Current Pharmaceutical Design - Volume 16, Issue 35, 2010

Angiogenesis has been described as one of the hallmarks of cancer, playing an essential role in tumor growth, invasion and metastasis. Antiangiogenic therapy was initially perceived as a “magic bullet” that could eventually be used for the treatment of any type of cancer. For this reason inhibition of angiogenesis has become a major challenge in the development of new anticancer agents, with a countless number of antiangiogenic strategies being tested in preclinical and clinical trials. Recent progress in combining antiangiogenic drugs, mainly acting on the VEGF/VEGFR (Vascular endothelial growth factor/ Vascular endothelial growth factor receptor) pathway, with chemotherapy and other conventional therapies are discussed in this issue. Strategies for the optimization of combination therapy and the selection of appropriate treatment regimens are examined. As new drugs are entering clinical trials, reliable biomarkers are needed to stratify patients for antiangiogenic therapy, to identify resistant patients and to monitor response to treatment. Moreover, recent experimental findings suggest that some antiangiogenic drugs could promote tumor invasiveness and metastasis. The success in the discovery and pharmacological development of future generations of angiogenesis inhibitors will benefit from further advances in the understanding of the mechanisms involved in human angiogenesis. There is a need of more molecules to be tested on and a special chapter dealing with recent developments in high-content screening (HCS) technologies, that represents an attractive alternative for anti-angiogenic drug discovery, is reported in the issue. HCS integrates highthroughput methodologies with automated multicolor fluorescence microscopy to collect quantitative morphological and molecular data from complex biological systems are also presented. The complex role of oxidative stress and redox signaling in cancer neovascularization have been reported on a process without which the tumor is unable to grow beyond few millimeters in size. Reactive oxygen species and nitric oxide affect cell responses to hypoxia, a major trigger of angiogenic switch in tumors and are important upstream regulators as well as downstream mediators of action of the most potent proangiogenic factor - vascular endothelial growth factor. Modulation of redox species production, signaling and metabolism and/or manipulating cellular antioxidant responses represents a multitargeted therapeutic approach which may possibly overcome the limitations of single-agent antiangiogenic treatments and potentiate effects of standard methods. The role of chemokines has been also discussed together with the chemokine stromal cell-derived factor-1 (SDF-1)/CXCL12 represents the single natural ligand for the chemokine receptor CXCR4. CXCL12 possesses angiogenic properties and is involved in the outgrowth and metastasis of CXCR4-expressing tumors and in certain inflammatory autoimmune disorders, such as rheumatoid arthritis. Here, we discuss the different aspects of CXCL12/CXCR4 biology as well as the development and anti-cancer/stem cell mobilizing activity of CXCR4 antagonists. In this issue, we indicate some key examples showing how alternative splicing decision may induce a switch from anti-angiogenic to proangiogenic functions and reciprocally. For some of these splicing events, the molecular mechanisms that trigger alternative splicing toward one or the other direction start to be elucidated. The emergence of strategies enabling to regulate alternative splicing opens new routes for anti-angiogenic therapies. We also add to the issue some interesting concepts on vascular smooth muscle cells (VSMC) respond to arterial wall injury by intimal proliferation and play a key role in atherogenesis by proliferating and migrating excessively in response to repeated injury, such as hypertension and atherosclerosis. In contrast, fully differentiated, quiescent VSMC allow arterial vasodilatation and vasoconstriction. Exaggerated and uncontrolled VSMC proliferation appears therefore to be a common feature of both atherosclerosis and hypertension. Attention is focused on subfamilies of MAPKs (Mitogen-activated protein kinases), the extracellular signal regulated kinases (ERKs). An overview of the work on ERKs 1 to 2, emphasizing when possible their biological activities in VSMC proliferation, is presented in this issue. It is clear from numerous studies, that ERK1/ERK2 pathway has an important role in VSMC proliferation induced by insulin (INS) and thrombin, even if thrombin regulation of VSMC's proliferation remains poorly understood. A chapter dedicated to Pim1 is also considered in this issue together with Human Pim1 (proviral integration site for Moloney murine leukemia virus) kinase is a 313-amino acid serine-threonine kinase that possesses several biological functions in cell survival, proliferation and differentiation, and its overexpression has been observed in a number of human cancers. This chapter is a clear example how the use of molecular modeling techniques is largely used for the identification and optimization of proteins inhibitors. This data collection, which to the best of our knowledge was not previously reviewed in such detail, could offer a useful tool for medicinal chemists working in the field of small molecule kinase inhibitors. This issue has been concepted inside the EU ‘COST ACTION CM0602 - ANGIOKEM: Inhibitors of angiogenesis: Design, synthesis and biological exploitation’. Anna Di Donna (University of Siena - Italy) is acknowledged for her help in collecting the manuscripts for the Guest Editor.

In eukaryotes, genes consist in coding sequences (exons) interspersed with non-coding ones (introns). The regulation of alternative inclusion/exclusion of exons, or part of exons, during the maturation of the pre-mRNA into mRNA (alternative splicing) allows a dramatic increase of the protein versus the gene repertoire. In a number of cases, alternative splicing decision generates proteins with distinct, sometimes opposite, functions from a given gene. Angiogenesis is the process of vascularisation in physiological conditions and a series of pathologies, including cancer where it favours tumour progression and dissemination of metastasis. In this issue, we discuss some key examples showing how alternative splicing may induce a switch from anti-angiogenic to pro-angiogenic functions reciprocally. For some of these splicing events, the molecular mechanisms that trigger alternative splicing toward one or the other direction start to be elucidated. The emergence of strategies enabling to regulate alternative splicing opens new routes for anti-angiogenic therapies.

Historically, oxidative stress was recognized to contribute to cancer development uniquely by induction of genomic instability. However, recent research has provided multiple evidence that reactive oxygen species and other free radicals, such as nitric oxide, often produced at elevated levels within tumor tissue, may function as signaling molecules that initiate and/or modulate the different regulatory pathways involved in tumorigenesis and metastasis. This review will focus on the complex role of oxidative stress and redox signaling in cancer neovascularization, a process without which the tumor is unable to grow beyond few millimeters in size. Reactive oxygen species and nitric oxide affect cell responses to hypoxia, a major trigger of angiogenic switch in tumors and are important upstream regulators as well as downstream mediators of action of the most potent proangiogenic factor - vascular endothelial growth factor. We will discuss targeting the redox-regulated mechanisms for antiangiogenic anticancer therapy and focus on recent developments in small-molecule agents that have either completed clinical trials or show a great promise to be subjected to them. Modulation of redox species production, signaling and metabolism and/or manipulating cellular antioxidant responses represents a multitargeted therapeutic approach which may possibly overcome the limitations of single-agent antiangiogenic treatments and potentiate effects of standard methods.

Vascular smooth muscle cells (VSMC) respond to arterial wall injury by intimal proliferation and play a key role in atherogenesis by proliferating and migrating excessively in response to repeated injury, such as hypertension and atherosclerosis. In contrast, fully differentiated, quiescent VSMC allow arterial vasodilatation and vasoconstriction. Exaggerated and uncontrolled VSMC proliferation appears therefore to be a common feature of both atherosclerosis and hypertension. Signal transduction pathways in eukaryotic cells integrate diverse extracellular signals, and regulate complex biological responses such as growth, differentiation and death. One group of proline-directed Ser/Thr protein kinases, the mitogen-activated protein kinases (MAPKs), plays a central role in these signalling pathways. Much attention has focused in recent years on subfamilies of MAPKs, the extracellular signal regulated kinases (ERKs). Here we overview the work on ERKs 1 to 2, emphasising when possible their biological activities in VSMC proliferation. It is clear from numerosus studies including our own, that ERK1/ERK2 pathway has an imoprtant role in VSMC proliferation induced by insulin (INS) and thrombin. Despite the physiological and pathophysiological importance of INS and thrombin, possible signal transduction pathways involved in INS and thrombin regulation of VSMC's proliferation remains poorly understood. Thus, this review examines recent findings in signalling mechanisms involved in INS and thrombin- triggered VSMC's proliferation with particular emphasis on ERK1/2 signalling pathways. Future investigations should now focus on the mechanisms of MAPK activation which might therefore represent a new mechanism involved in the antiproliferative effect revealed in this review.

Chemokines are key players in the attraction and activation of leukocytes and are thus implicated in the recruitment of immune cells at sites of infection and/or inflammation. They exert their action by binding to seven-transmembrane G protein-coupled receptors. The chemokine stromal cell-derived factor-1 (SDF-1)/CXCL12 represents the single natural ligand for the chemokine receptor CXCR4. CXCL12 possesses angiogenic properties and is involved in the outgrowth and metastasis of CXCR4-expressing tumors and in certain inflammatory autoimmune disorders, such as rheumatoid arthritis. CXCR4 expression on tumor cells is upregulated by hypoxia and angiogenic factors, such as vascular endothelial growth factor (VEGF). CXCR4 also acts as a co-receptor for entry of human immunodeficiency virus (HIV) in CD4+ T cells. Finally, CXCL12/CXCR4 interactions were shown to play an important role in the migration of hematopoietic stem cells and their progenitors from, and their retention within, the bone marrow, a site characterized by high CXCL12 expression. As such, CXCR4 inhibitors may be utilized to inhibit HIV-1 infection, tumor growth and metastasis and to mobilize hematopoietic stem cells from the bone marrow in the circulation, where they can be collected for autologous stem cell transplantation. Here, we discuss the different aspects of CXCL12/CXCR4 biology as well as the development and anti-cancer/stem cell mobilizing activity of CXCR4 antagonists.

Angiogenesis is associated with tumor development and malignancy and is a validated target for cancer treatment. Preclinical and clinical evidence substantiates the feasibility of combining angiogenesis inhibitors with conventional anticancer therapy. This review discusses recent progress in combining antiangiogenic drugs, mainly acting on the VEGF/VEGFR pathway, with chemotherapy and other conventional therapies. Strategies for the optimization of combination therapy and the selection of appropriate treatment regimens are examined. As new drugs are entering clinical trials, reliable biomarkers are needed to stratify patients for antiangiogenic therapy, to identify resistant patients and to monitor response to treatment.

Angiogenesis has been described as one of the hallmarks of cancer, playing an essential role in tumor growth, invasion, and metastasis. Antiangiogenic therapy was initially perceived as a “magic bullet” that could eventually be used for the treatment of any type of cancer. For this reason inhibition of angiogenesis has become a major challenge in the development of new anticancer agents, with a countless number of antiangiogenic strategies being tested in preclinical and clinical trials. The initial pessimism about the usefulness of the antiangiogenic therapeutic approach for cancer, derived from the poor results obtained in clinical trials, turned into euphoria after the approvals of the anti-VEGF monoclonal antibody bevacizumab and the multitargeted tyrosine kinase inhibitors sunitinib, sorafenib and pazopanib. Nowadays the clinical development of antiangiogenic therapies seems to be unstoppable, not only for cancer, but also for an increasing number of non-neoplasic angiogenesis-related diseases. Nevertheless, careful analysis of the clinical results reveals that therapy with angiogenesis inhibitors often does not prolong survival of cancer patients for more than months. This fact, combined with the high prices of the new antiangiogenic therapies have made a number of oncologists to doubt if they offer “good value”. Moreover, recent experimental findings suggest that some antiangiogenic drugs could promote tumor invasiveness and metastasis. The success in the discovery and pharmacological development of future generations of angiogenesis inhibitors will benefit from further advances in the understanding of the mechanisms involved in human angiogenesis.

Recent developments in high-content screening (HCS) technologies make it an attractive alternative for anti-angiogenic drug discovery. HCS integrates high-throughput methodologies with automated multicolor fluorescence microscopy to collect quantitative morphological and molecular data from complex biological systems. Organotypic systems based on primary vascular cells model many facets of angiogenesis. The adaptation of these complex in vitro assay systems to high-throughput HCS formats with automated image acquisition enables large-scale chemical library screening campaigns. These HCS principles can be extended further to allow small molecule compounds in in vivo model organisms such as zebrafish. In this review we discuss the latest developments within automated imagebased high-throughput screening of chemical libraries for anti-angiogenic compounds.

Human Pim1 (proviral integration site for Moloney murine leukemia virus) kinase is a 313-amino acid serine-threonine kinase that possesses several biological functions in cell survival, proliferation and differentiation, and its overexpression has been observed in a number of human cancers. Indeed, this kinase is a proto-oncogene that has been implicated in early transformation and tumor progression, especially in hematopoietic malignancies and prostate carcinoma where it is a marker of a poor prognosis. For these reasons, Pim1 is emerging as an important target in drug discovery, and many Pim1 inhibitors have been reported in the last three years. The challenge of this research is to obtain compounds that specifically inhibit only Pim1 and not Pim2 and Pim3, the other members of the Pim family, with the aim of providing selective inhibitors as potential therapeutic agents and also of studying the different roles of the three enzymes. In this review Pim1 functions and Pim1 role in human cancer are summarized, but the primary focus of the article is on the Pim1 threedimensional structure that was deeply analyzed by a detailed inspection of the available crystallographic data and all complexes of small molecule inhibitors reported in the literature to this point. Finally, the use of molecular modeling techniques for the identification and optimization of Pim1 inhibitors is extensively discussed. This data collection, which to the best of our knowledge was not previously reviewed in such detail, could offer a useful tool for medicinal chemists working in the field of small molecule kinase inhibitors.