Kinases comprise a large family of enzymes that mediate protein phosphorylation, one of the key mechanisms by which cell functions are regulated. Disruption of this regulation is associated with hundreds of diseases, including many cancers. It is therefore imperative that disease-related kinases are profiled, in order to inform the development of therapeutics and provide better outcomes for patients. Despite the great benefits to be gained from understanding how kinases function, little research has been aimed at elucidating their physiological substrates. Professor Bieberich has been working on developing ways of uncovering these details, devising several novel methods to open up the field for further research.
Kinases are a family of proteins that function as key regulators of cellular function. The genes encoding them make up one of the most functionally diverse and largest gene families in the human genome. They are enzymes that direct the activity, localisation and function of substrate proteins through the addition of phosphate groups, a process known as phosphorylation. Kinases are essential in many cellular signalling pathways, which involve a highly coordinated series of reactions to mediate complex functions, such as cell division. Although over 500 kinases are known to be encoded in the human genome, most remain to be functionally categorised.
Fighting Cancer Through Kinase Inhibition
Changes in kinase activity are associated with a multitude of devastating diseases, including many cancers. Inhibition of abnormal kinase activity using small molecule inhibitory drugs has now become the standard mode of therapeutic intervention for numerous cancers, with hundreds of such inhibitors currently in development. These drugs can be very effective as therapeutics, with patient response rates as high as 85%.
Despite their widespread use and potential effectiveness, knowledge of the physiological substrates of the kinases that these drugs target, and the downstream cellular effects of their activity, is limited. This has resulted in difficulties in accurately measuring the effectiveness of inhibitory drugs due to a lack of biomarkers, contributing to an unacceptably high failure rate in clinical trials. This lack of information also limits the potential to predict possible side effects of a drug and how inhibitor resistance may develop. Furthermore, a better informed approach to kinase inhibitor drug design would greatly decrease time and cost of development.
A New Era for Kinase ‘Substratomics’
Professor Bieberich is leading the way in what he terms kinase ‘substratomics’, the profiling of kinase substrates. He wants to help uncover what role individual kinases play in cells, which substrates they phosphorylate and what signalling pathways they are involved in regulating. Despite the wealth of information to be gained from investigating kinase substrates, research efforts to profile them have been limited. Although the drawing of a complete map of cellular kinases is an enormous task, Bieberich felt it was crucial to start somewhere.
In their quest to uncover kinase substrates, they found there was a need for a novel system for their identification. In 2007, Bieberich, together with Dr Li, developed a new approach known as the reverse in-gel kinase assay (RIKA), a powerful tool that has the capacity to rapidly profile kinase substrates. The system works with any kinase that can be incorporated into a polyacrylamide gel and catalytically reactivated after gel electrophoresis (gel electrophoresis is a technique used to resolve a tissue or cell protein extract).
The sample is run through a porous gel matrix with an electric current, which separates components of the sample in the gel according to the molecular weight of each protein. Kinase activity and substrate structure is then restored and an in-gel kinase reaction is carried out. The phosphorylation signal can then be detected to locate potential substrates, which can be sent for mass spectrometric analysis to be identified. In the team’s testing, the RIKA proved to have a much improved signal-to-noise ratio when compared to another existing system, as well as having the benefit of being highly accessible due to the simple experimental set-up.
Delving Deeper into Substrate Phosphorylation
Since the development of the RIKA, Bieberich and his team have been working on developing a method to enable the precise details of the reactions that occur between kinases and their substrates to be elucidated. There is almost nothing known about the phosphorylation stoichiometry (PS) for kinase substrates in the body; this is the measure of the extent of phosphorylation of a substrate that occurs. Building on the RIKA system by combining it with high resolution mass spectrometry technology, they are devising strategies for broadly measuring the PS of kinase substrates in vivo. The technique utilises mass tagging to differentiate between substrate molecules, allowing for the phosphorylated and total substrate molecules in a sample to be quantified. The ratio of non-phosphorylated substrate to that phosphorylated by the kinase is then calculated, revealing the substrate’s PS. Importantly, the PS can also be established following treatment of the substrate with a therapeutic inhibitor in order to measure drug efficacy.
This new system will provide an unprecedented opportunity for the identification of biomarkers by determining whether or not a defined set of direct kinase substrate responds to an inhibitor in a patient sample and, if so, to what extent. It will allow the development of a comprehensive panel of biomarkers for inhibitor response in cells, enabling accurate measurement of whether an inhibitory drug is effective. The project will also lead to the discovery of new substrate profiles for individual kinases, providing insights into the cellular roles they each play. This will provide the information required to develop combinations of drug therapies that co-target a particular pathway, which would greatly decrease the chances of drug resistance.
Refining Therapeutic Targets
In addition to this, many kinases are implicated in a wide range of cellular functions with a multitude of substrates. Therefore, a complete picture of each kinase’s substrate profile is crucial if we are to develop drugs that will only interfere with a specific cellular mechanism, rather than impairing all of the kinase’s functions. This would allow for very precise drug targeting with fewer side effects and less toxicity than current drugs that work through global kinase inhibition.
With Bieberich and his team’s contributions of novel methods for the discovery and analysis of kinase substrates, the unveiling of full kinase profiles is now more attainable than ever before. With over 500 human kinases to investigate, the concerted efforts of researchers in the field will be essential for us to obtain a complete map of kinase activity in cells and improve on kinase-targeted therapeutics.
What led you to focus your research on elucidating the substrates of cellular kinases in particular?
Our interest in kinases grew out of our attempts to understand how an important prostate tumour suppressor protein called NKX3.1 is regulated. As a PhD student, Dr Li discovered, using a classical method called an in-gel kinase assay, that protein kinase CK2 phosphorylates NKX3.1. He then came up with the brilliantly simple idea of running that assay backwards’ to use it as a kinase substrate profiling tool.
What do you think the biggest challenge to completing a profile of all kinase substrates will now be?
I think the biggest challenge will be to convince others that our approach provides physiologically relevant datasets that can be leveraged. There is a sort of knee-jerk reaction on the part of researchers in the field to say ‘Oh, a lot of the substrates identified in RIKAs are probably not real, the kinase would never see this or that protein in a live cell’, or ‘Kinases are so promiscuous in vitro that they can phosphorylate a lot of substrates that they wouldn’t in a physiological context’. While there will no doubt be some false positives, our published data show that for CK2, 97% of the substrates identified in a RIKA respond, in live cells, to a CK2-specific inhibitor. That’s pretty good evidence that most of the substrates are ‘real’. In terms of a kinase encountering a substrate in a RIKA that it would never encounter in a cell, we have not been able to think of an experiment to definitively show that two proteins never interact under any physiological circumstance. And we know for a fact that kinases can be mis-localised in diseases, especially cancer. So we’re willing to cast a wide net, and sort through the potential by-catch, because we firmly believe the ‘keepers’ are just too important for us to sit at the dock and design the perfect net.
With this knowledge being so crucial for optimal drug development, why has there not been a more concerted effort before now to discover the downstream effects of many kinase inhibitors?
That’s a really good question. Profiling kinase substrates and validating kinase inhibitor biomarkers is hard and expensive basic research. And there are just not a lot of options when it comes to facile enzyme substrate profiling methods.
Will your new technique for measuring inhibitor efficacy in vivo be employed in phase I drug trials?
We are very hopeful that our method of analysing inhibitor efficacy will be incorporated into clinical trials. Right now, many trials employ a single biomarker, usually using an anti-phosphoprotein antibody. We hope to provide a panel of biomarkers, comprised of tens or hundreds of direct kinase substrates.
What plans do you have for the future of your research?
We are already developing the next generation of the RIKA, designing it to work for kinases that are difficult to reactivate by refolding, and for multi-subunit enzymes. We are also exploring new approaches for separating and sequestering proteins that will get us away from polyacrylamide gels. But for now, we plan to plough through as much of the kinase ‘substratome’ as we can, using our current methods.
Dr Bieberich’s work aims to elucidate the physiological substrates of kinases. The reverse in-gel kinase assay (RIKA) that his team have developed is a powerful tool that can rapidly profile physiological substrates. This knowledge can be used to inform combination therapies.
- Innovative Molecular Analysis Technologies Program
- National Cancer Institute
- National Institutes of Health
- Dr Xiang Li, University of Maryland, Baltimore County
Dr Bieberich received his PhD from the Johns Hopkins University in 1987 and was a postdoctoral fellow at Yale from 1987–1990. From 1990–1997, he was a scientist at the Holland Laboratory, American Red Cross. Since 1997, Dr Bieberich has been a faculty member in the Department of Biological Sciences, UMBC.
Charles J Bieberich, PhD
Professor of Biological Sciences and
The Bearman Foundation Chair in
1000 Hilltop Circle
Baltimore, MD 21250
T: +1 410 455 3125