EXAMPLES OF APPLICATIONS 1 This presentation demonstrated the power of the Kinetworks™ approach to uncover significant research results in a cost effective and efficient manner. 1 Drug Target Discovery A major bottleneck in drug discovery is the identification and validation of drug targets Human genome project has identified over 5,000 cell signalling proteins as potential drug targets Less than 500 proteins have been investigated as drug targets by pharmaceutical industry in its entire history $5 billion spent annually screening non-ideal targets Kinexus is dedicated to identifying and validating drug targets and leads for our clients through offer of our unique Kinetworks™ proteomics services 2 Drug discovery is a very risky and expensive proposition. Only one in a hundred thousand compounds may become a successful drug, after the investment of over US $600 million on average and frequently more than 10 years in development. To discover a drug, the right target must be identified first. This target can be used as the bait to fish for small molecule compounds that are typically inhibitors of the chosen target by high throughput screening. Lead compounds are put sequentially through cell-based, then animal- based and finally human trials. Each subsequent step of the drug discovery and validation process is associated with dramatic increases in cost. Only about 1 in 10 lead compounds that look promising at the end of the animal trials make it successfully through Phase III human trials and receive FDA approval. It is this high rate of failure at the late stages of clinical trials that has made drug discovery so expensive. If the failures could be identified earlier in the drug discovery process, this would markedly reduce costs and lost opportunity. A common cause of failure in human trials is adverse drug reactions. The Kinexus Kinetworks™ screening platform for cell cycle, stress and apoptosis proteins can help to identify at an early stage possible toxic drug reactions and prevent the investment of millions of dollars on the testing of non-ideal targets. To have the greatest impact on drug discovery, it is critical to identify the most appropriate drug targets. This is a significant issue, because over $5 billion is spent annually by the pharmaceutical industry screening against targets that even if specific, potent and cell permeable inhibitors were uncovered , the side effects associated with knocking out these targets would mitigate their utility. With the identification of over 30,000 genes in the human genome, about 5000 cell signalling proteins have been designated as potential drug targets. However, the pharmaceutical industry does not have the capacity to evaluate all of these proteins for use in high throughput drug screening. In fact, less than 500 proteins have been examined extensively as potential drug targets by the pharmaceutical industry over the last century. 2 Kinetworks™ Services Multiple Steps in Drug Development Disease Marker/Drug Target Kinetworks™ Detection immunoblots Drug Target Discovery Drug Screening Toxicology Drug Discovery and Characterization Validation of Medicinal Chemistry Animal Models Preclinical Studies Patient Profiling Clinical Studies 3 An important outcome of the Kinetworks™ proteomics services offered by Kinexus is the generation of vast amounts of data about the regulation of specific proteins. This unique immunoblotting service is only one of many novel services that Kinexus plans to offer its clients. The data generated from the Kinetworks™ services is being merged into our Kinformatics™ functional proteomics database, which we are mining to establish the composition and architecture of protein kinase signalling networks in hundreds of different normal and pathological tissues and cells from diverse species. Our clients will also have Internet access to this database by subscription to KiNET™ bioinformatics services. The central objective of our endeavors is to identify protein kinase signalling pathways that link to specific diseases to identify diagnostic markers and protein kinase targets for drug discovery. Clients are able to query KiNET on-line about the expression patterns and phosphorylation states of hundreds of signalling proteins in hundreds of different model systems. 3 Kinetworks™ Analysis Provides quantitation of protein expression and phosphorylation levels with highly validated antibodies Highly sensitive, accurate, and reproducible Rapid turnaround Affordable pricing 4 The Kinetworks™ analysis utilizes commercial antibodies that have been highly validated by Kinexus to ensure accuracy. Enhanced chemoluminescence and densitiometric analysis with a FluorS Max Imager from Bio-Rad permits permits sensitive detection with as little as 350 µg of lysate cell/tissue protein. Data is linear over a 2000-fold range with standard deviations within 20% for high reproducibility. Results are returned to clients within 4 weeks. Substantial discounts are available with full disclosure of sample information or bulk orders. 4 Kinetworks™ Applications Signal transduction pathway mapping Protein-protein interactions Novel protein discovery Disease markers Drug targets Drug mechanisms of action Drug toxicity Animal model validation 5 Kinetworks™ has a broad range of applications. This presentation provides a sampling ofhow Kinetworks™ has already been successfully used for many of these purposes. 5 KPKS 1.2 Kinase Analysis Cells differ markedly in their protein kinase expression patterns 6 Kinexus has performed over 10,000 immunoblot analyses. These studies have revealed the vast differences between cell types in the expression patterns of protein kinases and other signalling proteins. 6 Tissue Differences in Protein Kinase Expression 0 2000 4000 6000 8000 0 2000 4000 6000 8000 0 2000 4000 6000 8000 0 2000 4000 6000 8000 0 2000 4000 6000 8000 BMX BMX BMX BMX BMX BTK BTK BTK BTK BTK CaMK1 Brain CaMK1 Heart CaMK1 Sk. Muscle CaMK1 Spleen CaMK1 Testes CaMK4 CaMK4 CaMK4 CaMK4 CaMK4 CaMKK Monkey CaMKK CaMKK CaMKK CaMKK CDK1 CDK1 CDK1 CDK1 CDK1 CDK2 Rat CDK2 CDK2 CDK2 CDK2 CDK4 CDK4 CDK4 CDK4 CDK4 CDK5 CDK5 CDK5 CDK5 CDK5 CDK6 CDK6 CDK6 CDK6 CDK6 CDK7 CDK7 CDK7 CDK7 CDK7 CDK9 CDK9 CDK9 CDK9 CDK9 CK1d CK1d CK1d CK1d CK1d CK1e CK1e CK1e CK1e CK1e CK2a CK2a CK2a CK2a CK2a CK2a' CK2a' CK2a' CK2a' CK2a' CK2a" CK2a" CK2a" CK2a" CK2a" COT COT COT COT COT CSK CSK CSK CSK CSK DAPK DAPK DAPK DAPK DAPK DNAPK DNAPK DNAPK DNAPK DNAPK ERK1 ERK1 ERK1 ERK1 ERK1 ERK2 ERK2 ERK2 ERK2 ERK2 ERK3 ERK3 ERK3 ERK3 ERK3 ERK6 ERK6 ERK6 ERK6 ERK6 FAK FAK FAK FAK FAK FYN FYN FYN FYN FYN GCK GCK GCK GCK GCK GRK2 GRK2 GRK2 GRK2 GRK2 GSK3a GSK3a GSK3a GSK3a GSK3a GSK3b GSK3b GSK3b GSK3b GSK3b HPK1 HPK1 HPK1 HPK1 HPK1 IKKa IKKa IKKa IKKa IKKa JAK1 JAK1 JAK1 JAK1 JAK1 JAK2 JAK2 JAK2 JAK2 JAK2 KSR1 KSR1 KSR1 KSR1 KSR1 LCK LCK LCK LCK LCK LYN LYN LYN LYN LYN MEK1 MEK1 MEK1 MEK1 MEK1 MEK2 MEK2 MEK2 MEK2 MEK2 MEK4 MEK4 MEK4 MEK4 MEK4 MEK6 MEK6 MEK6 MEK6 MEK6 MEK7 MEK7 MEK7 MEK7 MEK7 MNK2 MNK2 MNK2 MNK2 MNK2 MOS1 MOS1 MOS1 MOS1 MOS1 MST1 MST1 MST1 MST1 MST1 7 In the Science Magazine’s Signal Transduction Knowledge Environment (STKE) study, Kinetworks™ analysis(www.stke.org/cgi/content/full/sigtrans;2002/162/pe50), rThis and the following slide demonstrate the importance of tracking potential drug targets in diverse organs, since the expression patterns of protein kinases are not readily predictable. Furthermore, we have recently observed large differences in protein kinase expression patterns between male and female rats for the same somatic tissues. More than a third of the protein kinases showed expression changes of greater than 5-fold in the same types of organs. These unexpected differences between species may account in part for why drugs that perform well in pre-clinical animal trials fail in human clinical studies. 7 Tissue Differences in Protein Kinase Expression 0 2000 4000 6000 8000 0 2000 4000 6000 8000 0 2000 4000 6000 8000 0 2000 4000 6000 8000 0 2000 4000 6000 8000 NEK2 NEK2 NEK2 NEK2 NEK2 p38 MAPK p38 MAPK p38 MAPK p38 MAPK p38 MAPK PAKa Brain PAKa Heart PAKa Sk. Muscle PAKa PAKa PAKb PAKb PAKb PAKb Spleen PAKb Testes PDK1 Monkey PDK1 PDK1 PDK1 PDK1 Pim1 Rat Pim1 Pim1 Pim1 Pim1 PKA PKA PKA PKA PKA PKBa PKBa PKBa PKBa PKBa PKCa PKCa PKCa PKCa PKCa PKCb1 PKCb1 PKCb1 PKCb1 PKCb1 PKCd PKCd PKCd PKCd PKCd PKCe PKCe PKCe PKCe PKCe PKCg PKCg PKCg PKCg PKCg PKCl PKCl PKCl PKCl PKCl PKCm PKCm PKCm PKCm PKCm PKCq PKCq PKCq PKCq PKCq PKCz PKCz PKCz PKCz PKCz PKG1 PKG1 PKG1 PKG1 PKG1 PKR PKR PKR PKR PKR PYK2 PYK2 PYK2 PYK2 PYK2 RAF1 RAF1 RAF1 RAF1 RAF1 RAFB RAFB RAFB RAFB RAFB ROKa ROKa ROKa ROKa ROKa RSK1 RSK1 RSK1 RSK1 RSK1 RSK2 RSK2 RSK2 RSK2 RSK2 S6K p70 S6K p70 S6K p70 S6K p70 S6K p70 SAPKb SAPKb SAPKb SAPKb SAPKb SAPKb SAPKb SAPKb SAPKb SAPKb SRC SRC SRC SRC SRC SYK SYK SYK SYK SYK YES1 YES1 YES1 YES1 YES1 ZAP70 ZAP70 ZAP70 ZAP70 ZAP70 ZIPK ZIPK ZIPK ZIPK ZIPK ZIPK ZIPK ZIPK ZIPK ZIPK 8 8 Gender Differences in Protein Kinase Expression IKKa RAFB CSK MEK2 Mouse COT ZIP MEK1 BMX (Etk) CK1e Brain KSR1 PKA DAPK FAK RAF1 ERK6 JAK2 PKCz CaMKK PYK2 CK2a' PKG1 MOS1 PKCa PKCl RSK1 MEK6 PKBa S6K p70 IKKbeta CK2a" CK2a CDK5 ROKa PKCm PAK1 ERK3 PKCg Female SAPKb Female MEK4 Male RAF1 Male SAPKb PKCb CaMK4 ERK1 PKCm ERK2 JAK1 CDK9 p38 MAPK PKCe GRK2 PKCd GSK3b CDK7 GSK3a 0 5000 10000 15000 20000 25000 0 5000 10000 15000 20000 25000 30000 35000 40000 9 We have recently explored the effect of gender on patterns of protein kinase expression in somatic tissues from reproductive-competent male and female rats using the Kinetworks KPKS 1.3 Protein Kinase Screen. This results shown in slide for mouse brain and the following slide for mouse submadibular gland demonstrate that more than half of the protein kinases detected in each tissue displayed greater than 2- fold differences in expression for age-matched mice.