Current Pharmaceutical Design - Volume 10, Issue 16, 2004

The hematopoietic class III receptor tyrosine kinase (RTK) Flt3 (Flk2, STK1) has recently received much attention as a potential drug target. Activation of Flt3 by different types of mutations plays an important role for proliferation, resistance to apoptosis, and prevention of differentiation of leukemic blasts in acute myeloid leukemia (AML). At least one type of such mutations - an internal tandem duplication in the Flt3 juxtamembrane domain (Flt3-ITD) - has been associated with an unfavorable prognosis. Signal transduction of Flt3 involves activation of several conserved pathways, including the RAS / MAP-Kinase and the phosphoinositide-3-kinase / Akt signaling cascades. Transforming versions of Flt3 exhibit altered signaling, for example a very pronounced activation of STAT5, ultimately resulting in alternate profiles of gene expression and cell transformation. Selective inhibitors of Flt3 tyrosine kinase activity have the potential to suppress aberrant Flt3 signaling. Although highly homologous to other class III RTKs, Flt3 is resistant to the phenylaminopyrimidine STI571 (Gleevec, Imatinib), a potent inhibitor of other RTKs in the family, such as the PDGFb-receptor or c-Kit. STI571 binding to Flt3 is prevented by the phenylalanine 691 side-chain in the ATP binding center and mutating this site to threonine renders the corresponding Flt3 mutant sensitive to STI571. Compounds of several other structural families, including the quinoxaline AG1296, the bis(1H-2-indolyl)-1-methanone D-65476, the indolinones SU5416 and SU11248, the indolocarbazoles PKC412 and CEP-701, and the piperazonyl quinazoline CT53518, are potent inhibitors of Flt3 kinase. They exhibit different selectivity profiles, both with respect to other kinases and among wildtype Flt3 and its activated versions. These compounds hold promise as novel drugs against AML and as probes for understanding activation mechanisms and signaling pathways in the class III RTK family.

MAPK (Mitogen Activated Protein Kinase) pathways mediate fundamental biological processes and have moved into the limelight of drug discovery during the past decade. Here we review the biochemistry and biology of MAPK signalling with a focus on ERK, JNK and p38. We summarise current drug discovery efforts and clinical trials. Further, we critically discuss the rationale behind current strategies of using MAPK pathways as drug targets and suggest new approaches that take the complexity of MAPK signalling networks into account.

The Ras-Raf-MEK-ERK intracellular signaling cascade can be activated in response to a variety of extracellular stimuli. Growth factor binding to extracellular receptors results in activation of Ras, which in turn interacts with and activates Raf, leading to the phosphorylation of the dual specificity kinase MEK (MAP kinase kinase) on two distinct serine residues. MEK possesses a number of unique biochemical and biological features that make it an attractive target from an anticancer drug development perspective. The identification and subsequent testing of highly selective small molecule inhibitors of MEK have served to re-enforce the long held belief that the MEK / ERK module plays a critical role in controlling a number of cellular events that are critical to tumor cell growth and survival. We have witnessed advancement of the first MEK-targeted clinical drug candidate into clinical trials with the entry of CI-1040. The evaluation of sufficiently potent and selective MEK inhibitors in well-designed clinical trials is critical for ultimate validation of MEK as a molecular-based anticancer drug target.

Phosphoinositide 3-kinases (PI3K) represent a family of intracellular signaling proteins, which control a variety of important cellular functions such as proliferation, apoptosis and migration. Recent findings suggest an involvement of PI3K in the pathogenesis of numerous diseases including cancer, heart failure and autoimmune / inflammatory disorders. This review summarizes the current knowledge of the emerging therapeutic value of PI3K as targets for the intervention of several pathological disorders and in particular focusing on oncogenesis. A brief introduction on the molecular and biochemical features of these signaling proteins will be followed by a depiction of signaling interactions of PI3K in a cellular context. PI3K dependent signaling involved in the control of cell growth, proliferation, survival and cytoskeletal remodeling and the link to cellular dysfunctions will be discussed. Further we will summarize the phenotypic consequences by genetic targeting PI3K signaling in mice. In its final part this review outlines challenges and activities considering PI3K as targets for therapeutic intervention and progress in the development of first generation small molecule inhibitors.

As our understanding of tumorigenesis increases, interference with the various signaling pathways of tumor cells has become an attractive approach to arresting tumor cell growth and overcoming chemoresistance. Among many intracellular signaling proteins, protein kinase C (PKC) isoenzymes have been identified as possible targets to render tumor cells more susceptible to apoptosis and growth arrest. We review the known biology of the α-isoenzyme of PKC in different cancers to provide a rational approach for developing targeted therapies using PKC modulators, including aprinocarsen, an antisense oligonucleotide (ASO) against PKC-α.

In recent years, new strategies in cancer therapy have been developed targeting key signaling molecules in the receptor tyrosine kinase signal transduction pathway. In contrast, most therapeutical concepts to manipulate G protein-coupled receptors (GPCR)-mediated disorders are still limited to the use of receptor-specific agonists or antagonists. Visible progress in the understanding of GPCR signaling complexity, especially the detection of several families of highly target- and cell-specific regulator proteins of GPCRs, G proteins, and effector components may open new horizons to develop novel therapeutical concepts targeting GPCR signaling elements. Thus, this review will focus on different molecular levels that may be of particular interest in terms of new drug development such as: (i) GPCR subtypes, allosteric binding sites, dimerization and constitutive activity, the use of RAMPs (receptor-activity-modifying proteins) and RASSLs (receptor activated solely by synthetic ligands); (ii) AGS (activators of G protein signaling) and RGS (regulators of G protein signaling) proteins which modify G protein activity; (iii) the high diversity of isozymes involved in the generation, signal transmission, and degradation of second messenger molecules.

Gene expression profiling has become a versatile tool for biomedical research, which allows the assessment of a wide variety of basic questions in cellular regulation, in particular when a large number of molecular parameters have changed. There are various applications in drug research for which gene expression profiling is a very suitable approach. This includes: target identification, target validation, validation of drug specificity and monitoring of drug action during therapy. The focus of this article is the therapy monitoring and the interpretation of the gene expression profiles in respect to physiological differences of drug action. As an example, we will discuss changes in gene expression in blood samples from CML patients treated with the tyrosine kinase inhibitor (imatinib mesylate) and compare the observed effects on gene expression with the effects of IFNa treatment. In comparison with other examples of therapy monitoring the potential of this application of gene expression profiling for optimizing individual therapy will be discussed.

Reducing tumor load by therapeutic induction of cell death in the transformed phenotype is the desirable goal of most chemotherapeutic regimens. Despite the tremendous strides made in our understanding of mechanisms that endow tumor cells with the ability to evade execution signals, development of chemo-resistance is still a major obstacle in the successful management of the disease. A host of factors have been implicated in the acquisition of the resistant phenotype, such as activation of drug efflux pumps [1], overexpression of proteins that inhibit cell death [2-4], absence of critical members of the death circuitry [5, 6], and selective loss of cell cycle checkpoints [7]. Consequently, it is now well established that the process of carcinogenesis is not only a result of an increase in cells' proliferative capacity, but a product of increased proliferation and defective or diminished cell death signaling. To that end, one of the critical determinants of cellular response to exogenous stimuli is the cellular redox status. Intracellular generation of reactive oxygen species (ROS) is tightly regulated by the intrinsic anti-oxidant defense systems. Despite the conventional dogma that ROS are harmful to the cell, experimental evidence over the last decade or so bear witness to the fact that ROS also play an important role as signaling molecules in diverse physiological processes. Indeed, low levels of intracellular ROS have been linked to cellular proliferation and cell cycle progression, which provides an explanation for the pro-oxidant state invariably associated with the transformed phenotype. Coupled to that are recent observations implicating pro-oxidant intracellular milieu in tumor cells' resistance to cell death signals delivered through the cell surface receptor or upon exposure to chemotherapeutic drugs. These studies provide convincing evidence to support a direct or indirect role for intracellular superoxide anion in creating an intracellular milieu nonpermissive for cell death execution. Thus a novel approach to enhancing tumor cell sensitivity to chemotherapy-induced cell death would be to favourably tailor the cytosolic milieu to allow efficient apoptotic execution. Here we present a brief discussion on the role of ROS in cell growth and differentiation, and more specifically address the issue of chemoresistance from the standpoint of cellular redox status.

Basic studies on angiogenesis in normal and pathologic conditions, as well as research on drugs or genes / proteins that stimulate or regulate the angiogenic process, can rely on an increasing number of experimental models. Among non-mammalian models, Zebrafish is adopted by an increasing number of research groups. Moreover, angiogenesis and vasculogenesis in invertebrates like the leech Hirudo medicinalis share a high degree of similarity with the same processes occurring in humans, both under the structural / functional and biochemical points of view. Interestingly, Hirudo angiogenic growth factor receptors respond to corresponding human / mammalian recombinant growth factors and cytokines; in addition, Hirudo endogenous angiogenic growth factors and receptors react with antibodies against their human / mammalian counterparts. Furthermore, as it will be shown in this review, Hirudo has the unique advantage of having a virtually avascular muscular body wall, whereas the reliability of such a peculiar feature as a model for physiologically vascularised mammalian tissues has to be thoroughly investigated. Hirudo has proven so far to allow unambiguous, clear-cut studies on the angiogenic potential of gene-products or drugs, as well as on the antiangiogenic compounds. This article will review the biology of angiogenesis in Hirudo and the data so far collected on angiogenesis stimulation / modulation in this model; an example describing a study on the biological activity of a naked DNA vector for angiogenesis gene therapy will also be provided.