Kinex™ Protein Kinase Microarray Services

The Kinex™ Protein Kinase Microarray (KPKM) services are a convenient and very cost-effective solution to assist scientists in the evaluation of a wide panel of diverse protein kinases as targets for potential kinase inhibitors, as substrates for kinases and proteases, and binding partners for proteins and peptides. These services utilize our unique protein kinase microarrays with different detection methods depending on the specific application. Any of the results from these economical and rapid approaches can be further validated with complementary methods that are available with our discovery platform of integrated proteomics services.

Protein kinases consititute the single largest mammalian enzyme family with at least 515 members in the human proteome. They have been implicated in a wide range of complex cellular functions and pathways, ranging from metabolic regulation to tumorigenesis. Since many human diseases result from over-activation of protein kinases due to mutations and/or over-expression, they have emerged as attractive targets in the search for new therapeutic agents in the pharmaceutical industry. Approximately one third of protein targets under investigation in the pharmaceutical industry are protein kinases, underscoring the promise of protein kinase inhibitors for the treatment of human diseases. We believe that within the next decade, over half of the drugs in clinical trials will be kinase inhibitors. Currently, more than a dozen of small-molecule kinase inhibitors have been approved for various disease indications, and about 150 kinase-targeted drugs are in clinical development and many more are in various stages of pre-clinical development. Almost all of them are directed towards the adenosine triphosphate (ATP) binding site and display different selectivities, potencies and pharmacokinetic properties. Since the ATP-binding sites of protein kinases are highly conserved, the selectivity of kinase inhibitors becomes one of the most important issues in durg discovery process.

The selectivity of a kinase inhibitor was traditionally monitored only sporadically throughout the discovery process by in vitro kinase activity assays against a small subset of selected kinases. This appears to have contributed to the failure of many compounds during the late stage of development, and highlights the importance of comprehensive assessing of kinases-inhibitor selectivity during the early stage of drug discovery. Over the past few years, the size of the recombinant kinase panels has been increased dramatically to cover 300 or more protein kinases. Despite of the progress, it remained both technically and financially challenging to systematically assess the specificity of a compound against hundreds of kinases, as the assays are time-consuming and costly, and comprehensive knowledge of kinases and their substrates is still lacking.

To reduce the costs of kinase inhibitor counter screens, alternative methods have been developed to identify protein kinases that are potentially affected by candidate drug compounds. Ambit Biosciences (www.kinomescan.com) developed a high-throughput method named Kinomescan™ for screening small molecule kinase inhibitors against a panel of about 300 human kinases. It is based on a competition binding assay that quantitatively measures the ability of a compound to compete with an immobilized, active site-directed ligand to the bacterially expressed T7-phage tagged human kinases. The ability of the test compound to compete with the immobilized ligand is measured by quantitative PCR of the T7 tags of the displaced kinases. Each kinase is assayed individually for each compound, which requires relatively larger quantities of compounds. Kinases expressed as fusion proteins in E. coli may not be in their native conformations due to the lack of some post-translational modifications, which could potentially impact the binding of compounds. In contrast, an in situ kinase profiling platform, Kinativ™, offered by ActivX Biosciences, utilizes acyl phosphate-containing nucleotide probes that react selectively and covalently at the ATP binding sites in combination with a mass spectrometry-based detection method to assay the kinase binding affinity of compounds in protein lysates where native post-translational modifications and protein-protein interactions are preserved. However, the approach suffers from a few drawbacks including interference from other nucleotide-binding proteins, and binding bias towards kinases in high-abundance.

Recognizing the importance of elucidating the inhibition profiles of compounds, Kinexus has developed an innovative microarray-based profiling platform for screening small-molecule protein kinase inhibitors. The cores of this technology consist of a protein kinase micorarray and a biotinylated proprietary ATP probe developed at Kinexus. The probe has been shown to display affinity for the majority of protein kinases tested by our internal research work. Co-incubating a test compound with the biotinylated ATP probe on the protein kinase microarray allows simultaneous determination of the affinity of the compound against hundreds of protein kinases on the array on a competition binding basis. The kinases to which the compound exhibits binding will experience a reduction of the binding of the ATP probe, and the remaining ATP probe covalently bound to the kinases on the array can be detected with the fluorescently-labeled streptavidin conjugates. Compared to the aforementioned Kinomescan™ and Kinativ™, advantages of our microarray-based inhibitor profiling platform include higher throughput, small amounts of compound required, and the use of active protein kinases expressed in eukaryotes. Without the requirements for any sophisticated analytical instrument such as quantitative PCRs and mass spectometers, our technology presents a quick and effective approach to survey a broad spectrum of the human kinome for compound inhibition profiling prior to any in-depth assays to be perfomed. Thus, the protein kinase microarray-based inhibitor profiling complements well with our existing Kinase Inhibitor Profiling services that are based on measurement of phosphotransferase activity. Any hits from the Kinex™ Kinase Microarray can be confirmed with our Kinase Inhibitor Profiling services against the very same preparations of protein kinases.

The first generation Kinex™ Protein Kinase Microrarray-200 features 200 active recombinant human protein kinases originally expressed in an insect expression system. Each array consists of 3 identical fields to allow 3 assays with either 3 different compounds at single concentrations or a single compound of 3 different concentrations to be carried out simultaneously, with one field reserved for the probe-only control. Each kinase is spotted in triplicate within a field on a glass slide. In addition, multiple internal control samples have been incorporated onto the arrays during the array printing process for quality control purpose.

The 200 protein kinases spotted on the arrays cover most of the major groups of the human kineome and respective families. We expect to enhance the coverage once more active kinases become available to us in the near future. Thus far, the vast majority of the 200 kinases tested have shown affinity for our proprietary ATP analogue probe. Those kinases that display the strongest ATP analogue probe binding will yield the most reliable inhibitor screening results. Note that the ATP analogue probe is also useful for the detection of protein kinase activators as they may improve the binding of the ATP analogue probe. Therefore, it should be feasible to detect protein kinase activation as a consequence of phosphorylation in vitro on the chip by an added protein kinase or partial proteolysis by a protease that relieves inhibition by non-catalytic domains

The protein kinases on the Kinex™ Protein Kinase Microarray–200 can be used as test substrates for specific protein kinases of interest. Moreover, following their phosphorylation, it is feasible to determine whether the ATP binding ability of the phosphorylated kinases are altered using our ATP analogue probe.

Our clients can have Kinexus test specific kinases of interest to determine if any of the protein kinases on the Kinex™ Protein Kinase Microarray–200 are direct substrates and ascertain whether this affects the ATP binding ability of the phosphorylated proteins. Clients can supply their own preparations of protein kinases or choose from over 360 available kinases.

The protein kinases on the Kinex™ Protein Kinase Microarray–200 can be used for testing the specificity of antibodies. Following incubation with a primary rabbit antibody against one of the protein kinases on the Kinex™ Protein Kinase Microarray–200, the target kinase can be detected with fluorescent dye-conjugated anti-rabbit secondary antibody.

The Kinex™ Protein Kinase Microarray–200 can be used for testing the ability of proteins and peptides to bind with the protein kinases on the microarray. This can uncover new protein-protein interactions. Fluorescently tagged proteins of interest that bind to protein kinases on the Kinex™ Protein Kinase Microarray–200 can be detected.

Kinex™ Protein Kinase Microarray-based Small Molecule Inhibitor Profiling Service

To place an order for our microarray-based kinase inhibitor profiling services, clients will need to send separate aliquots of their compounds to Kinexus with a copy of the completed “Kinex™ Protein Kinase Microarray-based Small Molecule Inhibitor Profiling Service” order forms. Compounds can be submitted in either powder or solution. Compounds can be dissolved in either phosphate-free aqueous buffer or DMSO. The amount of compound powder to be sent to Kinexus should be sufficient for preparing 1 ml of the solution at the final testing concentration for each assay. The concentration of compound stock solution should be 100 times the final testing concentration. Compounds must be shipped via Fedex or other courier service providers at the expense of the clients. Compounds may be transported at room temperature or frozen, and it should be determined by the clients which is suitable.

Clients should complete the Client-Supplied Non-Confidential Sample Description Form (NSDF-C) provided our client information package for this service. If you do not wish to provide the requested information, then a Client-Supplied Confidential Sample Description Form (CSDF-C) must be completed, and full confidential pricing charges will be applied. Information requested in the order form is needed for proper handling of the compounds and carrying out the analyses. Completion of the forms to the best knowledge of clients is critical. Due to the presence of 3 incubation fields available for performing assays with the client’s compounds, clients may decide to either have the assays performed for the same compound at 3 different concentrations or 3 compounds at single concentrations to make best use of the array and to ensure the comparability of the data between assays. One of these fields could be used as the control, which is incubated with the ATP analogue probe in the absence of any other compounds.

Kinex™ Protein Kinase Microarray-based Kinase Substrate Profiling Service

To place an order for the Kinex™ Protein Kinase Microarray-based Kinase Substrate Profiling Service, the clients can choose one or more of the protein kinases available from Kinexus. Alternatively, the clients may send an aliquot of an active preparation of their protein kinase of interest to Kinexus. In this event, clients should complete the Client-Supplied Non-Confidential Sample Description Form (NKDF-P) provided in this client information package. If you do not wish to provide the requested information, then a Client-Supplied Confidential Sample Description Form (CSDF-P) must be completed, and full confidential pricing charges will be applied. We recognize that not all of the requested information about commercial protein kinases may be available to our clients, so please provide as much information as you are able to qualify for non-confidential pricing.

We recommend that client-supplied protein kinases are provided as concentrated as possible, preferably at around 10 mg/ml and in screw cap vials. We need approximately 2-5 µg of most protein kinases to create at least 750 µl of final testing solution. As there are large variations in the phosphotransferase activity of protein kinases, and every kinase is unique, Kinexus may be able to perform the Kinex™ Kinase Microarray Kinase Substrate Profiling Service with less kinase if it still works well at a lower concentration. The vials should be clearly labeled with an indelible marker with a unique identification number (recorded in the NSDF-P or CSDF-P forms), parafilmed, and then put into another support structure such as a 50-ml conical centrifuge tube to provide extra protection during shipping. Active preparations of test kinases must be shipped via Fedex or other courier service providers at the expense of the clients. The kinases must be transported frozen, and with sufficient dry ice for at least 4 days if shipped from outside North America.

Due to the presence of 3 incubation fields available for performing assays with the client’s kinases, the clients may decide to either have the assays performed for the same kinase at 3 different concentrations or time points or 3 kinases at single concentrations to make best use of the array and to ensure the comparability of the data between assays. One field may be reserved for use as the control, which is incubated with the ATP in the absence of any added kinases.

Kinex™ Protein Kinase Microarray-based Protein Interaction Profiling Service

To place an order for our microarray-based protein interaction screening services, clients should send an aliquot of the antibody, recombinant protein or peptide of interest to Kinexus with a copy of the completed “Kinex™ Protein Kinase Microarray-based Protein Interaction Profiling Service” order forms. Clients may also choose to have Kinexus produce recombinant proteins and peptides with our Custom Protein and Peptide Production Services. Proteins (~100 µg) and peptides sent to Kinexus should be sufficient for preparing at least 750 µl of the solution at the final testing concentration for each assay. The concentration of protein or peptide stock solution should be at least 10 times the final testing concentration. Proteins and peptides must be shipped via Fedex or other courier service providers at the expense of the clients. Proteins and peptides may be transported at room temperature or frozen, and it should be determined by the clients which is suitable.

Clients should complete the Client-Supplied Non-Confidential Sample Description Form (NSDF-AB or NSDF-P) provided in our client information package for this service. If you do not wish to provide the requested information, then a Client-Supplied Confidential Sample Description Form (CSDF-AB or CSDF-P) must be completed, and full confidential pricing charges will be applied. Information requested in the order form is needed for proper handling of the test proteins and peptides and carrying out the analyses. Completion of the forms to the best knowledge of clients will improve the prospects of success with this type of analysis. Due to the presence of 3 incubation fields available for performing assays with the client’s proteins and peptides, clients may decide to have the assays carried out for the same protein or peptide at 3 different concentrations or 3 proteins or peptides at single concentrations. One field may be reserved for use as the control, which is incubated in the absence of any added test proteins or peptides.

The turnaround time for these services is estimated to be 2 to 3 weeks from the date of receipt of client supplied compounds or proteins by Kinexus. However, this could vary slightly according to the size of the order and the demand of the service at the time when the order is placed. Results of each microarray ordered will be delivered in a comprehensive report consisting of a scanned array image in either jpeg or tiff format and a comparsion report for each compound at each concentration against each kinase on the array. Reports are normally delivered electronically to the email addresses of the contact person specified in the order forms.

Kinexus offers the Kinex™ Kinase Microarray Services at two different pricing levels depending on the level of confidentiality required. The Service Information Form provides the latest standard pricing levels for our Confidential and Non-Confidential Kinex™ Kinase Microarray Services. if the client provides their own compounds, protein kinases, other proteins and/or peptides, only partial information about these materials need to be disclosed in the CSDF forms. To receive a 40% discount off the regular price, Kinexus must be allowed after a minimum 6 month hold to possibly include the results in its on-line KiNET DataBank and SigNET KnowledgeBank, which will permit third parties to access the data.

For volume discounts or quotations for large orders, please contact the Director of Sales & Marketing at 1-866-KINEXUS (or 1-604-323-2547) (and select option 2 on the telephone directory) or e-mail sales@kinexus.ca.

The aforementioned procedure has been designed to reduce the use of shipping materials and courier costs, and to ensure that your precious compound, peptide or protein samples arrive in a safe and stable form at our laboratory facilities. Note that clients are responsible for payment of courier costs. The vials should be sent to the address listed below by any express courier. For dry ice containing shipments, we recommend Federal Express for shipments originating in North America, and World Express is the preferred courier choice outside of North America. Ship the compounds, peptides or proteins to the following address:

Kinexus Bioinformatics Corporation
Suite 1, 8755 Ash Street
Vancouver, B.C. Canada V6P 6T3
Telephone: (604) 323-2547
Facsimile: (604) 323-2548
E-mail info@kinexus.ca

Please ensure 3 copies of a signed commercial invoice (available near the end of the customer information package for this service) accompany your shipment which specifies your compound, peptide and protein samples are non hazardous and non infectious. Since the protein samples are not for resale, the value of your shipment should be priced at approximately $1.00 per sample. It is highly recommended that customers e-mail their courier airway bill number and the date of departure to info@kinexus.ca so we can track your shipment in transit and ensure it arrives in a timely manner. We will send a confirmation e-mail once your shipment arrives at our facility.

Kinexus offers several services to cost-effectively follow up the results from our Kinex™ Kinase Microarray Services. In particular, the Kinase Inhibitor Compound Profiling (KICP) Service is a convenient and cost-effective follow up for clients to further test the ability of their compounds to inhibit the enzymatic activities of protein kinases from the Kinex™ Kinase Microarray - 200 that demonstrate reduced binding of our ATP analogue probe. To examine the effects of compounds on selected kinases in animals and cells treated with these compounds, clients can utilize our Kinetworks™ Custom KCSS 1.0 (Multi-Sample) Protein Screen for analysis of 8 different cell or tissue lysates and choose up to 3 different phosphosite antibodies (provided the molecular weights are significantly separated by SDS-PAGE). Clients can correlate their phosphosite results with hundreds of other measurements of protein phosphorylation in hundreds of different model systems using our free KiNET DataBase. More information about specific phosphorylation sites is also freely available from our PhosphoNET KnowledgeBase. Clients can also investigate the regulation of specific phosphosites in hundreds of cell and tissue lysates with our KinexTM Reverse Lysate Microarray Service. With our IHC Immunohistochemistry Service, we can track the location of phosphorylation changes in tissue sections. For presentation and publication purposes with the results from this Custom Kinase Substrate Profiling Service, we can prepare figures and tables with our Custom Graphics Services. For more information about these services, please contact one of our customer service representatives at info@kinexus.ca.