The Experts below are selected from a list of 195 Experts worldwide ranked by ideXlab platform
J Lipowska - One of the best experts on this subject based on the ideXlab platform.
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Pyrimidine Biosynthesis in pathogens structures and analysis of dihydroorotases from yersinia pestis and vibrio cholerae
International Journal of Biological Macromolecules, 2019Co-Authors: J Lipowska, Charles D Miks, K Kwon, L Shuvalova, H Zheng, Krzysztof Lewinski, D R Cooper, I G Shabalin, W MinorAbstract:Abstract The de novo Pyrimidine Biosynthesis pathway is essential for the proliferation of many pathogens. One of the pathway enzymes, dihydroorotase (DHO), catalyzes the reversible interconversion of N-carbamoyl- l -aspartate to 4,5-dihydroorotate. The substantial difference between bacterial and mammalian DHOs makes it a promising drug target for disrupting bacterial growth and thus an important candidate to evaluate as a response to antimicrobial resistance on a molecular level. Here, we present two novel three-dimensional structures of DHOs from Yersinia pestis ( Yp DHO), the plague-causing pathogen, and Vibrio cholerae ( Vc DHO), the causative agent of cholera. The evaluations of these two structures led to an analysis of all available DHO structures and their classification into known DHO types. Comparison of all the DHO active sites containing ligands that are listed in DrugBank was facilitated by a new interactive, structure-comparison and presentation platform. In addition, we examined the genetic context of characterized DHOs, which revealed characteristic patterns for different types of DHOs. We also generated a homology model for DHO from Plasmodium falciparum .
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Pyrimidine Biosynthesis in pathogens – Structures and analysis of dihydroorotases from Yersinia pestis and Vibrio cholerae
International Journal of Biological Macromolecules, 2019Co-Authors: J Lipowska, Charles D Miks, K Kwon, L Shuvalova, H Zheng, D R Cooper, I G Shabalin, Krzysztof Lewiński, W MinorAbstract:The de novo Pyrimidine Biosynthesis pathway is essential for the proliferation of many pathogens. One of the pathway enzymes, dihydroorotase (DHO), catalyzes the reversible interconversion of N-carbamoyl-l-aspartate to 4,5-dihydroorotate. The substantial difference between bacterial and mammalian DHOs makes it a promising drug target for disrupting bacterial growth and thus an important candidate to evaluate as a response to antimicrobial resistance on a molecular level. Here, we present two novel three-dimensional structures of DHOs from Yersinia pestis (YpDHO), the plague-causing pathogen, and Vibrio cholerae (VcDHO), the causative agent of cholera. The evaluations of these two structures led to an analysis of all available DHO structures and their classification into known DHO types. Comparison of all the DHO active sites containing ligands that are listed in DrugBank was facilitated by a new interactive, structure-comparison and presentation platform. In addition, we examined the genetic context of characterized DHOs, which revealed characteristic patterns for different types of DHOs. We also generated a homology model for DHO from Plasmodium falciparum.
W Minor - One of the best experts on this subject based on the ideXlab platform.
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Pyrimidine Biosynthesis in pathogens structures and analysis of dihydroorotases from yersinia pestis and vibrio cholerae
International Journal of Biological Macromolecules, 2019Co-Authors: J Lipowska, Charles D Miks, K Kwon, L Shuvalova, H Zheng, Krzysztof Lewinski, D R Cooper, I G Shabalin, W MinorAbstract:Abstract The de novo Pyrimidine Biosynthesis pathway is essential for the proliferation of many pathogens. One of the pathway enzymes, dihydroorotase (DHO), catalyzes the reversible interconversion of N-carbamoyl- l -aspartate to 4,5-dihydroorotate. The substantial difference between bacterial and mammalian DHOs makes it a promising drug target for disrupting bacterial growth and thus an important candidate to evaluate as a response to antimicrobial resistance on a molecular level. Here, we present two novel three-dimensional structures of DHOs from Yersinia pestis ( Yp DHO), the plague-causing pathogen, and Vibrio cholerae ( Vc DHO), the causative agent of cholera. The evaluations of these two structures led to an analysis of all available DHO structures and their classification into known DHO types. Comparison of all the DHO active sites containing ligands that are listed in DrugBank was facilitated by a new interactive, structure-comparison and presentation platform. In addition, we examined the genetic context of characterized DHOs, which revealed characteristic patterns for different types of DHOs. We also generated a homology model for DHO from Plasmodium falciparum .
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Pyrimidine Biosynthesis in pathogens – Structures and analysis of dihydroorotases from Yersinia pestis and Vibrio cholerae
International Journal of Biological Macromolecules, 2019Co-Authors: J Lipowska, Charles D Miks, K Kwon, L Shuvalova, H Zheng, D R Cooper, I G Shabalin, Krzysztof Lewiński, W MinorAbstract:The de novo Pyrimidine Biosynthesis pathway is essential for the proliferation of many pathogens. One of the pathway enzymes, dihydroorotase (DHO), catalyzes the reversible interconversion of N-carbamoyl-l-aspartate to 4,5-dihydroorotate. The substantial difference between bacterial and mammalian DHOs makes it a promising drug target for disrupting bacterial growth and thus an important candidate to evaluate as a response to antimicrobial resistance on a molecular level. Here, we present two novel three-dimensional structures of DHOs from Yersinia pestis (YpDHO), the plague-causing pathogen, and Vibrio cholerae (VcDHO), the causative agent of cholera. The evaluations of these two structures led to an analysis of all available DHO structures and their classification into known DHO types. Comparison of all the DHO active sites containing ligands that are listed in DrugBank was facilitated by a new interactive, structure-comparison and presentation platform. In addition, we examined the genetic context of characterized DHOs, which revealed characteristic patterns for different types of DHOs. We also generated a homology model for DHO from Plasmodium falciparum.
Frederic Sigoillot - One of the best experts on this subject based on the ideXlab platform.
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Breakdown of the regulatory control of Pyrimidine Biosynthesis in human breast cancer cells
International Journal of Cancer, 2004Co-Authors: Frederic Sigoillot, Severine M SigoillotAbstract:The activity of the de novo Pyrimidine biosynthetic pathway in the MCF7 breast cancer cells was 4.4-fold higher than that in normal MCF10A breast cells. Moreover, while Pyrimidine Biosynthesis in MCF10A was tightly regulated, increasing as the culture matured and subsequently down-regulated in confluency, the biosynthetic rate in MCF7 cells remained elevated and invariant in all growth phases. The flux through the pathway is regulated by carbamoyl phosphate synthetase, a component of the multifunctional protein, CAD. The intracellular CAD concentration was 3.5- to 4-fold higher in MCF7 cells, an observation that explains the high rate of Pyrimidine Biosynthesis but cannot account for the lack of growth-dependent regulation. In MCF10A cells, up-regulation of the pathway in the exponential growth phase resulted from MAP kinase phosphorylation of CAD Thr456. The pathway was subsequently down-regulated by dephosphorylation of P approximately Thr456 and the phosphorylation of CAD by PKA. In contrast, the CAD P approximately Thr456 was persistently phosphorylated in MCF7 cells, while the PKA site remained unphosphorylated and consequently the activity of the pathway was elevated in all growth phases. In support of this interpretation, inhibition of MAP kinase in MCF7 cells decreased CAD P approximately Thr456, increased PKA phosphorylation and decreased Pyrimidine Biosynthesis. Conversely, transfection of MCF10A with constructs that elevated MAP kinase activity increased CAD P approximately Thr456 and the Pyrimidine biosynthetic rate. The differences in the CAD phosphorylation state responsible for unregulated Pyrimidine Biosynthesis in MCF7 cells are likely to be a consequence of the elevated MAP kinase activity and the antagonism between MAP kinase- and PKA-mediated phosphorylations.
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breakdown of the regulatory control of Pyrimidine Biosynthesis in human breast cancer cells
International Journal of Cancer, 2004Co-Authors: Frederic Sigoillot, Severine M SigoillotAbstract:The activity of the de novo Pyrimidine biosynthetic pathway in the MCF7 breast cancer cells was 4.4-fold higher than that in normal MCF10A breast cells. Moreover, while Pyrimidine Biosynthesis in MCF10A was tightly regulated, increasing as the culture matured and subsequently down-regulated in confluency, the biosynthetic rate in MCF7 cells remained elevated and invariant in all growth phases. The flux through the pathway is regulated by carbamoyl phosphate synthetase, a component of the multifunctional protein, CAD. The intracellular CAD concentration was 3.5- to 4-fold higher in MCF7 cells, an observation that explains the high rate of Pyrimidine Biosynthesis but cannot account for the lack of growth-dependent regulation. In MCF10A cells, up-regulation of the pathway in the exponential growth phase resulted from MAP kinase phosphorylation of CAD Thr456. The pathway was subsequently down-regulated by dephosphorylation of P∼Thr456 and the phosphorylation of CAD by PKA. In contrast, the CAD P∼Thr456 was persistently phosphorylated in MCF7 cells, while the PKA site remained unphosphorylated and consequently the activity of the pathway was elevated in all growth phases. In support of this interpretation, inhibition of MAP kinase in MCF7 cells decreased CAD P∼Thr456, increased PKA phosphorylation and decreased Pyrimidine Biosynthesis. Conversely, transfection of MCF10A with constructs that elevated MAP kinase activity increased CAD P∼Thr456 and the Pyrimidine biosynthetic rate. The differences in the CAD phosphorylation state responsible for unregulated Pyrimidine Biosynthesis in MCF7 cells are likely to be a consequence of the elevated MAP kinase activity and the antagonism between MAP kinase- and PKA-mediated phosphorylations. © 2004 Wiley-Liss, Inc.
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cell cycle dependent regulation of Pyrimidine Biosynthesis
Journal of Biological Chemistry, 2003Co-Authors: Frederic Sigoillot, Andrew J Berkowski, Severine M Sigoillot, Damian H KotsisAbstract:Abstract De novo Pyrimidine Biosynthesis is activated in proliferating cells in response to an increased demand for nucleotides needed for DNA synthesis. The Pyrimidine biosynthetic pathway in baby hamster kidney cells, synchronized by serum deprivation, was found to be up-regulated 1.9-fold during S phase and subsequently down-regulated as the cells progressed through the cycle. The nucleotide pools were depleted by serum starvation and were not replenished during the first round of cell division, suggesting that the rate of utilization of the newly synthesized nucleotides closely matched their rate of formation. The activation and subsequent down-regulation of the pathway can be attributed to altered allosteric regulation of the carbamoyl-phosphate synthetase activity of CAD (carbamoyl-phosphate synthetase-aspartate carbamoyltransferase-dihydroorotase), a multifunctional protein that initiates mammalian Pyrimidine Biosynthesis. As the culture approached S-phase there was an increased sensitivity to the allosteric activator, 5-phosphoribosyl-1-pyrophosphate, and a loss of UTP inhibition, changes that were reversed when cells emerged from S phase. The allosteric regulation of CAD is known to be modulated by MAP kinase (MAPK) and protein kinase A (PKA)-mediated phosphorylations as well as by autophosphorylation. CAD was found to be fully autophosphorylated in the synchronized cells, but the level remained invariant throughout the cycle. Although the MAPK activity increased early in G1, the phosphorylation of the CAD MAPK site was delayed until just before the onset of S phase, probably due to antagonistic phosphorylation by PKA that persisted until late G1. Once activated, Pyrimidine Biosynthesis remained elevated until rephosphorylation of CAD by PKA and dephosphorylation of the CAD MAPK site late in S phase. Thus, the cell cycle-dependent regulation of Pyrimidine Biosynthesis results from the sequential phosphorylation and dephosphorylation of CAD under the control of two important signaling cascades.
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autophosphorylation of the mammalian multifunctional protein that initiates de novo Pyrimidine Biosynthesis
Journal of Biological Chemistry, 2002Co-Authors: Frederic Sigoillot, David R. EvansAbstract:Abstract CAD, a large multifunctional protein that carries carbamoyl phosphate synthetase (CPSase), aspartate transcarbamoylase, and dihydroorotase activities, catalyzes the first three steps of de novo Pyrimidine Biosynthesis in mammalian cells. The CPSase component, which catalyzes the initial, rate-limiting step, exhibits complex regulatory mechanisms involving allosteric effectors and phosphorylation that control the flux of metabolites through the pathway. Incubation of CAD with ATP in the absence of exogenous kinases resulted in the incorporation of 1 mol of Pi/mol of CAD monomer. Mass spectrometry analysis of tryptic digests showed that Thr1037 located within the CAD CPS.B subdomain was specifically modified. The reaction is specific for MgATP, ADP was a competitive inhibitor, and the native tertiary structure of the protein was required. Phosphorylation occurred after denaturation, further purification of CAD by SDS gel electrophoresis, and renaturation on a nitrocellulose membrane, strongly suggesting that phosphate incorporation resulted from an intrinsic kinase activity and was not the result of contaminating kinases. Chemical modification with the ATP analog, 5′-p-fluorosulfonylbenzoyladenosine, showed that one or both of the active sites that catalyze the ATP-dependent partial reactions are also involved in autophosphorylation. The rate of phosphorylation was dependent on the concentration of CAD, indicating that the reaction was, at least in part, intermolecular. Autophosphorylation resulted in a 2-fold increase in CPSase activity, an increased sensitivity to the feedback inhibitor UTP, and decreased allosteric activation by 5-phosphoribosyl-1-pyrophosphate, functional changes that were distinctly different from those resulting from phosphorylation by either the protein kinase A or mitogen-activated protein kinase cascades.
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growth dependent regulation of mammalian Pyrimidine Biosynthesis by the protein kinase a and mapk signaling cascades
Journal of Biological Chemistry, 2002Co-Authors: Frederic Sigoillot, David R. EvansAbstract:Abstract The carbamoyl phosphate synthetase domain of the multifunctional protein CAD catalyzes the initial, rate-limiting step in mammalian de novo Pyrimidine Biosynthesis. In addition to allosteric regulation by the inhibitor UTP and the activator PRPP, the carbamoyl phosphate synthetase activity is controlled by mitogen-activated protein kinase (MAPK)- and protein kinase A (PKA)-mediated phosphorylation. MAPK phosphorylation, both in vivo and in vitro, increases sensitivity to PRPP and decreases sensitivity to the inhibitor UTP, whereas PKA phosphorylation reduces the response to both allosteric effectors. To elucidate the factors responsible for growth state-dependent regulation of Pyrimidine Biosynthesis, the activity of the de novoPyrimidine pathway, the MAPK and PKA activities, the phosphorylation state, and the allosteric regulation of CAD were measured as a function of growth state. As cells entered the exponential growth phase, there was an 8-fold increase in Pyrimidine Biosynthesis that was accompanied by a 40-fold increase in MAPK activity and a 4-fold increase in CAD threonine phosphorylation. PRPP activation increased to 21-fold, and UTP became a modest activator. These changes were reversed when the cultures approach confluence and growth ceases. Moreover, CAD phosphoserine, a measure of PKA phosphorylation, increased 2-fold in confluent cells. These results are consistent with the activation of CAD by MAPK during periods of rapid growth and its down-regulation in confluent cells associated with decreased MAPK phosphorylation and a concomitant increase in PKA phosphorylation. A scheme is proposed that could account for growth-dependent regulation of Pyrimidine Biosynthesis based on the sequential action of MAPK and PKA on the carbamoyl phosphate synthetase activity of CAD.
Daniel Dauzonne - One of the best experts on this subject based on the ideXlab platform.
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original chemical series of Pyrimidine Biosynthesis inhibitors that boost the antiviral interferon response
Antimicrobial Agents and Chemotherapy, 2017Co-Authors: Marianne Lucashourani, Daniel Dauzonne, Helene Munierlehmann, Samira Khiar, Sebastien Nisole, Julien Dairou, Olivier Helynck, Philippe V Afonso, Frederic Tangy, Pierreolivier VidalainAbstract:ABSTRACT De novo Pyrimidine Biosynthesis is a key metabolic pathway involved in multiple biosynthetic processes. Here, we identified an original series of 3-(1 H -indol-3-yl)-2,3-dihydro-4 H -furo[3,2- c ]chromen-4-one derivatives as a new class of Pyrimidine Biosynthesis inhibitors formed by two edge-fused polycyclic moieties. We show that identified compounds exhibit broad-spectrum antiviral activity and immunostimulatory properties, in line with recent reports linking de novo Pyrimidine Biosynthesis with innate defense mechanisms against viruses. Most importantly, we establish that Pyrimidine deprivation can amplify the production of both type I and type III interferons by cells stimulated with retinoic acid-inducible gene 1 (RIG-I) ligands. Altogether, our results further expand the current panel of Pyrimidine Biosynthesis inhibitors and illustrate how the production of antiviral interferons is tightly coupled to this metabolic pathway. Functional and structural similarities between this new chemical series and dicoumarol, which was reported before to inhibit Pyrimidine Biosynthesis at the dihydroorotate dehydrogenase (DHODH) step, are discussed.
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Respiratory syncytial virus infection in macaques is not suppressed by intranasal sprays of Pyrimidine Biosynthesis inhibitors.
Antiviral Research, 2016Co-Authors: Clément Grandin, Daniel Dauzonne, Marianne-lucas Hourani, Yves L Janin, Hélène Munier-lehmann, Adeline Paturet, Fabrice Taborik, Astrid Vabret, Hugues Contamin, Frederic TangyAbstract:There is imperious need for efficient therapies against ubiquitous and life-threatening respiratory viruses, foremost among them being the human respiratory syncytial virus (hRSV). Several research groups who performed functional screens for broad-spectrum antivirals identified compounds targeting the de novo Pyrimidine Biosynthesis pathway. Despite their strong antiviral activity in vitro, whether such antimetabolites are effective in vivo remains highly controversial. Here, we evaluated two potent Pyrimidine Biosynthesis inhibitors developed in our laboratory, IPPA17-A04 and GAC50, in a model of mild hRSV-infection in cynomolgus macaques. In this model, hRSV replication is restricted to the epithelium of the upper respiratory tract, and is compatible with a topical treatment by intranasal sprays. The local administration of palivizumab, a neutralizing anti-hRSV antibody used in clinics, significantly reduced virus replication. In contrast, Pyrimidine Biosynthesis inhibitors did not show any inhibitory effect on hRSV growth when delivered topically as experimented in our model. Our results should help to better define the potential applications of this class of antimetabolites in the treatment of viral infections.
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inhibition of Pyrimidine Biosynthesis pathway suppresses viral growth through innate immunity
PLOS Pathogens, 2013Co-Authors: Marianne Lucashourani, Daniel Dauzonne, Pierre Jorda, Gaelle Cousin, Alexandru Lupan, Olivier HelynckAbstract:Searching for stimulators of the innate antiviral response is an appealing approach to develop novel therapeutics against viral infections. Here, we established a cell-based reporter assay to identify compounds stimulating expression of interferon-inducible antiviral genes. DD264 was selected out of 41,353 compounds for both its immuno-stimulatory and antiviral properties. While searching for its mode of action, we identified DD264 as an inhibitor of Pyrimidine Biosynthesis pathway. This metabolic pathway was recently identified as a prime target of broad-spectrum antiviral molecules, but our data unraveled a yet unsuspected link with innate immunity. Indeed, we showed that DD264 or brequinar, a well-known inhibitor of Pyrimidine Biosynthesis pathway, both enhanced the expression of antiviral genes in human cells. Furthermore, antiviral activity of DD264 or brequinar was found strictly dependent on cellular gene transcription, nuclear export machinery, and required IRF1 transcription factor. In conclusion, the antiviral property of Pyrimidine Biosynthesis inhibitors is not a direct consequence of Pyrimidine deprivation on the virus machinery, but rather involves the induction of cellular immune response.
Megan L Shaw - One of the best experts on this subject based on the ideXlab platform.
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inhibition of arenavirus by a3 a Pyrimidine Biosynthesis inhibitor
Journal of Virology, 2014Co-Authors: Emilio Ortizriano, Stefanie Renee Devito, Dirk Eggink, Joshua Munger, Megan L Shaw, Juan Carlos De La Torre, Luis MartinezsobridoAbstract:Arenaviruses merit significant interest as important human pathogens, since several of them cause severe hemorrhagic fever disease that is associated with high morbidity and significant mortality. Currently, there are no FDA-licensed arenavirus vaccines available, and current antiarenaviral therapy is limited to an off-labeled use of the nucleoside analog ribavirin, which has limited prophylactic efficacy. The Pyrimidine Biosynthesis inhibitor A3, which was identified in a high-throughput screen for compounds that blocked influenza virus replication, exhibits a broad-spectrum antiviral activity against negative- and positive-sense RNA viruses, retroviruses, and DNA viruses. In this study, we evaluated the antiviral activity of A3 against representative Old World (lymphocytic choriomeningitis virus) and New World (Junin virus) arenaviruses in rodent, monkey, and human cell lines. We show that A3 is significantly more efficient than ribavirin in controlling arenavirus multiplication and that the A3 inhibitory effect is in part due to its ability to interfere with viral RNA replication and transcription. We document an additive antiarenavirus effect of A3 and ribavirin, supporting the potential combination therapy of ribavirin and Pyrimidine Biosynthesis inhibitors for the treatment of arenavirus infections.
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broad spectrum antiviral that interferes with de novo Pyrimidine Biosynthesis
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Hansheinrich Hoffmann, Andrea Kunz, Viviana Simon, Peter Palese, Megan L ShawAbstract:Compound A3 was identified in a high-throughput screen for inhibitors of influenza virus replication. It displays broad-spectrum antiviral activity, and at noncytotoxic concentrations it is shown to inhibit the replication of negative-sense RNA viruses (influenza viruses A and B, Newcastle disease virus, and vesicular stomatitis virus), positive-sense RNA viruses (Sindbis virus, hepatitis C virus, West Nile virus, and dengue virus), DNA viruses (vaccinia virus and human adenovirus), and retroviruses (HIV). In contrast to mammalian cells, inhibition of viral replication by A3 is absent in chicken cells, which suggests species-specific activity of A3. Correspondingly, the antiviral activity of A3 can be linked to a cellular protein, dihydroorotate dehydrogenase (DHODH), which is an enzyme in the de novo Pyrimidine Biosynthesis pathway. Viral replication of both RNA and DNA viruses can be restored in the presence of excess uracil, which promotes Pyrimidine salvage, or excess orotic acid, which is the product of DHODH in the de novo Pyrimidine Biosynthesis pathway. Based on these findings, it is proposed that A3 acts by depleting Pyrimidine pools, which are crucial for efficient virus replication.
