High Profiling

John Kozarich of ActivX Biosciences updates NGP on the development of KiNativ, a proprietary platform for High-Content Kinase Profiling, and its impact on the future of kinase inhibitor discovery.

John Kozarich, President of ActivX Biosciences in La Jolla, launched KiNativ last year on the pages of NGP. Since then, ActivX has built a growing list of customers for the platform and continues to expand the technology beyond protein kinases. NGP catches up with Kozarich to learn more about this novel approach and its future directions.

NGP. What exactly is KiNativ and how does it work?
JK. KiNativ is the product of seven years of research at ActivX to develop a simple set of chemical tools that could be used to interrogate the kinome, the full complement of protein kinases which, in humans, numbers over 500. Specifically, we designed a set of proprietary chemical probes based on ATP, the universal substrate for all protein kinases. These probes bind to the ATP site of kinases. Once bound, a reactive group in the probe is positioned in such a way to covalently tag lysines residues that are conserved in the active site of all kinases. Since we perform the reaction in cell and tissue lysates all kinases present (usually 150-200 kinases) in the sample are tagged simultaneously. These chemically-tagged kinases serve as the basis of the subsequent analysis.

NGP. How is this complex lysate of tagged kinases and thousands of other proteins deconvoluted into quantitative information on individual kinases?
JK. This is the exciting part. The lysate is specifically proteolyzed reducing all of the proteins present to peptide fragments. The specifically tagged kinases generate a set of specifically tagged peptides. Since the tag contains a recognition group for immuno-capture, the tagged peptides can be purified from the vast background of untagged peptides using an immuno-column separation. The resulting sample containing hundreds of tagged peptides is subjected to state-of-the-art liquid chromatography/mass spectrometry (LC-MS) techniques which separates the tagged peptides, determines their amino acid sequences, validates the sites of tag labeling and quantifies their signals. Ultimately, the amino acid sequences are related to known DNA sequences so a positive identification of the individual kinases present in the original sample can be made. Overall, we have validated over 400 human kinases for analysis by KiNativ.

NGP. It sounds quite complicated. How long does and analysis take?
JK. Well, the platform is nearly completely automated. The initial tagging reaction is performed on lysates containing a few milligrams of total protein and is over in five or 10 minutes. The downstream separation and LC-MS analysis is designed to handle 100s of samples a day. In general, the time from experiment through to final data analysis is a few days. For outside work, we normally quote a two-week data turnaround which is comparable to other kinase platforms.

NGP. How is information on kinase inhibitor selectivity obtained?
JK. The key is that most kinase inhibitors in research and development across the industry are designed to bind to the ATP site of specific kinases. So we can set up a competition between a kinase inhibitor and our chemical tagging reaction. The inhibitor can be added directly to a lysate or cells can be pretreated with the inhibitor prior to lysis. As the concentration of the inhibitor is increased, the tagging reaction is increasingly inhibited which ultimately leads to a reduction of tagged peptide signal in the LC-MS analysis. Thus, quantitative IC50s for the inhibitor can be obtained for not only the target kinase but also any off-target kinases that are present in the lysate.

NGP. Isn’t similar information obtainable using other kinases profiling platforms?
JK. Yes, similar information can be obtained but we have found many significant differences. Most other platforms rely on individual assays using recombinant kinases in simple enzyme-substrate format. The IC50s obtained represent an idealized system compared to the IC50s we obtain using the more physiologically relevant cells and lysates. With KiNativ, we get quantitative data on the full-length kinases in their native cellular context, which includes the network of protein-protein interactions that can affect their behavior toward some inhibitors; in a recombinant assay, using in most cases a fragment of the full-length kinase, the cellular context is missing and major differences can emerge. KiNativ provides additional dimensions of information that bring the platform closer to cellular reality than the others.

NGP. Are there other points of differentiation from recombinant kinase platforms?
JK. Yes. First, KiNativ is species-independent. Recombinant platforms require individual cloning of kinases so the focus is not surprisingly on human enzymes. Since our platform is driven by peptide sequence identification of tagged sites, as long as DNA sequence information is available for a species our platform can be used immediately. This is very useful for understanding the relevance of toxicity issues across specifics. If a kinase inhibitor shows toxicity in the dog, for example, we can profile the compound directly in dog tissue and potentially determine if the toxicity is idiosyncratic to the dog or relevant to human. Second, KiNativ can provide insight into the behavior of the target kinase across tissues in the same species. We have observed that many inhibitors show differential effects on the same kinase in different tissues. Recombinant assays cannot provide this information. Finally, KiNativ goes beyond protein kinases. The ATP-based chemical tags also recognize hundreds of other ATP binding proteins. For example, we recently launched our lipid kinase panel, which, we believe, is the most comprehensive panel available for these important enzymes. We are also developing other panels of pharmaceutical interest, such as a heat shock protein (HSP) panel. Since there are about 60 classes of ATP binding proteins, there is considerable potential to expand the platform.

NGP. How do profiling platforms based on affinity chromatography methods compare?
JK. There are several methods based on attaching ATP or kinase inhibitor(s) to a column as bait for capturing kinases from complex mixtures. In general, the heterogeneous systems employed and sometimes weak non-covalent affinities are problematic for getting a comprehensive readout of low abundance kinases. IC50 determinations can also be difficult when multiple inhibitors bound to a column compete for a common kinase. For KiNativ, the covalent tagging is carried out in undiluted lysates under homogeneous conditions. The tag is stable so it permits MS identification of kinases well below 50 copies per cell and has a dynamic range of at least four orders of magnitude. This is difficult to match for affinity column approaches.

NGP. How has your business developed since last year?
JK. The education of potential customers has gone on extremely well. We have a number of major customers spanning big pharma and biotech as well as some key academic collaboration. Once a customer understands the platform and sees the results, usually from a feasibility study, the probability of repeat business is very high.

NGP. What type of service options do customers have?
JK. Our most straightforward option is a fee-for-service access to our standard kinase panels, which have been developed for specific cell lines. As their appreciation of the technology grows, some customers request special projects, such as development of panels for specific cell lines and tissues of interest. Investigating inhibitor effects under various cellular stimuli is also a popular request. In certain cases, a collaboration or alliance to obtain access to KiNativ over a broad range of clinical and development programs can be more cost-effective.

NGP. What does the future hold for KiNativ?
JK. We believe that KiNativ will fill a new niche, which we call ‘High-Content Kinase Profiling’. KiNativ will be appreciated as providing an information-rich kinase inhibitor profile that will address issues that other platforms cannot deliver. Given the high probability of clinical failures, KiNativ profiles aid in the selection of the best compounds thereby increasing the chances of success. For more information, please visit www.kinativ.com.

About John Kozarich
John Kozarich is Chairman & President of ActivX Biosciences in La Jolla, a subsidiary of Kyorin Pharmaceutical Co. He is also Chairman of Ligand Pharmaceuticals and an Adjunct Professor at Scripps. Well known for his work on mechanisms of enzyme and drug action, John has spanned academia, Big Pharma and biotech.