Current Drug Targets - Volume 7, Issue 4, 2006

In continuatin of the previous issue, part II of the issue "Small molecules of natural origin for cancer therapy and chemoprevention" comprises of the following chapters. 3. From Pharmacogenomics and -proteomics to systems biology The recently developed genomic and proteomic technologies are thriving and fertilize many disciplines of life sciences, including pharmaceutical biology and pharmacognosy. The prospect is fascinating to gain mechanistic insights, how small molecules from natural origin act against cancer cells. The next wave - the construction of signalling and metabolic pathways with the help of data obtained from "-omics" technologies - is the task of systems biology, which will add another dimension to current pharmacology. Gabriele Zybarth and Nikolai Kley (Waltham, MA, USA) report the recent achievements in the field of genomics and proteomics, e.g., genome-wide transcription-profiling, genotyping of polymorphisms to predict drug efficacy, knock-out technology, chemical proteomics, target engineering, drug resistance and others. The chemical genomics-based target identification using anti-angiogenic agents as example is described by Ho Jeong Kwon (Seoul, Korea). Small molecules chemically synthesized or taken from natural sources are used to elucidate biological mechanisms and as lead compounds for the directed optimization of chemical compounds with drug-like properties. Sophisticated bioinformatics tools are applied to make this approach rapid and efficient. In analogy to classical genetics, one can distinguish forward from reverse chemical genomics. The forward approach aims to identify small molecule-induced cellular phenotypes and molecular target proteins affected. By reverse genomics, validated target proteins are used to searches for small molecules, which modulate the function of the target molecules. The article on artemisinin and its derivatives by Thomas Efferth (Heidelberg, Germany) gives a comprehensive overview of molecular pharmacology and pharmacogenomics of this drug class for anti-cancer treatment. Artemisinin is the active principle of the Chinese herb, Artemisia annua L., which shows profound cytotoxicity to cancer cells in addition to its anti-malarial properties. 4. Chemoprevention Chemopreventive agents inhibit the development of cancer, whereas chemotherapeutics are used to treat already established cancers. Since mechanisms of carcinogenesis frequently affect effectiveness of chemotherapy too, compounds which prevent carcinogenesis might also improve responsiveness of tumor cells to anti-tumor drugs. Dominique Delmas, Allan Lançon, Didier Colin, Brigitte Jannin and Norbert Latruffe (Dijon, France) report on resveratrol as promising chemopreventive agent. The beneficial effects of wine are associated with the physiological protection conferred by phenolic compounds such as resveratrol. It is a chemopreventive compound with multi-facetted features. It acts anti-carcinogenic, anti-angiogenic, anti-invasive, anti-metastatic, activates programmed cell death, arrests the cell cycle, and inhibits signal transduction pathways. Whereas it is not cytotoxic itself, it enhances the anti-tumor effect of anticancer drugs. Melinda C. Myzak and Roderick H. Dashwood (Corvallis, OR, USA) give an introduction in the field of histone deacetylases as targets for dietary cancer preventive agents and explain the relevance of aberrant histone acetylation for tumorigenesis. The authors also introduce the reader to histone deacetylase inhibitors of natural origin with chemopreventive activity and point to molecular mechanisms of action. Three examples are discussed in more detail, butyrate, diallyl disulfide, and sulphoraphane, all of which are constituents of relevant nutrients. 5. Biotechnology: plant cell culture for sustainable production of plant-derived drugs The knowledge on natural compounds and their molecular modes of action in cancer cells represents one side of the coin. The other side is the production of drugs for treatment of patients. There is an increasing demand in medicinal plants worldwide, most of which are collected from the wild. Although cultivation of medicinal plants is getting.........

The genomics and proteomics sciences have fundamentally changed the ways in which drug targets are being identified, characterized and validated. Here we review how genomics and proteomics research is improving our understanding of genetic determinants of drug susceptibility and response and, conversely, how organic small molecules mediate their pharmacological effects by modulating genome and proteome activities. We also examine the effect this improved understanding has on the drug discovery and development process.

Chemical genetics/genomics is an inter- and multi-disciplinary research engine, which utilizes small molecules to explore the function of genes and accelerate the drug discovery. Bioactive small molecules that are permeable to cellular membrane and bind to its cognate target protein can exert the phenotype changes of the cells or organisms. Functional target proteins of these small molecules have been successfully identified by affinity, genetics and genomics based target identification. The specific molecular recognition of small molecule with target protein has facilitated to decipher the mode of actions of small molecules and developed better drug based on their structure activity relationship. Based on this idea, we have applied chemical genomics to angiogenesis, a new blood vessel formation, resulting in the identification of new small molecules as well as targets. In this review, our application of chemical genomics towards a cellular phenotype, angiogenesis, will be demonstrated.

Secondary metabolites from plants can serve as defense against herbivores, microbes, viruses or competing plants. Many compounds from medicinal plants have pharmacological activities and thus may be a source for novel antitumor agents. We have analyzed natural products from traditional Chinese medicine during the past decade and focused our interest on the compound artemisinin from Artemisia annua L. (qinghao, sweet wormwood) and its derivatives. In addition to their anti-malarial properties, artemisinins are cytotoxic for cancer cells. The present review focuses on the mechanisms of action of artemisinins in cancer cells relating to: 1. anti-proliferative and anti-angiogenic effects, 2. induction of apoptosis, 3. oxidative stress, 4. oncogenes and tumor suppressor genes, and 5. multidrug resistance. Data on putative target molecules of artemisinins are presented and discussed, e.g. the translationally controlled tumor protein (TCTP). Emphasis is given to pharmacogenomic approaches to analyze the pleiotropic nature of mechanisms of artemisinins in cancer cells.

Resveratrol (3,4',5 tri-hydroxystilbene) is a phytoalexin produced in hudge amount in grapevine skin in response to infection by Bothrytis cinerea. This production of resveratrol blocks the proliferation of the pathogen, thereby acting as a natural antibiotic. Numerous studies have reported interesting properties of trans-resveratrol as a preventive agent against important pathologies i.e. vascular diseases, cancers, viral infection or neurodegenerative processes. Moreover, several epidemiological studies have revealed that resveratrol is probably one of the main microcomponents of wine responsible for its health benefits such as prevention of vaso-coronary diseases and cancer. Resveratrol acts on the process of carcinogenesis by affecting the three phases: tumor initiation, promotion and progression phases and suppresses the final steps of carcinogenesis, i.e. angiogenesis and metastasis. It is also able to activate apoptosis, to arrest the cell cycle or to inhibit kinase pathways. Interestingly, resveratrol does not present any cytotoxicity in animal models. Moreover, concentrations of resveratrol in blood seem to be sufficient for anti-invasive activity. The enterohepatic recirculation may contribute to a delayed elimination of the drug from the body and bring about a prolonged effect. By its binding to plasmatic proteins, resveratrol also exhibits a prolonged effect. Interestingly, low doses of resveratrol can sensitize to low doses of cytotoxic drugs and so provide an innovative strategy to enhance the efficacy of anticancer therapy in various human cancers. By these properties, resveratrol appears to be a good candidate in chemopreventive or chemotherapeutic strategies and is believed to be a novel weapon for new therapeutic strategies.

Cancer is a multi-factorial process involving genetic and epigenetic events which result in neoplastic transformation. Reversal of aberrant epigenetic events, including those that modulate the transcriptional activity of genes associated with various signaling pathways, holds the prospect of influencing multiple stages of tumorigenesis. Perturbation of normal histone acetylation status can result in undesirable phenotypic changes, including developmental disorders and cancer. Indeed, aberrant histone acetylation may be an etiological factor in several, if not all, types of cancer. In general, histone acetylation leads to chromatin remodeling and a de-repression of transcription. Histone deacetylase (HDAC) inhibitors may be useful for cancer prevention and therapy by virtue of their ability to 'reactivate' the expression of epigenetically silenced genes, including those involved in differentiation, cell cycle regulation, apoptosis, angiogenesis, invasion, and metastasis. Several natural and synthetic HDAC inhibitors have been shown to affect the growth and survival of tumor cells in vitro and in vivo. Interestingly, three dietary chemopreventive agents, butyrate, diallyl disulfide, and sulforaphane, also have HDAC inhibitory activity. This review discusses the role of aberrant histone acetylation in tumorigenesis and describes the potential for cancer chemoprevention and therapy with a particular emphasis on dietary HDAC inhibitors.

Medicinal plants are the most promising source for the development of drugs, and many types of active ingredients from the plant resources have been studied in order to clarify the relationship between the chemical structure and the activity. However, it is not easy to develop drugs from those active compounds, and in many cases, the supply of active compounds can have some problems: 1) limited quantity of active compounds in plant; 2) low plant growth rate; 3) the limited localization of active ingredients in the specific organs; and 4) from the perspective of the conservation of natural resources. Therefore, the stable supply of the compounds commercially is very difficult and contains risk hedge. Plant cell culture is an attractive technology to solve these problems by securing the stable supply of the active compounds without damage to the natural plant resources. Recently, an efficient production process of anticancer drug paclitaxel by Taxus cell suspension cultures was constructed. The established Taxus cell lines produced paclitaxel and related taxanes by specific external stimuli, such as methyl jasmonate. The time-course analysis revealed that there are two regulatory steps existing in the paclitaxel biosynthesis: the taxane-ring formation step that is up-regulated by MeJA, and the acylation step at the C-13 position. By applying the data from the two-stage culture and the high-density culture, a large-scale culture process was developed with a stable paclitaxel production in the range of 140-295 mg L-1, reaching 295mg L-1 at maximum.

Fungal infections constitute an ever-growing and significant medical problem in the world. The major fungal pathogens affecting humans include Candida species (such as C. albicans, C. glabrata, C. parapsilosis and C. tropicalis) and Aspergillus spp. (mainly A. fumigatus). Fungal diseases include simple toe nail infections, deepseated and recurrent vaginitis, as well as life-threatening systemic mycoses in patients with impaired immune systems. Hence, individuals with organ or bone marrow transplants, acquired immune deficiency syndrome, those undergoing intensive chemotherapy regimens, neonates and infants, as well as long-term hospitalized and intensive care unit patients are at very high risk to contract fungal infections. Candida species are the most frequent causes, representing the fourth leading cause of hospital-acquired diseases. Several hundred thousand cases are reported worldwide every year, about 70% of which are caused by Candida spp., and 20% by Aspergillus spp. Aspergillosis, affecting up to 25% of all leukemic patients, is the most frequent invasive mould worldwide with a mortality exceeding 90%. The overall mortality of about 35% just for candidemias, exceeds that of all Gram-negative bacterial septicaemias. Moreover, the incidence of systemic infections is rising, partly because improved clinical procedures are also leading to better survival of critically ill patients. The consequences and costs for health-care budgets are enormous, also since fungal infections significantly extend the average length of patient stays in hospitals. The stunning high mortalities of aspergilloses and candidemias are in part due to a lack of accurate and speedy diagnostic tests, but also because current antifungal therapies are not always effective enough. Diagnosis is further complicated by emergence of new and less prevalent pathogens, with Cryptococcus neoformans, Trichophyton spp., Microsporum spp. Epidermophyton spp. and Fusarium spp acting as the main culprits. Taken together, systemic mycoses are serious medical conditions, demanding permanent attention not only in a clinical setting, but also in antifungal drug discovery, as well as in basic research addressing the molecular pathology and mechanisms of disease progression. Therefore, this special issue, which includes six chapters, provides a comprehensive overview on the biology and pathology of major fungal pathogens, covering all aspects from epidemiology to molecular mechanisms of antifungal resistance and pathogenicity. One chapter shall discuss epidemiology related to the prevalence, distribution and frequency of fungal infections and their causative species. Another chapter will delineate state-ofthe- art methods for the clinical diagnosis and therapy of mycoses in various clinical settings. Further, the cell wall is a barrier not only protecting fungi, but also mediating adhesion to host cells, thereby enabling subsequent tissue penetration. Hence, one chapter shall discuss the role of cell wall and its components in pathogenicity, as well as suitability as target for drug discovery. Tissue penetration, invasion, the bypass of a residual immune surveillance and spread are major steps during manifestation of systemic disease, yet we have a limited understanding of how mycoses actually progress in vivo. Thus, one chapter describes the molecular routes of host cell invasion, including a description of animal models used for studies on pathogenicity. Moreover, biofilms, as formed by certain pathogens, can constitute serious clinical complications, since they display intrinsic resistance to antifungal drugs. Approaches to better understand their fomation and role in systemic disease are therefore discussed in another chapter. Finally, a chapter on molecular mechanisms of antifungal resistance phenomena, including those caused by membrane-bound drug efflux pumps, will complete this special section on the biology and pathology of fungal pathogens. Since all chapters were written by leaders in the respective field, I am confident that this all-in-one issue will be attractive for students, researchers, newcomers, as well as clinicians working in the field of infectious diseases caused by human fungal pathogens.

Fungal pathogens of the genus Candida form biofilms on catheters and prosthetic devices. These threedimensional structures composed of yeast and hyphal cells embedded in an extracellular matrix constitute an important pitfall in the management of disseminated Candida infections because of their intrinsic resistance to almost all antifungals in clinical use. Candida biofilms are especially resistant to azoles and amphotericin B but remain sensitive to the newly introduced echinocandins that target cell wall β-glucan biosynthesis. Antifungal resistance of biofilms results most probably from the conjunction of several mechanisms that act in a time-dependent manner. While drug efflux is likely to contribute to resistance during the early phases of biofilm formation, changes in the sterol composition of membranes might explain the resistance of mature biofilms. The original physiology of mature Candida biofilms is mirrored by specific gene expression patterns that may pinpoint genes important for the acquisition of pleiotropic antifungal resistance.

Pleiotropic drug resistance (PDR) is a well-described phenomenon occurring in fungi. PDR shares several similarities with processes in bacteria and higher eukaryotes. In mammalian cells, multidrug resistance (MDR) develops from an initial single drug resistance, eventually leading to a broad cross-resistance to many structurally and functionally unrelated compounds. Notably, a number of membrane-embedded energy-consuming ATP-binding cassette (ABC) transporters have been implicated in the development of PDR/MDR phenotypes. The yeast Saccharomyces cerevisiae genome harbors some 30 genes encoding ABC proteins, several of which mediate PDR. Therefore, yeast served as an important model organism to study the functions of evolutionary conserved ABC genes, including those mediating clinical antifungal resistance in fungal pathogens. Moreover, yeast cells lacking endogenous ABC pumps are hypersensitive to many antifungal drugs, making them suitable for functional studies and cloning of ABC transporters from fungal pathogens such as Candida albicans. This review discusses drug resistance phenomena mediated by ABC transporters in the model system S. cerevisiae and certain fungal pathogens.

Candida albicans is a commensal organism that lives as benign member of the microflora of healthy individuals. In response to changes in the host immune status or microflora, C. albicans ceases to be a commensal organism and infects a variety of host tissues. The capacity to shift from a commensal to pathogenic state requires a coordinated metabolic response that triggers discrete developmental programs and that induce the expression of specific virulence traits. Several virulence traits have been described in C. albicans including adhesion, morphological and phenotypic switching, and the production of secreted hydrolytic enzymes. These attributes contribute to host tissue recognition, tissue invasion and colonization, as well as evasion of the host immune response. Recent experimental progress has illuminated some of the cellular processes that enable Candida cells to sense and respond to changes in the host environment. Similarly, cells of the host innate immune system are able to recognize invading C. albicans cells and induce a complex immune response that ultimately determines the clinical outcome of the infection. In this review we describe the current understanding of the events taking place during systemic infections of C. albicans. The interplay between defined pathogen and host specific responses are discussed. Additionally, we provide experimental data on the pathological consequences resulting from acute systemic infections of C. albicans in mice.

Infections of the skin and the mucous membranes due to Candida species may occur either in immuncompromised or in non-immuncompromised patients. This is in contrast to systemic candidiasis (e.g. candidemia) which is only seen in severely immunocompromised patients. Bloodstream infections caused by Candida species are increasingly recognized in critical ill adult and pediatric individuals, with significant associated morbidity and mortality. Candida albicans is the single most common fungal species causing nosocomial infections. However, non-Candida albicans spp., including fluconazole-less-susceptible Candida glabrata, have become more common pathogens. In some patient populations such as hematological (neutropenic) patients Non-C. albicans species are detected much more frequently as compared to non-neutropenic patients in the intensive care. Non-C. albicans species are more likely to occur in patients, who receive or have received antifungal therapy with azoles (e.g. fluconazole). In this review the current epidemiological trends in mucosal and invasive candidiasis are discussed with regard to the role of non-Candida albicans species as the causative agent in immunocompromised patients.

The cell wall of fungi is a highly complex structure consisting of a network of polysaccharides in which a plethora of different proteins are embedded. It is one of the major organelles of the cell surrounding it like an armor which protects from environmental stresses like osmotic pressure and defines the shape and physical strength of the fungal cell. It is crucial for colonization and infection since it defines the interface between host and pathogen. No similar structure is present in the host, therefore it defines a prime target for drug development. In this context, it has been shown that cell surface proteins are required for adhesion to host cells. The fact, that both pathogenic fungi, like Candida albicans as well as non-pathogenic fungi, like Saccharomyces cerevisiae, in general, have a very similar polysaccharide structure but differ significantly in their protein composition which underscores the importance of cell wall proteins for pathogenesis. However, cell wall proteomics of fungi is a highly challenging task due to the complex biochemistry of these proteins. The extensive post-translational modifications and covalent attachment to the polysaccharide backbone of a large proportion of cell wall proteins makes it a demanding task to isolate and identify them. In this article, we describe the recent approaches that have been developed to describe cell wall dynamics and to isolate and identify cell wall proteins in the pathogenic yeast C. albicans.

Diagnosing fungal infections remains a problem, particularly in the immunocompromised patient. Symptoms are mostly non-specific and colonization is difficult to distinguish from invasive disease. Existing diagnostic tools often lack sensitivity. Thus, the combination of various diagnostic tools is mandatory to allow earlier diagnosis of systemic fungal infections. Microscopy, culture based methods, antigen detection, and PCR may help to facilitate and accelerate the diagnosis. Galactomannan and glucan are two promising antigens that may be useful for early detection of the infection, but also for therapeutic monitoring. Sensitive and specific PCR assays to detect fungal DNA are an important part of the diagnostic approach. But extensive validation and standardization is strongly needed, before PCR assays can be used in a routine laboratory. The tremendous increase in invasive fungal infections has led to an increased interest in new antifungal agents and the field of antifungal chemotherapy evolved even more rapidly than diagnostic assays. The development of less toxic formulations of amphotericin B, the introduction of improved azoles and the availability of the echinocandins are opening new opportunities for the treatment of fungal infections. However, continuing efforts in the laboratory and well-designed clinical trials are still needed.