Molekulare Charakterisierung der Liganden spezifischen Spannungsabhängigkeit der Opioid-Rezeptoren

G-Protein gekoppelte Rezeptoren sind einer der wichtigsten Angriffspunkte in der pharmakologischen Therapie. Hierbei stellen die Opioid-Rezeptoren, im speziellen der µ Opioid-Rezeptor, den wichtigsten Angriffspunkt in der Therapie von starken Schmerzen dar. Die schmerzstillende Wirkung der Opioide w...

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Bibliographic Details
Main Author: Kirchhofer, Sina Birgitt
Contributors: Bünemann, Moritz (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2021
Online Access:PDF Full Text
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G-Protein coupled receptors are one of the most important targets in pharmacological therapy. Here, the opioid receptors, in particular the µ opioid receptor, represent the most important target in therapy of severe pain. However, the pain killing effects are accompanied by severe side effects, like respiratory depression or addiction. The high risk for abuse or overdose led to the opioid crisis in the US, responsible for more than 50.000 caused deaths in 2019 alone. The majority of deaths here are due to synthetic opioids, such as fentanyl. A wide variety of opioids are used for pain therapy. These differ not only in their chemical structure. Their potency, efficacy and kinetics to activate Gi/o proteins is variable. There are already attempts to develop more effective and safer opioids. However, based on the aforementioned differences between opioids, it is important to better understand the different triggered effects of opioids. In this context it is crucial to evaluate the ligand-receptor interactions as well as to better understand the ligand-specific modulation of receptor activity upon voltage changes in the membrane potential. In this study, molecular docking was used to define ligand-receptor interactions. In addition, a fingerprint analysis of these ligand-binding poses was used to identify interaction patterns of different groups of opioids. These interactions could be verified experimentally via site-directed mutagenesis in FRET-based assays. This combination of in silico and in vitro analysis allowed a detailed characterization of the ligand-specific voltage dependence of the MOR. For this purpose, on the one hand, a large number of clinically relevant opioid agonists as well as antagonists were tested with regard to their voltage sensitivity at different levels of the GPCR signal cascade based on FRET-measurements under control of the membrane potential as well as measurements of K+-currents. The investigated ligands could be divided into different groups with respect to their voltage-induced behavior. One group displayed a strong increase in receptor activity induced by a depolarization. For this, it has been shown that there is a modulation of the efficacy of receptors activation. Another group of ligands showed a depolarization-induced decrease in receptor activity. However, ligands could also be found under which no voltage effect was detectable. These effects could be correlated with the interaction patterns of the ligands in the further course of this work. These correlations could again be supported by site-directed mutagenesis. This revealed interaction patterns and motifs in the receptor that define the direction and extend of the ligand-specific voltage effect of the MOR and allows conclusions to be drawn about a potential general voltage sensing mechanism of GPCRs. Furthermore, the voltage dependence of other opioid receptors was characterized. Again, a ligand-specific voltage dependence could be defined for the DOR and KOR. A pronounced voltage dependence could also be demonstrated for the nociception receptor. In addition, to enable further investigations of the opioid receptor family, FRET-based receptor conformations sensors for the MOR, KOR, and NOP were generated and characterized in this work. These newly established sensors were suitable for high-throughput measurements, providing a new, straightforward way to characterize opioids already in use in the clinic or to test newly developed opioids.