Identification Of Novel Drug Targets Research Articles

Identification and Structural-Functional Analysis of Cyclin-Dependent Kinases of the Cattle Tick Rhipicephalus (Boophilus) microplus

Cyclin-dependent kinases (CDKs) are a family of serine/threonine kinases essential for cell cycle progression. Herein, we describe the participation of CDKs in the physiology of Rhipicephalus microplus, the southern cattle tick and an important disease vector. Firstly, amino acid sequences homologous with CDKs of other organisms were identified from a R. microplus transcriptome database in silico. The analysis of the deduced amino acid sequences of CDK1 and CDK10 from R. microplus showed that both have caspase-3/7 cleavage motifs despite their differences in motif position and length of encoded proteins. CDK1 has two motifs (DKRGD and SAKDA) located opposite to the ATP binding site while CDK10 has only one motif (SLLDN) for caspase 3–7 near the ATP binding site. Roscovitine (Rosco), a purine derivative that inhibits CDK/cyclin complexes by binding to the catalytic domain of the CDK molecule at the ATP binding site, which prevents the transfer of ATP's γphosphoryl group to the substrate. To determine the effect of Rosco on tick CDKs, BME26 cells derived from R. microplus embryo cells were utilized in vitro inhibition assays. Cell viability decreased in the Rosco-treated groups after 24 hours of incubation in a concentration-dependent manner and this was observed up to 48 hours following incubation. To our knowledge, this is the first report on characterization of a cell cycle protein in arachnids, and the sensitivity of BME26 tick cell line to Rosco treatment suggests that CDKs are potential targets for novel drug design to control tick infestation.

SAT0181 Systems biology of SLE: Biochemical characterization of subgroups within SLE for improved diagnosis and treatment

BackgroundSystemic Lupus Erythematosus (SLE) is a systemic autoimmune disease with symptoms ranging from skin related problems to more severe cardiovascular effects. The heterogenous representation of the disease might be the...

Abstract 5503: Targeting thymidine metabolic pathways in cancer cells with a novel thymidine conjugate.

Abstract Cellular thymidine metabolism involves two pathways: the catabolic and salvage pathways. The catabolic pathway breaks down thymidine to carbon dioxide for energy while the salvage pathway converts thymidine to its 5′-triphosphate as one of building blocks for DNA synthesis. Cytosolic level of thymidine is regulated by both thymidine phosphorylase (TYMP) of the catabolic pathway and thymidine kinase (TK) of the salvage pathway. In tumor tissues and cancer cells, abnormally high levels of these two proteins have been consistently reported. Therefore, thymidine metabolic pathways in cancer cells are a potential therapeutic target for novel drug design and development. We recently developed a novel thymidine analog, 3′-deoxythymidine phenylquinoxaline conjugate (dT-QX) that selectively killed a series of cancer cell lines including human liver carcinoma HepG2 and Hep3B, breast adenocarcinoma MCF-7, rat brain glioma C6 and mouse liver cancer H22 cells and showed low cytotoxicity on the normal liver hepatocyte HL-7702 cells. dT-QX is a conjugate of 3′-deoxythymidine and a DNA intercalating moiety with a triazole linkage achieved by click chemistry. The selective killing of cancer cells was attributed to the selective inhibition of DNA synthesis in the cancer cells but not in normal liver HL-7702 cells, resulting in extensive production of mitochondrial superoxide stress. The conjugation of 3′-deoxythymidine was found to be critical for dT-QX to achieve the selective killing of cancer cells. The killing by dT-QX was revealed to be directly due to the expression levels of TYMP and TKs in cancer cells, confirming that dT-QX successfully targeted the thymidine metabolic pathways as designed. Finally, a preliminary mouse study with subcutaneous liver tumors confirmed that dT-QX effectively inhibited the growth of tumors in vivo. Citation Format: Qibing Zhou, Qiong Wei, Dejun Zhang. Targeting thymidine metabolic pathways in cancer cells with a novel thymidine conjugate. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5503. doi:10.1158/1538-7445.AM2013-5503

Pharmaceutical Perspectives of HECT-TYPE Ubiquitin Ligase Smurf1

Smad ubiquitylation regulatory factor-1 (Smurf1) is a HECT-type ubiquitin ligase. The role of Smurf1 in cell development and migration, viral autophagy and immune responses has been the subject of intensive study in recent years. Smurf1 regulates multiple biological networks, including TGF-β and BMP signaling pathways, the non-canonical pathway and the Toll-like receptor pathway, and is linked to certain diseases and disorders, such as bone formation and embryonic development disorders. Increasing evidence suggests that Smurf1 could be a good candidate for further translational studies and a potential target for novel drug design. In this review, we summarize the physiological functions of Smurf1 and its associated disorders; and discuss the current state of drug discovery in the context of the ubiquitin-proteasomal system, and feasible pharmaceutical strategies toward Smurf1 and its regulators, as well as RNA interference and structure-based chemical drug selection.

Lipoxygenase: An Emerging Target for Stroke Therapy

Neuroprotection as approach to stroke therapy has recently seen a revival of sorts, fueled in part by the continuing necessity to improve acute stroke care, and in part by the identification of novel drug targets. 12/15- Lipoxygenase (12/15-LOX), one of the key enzymes of the arachidonic acid cascade, contributes to both neuronal cell death and vascular injury. Inhibition of 12/15-LOX may thus provide multifactorial protection against ischemic injury. Targeting 12/15-LOX and related eicosanoid pathways is the subject of this brief review.

Identification of Novel Drug Targets in HpB38, HpP12, HpG27, Hpshi470, HpSJM180 Strains of Helicobacter pylori : An In Silico Approach for Therapeutic Intervention

Helicobacter species colonizes the stomach and are associated with the development of gastritis disease. Drugs for treatment of Helicobacter infection relieve pain or gastritis symptoms but they are not targeted specifically to Helicobacter pylori. Therefore, there is dire need for discovery of new drug targets and drugs for the treatment of H. pylori. The main objective of this study is to screen the potential drug targets by in silico analysis for the potent strains of H. pylori which include HpB38, HpP12, HpG27, Hpshi470 and HpSJM180. Genome and metabolic pathways of pathogen H. pylori and the host Homosapien sapiens are compared and genes which were unique to H. pylori were filtered and catalogued. These unique genes were subjected to gene property analysis to identify the potentiality of the drug targets. Among the total number of genes analysed in different strains of H. pylori nearly 558, 569, 539, 569, 567 number of genes in HpB38, HpP12, HpG27, Hpshi470 and HpSJM180 found qualified as unique molecules and among them 17 qualified as potential drug targets. Membrane fusion protein of hefABC efflux system, 50 S ribosomal protein L33, Hydrogenase expression protein/formation of HypD, Cag pathogenecity island protein X, Apolipoprotein N acyl transferase, DNA methyalse, Histone like binding protein, Peptidoglycan-associated lipoprotein OprL were found to be critical drug targets to H. pylori. Three (hefABC efflux system, Hydrogenase expression protein/formation of HypD, Cag pathogenecity island protein X) of the 17 predicted drug targets are already experimentally validated either genetically or biochemically lending credence to our unique approach.

Inferring Metabolic States in Uncharacterized Environments Using Gene-Expression Measurements

The large size of metabolic networks entails an overwhelming multiplicity in the possible steady-state flux distributions that are compatible with stoichiometric constraints. This space of possibilities is largest in the frequent situation where the nutrients available to the cells are unknown. These two factors: network size and lack of knowledge of nutrient availability, challenge the identification of the actual metabolic state of living cells among the myriad possibilities. Here we address this challenge by developing a method that integrates gene-expression measurements with genome-scale models of metabolism as a means of inferring metabolic states. Our method explores the space of alternative flux distributions that maximize the agreement between gene expression and metabolic fluxes, and thereby identifies reactions that are likely to be active in the culture from which the gene-expression measurements were taken. These active reactions are used to build environment-specific metabolic models and to predict actual metabolic states. We applied our method to model the metabolic states of Saccharomyces cerevisiae growing in rich media supplemented with either glucose or ethanol as the main energy source. The resulting models comprise about 50% of the reactions in the original model, and predict environment-specific essential genes with high sensitivity. By minimizing the sum of fluxes while forcing our predicted active reactions to carry flux, we predicted the metabolic states of these yeast cultures that are in large agreement with what is known about yeast physiology. Most notably, our method predicts the Crabtree effect in yeast cells growing in excess glucose, a long-known phenomenon that could not have been predicted by traditional constraint-based modeling approaches. Our method is of immediate practical relevance for medical and industrial applications, such as the identification of novel drug targets, and the development of biotechnological processes that use complex, largely uncharacterized media, such as biofuel production.

Wnt Secretion and Gradient Formation

Concentration gradients formed by the lipid-modified morphogens of the Wnt family are known for their pivotal roles during embryogenesis and adult tissue homeostasis. Wnt morphogens are also implicated in a variety of human diseases, especially cancer. Therefore, the signaling cascades triggered by Wnts have received considerable attention during recent decades. However, how Wnts are secreted and how concentration gradients are formed remains poorly understood. The use of model organisms such as Drosophila melanogaster has provided important advances in this area. For instance, we have previously shown that the lipid raft-associated reggie/flotillin proteins influence Wnt secretion and spreading in Drosophila. Our work supports the notion that producing cells secrete Wnt molecules in at least two pools: a poorly diffusible one and a reggie/flotillin-dependent highly diffusible pool which allows morphogen spreading over long distances away from its source of production. Here we revise the current views of Wnt secretion and spreading, and propose two models for the role of the reggie/flotillin proteins in these processes: (i) reggies/flotillins regulate the basolateral endocytosis of the poorly diffusible, membrane-bound Wnt pool, which is then sorted and secreted to apical compartments for long-range diffusion, and (ii) lipid rafts organized by reggies/flotillins serve as “dating points” where extracellular Wnt transiently interacts with lipoprotein receptors to allow its capture and further spreading via lipoprotein particles. We further discuss these processes in the context of human breast cancer. A better understanding of these phenomena may be relevant for identification of novel drug targets and therapeutic strategies.

An Ancient Protein Phosphatase, SHLP1, Is Critical to Microneme Development in Plasmodium Ookinetes and Parasite Transmission

SummarySignaling pathways controlled by reversible protein phosphorylation (catalyzed by kinases and phosphatases) in the malaria parasite Plasmodium are of great interest, for both increased understanding of parasite biology and identification of novel drug targets. Here, we report a functional analysis in Plasmodium of an ancient bacterial Shewanella-like protein phosphatase (SHLP1) found only in bacteria, fungi, protists, and plants. SHLP1 is abundant in asexual blood stages and expressed at all stages of the parasite life cycle. shlp1 deletion results in a reduction in ookinete (zygote) development, microneme formation, and complete ablation of oocyst formation, thereby blocking parasite transmission. This defect is carried by the female gamete and can be rescued by direct injection of mutant ookinetes into the mosquito hemocoel, where oocysts develop. This study emphasizes the varied functions of SHLP1 in Plasmodium ookinete biology and suggests that it could be a novel drug target for blocking parasite transmission.

Identification of Novel Drug Targets for the Treatment of Diabetic Retinopathy

Vision loss in diabetic retinopathy (DR) is attributable to retinal vascular disorders that result in macular edema and neoangiogenesis. In addition to laser photocoagulation therapy, intraocular injections of antivascular endothelial growth factor drugs have contributed to the treatment of these disease conditions. Nonetheless, the clinical feasibility of intraocular drug administration has raised an increasing demand to develop alternative drugs that can fundamentally ameliorate the retinal vascular dysfunctions in DR. For this purpose, experimental animal models that reproduce human DR would be of clinical benefit. Despite the unavailability of DR models in rats or mice, pharmacological and genetic manipulations without hyperglycemia have successfully recapitulated retinal edema and neoangiogenesis in postnatal mouse retinas, thereby enabling the understanding of the pathophysiology underlying DR. This article highlights the utility of experimental mouse models of retinal vascular abnormalities and discusses cellular and molecular mechanisms responsible for the onset and progression of DR. These approaches will lead to the identification of novel drug targets for the restoration of vascular integrity and regeneration of functional capillaries in DR.

The emerging field of chemo‐ and pharmacoproteomics

The emerging field of chemo- and pharmacoproteomics studies the mechanisms of action of bioactive molecules in a systems pharmacology context. In contrast to traditional drug discovery, pharmacoproteomics integrates the mechanism of a drug's action, its side effects including toxicity, and the discovery of new drug targets in a single approach. Thus, it determines early favorable (e.g. multiple kinase target in cancer drugs) and unfavorable (e.g. side effects) polypharmacology. Target profiling is accomplished using either active site-labeling probes or immobilized drugs. This strategy identifies direct targets and has in fact enabled even the determination of binding curves and half maximum inhibitory concentrations of these targets. In addition, the enrichment greatly reduces the complexity of the proteome to be analyzed by quantitative MS. Complementary to these approaches, global proteomics profiling studying drug treatement-induced changes in protein expression levels and/or post-translational modification status have started to become possible mostly due to significant improvements in instrumentation. Particularly, when using multidimensional separations, a considerable proteome depth of up to 10000 proteins can be achieved with current state-of-the-art mass spectrometers and bioinformatics tools. In summary, chemo- and pharmacoproteomics has already contributed significantly to the identification of novel drug targets and their mechanisms of action(s). Aided by further technological advancements, this interdisciplinary approach will likely be used more broadly in the future.

Abstract P4-06-02: Identification of novel G-protein coupled receptor targets in HER2-positive breast cancer.

Abstract Background: HER2 is a member of human epidermal growth factor receptor (HER) superfamily and HER2-overexpressing (HER2+) breast cancer (BC) is an aggressive tumor. Despite the clinical success of anti-HER2 therapies, de novo and acquired drug resistance occur in many patients. Identification of novel drug targets to overcome anti-HER2 therapy resistance is an unmet need. Since G-protein coupled receptors (GPCRs) are known to cross-talk with the HER superfamily, it is possible that some GPCRs may signal to modulate the HER2 pathway. GPCRs are considered excellent drug targets due to their plasma membrane localization, unique ligand-binding pocket, and availability of high throughput assays for drug screening. The expression and function of the majority of GPCRs are largely unknown in HER2+ BC. The goal of this study was to identify novel GPCR targets in HER2+ BC, in the context of anti-HER2 therapy resistance. Methods: We examined the differential GPCRs expression in BC stem cells, suggested to be involved in resistance, as well as in anti-HER2 treatment-resistant BT474 cell line model of HER2+ BC. BC stem cells were identified as aldehyde dehydrogenase-positive (ALDH+) cells using the Aldefluor assay. Brightly fluorescent ALDH+ cells were separated from ALDH- cells using FACS Aria II cell sorter. Anti-HER2 resistant derivatives of BT474 cells were established by long-term exposure to increasing drug concentration of trastuzumab (T), lapatinib (L), or their combination (T+L). RNA was isolated and subjected to profiling using TaqMan real time RT-PCR GPCR 384-well microarray to quantify the expression of mRNA encoding 343 GPCRs from 50 different subfamilies. Only overexpressed GPCRs were considered of interest as drug targets, and the overexpression of GPCRs was verified by RT-PCR and western blotting for the selected targets. Publically available TCGA dataset for mRNA expression was also interrogated to determine differential expression of selected GPCRs in HER2+ vs. other subtypes of BC. Results: Nine GPCRs [BAI3, EDNRA, GPR110, GPR116, GPR124, MTNR1A, EDG2, EMR2, GCGR] were upregulated in ALDH+ compared to ALDH- BT474 cells. In addition, 11 GPCRs [CCBP2, CCR9, F2RL1, GALR2, GPR1, GPR24, GPR87, GPR110, GPR183, LGR4, OXER1] were over-expressed in the resistant derivatives (T, L, and T+L) compared to the parental BT474 cells. Out of these, 13 belong to Class A and 6 to Class B, designated by The International Union of Basic and Clinical Pharmacology (IUPHAR). GPR110 was the only GPCR common to BC stem cells as well as resistant derivatives of BT474 cells. In TCGA dataset, GPR110 expression was significantly higher in HER2+ and basal subtypes of BC compared to ER+ luminal A and B subtypes. GPR110 as well as other differentially expressed GPCRs are currently being investigated as potential targets by determining the effects of their downregulation or exogenous over-expression on the growth and drug-sensitivity of these BC cells. Conclusions: We report for the first time the differential expression of a large panel of GPCRs in the BC stem cell population and in anti-HER2 resistant derivatives of the BT474 cell line model of HER2+ BC. Results of the functional studies will guide novel strategies to improve the treatment for HER2+ BC. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-06-02.

Overexpression of DRAM enhances p53‐dependent apoptosis

Tumor suppressor p53-dependent apoptosis is thought to be one of the most important tumor-suppressive mechanisms in human tumorigenesis. Till date, “super p53” mutants exhibiting more potent ability to induce apoptosis than wild-type p53 have been reported. These super p53s may provide a clue for development of novel therapeutic targets. However, the major mechanism underlying the super p53-dependent apoptosis remains unclear. To identify critical gene(s) in this mechanism, we performed a comprehensive and comparative expression analysis in p53-null Saos-2 cells with conditional expression of wild-type p53 and S121F, which was previously reported as a super p53 mutant. We identified damage-regulated autophagy modulator (DRAM) as one of the genes that were more upregulated by S121F than wild-type p53. Although knockdown of DRAM was not sufficient for reducing the ability of S121F to induce apoptosis, DRAM overexpression enhanced the ability in a wild-type p53-dependent manner. Here, we show that DRAM is an important gene for the enhancement of p53-dependent apoptosis. Additional analysis of the mechanism of super p53-dependent apoptosis may lead to the identification of novel drug targets for cancer therapy.

Recent advances in cardiovascular proteomics

Cardiovascular diseases (CVDs) are the major source of global morbidity and death and more people die annually from CVDs than from any other cause. These diseases can occur quickly, as seen in acute myocardial infarction (AMI), or progress slowly over years as with chronic heart failure. Advances in mass spectrometry detection and analysis, together with improved isolation and enrichment techniques allowing for the separation of organelles and membrane proteins, now allow for the indepth analysis of the cardiac proteome. Here we outline current insights that have been provided through cardiovascular proteomics, and discuss studies that have developed innovative technologies which permit the examination of the protein complement in specific organelles including exosomes and secreted proteins. We highlight these foundational studies and illustrate how they are providing the technologies and tools which are now being applied to further study cardiovascular disease; provide new diagnostic markers and potentially new methods of cardiac patient management with identification of novel drug targets. This article is part of a Special Issue entitled: From protein structures to clinical applications.

Envelope Glycoprotein of Arenaviruses

Arenaviruses include lethal human pathogens which pose serious public health threats. So far, no FDA approved vaccines are available against arenavirus infections, and therapeutic options are limited, making the identification of novel drug targets for the development of efficacious therapeutics an urgent need. Arenaviruses are comprised of two RNA genome segments and four proteins, the polymerase L, the envelope glycoprotein GP, the matrix protein Z, and the nucleoprotein NP. A crucial step in the arenavirus life-cycle is the biosynthesis and maturation of the GP precursor (GPC) by cellular signal peptidases and the cellular enzyme Subtilisin Kexin Isozyme-1 (SKI-1)/Site-1 Protease (S1P) yielding a tripartite mature GP complex formed by GP1/GP2 and a stable signal peptide (SSP). GPC cleavage by SKI-1/S1P is crucial for fusion competence and incorporation of mature GP into nascent budding virion particles. In a first part of our review, we cover basic aspects and newer developments in the biosynthesis of arenavirus GP and its molecular interaction with SKI-1/S1P. A second part will then highlight the potential of SKI-1/S1P-mediated processing of arenavirus GPC as a novel target for therapeutic intervention to combat human pathogenic arenaviruses.

Molecular characterization and ligand binding specificity of the PDZ domain-containing protein GIPC3 from Schistosoma japonicum

BackgroundSchistosomiasis is a serious global health problem that afflicts more than 230 million people in 77 countries. Long-term mass treatments with the only available drug, praziquantel, have caused growing concerns about drug resistance. PSD-95/Dlg/ZO-1 (PDZ) domain-containing proteins are recognized as potential targets for the next generation of drug development. However, the PDZ domain-containing protein family in parasites has largely been unexplored.MethodsWe present the molecular characteristics of a PDZ domain-containing protein, GIPC3, from Schistosoma japonicum (SjGIPC3) according to bioinformatics analysis and experimental approaches. The ligand binding specificity of the PDZ domain of SjGIPC3 was confirmed by screening an arbitrary peptide library in yeast two-hybrid (Y2H) assays. The native ligand candidates were predicted by Tailfit software based on the C-terminal binding specificity, and further validated by Y2H assays.ResultsSjGIPC3 is a single PDZ domain-containing protein comprised of 328 amino acid residues. Structural prediction revealed that a conserved PDZ domain was presented in the middle region of the protein. Phylogenetic analysis revealed that SjGIPC3 and other trematode orthologues clustered into a well-defined cluster but were distinguishable from those of other phyla. Transcriptional analysis by quantitative RT-PCR revealed that the SjGIPC3 gene was relatively highly expressed in the stages within the host, especially in male adult worms. By using Y2H assays to screen an arbitrary peptide library, we confirmed the C-terminal binding specificity of the SjGIPC3-PDZ domain, which could be deduced as a consensus sequence, -[SDEC]-[STIL]-[HSNQDE]-[VIL]*. Furthermore, six proteins were predicted to be native ligand candidates of SjGIPC3 based on the C-terminal binding properties and other biological information; four of these were confirmed to be potential ligands using the Y2H system.ConclusionsIn this study, we first characterized a PDZ domain-containing protein GIPC3 in S. japonicum. The SjGIPC3-PDZ domain is able to bind both type I and II ligand C-terminal motifs. The identification of native ligand will help reveal the potential biological function of SjGIPC3. These data will facilitate the identification of novel drug targets against S. japonicum infections.

Upregulation of the long non‐coding rna hotair promotes esophageal squamous cell carcinoma metastasis and poor prognosis

Recent studies of the individual functionalities of long non-coding RNAs (lncRNAs) in the development and progression of cancer have suggested that HOX transcript antisense RNA (HOTAIR) is capable of reprogramming chromatin organization and promoting cancer cell metastasis. In order to ascertain the expression pattern of the lncRNA HOTAIR and assess its biological role in the development and progression of esophageal squamous cell carcinoma (ESCC), HOTAIR expression in ESCC tissues and adjacent noncancerous tissues were collected from 78 patients and measured by real-time reverse transcription-polymerase chain reaction (RT-PCR). HOTAIR correlation with clinicopathological features and prognosis was also analyzed. Suppression of HOTAIR using siRNA treatment was performed in order to explore its role in tumor progression. Notably elevated HOTAIR expression levels were observed in cancerous tissues compared to adjacent noncancerous tissues (96%, P < 0.01), showing a high correlation with cancer metastasis (P < 0.01), elevated TNM (2009) stage classification (P < 0.01), and lowered overall survival rates (P = 0.003). Multivariate analysis revealed that HOTAIR expression (P = 0.003) is also an independent prognostic factor for comparison of TNM stage (P = 0.024) and lymph node metastasis (P = 0.010). Furthermore, in vitro assays of the ESCC cell line KYSE30 demonstrated that knockdown of HOTAIR reduced cell invasiveness and migration while increasing the response of cells to apoptosis. Thus, HOTAIR is a novel molecule involved in both ESCC progression and prognosis. Full elucidation of HOTAIR functionality relevant to ESCC may open avenues for the use of lncRNAs in identification of novel drug targets and therapies for ESCC and other prevalent cancers.

Chemical Genomics and Emerging DNA Technologies in the Identification of Drug Mechanisms and Drug Targets

Chemical genomics combines chemistry with molecular biology as a means of exploring the function of unknown proteins or identifying the proteins responsible for a particular phenotype induced by a small cell-permeable bioactive molecule. Chemical genomics therefore has the potential to identify and validate therapeutic targets and to discover drug candidates for rapidly and effectively generating new interventions for human diseases. The recent emergence of genomic technologies and their application on genetically tractable model organisms like Drosophila melanogaster, Caenorhabditis elegans and Saccharomyces cerevisiae have provided momentum to cell biological and biomedical research, particularly in the functional characterization of gene functions and the identification of novel drug targets. We therefore anticipate that chemical genomics and the vast development of genomic technologies will play critical roles in the genomic age of biological research and drug discovery. In the present review we discuss how simple biological model organisms can be used as screening platforms in combination with emerging genomic technologies to advance the identification of potential drugs and their molecular mechanisms of action.

Abstract LB-77: Crm1-mediated nucleocytoplasmic transport inhibition perturbs multiple cellular pathways to trigger melanoma cell apoptosis

Abstract Development of multiple resistance mechanisms in melanomas has necessitated the identification of novel drug targets, which when inhibited could impact multiple cellular pathways thus circumventing potential resistance mechanisms. By performing cDNA-microarray analysis, we identified four key components of the nucleocytoplasmic transport machinery, Chromosome Region Maintenance 1 (Crm1), Ran-GTPase, Ran-GTPase Activating Protein 1 (RanGAP1) and Ran Binding Protein 1 (RanBP1) to be overexpressed in human melanoma metastases. Crm1 inhibition, employing the highly specific Crm1 inhibitor Leptomycin B (LMB) induced a dramatic depletion of pro-survival/cytoplasmic Erk1/2 and p90RSK1 and mediated persistent Erk signaling hyperactivation. Consistently, Crm1 inhibition inflicted extensive apoptosis in melanoma cells while sparing immortalized melanocytes and primary lung fibroblasts. Apoptosis required both the intrinsic and extrinsic apoptotic pathways and was associated with a nuclear entrapment and down-regulation of anti-apoptotic Survivin. Apoptosis was preceded by a G1 cell-cycle arrest and even though Crm1 inhibition mediated dramatic p53 and p21 induction exclusively in wild-type p53 melanoma cells, latter's silencing or inactivation failed to alleviate ensuing apoptosis. Notably, Crm1 inhibition elicited cell line specific, G1 to S progression-retarding changes in the expression of multiple cell-cycle regulatory proteins (Cdk2, Cdk4, p27, p16 and p18) thus potentially explaining p53 dispensability. Based on our results, we propose Crm1 as a potential therapeutic target in human melanoma, whose inhibition initiates a multitude of cell context-dependent molecular events to trigger G1-arrest followed by massive apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-77. doi:1538-7445.AM2012-LB-77

Redox Regulation in Malaria: Current Concepts and Pharmacotherapeutic Implications

Malaria imposes a serious threat to human and becomes more prevalent due to the emergence of drug resistant parasite. Understanding of the underlying mechanism of drug resistance and identification of novel drug targets are key effective processes for the management of malaria. Malaria parasite is highly susceptible to oxidative stress but lives in a pro-oxidant rich environment containing oxygen and iron, which produce a large amount of reactive oxygen species. Management of oxidative stress in malaria parasite is tightly regulated through active redox and antioxidant defense systems. The elevation of oxidative stress as a result of inhibition of any component of this defense system leads to redox imbalance and ultimately parasite death. Therefore, redox system plays an indispensable role for the survival of parasite within the host. Identification of key molecules, which disrupt parasite redox balance by altering key redox reactions and promote oxidative stress in parasites, would be an effective approach to develop novel antimalarial drugs. During the last few decades, contributions by researchers around the globe provide even better understanding of redox biology of malaria parasite. Here, in this review, we are highlighting the knowledge gathered so far regarding the essential redox-active processes and their components in malaria parasite to overcome elevated oxidative insults. We have also given maximum efforts to enlist currently used redox-active antimalarials, their mode of action and pharmacotherapeutic implications.