Modulation of ion channels by natural products ‒ Identification of hERG channel inhibitors and GABAA receptor ligands from plant extracts Inauguraldissertation zur Erlangung der Würde eines Doktors der Philosophie vorgelegt der Philosophisch-Naturwissenschaftlichen Fakultät der Universität Basel von Anja Schramm aus Wiedersbach (Thüringen), Deutschland Basel, 2014 Original document stored on the publication server of the University of Basel edoc.unibas.ch This work is licenced under the agreement „Attribution Non-Commercial No Derivatives – 3.0 Switzerland“ (CC BY-NC-ND 3.0 CH). The complete text may be reviewed here: creativecommons.org/licenses/by-nc-nd/3.0/ch/deed.en Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät auf Antrag von Prof. Dr. Matthias Hamburger Prof. Dr. Judith Maria Rollinger Basel, den 18.02.2014 Prof. Dr. Jörg Schibler Dekan Attribution-NonCommercial-NoDerivatives 3.0 Switzerland (CC BY-NC-ND 3.0 CH) You are free: to Share — to copy, distribute and transmit the work Under the following conditions: Attribution — You must attribute the work in the manner specified by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). Noncommercial — You may not use this work for commercial purposes. No Derivative Works — You may not alter, transform, or build upon this work. With the understanding that: Waiver — Any of the above conditions can be waived if you get permission from the copyright holder. Public Domain — Where the work or any of its elements is in the public domain under applicable law, that status is in no way affected by the license. Other Rights — In no way are any of the following rights affected by the license: o Your fair dealing or fair use rights, or other applicable copyright exceptions and limitations; o The author's moral rights; o Rights other persons may have either in the work itself or in how the work is used, such as publicity or privacy rights. Notice — For any reuse or distribution, you must make clear to others the license terms of this work. The best way to do this is with a link to this web page. Quelle: creativecommons.org/licenses/by-nc-nd/3.0/ch/deed.en Datum: 12.11.2013 Für meine Familie In den frühen Zeiten unserer Erde waren Pflanzen der Menschen natürliche Nahrung und blieben es seither als lebenserhaltendes Mittel und Medizin zur Wiederherstellung der Gesundheit. John Gerard, The Herbal, 1597 Table of Contents Summary 10 Zusammenfassung 12 1. Aim of the work 15 2. Introduction 19 2.1. The hERG channel 20 Structure and gating of the hERG channel 20 Localization and physiological role of the hERG current IKr 23 Pharmacology of hERG channels 24 Drug-binding site 26 2.2. Preclinical strategies for assessing the cardiac safety profile 29 2.3. Plant-derived natural products as hERG channel inhibitors 33 Alkaloids tested for hERG channel inhibition 35 Flavonoids tested for hERG channel inhibition 43 Miscellaneous structural classes tested for hERG channel inhibition 47 2.4. Identification of ion channel ligands from plant extracts 56 3. Results and discussion 61 3.1. Natural products as potential hERG channel inhibitors ‒ Outcomes from a screening of widely used herbal medicines and edible plants 62 3.2. hERG channel inhibitors in extracts of Coptidis rhizoma 90 3.3. Natural products as potential human ether-a-go-go-related gene channel inhibitors – Screening of plant-derived alkaloids 97 3.4. Gram-scale purification of dehydroevodiamine from Evodia rutaecarpa fruits, and a procedure for selective removal of quaternary indoloquinazoline alkaloids from Evodia extracts 112 3.5. Phytochemical profiling of Curcuma kwangsiensis rhizome extract, and identification of labdane diterpenoids as positive GABAA receptor modulators 136 4. Conclusions and outlook 191 Acknowledgments 195 Summary Ion channels are expressed in virtually all cell types in the human body and are involved in various physiological processes. Hence, it is not surprising that ion channels play an important role in modern drug discovery. Lead compounds are nowadays routinely tested against a panel of ion channels to evaluate selectivity and potential off-target activities. The human ether-à-go-go-related gene (hERG) channel, a voltage-gated potassium channel, is the currently most critical antitarget with respect to cardiac safety. Inhibition of the hERG channel can prolong the QT interval on the electrocardiogram (ECG) and, as a consequence, lead to life-threatening arrhythmia. Considering the daily intake of plant-derived foods and herbal products, surprisingly few natural products have been tested for hERG blocking properties. In the course of an interdisciplinary hERG project, a selection of widely used herbal drugs and dietary plants was screened by means of a two-microelectrode voltage-clamp assay with Xenopus oocytes. Moderate hERG block was observed for the traditional Chinese herbal drug Coptidis rhizoma and black pepper fruits, and successfully tracked by HPLC-based activity profiling to dihydroberberine and piperine, respectively. The hERG blocking activity of cinnamon, guarana, and nutmeg, in contrast, was attributed to tannins. Our screening data suggest that major European medicinal plants and frequently consumed food plants are associated with a low risk for hERG inhibition. However, the case of Coptidis rhizoma pointed towards a need for a more thorough assessment of herbal drugs of the traditional Chinese medicine (TCM). Subsequent screening of a plant-derived alkaloid library led to the identification of several potent hERG blockers. Further investigations are certainly warranted to assess the cardiac safety profile of these alkaloids. Dehydroevodiamine (DHE), a major bioactive constituent of the traditional Chinese herbal drug Evodiae fructus, has been previously shown to inhibit several cardiac ion currents in vitro. 10 For further evaluation of its in vivo pharmacological and toxicological properties, gram amounts of DHE were needed. Since DHE is not commercially available, we developed an efficient method for its gram-scale isolation from a crude Evodia extract. Our approach is based on a combination of cation-exchange chromatography and preparative RP-HPLC. Moreover, the DHE content in commercially available Evodia products was assessed by HPLC-PDA analysis. A daily intake of up to mg amounts of DHE was calculated from recommended doses of these products. We also devised a procedure for the production of DHE-depleted Evodia products, should DHE indeed turn out to be toxicologically relevant. The gamma-aminobutyric acid type A (GABAA) receptor, a ligand-gated chloride channel, mediates fast inhibitory neurotransmission in the central nervous system (CNS), and is thus a clinically important drug target. In the search for positive α1β2γ2S GABAA receptor modulators of plant origin, we investigated an extract of Curcuma kwangsiensis rhizomes. HPLC-based activity profiling was used in combination with a two-microelectrode voltage-clamp assay on Xenopus oocytes to identify the active constituents. Targeted isolation afforded a series of 11 structurally related labdane diterpenoids, including four new natural products. Structure elucidation was achieved by comprehensive analysis of HR-ESI-TOF-MS and NMR data. The absolute configuration of the compounds was assigned by electronic circular dichroism (ECD). The highest GABAA receptor modulating activity was observed for zerumin A. From a more general perspective, this study demonstrates that HPLC-based activity profiling is an effective strategy to characterize bioactive compounds in crude natural extracts. 11 Zusammenfassung Ionenkanäle werden in nahezu allen Zelltypen des menschlichen Körpers exprimiert und sind in verschiedene physiologische Prozesse involviert. Daher ist es nicht verwunderlich, dass Ionenkanäle eine wichtige Rolle in der modernen Arzneimittelforschung spielen. Um die Selektivität und mögliche Off-Target-Aktivitäten von Leitsubstanzen zu beurteilen, werden diese heutzutage routinemässig an einer Vielzahl von Ionenkanälen auf ihre Aktivität hin untersucht. Der hERG Kanal, ein spannungsgesteuerter Kaliumkanal, ist das derzeit bedeutenste Antitarget hinsichtlich der kardialen Sicherheit von Arzneistoffen. Eine Blockade des hERG Kanals kann zu einer Verlängerung des QT-Intervalls im Elektrokardiogramm (EKG) führen und infolgedessen lebensbedrohliche Herzrhythmusstörungen hervorrufen. Trotz der steigenden Popularität von Naturheilmitteln und des täglichen Verzehrs von pflanzlichen Nahrungsmitteln ist die Interaktion von Naturstoffen mit dem hERG Kanal bisher wenig erforscht. Im Rahmen eines interdisziplinären hERG Projektes wurden sowohl bedeutende Medizinalpflanzen als auch gebräuchliche Nahrungspflanzen in einem Zwei-Mikroelektroden-Spannungsklemm-Assay an Xenopus Oozyten getestet. Die chinesische Arzneidroge Coptidis Rhizoma und der schwarze Pfeffer riefen beide eine moderate, aber nennenswerte hERG Blockade hervor. Mittels HPLC-basiertem Aktivitätsprofiling konnten die entsprechenden aktiven Inhaltsstoffe als Dihydroberberine und Piperin identifiziert werden. Die hERG-blockierende Wirkung von Zimt, Guarana und Muskatnuss wurde hingegen auf Tannine zurückgeführt. Die Screeningergebnisse zeigen, dass die wichtigsten europäischen Heilpflanzen und pflanzlichen Nahrungsmittel ein geringes Risiko für eine Blockade des hERG Kanals aufweisen. Das Beispiel von Coptidis Rhizoma weist jedoch darauf hin, dass pflanzliche Arzneidrogen der traditionellen chinesischen Medizin (TCM) gründlich