Protein Kinase Inhibitors: The Tyrosine-Specific Protein Kinases

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Abstract

Inhibitors for tyrosine-specific protein kinases ultimately may constitute a novel family of medicinally active agents. Unfortunately, the challenges associated with the acquisition of inhibitors for these enzyme targets are unlike any that have ever been encountered in medicinal chemistry. Protein kinases pose a variety of obstacles in regard to inhibitor design, nearly all of which deal with, in one fashion or another, the issue of specificity. The protein kinase family is extraordinarily large, with estimates that the human genome codes for as many as 2000 protein kinases. Furthermore, inhibitors that are directed to the ATP-binding sites of these enzymes must contend with the presence of a large number of other ATP-utilizing proteins and, in addition, must compete with the high intracellular concentrations of ATP. Although specificity ultimately may prove to be less of a concern with peptide-based inhibitors, these agents neither are readily bioavailable nor do they bind with the requisite affinity to the protein-binding domains of protein kinases. In the face of these challenges, an enormous number of inhibitors have been synthesized and evaluated for the tyrosine-specific protein kinases. The advantages and disadvantages associated with inhibitors that are targeted to the ATP-binding site, the protein-binding site, and nonactive site regions required for appropriate subcellular localization are discussed. The handful of tyrosine-specific protein kinases that have been selected as targets to date and their roles in various disease processes are described as well.

Introduction

Kinase-mediated protein phosphorylation is a crucial component of the signal transduction pathways by which extracellular signaling molecules influence their target cells. Such processes as mitosis are ultimately dependent upon the interaction of extracellular growth factors with their appropriate membrane-embedded receptors, an interaction that is transmitted to the cell nucleus through protein kinases (Lewin 1990). Furthermore, passage of the cell through the individual phases of the cell-division cycle also is regulated by protein kinases. Consequently, it is not surprising that genetic changes, which result in the formation of unregulated and, therefore, constitutively active protein kinases, are frequently responsible for carcinogenesis. Furthermore, over-expression of otherwise normal protein kinases has been strongly tied to specific cancers in humans as well. One might therefore expect that agents which are able to shut down the catalytic activity of these oncogenic or overexpressed protein kinases should block uncontrolled cell growth. Indeed, neu-oncogene transformed cells, when exposed to monoclonal antibodies directed against the neu gene product, exhibit a reversion to a nontransformed phenotype (Drebin et al. 1985). In addition to cancer, protein kinases have now been implicated in a wide variety of other disorders, including Alzheimer’s disease, diabetes, and restenosis. As a consequence, inhibitors of this family of enzymes ultimately may serve as new weapons in our arsenal for battling human disease and suffering.

Section snippets

The challenge: potent protein kinase-specific inhibitors

Protein kinases, and the signal transduction pathways that they constitute, are medicinally attractive targets of opportunity. However, in many ways, the challenges associated with constructing powerful protein kinase-specific inhibitors are daunting:

  • 1.

    The protein kinase family of enzymes is extraordinarily large, with estimates that the mammalian genome codes for as many as 2000 different protein kinases (Hunter 1994). Is it reasonable to expect that inhibitors can be designed that specifically

Biological control of protein kinase specificity

Protein kinases select their in vivo substrate targets with exquisite precision, in spite of the fact that the members of this very large enzyme family are similar in structure, utilize an identical substrate (ATP), and phosphorylate the same limited number of alcohol-containing amino acid residues in proteins. The in vivo substrate specificity of these enzymes is controlled in a number of ways. First, protein kinases are generally divided into two subfamilies, based upon their ability to

Tyrosine kinase targets: a brief introduction

Although the human genome has been estimated to code for as many as 2000 different protein kinases (Hunter 1994), the number of tyrosine kinases that thus far have served as targets for inhibitor design is barely more than a handful. This is partly due to the fact that only a fraction of the estimated total number of protein kinases has been identified, and a significantly smaller fraction is readily available. Furthermore, the biochemical roles played by most tyrosine kinases, not to mention

Inhibitors Competitive with ATP

Yuan et al. 1990 reported that the d-tetrafluorotyrosine (F4Tyr)-containing gastrin analog Arg-Arg-Leu-Glu-Glu-Glu-Glu-Glu-Ala-(d)F4Tyr-Gly acts as an inhibitor of the insulin receptor (InsR) kinase with a Ki of 20 μM. Two years later, quercitin (1) was shown to be an inhibitor of the same enzyme, but with

an IC50 of 2 μM (Shisheva and Shechter 1992). Which compound is most effective at reducing InsR kinase activity? Quercetin appears to be the obvious answer (assuming that an IC50 of 2 μM

Assay conditions: absolute and relative inhibitor potency

The high intracellular levels of ATP can have a profound effect on the absolute concentration of inhibitor required to produce a reduction in protein kinase activity. What other physicochemical properties influence inhibitory efficacy? Hangauer and colleagues (Choi et al., 1996*; see also Nair et al. 1995) addressed this question by assessing the effectiveness of various inhibitors under cellular mimetic and standard literature conditions. These assay conditions differ in a variety of ways. The

Tyrosine-specific protein kinase inhibitors

For earlier reviews on protein kinase inhibitors see Blumberg et al. 1995, Boutin 1994, Brunton and Workman 1993, Burke 1994, Casnellie 1992, Chang and Geahlen 1992, Chorvath and Sedlak 1996, Dobrusin and Fry 1992, Dow 1994, Fry 1994, Fry and Bridges 1995, Hidaka and Kobayashi 1994, Jacquemin-Sablon et al. 1995, Langdon and Smyth 1995, Levitzki and Gazit 1995, MacKintosh and MacKintosh 1995, Pang and Guo 1996, Philip and Harris 1995, Powis 1995, Powis and Alberts 1994, and Shugar 1995.

Summary

Based on the enormous number of compounds that have been developed in the past few years, it is evident that the area of protein kinase inhibitor design is an active one. Indeed, by our count, this field has been covered by more than 20 reviews since 1992. Where do we stand? Clearly, there is the problem of specificity, an issue that has generated significant discussion in previous reviews as well (Boutin 1994). The concern here is self-evident. The overwhelming majority of inhibitors developed

Acknowledgements

Research cited from the authors’ laboratory has been supported by the National Institutes of Health and the Breast Cancer Research Initiative funded by the United States Army Medical Research and Development Command. We apologize, in advance, to those investigators whose work was inadvertently omitted.

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