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  • Do the binding sites of substrates and tricyclic antidepressants overlap on the

    human serotonin transporter?

    Doctoral thesis at the Medical University of Vienna in partial fulfillment for the degree of

    Doctor of Philosophy (PhD)

    Submitted by

    Subhodeep Sarker

    Supervisor:

    Univ. Prof. Dr. Michael Freissmuth, MD Professor and Chairman Institute of Pharmacology

    Center for Physiology and Pharmacology Medical University of Vienna

    Waehringer Strasse 13a A-1090 Vienna, Austria

    Vienna, August 2010

  • With utmost reverence and humility within, I dedicate this sojourn to my Guru, Misha and dearest friend Sonja, who symbolize to me the quintessential epitome of human

    values

    1

  • ACKNOWLEDGEMENTS

    Albeit having inherited the oldest living tradition on Earth, with roots reaching

    back into prehistory, one of its very few customs I follow blindfolded, without ever

    questioning its very rational basis, is that of the ancient Gurukul, wherein ones teacher is

    held Supreme in all conceivable realms of conscious existence.

    Without the unstinted faith of my Guru, Prof. Michael Freissmuth, I may never

    have walked the conglomerate steps I did during the course of this thesis. He inspired me

    every moment I breathed during this sojourn. I shall forever be grateful to him for

    providing me with the necessary leverage I needed to come out of my shy, inhibited and

    reclusive nature and for setting to me a benchmark for self-accomplishment. The doors to

    his mind and magnanimous heart were perennially left open to me and he was never

    imposing a lesson, to which I will fall back upon for inspiration to leave with posterity

    to ponder upon. Perhaps his greatest legacy that I will carry within is the knack of asking

    questions - questions perennially reframed to testable hypotheses and amenably

    addressed through scientific rigor. He leaves the indelible impression of a great man

    within.

    With equal sentiment, I am inexplicably indebted for life, to Dr. Sonja Sucic. Her

    inimitable selflessness, contagious enthusiasm and honest passion for her life and work

    inspired me to emulate her ethic and incorporate in my own. Her steadfast friendship and

    contribution to every conceivable aspect of my life crystallized to be the definitive

    inspiration towards fruitful execution of my work and in completion of this thesis. The

    joy of discovery and experimentation was demonstrated to me by my parents and Guru

    but it is what I cherished exploring most, with Dr. Sucic. I, especially am grateful to her

    for having refurbished, with a pinch of satire, my very perception towards the ironical

    vagaries of life. She commands the most profound influence on my philosophical

    underpinnings in the way I approach life today.

    2

  • Besides, I am grateful to Prof. Harald H. Sitte and his group who helped expand

    my work to include a more holistic understanding of monoamine transporter inhibition. I

    also express my gratitude to Prof. Gerhard F. Ecker and his graduate student Mag. Ren

    Weissensteiner, for their insights from computational biology enabled me to generate

    hypotheses towards designing experiments in an iterative approach. I wish to congratulate

    all my colleagues in the laboratory for providing me with a productive atmosphere

    necessary to work in an efficient manner. Especially, Mr. Ali El-Kasaby with whom I

    enjoyed my humble origins in the laboratory and developed a formidable friendship to

    last for a lifetime. I am thankful to Mrs. Ludmilla Hertting and late Mrs. Alice Zafaurek

    for their kind support with administrative affairs and extend my gratitudes to Prof.

    Werner Sieghart (Medical University of Vienna, Austria), Prof. Stefan Bhm (Medical

    University of Vienna, Austria), Prof. Nicholas Singewald (Medical University of

    Innsbruck, Austria), Prof. Cord-Michael Becker (University of Erlangen-Nrnberg,

    Germany) and Prof. Menahem Segal (The Weizmann Institute of Science, Rehovot,

    Israel), for their critique and encouragement served to maintain direction during my

    research.

    In addition, I humbly acknowledge the financial support received for my research

    from the Austrian Science Fund (FWF) and the Medical University of Vienna in the Cell

    Communication in Health and Disease (CCHD) Doctoral Program.

    Finally, my endearing set of parents, is the very reason I ever had the opportunity

    to walk in pursuit of my childhood fascination to discover the world I inherited in a

    dedicated endeavor. Their steadfast friendship, love, affection, belief and towering

    inspiration helped me to withstand rigors in between. I am grateful to life for letting me

    know my parents closely!

    Shubho

    August 6, 2010

    Vienna, Austria

    3

  • ORIGINAL PUBLICATIONS

    Sarker, S., Weissensteiner, R., Steiner, I., Sitte, HH., Ecker, GF., Freissmuth, M., Sucic,

    S. (2010) The high-affinity binding site for tricyclic antidepressants resides in the outer

    vestibule of the serotonin transporter (in revision).

    MEETING ABSTRACTS

    Sarker, S., Steiner, I. (2008) Do the binding sites of substrates and tricyclic

    antidepressants overlap on the human serotonin transporter? BMC Pharmacol.8

    (Suppl.):A9. 14th Scientific Symposium of the Austrian Pharmacological Society

    (APHAR), Innsbruck, Austria, 21-22 November, 2008.

    4

  • To: Michael Freissmuth

    From: molpharm@aspet.org

    ReplyTo: Richard Kim , molpharm@aspet.org

    Subject: MOLPHARM/2010/067538-Revision Invitation

    Cc: Richard Kim

    RE: MOLPHARM/2010/067538

    Dear Dr. Freissmuth:

    Your manuscript entitled "THE HIGH-AFFINITY BINDING SITE FOR TRICYCLIC

    ANTIDEPRESSANTS RESIDES IN THE OUTER VESTIBULE OF THE SEROTONIN

    TRANSPORTER" has been reviewed by two experts familiar with this field. Please visit

    http://submit-molpharm.aspetjournals.org to retrieve their comments and the revision

    checklist. We would be willing to consider a revised manuscript; however, it will have to

    address all the concerns of the reviewers. Please be aware that the revised manuscript

    will be sent back to the original reviewers and is not assured of acceptance. When

    uploading the revised version, please be sure to include in the Response to Reviewers box

    an itemized list of all changes made, or your rebuttal, in response to each of the readers'

    suggestions. If this submission is accepted, the final PDF will be posted to Molecular

    Pharmacology website as the published article while your paper is in press. DO NOT

    NEGLECT to retrieve the Color Authorization form and the Revision Checklist, using the

    REQUIRED DOCUMENTS link in your "Manuscripts with Decisions in Past 12

    Months" area, to correct any publishing format deficiencies pertaining to your specific

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    analysis was run on your previous submission; this reporthttp://submit-

    molpharm.aspetjournals.org/cgi/information?type=deau&msid=MOLPHARM/2010/0675

    5

  • 38 will identify any problems affecting the production of your figures. Note that the

    revised text must be uploaded within the next 60 days. A manuscript received after that

    time will be treated as a new submission; in that case, the revised manuscript will need to

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    MANUSCRIPTS THAT DO NOT INCORPORATE CHANGES OUTLINED IN THE

    "REVISION CHECKLIST" WILL NOT BE FORWARDED TO THE PRINTER.

    Thank you for submitting your work to Molecular Pharmacology.

    Sincerely,

    Richard B. Kim M.D.

    Associate Editor

    Molecular Pharmacology

    ASPET

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    Bethesda, MD. 20814-3995

    Phone:301-634-7067 (Erin Salb) 301-634-7978 (Jill Filler)

    Fax:301-634-7158

    molpharm@aspet.org

    6

    mailto:molpharm@aspet.org

  • Reviewer 1 Comments for the Author...

    This manuscript presents the results of an investigation into the interaction of tricyclic

    antidepressants (TCAs) with the serotonin transporter (SERT). Crystallographic analysis

    of the bacterial LeuT homolog identified specific binding sites for TCAs, and some

    authors have postulated that the same regions and types of interactions apply to SERTs.

    However, while TCAs are competitive inhibitors of SERTs, they are low-affinity,

    noncompetitive inhibitors of LeuT, suggesting a different type and site of interaction. In

    this work Sarker et al combine molecular modeling with functional measurements

    (including mutagenesis) to provide a working model for the TCA binding site of SERT.

    The work is carefully done, and the results support the conclusions. However, there are

    several aspects of the work that need expansion, as detailed below.

    p. 7: We also verified that the model was consistent with the results from previous

    mutagenesis studies.. Please elaborate on what the criteria were and how th

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