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2. Inhibitors of Rho kinases (ROCK) induce multiple mitotic defects and synthetic lethality in BRCA2-deficient cells

Author

Julieta Martino, Sebastián Omar Siri, Nicolás Luis Calzetta, Natalia Soledad Paviolo, Cintia Garro, Maria F Pansa, Sofía Carbajosa, Aaron C Brown, José Luis Bocco, Israel Gloger, Gerard Drewes, Kevin P Madauss, Gastón Soria, and Vanesa Gottifredi

Subjects
cancer cell lines, ROCK, synthetic lethality, BRCA2, cytokinesis failure, mitosis, Medicine, Science, Biology (General), QH301-705.5
Abstract

The trapping of Poly-ADP-ribose polymerase (PARP) on DNA caused by PARP inhibitors (PARPi) triggers acute DNA replication stress and synthetic lethality (SL) in BRCA2-deficient cells. Hence, DNA damage is accepted as a prerequisite for SL in BRCA2-deficient cells. In contrast, here we show that inhibiting ROCK in BRCA2-deficient cells triggers SL independently from acute replication stress. Such SL is preceded by polyploidy and binucleation resulting from cytokinesis failure. Such initial mitosis abnormalities are followed by other M phase defects, including anaphase bridges and abnormal mitotic figures associated with multipolar spindles, supernumerary centrosomes and multinucleation. SL was also triggered by inhibiting Citron Rho-interacting kinase, another enzyme that, similarly to ROCK, regulates cytokinesis. Together, these observations demonstrate that cytokinesis failure triggers mitotic abnormalities and SL in BRCA2-deficient cells. Furthermore, the prevention of mitotic entry by depletion of Early mitotic inhibitor 1 (EMI1) augmented the survival of BRCA2-deficient cells treated with ROCK inhibitors, thus reinforcing the association between M phase and cell death in BRCA2-deficient cells. This novel SL differs from the one triggered by PARPi and uncovers mitosis as an Achilles heel of BRCA2-deficient cells.

Published
2023
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3. NK cells with decreased expression of multiple activating receptors is a dominant phenotype in pediatric patients with acute lymphoblastic leukemia

Author

Lucero Valenzuela-Vázquez, Juan Carlos Nuñez-Enriquez, Jacqueline Sánchez-Herrera, Aurora Medina-Sanson, María Luisa Pérez-Saldivar, Elva Jiménez-Hernández, Jorge Alfonso Martiín-Trejo, María de Los Ángeles Del Campo-Martínez, Janet Flores-Lujano, Raquel Amador-Sánchez, Félix Gustavo Mora-Ríos, José Gabriel Peñaloza-González, David Aldebarán Duarte-Rodríguez, José Refugio Torres-Nava, Rosa Martha Espinosa-Elizondo, Beatriz Cortés-Herrera, Luz Victoria Flores-Villegas, Laura Elizabeth Merino-Pasaye, Carolina Almeida-Hernández, Rosario Ramírez-Colorado, Karina Anastacia Solís-Labastida, Francisco Medrano-López, Jessica Arleet Pérez-Gómez, Martha Margarita Velázquez-Aviña, Annel Martínez-Ríos, Antonio Aguilar-De los Santos, Jessica Denisse Santillán-Juárez, Alma Gurrola-Silva, Alejandra Jimena García-Velázquez, Minerva Mata-Rocha, Gabriela Alicia Hernández-Echáurregui, Omar Alejandro Sepúlveda-Robles, Haydeé Rosas-Vargas, Ismael Mancilla-Herrera, Silvia Jimenez-Morales, Alfredo Hidalgo-Miranda, Ivan Martinez-Duncker, Jeremy D. Waight, Kenneth W. Hance, Kevin P. Madauss, Juan Manuel Mejía-Aranguré, and Mario Ernesto Cruz-Munoz

Subjects
NK cells, acute lymphoblastic leukemia, immunooncology, cancer, immune system, Neoplasms. Tumors. Oncology. Including cancer and carcinogens, RC254-282
Abstract

NK cells have unique attributes to react towards cells undergoing malignant transformation or viral infection. This reactivity is regulated by activating or inhibitory germline encoded receptors. An impaired NK cell function may result from an aberrant expression of such receptors, a condition often seen in patients with hematological cancers. Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer worldwide and NK cells have emerged as crucial targets for developing immunotherapies. However, there are important gaps concerning the phenotype and behavior of NK cells during emergence of ALL. In this study we analyze the phenotype and function of NK cells from peripheral blood in pediatric patients with ALL at diagnosis. Our results showed that NK cells exhibited an altered phenotype highlighted by a significant reduction in the overall expression and percent representation of activating receptors compared to age-matched controls. No significant differences were found for the expression of inhibitory receptors. Moreover, NK cells with a concurrent reduced expression in various activating receptors, was the dominant phenotype among patients. An alteration in the relative frequencies of NK cells expressing NKG2A and CD57 within the mature NK cell pool was also observed. In addition, NK cells from patients displayed a significant reduction in the ability to sustain antibody-dependent cellular cytotoxicity (ADCC). Finally, an aberrant expression of activating receptors is associated with the phenomenon of leukemia during childhood.

Published
2022
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4. Supplementary Methods from Polo-like Kinase 1 Inhibition as a Therapeutic Approach to Selectively Target BRCA1-Deficient Cancer Cells by Synthetic Lethality Induction

Author

Gastón Soria, Vanesa Gottifredi, José Luis Bocco, Germán A. Gil, Elmer Fernandez, Israel Gloger, Kevin P. Madauss, Gerard Drewes, Beatriz L. Caputto, María Celeste Rodríguez-Baili, María Belén Federico, Florencia Villafañez, Laura Guantay, Virginia Angiolini, Lucía Rodriguez-Berdini, Ana C. Racca, Iris Alejandra García, Ayelén D. Nigra, Diego L. Andino, Andrés M. Castellaro, Natalia S. Paviolo, María Florencia Pansa, and Sofía Carbajosa

Abstract

Extended methods including bionformatic analysis details and scripts used

Published
2023
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5. Figure S2 from Polo-like Kinase 1 Inhibition as a Therapeutic Approach to Selectively Target BRCA1-Deficient Cancer Cells by Synthetic Lethality Induction

Author

Gastón Soria, Vanesa Gottifredi, José Luis Bocco, Germán A. Gil, Elmer Fernandez, Israel Gloger, Kevin P. Madauss, Gerard Drewes, Beatriz L. Caputto, María Celeste Rodríguez-Baili, María Belén Federico, Florencia Villafañez, Laura Guantay, Virginia Angiolini, Lucía Rodriguez-Berdini, Ana C. Racca, Iris Alejandra García, Ayelén D. Nigra, Diego L. Andino, Andrés M. Castellaro, Natalia S. Paviolo, María Florencia Pansa, and Sofía Carbajosa

Abstract

Legends included in the figure file

Published
2023
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6. Data from Polo-like Kinase 1 Inhibition as a Therapeutic Approach to Selectively Target BRCA1-Deficient Cancer Cells by Synthetic Lethality Induction

Author

Gastón Soria, Vanesa Gottifredi, José Luis Bocco, Germán A. Gil, Elmer Fernandez, Israel Gloger, Kevin P. Madauss, Gerard Drewes, Beatriz L. Caputto, María Celeste Rodríguez-Baili, María Belén Federico, Florencia Villafañez, Laura Guantay, Virginia Angiolini, Lucía Rodriguez-Berdini, Ana C. Racca, Iris Alejandra García, Ayelén D. Nigra, Diego L. Andino, Andrés M. Castellaro, Natalia S. Paviolo, María Florencia Pansa, and Sofía Carbajosa

Abstract

Purpose:BRCA1 and BRCA2 deficiencies are widespread drivers of human cancers that await the development of targeted therapies. We aimed to identify novel synthetic lethal relationships with therapeutic potential using BRCA-deficient isogenic backgrounds.Experimental Design:We developed a phenotypic screening technology to simultaneously search for synthetic lethal (SL) interactions in BRCA1- and BRCA2-deficient contexts. For validation, we developed chimeric spheroids and a dual-tumor xenograft model that allowed the confirmation of SL induction with the concomitant evaluation of undesired cytotoxicity on BRCA-proficient cells. To extend our results using clinical data, we performed retrospective analysis on The Cancer Genome Atlas (TCGA) breast cancer database.Results:The screening of a kinase inhibitors library revealed that Polo-like kinase 1 (PLK1) inhibition triggers strong SL induction in BRCA1-deficient cells. Mechanistically, we found no connection between the SL induced by PLK1 inhibition and PARP inhibitors. Instead, we uncovered that BRCA1 downregulation and PLK1 inhibition lead to aberrant mitotic phenotypes with altered centrosomal duplication and cytokinesis, which severely reduced the clonogenic potential of these cells. The penetrance of PLK1/BRCA1 SL interaction was validated using several isogenic and nonisogenic cellular models, chimeric spheroids, and mice xenografts. Moreover, bioinformatic analysis revealed high-PLK1 expression in BRCA1-deficient tumors, a phenotype that was consistently recapitulated by inducing BRCA1 deficiency in multiple cell lines as well as in BRCA1-mutant cells.Conclusions:We uncovered an unforeseen addiction of BRCA1-deficient cancer cells to PLK1 expression, which provides a new means to exploit the therapeutic potential of PLK1 inhibitors in clinical trials, by generating stratification schemes that consider this molecular trait in patient cohorts.

Published
2023
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7. Table S1 from Polo-like Kinase 1 Inhibition as a Therapeutic Approach to Selectively Target BRCA1-Deficient Cancer Cells by Synthetic Lethality Induction

Author

Gastón Soria, Vanesa Gottifredi, José Luis Bocco, Germán A. Gil, Elmer Fernandez, Israel Gloger, Kevin P. Madauss, Gerard Drewes, Beatriz L. Caputto, María Celeste Rodríguez-Baili, María Belén Federico, Florencia Villafañez, Laura Guantay, Virginia Angiolini, Lucía Rodriguez-Berdini, Ana C. Racca, Iris Alejandra García, Ayelén D. Nigra, Diego L. Andino, Andrés M. Castellaro, Natalia S. Paviolo, María Florencia Pansa, and Sofía Carbajosa

Abstract

Full list of hits of the BRCA1-deficient population in the screening performed with the PKIS2 library

Published
2023
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9. Inhibitors of ROCK kinases induce multiple mitotic defects and synthetic lethality in BRCA2-deficient cells

Author

Julieta Martino, Sebastián O. Siri, Natalia S. Paviolo, Cintia Garro, María F. Pansa, Sofía Carbajosa, Aaron C. Brown, José L. Bocco, Israel Gloger, Gerard Drewes, Kevin P. Madauss, Gastón Soria, and Vanesa Gottifredi

Abstract

BRCA2-deficient cells are highly sensitive to poly-ADP-ribose polymerase inhibitors (PARPi) due to their impaired homologous recombination repair. This increased cytotoxicity is triggered by DNA replication stress induced by PARP trapping on DNA. Thus, it is broadly assumed that DNA damage is a prerequisite for BRCA2 synthetic lethality (SL). Here we show that inhibiting ROCK kinases in BRCA2 deficient cells, triggers SL independently from acute replication stress. In contrast, such SL is preceded by enhanced M-phase defects such as anaphase bridges, and abnormal mitotic figures, which were associated with multipolar spindles, supernumerary centrosomes and multinucleation. SL was also triggered by inhibiting Citron Rho-interacting kinase, another enzyme which, similarly to ROCK kinases, regulates cytokinesis. Together, these observations suggest cytokinesis failure as trigger of mitotic abnormalities and SL in BRCA2 cells. Furthermore, preventing mitotic entry by Early mitotic inhibitor 1 (EMI1) depletion promoted survival of BRCA2 deficient cells treated with inhibitors of ROCK kinases, thus reinforcing the association between M-phase and the cell death in BRCA2 deficient cells. This novel mechanism of SL induction is in contrast to the one triggered by PARPi and uncovers mitosis as an Achilles heel of BRCA2 deficient cells.

Published
2022
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11. Restoration of antitumor immunity through anti-MICA antibodies elicited with a chimeric protein

Author

Carolina Ines Domaica, Kenneth W. Hance, Constanza Lauché, Kevin P. Madauss, Florencia Secchiari, Andrea Ziblat, Ximena Lucía Raffo Iraolagoitia, Fernando Alberto Goldbaum, Maria Cecilia Santilli, Nicolas Gilio, Sol Yanel Nuñez, Norberto Walter Zwirner, Evangelina Almada, Adrián Friedrich, Vanesa Zylberman, Mercedes Beatriz Fuertes, Jessica Mariel Sierra, Israel S. Gloger, Raul German Spallanzani, María Victoria Regge, Nicolás Torres, and Romina Paola Pardo

Subjects
Male, Cancer Research, CIENCIAS MÉDICAS Y DE LA SALUD, Lymphoma, Recombinant Fusion Proteins, Inmunología, chemical and pharmacologic phenomena, IMMUNITY, immunology, Mice, TUMOR, Immunity, Multienzyme Complexes, Tumor Cells, Cultured, Immunology and Allergy, Animals, RC254-282, Pharmacology, biology, Antitumor immunity, Histocompatibility Antigens Class I, Antibody-Dependent Cell Cytotoxicity, Antibodies, Monoclonal, Neoplasms. Tumors. Oncology. Including cancer and carcinogens, Basic Tumor Immunology, purl.org/becyt/ford/3.1 [https], Virology, Fusion protein, Brucella, Mice, Inbred C57BL, Medicina Básica, stomatognathic diseases, Oncology, Urinary Bladder Neoplasms, NK Cell Lectin-Like Receptor Subfamily K, MICA, biology.protein, Molecular Medicine, purl.org/becyt/ford/3 [https], Female, Antibody, CHIMERIC PROTEIN
Abstract

Background Natural killer and cytotoxic CD8+ T cells are major players during antitumor immunity. They express NKG2D, an activating receptor that promotes tumor elimination through recognition of the MHC class I chain-related proteins A and B (MICA and MICB). Both molecules are overexpressed on a great variety of tumors from different tissues, making them attractive targets for immunotherapy. However, tumors shed MICA and MICB, and the soluble forms of both (sMICA and sMICB) mediate tumor-immune escape. Some reports indicate that anti-MICA antibodies (Ab) can promote the restoration of antitumor immunity through the induction of direct antitumor effects (antibody-dependent cell-mediated cytotoxicity, ADCC) and scavenging of sMICA. Therefore, we reasoned that an active induction of anti-MICA Ab with an immunogenic protein might represent a novel therapeutic and prophylactic alternative to restore antitumor immunity. Methods We generated a highly immunogenic chimeric protein (BLS-MICA) consisting of human MICA fused to the lumazine synthase from Brucella spp (BLS) and used it to generate anti-MICA polyclonal Ab (pAb) and to investigate if these anti-MICA Ab can reinstate antitumor immunity in mice using two different mouse tumors engineered to express MICA. We also explored the underlying mechanisms of this expected therapeutic effect. Results Immunization with BLS-MICA and administration of anti-MICA pAb elicited by BLS-MICA significantly delayed the growth of MICA-expressing mouse tumors but not of control tumors. The therapeutic effect of immunization with BLS-MICA included scavenging of sMICA and the anti-MICA Ab-mediated ADCC, promoting heightened intratumoral M1/proinflammatory macrophage and antigen-experienced CD8+ T cell recruitment. Conclusions Immunization with the chimeric protein BLS-MICA constitutes a useful way to actively induce therapeutic anti-MICA pAb that resulted in a reprogramming of the antitumor immune response towards an antitumoral/proinflammatory phenotype. Hence, the BLS-MICA chimeric protein constitutes a novel antitumor vaccine of potential application in patients with MICA-expressing tumors. Fil: Torres, Nicolas. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Regge, María Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Secchiari, Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Friedrich, Adrián David. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Spallanzani, Raúl Germán. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Raffo Iraolagoitia, Ximena Lucía. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Nuñez, Sol Yanel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Sierra, Jessica Mariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Ziblat, Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Santilli, Maria Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Gilio, Nicolas. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Almada, Evangelina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Lauché, Constanza Elena. Inmunova S.A; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Pardo, Romina Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Inmunova S.A; Argentina Fil: Domaica, Carolina Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Fuertes, Mercedes Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Madauss, Kevin Patrick. Glaxo Smith Kline; Estados Unidos Fil: Hance, Kenneth W.. Glaxo Smith Kline; Estados Unidos Fil: Gloger, Israel S.. Glaxo Smith Kline; Estados Unidos Fil: Zylberman, Vanesa. Inmunova S.A; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Goldbaum, Fernando Alberto. Inmunova S.A; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Zwirner, Norberto Walter. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; Argentina

Published
2020

12. Trust in Science: a novel research partnership model in Latin America

Author

Rosana Felice, Kevin P. Madauss, and Israel S. Gloger

Subjects
0301 basic medicine, Pharmacology, Latin Americans, business.industry, Partnership model, General Medicine, Public relations, Talent development, 03 medical and health sciences, 030104 developmental biology, 0302 clinical medicine, 030220 oncology & carcinogenesis, Political science, Drug Discovery, business
Abstract

Ten years ago, GlaxoSmithKline established an innovative model for public–private research partnerships in Latin America known as Trust in Science. Here, we summarize this novel initiative, illustrate its achievements in terms of scientific advances and local talent development, and highlight the key learnings that could be applied to similar initiatives. Ten years ago, GlaxoSmithKline established an innovative model for public–private research partnerships in Latin America known as Trust in Science. Here, we summarize this novel initiative, illustrate its achievements in terms of scientific advances and local talent development, and highlight the key learnings that could be applied to similar initiatives.

Published
2021
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13. AKT inhibition impairs PCNA ubiquitylation and triggers synthetic lethality in homologous recombination-deficient cells submitted to replication stress

Author

José Luis Bocco, María Florencia Pansa, Heinz Jacobs, Sabrina F. Mansilla, Kevin P. Madauss, Virginia Angiolini, Laura Guantay, Gastón Soria, Florencia Villafañez, Iris Alejandra García, Sofía Carbajosa, Vanesa Gottifredi, María Candelaria Llorens, and Israel S. Gloger

Subjects
DNA Replication, 0301 basic medicine, Cancer Research, Phenotypic screening, DNA damage, Ubiquitin-Protein Ligases, DNA-Directed DNA Polymerase, Synthetic lethality, Biology, Article, Cell Line, purl.org/becyt/ford/1 [https], 03 medical and health sciences, 0302 clinical medicine, Cell Line, Tumor, Proliferating Cell Nuclear Antigen, Genetics, Humans, PCNA, LETHALITY, Homologous Recombination, Translesion synthesis, purl.org/becyt/ford/1.6 [https], Molecular Biology, Protein kinase B, DNA-PKcs, Cell Death, DNA synthesis, AKT, Ubiquitination, DNA replication, DNA, HCT116 Cells, 3. Good health, Proliferating cell nuclear antigen, Cell biology, HEK293 Cells, 030104 developmental biology, 030220 oncology & carcinogenesis, biology.protein, Homologous recombination, SYNTHETIC, Proto-Oncogene Proteins c-akt, DNA Damage
Abstract

Translesion DNA synthesis (TLS) and homologous recombination (HR) cooperate during S-phase to safeguard replication forks integrity. Thus, the inhibition of TLS becomes a promising point of therapeutic intervention in HR-deficient cancers, where TLS impairment might trigger synthetic lethality (SL). The main limitation to test this hypothesis is the current lack of selective pharmacological inhibitors of TLS. Herein, we developed a miniaturized screening assay to identify inhibitors of PCNA ubiquitylation, a key post-translational modification required for efficient TLS activation. After screening a library of 627 kinase inhibitors, we found that targeting the pro-survival kinase AKT leads to strong impairment of PCNA ubiquitylation. Mechanistically, we found that AKT-mediated modulation of Proliferating Cell Nuclear Antigen (PCNA) ubiquitylation after UV requires the upstream activity of DNA PKcs, without affecting PCNA ubiquitylation levels in unperturbed cells. Moreover, we confirmed that persistent AKT inhibition blocks the recruitment of TLS polymerases to sites of DNA damage and impairs DNA replication forks processivity after UV irradiation, leading to increased DNA replication stress and cell death. Remarkably, when we compared the differential survival of HR-proficient vs HR-deficient cells, we found that the combination of UV irradiation and AKT inhibition leads to robust SL induction in HR-deficient cells. We link this phenotype to AKT ability to inhibit PCNA ubiquitylation, since the targeted knockdown of PCNA E3-ligase (RAD18) and a non-ubiquitylable (PCNA K164R) knock-in model recapitulate the observed SL induction. Collectively, this work identifies AKT as a novel regulator of PCNA ubiquitylation and provides the proof-of-concept of inhibiting TLS as a therapeutic approach to selectively kill HR-deficient cells submitted to replication stress. Fil: Villafañez, Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: García, Iris Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Carbajosa González, Sofía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Pansa, Maria Florencia. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Mansilla, Sabrina Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Llorens de Los Ríos, María Candelaria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Angiolini, Virginia Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones y Transferencia de Villa María. Universidad Nacional de Villa María. Centro de Investigaciones y Transferencia de Villa María; Argentina Fil: Guantay, Maria Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Jacobs, Heinz. Tumor Biology And Immunology-netherlands Cancer Institu; Países Bajos Fil: Madauss, Kevin P.. Glaxosmithkline; Reino Unido Fil: Gloger, Israel. Glaxosmithkline; Reino Unido Fil: Gottifredi, Vanesa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Bocco, Jose Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Soria, Ramiro Gaston. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina

Published
2019

14. Polo-like kinase 1 inhibition as a therapeutic approach to selectively target BRCA1-deficient cancer cells by synthetic lethality induction

Author

Gastón Soria, Germán A. Gil, Elmer Andrés Fernández, Andrés Marcos Castellaro, Iris Alejandra García, Diego L Andino, José Luis Bocco, Ayelén D Nigra, Vanessa Gottifredi, Natalia S. Paviolo, Ana Cristina Racca, María Belén Federico, Laura Guantay, Sofía Carbajosa, María Celeste Rodríguez-Baili, Gerard Drewes, Kevin P. Madauss, Virginia Angiolini, María Florencia Pansa, Beatriz L. Caputto, Lucia Rodríguez-Berdini, Israel S. Gloger, and Florencia Villafañez

Subjects
0301 basic medicine, Cancer Research, letalidad sintetica, DNA damage, Gene Expression, Apoptosis, Cell Cycle Proteins, Synthetic lethality, Polo-like kinase, Biology, Protein Serine-Threonine Kinases, expresion de genes, purl.org/becyt/ford/1 [https], 03 medical and health sciences, Mice, 0302 clinical medicine, Cell Line, Tumor, Proto-Oncogene Proteins, Gene expression, Gene Knockdown Techniques, medicine, Animals, Humans, cancer, purl.org/becyt/ford/1.6 [https], Protein Kinase Inhibitors, Cells, Cultured, BRCA2 Protein, Chromosome Aberrations, BRCA1 Protein, Cell Cycle, Cancer, medicine.disease, Xenograft Model Antitumor Assays, Disease Models, Animal, 030104 developmental biology, Oncology, Cell culture, 030220 oncology & carcinogenesis, Cancer cell, Cancer research, Synthetic Lethal Mutations, DNA Damage
Abstract

Purpose: BRCA1 and BRCA2 deficiencies are widespread drivers of human cancers that await the development of targeted therapies. We aimed to identify novel synthetic lethal relationships with therapeutic potential using BRCA-deficient isogenic backgrounds. Experimental Design: We developed a phenotypic screening technology to simultaneously search for synthetic lethal (SL) interactions in BRCA1- and BRCA2-deficient contexts. For validation, we developed chimeric spheroids and a dualtumor xenograft model that allowed the confirmation of SL induction with the concomitant evaluation of undesired cytotoxicity on BRCA-proficient cells. To extend our results using clinical data, we performed retrospective analysis on The Cancer Genome Atlas (TCGA) breast cancer database. Results: The screening of a kinase inhibitors library revealed that Polo-like kinase 1 (PLK1) inhibition triggers strong SL induction in BRCA1-deficient cells. Mechanistically, we found no connection between the SL induced by PLK1 inhibition and PARP inhibitors. Instead, we uncovered that BRCA1 downregulation and PLK1 inhibition lead to aberrant mitotic phenotypes with altered centrosomal duplication and cytokinesis, which severely reduced the clonogenic potential of these cells. The penetrance of PLK1/BRCA1 SL interaction was validated using several isogenic and nonisogenic cellular models, chimeric spheroids, and mice xenografts. Moreover, bioinformatic analysis revealed high-PLK1 expression in BRCA1-deficient tumors, a phenotype that was consistently recapitulated by inducing BRCA1 deficiency in multiple cell lines as well as in BRCA1-mutant cells. Conclusions: We uncovered an unforeseen addiction of BRCA1-deficient cancer cells to PLK1 expression, which provides a new means to exploit the therapeutic potential of PLK1 inhibitors in clinical trials, by generating stratification schemes that consider this molecular trait in patient cohorts. Fil: Carbajosa González, Sofía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Pansa, Maria Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Paviolo, Natalia Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Fundación Instituto Leloir; Argentina Fil: Castellaro, Andrés Marcos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Andino, Diego Leonardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigación y Desarrollo en Inmunología y Enfermedades Infecciosas. Universidad Católica de Córdoba. Centro de Investigación y Desarrollo en Inmunología y Enfermedades Infecciosas; Argentina Fil: Nigra, Ayelén Denise. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: García, Iris Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Racca, Ana Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Rodriguez, María Celeste. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Angiolini, Virginia Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Guantay, Maria Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Villafañez, Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Federico, Maria Belén. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Rodríguez, Lucía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Caputto, Beatriz Leonor. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Drewes, Gerard. Cellzome AG; Alemania Fil: Madauss, Kevin P.. Global Observatory on Health Research and Development; Estados Unidos Fil: Gloger, Israel. Global Observatory on Health Research and Development; Estados Unidos Fil: Fernandez, Elmer Andres. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigación y Desarrollo en Inmunología y Enfermedades Infecciosas. Universidad Católica de Córdoba. Centro de Investigación y Desarrollo en Inmunología y Enfermedades Infecciosas; Argentina Fil: Gil, German Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Bocco, Jose Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina Fil: Gottifredi, Vanesa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Soria, Ramiro Gaston. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina

Published
2019

15. Aberrant O-glycosylation modulates aggressiveness in neuroblastoma

Author

Daniel F. Alonso, Marina Albertó, Mariano R. Gabri, Cynthia A. Gulino, Sandra Camarero, Fabiana Lubieniecki, Laura Galluzzo Mutti, Kevin P. Madauss, Héctor Adrián Cuello, Valeria Inés Segatori, and Rosario Aschero

Subjects
0301 basic medicine, Glycan, Glycosylation, medicine.drug_class, glycophenotype, 03 medical and health sciences, chemistry.chemical_compound, neuroblastoma, 0302 clinical medicine, Neuroblastoma, MYCN, medicine, Gene silencing, O-glycans, Cell adhesion, neoplasms, biology, Chemistry, Histone deacetylase inhibitor, histone acetylation, medicine.disease, 030104 developmental biology, Trichostatin A, Oncology, Cell culture, 030220 oncology & carcinogenesis, biology.protein, Cancer research, medicine.drug, Research Paper
Abstract

Neuroblastoma (NB) is the most common pediatric malignancy diagnosed before the first birthday in which MYCN oncogene amplification is associated with poor prognosis. Although aberrant glycosylation is an important actor in cell biology, little is known about its role in pediatric cancers such as NB. In this work we characterized the glycophenotype and the enzyme expression involved in glycans biosynthesis in five established human NB cell lines and in patient-derived primary tumors with different MYCN status. Our results show a high expression of Lewis glycan family both in MYCN-amplified cell lines and patient samples. Additionally, we report that MYCN-amplified cells overexpressed Core 2-initiating glycosyltransferase C2GNT1 in association with specific ST3Gals and FUTs, and showed increased binding to E- and P- selectins. Silencing of C2GNT1 expression in NB cells diminished expression of Lewis glycans, decreased the E- and P-selectin binding, and reduced cell adhesion, migration and proliferation in vitro. Treatment of MYCN-non-amplified cells with Trichostatin A (TSA), an histone deacetylase inhibitor, increased the expression of Lewis glycans and the enzymes involved in their biosynthesis. Our results demonstrate that MYCN-amplified NB cells overexpress Lewis family glycans, which belong to the Core 2 O-glycans group. Their expression plays a key role in the malignant behaviour of the NB cells and it is modulated by epigenetic mechanisms.

Published
2018

16. Novel scaffolds for inhibition of Cruzipain identified from high-throughput screening of anti-kinetoplastid chemical boxes

Author

Vanina E. Alvarez, Fernán Agüero, Lionel Urán Landaburu, Joel Karpiak, Emir Salas-Sarduy, Juan José Cazzulo, and Kevin P. Madauss

Subjects
0301 basic medicine, Chagas disease, Trypanosoma cruzi, Otras Ciencias Biológicas, 030106 microbiology, Antiprotozoal Agents, Protozoan Proteins, lcsh:Medicine, Cruzipain, Computational biology, Article, Host-Parasite Interactions, purl.org/becyt/ford/1 [https], Ciencias Biológicas, 03 medical and health sciences, Protein Domains, Drug Discovery, medicine, Humans, Chagas Disease, Kinetoplastida, Author Correction, lcsh:Science, purl.org/becyt/ford/1.6 [https], Nifurtimox, Trypanosoma Cruzi, Multidisciplinary, Molecular Structure, biology, Drug discovery, lcsh:R, medicine.disease, biology.organism_classification, chEMBL, High-Throughput Screening Assays, Molecular Docking Simulation, Cysteine Endopeptidases, 030104 developmental biology, Nitroimidazoles, Benznidazole, Docking (molecular), Immunology, lcsh:Q, Proteinase Inhibitors, CIENCIAS NATURALES Y EXACTAS, medicine.drug
Abstract

American Trypanosomiasis or Chagas disease is a prevalent, neglected and serious debilitating illness caused by the kinetoplastid protozoan parasite Trypanosoma cruzi. The current chemotherapy is limited only to nifurtimox and benznidazole, two drugs that have poor efficacy in the chronic phase and are rather toxic. In this scenario, more efficacious and safer drugs, preferentially acting through a different mechanism of action and directed against novel targets, are particularly welcome. Cruzipain, the main papain-like cysteine peptidase of T. cruzi, is an important virulence factor and a chemotherapeutic target with excellent pre-clinical validation evidence. Here, we present the identification of new Cruzipain inhibitory scaffolds within the GlaxoSmithKline HAT (Human African Trypanosomiasis)and Chagas chemical boxes, two collections grouping 404 non-cytotoxic compounds with high antiparasitic potency, drug-likeness, structural diversity and scientific novelty. We have adapted a continuous enzymatic assay to a medium-throughput format and carried out a primary screening of both collections, followed by construction and analysis of dose-response curves of the most promising hits. Using the identified compounds as a starting point a substructure directed search against CHEMBL Database revealed plausible common scaffolds while docking experiments predicted binding poses and specific interactions between Cruzipain and the novel inhibitors. Fil: Salas Sarduy, Emir. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Urán Landaburu, Héctor Lionel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Karpiak, Joel. GlaxoSmithKline R&D; Estados Unidos Fil: Madauss, Kevin P.. GlaxoSmithKline R&D; Estados Unidos Fil: Cazzulo, Juan Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Agüero, Fernan Gonzalo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Alvarez, Vanina Eder. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina

Published
2017

17. Author Correction: Novel scaffolds for inhibition of Cruzipain identified from high-throughput screening of anti-kinetoplastid chemical boxes

Author

Fernán Agüero, Joel Karpiak, Lionel Urán Landaburu, Vanina E. Alvarez, Kevin P. Madauss, Juan José Cazzulo, and Emir Salas-Sarduy

Subjects
Multidisciplinary, Computer science, High-throughput screening, lcsh:R, lcsh:Medicine, Cruzipain, Computational biology, 030226 pharmacology & pharmacy, 03 medical and health sciences, 0302 clinical medicine, 030220 oncology & carcinogenesis, ComputingMethodologies_DOCUMENTANDTEXTPROCESSING, lcsh:Q, lcsh:Science
Abstract

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Published
2018
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18. PO-232 Lewis glycans and their epigenetic regulation are associated with neuroblastoma aggressiveness

Author

Daniel F. Alonso, Cynthia A. Gulino, Marina Albertó, Héctor Adrián Cuello, Valeria Inés Segatori, L. Galluzzo Mutti, Mariano R. Gabri, Fabiana Lubieniecki, Kevin P. Madauss, and Rosario Aschero

Subjects
Cancer Research, Glycan, biology, medicine.drug_class, Chemistry, Histone deacetylase inhibitor, Trichostatin A, Oncology, Cell culture, medicine, Cancer research, biology.protein, Gene silencing, Histone deacetylase, Epigenetics, Cell adhesion, neoplasms, medicine.drug
Abstract

Introduction Glycans are diverse and complex molecules with a crucial participation in critical events of cell biology. Their implications in adult cancer are widely reported, but little is known about their role in paediatric cancer such as Neuroblastoma (NB). NB is the most common paediatric malignancy diagnosed before the first birthday in which MYCN oncogene amplification is associated with poor prognosis. Hence, the study of aberrant glycosylation is a promising field to understand NB transformation and to find new therapeutic targets. Material and methods This study was carried out over five established human NB cell lines and patients´ tumour samples with different MYCN status. Terminal glycans expression and selectin binding capacity was characterised by flow cytometry. Expression of enzymes involved in glycans biosynthesis was measured by qRT-PCR. The role of Core2-O glycans was studied by C2GNT1 silencing and in vitro cell adhesion, proliferation and migration. Epigenetic regulation was evaluated using Trichostatin A (TSA) histone deacetylase inhibitor. Results and discussions Minimal expression of truncated glycans (T, Tn and STn) was found in all cell lines tested. In contrast, an association between MYCN amplification and Lewis glycan family (mainly SleX and LeY) expression was observed both in cell lines and patients´ samples. In accordance, MYCN-amplified cells showed overexpression of C2GNT1, the glycoenzyme responsible for initiating the Core 2 O-glycans branch that could be extended to the Lewis antigens. Furthermore, we detected overexpression of ST3Gal4, FUT4, 7, 9 and 11, terminal enzymes of this biosynthetic pathway. In the same light, since selectins binding depends on Lewis glycans presence, we observed an increase on E- and P-selectin binding in MYCN-amplified cell lines. Silencing of C2GNT1 provoked a reduction in Lewis glycans expression and decreased in vitro cell adhesion, migration and proliferation in both MYCN status cells. Interestingly, C2GNT1 silencing of MYCN-amplified cells also showed a decreased in E- and P-selectin binding. Finally, inhibition of histone deacetylase in MYCN-non amplified cells produced a significant increase in the expression of Lewis glycans and enzymes FUT3 and FUT7. Conclusion In this work we highlight the impact of Core-2 Lewis family as part of O-glycans in malignant NB cell behaviour, the enzymes involved and the epigenetic regulation in relation to MYCN status.

Published
2018
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19. FXR agonist activity of conformationally constrained analogs of GW 4064

Author

Harry B. Marr, Kevin P. Madauss, Jonathan Y. Bass, Shawn P. Williams, Dan Todd, Paul Kenneth Spearing, Adwoa Akwabi-Ameyaw, Lihong Chen, Robert B. McFadyen, Justin A. Caravella, Derek J. Parks, Frank Navas, David N. Deaton, Katrina L. Creech, G. Bruce Wisely, Richard Dana Caldwell, and Aaron B. Miller

Subjects
Agonist, Stereochemistry, medicine.drug_class, Clinical Biochemistry, Receptors, Cytoplasmic and Nuclear, Pharmaceutical Science, Thiophenes, Naphthalenes, Crystallography, X-Ray, Biochemistry, Chemical synthesis, Structure-Activity Relationship, chemistry.chemical_compound, Drug Discovery, medicine, Animals, Humans, Computer Simulation, Molecular Biology, Binding Sites, Organic Chemistry, Benzothiophene, Isoxazoles, Rats, Nuclear receptor, chemistry, Molecular Medicine, Farnesoid X receptor
Abstract

Two series of conformationally constrained analogs of the FXR agonist GW 4064 1 were prepared. Replacement of the metabolically labile stilbene with either benzothiophene or naphthalene rings led to the identification of potent full agonists 2a and 2g.

Published
2009
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20. Discovery of orally active, pyrrolidinone-based progesterone receptor partial agonists

Author

Rakesh Nagilla, Sharada Manns, Eugene L. Stewart, Marlys Hammond, Tram H. Hoang, Scott K. Thompson, Walter Trizna, Chaya Duraiswami, Eugene T. Grygielko, Thuy B. Tran, James S. Frazee, Kevin P. Madauss, Johnson Latisha C, Shawn P. Williams, Lindsay E. Glace, David G. Washburn, and Jeffrey D. Bray

Subjects
Agonist, medicine.drug_class, Stereochemistry, Clinical Biochemistry, hERG, Administration, Oral, Pharmaceutical Science, Pharmacology, Biochemistry, Partial agonist, Structure-Activity Relationship, chemistry.chemical_compound, In vivo, Drug Discovery, Progesterone receptor, medicine, Animals, Humans, Pyrrolidinones, Molecular Biology, Binding Sites, biology, Organic Chemistry, Haplorhini, Ether-A-Go-Go Potassium Channels, Rats, Nuclear receptor, chemistry, biology.protein, Molecular Medicine, Receptors, Progesterone, Lead compound
Abstract

We have designed and synthesized a novel series of pyrrolidinones as progesterone receptor partial agonists. Compounds from this series had improved AR selectivity, rat pharmacokinetic properties, and in vivo potency compared to the lead compound. In addition, these compounds had improved selectivity against hERG channel inhibition.

Published
2009
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21. Pyrrolidinyl pyridone and pyrazinone analogues as potent inhibitors of prolyl oligopeptidase (POP)

Author

Mary H. Hanlon, Aaron B. Miller, David J.T. Porter, Kevin P. Madauss, J. David Becherer, Robert A. Reid, Caroline J. Diaz, Luke H. Carter, A.M. Hassell, and Curt D. Haffner

Subjects
Pyrrolidines, Serine Proteinase Inhibitors, genetic structures, Pyridones, Stereochemistry, Clinical Biochemistry, Molecular Conformation, Pharmaceutical Science, Oligopeptidase, Crystallography, X-Ray, Biochemistry, Chemical synthesis, Pyrrolidine, chemistry.chemical_compound, Prolyl endopeptidase, Drug Discovery, Hydrolase, medicine, Combinatorial Chemistry Techniques, Humans, Molecular Biology, chemistry.chemical_classification, Serine protease, Molecular Structure, biology, Serine Endopeptidases, Organic Chemistry, Brain, Enzyme, chemistry, Enzyme inhibitor, Drug Design, biology.protein, Molecular Medicine, Prolyl Oligopeptidases, medicine.drug
Abstract

We report the synthesis and in vitro activity of a series of novel pyrrolidinyl pyridones and pyrazinones as potent inhibitors of prolyl oligopeptidase (POP). Within this series, compound 39 was co-crystallized within the catalytic site of a human chimeric POP protein which provided a more detailed understanding of how these inhibitors interacted with the key residues within the catalytic pocket.

Published
2008
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22. Conformationally constrained farnesoid X receptor (FXR) agonists: Naphthoic acid-based analogs of GW 4064

Author

Lihong Chen, Jonathan Y. Bass, David N. Deaton, Aaron B. Miller, Justin A. Caravella, Istvan Kaldor, Robert B. McFadyen, Stacey A. Jones, Dan Todd, Paul Kenneth Spearing, Kevin P. Madauss, Adwoa Akwabi-Ameyaw, Derek J. Parks, Richard Dana Caldwell, Frank Navas, Yaping Liu, Katrina L. Creech, G. Bruce Wisely, Harry B. Marr, and Shawn P. Williams

Subjects
Agonist, Molecular model, medicine.drug_class, Stereochemistry, Clinical Biochemistry, Carboxylic Acids, Molecular Conformation, Administration, Oral, Receptors, Cytoplasmic and Nuclear, Pharmaceutical Science, Naphthalenes, Crystallography, X-Ray, Biochemistry, Chemical synthesis, Mice, Structure-Activity Relationship, Dogs, Drug Discovery, medicine, Animals, Structure–activity relationship, Receptor, Molecular Biology, Cholestasis, Molecular Structure, Chemistry, Stomach, Organic Chemistry, Haplorhini, Isoxazoles, G protein-coupled bile acid receptor, Rats, DNA-Binding Proteins, Disease Models, Animal, Nuclear receptor, Gastric Mucosa, Molecular Medicine, Farnesoid X receptor, Transcription Factors
Abstract

Starting from the known FXR agonist GW 4064 1a, a series of stilbene replacements were prepared. The 6-substituted 1-naphthoic acid 1b was an equipotent FXR agonist with improved developability parameters relative to 1a. Analog 1b also reduced the severity of cholestasis in the ANIT acute cholestatic rat model.

Published
2008
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23. A Structural and in Vitro Characterization of Asoprisnil: A Selective Progesterone Receptor Modulator

Author

Eugene T. Grygielko, Eugene L. Stewart, Jeffrey D. Bray, Nicholas J. Laping, Charlene Wu, Shawn P. Williams, Thomas B. Stanley, Kevin P. Madauss, Su-Jun Deng, Steve A. Short, Anthony Sulpizio, and Scott K. Thompson

Subjects
Models, Molecular, Protein Conformation, medicine.drug_class, Breast Neoplasms, Biology, Pharmacology, Crystallography, X-Ray, Transfection, Polymerase Chain Reaction, chemistry.chemical_compound, Endocrinology, Cell Line, Tumor, Oximes, Selective progesterone receptor modulator, Progesterone receptor, medicine, Humans, Estrenes, Receptor, Molecular Biology, Estradiol, General Medicine, Asoprisnil, Gene Expression Regulation, Neoplastic, Mechanism of action, Nuclear receptor, chemistry, Estrogen, Female, medicine.symptom, Receptors, Progesterone, Corepressor, hormones, hormone substitutes, and hormone antagonists, Plasmids
Abstract

Selective progesterone receptor modulators (SPRMs) have been suggested as therapeutic agents for treatment of gynecological disorders. One such SPRM, asoprisnil, was recently in clinical trials for treatment of uterine fibroids and endometriosis. We present the crystal structures of progesterone receptor (PR) ligand binding domain complexed with asoprisnil and the corepressors nuclear receptor corepressor (NCoR) and SMRT. This is the first report of steroid nuclear receptor crystal structures with ligand and corepressors. These structures show PR in a different conformation than PR complexed with progesterone (P4). We profiled asoprisnil in PR-dependent assays to understand further the PR-mediated mechanism of action. We confirmed previous findings that asoprisnil demonstrated antagonism, but not agonism, in a PR-B transfection assay and the T47D breast cancer cell alkaline phosphatase activity assay. Asoprisnil, but not RU486, weakly recruited the coactivators SRC-1 and AIB1. However, asoprisnil strongly recruited the corepressor NCoR in a manner similar to RU486. Unlike RU486, NCoR binding to asoprisnil-bound PR could be displaced with equal affinity by NCoR or TIF2 peptides. We further showed that it weakly activated T47D cell gene expression of Sgk-1 and PPL and antagonized P4-induced expression of both genes. In rat leiomyoma ELT3 cells, asoprisnil demonstrated partial P4-like inhibition of cyclooxygenase (COX) enzymatic activity and COX-2 gene expression. In the rat uterotrophic assay, asoprisnil demonstrated no P4-like ability to oppose estrogen. Our data suggest that asoprisnil differentially recruits coactivators and corepressors compared to RU486 or P4, and this specific cofactor interaction profile is apparently insufficient to oppose estrogenic activity in rat uterus.

Published
2007
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24. Discovery of non-steroidal mifepristone mimetics: Pyrazoline-based PR antagonists

Author

Eugene L. Stewart, Xi Liang, David W. Gray, Shawn P. Williams, William J. Hoekstra, David G. Jones, Kevin P. Madauss, Lara S. Kallander, Scott K. Thompson, and Robert A. Noe

Subjects
Models, Molecular, Stereochemistry, Clinical Biochemistry, Pharmaceutical Science, Pyrazoline, Pharmacology, Ligands, Transfection, Biochemistry, Chemical synthesis, Cell Line, chemistry.chemical_compound, Drug Discovery, Progesterone receptor, medicine, Humans, Homology modeling, Receptor, Molecular Biology, Abortifacient, Fluorescent Dyes, Chemistry, Molecular Mimicry, Organic Chemistry, Antagonist, General Medicine, Mifepristone, Docking (molecular), Pyrazoles, Molecular Medicine, Receptors, Progesterone, Protein Binding, medicine.drug
Abstract

Mifepristone is a non-selective antagonist of 3-oxosteroid receptors with both abortifacient and anti-endometriotic activities. Non-steroidal mimetics of mifepristone and progesterone are important templates for modulation of the progesterone receptor (PR). For our PR program, we sought an unexplored, synthetically accessible non-steroidal mimetic of mifepristone, suitable for parallel synthesis of analogues. Docking of compounds into a PR homology model identified 4-substituted pyrazolines, which, when synthesized and tested, exhibited functional antagonism of PR.

Published
2005
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25. Progesterone Receptor Ligand Binding Pocket Flexibility: Crystal Structures of the Norethindrone and Mometasone Furoate Complexes

Author

Shawn P. Williams, Eugene L. Stewart, Iain M. McLay, Robert J. H. Austin, Steven A. Short, Ian J. Uings, John B. Pritchard, Millard H. Lambert, Kevin P. Madauss, and Su-Jun Deng

Subjects
Models, Molecular, Binding Sites, Molecular model, Chemistry, Stereochemistry, Metribolone, medicine.medical_treatment, Mometasone furoate, Crystallography, X-Ray, Ligands, Ligand (biochemistry), Steroid, chemistry.chemical_compound, Drug Discovery, Progesterone receptor, medicine, Molecular Medicine, Norethindrone, Binding site, Receptors, Progesterone, Mometasone Furoate, Pregnadienediols, Conformational isomerism, medicine.drug
Abstract

Although progesterone, the natural ligand of the progesterone receptor (PR), has a hydrogen atom at the 17alpha position, other potent steroid agonists such as norethindrone and mometasone furoate have larger substituents at this position that are accommodated by the PR ligand binding pocket. Crystallographic analysis of PR ligand binding domain complexes clearly demonstrated that these moieties were accommodated by local shifts of the protein main chain and by adoption of alternative side chain rotamer conformations of ligand-proximal amino acids. These conformational changes imparted a ligand-specific volume to the binding pocket, from 490 A3 in the metribolone complex to 520 A3 in the norethindrone complex, 565 A3 in the progesterone complex, and 730 A3 in the mometasone furoate complex. Despite these marked alterations in binding pocket volume, critical interactions essential for establishment of an active AF2 conformation were maintained.

Published
2004
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26. Conformationally constrained farnesoid X receptor (FXR) agonists: alternative replacements of the stilbene

Author

Aaron B. Miller, Harry B. Marr, Katrina L. Creech, G. Bruce Wisely, Shawn P. Williams, Adwoa Akwabi-Ameyaw, Lihong Chen, David N. Deaton, Justin A. Caravella, Paul Kenneth Spearing, Kevin P. Madauss, Derek J. Parks, Frank Navas, and Dan Todd

Subjects
Agonist, Stereochemistry, medicine.drug_class, Clinical Biochemistry, Molecular Sequence Data, Molecular Conformation, Pharmaceutical Science, Receptors, Cytoplasmic and Nuclear, Biochemistry, Cell Line, chemistry.chemical_compound, Drug Discovery, Stilbenes, medicine, Moiety, Animals, Humans, Carboxylate, Amino Acid Sequence, Molecular Biology, Indole test, Organic Chemistry, Benzothiophene, Isoxazoles, G protein-coupled bile acid receptor, chemistry, Molecular Medicine, Farnesoid X receptor, Transcription regulator
Abstract

To further explore the optimum placement of the acid moiety in conformationally constrained analogs of GW 4064 1a, a series of stilbene replacements were prepared. The benzothiophene 1f and the indole 1g display the optimal orientation of the carboxylate for enhanced FXR agonist potency.

Published
2011

27. Conformationally constrained farnesoid X receptor (FXR) agonists: heteroaryl replacements of the naphthalene

Author

Justin A. Caravella, Terrence L. Smalley, Jonathan Y. Bass, Kevin P. Madauss, Aaron B. Miller, Robert B. McFadyen, Katrina L. Creech, G. Bruce Wisely, Harry B. Marr, Lihong Chen, Dan Todd, Shawn P. Williams, David N. Deaton, Paul Kenneth Spearing, Derek J. Parks, Frank Navas, and Wendy Y. Mills

Subjects
Agonist, Blood Glucose, medicine.drug_class, Stereochemistry, Clinical Biochemistry, Molecular Conformation, Pharmaceutical Science, Receptors, Cytoplasmic and Nuclear, Naphthalenes, Crystallography, X-Ray, Ligands, Weight Gain, Biochemistry, Chemical synthesis, Diabetes Mellitus, Experimental, chemistry.chemical_compound, Mice, Dogs, Drug Discovery, medicine, Fluorescence Resonance Energy Transfer, Animals, Humans, Receptor, Molecular Biology, Binding Sites, Bicyclic molecule, Chemistry, Organic Chemistry, Quinoline, Isoxazoles, G protein-coupled bile acid receptor, Protein Structure, Tertiary, Rats, Nuclear receptor, Quinolines, Molecular Medicine, Farnesoid X receptor
Abstract

To improve on the drug properties of GSK8062 1b, a series of heteroaryl bicyclic naphthalene replacements were prepared. The quinoline 1c was an equipotent FXR agonist with improved drug developability parameters relative to 1b. In addition, analog 1c lowered body weight gain and serum glucose in a DIO mouse model of diabetes.

Published
2010

28. Design and x-ray crystal structures of high-potency nonsteroidal glucocorticoid agonists exploiting a novel binding site on the receptor

Author

Diane M. Coe, Thuy B. Tran, Keith Biggadike, Simon J. F. Macdonald, David House, Iain M. McLay, Randy K. Bledsoe, Tony W. J. Cooper, Simon Taylor, Kevin P. Madauss, Victoria Weller, Tracy Jane Shipley, Iain Uings, Shawn P. Williams, and Marie A. Iannone

Subjects
Models, Molecular, Indazole, Multidisciplinary, Binding Sites, Chemistry, Ligand, Stereochemistry, NF-kappa B, Crystallography, X-Ray, Ligands, Combinatorial chemistry, Amides, Protein Structure, Tertiary, chemistry.chemical_compound, Glucocorticoid receptor, Receptors, Glucocorticoid, Amide, Cell Line, Tumor, Drug Design, Physical Sciences, Humans, Binding site, Receptor, Selectivity, Structural motif
Abstract

Crystallography and computer modeling have been used to exploit a previously unexplored channel in the glucocorticoid receptor (GR). Highly potent, nonsteroidal indazole amides showing excellent complementarity to the channel were designed with the assistance of the computational technique AlleGrow. The accuracy of the design process was demonstrated through crystallographic structural determination of the GR ligand-binding domain–agonist complex of the D-prolinamide derivative 11 . The utility of the channel was further exemplified through the design of a potent phenylindazole in which structural motifs, seen to interact with the traditional GR ligand pocket, were abandoned and replaced by interactions within the new channel. Occupation of the channel was confirmed with a second GR crystal structure of this truncated D-alaninamide derivative 13 . Compound 11 displays properties compatible with development as an intranasal solution formulation, whereas oral bioavailability has been demonstrated with a related truncated exemplar 14 . Data with the pyrrolidinone amide 12 demonstrate the potential for further elaboration within the “meta” channel to deliver compounds with selectivity for the desired transrepressive activity of glucocorticoids. The discovery of these interactions with this important receptor offers significant opportunities for the design of novel GR modulators.

Published
2009

29. Improving the developability profile of pyrrolidine progesterone receptor partial agonists

Author

Eugene T. Grygielko, Patrick Stoy, Eugene L. Stewart, Tram H. Hoang, Johnson Latisha C, Jaclyn R. Patterson, Marlys Hammond, Qing Lu, Linda S. Barton, Rakesh Nagilla, David G. Washburn, Shawn P. Williams, Lara S. Kallander, Jeffrey D. Bray, Leonard M. Azzarano, Scott K. Thompson, Chaya Duraiswami, James S. Frazee, Kevin P. Madauss, Nicholas J. Laping, and Xiaoping Xu

Subjects
ERG1 Potassium Channel, Pyrrolidines, medicine.drug_class, Stereochemistry, Clinical Biochemistry, hERG, Endometriosis, Pharmaceutical Science, Carboxamide, Crystallography, X-Ray, Biochemistry, Chemical synthesis, Partial agonist, Pyrrolidine, chemistry.chemical_compound, In vivo, Drug Discovery, Progesterone receptor, medicine, Animals, Humans, Receptor, Molecular Biology, Sulfonamides, Binding Sites, biology, Chemistry, Organic Chemistry, Ether-A-Go-Go Potassium Channels, Rats, biology.protein, Molecular Medicine, Female, Carbamates, Receptors, Progesterone
Abstract

The previously reported pyrrolidine class of progesterone receptor partial agonists demonstrated excellent potency but suffered from serious liabilities including hERG blockade and high volume of distribution in the rat. The basic pyrrolidine amine was intentionally converted to a sulfonamide, carbamate, or amide to address these liabilities. The evaluation of the degree of partial agonism for these non-basic pyrrolidine derivatives and demonstration of their efficacy in an in vivo model of endometriosis is disclosed herein.

Published
2009

30. Rational design of orally-active, pyrrolidine-based progesterone receptor partial agonists

Author

Eugene T. Grygielko, Shawn P. Williams, Lindsay E. Glace, Jeffrey D. Bray, Chaya Duraiswami, James S. Frazee, Kevin P. Madauss, David G. Washburn, Tram H. Hoang, Leahann Lapinski, Walter Trizna, Nicholas J. Laping, and Scott K. Thompson

Subjects
Agonist, Pyrrolidines, medicine.drug_class, Stereochemistry, Clinical Biochemistry, Pharmaceutical Science, Administration, Oral, Crystallography, X-Ray, Biochemistry, Partial agonist, Pyrrolidine, chemistry.chemical_compound, Drug Discovery, Progesterone receptor, medicine, Animals, Computer Simulation, Molecular Biology, Hormone binding protein, Binding Sites, Organic Chemistry, Rational design, Protein Structure, Tertiary, Rats, chemistry, Nuclear receptor, Estrogen, Drug Design, Models, Animal, Molecular Medicine, Receptors, Progesterone
Abstract

Using the X-ray crystal structure of an amide-based progesterone receptor (PR) partial agonist bound to the PR ligand binding domain, a novel PR partial agonist class containing a pyrrolidine ring was designed. Members of this class of N-alkylpyrrolidines demonstrate potent and highly selective partial agonism of the progesterone receptor, and one of these analogs was shown to be efficacious upon oral dosing in the OVX rat model of estrogen opposition.

Published
2009

31. The first X-ray crystal structure of the glucocorticoid receptor bound to a non-steroidal agonist

Author

Iain M. McLay, Gordon G. Weingarten, Eugene L. Stewart, Kevin P. Madauss, Shawn P. Williams, Randy K. Bledsoe, and Iain Uings

Subjects
Agonist, Models, Molecular, Molecular model, medicine.drug_class, Stereochemistry, Clinical Biochemistry, Molecular Conformation, Pharmaceutical Science, Carboxamide, Crystallography, X-Ray, Biochemistry, Dexamethasone, chemistry.chemical_compound, Structure-Activity Relationship, Glucocorticoid receptor, Receptors, Glucocorticoid, Drug Discovery, medicine, Structure–activity relationship, Receptor, Benzamide, Molecular Biology, Molecular Structure, Organic Chemistry, chemistry, Benzamides, Molecular Medicine, Pyrazoles, hormones, hormone substitutes, and hormone antagonists, Glucocorticoid, medicine.drug
Abstract

The amino-pyrazole 2,6-dichloro-N-ethyl benzamide 1 is a selective GR agonist with dexamethasone-like in vitro potency. Its X-ray crystal structure in the GR LBD (Glucocorticoid ligand-binding domain) is described and compared to other reported structures of steroidal GR agonists in the GR LBD (3E7C).

Published
2008

32. Crystallization of protein-ligand complexes

Author

Robert X. Xu, Lisa M. Shewchuk, Robert T. Gampe, Tamara E. Grisard, Shawn P. Williams, Gang An, Liping Wang, Su-Jun J. Deng, Warren J. Rocque, Anne M. Hassell, Robert T. Nolte, Kurt Weaver, Kevin P. Madauss, H. Luke Carter, Jane Bynum, G. Bruce Wisely, and Randy K. Bledsoe

Subjects
crystallization, Stereochemistry, Ab initio, Molecular Conformation, Crystallography, X-Ray, Ligands, Cofactor, Receptors, Glucocorticoid, Structural Biology, Protein purification, Molecule, Animals, Humans, Binding site, Binding Sites, biology, Chemistry, Temperature, Proteins, General Medicine, Small molecule, Research Papers, Receptors, Mineralocorticoid, Receptors, Androgen, Liposomes, Mutation, biology.protein, Nucleic acid, protein–ligand complexes, Carrier Proteins, Protein ligand
Abstract

Methods presented for growing protein–ligand complexes fall into the categories of co-expression of the protein with the ligands of interest, use of the ligands during protein purification, cocrystallization and soaking the ligands into existing crystals., Obtaining diffraction-quality crystals has long been a bottleneck in solving the three-dimensional structures of proteins. Often proteins may be stabilized when they are complexed with a substrate, nucleic acid, cofactor or small molecule. These ligands, on the other hand, have the potential to induce significant conformational changes to the protein and ab initio screening may be required to find a new crystal form. This paper presents an overview of strategies in the following areas for obtaining crystals of protein–ligand complexes: (i) co-expression of the protein with the ligands of interest, (ii) use of the ligands during protein purification, (iii) cocrystallization and (iv) soaks.

Published
2006

33. A ligand-mediated hydrogen bond network required for the activation of the mineralocorticoid receptor

Author

Randy K. Bledsoe, Timothy M. Willson, Ryan P. Trump, Jason A. Holt, Eugene L. Stewart, Kevin P. Madauss, Thomas B. Stanley, Millard H. Lambert, Shawn P. Williams, Kenneth H. Pearce, and Christopher J. Apolito

Subjects
Agonist, Models, Molecular, Transcriptional Activation, medicine.drug_class, Stereochemistry, Crystallography, X-Ray, Ligands, Biochemistry, Protein Structure, Secondary, Protein structure, Mineralocorticoid receptor, medicine, Humans, Receptor, Molecular Biology, Binding selectivity, Molecular Structure, Chemistry, Hydrogen bond, Hydrogen Bonding, Cell Biology, Ligand (biochemistry), Protein Structure, Tertiary, Receptors, Mineralocorticoid, Mutation, Steroids, Alpha helix, Protein Binding
Abstract

Ligand binding is the first step in hormone regulation of mineralocorticoid receptor (MR) activity. Here, we report multiple crystal structures of MR (NR3C2) bound to both agonist and antagonists. These structures combined with mutagenesis studies reveal that maximal receptor activation involves an intricate ligand-mediated hydrogen bond network with Asn770 which serves dual roles: stabilization of the loop preceding the C-terminal activation function-2 helix and direct contact with the hormone ligand. In addition, most activating ligands hydrogen bond to Thr945 on helix 10. Structural characterization of the naturally occurring S810L mutant explains how stabilization of a helix 3/helix 5 interaction can circumvent the requirement for this hydrogen bond network. Taken together, these results explain the potency of MR activation by aldosterone, the weak activation induced by progesterone and the antihypertensive agent spironolactone, and the binding selectivity of cortisol over cortisone.

Published
2005

34. Generation and characterization of human steroidogenic factor 1 LBD crystals with and without bound cofactor peptide

Author

Kevin P. Madauss, Greg M. Waitt, Dalia Juzumiene, Jon D. Williams, and Shawn P. Williams

Subjects
Steroidogenic factor 1, Models, Molecular, endocrine system, Subfamily, Stereochemistry, Molecular Conformation, Receptors, Cytoplasmic and Nuclear, Peptide, Endogeny, Ligands, Steroidogenic Factor 1, Cofactor, Nuclear Receptor Coactivator 2, Endocrinology, Humans, Receptor, Ternary complex, chemistry.chemical_classification, Homeodomain Proteins, biology, General Medicine, Protein Structure, Tertiary, DNA-Binding Proteins, Nuclear receptor, chemistry, Biochemistry, biology.protein, Crystallization, Transcription Factors
Abstract

The nuclear receptor Steroidogenic Factor 1 (SF1) plays a critical role in the development of the adrenal gland and gonads, and in sexual differentiation. SF1 performs this pivotal function through the regulation of hormone expression that is essential for organogenesis and endocrine homeostasis. SF1 is a member of a nuclear receptor subclass that contains LRH1 and the Drosophila receptor FTZ-F1. To date, a natural ligand has not been reported for any member of this subfamily. Here we report the crystallization and characterization of the ligand-binding domain (LBD) of human SF1 from two different crystal forms: a binary complex with fortuitous ligand and a ternary complex with the same ligand and a peptide containing a motif of a nuclear receptor cofactor. The structural determination of the binary complex required the use of sulfur SAD phasing, a relatively new technique that uses anomalous diffraction from the endogenous sulfur atoms present in the protein. The structure of the ternary complex was determined by multiple wavelength anomalous diffraction (MAD) using seleno-methionine substituted SF1. Preliminary analysis suggested SF1 contained a fortuitous ligand in the binding pocket. This ligand may account for the relatively high basal activity observed for SF1 in cofactor recruitment and cell-based assays.

Published
2005

35. Structural analyses reveal phosphatidyl inositols as ligands for the NR5 orphan receptors SF-1 and LRH-1

Author

Holly A. Ingraham, Kevin P. Madauss, Robert J. Fletterick, Valerie G. Montana, Elena P. Sablin, Joseph W. Thornton, Miyuki Suzawa, Shawn P. Williams, Gregory M. Waitt, Rodney K. Guy, Timothy M. Willson, Robert X. Xu, Jamie Moore, Irina N. Krylova, Jane Bynum, Jon D. Williams, Lioudmila A. Lebedeva, Dalia Juzumiene, and J. Andrew MacKay

Subjects
Steroidogenic factor 1, Models, Molecular, Subfamily, Receptors, Cytoplasmic and Nuclear, Biology, Crystallography, X-Ray, Ligands, Phosphatidylinositols, Steroidogenic Factor 1, General Biochemistry, Genetics and Molecular Biology, Evolution, Molecular, 03 medical and health sciences, Mice, 0302 clinical medicine, Phosphatidylinositol Phosphates, Cell Line, Tumor, Gene expression, Animals, Humans, Receptor, Transcription factor, Phylogeny, 030304 developmental biology, Homeodomain Proteins, 0303 health sciences, Binding Sites, Biochemistry, Genetics and Molecular Biology(all), Liver receptor homolog-1, Protein Structure, Tertiary, DNA-Binding Proteins, Biochemistry, Nuclear receptor, 030220 oncology & carcinogenesis, Second messenger system, Protein Binding, Signal Transduction, Transcription Factors
Abstract

SummaryVertebrate members of the nuclear receptor NR5A subfamily, which includes steroidogenic factor 1 (SF-1) and liver receptor homolog 1 (LRH-1), regulate crucial aspects of development, endocrine homeostasis, and metabolism. Mouse LRH-1 is believed to be a ligand-independent transcription factor with a large and empty hydrophobic pocket. Here we present structural and biochemical data for three other NR5A members—mouse and human SF-1 and human LRH-1—which reveal that these receptors bind phosphatidyl inositol second messengers and that ligand binding is required for maximal activity. Evolutionary analysis of structure-function relationships across the SF-1/LRH-1 subfamily indicates that ligand binding is the ancestral state of NR5A receptors and was uniquely diminished or altered in the rodent LRH-1 lineage. We propose that phospholipids regulate gene expression by directly binding to NR5A nuclear receptors.

Published
2004

36. Structure of neurolysin reveals a deep channel that limits substrate access

Author

W. David Tolbert, Kevin P. Madauss, Wei Lian, David W. Rodgers, Moriah R. Beck, and C. Kent Brown

Subjects
Models, Molecular, Multidisciplinary, Binding Sites, biology, Chemistry, Stereochemistry, Active site, Substrate (chemistry), Metalloendopeptidases, Biological Sciences, Crystallography, X-Ray, Protein Structure, Tertiary, Substrate Specificity, Crystallography, Zinc metallopeptidase, Neurolysin, Cleave, Hydrolase, biology.protein, Binding site, Communication channel
Abstract

The zinc metallopeptidase neurolysin is shown by x-ray crystallography to have large structural elements erected over the active site region that allow substrate access only through a deep narrow channel. This architecture accounts for specialization of this neuropeptidase to small bioactive peptide substrates without bulky secondary and tertiary structures. In addition, modeling studies indicate that the length of a substrate N-terminal to the site of hydrolysis is restricted to approximately 10 residues by the limited size of the active site cavity. Some structural elements of neurolysin, including a five-stranded β-sheet and the two active site helices, are conserved with other metallopeptidases. The connecting loop regions of these elements, however, are much extended in neurolysin, and they, together with other open coil elements, line the active site cavity. These potentially flexible elements may account for the ability of the enzyme to cleave a variety of sequences.

Published
2001

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