KINAXO Cellular Target Profiling®

The KINAXO Cellular Target Profiling® service provides powerful insights into small molecule target interactions and binding affinities across the proteome of any given cell line or tissue sample. The compound’s protein targets are determined in a physiological context. Subsequent ranking from high to low affinity distinguishes the most sensitive cellular targets from weak interactions.

This information supports decision making significantly at many stages of the drug development process, e.g. lead compound selection, target deconvolution, drug re-profiling, as well as helping assess off-target toxicity. KINAXO's chemical proteomics technology was recently featured in EuroBiotechNews (pdf).

When a compound’s native protein target profile is known, development programs can be much more confident about in vivo selectivity and mode of action. Now, KINAXO makes it easy to take advantage of this with its patented KINAXO Cellular Target Profiling® technology.   

The KINAXO Cellular Target Profiling® service combines state-of-the-art chemical proteomics methods with the latest quantitative mass spectrometry techniques to deliver a complete picture of your compound's proteomic target interactions:

  • Identify all protein targets that interact with a small molecule within any given cell line or tissue proteome
  • Rank cellular compound/target binding affinities across a broad dynamic range
  • Profile small molecules against native cellular derived proteins
  • Correlate pre-clinical and clinical data with your compound’s native target spectrum

KINAXO Cellular Target Profiling® can easily be adapted to a vast range of small molecule compounds, providing customized solutions for numerous applications, including:

  • Identifying previously unknown disease-relevant targets
  • Identifying potential compound-related off-target toxicity effects
  • Assessing a drug’s therapeutic potential in other medical indications
  • Identifying targets and determining modes of action for compounds found in cell-based drug screening

Figure 1 (left): Cellular targets are captured by the bead-coupled compound, eluted, then identified and quantified by mass spectrometry.

Figure 2 (right): An equilibrium binding reaction is established between the cell lysate, bead-coupled compound and ‘free’ compound. The targets that remain bound to the bead-coupled compound are eluted, then identified and quantified by mass spectrometry.

KINAXO Cellular Target Profiling® Technology

KINAXO has extensive experience and world-class expertise in the drug profiling applications of chemical proteomics based on mass spectrometry, the core platform that underpins the KINAXO Cellular Target Profiling® service.

SILAC (Stable Isotope Labeling by Amino acids in Cell culture), iTRAQ (Isobaric Tags for Relative and Absolute Quantification) and TMT (Tandem Mass Tags) are proteome labeling technologies used for quantitative mass spectrometry analysis of cell line and tissue samples. By combining these technologies with state-of-the-art proprietary affinity-based separation and bioinformatic methods, one can determine free compound affinities to all cellular targets.

Determination of cellular target binding affinities

The Kd, free values define the binding affinity of the free (non bead-coupled) compound to each of the targets identified from the cellular proteome. These values are derived by a two step process.

Firstly, the affinities are determined for each of the protein targets binding to the bead-coupled compound (Figure 1). Secondly, in competition experiments the free compound displaces targets bound to the bead-coupled compound (Figure 2).

These experiments must be performed under multiple test conditions to generate the data for binding and competition curves. Relative quantification is achieved by metabolic (SILAC) or chemical (iTRAQ, TMT) labeling of the cell line or tissue sample proteomes, combined with proprietary experimental and analytical methods. The final Kd, free values are derived by applying algorithms to these two data sets.

Ensuring the highest quality data

Every customer project includes a dedicated series of control experiments to obtain the highest quality results and appropriately annotated data. The controls ensure that the data exclude any background binding signal, avoid bead saturation and define the binding equilibrium status for each target/compound interaction.

For further information please download our technical application note TN1.

KINAXO Cellular Target Profiling® is a highly validated, reproducible technology that has been used to profile numerous small molecules. For further information on the applicability of KINAXO Cellular Target Profiling®, please download our project report about profiling p38 and PI3K kinase inhibitors (application Note AN1)  as well as the Hsp90 inhibitor geldanamycin (application note AN2).

Drug re-profiling

As traditional drug development approaches become increasingly expensive, yet fail to fill the shrinking pipelines, repositioning of known substances is gaining attention as a promising possibility to identify alternative medical indications. Re-profiling is an elegant method to reveal new application areas for drugs that are either already approved, or for compounds that have cleared the basic requirements for safety and chemistry, have proven bioavailability, but failed for their intended indications. Given the significant investment already made in these drugs, a quest for alternative applications is worthwhile.  

KINAXO Cellular Target Profiling® has proved to be a powerful tool for drug re-profiling. For example, it helped identify previously unknown target proteins for Pfizer’s approved anti-inflammatory drug Celebrex®, demonstrating how effective this approach is for re-profiling small molecules.

For further information, please download our project report application note AN3.

Target deconvolution

Over the past decade, many biopharmaceutical companies have designed their drug development programs around a high throughput screening approach, testing large libraries of chemical entities against a few validated protein targets. So far, target-based drug discovery has shown rather disappointing results in terms of delivering efficient new drugs. In oncology programs, the ability of human cancers to ‘mutate’ and consequently overcome single target inhibition strategies means many drug development companies are taking a ‘phenotypic’ approach. In other words, screening compounds for a particular desired cellular response prior to honing in on the specific target(s).

Phenotypic effects are measured as an integral of the response of an entire system, and match the multi-factorial impact of a compound. Defining the mode of action of promising new compounds in the native cellular environment is invaluable for further developing a therapeutic strategy.

KINAXO Cellular Target Profiling® technology supports phenotypic-based drug discovery not only by defining the compound target(s), but also the affinities of these interactions, a key to ranking their biological relevance.

Several customer projects applying KINAXO Cellular Target Profiling® for target deconvolution have already been completed successfully.