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Understanding the Function of the Membrane

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Understanding the Function of the Membrane Departamento de Ciencias Biomédicas Área de Fisiología UNDERSTANDING THE FUNCTION OF THE MEMBRANE TRANSPORTER ABCG2 BY COMPARISON WITH P- GLYCOPROTEIN: INTERACTION WITH ANTITUMORALS, ANTIBIOTICS, HORMONES AND OTHER COMPOUNDS “ESTUDIO DE LA FUNCIÓN DEL TRANSPORTADOR DE MEMBRANA ABCG2 MEDIANTE COMPARACIÓN CON LA GLICOPROTEÍNA-P: INTERACCIÓN CON ANTITUMORALES, ANTIBIÓTICOS, HORMONAS Y OTROS COMPUESTOS” Memoria para la obtención del grado de Doctor Estefanía Egido de Frutos León, 2014 INFORME DE LAS DIRECTORAS DE LA TESIS (Art. 11.3 del R.D. 56/2005) La Dra. Dña. Gracia Merino Peláez y la Dra. Dña. Anna Seelig, como Directoras de la Tesis Doctoral titulada “Understanding the function of the membrane transporter ABCG2 by comparison with P-glycoprotein: interaction with antitumorals, antibiotics, hormones and other compounds” realizada por Dña. Estefanía Egido de Frutos en el programa de doctorado Biomedicina en el departamento de Ciencias Biomédicas, informan favorablemente el depósito de la misma, dado que reúne las condiciones necesarias para su presentación y defensa. Lo que firman para dar cumplimiento del artículo 11.3 del R.D. 56/2005, en León a___ de ____________de 2014. Fdo: Dra. Merino Peláez Fdo: Dra. Anna Seelig ADMISIÓN A TRÁMITE DEL DEPARTAMENTO (Art. 11.3 del R.D. 56/2005 y 7ª norma complementaria de la ULE) El órgano responsable del programa de doctorado en Biomedicina, en su reunión celebrada el día___de ___________ de 2014, ha acordado dar su conformidad a la admisión a trámite de lectura de la Tesis Doctoral titulada “Estudio de la función del transportador de membrana ABCG2 mediante comparación con la Glicoproteína-P: interacción con antitumorales, antibióticos, hormonas y otros compuestos“, dirigida por la Dra. Dña. Gracia Merino Peláez Fernández y la Dra. Dña. Anna Seelig, elaborada por Dña. Estefanía Egido de Frutos y cuyo título en inglés es “Understanding the function of the membrane transporter ABCG2 by comparison with P-glycoprotein: interaction with antitumorals, antibiotics, hormones and other compounds”. Lo que firman para dar cumplimiento del artículo 11.3 del R.D. 56/2005, en León a___ de ____________de 2014. El Director del Departamento, La Secretaria, Fdo: Juan José García Vieítez Fdo: Pilar Sánchez Collado Parte de la presente memoria será objeto de las siguientes: Publicaciones en preparación - Estefanía Egido, Anna Seelig and Gracia Merino. BCRP/ABCG2 interaction of the antibiotics pefloxacin, norfloxacin and moxifloxacin: ATPase activity correlation with other in vitro and in vivo experiments using fluoroquinolones. Manuscript in preparation. - Estefanía Egido, Rita Müller, Xiaochun Li-Blatter, Gracia Merino and Anna Seelig. The Breast Cancer Resistance Protein (ABCG2) and P-Glycoprotein (ABCB1) - Two Uneven Cousins yet with Related Skills. Manuscript in preparation. - Estefanía Egido, Rita Müller, Xiaochun Li-Blatter, Gracia Merino and Anna Seelig. ABCG2/BCRP and P-glycoprotein – hormone analysis of ATPase activity and transport. Manuscript in preparation. Parte de la presente memoria ha sido objeto de las siguientes: Comunicaciones a Congresos - Egido E, González-Lobato L, Barrera B , Real R , Prieto JG , Álvarez AI, Merino G “In vitro interaction of the fluoroquinolone Pefloxacin with Breast Cancer Resistance Protein (BCRP/ABCG2)”. 3rd FEBS Meeting: ATP-binding cassette (ABC) proteins: from multidrug resistance to genetic disease. Innsbruck, Austria 2010. - Egido E, Li-Blatter X, Müller R, Merino G, Seelig A. “P-Glycoprotein (MDR1, ABCB1) and Brest Cancer Resistance Protein (BCRP, ABCG2) – Two ABC Transporters with Different Substrate Specificities”. Biozentrum Symposium. Basilea, Suiza 2011. - Egido E, Müller R, Li-Blatter X, Merino G, Seelig A. “The function of the ATP binding cassette transporters P-glycoprotein and BCRP in hormone and peptide transport across the membrane”. 13th edition of the Naples Workshop on Bioactive Peptides “Conformation and Activity in Peptides: Relationships and Interactions”. Nápoles, Italia 2012. - Egido E, Müller R, Li-Blatter X, Merino G, Seelig A. “BCRP and P-glycoprotein – Predicting the transport rate on the basis of ATPase activity measurements”. 4th FEBS Meeting: ATP-binding cassette (ABC) proteins: from multidrug resistance to genetic disease. Innsbruck, Austria 2012. Esta Tesis Doctoral se ha desarrollado gracias a la concesión de una Ayuda para la “Contratación de Personal Investigador de Reciente Titulación Universitaria (PIRTU)” de la Consejería de Educación (Junta de Castilla y León) y del Fondo Social Europeo (2008-2012). Gran parte del desarrollo de la presente Tesis Doctoral se ha realizado en el centro de investigación internacional en el laboratorio de la Prof. Dr. Anna Seelig (Biophysical Chemistry Deparment del Biozentrum, University of Basel, Suiza), siendo beneficiaria de la ayudas para movilidad de estudiantes en 2011 de la convocatoria Orden EDU/2719/2011, Ministerio de Educación y de las ayudas para estancias breves en centros de investigación nacionales o extranjeros a realizar en 2010 de la convocatoria Orden EDU/2307/2009, Junta de Castilla y León. Para el desarrollo de algunas partes también se ha contado con la financiación de diversas entidades: - JUNTA DE CASTILLA Y LEÓN. Título del proyecto: “Evaluación de nuevos candidatos antitumorales: Estudios in vitro de transporte mediado por la BCRP y estudios in vivo de toxicidad y farmacocinética”. REF. SAN673/LE04/08. Duración: 01/01/2008-31/12/2008. Investigadora Principal: Dra. Gracia Merino Peláez. - INSTITUTO BIOMAR SA. Título del proyecto: “Estudio de la biodisposición y toxicidad de nuevos tratamientos antitumorales”. Duración: 2008-2012. Investigadora Principal: Dra. Gracia Merino Peláez. - SWISS NATIONAL SCIENCE FOUNDATION. REF. 3100AO-107793. La experiencia no es lo que sucede, si no lo que se hace con lo que sucede. (A. Huxley) GENERAL INDEX Page LIST OF FIGURES AND TABLES…………………………………………………………… … I LIST OF ABBREVIATIONS…………………………………………………………………………… IX INTRODUCTION……………………………………………………………………………………………… 1 BIBLIOGRAPHIC REVISION………………………………………………………………………… 7 1. Membrane transporters……………………………………………………………..… 9 2. ABC transporters………………………………………………………………………........ 11 2.1. Classification…………………………………………………………………………….. 11 2.2. Structure…………………………………………………………………………............ 12 2.3. Mechanism of action………………………………………………………………... 13 2.4. Distribution, function and implications………………………………………. 15 2.4.1. Implications in organism protection and drug disposition (ADME)……………………………………………………………......................... 16 2.4.2. The MultiDrug Resistance (MDR) phenomenon: use of ABC transporters inhibitors…………………………………………………………….…. 17 2.4.3. Other implications in cancer………………………………………………………... 21 2.4.4. Mutations and diseases……………………………………………………………... 21 2.4.5. Other physiological functions………………………………………………………. 22 2.5. Influence in drug development: approaches to identify new drugs………………………………………………………………….... 23 2.6. Perspectives…………………………………………………………………………..… 23 3. BCRP/ABCG2…………………………………………………………………………………….. 25 3.1. Discovery……………………………………………………………………………….... 25 3.1.1. ABCG family classification context………………………………….. 25 3.2. Structure, membrane topology and mechanism of transport………………………………………………………………………………….. 27 3.3. Regulation of ABCG2 expression……………………………………………… 32 3.4. Distribution……………………………………………………………………………… 33 3.5. Function and implications............................................................. 35 3.5.1. Protection limiting substrate penetration and tissue distribution……………………………………………………………………………… 35 3.5.2. Clinical implications………………………………………………………………….. 39 3.5.2.1. In ADME…………………………………………………………………. 39 3.5.2.2. In Cancer and MDR…………………………………………………... 40 3.5.2.3. In polymorphisms and mutants……………………………………. 42 3.5.2.4. In others………………………………………………………………… 43 3.6. Interaction with compounds…………………………………………………… 44 3.6.1. Substrates and inhibitors…………………………………………………………… 44 3.6.2. Chemical properties of the compounds……………………………………….. 49 3.6.3. Structure-activity relationships (SARs)………………………………………….. 50 4. P-glycoprotein………………………………………………………………………………. 52 4.1. Discovery……………………………………………………………………………..… 52 4.2. Structure, membrane topology and mechanism of transport………………………………………………………………………………… 52 4.3. Distribution………………………………………………………………………….… 57 4.4. Function and implications............................................................ 58 4.4.1. Protection limiting substrate penetration and tissue distribution…………………………………………………………………………... 58 4.4.2. Clinical implications……………………………………………………………………. 58 4.4.2.1. In ADME…………………………………………………………………… 58 4.4.2.2. In Cancer and MDR…………………………………………………….. 59 4.4.2.3. In polymorphisms and mutants……………………………………... 60 4.5. Interaction with compounds…………………………………………………….… 60 4.5.1. Substrates and inhibitors…………………………………………………………….… 60 4.5.2. Chemical properties of the compounds……………………………………………. 62 4.5.3. Structure-activity relationships (SARs)……………………………………………… 63 5. Important compounds for this study……………………………………………. 64 5.1. Steroid hormones……………………………………………………………………….. 64 5.1.1. Steroid hormones used in this study………………………………………………… 64 5.1.2. Interaction of steroid hormones with ABCG2 and P-gp…………………………. 66 5.2. Fluoroquinolones………………………………………………………………………… 71 5.2.1. Fluoroquinolones used in this study………………………………………………… 73 5.2.2. Interaction of fluoroquinolones with ABCG2 and P-gp…………………………. 75 6. Methodological bases for the study of drug interactions with ABC transporters: advantages and disadvantages…………………………………………………………………………………….
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