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Elucidation of the Specificity of Neuroactive Steroids and Related Compounds at the at the Vesicular Glutamate Transporter

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Elucidation of the Specificity of Neuroactive Steroids and Related Compounds at the at the Vesicular Glutamate Transporter University of Montana ScholarWorks at University of Montana Graduate Student Theses, Dissertations, & Professional Papers Graduate School 2007 Elucidation of the Specificity of Neuroactive Steroids and Related Compounds at the at the Vesicular Glutamate Transporter Wesley Edward Smith The University of Montana Follow this and additional works at: https://scholarworks.umt.edu/etd Let us know how access to this document benefits ou.y Recommended Citation Smith, Wesley Edward, "Elucidation of the Specificity of Neuroactive Steroids and Related Compounds at the at the Vesicular Glutamate Transporter" (2007). Graduate Student Theses, Dissertations, & Professional Papers. 370. https://scholarworks.umt.edu/etd/370 This Dissertation is brought to you for free and open access by the Graduate School at ScholarWorks at University of Montana. It has been accepted for inclusion in Graduate Student Theses, Dissertations, & Professional Papers by an authorized administrator of ScholarWorks at University of Montana. For more information, please contact [email protected]. ELUCIDATION OF THE SPECIFICITY OF NEUROACTIVE STEROIDS AND RELATED COMPOUNDS AT THE VESICULAR GLUTAMATE TRANSPORTER By Wesley Edward Smith Bachelor of Science, Bridgewater State College, Bridgewater, MA, 2000 Dissertation presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy Biomedical Sciences The University of Montana Missoula, MT Spring 2007 Approved by: Dr. David A. Strobel, Dean Graduate School Dr. Richard J. Bridges, Co-chair Department of Biomedical and Pharmaceutical Sciences Dr. John M. Gerdes, Co-chair Department of Biomedical and Pharmaceutical Sciences Dr. Keith K. Parker Department of Biomedical and Pharmaceutical Sciences Dr. David J. Poulsen Department of Biomedical and Pharmaceutical Sciences Dr. Charles M. Thompson Department of Biomedical and Pharmaceutical Sciences Dr. Kent Sugden Department of Chemistry Smith, Wesley, Ph.D., January 2007 Biomedical Sciences Elucidation of the Specificity of Neuroactive Steroids and Related Compounds at the at the Vesicular Glutamate Transporter Chairperson: Dr. Richard J. Bridges As the primary excitatory amino acid, glutamate is essential to proper functioning of the mammalian CNS. Proper regulation of the synaptic release of glutamate, potentially regulated by synaptic vesicle content, is one of many critical aspects to normal excitatory functioning. In particular, the vesicular glutamate transporters (VGLUTs), which load synaptic vesicles with glutamate prior to presynaptic release of neurotransmitter, are distinct from that of the plasma membrane excitatory amino acid transporters (EAAT). The development of a library of compounds which selectively inhibit the uptake of [3H]-L- glutamate into the VGLUTs, has revealed importance of particular structural motifs. Among these structural motifs, one of the most important is that of the “embedded glutamate” which mimics the endogenous substrate of the transporter. With respect to potency, the substitution of a lipophilic moiety at the C6 position seems to be the most important to date, as illustrated by 5,6-napthyl quinoline dicarboxylic acid (5,6-QDC). The structure of this compound strongly resembles that of a steroid molecule. In light of recent research suggesting steroids act within the CNS in a non-genomic manner, this observation prompted the testing of a panel of steroid molecules at VGLUT. These compounds, known as “neuroactive steroids” have been shown to be synthesized, modified, and/or active within the brain. Research from our lab, as well as from the Thompson lab, shows that certain sulfated neuroactive steroids are potent inhibitors of [3H]-L-glutamate uptake into synaptic vesicles. This work identifies pregnenolone sulfate, along with 5,6-QDC, as competitive inhibitors of VGLUT (Ki values of 107 and 228 µM, respectively). These two molecules display specificity for VGLUT, with respect to other sites on the synaptic vesicle (i.e., electrochemical gradient), and among other vesicular neurotransmitter transporters (i.e., VGAT, VMAT). Two molecules, 5,6-QDC and Congo Red Fragment (CRF) were aligned to the VGLUT Pharmacophore to illustrate the SAR of these compounds. Biochemical studies have also been conducted to delineate substrate activity of neuroactive steroids and related compounds at VGLUT. The specificity of certain sulfated neuroactive steroids suggest that they could be endogenous regulators of vesicular glutamate uptake. ii ACKNOWLEDGEMENTS This work would not have been possible without the help of some very patient folks. I would like to acknowledge all of those people who helped bring this work to its final form. First and foremost, I would like to thank my research advisor Dr. Richard Bridges who served as both an advocate and inspiration on my behalf. Secondly, none of this would have come to fruition without the generosity of Dr. Vernon Grund, who gave me the opportunity to start this work. Thanks to Dr. John Gerdes, Dr. Charles Thompson, Dr. Keith Parker, Dr. David Poulsen, and Dr. Kent Sugden for advice regarding the development and culmination of this project. I also give special acknowledgement to those who helped in generating experimental data: Dr. Holly Cox, Dr. Kim Cybulski, and Dr. Erin Bolstad. To the members of the Bridges Lab, past and present, who never let a single moment be dull, I give special thanks: Shailesh Agarwal, Dr. Sarj Patel, Fred Rhoderick, Todd Seib, Dr. Brady Warren, and Ran Ye. I would like to thank my fellow graduate students, faculty, and staff of the Department of Biomedical and Pharmaceutical Sciences for the support and opportunities which were provided to me. I would also like to thank friends outside of the lab: JC Schneider, Rob and Jana Greenwell, and Glen Sundberg. I would especially like to thank my parents, Gary and Jane Smith, for supporting me throughout my academic career. Also, I extend my gratitude to my surrogate family in Missoula: Natalie, Dave, and Rose. Lastly, I give my utmost appreciation to the members of my personal lab: my wife, Tara, who was instrumental in keeping me focused, but also encouraging me to take a day off when necessary, and also, our two dogs, Hank and Quinn, for their enthusiasm when I just wanted one last run. iii TABLE OF CONTENTS ABSTRACT………………………………………………………………………...…......ii ACKNOWLEDGEMENTS…………………………………………………...………….iii LIST OF TABLES…….………………………………………………………………..…v LIST OF FIGURES...........….....……………………………………………..………..…vi CHAPTER 1: BACKGROUND AND SIGNIFICANCE…..……………………………1 CHAPTER 2: MATERIALS AND METHODS……………………..…………………21 CHAPTER 3: RESULTS Section I: Specificity of inhibition elicited by Sulfated neuroactive steroids, substituted QDCs, and derivatives of naphthalene disulfonic acids at VLGUT……………………………………………………………....…….27 Section II: Development of a VLGUT pharmacophore model and alignments with identified, competitive inhibitors………………...…………………………...37 Section III: Influence of neuroactive steroids and related compounds on 3H-L-glutamate efflux from synaptic vesicles………………………………………………………..…..…58 Section IV: Specificity of sulfated neuroactive steroids and related compounds at other sites on the synaptic vesicle…………………..……………………………….……69 Section V: Specificity of sulfated neuroactive steroids and related compounds at other vesicular transporters (i.e. VMAT, VGAT)…………………………………..79 CHAPTER 4: DISCUSSION………...…………………………………………...…….88 iv LIST OF TABLES Table 3.I.1. Inhibition of VGLUT by sulfated neuroactive steroids………………………………......……….....……31 Table 3.II.1. Ligands used in the generation and testing of the VGLUT Pharmacophore …….…………..….………………...………………..45 Table 3.V.1. Pharmacological specificity of neuroactive steroids……………………………………………….….……………83 Table 3.V.2. Pharmacological specificity of sulfated-neuroactive steroids as inhibitors of VGLUT, VMAT, and VGAT…………..…………....86 v LIST OF FIGURES Figure 1.1. Excitatory amino acid synapse…………………..…………………………….…………….…...2 Figure 1.2. Previously identified VGLUT inhibitors…………………………………………….………..................11 Figure 1.3. Commonalities of various VGLUT inhibitors………………………………………………….……….........15 Figure 1.4 Depiction of a steroid nucleus…………………………………………..17 Figure 1.5. Structures of inhibitory and excitatory neuroactive steroids……………….…………………………….............18 Figure 3.I.1. Concentration-response analysis of potent sulfated-neuroactive steroids at VGLUT…………………..…….…......30 Figure 3.I.2. Demonstration of the competitive inhibition by PREGS on 3H-L-Glutamate uptake into synaptic vesicles..…………………………………….………………...34 Figure 3.I.3. Demonstration of the competitive inhibition by 5,6-QDC on 3H-L-Glutamate uptake into synaptic vesicles…………....……………………………………...35 Figure 3.I.4. Structures of identified competitive inhibitors……..……………….….36 Figure 3.II.1. Illustration of the superpostition of CSB, BCT, and 6-(4’)-QDC………………...………………...…………….....40 Figure 3.II.2. Identified regions and measures of the VGLUT Pharmacophore………………………………………...............41 Figure 3.II.3. Alignment of VGLUT Pharmacophore with PREGS…...………………………….…...………………………..43 Figure 3.II.4. Diagram of CRF alignment with the VGLUT Pharmacophore (A1)……..………………..………….………48 Figure 3.II.5. Demonstration of pharmacophore alignment (A1) versus conformer number ……………………………...…………49 Figure 3.II.6. Alignment (A1) of VGLUT pharmacophore with CRF………..…………………………………………....................50 vi Figure 3.II.7. Diagram of CRF alignment with the VGLUT Pharmacophore (A2) …………...…….…………….........…...51 Figure 3.II.8. Demonstration of
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