Protein kinases are enzymes which transfer phosphate groups from donors such as ATP to proteins in a process call “phosphorylation”. This process is usually reversible; phosphatases are the enzymes which “dephosphorylate” or remove phosphate groups from proteins. Each phosphate group carries two negative charges. The addition or removal of a phosphate group can change the three-dimensional shape of the protein. Such “conformational changes” alter the biological activity of the protein, changing the way it interacts with other proteins in a complex communication network within the cell. Many cellular receptors use phosphorylation as a means of signal transduction. For example, various receptors for growth factors, such as insulin-like growth factor (IGF) and epidermal growth factor (EGF), use phosphorylation to induce cellular growth and proliferation. Kinases and phosphatases can be thought of as “on” and “off” switches which are activated by such extracellular signaling molecules.
Cancer Treatment or Holy Grail?
In the 1980s it was observed that, unlike normal cells, many cancer cells proliferate in the absence of extracellular growth factors. Many tumors were found to overexpress EGF. Often phosphorylation signaling pathways were dysregulated, meaning that the “switch” was always stuck in the “on” position. (ref, ref) Kinase inhibition became a promising target to “turn off” these dysfunctional switches. Kinases are also involved in the replication of HIV and other pathogens, as well as virtually all physiological processes, including many pathways associated with aging (e.g. PI3-kinase, AKT, mTOR, MAPK, etc.) The prospect of being able to selectively modulate kinase activity seemed to hold the key for treating a wide-range of conditions. Thus, the quest began. It has been estimated that one-third of pharmaceutical company research efforts have been focused on kinase inhibition. Spurred on by the spectacular success of imatinib, the quest continues today.
Imatinib – the “Magic Bullet”.
In 2001, Imatinib (Gleevec) was featured on the cover of TIME magazine, as “the magic bullet” for treating cancer. In 2009, the developers received the prestigious Lasker Award (known as the “American Nobel Prize”) for “converting a fatal cancer into a manageable chronic condition”.(ref) Imatinib is a specific kinase inhibitor, which induces complete remission in the great majority of patients with chronic-phase CML, a common form of leukemia, with very few side effects. Imatinib, and its successors, have also been widely heralded as a model of rational drug design, using a targeted approach, and a new paradigm for the development of “next-generation” kinase inhibitors. (ref).
Taking a Closer Look.
It would appear that we are on the verge of a new age, not just in cancer treatment, but in pharmacological development. Imatinib was developed over a decade ago. The hype continues. But is it justified? The real question is whether the success of imatinib is repeatable with other conditions, or was it a special case. The answer is that the remarkable success of imatinib was a very exceptional case, unlikely to be repeated with very many other conditions.
Pathophysiology of CML:
“CML was the first malignancy to be linked to a clear genetic abnormality, the chromosomal translocation known as the Philadelphia chromosome. In this translocation, parts of two chromosomes (the 9th and 22nd by conventional karyotypic numbering) switch places. As a result, part of the BCR (“breakpoint cluster region”) gene from chromosome 22 is fused with the ABL gene on chromosome 9. This abnormal “fusion” gene generates a protein of p210 or sometimes p185 weight (p is a weight measure of cellular proteins in kDa). Because abl carries a domain that can add phosphate groups to tyrosine residues (a tyrosine kinase), the bcr-abl fusion gene product is also a tyrosine kinase. The fused BCR-ABL protein interacts with the interleukin 3beta(c) receptor subunit. The BCR-ABL transcript is continuously active and does not require activation by other cellular messaging proteins. In turn, BCR-ABL activates a cascade of proteins which control the cell cycle, speeding up cell division. Moreover, the BCR-ABL protein inhibits DNA repair, causing genomic instability and making the cell more susceptible to developing further genetic abnormalities. The action of the BCR-ABL protein is the pathophysiologic cause of chronic myelogenous leukemia. With improved understanding of the nature of the BCR-ABL protein and its action as a tyrosine kinase, targeted therapies have been developed (the first of which was imatinib mesylate) which specifically inhibit the activity of the BCR-ABL protein. These tyrosine kinase inhibitors can induce complete remissions in CML, confirming the central importance of bcr-abl as the cause of CML.”
CML is unique.
- Very few conditions are the result of such a single genetic abnormality.
- The BCR-ABL fusion protein represents the ideal molecular target. Since it is an abnormal protein that does not ordinarily even exist in healthy humans, inhibiting its activity does not interfere with normal processes, or cause unwanted side effects.
Very few conditions are the result of a single well-known, abnormal, protein, which can be readily targeted without interfering with normal physiological processes. The success of imatinib, and similar compounds, does not represent a paradigm shift in drug development. On the contrary, the complexity of phosporylation networks will require robust systems biology approaches, not a single-target reductionist approach.
The Neglected Phosphatases
In the rush to investigate kinase inhibitors, the important role of the other “off” switch has been largely ignored. Just as dysregulated kinase activity is often associated with cancer, many phosphatases act as tumor suppressors (ref). In fact, mutations reducing phosphatase activity appear to play a much more important role in tumorigenesis than do mutations affecting kinase activity. One important phosphatase, PTEN, is the second most frequently mutated gene in human cancers, following p53. Given the fact that PTEN also regulates p53 levels (ref), some have even called PTEN “the new guardian of the genome”. (ref) For more information on p53, see: p53 and Longevity.
What has been the track record of target-based approaches?
Despite advances in our understanding of genomics, and the development of ever-more advanced methodological technology, the discovery of effective pharmaceuticals has declined.
“Investment in drug research and development (R&D) has increased substantially in recent decades, but the annual number of truly innovative new medicines approved by the US Food and Drug Administration (FDA) has not increased accordingly, and attrition rates are very high1. Indeed, in a recent analysis2 it was noted that without a dramatic improvement in R&D productivity, the pharmaceutical industry cannot sustain sufficient innovation to replace the loss of revenues due to patent expirations for successful products. …Since the dawn of the genomics era in the 1990s, the main focus of drug discovery has been on drug targets, which are typically proteins that appear to have a key role in disease pathogenesis3, 4, 5.Modification of target activity provides a rational basis for the discovery of new medicines; a target-centric approach provides a specific biological hypothesis to be tested and a starting point for the identification of molecules to do this with. Tremendous advances have been made in the development of new tools to identify targets and compounds that interact with these targets (for example, high-throughput target-based screening assays that are applicable to key protein families such as G protein-coupled receptors and kinases). Structure-based tools that can be used to aid lead identification and optimization for some targets have also been developed, including X-ray crystallography and computational modeling and screening (virtual screening).
However, despite the power of these tools to identify potential drug candidates, R&D productivity remains a crucial challenge for the pharmaceutical industry, which raises questions about the possible limitations of a target-centric approach to drug discovery…The increased reliance on hypothesis-driven target-based approaches in drug discovery has coincided with the sequencing of the human genome and an apparent belief by some that every target can provide the basis for a drug. As such, research across the pharmaceutical industry as well as academic institutions has increasingly focused on targets, arguably at the expense of the development of preclinical assays that translate more effectively into clinical effects in patients with a specific disease.”
Multiple Targets: Systems Pharmacology
Despite the disappointing track record of single-target approaches, I do not believe that target-based approaches should be abandoned. What is needed is a multiple-target approach, which can be combined with phenotypic assays. Whole systems approaches are needed to effectively model the interconnected complexity of human physiology. Such approaches will need to use methods discussed in Systems Biology and its tools. Protein phosphorylation networks comprise a complex system. Alterations can be compensated for in unexpected ways; and small indirect effects can have large unexpected consequences.
“Our results show that, at steady state, inactivation of most kinases and phosphatases affected large parts of the phosphorylation-modulated signal transduction machinery, and not only the immediate downstream targets. The observed cellular growth phenotype was often well maintained despite the perturbations, arguing for considerable robustness in the system. Our results serve to constrain future models of cellular signaling and reinforce the idea that simple linear representations of signaling pathways might be insufficient for drug development and for describing organismal homeostasis… Another finding of this study was the unexpectedly strong dominance of indirect effects (as opposed to direct molecular target effects), which were often without a resulting strong cellular phenotype. To some extent, this observation fits with a view of signaling networks having to be highly flexible and redundant to respond to an ever-changing environment while maintaining stable cellular states (44). This constrains the architecture of the system, as described by the “law of requisite variety” (45, 46), a fundamental law in systems control theory. It states that stable systems have to encode a number of control states that is higher than or equal to the number of states to be controlled. Considering that for each cell the space of “environmental states” is enormous, consequently, also the cellular “control variable space” must have an equal or greater size. The combinatorial possibilities of the phosphoproteome seem to ideally fulfill this demand (44).”
Clearly, kinase inhibition makes a very attractive pharmaceutical target. Many small-molecule inhibitors have been approved for cancer treatment. However, the current pharmaceutical company obsession with single-target, kinase-inhibiting drugs highlights the problem of too much research being driven by the quick profit-incentive, at the expensive of truly understanding the dynamics of the underlying physiological processes. Since kinases have such a wide-range of effects in multiple tissues types, simply inhibiting them is likely to have many detrimental off-target effects. We, really, first need to better understand what is happening at the molecular and organismal level in these signaling pathways in order to increase therapeutic efficacy and specificity. Gone are the days in which a single researcher, or small group, could enter a laboratory and develop breakthrough results. Future advances will require greater interdisciplinary collaboration, relying on teams with experts in many different fields, such as physics, genetics, enzymology, mathematics, proteomics, etc. Such multidisciplinary, whole systems approaches will be challenging, but necessary to make effective use of currently available technology, and to meaningfully interpret the resulting datasets, in order to take our understanding of complex biological processes, and the development of therapeutics to the next level.