FREQUENTLY ASKED QUESTIONS

 

1. What is KINAXO Cellular Target Profiling^®?

KINAXO Cellular Target Profiling^® reveals the interactions of a small molecule with the complete proteome in the physiological context of a cell line or tissue. At the same time it provides affinity data for these interactions in the cell, together with a profile of the compound’s native cellular target. The compound’s protein targets can be ranked from high to low affinity, so the most sensitive cellular targets can be distinguished from weak interactions. The client selects which cell lines or tissues are used.

2. How does KINAXO Cellular Target Profiling^® work?

KINAXO determines a compound’s cellular target profile, including affinities, using sophisticated affinity-based separation procedures in combination with ultra-sensitive mass spectrometry. We use immobilized compounds to detect their protein binding partners from cell lines or tissue samples. The target affinities for unmodified, parental compounds are measured with quantitative mass spectrometry using stable isotope labeling of amino acids in cell culture (SILAC) or chemical labeling (iTRAQ /TMT). Co-crystallization data, homology models or SAR (Structure Activity Relationship) data support the choice of an appropriate site for immobilizing the client’s drug candidate. Naturally, we confirm that the linked compound binds and inhibits known targets just like the free inhibitor.

KINAXO Cellular Target Profiling^® services can easily be adapted to a broad range of small-molecule compounds. To date, we have successfully profiled dozens of structurally diverse and functionally unrelated kinase inhibitors, as well as other small molecules such as Hsp90 inhibitors or antibiotics, to provide our clients with tailor-made solutions. For further information on KINAXO Cellular Target Profiling^®, please see our technical note TN1. To learn more about how it can be used to profile kinase inhibitors, download our application note AN1. For further information about profiling various other small molecules, see application notes AN2 and AN3.

3.  What is KinAffinity™? How does it work?

KinAffinity™ can rapidly, precisely and efficiently profile targets of small molecule kinase inhibitors across the native kinome of any given cell line or tissue. It simultaneously determines a kinase inhibitor’s affinity to more than 340 endogenously expressed kinases. This means it delivers detailed information about an inhibitor’s selectivity and potential off-kinase target effects.

KinAffinity™’s ready-to-use matrix captures a cell’s or tissue’s expressed kinome, so synthesis of individual linker compounds is not necessary. Target affinities for the kinase inhibitor being profiled are measured using quantitative mass spectrometry. For further information, please see our technical note TN2.

KinAffinity™ is a highly validated, dependable and reproducible technology that has been used to successfully profile several small molecules. For further information on its capability and applicability, please download our project report AN4.

4.  Has the technology been published?

KINAXO’s underlying technology has been published by KINAXO’s founder Dr. Henrik Daub (Sharma et al., Proteomics strategy for quantitative protein interaction profiling in cell extracts, Nature Methods 2009, in press).

Several studies have been published in PNAS, Cancer Research and other peer-reviewed journals. Publications include profiling of clinical candidates such as the Sutent^® derivative SU6668 (Godl, K. et al. Cancer Res. 2005, 65, 6919-6926) or Iressa/Gefitinib (Brehmer, D. et al. Cancer Res. 2005, 65, 379-382). In 2007, we significantly enhanced our KINAXO Cellular Target Profiling^® technology by adding quantitative analyses of the free, unmodified compound to our range of services (Godl, K. Euro Biotech News 2008, 7, 36-37).

5. What validation data can be shown concerning the applications?

KINAXO Cellular Target Profiling^® Technology has been validated in collaborations with leading pharmaceutical and biotechnology companies, e.g. JNJ, Bayer HealthCare, UCB and Onconova. Examples include clinical candidate optimization and selection, target identification, drug re-profiling and drug rescue. KINAXO Cellular Target Profiling^® results have been presented at international conferences (e.g. AACR) and will be jointly published with our clients in peer-reviewed journals.

For technical validation, we have performed KINAXO Cellular Target Profiling^® in-house case studies on marketed drugs (e.g. Nexavar^®, Sprycel^®). These studies demonstrate that KINAXO Cellular Target Profiling^® correlates very well with the observed biological effects and published data.

KINAXO’s KinAffinity™ Technology has been validated by in-house case studies on well-characterized multi-specific kinase inhibitors (e.g. SKI-606, Sprycel^®), delivering results that are consistent with published data. Collaboration projects with several pharmaceutical and biotechnology companies further confirm KinAffinity’s™ capabilities and reliability.

In direct comparison to KINAXO Cellular Target Profiling^® Technology, the data gained by KinAffinity™ show good overall correlation.

6. Which tissue or cell lines can be used for Cellular Target Profiling^® / KinAffinity™?

Any tissue or cell line of your choice can be used. Most studies aim to determine the compound’s on- / off-targets in a cell system where the compound exhibits biological activity. A typical project involves analyzing several cell lines and/or tissues from (pre-)clinical studies in order to obtain in-depth knowledge about the compound’s mode of action in diseased and healthy states.

7. What do I have to contribute to a KINAXO Cellular Target Profiling^® / KinAffinity™ project?

For KINAXO Cellular Target Profiling^®, KINAXO requires 50 mg of linker compound for immobilization. Although KINAXO does not offer any synthesis services, our scientists are highly experienced in designing linker compounds, and if requested can offer assistance. The chemical linker should be attached in a way that does not interfere with target binding. If the binding mode is unknown, we suggest attaching chemical linkers to two or three different sites so the compound can interact with its target proteins via different binding mechanisms. KINAXO offers several cell lines for testing in KINAXO Cellular Target Profiling^® and KinAffinity™ (please inquire which cell lines are available). You would have to provide any other cell line or tissue (for detailed amounts please see 13).

8. How can KINAXO’s technologies help me understand my compound’s biological effects and predict its clinical outcome?

Understanding the mode of action is a key issue in any drug development program. Experience shows that the cellular interplay of a drug’s protein interactions ultimately contributes to the observed biological effects. Therefore, it is crucial to know about a drug candidate’s direct cellular target interactions before commencing costly clinical trials.

Our KINAXO Cellular Target Profiling^® and KinAffinity™ Technologies determine a compound’s protein target profile in cells or tissue, thus reflecting the native conformation and activity of the target protein in an unbiased manner in its physiological context. As a consequence, the derived target and off-target profile supports predictions about the compound’s effects as well as side-effects on cells in vivo. This helps one understand the therapeutic potential of a drug as well as toxic side-effects.

9. At which stage in drug development should I conduct KINAXO Cellular Target Profiling^® / KinAffinity™?

Depending on the client’s specific questions and challenges, KINAXO Cellular Target Profiling^® / KinAffinity™ will help those responsible for development make more profound decisions at various development stages, from lead identification to clinical development. In addition to these applications, KINAXO’s technologies can be used to identify additional potential therapeutic options at all stages of development, as well as for already marketed drugs.

10. Can you describe a typical basic or comprehensive project with KINAXO?

KINAXO provides customized services according to our client’s needs. A typical basic KINAXO Cellular Target Profiling^® Project would involve analyzing one compound using 1-2 cellular or tissue extracts. A comprehensive study could comprise a series of lead candidates analyzed with a representative selection of 4-5 different cell types.

A standard KinAffinity™ project includes analyzing a kinase inhibitor using cell lines or tissue extract chosen by our customer.

Whatever the client’s needs are concerning cell lines or tissues, our technology allows us to customize the service to these cell lines or tissues.

11. How does KinAffinity™ compare with commercially available in vitro kinase panels?

Hit profiling against a limited panel of recombinant kinases is routinely used to determine target activities in early drug discovery. Beyond the limitations of the kinase spectrum, these biochemical assays assess the activity in settings using isolated targets, regular recombinant protein enzymes or protein fragments, and in a non-physiological manner.

In contrast to target based-screening, our chemical proteomics approach quantitatively determines compound-target profiles in an unbiased manner, using a ready-to-use matrix to capture and enrich a cell's or tissue's expressed kinome: In other words, interactions of a kinase inhibitor with endogenously expressed, full length and post-translationally modified target proteins in a physiological context, and in the presence of cellular co-factors and native partners.

Furthermore, KinAffinity™ uses cells or tissue, and can therefore detect interactions with kinases not included in any commercially available kinase panel. So far, 341 non-redundant kinases can be enriched for target profiling - which exceeds the number of kinases one can screen using many commercially available panels.

This improved knowledge about drug-proteome interactions leads to a deeper level and broader spectrum of information on specific questions arising at various stages of the R&D process.

12. Who will benefit from the information gained from KINAXO Cellular Target Profiling^® and KinAffinity™?

Selection of the most promising compounds to continue into advanced pre-clinical studies is one of the most challenging decisions facing directors of drug development programs. Multiple parameters must be considered in such decision-making, including not only the compound’s potency against primary target(s) but also potential off-target drug interactions.

KINAXO’s services permit rational decisions based on unbiased, proteome-wide selectivity data, helping enormously in the development of drugs with well-characterized inhibition profiles. Those most likely to benefit from KINAXO Cellular Target Profiling^® and KinAffinity™ include:

• Project directors in charge of nominating clinical candidates
• Medicinal chemists involved in lead optimization
• Pharmacologists overseeing pre-clinical and clinical programs
• Senior management responsible for the company’s drug pipeline

13. How much cell / tissue material is required for KINAXO Cellular Target Profiling^® / KinAffinity™?

For KINAXO Cellular Target Profiling^®, KINAXO uses metabolic labeling (SILAC) for cells, as well as isotope coded covalent tags (iTRAQ / TMT) to label cells / tissue. KinAffintiy™ is performed using SILAC, iTRAQ or TMT labeled cells / tissue.

In all cases, KINAXO can take on the labeling of cells / tissue. For SILAC labeling, we require a cryostock of cells, which are transferred into culture and subsequently labeled.

For iTRAQ / TMT labeling, we require a cryostock of cells or a certain protein amount of frozen cell / tissue samples. Although the protein content greatly varies between different cell lines / tissue - depending on its origin and quality - some rough guides are given in the table below.

If preferred, you can also label your cells / tissue yourself. In this case, please contact us to match labeling conditions to our measurement setup.

	Amount of protein required	Approximate amount of cells/ tissue required		Biological material requested
		Cells	Tissue	SILAC	iTRAQ/TMT
KINAXO Cellular Target Profiling®	120mg	6x108	1-6g	Cryostock Cell / tissue pellet Labeled cells	Cryostock Cell / tissue pellet Labeled cells
				iTRAQ / TMT
KinAffinity™	100mg	5x108	1-5 g	Cryostock Cell / tissue pellet

14. How will the results be delivered, which data are reported? How long is a typical project?

For each project we deliver a full and detailed study report including a presentation at our client’s site.

Our KINAXO Cellular Target Profiling^® services list the compound’s cellular targets ranked from high to low affinity to distinguish between the most sensitive cellular targets and weak interactions.

KinAffinity™ shows a compound’s cellular kinase targets including all their binding affinities.

We also provide data interpretation services and further bioinformatic analyses if requested. The time required for a basic KINAXO Cellular Target Profiling^® project varies from four to six weeks, a KinAffinity™ project takes about two weeks.