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Protein and lipid kinases fulfill essential roles in many signaling pathways that regulate normal cell functions. Deregulation of these kinase activities lead to a variety of pathologies ranging from cancer to inflammatory diseases, diabetes, infectious diseases, cardiovascular disorders, cell growth and survival. 518 protein kinases and about 20 lipid-modifying kinases are encoded by the human genome, and a much larger proportion of additional kinases are present in parasite, bacterial, fungal, and viral genomes that are susceptible to exploitation as drug targets. Since many human diseases result from overactivation of protein and lipid kinases due to mutations and/or overexpression, this enzyme class represents an important target for the pharmaceutical industry. Approximately one third of all protein targets under investigation in the pharmaceutical industry are protein or lipid kinases.The kinase inhibitors that have been launched, thus far, are mainly in oncology indications and are directed against a handful of protein and lipid kinases. With one exception, all of these registered kinase inhibitors are directed toward the ATP-site and display different selectivities, potencies, and pharmacokinetic properties. At present, about 150 kinase-targeted drugs are in clinical development and many more in various stages of preclinical development. Kinase inhibitor drugs that are in clinical trials target all stages of signal transduction from the receptor protein tyrosine kinases that initiate intracellular signaling, through second-messenger-dependent lipid and protein kinases, and protein kinases that regulate the cell cycle.This review provides an insight into protein and lipid kinase drug discovery with respect to achievements, binding modes of inhibitors, and novel avenues for the generation of second-generation kinase inhibitors to treat cancers.

Using the reverse protein array platform in combination with planar waveguide technology, which allows detection of proteins in spotted cell lysates with high sensitivity in a 96-well microtiter-plate format for growing, treating, and lysing cells was shown to be suitable for this approach and indicates the usefulness of the technology as a screening tool for characterization of large numbers of kinase inhibitors. In this study, we have used reverse protein arrays to profile kinase inhibitors in various cellular pathways in order to unravel their MoA. Multiplexing and simultaneous analysis of several phospho-proteins within the same lysate allows (1) the estimation of inhibitor concentrations needed to shut down an entire pathway, (2) the estimation of inhibitor selectivity, and (3) the comparison of inhibitors of different kinases within one assay. For example, parallel analysis of p-InsR, p-PKB, p-GSK-3, p-MEK, p-ERK, and p-S6rp in insulin treated A14 cells allows profiling for inhibitors of the InsR, PI3K, PKB, mTor, RAF, and MEK. Selective kinase inhibitors revealed different specific inhibitory pattern of the analyzed phospho-read outs. Altogether, multiplexed analysis of reverse (phase) protein arrays is a powerful tool to characterize kinase inhibitors in a semi-automated low to medium throughput assay format.

The identification and characterization of the members of individual signal transduction cascades, and advances in understanding how these signals are integrated in normal and pathological conditions have provided new strategies for therapeutic intervention. Rapid progress has occurred in last few years in the development of inhibitors that target protein tyrosine kinases (PTKs), enzymes that transfer the γ-phosphate group of adenosine triphosphate (ATP) to the hydroxyl group of tyrosine residues on target proteins. Although PTKs represent a small percentage of the total number of kinases in the “kinome,” 90 of 518, a disproportional number of inhibitors currently in clinical trials are directed against them; e.g., more than 20 different tyrosine kinases are being evaluated as potential targets in oncology. There are a number of reasons why tyrosine kinases have been considered to be good targets. Epistatically, PTKs are located upstream and downstream of tumor suppressor genes or oncogenes and have been demonstrated to play central roles in apoptosis, proliferation, invasion, and differentiation (1). Aberrant activation of tyrosine kinases, owing to mutation or overexpression, is sufficient for them to become transforming in cellular and animal models. The majority of targets are receptor protein tyrosine kinases (RPTKs), as deregulating mutations of over half of the known RPTKs have been associated with different human malignancies; see Table 1 for examples. Finally, and equally as important as the epidemiological and biochemical data, the prevalence of PTKs as targets is because of the fact that they are considered druggable.

The progress made in understanding the molecular basis of mammalian cell transformation has led to the unifying concept of growth regulation and its disorders in cancer cells. Today it is well recognized that many products of "cancer genes" encode for proteins that regulate normal mitogenesis and apoptosis. Taken together, this indicates that the carcinogenic process may be viewed as a progressive disorder of signal transduction (1-6). In fact, many of the genes that are mutated or lost in cancer cells, including both the oncogenes and tumor suppressors, encode proteins that are crucial regulators for intra- as well as intercellular signal transduction (1-6). This conceptual framework has provided a basis for the development of novel anticancer strategies and therapeutic modalities with the aim to inhibit cancer growth either by blocking mitogenic signal transduction or to specifically induce apoptosis of cancer cells. Although these various approaches have not been validated clinically, these strategies are likely to identify compounds with less side effects compared to standard chemotherapeutic agents.