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Drug Targets (drug + target)
Kinds of Drug Targets Selected AbstractsRNA as a Drug Target: The Case of AminoglycosidesCHEMBIOCHEM, Issue 10 2003Quentin Vicens Dr. How to prevent antibiotic therapies from hitting a snag: Most antibiotics target molecular switches on the ribosome. The binding sites are discrete and made of conserved RNA residues rather than ribosomal proteins (see representation of paromycin binding to the ribosome). However, bacteria have evolved and mutated so that the antibiotics cannot bind. At least two types of new molecules could be designed to keep up in this race against evolution: 1) drugs that would bind to the mutated bacterial sites but not (to avoid toxicity) with human cytoplasmic and mitochondrial ribosomes, and 2) drugs that would interfere with other molecular switches involved in translation or regulation pathways. [source] Sequence of the Mitochondrial Genome of Pneumocystis carinii: Implications for Biological Function and Identification of Potential Drug TargetsTHE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 2006THOMAS M. SESTERHENN [source] Are MAP Kinases Drug Targets?CHEMMEDCHEM, Issue 8 2007but Difficult Ones Abstract Pharmaceutical companies are facing an increasing interest in new target identification and validation. In particular, extensive efforts are being made in the field of protein kinase inhibitors research and development, and the past ten years of effort in this field have altered our perception of the potential of kinases as drug targets. Therefore, in the drug discovery process, the selection of relevant, susceptible protein kinase targets combined with searches for leads and candidates have become a crucial approach. The success of recent launches of protein kinase inhibitors (Gleevec, Imatinib, Sutent, Iressa, Nexavar, Sprycel) gave another push to this field. Numerous other kinase inhibitors are currently undergoing clinical trials or clinical development. Some questions are nevertheless unanswered, mostly related to the great number of known kinases in the human genome, to their similarity with each other, to the existence of functionally redundant kinases for specific pathways, and also because the connection between particular pathways and diseases is not always clear. The review is leading the reader through a panoramic view of protein kinase inhibition with a major focus on MAPK, successful examples and clinical candidates. [source] How to Achieve Confidence in Drug Discovery and Development: Managing Risk (from a Reductionist to a Holistic Approach)CHEMMEDCHEM, Issue 6 2009Annette Bakker Dr. Abstract Confidence in mechanism: Creating a more holistic understanding of disease pathophysiology and an early confidence in the mechanism under investigation could help facilitate the selection of not only the most appropriate targets but also the best mechanisms for disease intervention and how to select and optimise the best compounds. Drug target and candidate selection are two of the key decision points within the drug discovery process for which all companies use certain selection criteria to make decisions on which targets to accept into their discovery pipelines and which compounds will pass into development. These steps not only help define the overall productivity of every company but they are also decisions taken without full predictive knowledge of the risks that lie ahead or how best to manage them. In particular, the process of selecting new targets does not normally involve full evaluation of the risk(s) in the mechanism under investigation (the modulation of the target), which may result in an inability to fully connect in,vitro and animal model results to the disease (clinical) setting. The resulting poor progression statistics of many compounds in the clinic is at least partially the result of a lack of understanding of disease pathophysiology. Notably, the lack of efficacy is still a major reason for failure in the clinic.1 Creating a more holistic understanding of disease pathophysiology and an early confidence in the mechanism under investigation could help facilitate the selection of not only the most appropriate targets but also the best mechanisms for disease intervention and how to select and optimise the best compounds. [source] Viral proteinases: targets of opportunityDRUG DEVELOPMENT RESEARCH, Issue 6 2006Chelsea M. Byrd Abstract During antiviral drug development, any essential stage of the viral life cycle can serve as a potential drug target. Since most viruses encode specific proteases whose cleavage activity is required for viral replication, and whose structure and activity are unique to the virus and not the host cell, these enzymes make excellent targets for drug development. Success using this approach has been demonstrated with the plethora of protease inhibitors approved for use against HIV. This discussion is designed to review the field of antiviral drug development, focusing on the search for protease inhibitors, while highlighting some of the challenges encountered along the way. Protease inhibitor drug discovery efforts highlighting progress made with HIV, HCV, HRV, and vaccinia virus as a model system are included. Drug Dev. Res. 67:501,510, 2006. © 2006 Wiley-Liss, Inc. [source] Novel estrogen receptor ligands and their structure,activity relationship evaluated by scintillation proximity assay for high-throughput screeningDRUG DEVELOPMENT RESEARCH, Issue 4 2005Ling He Abstract The estrogen receptor (ER) is an important drug target with allosteric characteristics that binds orthotopic hormones and other ligands. A recently developed scintillation proximity (SPA)-based assay for high-throughput screening (HTS) of compound libraries was used to identify novel estrogen receptor ligands that might have ER subtype selective binding activity. Radioligand binding was determined in a multi-detector scintillation counter designed for microtitration plates. Equilibrium binding experiments and kinetic competition tests were performed with [3H]-estradiol and human ER, and ER, receptors. A library of 6,000 structurally diverse compounds was screened. From this, several novel ligands were identified that showed pronounced subtype-selective differences in ligand binding for ER, and ER,. The observed equilibrium dissociation constant (Kd) for the binding of [3H]estradiol to ER, and ER, receptors were approximately 0.25 and 0.64 nM, respectively. When 17,-estradiol, raloxifene and daidzein were tested for binding affinity to ER, in a competition assay, the IC50 values were 0.34, 1.31, and 75.6 nM, respectively. When tested for binding affinity to ER,, the IC50 values were 1.05, 11.4, and 10.6 nM, respectively. The results obtained show that the methodology is valid in comparison to previously published data regarding estradiol and other standard compounds (raloxifene and daidzein) binding characteristics of estrogen receptors. The assay is also well suited to applied research as a tool in HTS of compound libraries in the search of ER ligands. Several novel active compounds were identified and selected as potent ER subtype ligands. Drug Dev Res 64:203,212, 2005. © 2005 Wiley-Liss, Inc. [source] The clinical impact of pharmacogenetics on the treatment of epilepsyEPILEPSIA, Issue 1 2009Wolfgang Löscher Summary Drug treatment of epilepsy is characterized by unpredictability of efficacy, adverse drug reactions, and optimal doses in individual patients, which, at least in part, is a consequence of genetic variation. Since genetic variability in drug metabolism was reported to affect the treatment with phenytoin more than 25 years ago, the ultimate goal of pharmacogenetics is to use the genetic makeup of an individual to predict drug response and efficacy, as well as potential adverse drug events. However, determining the practical relevance of pharmacogenetic variants remains difficult, in part because of problems with study design and replication. This article reviews the published work with particular emphasis on pharmacogenetic alterations that may affect efficacy, tolerability, and safety of antiepileptic drugs (AEDs), including variation in genes encoding drug target (SCN1A), drug transport (ABCB1), drug metabolizing (CYP2C9, CYP2C19), and human leucocyte antigen (HLA) proteins. Although the current studies associating particular genes and their variants with seizure control or adverse events have inherent weaknesses and have not provided unifying conclusions, several results, for example that Asian patients with a particular HLA allele, HLA-B*1502, are at a higher risk for Stevens-Johnson syndrome when using carbamazepine, are helpful to increase our knowledge how genetic variation affects the treatment of epilepsy. Although genetic testing raises ethical and social issues, a better understanding of the genetic influences on epilepsy outcome is key to developing the much needed new therapeutic strategies for individuals with epilepsy. [source] Kinetic mechanism for p38 MAP kinase ,FEBS JOURNAL, Issue 18 2005A partial rapid-equilibrium random-order ternary-complex mechanism for the phosphorylation of a protein substrate p38 Mitogen-activated protein kinase alpha (p38 MAPK,) is a member of the MAPK family. It is activated by cellular stresses and has a number of cellular substrates whose coordinated regulation mediates inflammatory responses. In addition, it is a useful anti-inflammatory drug target that has a high specificity for Ser-Pro or Thr-Pro motifs in proteins and contains a number of transcription factors as well as protein kinases in its catalog of known substrates. Fundamental to signal transduction research is the understanding of the kinetic mechanisms of protein kinases and other protein modifying enzymes. To achieve this end, because peptides often make only a subset of the full range of interactions made by proteins, protein substrates must be utilized to fully elucidate kinetic mechanisms. We show using an untagged highly active form of p38 MAPK,, expressed and purified from Escherichia coli[Szafranska AE, Luo X & Dalby KN (2005) Anal Biochem336, 1,10) that at pH 7.5, 10 mm Mg2+ and 27 °C p38 MAPK, phosphorylates ATF2,115 through a partial rapid-equilibrium random-order ternary-complex mechanism. This mechanism is supported by a combination of steady-state substrate and inhibition kinetics, as well as microcalorimetry and published structural studies. The steady-state kinetic experiments suggest that magnesium adenosine triphosphate (MgATP), adenylyl (,,,-methylene) diphosphonic acid (MgAMP-PCP) and magnesium adenosine diphosphate (MgADP) bind p38 MAPK, with dissociation constants of KA = 360 µm, KI = 240 µm, and KI > 2000 µm, respectively. Calorimetry experiments suggest that MgAMP-PCP and MgADP bind the p38 MAPK,,ATF2,115 binary complex slightly more tightly than they do the free enzyme, with a dissociation constant of Kd , 70 µm. Interestingly, MgAMP-PCP exhibits a mixed inhibition pattern with respect to ATF2,115, whereas MgADP exhibits an uncompetitive-like pattern. This discrepancy occurs because MgADP, unlike MgAMP-PCP, binds the free enzyme weakly. Intriguingly, no inhibition by 2 mm adenine or 2 mm MgAMP was detected, suggesting that the presence of a ,-phosphate is essential for significant binding of an ATP analog to the enzyme. Surprisingly, we found that inhibition by the well-known p38 MAPK, inhibitor SB 203580 does not follow classical linear inhibition kinetics at concentrations >,100 nm, as previously suggested, demonstrating that caution must be used when interpreting kinetic experiments using this inhibitor. [source] The type 1 equilibrative nucleoside transporter regulates anxiety-like behavior in miceGENES, BRAIN AND BEHAVIOR, Issue 8 2007J. Chen Activation of adenosine receptors in the brain reduces anxiety-like behavior in animals and humans. Because nucleoside transporters regulate adenosine levels, we used mice lacking the type 1 equilibrative nucleoside transporter (ENT1) to investigate whether ENT1 contributes to anxiety-like behavior. The ENT1 null mice spent more time in the center of an open field compared with wild-type littermates. In the elevated plus maze, ENT1 null mice entered more frequently into and spent more time exploring the open arms. The ENT1 null mice also spent more time exploring the light side of a light,dark box compared with wild-type mice. Microinjection of an ENT1-specific antagonist, nitrobenzylthioinosine (nitrobenzylmercaptopurine riboside), into the amygdala of C57BL/6J mice reduced anxiety-like behavior in the open field and elevated plus maze. These findings show that amygdala ENT1 modulates anxiety-like behavior. The ENT1 may be a drug target for the treatment of anxiety disorders. [source] Nuclear and membrane expression of the angiogenesis regulator delta-like ligand 4 (DLL4) in normal and malignant human tissuesHISTOPATHOLOGY, Issue 5 2009Juan Carlos Martinez Aims:, Delta-like ligand 4 (DLL4) is one of five known Notch ligands in mammals and interacts predominantly with Notch 1. DLL4 is induced by vascular endothelial growth factor (VEGF) and acts downstream of VEGF as a ,brake' on VEGF-induced vessel growth, forming an autoregulatory negative feedback loop inactivating VEGF. This action was believed to occur only in vascular development, raising hopes that DLL4 could be a specific drug target for controlling vessel growth in tumours and other pathological conditions. Our aim was to pursue this by raising a monoclonal antibody to the internal domain of DLL4 and assess its distribution in normal and malignant tissues in comparison with antibodies against the external domain of DLL4. Methods and results:, The anti-DLL4 monoclonal antibody was raised using conventional mouse hybridoma techniques. The antibody has been fully characterized by Western blotting and transfectant immunostaining. It has also been comprehensively compared with other antibodies against both the internal and external domains of DLL4. The antigen is widely expressed on human tissues not only on endothelium but also on epithelium and stromal cells. Indeed, in our comprehensive survey only pulmonary alveoli failed to express DLL4. Of a wide range of malignancies, most also expressed DLL4 on tumour cells with a predominantly cytoplasmic pattern, although a number also displayed nuclear positivity. Conclusions:, Contrary to previous beliefs, DLL4 is widely distributed in tissues other than vessels including many malignancies. Furthermore, the molecule is internalized on binding its receptor and often transported to the nucleus. These findings raise many interesting possibilities for further study of DLL4 and its potential as a therapeutic target. [source] Neuronal cholesterol esterification by ACAT1 in Alzheimer's diseaseIUBMB LIFE, Issue 4 2010Ta-Yuan Chang Abstract Cholesterol has been implicated in various neurodegenerative diseases. Here we review the connection between cholesterol and Alzheimer's disease (AD), focusing on a recent study that links neuronal cholesterol esterification with biosynthesis of 24(S)-hydroxycholesterol and the fate of human amyloid precursor protein in a mouse model of AD. We also briefly evaluate the potential of ACAT1 as a drug target for AD. © 2010 IUBMB IUBMB Life, 62(4): 261,267, 2010 [source] Methionine gamma-lyase: The unique reaction mechanism, physiological roles, and therapeutic applications against infectious diseases and cancersIUBMB LIFE, Issue 11 2009Dan Sato Abstract Sulfur-containing amino acids (SAAs) are essential components in many biological processes and ubiquitously distributed to all organisms. Both biosynthetic and catabolic pathways of SAAs are heterogeneous among organisms and between developmental stages, and regulated by the environmental changes. Limited lineage of organisms ranging from archaea to plants, but not human, possess a unique enzyme methionine gamma-lyase (MGL, EC 4.4.1.11) to directly degrade SAA to ,-keto acids, ammonia, and volatile thiols. The reaction mechanisms and the physiological roles of this enzyme are partially demonstrated by the enzymological analyzes, structure determination, isotopic labeling of the intermediate metabolites, and functional analyzes of deficient mutants. MGL has been exploited as a drug target for the infectious diseases caused by parasitic protozoa and anaerobic periodontal bacteria. In addition, MGL has been utilized to develop therapeutic interventions of various cancers, by introducing recombinant proteins to deplete methionine essential for the growth of cancer cells. In this review, we discuss the current understanding of enzymological properties, putative physiological roles, and therapeutic applications of MGL. © 2009 IUBMB IUBMB Life, 61(11): 1019,1028, 2009 [source] Crossing the barrier: oxysterols as cholesterol transporters and metabolic modulators in the brainJOURNAL OF INTERNAL MEDICINE, Issue 6 2006I. BJÖRKHEM Abstract. A normal brain function requires constant levels of cholesterol, and the need for constancy seems to be higher here than in any other organ. Nature has met this need by isolation of brain cholesterol by a highly efficient blood,brain barrier. As a low synthesis of cholesterol is present in the brain, a mechanism for compensatory elimination is required. A decade ago we made the unexpected finding that the favoured mechanism for this involves conversion into 24S-hydroxycholesterol, followed by diffusion over the blood,brain barrier. Recent studies by us and others on this new pathway have given new insights into the mechanisms by which cholesterol homeostasis is maintained in the brain. We recently demonstrated a flux of another oxygenated product of cholesterol, 27-hydroxycholesterol, in the opposite direction. The latter flux may be important for neurodegeneration, and may be the link between hypercholesterolaemia and Alzheimer's disease. An overview of the above studies is presented and the possibility that the cholesterol 24S-hydroxylase in the brain may be important for memory and learning and that it may be a new drug target is discussed. [source] The persisting challenge of selective and specific proteasome inhibition,JOURNAL OF PEPTIDE SCIENCE, Issue 2 2009Michael Groll Abstract Since the discovery of the proteasome and its structure elucidation intensive research programs in academic institutions and pharmaceutical industries led to identification of a wide spectrum of synthetic and natural small proteasomal inhibitors. Activity studies with these small molecules helped to deeply understand the complex biochemical organization and functioning of the proteasome. The new structural and biochemical insights placed the proteasome as an important anti-cancer drug target, as revealed by the dipeptide boronate proteasome inhibitor, bortezomib, which is currently used for treatment of multiple myeloma. Serious side effects and partial cell resistance against bortezomib demand creation and discovery of new improved generations of more specific and potent proteasomal inhibitors. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd. [source] Triclosan inhibition of mycobacterial InhA in Saccharomyces cerevisiae: yeast mitochondria as a novel platform for in vivo antimycolate assaysLETTERS IN APPLIED MICROBIOLOGY, Issue 4 2010A. Gurvitz Abstract Aims:, To demonstrate the suitability of yeast to act as a novel biotechnological platform for conducting in vivo inhibition assays using drugs with low efficacies towards their mycobacterial targets, such as occurs in the situation with triclosan and InhA. Methods and Results:, A surrogate yeast host represented by Saccharomyces cerevisiae etr1, cells lacking Etr1p, the 2- trans -enoyl-thioester reductase of mitochondrial type 2 fatty acid synthase (FASII), was designed to rely on the Mycobacterium tuberculosis FASII enzyme InhA. Although InhA is 10 000 times less sensitive to the antimicrobial drug triclosan than is bacterial FabI, the respiratory growth of yeast cells depending on InhA was severely affected on glycerol medium containing triclosan. Conclusions:, The yeast system could detect enzyme inhibition despite the use of a drug with only low efficacy. Significance and Impact of the Study:, Tuberculosis affects a third of the human population, and InhA is a major drug target for combating this disease. InhA is inhibited by isoniazid, but triclosan-derived compounds are presently being developed as antimycolates. A demonstration of triclosan inhibition of InhA in yeast represents a meaningful variation in studying this effect in mycobacteria, because it occurred without the potentially confusing aspects of perturbing protein,protein interactions which are presumed vital to mycobacterial FASII, inactivating other important enzymes or eliciting a dedicated transcriptional response in Myco. tuberculosis. [source] The evolution of progesterone receptor ligandsMEDICINAL RESEARCH REVIEWS, Issue 3 2007Kevin P. Madauss Abstract Progesterone is one of the first nuclear receptor hormones to be described functionally and subsequently approached as a drug target. Because progesterone (1) affects both menstruation and gestation via the progesterone receptor (PR), research aimed at modulating its activity is usually surrounded by controversy. However, ligands for PR were developed into drugs, and their evolution can be crudely divided into three periods: (1) drug-like steroids that mimic the gestational properties of progesterone; (2) drug-like steroids with different properties from progesterone and expanded therapeutic applications; and (3) non-steroidal PR ligands with improved selectivity and modulator properties and further expanded therapeutic applications. Although the latter have yet to see widespread clinical applications, their development is founded on a half century of research, and they represent the future for this drug target. © 2006 Wiley Periodicals, Inc. Med Res Rev, 27, No. 3, 374,400, 2007 [source] Trypanosoma brucei pteridine reductase 1 is essential for survival in vitro and for virulence in miceMOLECULAR MICROBIOLOGY, Issue 3 2010Natasha Sienkiewicz Summary Gene knockout and knockdown methods were used to examine essentiality of pteridine reductase (PTR1) in pterin metabolism in the African trypanosome. Attempts to generate PTR1 null mutants in bloodstream form Trypanosoma brucei proved unsuccessful; despite integration of drug selectable markers at the target locus, the gene for PTR1 was either retained at the same locus or elsewhere in the genome. However, RNA interference (RNAi) resulted in complete knockdown of endogenous protein after 48 h, followed by cell death after 4 days. This lethal phenotype was reversed by expression of enzymatically active Leishmania major PTR1 in RNAi lines (oeRNAi) or by addition of tetrahydrobiopterin to cultures. Loss of PTR1 was associated with gross morphological changes due to a defect in cytokinesis, resulting in cells with multiple nuclei and kinetoplasts, as well as multiple detached flagella. Electron microscopy also revealed increased numbers of glycosomes, while immunofluorescence microscopy showed increased and more diffuse staining for glycosomal matrix enzymes, indicative of mis-localisation to the cytosol. Mis-localisation was confirmed by digitonin fractionation experiments. RNAi cell lines were markedly less virulent than wild-type parasites in mice and virulence was restored in the oeRNAi line. Thus, PTR1 may be a drug target for human African trypanosomiasis. [source] Characterization of a Leishmania stage-specific mitochondrial membrane protein that enhances the activity of cytochrome c oxidase and its role in virulenceMOLECULAR MICROBIOLOGY, Issue 2 2010Ranadhir Dey Summary Leishmaniasis is caused by the dimorphic protozoan parasite Leishmania. Differentiation of the insect form, promastigotes, to the vertebrate form, amastigotes, and survival inside the vertebrate host accompanies a drastic metabolic shift. We describe a gene first identified in amastigotes that is essential for survival inside the host. Gene expression analysis identified a 27 kDa protein-encoding gene (Ldp27) that was more abundantly expressed in amastigotes and metacyclic promastigotes than in procyclic promastigotes. Immunofluorescence and biochemical analysis revealed that Ldp27 is a mitochondrial membrane protein. Co-immunoprecipitation using antibodies to the cytochrome c oxidase (COX) complex, present in the inner mitochondrial membrane, placed the p27 protein in the COX complex. Ldp27 gene-deleted parasites (Ldp27,/,) showed significantly less COX activity and ATP synthesis than wild type in intracellular amastigotes. Moreover, the Ldp27,/, parasites were less virulent both in human macrophages and in BALB/c mice. These results demonstrate that Ldp27 is an important component of an active COX complex enhancing oxidative phosphorylation specifically in infectious metacyclics and amastigotes and promoting parasite survival in the host. Thus, Ldp27 can be explored as a potential drug target and parasites devoid of the p27 gene could be considered as a live attenuated vaccine candidate against visceral leishmaniasis. [source] The essential neutral sphingomyelinase is involved in the trafficking of the variant surface glycoprotein in the bloodstream form of Trypanosoma bruceiMOLECULAR MICROBIOLOGY, Issue 6 2010Simon A. Young Summary Sphingomyelin is the main sphingolipid in Trypanosoma brucei, the causative agent of African sleeping sickness. In vitro and in vivo characterization of the T. brucei neutral sphingomyelinase demonstrates that it is directly involved in sphingomyelin catabolism. Gene knockout studies in the bloodstream form of the parasite indicate that the neutral sphingomyelinase is essential for growth and survival, thus highlighting that the de novo biosynthesis of ceramide is unable to compensate for the loss of sphingomyelin catabolism. The phenotype of the conditional knockout has given new insights into the highly active endocytic and exocytic pathways in the bloodstream form of T. brucei. Hence, the formation of ceramide in the endoplasmic reticulum affects post-Golgi sorting and rate of deposition of newly synthesized GPI-anchored variant surface glycoprotein on the cell surface. This directly influences the corresponding rate of endocytosis, via the recycling endosomes, of pre-existing cell surface variant surface glycoprotein. The trypanosomes use this coupled endocytic and exocytic mechanism to maintain the cell density of its crucial variant surface glycoprotein protective coat. TbnSMase is therefore genetically validated as a drug target against African trypanosomes, and suggests that interfering with the endocytic transport of variant surface glycoprotein is a highly desirable strategy for drug development against African trypanosomasis. [source] Dissecting the essentiality of the bifunctional trypanothione synthetase-amidase in Trypanosoma brucei using chemical and genetic methodsMOLECULAR MICROBIOLOGY, Issue 3 2009Susan Wyllie Summary The bifunctional trypanothione synthetase-amidase (TRYS) comprises two structurally distinct catalytic domains for synthesis and hydrolysis of trypanothione (N1,N8 - bis(glutathionyl)spermidine). This unique dithiol plays a pivotal role in thiol-redox homeostasis and in defence against chemical and oxidative stress in trypanosomatids. A tetracycline-dependent conditional double knockout of TRYS (cDKO) was generated in bloodstream Trypanosoma brucei. Culture of cDKO parasites without tetracycline induction resulted in loss of trypanothione and accumulation of glutathione, followed by growth inhibition and cell lysis after 6 days. In the absence of inducer, cDKO cells were unable to infect mice, confirming that this enzyme is essential for virulence in vivo as well as in vitro. To establish whether both enzymatic functions were essential, an amidase-dead mutant cDKO line was generated. In the presence of inducer, this line showed decreased growth in vitro and decreased virulence in vivo, indicating that the amidase function is not absolutely required for viability. The druggability of TRYS was assessed using a potent small molecule inhibitor developed in our laboratory. Growth inhibition correlated in rank order cDKO, single KO, wild-type and overexpressing lines and produced the predicted biochemical phenotype. The synthetase function of TRYS is thus unequivocally validated as a drug target by both chemical and genetic methods. [source] The glycosylphosphatidylinositol (GPI) biosynthetic pathway of bloodstream-form Trypanosoma brucei is dependent on the de novo synthesis of inositolMOLECULAR MICROBIOLOGY, Issue 1 2006Kirstee L. Martin Summary In bloodstream-form Trypanosoma brucei (the causative agent of African sleeping sickness) the glycosylphosphatidylinositol (GPI) anchor biosynthetic pathway has been validated genetically and chemically as a drug target. The conundrum that GPI anchors could not be in vivo labelled with [3H]-inositol led us to hypothesize that de novo synthesis was responsible for supplying myo -inositol for phosphatidylinositol (PI) destined for GPI synthesis. The rate-limiting step of the de novo synthesis is the isomerization of glucose 6-phosphate to 1- d -myo -inositol-3-phosphate, catalysed by a 1- d -myo -inositol-3-phosphate synthase (INO1). When grown under non-permissive conditions, a conditional double knockout demonstrated that INO1 is an essential gene in bloodstream-form T. brucei. It also showed that the de novo synthesized myo -inositol is utilized to form PI, which is preferentially used in GPI biosynthesis. We also show for the first time that extracellular myo- inositol can in fact be used in GPI formation although to a limited extent. Despite this, extracellular inositol cannot compensate for the deletion of INO1. Supporting these results, there was no change in PI levels in the conditional double knockout cells grown under non-permissive conditions, showing that perturbation of growth is due to a specific lack of de novo synthesized myo -inositol and not a general inositol-less death. These results suggest that there is a distinction between de novo synthesized myo -inositol and that from the extracellular environment. [source] A new role for P2 receptors: talking with calcium-activated potassium channelsNEUROGASTROENTEROLOGY & MOTILITY, Issue 11 2007P. P. Bertrand Abstract Purinergic fast synaptic transmission may play a very subtle role in regulating the excitability of enteric circuits. That is one of the important findings in a new paper by Ren and Galligan in the current issue of this Journal. They first provide compelling evidence that P2X3 receptors (ionotropic purine receptors) are expressed by guinea-pig motor and interneurons and that these subtypes mediate the purinergic fast excitatory postsynaptic potential (EPSP). They also found that the P2X3 -mediated depolarization was often followed by a hyperpolarization. This is an intriguing finding because if the purinergic fast EPSPs are also followed by a hyperpolarization, then it could play a role in truncating bursts of synaptic potentials or in shaping periodic synaptic input. The hyperpolarization is caused by calcium entry through the P2X3 receptor which then activates a calcium-activated potassium (KCa) channel. Surprisingly, the hyperpolarization was not affected by any of the standard blockers of calcium- or voltage-activated K+ channels suggesting that a novel KCa channel is present in the enteric neurons. Such a wide-spread channel could well have an important physiological role and could be an important new drug target for regulating reflex activity in the enteric nervous system. [source] A critical review of the cannabinoid receptor as a drug target for obesity managementOBESITY REVIEWS, Issue 1 2009F. Akbas Summary The discovery of cannabinoids, with the well-known stimulatory effect of Cannabis sativa on appetite, has offered a new drug target for obesity treatment. Cannabinoids act on two different receptors: CB1 receptors which are sited in the brain and many peripheral tissues, and CB2 receptors which are primarily found in immune system cells. Cannabinoid receptor antagonists act centrally by blocking CB1 receptors, thereby reducing food intake. Moreover, they probably also act peripherally by increasing thermogenesis and therefore energy expenditure, as has been suggested by animal experiments. Despite these promising mechanisms of action, recent clinical studies examining the effect of the two CB1 receptor antagonists rimonabant and taranabant showed that the attained weight loss did not exceed that attained with other currently approved anti-obesity medications. Moreover, potentially severe psychiatric adverse effects limit their clinical use. As several new CB1 receptor antagonists are presently undergoing development, it remains to be elucidated to what extent they differ in terms of efficacy and safety. This review primarily discusses how close cannabinoid receptor antagonists are to the ideal anti-obesity drug, with respect to their mechanisms of action, clinical effectiveness and safety. [source] Stearoyl-CoA desaturase as a new drug target for obesity treatmentOBESITY REVIEWS, Issue 2 2005A. Dobrzyn Summary Stearoyl-CoA desaturase (SCD), the rate-limiting enzyme in monounsaturated fatty acid synthesis, has recently been shown to be the critical control point regulating hepatic lipogenesis and lipid oxidation. As several manifestations of the metabolic syndrome and type 2 diabetes mellitus are associated with alterations in intracellular lipid partitioning, we propose that SCD1 may be a potential therapeutic target in the treatment of obesity and the metabolic syndrome. In support of this notion, we have shown that SCD1-deficient mice have increased energy expenditure, reduced body adiposity, increased insulin sensitivity and are resistant to diet-induced obesity and liver steatosis. Furthermore, SCD1 was found to be specifically repressed during leptin-mediated weight loss, and leptin-deficient ob/ob mice lacking SCD1 showed marked correction of the hypometabolic phenotype and hepatic steatosis. Much evidence indicates that the direct anti-steatotic effect of SCD1 deficiency stems from increased fatty acid oxidation and decreased lipid synthesis. All of these findings reveal that pharmacological manipulation of SCD activity might be of benefit in the treatment of obesity, diabetes, liver steatosis and other diseases of the metabolic syndrome. [source] Crystal structure of a major secreted protein of Mycobacterium tuberculosis,MPT63 at 1.5-Å resolutionPROTEIN SCIENCE, Issue 12 2002Celia W. Goulding Abstract MPT63 is a small, major secreted protein of unknown function from Mycobacterium tuberculosis that has been shown to have immunogenic properties and has been implicated in virulence. A BLAST search identified that MPT63 has homologs only in other mycobacteria, and is therefore mycobacteria specific. As MPT63 is a secreted protein, mycobacteria specific, and implicated in virulence, MPT63 is an attractive drug target against the deadliest infectious disease, tuberculosis (TB). As part of the TB Structural Genomics Consortium, the X-ray crystal structure of MPT63 was determined to 1.5-Ångstrom resolution with the hope of yielding functional information about MPT63. The structure of MPT63 is an antiparallel ,-sandwich immunoglobulin-like fold, with the unusual feature of the first ,-strand of the protein forming a parallel addition to the small antiparallel ,-sheet. MPT63 has weak structural similarity to many proteins with immunoglobulin folds, in particular, Homo sapiens ,2-adaptin, bovine arrestin, and Yersinia pseudotuberculosis invasin. Although the structure of MPT63 gives no conclusive evidence to its function, structural similarity suggests that MPT63 could be involved in cell-host interactions to facilitate endocytosis/phagocytosis. [source] Self-association of an amphipathic helix peptide inhibitor of HIV-1 integrase assessed by electro spray ionization mass spectrometry in trifluoroethanol/water mixturesRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 5 2001S. Fermandjian Establishing the auto-associative properties of a molecule in solution can be important for determination of its structure and function. EAA26 (VESMNEELKKIIAQVRAQAEHLKTAY) has been designed to inhibit HIV-1 integrase via formation of a stable coiled-coil structure with a nearly homologous segment in the enzyme. The latter catalyzes the permanent incorporation of a DNA copy of the retrovirus genome into host cell DNA, and is thus essential to the life of the retrovirus. This makes integrase an obvious drug target in the therapy of AIDS. The present work has demonstrated, using electrospray ionization mass spectrometry (ESI-MS), that EAA26 is monomeric in pure water, and tetrameric and dimeric at respectively low and medium concentrations of 2,2,2-trifluoroethanol (TFE), and again monomeric at higher TFE concentrations. Thus, the apolar solvent TFE may contribute to either stabilization or disruption of the intermolecular hydrophobic contacts depending on its concentration in aqueous solution. Previous NMR and ultracentifugation results are thus confirmed, indicating the reliability of ESI-MS for defining the self-association state of biologically relevant peptides in both water and organic-water solutions. Copyright © 2001 John Wiley & Sons, Ltd. [source] Signal Transduction Pathways Involved in Brain Death-Induced Renal InjuryAMERICAN JOURNAL OF TRANSPLANTATION, Issue 5 2009H. R. Bouma Kidneys derived from brain death organ donors show an inferior survival when compared to kidneys derived from living donors. Brain death is known to induce organ injury by evoking an inflammatory response in the donor. Neuronal injury triggers an inflammatory response in the brain, leading to endothelial dysfunction and the release of cytokines in the circulation. Serum levels of interleukin-6, -8, -10, and monocyte chemoattractant protein-1 (MCP-1) are increased after brain death. Binding with cytokine-receptors in kidneys stimulates activation of nuclear factor-kappa B (NF-,B), selectins, adhesion molecules and production of chemokines leading to cellular influx. Mitogen-activated protein kinases (MAP-kinases) mediate inflammatory responses and together with NF-,B they seem to play an important role in brain death induced renal injury. Altering the activation state of MAP-kinases could be a promising drug target for early intervention to reduce cerebral injury related donor kidney damage and improve outcome after transplantation. [source] The X-ray structure of Salmonella typhimurium uridine nucleoside phosphorylase complexed with 2,2,-anhydrouridine, phosphate and potassium ions at 1.86,Å resolutionACTA CRYSTALLOGRAPHICA SECTION D, Issue 1 2010Alexander A. Lashkov Uridine nucleoside phosphorylase is an important drug target for the development of anti-infective and antitumour agents. The X-ray crystal structure of Salmonella typhimurium uridine nucleoside phosphorylase (StUPh) complexed with its inhibitor 2,2,-anhydrouridine, phosphate and potassium ions has been solved and refined at 1.86,Å resolution (Rcryst = 17.6%, Rfree = 20.6%). The complex of human uridine phosphorylase I (HUPhI) with 2,2,-anhydrouridine was modelled using a computational approach. The model allowed the identification of atomic groups in 2,2,-anhydrouridine that might improve the interaction of future inhibitors with StUPh and HUPhI. [source] Structure and function of GlmU from Mycobacterium tuberculosisACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2009Zhening Zhang Antibiotic resistance is a major issue in the treatment of infectious diseases such as tuberculosis. Existing antibiotics target only a few cellular pathways and there is an urgent need for antibiotics that have novel molecular mechanisms. The glmU gene is essential in Mycobacterium tuberculosis, being required for optimal bacterial growth, and has been selected as a possible drug target for structural and functional investigation. GlmU is a bifunctional acetyltransferase/uridyltransferase that catalyses the formation of UDP-GlcNAc from GlcN-1-P. UDP-GlcNAc is a substrate for two important biosynthetic pathways: lipopolysaccharide and peptidoglycan synthesis. The crystal structure of M. tuberculosis GlmU has been determined in an unliganded form and in complex with GlcNAc-1-P or UDP-GlcNAc. The structures reveal the residues that are responsible for substrate binding. Enzyme activities were characterized by 1H NMR and suggest that the presence of acetyl-coenzyme A has an inhibitory effect on uridyltransferase activity. [source] The 1.25,Å resolution structure of phosphoribosyl-ATP pyrophosphohydrolase from Mycobacterium tuberculosisACTA CRYSTALLOGRAPHICA SECTION D, Issue 6 2008Farah Javid-Majd Phosphoribosyl-ATP pyrophosphohydrolase is the second enzyme in the histidine-biosynthetic pathway, irreversibly hydrolyzing phosphoribosyl-ATP to phosphoribosyl-AMP and pyrophosphate. It is encoded by the hisE gene, which is present as a separate gene in many bacteria and archaea but is fused to hisI in other bacteria, fungi and plants. Because of its essentiality for growth in vitro, HisE is a potential drug target for tuberculosis. The crystal structures of two native (uncomplexed) forms of HisE from Mycobacterium tuberculosis have been determined to resolutions of 1.25 and 1.79,Å. The structure of the apoenzyme reveals that the protein is composed of five ,-helices with connecting loops and is a member of the ,-helical nucleoside-triphosphate pyrophosphatase superfamily. The biological unit of the protein is a homodimer, with an active site on each subunit composed of residues exclusively from that subunit. A comparison with the Campylobacter jejuni dUTPase active site allowed the identification of putative metal- and substrate-binding sites in HisE, including four conserved glutamate and glutamine residues in the sequence that are consistent with a motif for pyrophosphohydrolase activity. However, significant differences between family members are observed in the loop region between ,-helices H1 and H3. The crystal structure of M. tuberculosis HisE provides insights into possible mechanisms of substrate binding and the diversity of the nucleoside-triphosphate pyrophosphatase superfamily. [source] | ||||||||||||||||||||||||||||||||||