number of compounds against a single target to determine lead hits that are then characterized further in selectivity and potency assays to validate and prioritize these hits as part of a drug discovery cascade. The primary assay and even the follow-up assays are typically run as an endpoint format to reduce the complexity. Determining the full-kinome selectivity profile for kinase inhibitors is a common need, but this can be very challenging since this requires the screening of lead compounds against hundreds of kinases run at different concentrations, with varying stability and that require different substrates or additives, as part of specific assay conditions including measuring inhibition at the ATP Km concentration for each kinase in the panel.

We will present the development of a semi-automated assay and then to fully automated assay workflow to address the complexity encountered with selectivity profiling.  Using the PhosphoSens® continuous assay format for profiling provides a more robust and informative readout of the target biology (detection of any enzyme lag or instability) and inhibitor function (determination of initial and final reaction rates provides a more accurate assessment of inhibition and allows detection of time-dependent inhibition).  This involves the automation of three types of samples: enzymes, substrates, compounds, and their individual properties and processing needs. It also involves new process development and the automation of unique liquid handling. New techniques for stability and solubility of the samples and reagents were paramount to achieve consistent and highly reproducible results. We will present data on full-kinase profiles of known specific and non-specific inhibitors validating the continuous assay format as well as the fully automated process.

Key Takeaways

• The information-rich Full-Kinome Profiling assay was successfully converted to a semi-automated system including improved sample management and assay analysis. The semi-automated system and new assay was validated by using a promiscuous standard, Staurosporine, and comparing multiple manual runs to multiple semi-automated runs.
  
  
• The manual assay was modified to allow for better and more efficient processing and automation.
  
  
• The manual throughout was increased from 14 to 84 compounds/month by using semi-automation and will be increased further by using full automation.
  
  
• Redundancy in automation, efficiency in processing, and adapting new technologies will improve the chances of meeting projected AssayQuant Technologies growth.
  
  
• Most importantly, having the team’s dedication and support at all levels helped achieve our automation goals.