2012-01-Thesis Final
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The Pennsylvania State University The Graduate School College of Medicine LIGAND-DIRECTED FUNCTIONAL SELECTIVITY AT THE OPIOID RECEPTOR FAMILY: AN EPIC APPROACH TO UNDERSTANDING OPIOID RECEPTOR SIGNALING A Dissertation in Neuroscience by Megan Morse Copyright 2012 Megan Morse Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy May 2012 The Dissertation of Megan Morse was reviewed and approved* by the following: Robert G. Levenson Professor of Pharmacology Co-director, MD/PhD Program Dissertation Advisor Chair of Committee Patricia S. Grigson Professor of Neural and Behavioral Sciences Co-Chair graduate program in Neuroscience Victor Ruiz-Velasco Associate Professor of Anesthesiology Kevin Alloway Professor of Neural and Behavioral Sciences *Signatures are on file in the Graduate School i Abstract Opioid receptors are G-protein coupled receptors (GPCRs) that are activated by opioid ligands. These ligands offer powerful medical benefits due to their analgesic properties, but activation of opioid receptors can also lead to many negative side effects, including tolerance and dependence. Conventional theory ascribes most of the analgesic and addictive effects of opioids to the activation of the mu opioid receptor subtype (MOR). However, it has recently been suggested that the other classic opioid receptors, the delta opioid receptor (DOR) and the kappa opioid receptor (KOR), may also contribute important functionality. We hypothesize that all of the classic opioid receptors play a vital role in understanding the full functionality of the opioid signaling system. Directed drug design is the future of therapeutic development, and in order to be capable of creating targeted opioid analgesics, we must be able to break down the signaling pathway. Using dynamic mass redistribution (DMR) assays and biosensor technology, we characterized all three classic opioid receptors using a library of known opioid ligands. We also studied the roles of cellular context in opioid receptor signaling by characterizing the endogenous population of opioid receptors found in SH-SY5Y neuroblastoma cells. Utilizing 13 different assay formats, this technology allowed us to examine receptor specificity, G-protein coupling, and downstream pathway selectivity. It has been suggested in the literature that ligand-directed functional selectivity provides a sufficient basis for understanding GPCR activity and molding the future of drug design. We hypothesize that the use of biosensor high throughput technology presents a viable opportunity to fully decipher the complexities of the opioid signaling cascade. ii Table of Contents List of Figures………………………………………………………………………...iv List of Tables……………………………………………………………………….....v List of Abbreviations…………………………………………………………………vi Chapter 1: Literature Review……………………………………………………….1 Opioid Drugs…………………………………………………………………..1 Endogenous Opioids………………………….……………………………….3 Opioid Receptor Structure and Function……………………………………...4 MOR…………………………………………………………………………..8 KOR……………………………………………………………….…………..9 DOR…………………………………………………………………………..10 ORL-1…………………………….…………………………………………..11 Cellular and Anatomical Distribution………………………………………...11 G-protein Coupling and Signaling…………………………………………....12 Opioid Receptor Specificity and Functional Selectivity…………………..….14 Receptor Internalization: First Implication for Functional Selectivity…….....15 Opioid Receptor Dimerization………………………………………..………19 Introduction to Biosensors………………………………………………..…..21 Theory behind Resonance Biosensors…………………………………….….22 Dynamic Mass Redistribution…………………………………………….….23 Biosensors: A revolutionary way to study the opioid receptors? ………..…..25 Rationale and Hypothesis………………………………………………..…...26 Chapter 2: Ligand-directed functional selectivity at the mu opioid receptor revealed by label-free integrative pharmacology on-target…………...…28 Introduction……………………………………………………………...…...28 Experimental Procedures………………………………………………….....30 Results…………………………………………………………………......…39 Discussion…………………………………………………………….…...…56 Chapter 3: Label-free integrative pharmacology on-target of opioid ligands at the opioid receptor family …………………………….…….….59 Introduction………………………………………………………………......59 Experimental Procedures………………………………………………...…..60 Results…………………………………………………………………....…..64 Discussion………………………………………………………………..…..85 Chapter 4: Closing Discussion…………………………………………………......89 References…………………………………………………………………………...103 Appendix…………………………………………………………………………….116 iii List of Figures 1.1: Structure Homology of classic opioid receptors…………………………...…………..…6 1.2: Schematic overview of genetic knockdown……………………………...…………….....8 1.3: Anatomical distribution of opioid receptors in the rodent brain………......……………..12 1.4: 25 years of understanding in GPCR advancements…………..……………………...…..15 1.5: GPCR signaling: G-protein vs β-arrestin pathways………………………………..….....16 1.6: Principles of two types of label-free biosensors…………………………………..…......22 1.7: Principles of DMR……………………………………………………….…………........24 2.1: Dose dependent DMR responses of ligands in HEK-MOR cells……..………………....40 2.2: Blockage of MOR agonist-induced DMR by naloxone…………………..…………..…42 2.3: Numerical descriptor of opioid ligand pharmacology………………………………..….43 2.4: False colored heat map of 42 opioid ligands on HEK-293 and HEK-MOR……….........45 2.5: The DMR characteristics of BNTX and 0.1% DMSO………………………………..…47 2.6: DMR response of levallorphan, β-funaltrexamine, and naltrindole………….……….....47 2.7: ICI 199,441 induced DMR responses…………………………………….………….….50 2.8: Comparison on DMR and cAMP responses induced by ligands………………….….…51 2.9: Sensitivity of opioid ligand-induced DMR to PTx…………………….………….…….52 2.10: Sensitivity of opioid ligand-induced DMR to CTx and Fsk………………………..….53 2.11: Comparison of immediate responses of buffer and inhibitor pretreated HEK-MOR cells……………………………………………………….……….……54 2.12: Comparison of 30 min poststimulation responses of buffer and inhibitor pretreated HEK-MOR cells……………………………….………….……55 3.1: Extracting DMR parameters for effective similarity analysis…………………….…..…65 3.2: A false colored heat map of DMRs of opioid ligands in five cell lines……………...….68 3.3: Dose-dependent responses of agonists in distinct cell lines…………………..................70 3.4: A false colored heat map based on the selectivity of opioid ligands to the opioid receptor family…………………………………………………...…....71 3.5: The inhibition pattern by opioid ligands………………………………………………...74 3.6: A false colored heat map of functional selectivity of opioid ligands at the DOR…….....75 3.7: A false colored heat map of functional selectivity of opioid ligands at the KOR…….....79 3.8: A false colored heat map of functional selectivity of opioid ligands at the endogenous receptors in SH-SY5Y………………………………………….…...….81 3.9: Dose responses of a panel of opioid ligands in HEK-DOR cells…………….....…….…82 3.10: Dose responses of a panel of opioid ligands in HEK-KOR cells…………….….....…..83 3.11: Dose responses of a panel of opioid ligands in SH-SY5Y cells………………........…..85 Appendix A: DMR responses of ligands with activity in parental HEK293 cells…………..116 iv List of Tables 2.1: Assay layout for MOR cells……………………………………………………..………38 3.1: Assay design for all cells………………………………………………………......…….63 Appendix B…………………………………………………………………………………..117 Appendix C…………………………………………………………………………………..119 Appendix D…………………………………………………………………………………..121 v List of Common Abbreviations GPCR G-protein coupled receptor DOR delta-opioid receptor KOR kappa opioid receptor MOR mu opioid receptor ORL1 opioid receptor-like 1 DMR dynamic mass redistribution P-DMR positive DMR N-DMR negative DMR iPOT label-free integrative pharmacology on-target HEK human embryonic kidney CTx cholera toxin PTx pertussis toxin TCT tissue culture treated DMSO dimethyl sulfoxide DMEM Dulbecco’s modified Eagle’s medium HBSS Hank’s Balanced Salt Solution HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid KO Knockout CNS Central Nervous System cAMP cyclic adenosine monophosphate vi Chapter 1 Literature Review Opioid Drugs Opioids are powerful compounds that have numerous, unparalleled medicinal benefits. These compounds can be endogenous or exogenous, natural or derived. In clinical settings, opioid-based drugs are currently the best choice for the treatment of severe, chronic, and unremitting pain. Unfortunately, alongside the medicinal benefits of opioids are a number of side effects caused by their use and misuse. These side effects, which can by physical or behavioral, are often as mild as constipation and nausea, but have the potential to be as severe as tolerance, withdrawal, and ultimately addiction (Neve, 2009; Kieffer & Evans, 2002; Palos et al., 2004; Stein et al., 2003). With this in mind, the current direction of opioid research is focused on separating the beneficial functions of opioids from their detrimental counterparts. (Kieffer & Evans, 2002; Corbett et al., 2006). This ‘holy grail’ of addiction research has thus far been elusive, but a growing body of knowledge regarding opioids and their activity is beginning to open the door on a potential breakthrough in drug discovery. The medicinal properties of opioids have been known and applied for over 5000 years. The early opioids were derived from the poppy plant, Papaver somniferum, to relieve pain and produce euphoria (Waldhoer et al., 2004). Currently, morphine is the most common medicinally used opioid derivative. First isolated in 1806 from opium alkaloids, the drug was named for Morpheus, the Greek god of dreams. In the