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Mechanism, Function, and Inhibition of Peptide Deformylase

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MECHANISM, FUNCTION, AND INHIBITION OF PEPTIDE DEFORMYLASE DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Kiet T. Nguyen ***** The Ohio State University 2005 Dissertation Committee: Approved by Professor Dehua Pei, Adviser Professor Ross Dalbey Dehua Pei Professor Sean Taylor The Ohio State Biochemistry Graduate Program ABSTRACT Peptide deformylase (PDF) was originally thought as unique and essential only to the prokaryotes and apparently was absent from the eukaryotes. In this work, two peptide deformylase homologs from the eukaryotic Plasmodium falciparum (PfPDF) and Homo Sapiens (HsPDF) were cloned and characterized. Both proteins were found to be active and contained similar properties to the Escherichia coli peptide deformylase. Expression of the PfPDF protein was detected inside the Plasmodium cell, and potent PDF inhibitors were found to reduce cell growth. The HsPDF was fused with green fluorescence protein (GFP) and revealed mitochondrial localization. Human cell growth study was carried out and was unaffected by potent PDF inhibitors. This work provides evidence that PDF indeed exists in the eukaryotes. However, the role for deformylation in humans was apparently unnecessary, and this work may validate the support for a PDF drug target, as well as providing some preliminary evidence that PDF inhibitors are not toxic to human cells. The next project involved the identification of a novel class of macrocyclic PDF inhibitors, which tailored from the traditional PDF inhibitors in being acyclic and peptide-like in nature. This new class of PDF inhibitors exhibited potent inhibition and antibacterial properties, against both the Gram-negative and Gram-positive bacteria. The cyclization effect improved the PDF inhibition selectivity and biostability. Here, this ii work may provide a lead toward more effective PDF inhibitors more resistant to proteolysis and demonstrating improved specificity. The final part of this work involved the purification and identification enzymes degrading chemotactic f-Met-Leu-Phe peptide (fMLF) released from bacteria. This area of study encompasses the PDF drug effect presented by the commensal bacteria within the mammalian intestines. N-acylpeptide hydrolase and N-acylase 1A from rat small intestine mucosal layer were isolated and identified to degrade the fMLF chemopeptide and other N-formylmethionine peptides. The results may provide support for better understanding the host defense mechanism against the commensal bacteria and to identify the potential problems that may encounter as PDF makes an attractive novel therapeutic drug target. iii Dedicated to my parents and foster parents iv ACKNOWLEDGMENTS I wish to thank my adviser, Professor Dehua Pei, for his intellectual support, professional guidance, and for having patience with me over the years. I also thank him for the environment in which he had created for a suitable professional training. I am deeply thankful for my former and present lab members for their stimulating discussion and providing support on multiple levels in the lab, especially Dr. Kirk Beebe, Mike Sweeney, Grace Zhu, Junguk Park, and Dr. Xubo Hu. As friends and colleagues, they have motivated me and provided a special place to conduct my research, an environment that I embrace with a desire to work long hours and to be able to show up the next day with the same passion. Dr. Xubo Hu was exceptionally helpful in providing the majority of the PDF inhibitors. I am grateful and dedicated my life to my wife, Valerie, who during my years of study had believed in me and provided emotional support that could only stem from a loved one. Her understanding and sacrifice had tremendously driven me to succeed and provided a place for refuge whenever I need to resolve research frustrations. This research was supported by a grant from the National Institute of Health. v VITA 1997……………………………………...B.A. Biology, Wittenberg University 1997-1999……………………………….Consultant, Materials Directorate, Wright- Patterson Air Force Research Lab 2000-present……………………………. Graduate Teaching and Research Associate, The Ohio State University PUBLICATIONS 1. Nguyen, K.T., and Pei, D. Purification and Characterization of Enzymes Involved in the Degradation of Chemotactic N-Formyl Peptides. (2005) Biochemistry, submitted. 2. Lucas, R.W., Ingasson, B.P., Krumm, B.E., Hu, X., Nguyen, K.T., Verlinde, C.L.M.J., Pei, D., and Hol, W.G.J. Rational Drug Design of Polypeptide Deformylase Inhibitors in Hemophilus Influenzae: Species Variations in Drug Design. (2005) Protein Science, in preparation. 3. Ingasson, B.P., Lucas, RW., Krumm, B.E., Hu, X., Nguyen, K.T., Verlinde, C.L.M.J., Pei, D., and Hol, W.G.J. Structural Basis for the Inhibtion of Plasmodium Falciparum Deformylase by Macrocyclic peptidomimetics. (2005) Protein Science, in preparation. 4. Nguyen, K.T., Hu, X., and Pei, D. (2004) Slow-Binding Inhibition of Peptide Deformylase by Cyclic Peptidomimetics as Revealed by a New Spectrophotometric Assay. Bioorg. Chem. 32,178-191. 5. Robien, M.A, Nguyen, K.T, Kumar, A., Hirsh, I., Turley, S., Pei, D., and Hol, W. G. J. (2004) An Improved Crystal Form of Plasmodium falciparum Peptide Deformylase. Protein Science 13, 1155-1163. 6. Hu, X., Nguyen, K.T., Vernon, C.J., Lofland, D., Moser, H.E., and Pei, D. (2004) Macrocyclic Inhibitors for Peptide Deformylase: A Structure-Activity Relationship Study of the Ring Size. J. Med. Chem. 47, 4941-4949. vi 7. Nguyen, K.T., Hu, X., Colton, C., Chakrabarti, R., Zhu, M.X., and Pei, D. (2003) Characterization of a Human Peptide Deformylase: Implications for Antibacterial Drug Design. Biochemistry 42, 9952-9958. 8. Hu, X., Nguyen, K. T., Verlinde, C. L. M. J., Hol, W. G. J., and Pei, D. (2003) Structure-Based Design of a Macrocyclic Inhibitor for Peptide Deformylase. J. Med. Chem. 46, 3771-3774. 9. Deng, H., Callender, R., Zhu, J., Nguyen, K. T., and Pei, D. (2003) Determination of the Ionization State and Catalytic Function of Glu-133 in Peptide Deformylase by Difference FTIR Spectroscopy. Biochemistry 41, 10563-10569. 10. Kumar, A., Nguyen, K. T., Srivathsan, S., Ornstein, B., Turley, S., Hirsh, I., Pei, D., and Hol, W. G. J. (2002) Crystals of Peptide Deformylase from Plasmodium falciparum Reveal Critical Characteristics of the Active Site for Drug Design. Structure 10, 357-367. 11. Bracchi-Ricard, V., Nguyen, K. T., Zhou, Y., Rajagopalan, P. T. R., Chakrabarti, D., and Pei, D. (2001) Characterization of an Eukaryotic Peptide Deformylase from Plasmodium falciparum. Arch. Biochem. Biophys. 396, 162–170. 12. Su, W., Cooper, T. M., Nguyen, K. T., Brandt, M. C., Brandelik, D. M., and McLean, D. G. (1998) Structure-optical Property Relationships of Porphyrins. Proc. SPIE, 3472, 136-143. FIELDS OF STUDY Major Field: Ohio State Biochemistry Program vii TABLE OF CONTENTS P a g e Abstract................................................................................................................................ii Dedication...........................................................................................................................iv Acknowledgments....................................................................................................... ........v Vita......................................................................................................... ............................vi List of Tables.....................................................................................................................xii List of Figures...................................................................................................................xiii List of Abbreviations.................................................................. .......................................xv Chapters: 1. General Introduction................................................................................................1 1.1 Biological Function of Peptide deformylase............ ...................................1 1.2 Peptide Deformylase in Eukaryotes.............................................................3 1.3 Structure and Mechanism...................................................................... ......5 1.4 Peptide Deformylase as Novel Drug Target................................................8 2. PDF Assays............................................................................................................15 2.1 Introduction................................................................................................15 2.2 FDH-coupled Spectrophotometry Assay (Method A)............... ...............16 2.3 AAP-coupled Spectrophotometry Assay (Method B)......................... ......17 2.4 Direct-spectrophotometry Assay (Method C)........................................ ...18 2.5 Development of a New Spectrophotometric Assay (Method D)...............19 2.5.1 Analytical Methods........................................................................19 2.5.2 Continuous PDF Assay with DPPI................................................20 2.5.3 PDF Inhibition Assay.....................................................................21 2.6 Results and Discussion......................................................................... .....21 2.6.1 Assay Design............................................................ .....................21 2.6.2 Continuous DPPI Coupled Assay..................................................22 2.6.3 Application
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