37 results on '"Nonato, Maria Cristina"'
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2. Introducing CRAFT: The Center for Research and Advancement in Fragments and molecular Targets.
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Andrade, Carolina Horta, Nonato, Maria Cristina, and da Silva Emery, Flavio
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- 2024
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3. Druggable hot spots in trypanothione reductase: novel insights and opportunities for drug discovery revealed by DRUGpy
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Teixeira, Olivia, Lacerda, Pedro, Froes, Thamires Quadros, Nonato, Maria Cristina, and Castilho, Marcelo Santos
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- 2021
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4. Women in science symposium
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Crossman, David J. and Nonato, Maria Cristina
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- 2021
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5. Evaluation of 7-arylaminopyrazolo[1,5-a]pyrimidines as anti-Plasmodium falciparum, antimalarial, and Pf-dihydroorotate dehydrogenase inhibitors
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Azeredo, Luís Felipe S.P., Coutinho, Julia P., Jabor, Valquiria A.P., Feliciano, Patricia R., Nonato, Maria Cristina, Kaiser, Carlos R., Menezes, Carla Maria S., Hammes, Amanda S.O., Caffarena, Ernesto Raul, Hoelz, Lucas V.B., de Souza, Nicolli B., Pereira, Glaécia A.N., Cerávolo, Isabela P., Krettli, Antoniana U., and Boechat, Nubia
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- 2017
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6. Anti- Trypanosoma cruzi Activity, Mutagenicity, Hepatocytotoxicity and Nitroreductase Enzyme Evaluation of 3-Nitrotriazole, 2-Nitroimidazole and Triazole Derivatives.
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Menozzi, Cheyene Almeida Celestino, França, Rodolfo Rodrigo Florido, Luccas, Pedro Henrique, Baptista, Mayara dos Santos, Fernandes, Tácio Vinício Amorim, Hoelz, Lucas Villas Bôas, Sales Junior, Policarpo Ademar, Murta, Silvane Maria Fonseca, Romanha, Alvaro, Galvão, Bárbara Verena Dias, Macedo, Marcela de Oliveira, Goldstein, Alana da Cunha, Araujo-Lima, Carlos Fernando, Felzenszwalb, Israel, Nonato, Maria Cristina, Castelo-Branco, Frederico Silva, and Boechat, Nubia
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TRYPANOSOMA cruzi ,TRIAZOLE derivatives ,CHAGAS' disease ,PARASITIC diseases ,MOLECULAR docking ,ERGOSTEROL - Abstract
Chagas disease (CD), which is caused by Trypanosoma cruzi and was discovered more than 100 years ago, remains the leading cause of death from parasitic diseases in the Americas. As a curative treatment is only available for the acute phase of CD, the search for new therapeutic options is urgent. In this study, nitroazole and azole compounds were synthesized and underwent molecular modeling, anti-T. cruzi evaluations and nitroreductase enzymatic assays. The compounds were designed as possible inhibitors of ergosterol biosynthesis and/or as substrates of nitroreductase enzymes. The in vitro evaluation against T. cruzi clearly showed that nitrotriazole compounds are significantly more potent than nitroimidazoles and triazoles. When their carbonyls were reduced to hydroxyl groups, the compounds showed a significant increase in activity. In addition, these substances showed potential for action via nitroreductase activation, as the substances were metabolized at higher rates than benznidazole (BZN), a reference drug against CD. Among the compounds, 1-(2,4-difluorophenyl)-2-(3-nitro-1H-1,2,4-triazol-1-yl)ethanol (8) is the most potent and selective of the series, with an IC
50 of 0.39 µM and selectivity index of 3077; compared to BZN, 8 is 4-fold more potent and 2-fold more selective. Moreover, this compound was not mutagenic at any of the concentrations evaluated, exhibited a favorable in silico ADMET profile and showed a low potential for hepatotoxicity, as evidenced by the high values of CC50 in HepG2 cells. Furthermore, compared to BZN, derivative 8 showed a higher rate of conversion by nitroreductase and was metabolized three times more quickly when both compounds were tested at a concentration of 50 µM. The results obtained by the enzymatic evaluation and molecular docking studies suggest that, as planned, nitroazole derivatives may utilize the nitroreductase metabolism pathway as their main mechanism of action against Trypanosoma cruzi. In summary, we have successfully identified and characterized new nitrotriazole analogs, demonstrating their potential as promising candidates for the development of Chagas disease drug candidates that function via nitroreductase activation, are considerably selective and show no mutagenic potential. [ABSTRACT FROM AUTHOR]- Published
- 2023
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7. Untargeted LC–MS metabolomic studies of Asteraceae species to discover inhibitors of Leishmania major dihydroorotate dehydrogenase
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Chibli, Lucas A., Rosa, Annylory L., Nonato, Maria Cristina, and Da Costa, Fernando B.
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- 2019
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8. Welcoming Alejandro Buschiazzo, Dorothee Liebschner and Stephen Muench as Co‐editors of Acta Crystallographica F – Structural Biology Communications.
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Agirre, Jon, Nonato, Maria Cristina, and van Raaij, Mark J.
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COMPUTATIONAL biology , *MICROBIOLOGY , *MICROSCOPY , *EXPERTISE , *BIOLOGY - Abstract
The Section Editors welcome and introduce three new Co‐editors to the journal and summarize their expertise in computational structural biology, experimental structural biology and cryo‐electron microscopy. [ABSTRACT FROM AUTHOR]
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- 2024
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9. Kinetic mechanism and catalysis of Trypanosoma cruzi dihydroorotate dehydrogenase enzyme evaluated by isothermal titration calorimetry
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Cheleski, Juliana, Wiggers, Helton José, Citadini, Ana Paula, da Costa Filho, Antônio José, Nonato, Maria Cristina, and Montanari, Carlos Alberto
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- 2010
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10. 3-Epiabruslactone A, a New Triterpene Lactone Isolated from Austroplenckia populnea
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Silva Grácia Divina de Fátima, Duarte Lucienir Pains, Paes Helena Clara da Silva, Sousa José Rêgo de, Nonato Maria Cristina, Portezani Paulo José, and Mascarenhas Yvonne Primerano
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Austroplenckia populnea ,Celastraceae ,3-epiabruslactone A ,structure elucidation ,Chemistry ,QD1-999 - Abstract
A new lactonic triterpene isolated from the heartwood of Austroplenckia populnea (Celastraceae) was characterized as 3alpha-hydroxyolean-12-en-29,22alpha-olide (the gamma-lactone of the 3alpha,22alpha-dihydroxyolean-12-en-29alpha-oic acid), the 3-epimer of the abruslactone A, on the basis of its spectral data, chemical transformations, and single crystal X-ray analysis.
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- 1998
11. The importance of international collaborations in science and structural biology.
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Nonato, Maria Cristina, van Raaij, Mark J., and Agirre, Jon
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COOPERATIVE research , *BIOLOGY - Abstract
The Acta Cryst. F – Structural Biology Communications Editors explain how important international collaborations are in science and structural biology. [ABSTRACT FROM AUTHOR]
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- 2024
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12. Isolation and biochemical, functional and structural characterization of a novel l-amino acid oxidase from Lachesis muta snake venom
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Bregge-Silva, Cristiane, Nonato, Maria Cristina, de Albuquerque, Sérgio, Ho, Paulo Lee, de Azevedo, Inácio L.M. Junqueira, Diniz, Marcelo Ribeiro Vasconcelos, Lomonte, Bruno, Rucavado, Alexandra, Díaz, Cecilia, Gutiérrez, José María, and Arantes, Eliane Candiani
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- 2012
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13. Beyond publishing: introducing Interviews with authors.
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Nonato, Maria Cristina, van Raaij, Mark J., and Agirre, Jon
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PERIODICAL publishing , *PUBLISHING , *AUTHORS , *RESEARCH personnel - Abstract
To find out what lies behind the articles published in Acta Cryst. F – Structural Biology Communications the journal now publishes interviews with its authors. [ABSTRACT FROM AUTHOR]
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- 2024
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14. Shaping the future of Acta Crystallographica F: unveiling our vision.
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Nonato, Maria Cristina
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BIOLOGY - Abstract
One of the Editors of Acta Cryst. F – Structural Biology Communications describes what the future holds for the journal. [ABSTRACT FROM AUTHOR]
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- 2023
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15. Structural, biochemical and biophysical characterization of recombinant human fumarate hydratase.
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Ajalla Aleixo, Mariana A., Rangel, Victor L., Rustiguel, Joane K., de Pádua, Ricardo A. P., and Nonato, Maria Cristina
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ENZYME kinetics ,CHEMICAL kinetics ,ALLOSTERIC regulation ,DNA repair ,X-ray crystallography ,CIRCULATING tumor DNA - Abstract
Fumarate hydratases (FHs, fumarases) catalyze the reversible conversion of fumarate into l‐malate. FHs are distributed over all organisms and play important roles in energy production, DNA repair and as tumor suppressors. They are very important targets both in the study of human metabolic disorders and as potential therapeutic targets in neglected tropical diseases and tuberculosis. In this study, human FH (HsFH) was characterized by using enzyme kinetics, differential scanning fluorimetry and X‐ray crystallography. For the first time, the contribution of both substrates was analyzed simultaneously in a single kinetics assay allowing to quantify the contribution of the reversible reaction for kinetics. The protein was crystallized in the spacegroup C2221, with unit‐cell parameters a = 125.43, b = 148.01, c = 129.76. The structure was solved by molecular replacement and refined at 1.8 Å resolution. In our study, a HEPES molecule was found to interact with HsFH at the C‐terminal domain (Domain 3), previously described as involved in allosteric regulation, through a set of interactions that includes Lys 467. HsFH catalytic efficiency is higher when in the presence of HEPES. Mutations at residue 467 have already been implicated in genetic disorders caused by FH deficiency, suggesting that the HEPES‐binding site may be important for enzyme kinetics. This study contributes to the understanding of the HsFH structure and how it correlates with mutation, enzymatic deficiency and pathology. [ABSTRACT FROM AUTHOR]
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- 2019
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16. Crystal Structures of Fumarate Hydratases from Leishmania major in a Complex with Inhibitor 2‑Thiomalate.
- Author
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Feliciano, Patricia R., Drennan, Catherine L., and Nonato, Maria Cristina
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- 2019
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17. Oxidases, Dehydrogenases and Related Systems
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Miller, Susan, Mattevi, Andrea, Medina, M., Nonato, Maria Cristina, Macheroux, Peter, Hemmi, Hisashi, Jentoft Olsen, Rikke Katrine, Kim, Jung-Ja, Vanoni, Maria A., Wallner, Silvia, Wongnate, Thanyaporn, Tu, Shiao-Chun, Schuman Jorns, Marilyn, Tanner, John J., Thorpe, Colin, Ghisla, S., Palfey, Bruce, Binda, Claudia, Ceccarelli, Eduardo, Hille, Russ, Gregersen, Niels Henrik, Rokita, Steven Edward, Becker, Donald, Edmondson, D. E., Fitzpatrick, Paul, Gadda, Giovanni, Dully, Corinna, Chaiyen, Pimchai, Costa Filho, Antonio J., Daniel, Bastian, Miller, Susan, Mattevi, Andrea, Medina, M., Nonato, Maria Cristina, Macheroux, Peter, Hemmi, Hisashi, Jentoft Olsen, Rikke Katrine, Kim, Jung-Ja, Vanoni, Maria A., Wallner, Silvia, Wongnate, Thanyaporn, Tu, Shiao-Chun, Schuman Jorns, Marilyn, Tanner, John J., Thorpe, Colin, Ghisla, S., Palfey, Bruce, Binda, Claudia, Ceccarelli, Eduardo, Hille, Russ, Gregersen, Niels Henrik, Rokita, Steven Edward, Becker, Donald, Edmondson, D. E., Fitzpatrick, Paul, Gadda, Giovanni, Dully, Corinna, Chaiyen, Pimchai, Costa Filho, Antonio J., and Daniel, Bastian
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- Flavoproteins
- Abstract
The dynamic field of flavin and flavoprotein biochemistry has seen rapid advancement in recent years. This comprehensive two volume set provides an overview of all aspects of contemporary research in this important class of enzymes. Topics treated include flavoproteins involved in energy generation, signal transduction and electron transfer (including respiration); oxygen activation by flavoproteins; the biology and biochemistry of complex flavoproteins; flavin and flavoprotein photochemistry/photophysics as well as biotechnological applications of flavoproteins. Recent developments in this field include new structures (including those of large membrane-integral electron transfer complexes containing FMN or FAD), elucidation of the role of flavoproteins in cell signalling pathways (including both phototaxis and the circadian cycle) and important new insights into the reaction mechanisms of flavin-containing enzymes. This volume focussing on oxidases, dehydrogenases and related systems is an essential reference for all researchers in biochemistry, chemistry, photochemistry and photophysics working on flavoenzymes.
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- 2012
18. Correction: Lesbon et al. Nucleocapsid (N) Gene Mutations of SARS-CoV-2 Can Affect Real-Time RT-PCR Diagnostic and Impact False-Negative Results. Viruses 2021, 13 , 2474.
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Lesbon, Jéssika Cristina Chagas, Poleti, Mirele Daiana, de Mattos Oliveira, Elisângela Chicaroni, Patané, José Salvatore Leister, Clemente, Luan Gaspar, Viala, Vincent Louis, Ribeiro, Gabriela, Giovanetti, Marta, de Alcantara, Luiz Carlos Junior, Teixeira, Olivia, Nonato, Maria Cristina, de Lima, Loyze Paola Oliveira, Martins, Antonio Jorge, dos Santos Barros, Claudia Renata, Marqueze, Elaine Cristina, de Souza Todão Bernardino, Jardelina, Moretti, Debora Botequio, Brassaloti, Ricardo Augusto, de Lello Rocha Campos Cassano, Raquel, and Mariani, Pilar Drummond Sampaio Correa
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SARS-CoV-2 ,REVERSE transcriptase polymerase chain reaction ,PROTEIN crystallography ,PROTEIN structure ,GENETIC mutation - Abstract
Nucleocapsid (N) Gene Mutations of SARS-CoV-2 Can Affect Real-Time RT-PCR Diagnostic and Impact False-Negative Results. The authors hereby request the inclusion of two authors (Olivia Teixeira and Maria Cristina Nonato) in the recently published article in I Viruses i entitled "Nucleocapsid (N) gene mutations of SARS-CoV-2 can affect real-time RT-PCR diagnostic and impact false-negative results" [[1]]. Insert New Author Contributions Statement The contributions of Olivia Teixeira and Maria Cristina Nonato were not included in the original publication. [Extracted from the article]
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- 2022
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19. Recombinant production, crystallization and crystal structure determination of dihydroorotate dehydrogenase from Leishmania (Viannia) braziliensis.
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Reis, Renata Almeida Garcia, Lorenzato, Eder, Silva, Valeria Cristina, and Nonato, Maria Cristina
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DIHYDROOROTATE dehydrogenase ,RECOMBINANT antibodies ,LEISHMANIASIS ,CRYSTALLIZATION ,CRYSTAL structure - Abstract
The enzyme dihydroorotate dehydrogenase (DHODH) is a flavoenzyme that catalyses the oxidation of dihydroorotate to orotate in the de novo pyrimidine-biosynthesis pathway. In this study, a reproducible protocol for the heterologous expression of active dihydroorotate dehydrogenase from Leishmania (Viannia) braziliensis ( LbDHODH) was developed and its crystal structure was determined at 2.12 Å resolution. L. (V.) braziliensis is the species responsible for the mucosal form of leishmaniasis, a neglected disease for which no cure or effective therapy is available. Analyses of sequence, structural and kinetic features classify LbDHODH as a member of the class 1A DHODHs and reveal a very high degree of structural conservation with the previously reported structures of orthologous trypanosomatid enzymes. The relevance of nucleotide-biosynthetic pathways for cell metabolism together with structural and functional differences from the respective host enzyme suggests that inhibition of LbDHODH could be exploited for antileishmanicidal drug development. The present work provides the framework for further integrated in vitro, in silico and in vivo studies as a new tool to evaluate DHODH as a drug target against trypanosomatid-related diseases. [ABSTRACT FROM AUTHOR]
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- 2015
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20. Discovery of Antimalarial Azetidine-2-carbonitriles That Inhibit P. falciparum Dihydroorotate Dehydrogenase
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Maetani, Micah, Kato, Nobutaka, Jabor, Valquiria A. P., Calil, Felipe A., Nonato, Maria Cristina, Scherer, Christina A., and Schreiber, Stuart L.
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BRD7539 ,BRD9185 ,DHODH ,malaria ,diversity-oriented synthesis ,Plasmodium falciparum - Abstract
Dihydroorotate dehydrogenase (DHODH) is an enzyme necessary for pyrimidine biosynthesis in protozoan parasites of the genus Plasmodium, the causative agents of malaria. We recently reported the identification of novel compounds derived from diversity-oriented synthesis with activity in multiple stages of the malaria parasite life cycle. Here, we report the optimization of a potent series of antimalarial inhibitors consisting of azetidine-2-carbonitriles, which we had previously shown to target P. falciparum DHODH in a biochemical assay. Optimized compound BRD9185 (27) has in vitro activity against multidrug-resistant blood-stage parasites (EC50 = 0.016 μM) and is curative after just three doses in a P. berghei mouse model. BRD9185 has a long half-life (15 h) and low clearance in mice and represents a new structural class of DHODH inhibitors with potential as antimalarial drugs.
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- 2017
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21. A rational protocol for the successful crystallization of l-amino-acid oxidase from Bothrops atrox.
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Alves, Raquel Melo, Feliciano, Patricia Rosa, Sampaio, Suely Vilela, and Nonato, Maria Cristina
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CRYSTALLIZATION ,AMINO acids ,OXIDASES ,FER-de-lance ,X-ray diffraction measurement ,FLAVOPROTEINS - Abstract
Despite the valuable contributions of robotics and high-throughput approaches to protein crystallization, the role of an experienced crystallographer in the evaluation and rationalization of a crystallization process is still crucial to obtaining crystals suitable for X-ray diffraction measurements. In this work, the difficult task of crystallizing the flavoenzyme l-amino-acid oxidase purified from Bothrops atrox snake venom was overcome by the development of a protocol that first required the identification of a non-amorphous precipitate as a promising crystallization condition followed by the implementation of a methodology that combined crystallization in the presence of oil and seeding techniques. Crystals were obtained and a complete data set was collected to 2.3 Å resolution. The crystals belonged to space group P2
1 , with unit-cell parameters a = 73.64, b = 123.92, c = 105.08 Å, β = 96.03°. There were four protein subunits in the asymmetric unit, which gave a Matthews coefficient VM of 2.12 Å3 Da−1 , corresponding to 42% solvent content. The structure has been solved by molecular-replacement techniques. [ABSTRACT FROM AUTHOR]- Published
- 2011
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22. Crystallization and preliminary X-ray diffraction analysis of recombinant chlorocatechol 1,2-dioxygenase from Pseudomonas putida.
- Author
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Rustiguel, Joane Kathelen, Pinheiro, Matheus Pinto, Araújo, Ana Paula Ulian, and Nonato, Maria Cristina
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X-ray diffraction ,PSEUDOMONAS putida ,CHLOROCATECHOLS ,DIOXYGENASES ,CRYSTALLIZATION - Abstract
Chlorocatechol 1,2-dioxygenase from the Gram-negative bacterium Pseudomonas putida (Pp 1,2-CCD) is considered to be an important biotechnological tool owing to its ability to process a broad spectrum of organic pollutants. In the current work, the crystallization, crystallographic characterization and phasing of the recombinant Pp 1,2-CCD enzyme are described. Reddish-brown crystals were obtained in the presence of polyethylene glycol and magnesium acetate by utilizing the vapour-diffusion technique in sitting drops. Crystal dehydration was the key step in obtaining data sets, which were collected on the D03B-MX2 beamline at the CNPEM/MCT - LNLS using a MAR CCD detector. Pp 1,2-CCD crystals belonged to space group P6
1 22 and the crystallographic structure of Pp 1,2-CCD has been solved by the MR-SAD technique using Fe atoms as scattering centres and the coordinates of 3-chlorocatechol 1,2-dioxygenase from Rhodococcus opacus (PDB entry ) as the search model. The initial model, which contains three molecules in the asymmetric unit, has been refined to 3.4 Å resolution. [ABSTRACT FROM AUTHOR]- Published
- 2011
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23. Full-length model of the human galectin-4 and insights into dynamics of inter-domain communication.
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Rustiguel, Joane K., Soares, Ricardo O. S., Meisburger, Steve P., Davis, Katherine M., Malzbender, Kristina L., Ando, Nozomi, Dias-Baruffi, Marcelo, and Nonato, Maria Cristina
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- 2016
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24. Novel quaternary structures of the human prion protein globular domain.
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Bortot, Leandro Oliveira, Rangel, Victor Lopes, Pavlovici, Francesca A., El Omari, Kamel, Wagner, Armin, Brandao-Neto, Jose, Talon, Romain, von Delft, Frank, Reidenbach, Andrew G., Vallabh, Sonia M., Minikel, Eric Vallabh, Schreiber, Stuart, and Nonato, Maria Cristina
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QUATERNARY structure , *GLOBULAR proteins , *PROTEIN domains , *MOLECULAR dynamics , *PRIONS , *PRION diseases - Abstract
Prion disease is caused by the misfolding of the cellular prion protein, PrPC, into a self-templating conformer, PrPSc. Nuclear magnetic resonance (NMR) and X-ray crystallography revealed the 3D structure of the globular domain of PrPC and the possibility of its dimerization via an interchain disulfide bridge that forms due to domain swap or by non-covalent association of two monomers. On the contrary, PrPSc is composed by a complex and heterogeneous ensemble of poorly defined conformations and quaternary arrangements that are related to different patterns of neurotoxicity. Targeting PrPC with molecules that stabilize the native conformation of its globular domain emerged as a promising approach to develop anti-prion therapies. One of the advantages of this approach is employing structure-based drug discovery methods to PrPC. Thus, it is essential to expand our structural knowledge about PrPC as much as possible to aid such drug discovery efforts. In this work, we report a crystallographic structure of the globular domain of human PrPC that shows a novel dimeric form and a novel oligomeric arrangement. We use molecular dynamics simulations to explore its structural dynamics and stability and discuss potential implications of these new quaternary structures to the conversion process. • Prion disease is caused by the misfolding of the cellular prion protein PrPc. • Compounds that inhibit the misfolding of PrPC could provide anti-prion therapies. • A new crystallographic structure of PrPC shows novel oligomeric arrangements. • Molecular dynamics simulations were used to correlate the new quaternary structures to the misfolding process. [ABSTRACT FROM AUTHOR]
- Published
- 2021
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25. Glutathione reductase: A cytoplasmic antioxidant enzyme and a potential target for phenothiazinium dyes in Neospora caninum.
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Venancio-Brochi, Jade Cabestre, Pereira, Luiz Miguel, Calil, Felipe Antunes, Teixeira, Olívia, Baroni, Luciana, Abreu-Filho, Péricles Gama, Braga, Gilberto Úbida Leite, Nonato, Maria Cristina, and Yatsuda, Ana Patrícia
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NEOSPORA caninum , *GLUTATHIONE reductase , *TOLUIDINE blue , *ANTIOXIDANTS , *METHYLENE blue , *IONIC strength - Abstract
Neospora caninum causes heavy losses related to abortions in bovine cattle. This parasite developed a complex defense redox system, composed of enzymes as glutathione reductase (GR). Methylene blue (MB) impairs the activity of recombinant form of Plasmodium GR and inhibits the parasite proliferation in vivo and in vitro. Likewise, MB and its derivatives inhibits Neospora caninum proliferation, however, whether the MB mechanism of action is correlated to GR function remains unclear. Therefore, here, N. caninum GR (NcGR) was characterized and its potential inhibitors were determined. NcGR was found in the tachyzoite cytosol and has a similar structure and sequence compared to its homologs. We verified the in vitro activity of rNcGR (875 nM) following NADPH absorbance at 340 nM (100 mM KH 2 PO 4 , pH 7.5, 1 mM EDTA, ionic strength: 600 mM, 25 °C). rNcGR exhibited a Michaelian behavior (K m(GSSG) :0.10 ± 0.02 mM; k cat(GSSG) :0.076 ± 0.003 s-1; K m(NADPH) :0.006 ± 0.001 mM; k cat(NADPH) : 0.080 ± 0.003 s-1). The IC 50 of MB,1,9-dimethyl methylene blue, new methylene blue, and toluidine blue O on rNcGR activity were 2.1 ± 0.2 μM, 11 ± 2 μ M, 0.7 ± 0.1 μM, and 0.9 ± 0.2 μ M, respectively. Our results suggest the importance of NcGR in N. caninum biology and antioxidant mechanisms. Moreover, data presented here strongly suggest that NcGR is an important target of phenothiazinium dyes in N. caninum proliferation inhibition. [ABSTRACT FROM AUTHOR]
- Published
- 2021
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26. Unraveling the structure and function of CdcPDE: A novel phosphodiesterase from Crotalus durissus collilineatus snake venom.
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Oliveira, Isadora Sousa de, Pucca, Manuela Berto, Wiezel, Gisele Adriano, Cardoso, Iara Aimê, Bordon, Karla de Castro Figueiredo, Sartim, Marco Aurélio, Kalogeropoulos, Konstantinos, Ahmadi, Shirin, Baiwir, Dominique, Nonato, Maria Cristina, Sampaio, Suely Vilela, Laustsen, Andreas Hougaard, auf dem Keller, Ulrich, Quinton, Loïc, and Arantes, Eliane Candiani
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SNAKE venom , *CROTALUS , *BLOOD platelet aggregation , *VENOM , *ADENOSINE diphosphate , *ANTIVENINS , *CONOTOXINS , *ETHYLENEDIAMINETETRAACETIC acid - Abstract
This study reports the isolation, structural, biochemical, and functional characterization of a novel phosphodiesterase from Crotalus durissus collilineatus venom (Cdc PDE). Cdc PDE was successfully isolated from whole venom using three chromatographic steps and represented 0.7% of total protein content. Cdc PDE was inhibited by EDTA and reducing agents, demonstrating that metal ions and disulfide bonds are necessary for its enzymatic activity. The highest enzymatic activity was observed at pH 8–8.5 and 37 °C. Kinetic parameters indicated a higher affinity for the substrate bis (p -nitrophenyl) phosphate compared to others snake venom PDEs. Its structural characterization was done by the determination of the protein primary sequence by Edman degradation and mass spectrometry, and completed by the building of molecular and docking-based models. Functional in vitro assays showed that Cdc PDE is capable of inhibiting platelet aggregation induced by adenosine diphosphate in a dose-dependent manner and demonstrated that Cdc PDE is cytotoxic to human keratinocytes. Cdc PDE was recognized by the crotalid antivenom produced by the Instituto Butantan. These findings demonstrate that the study of snake venom toxins can reveal new molecules that may be relevant in cases of snakebite envenoming, and that can be used as molecular tools to study pathophysiological processes due to their specific biological activities. • Cdc PDE is the first phosphodiesterase isolated from C. d. collilineatus snake venom. • Metal ions and disulfide bonds are essential for its enzymatic activity. • Crotalid antivenom is able to recognize Cdc PDE. • Cdc PDE inhibits platelet ADP-induced aggregation. • Cdc PDE is cytotoxic to human keratinocytes. [ABSTRACT FROM AUTHOR]
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- 2021
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27. Characterization of class II fumarase from Schistosoma mansoni provides the molecular basis for selective inhibition.
- Author
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Cardoso, Iara Aimê, de Souza, Aline Kusumota Luiz, Burgess, Adam Muslem George, Chalmers, Iain Wyllie, Hoffmann, Karl Francis, and Nonato, Maria Cristina
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SCHISTOSOMA mansoni , *TURNOVER frequency (Catalysis) , *SCHISTOSOMIASIS , *FLUORIMETRY , *FELIDAE - Abstract
Schistosomiasis is a neglected tropical disease that affects more than 250 million people worldwide. The only drug available for its treatment undergoes first-pass hepatic metabolism and is not capable of preventing reinfection, which makes the search of new therapies urgently needed. Due to the essential role of fumarases in metabolism, these enzymes represent potential targets for developing novel schistosomiasis treatments. Here, we evaluate the expression profiles for class I and class II fumarases from Schistosoma mansoni (Sm FH I and Sm FH II , respectively), and report the complete characterization of Sm FH II. The first Sm FH II structure in complex with L-malate was determined at 1.85 Å resolution. The significant thermoshift observed for Sm FH II in the presence of identified ligands makes the differential scanning fluorimetry an adequate technique for ligand screening. A complete kinetic characterization of Sm FH II was performed, and comparison with the human fumarase (Hs FH) revealed differences regarding the turnover number (k cat). Structural characterization allowed us to identify differences between Sm FH II and Hs FH that could be explored to design new selective inhibitors. This work represents the very first step towards validate the fumarases as drug targets to treat schistosomiasis. Our results provide the structural basis to rational search for selective ligands. • The first Sm FH II structure in complex with L-malate was determined at 1.85 Å resolution. • An addition portion located at the central domain proved to be essential for structural stability. • Differential scanning fluorimetry was found to be an appropriate technique for ligand screening. • Hs FH and Sm FH II presented similar K m values, but k cat values were much higher for Hs FH. • Differences between Sm FH II and Hs FH could be explored in the design of selective inhibitors. [ABSTRACT FROM AUTHOR]
- Published
- 2021
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28. Kinetic and structural studies of Mycobacterium tuberculosis dihydroorotate dehydrogenase reveal new insights into class 2 DHODH inhibition.
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Teixeira, Olívia, Martins, Ingrid Bernardes Santana, Froes, Thamires Quadros, de Araujo, Alexandre Suman, and Nonato, Maria Cristina
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MYCOBACTERIUM tuberculosis , *DIHYDROOROTATE dehydrogenase , *MOLECULAR dynamics , *MENADIONE , *PATIENT compliance , *COMMUNICABLE diseases , *DEHYDROGENASES - Abstract
Tuberculosis (TB) is a leading cause of death worldwide. TB represents a serious public health threat, and it is characterized by high transmission rates, prevalence in impoverished regions, and high co-infection rates with HIV. Moreover, the serious side effects of long-term treatment that decrease patient adherence, and the emergence of multi-resistant strains of Mycobacterium tuberculosis , the causing agent of TBs, pose several challenges for its eradication. The search for a new TB treatment is necessary and urgent. Dihydroorotate dehydrogenase (DHODH) is responsible for the stereospecific oxidation of (S)-dihydroorotate (DHO) to orotate during the fourth and only redox step of the de novo pyrimidine nucleotide biosynthetic pathway. DHODH has been considered an attractive target against infectious diseases. As a first step towards exploiting DHODH as a drug target against TB, we performed a full kinetic characterization of both bacterial MtDHODH and its human ortholog (HsDHDOH) using both substrates coenzyme Q0 (Q0) and vitamin K3 (K3). MtDHODH follows a ping-pong mechanism of catalysis and shares similar catalytic parameters with the human enzyme. Serendipitously, Q0 was found to inhibit MtDHODH (K I (Q0) = 138 ± 31 μM). To the best of our knowledge, Q0 is the first non-orotate like dihydroorotate-competitive inhibitor for class 2 DHODHs ever described. Molecular dynamics simulations along with in silico solvent mapping allowed us to successfully probe protein flexibility and correlate it with the druggability of binding sites. Together, our results provide the starting point for the design of a new generation of potent and selective inhibitors against MtDHODH. [Display omitted] • MtDHODH catalyzes dihydroorotate (DHO) oxidation by a ping-pong mechanism. • MtDHODH was evaluated by in silico solvent mapping and molecular dynamics studies. • Coenzyme Q0 (Q0) was identified as a DHO-competitive inhibitor for class 2 DHODHs. • Inhibitor selectivity for MtDHODH over HsDHODH can be achieved. [ABSTRACT FROM AUTHOR]
- Published
- 2023
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29. A soluble recombinant form of human leucocyte antigen-G 6 (srHLA-G6).
- Author
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Pelá, Flávia Porto, Rustiguel, Joane Kathelen, Rodrigues, Lilian Cataldi, Mendonça, Jacqueline Nakau, Andrade, Camillo Del Cistia, Lopes, Norberto Peporine, Rosa, José Cesar, Nonato, Maria Cristina, Favier, Benoit, Donadi, Eduardo Antônio, and Dias-Baruffi, Marcelo
- Subjects
- *
HUMAN leucocytes , *MAJOR histocompatibility complex , *RNA splicing , *IMMUNOSUPPRESSION , *ENZYME-linked immunosorbent assay , *MONOCLONAL antibodies - Abstract
Human Leucocyte Antigen-G (HLA-G) is a non classical major histocompatibility complex (MHC) molecule that through RNA splicing can encode seven isoforms which are membrane bound (-G1, -G2, -G3 and –G4) and soluble (–G5, -G6 and –G7). HLA-G is described as important immune suppressor endogenous molecule to favor maternal-fetal tolerance, transplant survival and tumor immune scape. HLA-G shows low protein variability and a unique structural complexity that is related with the expression of different isoforms followed by biochemical processes, such as, proteolytic cleavage, molecular interactions, and protein ubiquitination. Studies with HLA-G have shown difficult to assess the role of the individual isoforms. Thus, the aim of this work was to obtain a HLA-G6 recombinant form. The results indicated the production of high homogeneous preparations of soluble recombinant HLA-G6 (srHLA-G6) with molecular mass 23,603.76 Da, determined by MALD-TOF/TOF. In addition, native and denatured srHLA-G6 were detected by ELISA, using commercial monoclonal antibodies. Finally, we developed a suitable methodology to express srHLA-G6 that could contribute in structural and functional studies involving specific isoforms. [ABSTRACT FROM AUTHOR]
- Published
- 2017
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30. Recombinant expression, purification and preliminary biophysical and structural studies of C-terminal carbohydrate recognition domain from human galectin-4.
- Author
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Rustiguel, Joane K., Kumagai, Patricia S., Dias-Baruffi, Marcelo, Costa-Filho, Antonio J., and Nonato, Maria Cristina
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- *
RECOMBINANT proteins , *PROTEIN expression , *PROTEIN structure , *C-terminal residues , *CARBOHYDRATES , *GALECTINS , *GASTROINTESTINAL system , *DRUG development - Abstract
Galectin-4 (Gal4), a tandem-repeat type galectin, is expressed in healthy epithelium of the gastrointestinal tract. Altered levels of Gal4 expression are associated with different types of cancer, suggesting its usage as a diagnostic marker as well as target for drug development. The functional data available for this class of proteins suggest that the wide spectrum of cellular activities reported for Gal4 relies on distinct glycan specificity and structural characteristics of its two carbohydrate recognition domains. In the present work, two independent constructs for recombinant expression of the C-terminal domain of human galectin-4 (hGal4-CRD2) were developed. His 6 -tagged and untagged recombinant proteins were overexpressed in Escherichia coli, and purified by affinity chromatography followed by gel filtration. Correct folding and activity of hGal4-CRD2 were assessed by circular dichroism and fluorescence spectroscopies, respectively. Diffraction quality crystals were obtained by vapor-diffusion sitting drop setup and the crystal structure of CRD2 was solved by molecular replacement techniques at 1.78 Å resolution. Our work describes the development of important experimental tools that will allow further studies in order to correlate structure and binding properties of hGal4-CRD2 and human galectin-4 functional activities. [ABSTRACT FROM AUTHOR]
- Published
- 2016
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31. Protein-metabolite interactomics of carbohydrate metabolism reveal regulation of lactate dehydrogenase.
- Author
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Hicks KG, Cluntun AA, Schubert HL, Hackett SR, Berg JA, Leonard PG, Ajalla Aleixo MA, Zhou Y, Bott AJ, Salvatore SR, Chang F, Blevins A, Barta P, Tilley S, Leifer A, Guzman A, Arok A, Fogarty S, Winter JM, Ahn HC, Allen KN, Block S, Cardoso IA, Ding J, Dreveny I, Gasper WC, Ho Q, Matsuura A, Palladino MJ, Prajapati S, Sun P, Tittmann K, Tolan DR, Unterlass J, VanDemark AP, Vander Heiden MG, Webb BA, Yun CH, Zhao P, Wang B, Schopfer FJ, Hill CP, Nonato MC, Muller FL, Cox JE, and Rutter J
- Subjects
- Humans, Fatty Acids metabolism, Organ Specificity, Mass Spectrometry methods, Allosteric Regulation, Carbohydrate Metabolism, L-Lactate Dehydrogenase metabolism, Metabolome
- Abstract
Metabolic networks are interconnected and influence diverse cellular processes. The protein-metabolite interactions that mediate these networks are frequently low affinity and challenging to systematically discover. We developed mass spectrometry integrated with equilibrium dialysis for the discovery of allostery systematically (MIDAS) to identify such interactions. Analysis of 33 enzymes from human carbohydrate metabolism identified 830 protein-metabolite interactions, including known regulators, substrates, and products as well as previously unreported interactions. We functionally validated a subset of interactions, including the isoform-specific inhibition of lactate dehydrogenase by long-chain acyl-coenzyme A. Cell treatment with fatty acids caused a loss of pyruvate-lactate interconversion dependent on lactate dehydrogenase isoform expression. These protein-metabolite interactions may contribute to the dynamic, tissue-specific metabolic flexibility that enables growth and survival in an ever-changing nutrient environment.
- Published
- 2023
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32. Nucleocapsid (N) Gene Mutations of SARS-CoV-2 Can Affect Real-Time RT-PCR Diagnostic and Impact False-Negative Results.
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Lesbon JCC, Poleti MD, de Mattos Oliveira EC, Patané JSL, Clemente LG, Viala VL, Ribeiro G, Giovanetti M, de Alcantara LCJ, Teixeira O, Nonato MC, de Lima LPO, Martins AJ, Dos Santos Barros CR, Marqueze EC, de Souza Todão Bernardino J, Moretti DB, Brassaloti RA, de Lello Rocha Campos Cassano R, Mariani PDSC, Slavov SN, Dos Santos RB, Rodrigues ES, Santos EV, Borges JS, de La Roque DGL, Kitajima JP, Santos B, Assato PA, da Silva da Costa FA, Banho CA, Sacchetto L, Moraes MM, Palmieri M, da Silva FEV, Grotto RMT, Souza-Neto JA, Nogueira ML, Coutinho LL, Calado RT, Neto RM, Covas DT, Kashima S, Elias MC, Sampaio SC, and Fukumasu H
- Subjects
- Brazil epidemiology, COVID-19 epidemiology, Coronavirus RNA-Dependent RNA Polymerase genetics, DNA Primers, False Negative Reactions, Genome, Viral genetics, Humans, Mutation, Phosphoproteins genetics, RNA, Viral genetics, SARS-CoV-2 genetics, COVID-19 diagnosis, COVID-19 Nucleic Acid Testing, Coronavirus Nucleocapsid Proteins genetics, SARS-CoV-2 isolation & purification
- Abstract
The current COVID-19 pandemic demands massive testing by Real-time RT-PCR (Reverse Transcription Polymerase Chain Reaction), which is considered the gold standard diagnostic test for the detection of the SARS-CoV-2 virus. However, the virus continues to evolve with mutations that lead to phenotypic alterations as higher transmissibility, pathogenicity or vaccine evasion. Another big issue are mutations in the annealing sites of primers and probes of RT-PCR diagnostic kits leading to false-negative results. Therefore, here we identify mutations in the N (Nucleocapsid) gene that affects the use of the GeneFinder COVID-19 Plus RealAmp Kit. We sequenced SARS-CoV-2 genomes from 17 positive samples with no N gene detection but with RDRP (RNA-dependent RNA polymerase) and E (Envelope) genes detection, and observed a set of three different mutations affecting the N detection: a deletion of 18 nucleotides (Del28877-28894), a substitution of GGG to AAC (28881-28883) and a frameshift mutation caused by deletion (Del28877-28878). The last one cause a deletion of six AAs (amino acids) located in the central intrinsic disorder region at protein level. We also found this mutation in 99 of the 14,346 sequenced samples by the Sao Paulo state Network for Pandemic Alert of Emerging SARS-CoV-2 variants, demonstrating the circulation of the mutation in Sao Paulo, Brazil. Continuous monitoring and characterization of mutations affecting the annealing sites of primers and probes by genomic surveillance programs are necessary to maintain the effectiveness of the diagnosis of COVID-19.
- Published
- 2021
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33. Synthesis and Biological Evaluation of Natural-Product-Inspired, Aminoalkyl-Substituted 1-Benzopyrans as Novel Antiplasmodial Agents.
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Uth JF, Börgel F, Lehmkuhl K, Schepmann D, Kaiser M, Jabor VAP, Nonato MC, Krauth-Siegel RL, Schmidt TJ, and Wünsch B
- Subjects
- Alkylation, Animals, Antimalarials chemical synthesis, Benzopyrans chemical synthesis, Biomimetic Materials chemical synthesis, Biomimetic Materials chemistry, Biomimetic Materials pharmacology, Chemistry Techniques, Synthetic, Erythrocytes drug effects, Erythrocytes parasitology, Kinetics, Mice, Structure-Activity Relationship, Antimalarials chemistry, Antimalarials pharmacology, Benzopyrans chemistry, Benzopyrans pharmacology, Biological Products chemistry, Plasmodium drug effects
- Abstract
Herein, relationships between the structures of 1-aminoethyl-substituted chromenes and their antimalarial activities were thoroughly investigated. At first, the methyl moiety in the side chain was removed to eliminate chirality. The hydrogenation state of the benzopyran system, the position of the phenolic OH moiety, and the distance of the basic amino moiety toward both aromatic rings were varied systematically. 1-Benzopyran-5-ol 8b (IC
50 = 10 nM), 1-benzopyran-7-ol 9c (IC50 = 38 nM), and the aminoalcohol 19c (IC50 = 17 nM) displayed antiplasmodial activity with IC50 values below 50 nM. To identify the mechanism of action, inhibition of three key enzymes by 9c was investigated. 9c was not able to reduce the number of Plasmodia in erythrocytes of mice. This low in vivo activity was explained by fast clearance from blood plasma combined with rapid biotransformation of 9c . Three main metabolites of 9c were identified by liquid chromatography-mass spectrometry (LC-MS) methods.- Published
- 2021
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34. DHODH Hot Spots: An Underexplored Source to Guide Drug Development Efforts.
- Author
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Froes TQ, Zapata LCC, Akamine JS, Castilho MS, and Nonato MC
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- Antiviral Agents, Dihydroorotate Dehydrogenase metabolism, Humans, Trypanosoma cruzi enzymology, Dihydroorotate Dehydrogenase antagonists & inhibitors, Dihydroorotate Dehydrogenase chemistry, Drug Development trends
- Abstract
Background: Dihydroorotate dehydrogenase (DHODH) has long been recognized as an important drug target for proliferative and parasitic diseases, including compounds that exhibit trypanocidal action and broad-spectrum antiviral activity. Despite numerous and successful efforts in structural and functional characterization of DHODHs, as well as in the development of inhibitors, DHODH hot spots remain largely unmapped and underexplored., Objective: This review describes the tools that are currently available for the identification and characterization of hot spots in protein structures and how freely available webservers can be exploited to predict DHODH hot spots. Moreover, it provides for the first time a review of the antiviral properties of DHODH inhibitors., Methods: X-ray structures from human (HsDHODH) and Trypanosoma cruzi DHODH (TcDHODH) had their hot spots predicted by both FTMap and Fragment Hotspot Maps web servers., Results: FTMap showed that hot spot occupancy in HsDHODH is correlated with the ligand efficiency (LE) of its known inhibitors, and Fragment Hotspot Maps pointed out the contribution of selected moieties to the overall LE. The conformational flexibility of the active site loop in TcDHODH was found to have a major impact on the druggability of the orotate binding site. In addition, both FTMap and Fragment Hotspot Maps servers predict a novel pocket in TcDHODH dimer interface (S6 site)., Conclusion: This review reports how hot spots can be exploited during hit-to-lead steps, docking studies or even to improve inhibitor binding profile and by doing so using DHODH as a model, points to new drug development opportunities., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)
- Published
- 2021
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35. Multimodal small-molecule screening for human prion protein binders.
- Author
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Reidenbach AG, Mesleh MF, Casalena D, Vallabh SM, Dahlin JL, Leed AJ, Chan AI, Usanov DL, Yehl JB, Lemke CT, Campbell AJ, Shah RN, Shrestha OK, Sacher JR, Rangel VL, Moroco JA, Sathappa M, Nonato MC, Nguyen KT, Wright SK, Liu DR, Wagner FF, Kaushik VK, Auld DS, Schreiber SL, and Minikel EV
- Subjects
- Benzimidazoles chemistry, Drug Discovery, Drug Evaluation, Preclinical, Humans, Magnetic Resonance Spectroscopy, Prion Diseases metabolism, Prion Proteins metabolism, Small Molecule Libraries chemistry, Benzimidazoles pharmacology, Prion Diseases drug therapy, Prion Proteins antagonists & inhibitors, Small Molecule Libraries pharmacology
- Abstract
Prion disease is a rapidly progressive neurodegenerative disorder caused by misfolding and aggregation of the prion protein (PrP), and there are currently no therapeutic options. PrP ligands could theoretically antagonize prion formation by protecting the native protein from misfolding or by targeting it for degradation, but no validated small-molecule binders have been discovered to date. We deployed a variety of screening methods in an effort to discover binders of PrP, including
19 F-observed and saturation transfer difference (STD) NMR spectroscopy, differential scanning fluorimetry (DSF), DNA-encoded library selection, and in silico screening. A single benzimidazole compound was confirmed in concentration-response, but affinity was very weak ( Kd > 1 mm), and it could not be advanced further. The exceptionally low hit rate observed here suggests that PrP is a difficult target for small-molecule binders. Whereas orthogonal binder discovery methods could yield high-affinity compounds, non-small-molecule modalities may offer independent paths forward against prion disease., Competing Interests: Conflict of interest—E. V. M. has received consulting fees from Deerfield Management and Guidepoint and has received research support in the form of unrestricted charitable contributions from Charles River Laboratories and Ionis Pharmaceuticals. S. L. S. serves on the Board of Directors of the Genomics Institute of the Novartis Research Foundation (“GNF”); is a shareholder and serves on the Board of Directors of Jnana Therapeutics; is a shareholder of Forma Therapeutics; is a shareholder and advises Kojin Therapeutics, Kisbee Therapeutics, Decibel Therapeutics, and Eikonizo Therapeutics; serves on the Scientific Advisory Boards of Eisai Co., Ltd., Ono Pharma Foundation, Exo Therapeutics, and F-Prime Capital Partners; and is a Novartis Faculty Scholar. S. M. V. has received speaking fees from Illumina and Biogen and has received research support in the form of unrestricted charitable contributions from Charles River Laboratories and Ionis Pharmaceuticals. D. R. L. is a consultant for and cofounder of Exo Therapeutics, which uses DNA-encoded libraries for drug development. D. C., D. S. A., O. K. S., and S. K. W. are employees of Novartis. K. T. N. is an employee of Atomwise., (© 2020 Reidenbach et al.)- Published
- 2020
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36. Development of ML390: A Human DHODH Inhibitor That Induces Differentiation in Acute Myeloid Leukemia.
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Lewis TA, Sykes DB, Law JM, Muñoz B, Rustiguel JK, Nonato MC, Scadden DT, and Schreiber SL
- Abstract
Homeobox transcription factor A9 (HoxA9) is overexpressed in 70% of patients diagnosed with acute myeloid leukemia (AML), whereas only a small subset of AML patients respond to current differentiation therapies. A cell line overexpressing HoxA9 was derived from the bone marrow of a lysozyme-GFP mouse. In this fashion, GFP served as an endogenous reporter of differentiation, permitting a high-throughput phenotypic screen against the MLPCN library. Two chemical scaffolds were optimized for activity yielding compound ML390, and genetic resistance and sequencing efforts identified dihydroorotate dehydrogenase (DHODH) as the target enzyme. The DHODH inhibitor brequinar works against these leukemic cells as well. The X-ray crystal structure of ML390 bound to DHODH elucidates ML390s binding interactions.
- Published
- 2016
- Full Text
- View/download PDF
37. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of recombinant human fumarase.
- Author
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Pereira de Pádua RA and Nonato MC
- Subjects
- Amino Acid Sequence, Cloning, Molecular, Crystallization, Electrophoresis, Polyacrylamide Gel, Humans, Molecular Sequence Data, Fumarate Hydratase chemistry, Fumarate Hydratase isolation & purification, Recombinant Proteins chemistry, Recombinant Proteins isolation & purification, X-Ray Diffraction
- Abstract
Human fumarase (HsFH) is a well-known citric acid cycle enzyme and is therefore a key component in energy metabolism. Genetic studies on human patients have shown that polymorphisms in the fumarase gene are responsible for diseases such as hereditary leiomyomatosis and renal cell cancer. As a first step in unravelling the molecular basis of the mechanism of fumarase deficiency in genetic disorders, the HsFH gene was cloned in pET-28a, heterologously expressed in Escherichia coli, purified by nickel-affinity chromatography and crystallized using the vapour-diffusion technique. X-ray diffraction experiments were performed at a synchrotron source and the structure was solved at 2.1 Å resolution by molecular replacement.
- Published
- 2014
- Full Text
- View/download PDF
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