CHARACTERIZATION OF INHIBITORS OF ALDEHYDE DEHYDROGENASE IDENTIFIED THROUGH A HIGH-THROUGHPUT DOCKING APPROACH Yvelina Tsvetanova Ivanova Submitted to the faculty of the University Graduate School in partial fulfillment of the requirements for the degree Master of Science in the Department of Biochemistry and Molecular Biology, Indiana University May 2013 Accepted by the Faculty of Indiana University, in partial fulfillment of the requirements for the degree of Master of Science Thomas D Hurley, Ph.D., Chair Mark G Goebl, Ph.D Master’s Thesis Committee _ Edward F Srour, Ph.D ii Dedication This is dedicated to my husband, and my parents… “When you nothing, you feel overwhelmed and powerless But when you get involved, you feel the sense of hope and accomplishment that comes from knowing you are working to make things better.” - Ralph Waldo Emerson iii Acknowledgments ‘Equipped with his five senses, man explores the universe around him and calls the adventure Science.’’ ~Edwin Powell Hubble, The Nature of Science, 1954 I would like to thank Dr Tom Hurley for his continuous mentorship, understanding, patience and confidence in me Thank you for the opportunity to be part of your group and be able to work on one of your research projects I would also like to thank all the PhD students in Dr Hurley’s lab for the continuous encouragement and help I am very privileged to have brilliant mentors guiding me and ready to let me disrupt their work with questions Finally, I would like to thank all the members of the different labs where I fulfilled my project gratefully to the instrumentation that they shared with me iv Table of Contents List of Tables vi List of Figures vii Abbreviations viii Introduction Materials and Methods 16 Results and Discussion 20 Tables 26 Figures 30 References 40 Curriculum Vitae v List of Tables Table The ALDH Superfamily of Enzymes Table Known inhibitors of Aldehyde Dehydrogenases Table Comparison between parent inhibitors #32, 72 and 78 to different structural analogs vi List of Figures Fig ALDH2 3D structure with bound Alda-1(gray atoms) Fig Aldehyde Oxidation and Esterase Hydrolysis Fig Surface representations of the substrate binding topologies of the three human ALDH isoenzymes Fig Predicted binding mode for Compound I72 bound within the substrate binding channel of ALDH2 Fig Structures of the inhibitors identified as possible hits from the computational screening Fig IC50 curves for Inhibitors I32, I72, I74 and I78 on the dehydrogenase activity of ALDH2 Fig IC50 curves for Inhibitors I32, I72, I74, I76 and I78 on the esterase activity for ALDH2 Fig Competition for the NAD-catalyzed activation of the esterase activity for ALDH2 by I72, I74, I76, and I78 Fig Ultraviolet/Visible spectral scans for compounds I72, I76, and I78 Fig 10 The active site of ALDH2 with bound Alda-1 and daidzin superimposed in the substrate-binding channel vii Abbreviations ALDH2 aldehyde dehydrogenase ALDH aldehyde dehydrogenase hALDH2 human aldehyde dehydrogenase RA retinoic acid DEAB 4-Diethylamino benzaldehyde ACP 1-amino cyclopropanol DBH dopamine -hydroxylase 4HNE 4-hydroxynonenal viii Introduction Aldehyde dehydrogenases (ALDH) are a family of seventeen homologous enzymes that catalyze the oxidation of an assortment of endogenous and exogenous aldehydes (1) ALDHs serve as enzymes intended to protect the cells from the damaging effects of these highly reactive compounds Constituents of the aldehyde dehydrogenase (ALDH) superfamily catalyze the oxidation of cytotoxic aldehydes by using NAD(P)+ as the electron acceptor (2, 3) ALDH family members participate in the metabolism of amino acids, steroids, biogenic amines, lipids and vitamins as well as in ethanol, retinol, dopamine and GABA metabolism (4) Table represents the ALDH gene superfamily The gene nomenclature is based on sequence identity within the superfamily such that enzymes sharing less than 40% sequence identity are classified in separate families and those sharing greater than 60% sequence identity constitute distinct subfamilies (28) For instance, members of the ALDH1 family share less than 30% sequence identity with the ALDH3 family, whereas ALDH1A1, ALDH1A2 and ALDH1A3 share approximately 75% sequence identity The only exception to this nomenclature system is ALDH2, which by sequence identity belongs in the ALDH1 family (approximately 68% to ALDH1A1), but the extensive publication record on ALDH2 and its role in ethanol metabolism, made it simpler to retain its original gene designation Recent work has shown that specific ALDH isoenzymes can contribute to the underlying pathology of different diseases Many ALDH isozymes are important in oxidizing reactive aldehydes resulting from lipid peroxidation, and, thus, help maintain cellular homeostasis Increased expression and activity of ALDH isozymes are found in many human cancers and are often associated with poor prognosis (95, 96) Therefore, the development of inhibitors of the different ALDH enzymes is of interest as means to treat some of these disease states The three most extensively studied isoenzymes are ALDH1A1, ALDH2 and ALDH3A1 Each of the isoenzymes displays individual physical and enzymatic properties, such as quarternary structure and substrate specificity The closest homolog of human ALDH2 is human ALDH1B1 with 72% sequence identity, but with less than 60% sequence identity within the substrate binding site (104) In addition to ALDH1B1, the human ALDH1 family also contains the ALDH1A1, ALDH1A2 and ALDH1A3 subfamilies These ALDH1A isozymes are the primary isoenzymes that synthesize Retinoic Acid (RA) from retinal and it is critical in regulating RA signaling and as a consequence is strongly implicated in embryogenesis and development The ALDH1A1 gene product is a cytosolic homo-tetramer (~55 kDa subunits) that is expressed in brain, cornea, lens, retina, Esterase Assay - Inhibitor # 32 100 % activity 80 60 40 20 0.1 Esterase Assay - Inhibitor # 72 10 conc Esterase Assay - Inhibitr #74 100 100 80 60 % activity % activity 80 60 40 40 20 20 0 0.1 10 100 0.1 10 conc 100 1000 conc Esterase Assay - Inhibitor # 76 Esterase Assay - Inhibitor # 78 100 100 80 60 % activity % activity 80 40 20 60 40 20 0 0.1 10 100 0.1 conc 10 100 conc Fig IC50 curves for Inhibitors I32, I72, I74, I76 and I78 on the esterase activity for ALDH2 Note the relative lack of potency for these compounds 36 Inhibitor # 74 Inhibitor # 72 2.5 2.5 1.5 50uM inhib 20uM counts counts 1.5 50uM inhib 20uM 10uM 10uM 5uM 5uM 0.5 0.5 0 0.1 0.2 0.3 0.4 NAD concentration 0.5 0.6 0.1 Inhibitor # 76 0.5 0.6 Inhibitor # 78 1.4 1.6 1.2 1.4 1.2 0.8 50uM inhib 0.6 20uM 10uM 0.4 5uM 0.2 counts counts 0.2 0.3 0.4 NAD concentration 50uM inhib 0.8 20uM 0.6 10uM 0.4 5uM 0.2 0 0.1 0.2 0.3 0.4 NAD concentration 0.5 0.6 0.1 0.2 0.3 0.4 NAD concentration 0.5 0.6 Fig Competition for the NAD-catalyzed activation of the esterase activity for ALDH2 by I72, I74, I76, and I78 Note that the extent of activation is decreased upon increasing concentrations of inhibitors 74 and 76 37 I76 Spectra I72 Spectra Absorbance Absorbance of Nitrophenol 80000 35000 Absorbance 70000 Absorbance of NADH 30000 60000 25000 Intensity (U) 15000 Absorbance of NADH 10000 40000 30000 Absorbance of Nitrophenol 20000 5000 10000 240 340 440 540 Wavelength (nm) 640 740 240 340 440 540 Wavelength (nm) 640 740 I78 Spectra Absorbance 50000 Absorbance of Nitrophenol 45000 40000 Absorbance of NADH 35000 Intensity (U) Intensity (U) 50000 20000 30000 25000 20000 15000 10000 5000 240 340 440 540 Wavelength (nm) 640 740 Fig Ultraviolet/Visible spectral scans for compounds I72, I76 and I78 38 Fig 10 The active site of ALDH2 with bound Alda-1 (reference, yellow sticks) and daidzin (reference, dark grey sticks) superimposed in the substrate-binding channel The structure displayed was generated using PyMoL and the surface displayed represents the accessible surface of the substrate site Atom-type coloring is used to 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S., Hurley T Discovery of novel regulators of aldehyde dehydrogenase isoenzymes Chem Biol Inter 191(1-3), 153-8, 2011 48 Curriculum Vitae Yvelina Tsvetanova Ivanova Education Master of Science in Biochemistry and Molecular Biology Indiana University, Indianapolis, IN May 2013 Graduate Certificate in Biotechnology Indiana University, Indianapolis, IN December 2009 Bachelor of Science in Biotechnology Purdue University, Indianapolis, IN August 2007 Work Experience Scientist Eli Lilly and Company – Indianapolis, IN – Sep 2007 to Present Laboratory Technician Indiana University School of Medicine – Indianapolis, IN – Sep 2005 to Sep 2007 Lab Experience and Job Responsibilities Research Experience Scientist: Eli Lilly and Company, 2007-Present Research related to immunogenicity and special biomarkers Developed and characterized biomarker assays to support clinical drug development Thesis Research: Biochemistry Department, Indiana University School of Medicine, 2010-2012 (Research advisor: Dr Thomas Hurley) Developed a high-throughput screening assay to identify a number of unique compounds that function as inhibitors of ALDH2 Laboratory Technician: Biochemistry Department, Indiana University School of Medicine, 2005-2007 (Supervisor: Dr William Sullivan) Dr Sullivan’s laboratory studies epigenetics and the regulation of gene expression in protozoan parasites My research was on Toxoplasma gondii and mapping the nuclear localization signal of GCN5 B – HAT I achieved proficiency in experimental design, statistical data analysis, and technical writing skills Technology Statistical analysis tools including GraphPad Prism 4.0 and Microsoft Excel Internet databases Utilization of internet resources Professional Strengths and Responsibilities Experience in the field of proteomics Experience in mentoring team members and working with CRO’s, while ensuring timely delivery of results Ability to handle multiple projects and meet deadlines Good communication skills and strong scientific writing Fluent in French and Bulgarian Activities and Volunteering Translator – Indy Translators – Indianapolis, IN – 2010 to Present Volunteer –The United Ways – Indianapolis, IN – 2011 Professional Training and Seminars AAPS American Association of Pharmaceutical Scientists – attended days Immunogenicity training – Seattle, WA 9th Annual Bioassays and Bioanalytical Method Development – attended days Bioassays method development training – Berkeley, CA Publications Parajuli B., Kimble-Hill A., Khanna M., Ivanova I., Meroueh S., Hurley T Discovery of novel regulators of aldehyde dehydrogenase isoenzymes Chem Biol Inter 191, 153-158, 2011 Sloan S., Siegel R., Ivanova-Cox Y., Watson D., Deeg M., Konrad R A novel high-sensitivity electrochemiluminescence (ECL) sandwich immunoassay for the specific quantitative measurement of plasma glucagon Clin Biochem 4, 8-241, 2012 ... ALDH 8A1 ALDH 6A1 9q21.13 15q 22. 1 15q26.3 9p11.1 12q24 .2 3q21 .2 2q23.3 1q23.1 6p 22. 2 6q23 .2 14q24.3 ALDH 3A1 ALDH 3A2 ALDH3B1 ALDH3B2 ALDH1 8A1 ALDH1 6A1 17p11 .2 17p11 .2 11q13 .2 11q13 .2 10q24.3 19q13.33... database (114, Adapted from Strickland et al., 20 11) Gene Name ALDH 4A1 ALDH 7A1 Chromosome location 1p36.13 5q31 ALDH 1A1 ALDH 1A2 ALDH 1A3 ALDH1B1 ALDH2 ALDH1L1 ALDH1L2 ALDH 9A1 ALDH 5A1 ALDH 8A1 ALDH 6A1 ... substrate-binding channel vii Abbreviations ALDH2 aldehyde dehydrogenase ALDH aldehyde dehydrogenase hALDH2 human aldehyde dehydrogenase RA retinoic acid DEAB 4-Diethylamino benzaldehyde ACP 1-amino