Identification and application of novel and selective blockers for the heat-activated cation channel TRPM3

Der Fakult¨atf¨urBiowissenschaften, Pharmazie und Psychologie

der Universit¨atLeipzig

eingereichte

DISSERTATION

zur Erlangung des akademischen Grades

doctor rerum naturalium

Dr. rer. nat.

vorgelegt

von Diplom Biologin Isabelle Straub

geboren am 18.01.1980 in Neunkirchen

Leipzig, den 23.05.2014

Bibliography Isabelle Straub Identification and application of novel and selective blockers for the heat-activated cation channel TRPM3 Fakult¨atf¨urBiowissenschaften, Pharmazie und Psychologie Universit¨atLeipzig Dissertation 86 Seiten, 165 Literaturangaben, 38 Abbildungen, 0 Tabellen

TRPM3 (melastatin-related transient receptor potential 3) is a calcium-permeable nonselective cation channel that is expressed in various tissues, including insulin- secreting β-cells and a subset of sensory neurons from trigeminal and dorsal root ganglia (DRG). TRPM3 can be activated by the neurosteroid pregnenolone sul- phate (PregS) or heat. TRPM3−/− mice display an impaired sensation of nox- ious heat and inflammatory thermal hyperalgesia. A calcium-based screening of a compound library identified four natural compounds as TRPM3 blockers. Three of the natural compounds belong to the citrus fruit flavanones (hesperetin, erio- dictyol and naringenin), the forth compound is a deoxybenzoin that can be syn- thesized from an isoflavone of the root of Ononis spinosa (ononetin). The IC50 for the substances ranged from upper nanomolar to lower micromolar concentra- tions. Electrophysiological whole-cell measurements as well as calcium measure- ments confirmed the potency of the compounds to block TRPM3 in DRG neu- rones. To further improve the potency and the selectivity of TRPM3 block and to identify the pharmacophore within the flavanone structure, we conducted a hit optimisation procedure by re-screening a focussed library. The library composed of several flavanones with different substitutions on relevant chemical positions and of representatives from different flavonoid subgroups. Within this secondary screen, we identified isosakuranetin and liquiritigenin as active blockers of PregS-induced Ca2+ entry through TRPM3. Isosakuranetin, a flavanone that can be found in blood oranges and grapefruits, displayed an IC50 of 50 nM, and is the most potent inhibitor of TRPM3 identified so far. The novel compounds exhibited a marked specificity for TRPM3 compared with other thermosensitive TRP channels, and

i 2+ blocked PregS-induced [Ca ]i signals and ionic currents in freshly isolated DRG neurones. Furthermore, isosakuranetin and hesperetin reduced the sensitvity of mice to noxious heat and PregS-induced chemical pain. Since the physiological functions of TRPM3 channels are still poorly defined, the development and vali- dation of potent and selective blockers is expected to contribute to clarifying the role of TRPM3 in vivo. Considering the involvement of TRPM3 in nociception, TRPM3 blockers may represent a novel concept for analgesic treatment.

ii Contents

Nomenclature 1

1 Abstract 3

2 Zusammenfassung 7

3 Introduction 13 3.1 Helix-loop-helix superfamily of cation channels ...... 13 3.2 Transient receptor potential channels ...... 14 3.3 TRP channels as temperature sensors ...... 17 3.4 TRPM3 ...... 19 3.5 Pharmacological modulation of TRPM3 ...... 21 3.6 Aim of the thesis and systematic approach ...... 22 3.7 Relationship of the publications ...... 24

4 Citrus fruit and fabacea secondary metabolites potently and selectively block TRPM3 27

5 Flavanones that selectively inhibit TRPM3 attenuate thermal nocicep- tion in vivo 51

6 References 69

iii

Nomenclature

2-APB ...... 2-aminoethoxydiphenyl borate aa ...... Amino acid BAA ...... Bisandrographolide CGRP ...... Calcitonin gene related peptide CNG ...... Cyclic nucleotide gated DRG ...... Dorsal root ganglia nompC ...... No mechanoreceptor potential C NSAIDs ...... Nonsteroidal anti-inflammatory drugs PPAR-γ ...... Peroxisome-proliferator-activated receptor-γ PregS ...... Pregnenolone sulphate THC ...... Tetrahydrocannabinol TM ...... Transmembrane TRP ...... Transient receptor potential TRPA ...... Transient receptor potential ankyrin TRPC ...... Transient receptor potential canonical TRPM ...... Transient receptor potential melastatin-related TRPML ...... Transient receptor potential mucolipin TRPP ...... Transient receptor potential polycystein TRPV ...... Transient receptor potential vanilloid VR1 ...... Vanilloid receptor 1

1

1 Abstract

Transient receptor potential (TRP) channels are a ubiquitously expressed family of ion channels that fulfil a plethora of different functions in the organism. They participate, amongst other functions, in the perception of pain, temperature and taste, and they are important players in the calcium and magnesium homeosta- sis. In contrary to other ion channels that have been identified by means of their ion selectivity or ligands, most TRP channels have been identified on the basis of sequence homologies. Therefore, specific activators and blockers are lacking for most TRP channels. In the meantime, functional data obtained from TRP channel knock-out mouse models helped to identify the physiological function of some, but not all TRP channels. As a possible drawback of knock-out strate- gies, proteins with redundant functions might obscure or compensate the missing protein. Thus, specific modulators are indispensable for the characterisation of TRP channel functions, especially in in vivo experiments. Genome-wide associa- tion studies provided evidence that TRP channel mutations are linked to different diseases. Thus, specific modulators of TRP channels are not only essential for the characterisation for the TRP channel function but may also bear the potential to develop novel therapeutic strategies. TRPM3, the third member of the melastatin-related TRP channel subfamily, is one of the poorly characterized TRP channels. The TRPM3 gene is transcribed into different splice variants that differ with respect to their biophysiological prop- erties. TRPM3α2 is a Ca2+-permeable ion channel that is, together with the closely related TRPM1, activated by the neurosteroid pregnenolone sulfate (PregS). In contrast to TRPM3α2, TRPM3α1 is a Ca2+-impermeable, monovalent selective splice variant, and no activators are known to date. TRPM3α2 is functionally expressed in the endocrine pancreas, and activation of TRPM3α2 enhances the insulin secretion. TRPM3α2 is also expressed in neurones of the trigeminal and

3 dorsal root ganglia (DRG) and can be activated by temperatures above 37 ◦C. Data obtained with TRPM3-deficient mice revealed that TRPM3α2 is involved in the perception of noxious heat and in the development of inflammatory thermal hyperalgesia. The role of TRPM3α2 in the insulin secretion has yet not been confirmed in vivo. The aim of this thesis was to identify modulators of TRPM3α2 that can be used as pharmacological tools to help to unravel the physiological functions of TRPM3α2 in vitro and in vivo. Specifically, the function of TRPM3α2 as a puta- tive heat-sensing nociceptive receptor was investigated in recombinant expression systems, in primary cells that natively express TRPM3α2 and at the organismic level in vivo, applying the newly identified blockers. All experiments described in this thesis have been performed with the Ca2+-permeable TRPM3α2 channel. A common feature of thermo-sensitive TRP channels is their modulation by nat- ural compounds. Therefore, we screened a compound library that comprises not only drugs and drug-like compounds, but also molecularly defined natural com- pounds and toxins. HEK293 cells stably expressing TRPM3 were loaded with the calcium indicator dye Fluo-4, dispensed into 384-well plates that were preloaded with the compounds of the library, and the PregS-induced Ca2+ entry was detected. As a result of the primary screen, we identified four natural compounds as potent TRPM3 blockers (Chapter 4). Three of the newly identified inhibitors belong to the citrus fruit flavanones (hesperetin, eriodictyol and naringenin), the forth com- pound is a deoxybenzoin that was originally synthesized from an isoflavone of the root of Ononis spinosa (ononetin). The IC50 for the substances ranged between 0.3 µM for ononetin and 2.0 µM for hesperetin. Citrus fruit flavanones are glycosy- lated secondary plant metabolites. After consumption of flavanone glycosides, the sugar moiety is cleaved by intestinal bacteria and the resulting flavanone aglycon can be resorbed. Naringenin derives from the bitter-tasting naringin that is mainly found in grapefruits. Hesperetin arose from hesperedin a flavanone that predom- inantly occurs in oranges. Neither naringin nor hesperedin, the two flavanone glycosides, had any blocking effect on TRPM3. Whole-cell electrophysiological measurements were performed to further characterise the potency of naringenin and ononetin to block TRPM3. This ongoing electrophysiological characterisa- tion revealed that naringenin and ononetin also block nifedipine- or heat-induced

4 TRPM3 currents. Furthermore, the newly identified blockers showed a high speci- ficity for TRPM3. Neither TRPM1, the most closely related TRP channel, nor other thermo-sensitive TRP channels (TRPV1, TRPM8, TRPA1) that are ex- pressed in DRG neurones were blocked by concentrations of the compounds that are sufficient to completely abrogate TRPM3 activity. Single-cell fluorescence mea- surements in freshly isolated DRG neurones showed that naringenin and ononetin also block endogenously expressed TRPM3. Despite low bioavailability and exten- sive phase 1 and phase 2 metabolism, some studies demonstrated that hesperetin and naringenin may reach plasma concentrations that would be sufficient to inhibit TRPM3 in vitro. The flavonoid substructure comprises two aromatic rings (A and B ring) that are linked with a heterocycle (C ring). Flavonoids can be divided into different subgroups, depending on the degree of the oxidation and the substitutions in the C ring. Based on the compounds identified in the primary screen, we next aimed at the identification of the pharmacophore within the flavanone structure. To this end, we constructed a focussed library that contains several flavanones with different substitutions on relevant chemical positions, and several representative compounds from other flavonoid subgroups (Chapter 5). At a concentration of 25 µM, flavonoids that do not share the flavanone backbone failed to completely block the PregS-induced TRPM3 activation. Thus, within the chemical class of flavonoids, the flavanone substructure represents an optimized substructure. Since pinocembrin, the unsubstituted flavanone substructure, was only poorly active, substitutions on the A and B ring are essential to efficiently block TRPM3. In the secondary screen we identified two novel flavanones that potently blocked TRPM3: isosakuranetin with an IC50 of 50 nM and liquiritigenin with an IC50 of 500 nM. Electrophysiological characterisation revealed that isosakuranetin inhibits both, the PregS- and the heat-induced activation of TRPM3α2. In addition, isosaku- ranetin can act from either side of the membrane, but the slow onset of block found upon extracellular application points to an intracellular binding site. Considering the specificity, liquiritigenin strongly discriminated against other members of the TRP channel subfamily. High concentrations of isosakuranetin activated recombi- nantly overexpressed TRPA1, but its impact on endogenously expressed TRPA1 was low. Isosakuranetin blocked TRPM3-induced currents in freshly isolated DRG

5 neurones, but had no inhibitory effect on endogenously expressed TRPA1, TRPM8 and TRPV1. Cytotoxicity tests revealed a marked cytotoxic effect of naringenin, whereas all other TRPM3-blocking flavanones were well tolerated. For this reason we chose isosakuranetin and hesperetin to investigate a possible pain-alleviating effect of the TRPM3 blockers in animal experiments. Mice that were intraperi- toneally injected with 2 mg/kg or 10 mg/kg of isosakuranetin and placed on a hot plate (52 ◦C) showed a significantly prolonged latency of nocifensive behaviour compared to solvent-injected control animals. Intraperitoneal injection of hes- peretin was also effective at a dose of 10 mg/kg, but not at 2 mg/kg. Intraplantar injection of PregS elicited a nocifensive behaviour that could be significantly dimin- ished by previous intraperitoneal injection of 2 mg/kg or 10 mg/kg isosakuranetin. Plasma concentrations of isosakuranetin after intraperitoneal injection of 2 mg/kg already reached concentrations that were sufficient to block TRPM3 in vitro. In the context of this thesis citrus fruit flavanones were identified as potent and selective TRPM3 blockers. The here identified blockers can be used to in- vestigate the function of TRPM3 in vitro and in vivo. Furthermore, these novel pharmacological tools may help to solve the discrepancy between in vitro and in vivo experiments concerning the role of TRPM3 in insulin secretion. The function of TRPM3 in DRG neurones is reminiscent of that of the pain-relevant vanil- loid receptor TRPV1. TRPV1 blockers have been tested in clinical studies as potentially analgesic drugs. However, inhibition of TRPV1 not only alleviates heat-induced pain but also increases the body temperature of the subjects. This body temperature-elevating effect forced the early termination of some clinical tri- als. Contrary to TRPV1 blockers, isosakuranetin and hesperetin did not elevate the body temperature of systemically treated mice. Besides its function in the perception of noxious heat, TRPM3 is involved in the inflammatory thermal hy- peralgesia. Thus, further experiments are necessary to show whether the novel TRPM3 blockers are effective in blocking TRPM3-induced thermal hyperalgesia and may get in focus for the development of novel analgesic therapies.

6 2 Zusammenfassung

Transient Receptor Potential (TRP)-Kan¨ale sind ubiquit¨ar exprimierte Kationen- kan¨ale, die eine Vielzahl unterschiedlicher Funktionen im Organismus erfullen.¨ Sie sind unter anderem an der Wahrnehmung von Schmerz, Temperatur und Ge- schmack beteiligt, und spielen eine wichtige Rolle bei der Calcium- und Magnesium- Hom¨oostase. Viele TRP-Kan¨ale sind anders als die meisten anderen Ionenkan¨ale nicht durch ihre Liganden oder ihre Selektivit¨at, sondern uber¨ die Homologie ihrer Sequenzen identifiziert worden. Dies hat zur Folge, dass nicht fur¨ alle TRP-Kan¨ale spezifische Aktivatoren oder Blocker bekannt sind, und somit die Funktion einzel- ner TRP-Kan¨ale noch weitgehend unbekannt ist. Mit Hilfe transgener knock-out Mausmodelle konnten zwischenzeitlich einige TRP-Kanalfunktionen gekl¨art wer- den. Ein Nachteil der knock-out-Strategie ist allerdings, dass andere, funktionell red- undante Proteine teilweise oder vollst¨andig den Wegfall des TRP-Kanals kompen- sieren k¨onnen und somit m¨oglicherweise der eigentliche Funktionsverlust verschlei- ert wird. Aus diesem Grund sind spezifische Modulatoren fur¨ die Charakterisierung der TRP-Kanalfunktion, besonders bei in vivo-Versuchen, unerl¨asslich. Spezifische Modulatoren sind nicht nur fur¨ die Charakterisierung der TRP-Kanalfunktionen notwendig, sondern sie rucken¨ auch immer mehr in den Mittelpunkt pharmako- logischer Forschung, da bei der Kartierung krankheitsrelevanter Loci in genom- weiten Assoziationsstudien gezeigt wurde, dass Mutationen von TRP-Kan¨alen mit verschiedenen Erkrankungen assoziiert sind. Ein bislang relativ schlecht charakte- risierter TRP-Kanal ist TRPM3. Das TRPM3-Gen codiert fur¨ eine Vielzahl un- terschiedlicher Splicevarianten, die sich unter anderem in ihren biophysikalischen Eigenschaften unterscheiden. Beispielsweise ist die Ca2+-permeable Splicevariante TRPM3α2, wie ihr engster Verwandter TRPM1, durch das Neurosteroid Pregneno- lonsulfat (PregS) aktivierbar. Fur¨ die Ca2+-impermeable Splicevariante TRPM3α1

7 hingegen sind noch keine Modulatoren bekannt. Entsprechend ist sie daher auch noch nicht funktionell charakterisiert. Mittels in vitro-Versuchen konnte gezeigt werden, dass TRPM3α2 funktionell im endokrinen Pankreas exprimiert ist und es nach Aktivierung des Kanals zu einer verst¨arkten Insulinausschuttung¨ kommt. TRPM3α2 ist in Neuronen der Trigeminal- und Hinterwurzelganglien exprimiert und wird durch Temperaturen uber¨ 37 ◦ aktiviert. Experimente mit TRPM3 knock- out-M¨ausen zeigten, dass TRPM3 an der Wahrnehmung von schmerzhafter Hitze sowie inflammatorischer Hyperalgesie beteiligt ist. Eine Relevanz von TRPM3α2 bei der Insulinausschuttung¨ konnte in den knock-out-Tieren in vivo noch nicht best¨atigt werden. Ziel dieser Arbeit war es, mittels eines Screenings Modulatoren fur¨ den Ionenka- nal TRPM3α2 zu identifizieren um pharmakologische Werkzeuge zu erhalten, die zur Aufkl¨arung der physiologischen Funktion von TRPM3α2 beitragen k¨onnen. Weiterhin sollte mit den identifizierten Blockern die Funktion von TRPM3α2 als Schmerz- und Hitzerezeptor in vivo untersucht und die Frage gekl¨art werden, ob sich TRPM3α2 als potenzielle Zielstruktur fur¨ die pharmakologische Forschung zur Entwicklung von Analgetika eignet. Alle Experimente mit rekombinantem TRPM3, die im Rahmen dieser Arbeit dargestellt sind, wurden mit der Ca2+- permeablen TRPM3α2-Variante durchgefuhrt.¨ Eine Gemeinsamkeit der thermo-aktivierbaren TRP-Kan¨ale ist ihre Modulier- barkeit durch Naturstoffe. Daher wurde eine Substanzbibliothek zum Screening ausgew¨ahlt, die neben zahlreichen zugelassenen Medikamenten auch Naturstof- fe und Toxine enth¨alt. In Chapter 4 sind die Ergebnisse des prim¨aren Screens dargestellt. Eine HEK293-Zelllinie, die stabil TRPM3 exprimiert, wurde mit den verschiedenen Substanzen der Bibliothek inkubiert und der Ca2+-Einstrom, aus- gel¨ost durch den TRPM3-Aktivator PregS, fluorometrisch gemessen. Durch weite- re Optimierungsschritte wurden vier verschiedene Naturstoffe als TRPM3-Blocker identifiziert. Darunter befanden sich drei Flavanone aus Zitrusfruchten¨ (Hespe- retin, Eriodictyol und Naringenin) und ein Deoxybenzoin, welches ursprunglich¨ aus einem Isoflavon der Wurzel von Ononis spinosa gewonnen wurde (Ononetin).

Die IC50-Werte der Substanzen lagen zwischen 0,3 µM fur¨ Ononetin und 2,0 µM fur¨ Hesperetin. Flavanone kommen in der Natur nur als Glykosid vor. Erst im Darm wird der Zucker durch Bakterien abgespalten, so dass das entsprechende

8 Flavanon-Aglykon entsteht und resorbiert werden kann. Naringenin entsteht so aus dem in Grapefruit vorkommenden, bitter schmeckenden Naringin. Hesperetin entsteht aus Hesperedin, einem vor allem in Orangen vorkommenden Flavanon- Glycosid. Die Flavanon-Glykoside selbst (Naringin und Hesperedin) zeigten kei- nerlei hemmende Wirkung auf TRPM3. Eine weiterfuhrende¨ elektrophysiologische Charakterisierung zeigte, dass auch die durch Nifedipin oder Hitze ausgel¨osten TRPM3-Str¨ome durch Naringenin und Ononetin blockierbar sind. Weiterhin zeig- ten die neu entdeckten TRPM3-Blocker eine hohe Spezifit¨at. Weder TRPM1, der engste Verwandte von TRPM3, noch andere, in Neuronen der Hinterwurzelgangli- en exprimierte, thermo-sensitive TRP-Kan¨ale (TRPV1, TRPM8, TRPA1) wurden durch die Modulatoren in den fur¨ die TRPM3-Blockade notwendigen Konzentra- tionen blockiert. Einzelzell-Fluoreszenzmessungen zeigten, dass Naringenin und Ononetin auch den in sensorischen Neuronen des Hinterwurzelganglions endogen exprimierten TRPM3 blockieren. Trotz geringer Bioverfugbarkeit¨ von Naringenin und Hesperetin gelang es bei einigen Studien im Plasma einzelner Individuen, nach deren Konsum von sehr großen Mengen an Orangensaft oder Grapefruit- saft, bereits Konzentrationen von Naringenin und Hesperetin nachzuweisen, die ausreichend w¨aren um TRPM3 in vitro zu blockieren. Die Flavonoid-Grundstruktur besteht aus zwei aromatischen Ringen (A und B), die uber¨ eine heterozyklische Brucke¨ (C-Ring) miteinander verbunden sind. Es existieren verschiedene Flavonoid-Untergruppen, die sich in ihrem Oxidations- grad, sowie den Substituenten am C-Ring unterscheiden. Ausgehend von den im Prim¨arscreen identifizierten Substanzen wurden zur Identifizierung des Phama- kophors verschiedene Flavanone ausgesucht, welche sich durch unterschiedliche Substituenten an relevanten Stellen der Flavanon-Grundstruktur unterscheiden. Zus¨atzlich wurden einzelne Vertreter unterschiedlicher Flavonoid-Untergruppen ausgew¨ahlt, und die Potenz TRPM3 zu blockieren, wurde fur¨ diese chemisch unter- schiedlichen Entit¨aten fluorometrisch bestimmt (Chapter 5). Keine der zus¨atzlich untersuchten Flavonoid-Untergruppen zeigte bei einer Konzentration von 25 µM einen vollst¨andigen Block der PregS-induzierten TRPM3-Aktivit¨at, sodass die Flavanon-Grundstruktur innerhalb dieser chemischen Klasse bereits das Optimum darstellt. Weiterhin zeigte sich, dass die Flavanone Grundstruktur allein noch nicht ausreicht, um TRPM3 vollst¨andig zu blockieren. Sowohl Substituenten am A-

9 Ring, als auch am B-Ring sind fur¨ eine effektive Inhibition n¨otig. Es konnten zwei weitere Flavanone identifiziert werden, die TRPM3 mit hoher Potenz blockieren:

Isosakuranetin mit einer IC50 von 50 nM und Liquiritigenin mit einer IC50 von 500 nM. Isosakuranetin wurde elektrophysiologisch charakterisiert, und es konn- te gezeigt werden, dass neben PregS-induzierten TRPM3-Str¨omen auch Hitze- induzierte TRPM3-Str¨ome blockierbar sind. Die Bindestelle fur¨ Isosakuranetin ist von beiden Seiten der Membran zug¨anglich, wobei die langsame Anfangskinetik des Blocks auf eine intrazellul¨are Bindestelle hinweist. Hinsichtlich der Spezifit¨at zeigte sich, dass Liquiritigenin am st¨arksten zwischen den einzelnen TRP-Kan¨alen diskri- miniert. Hohe Konzentrationen von Isosakuranetin aktivieren hingegen TRPA1 in uberexprimierenden¨ Zellen. Dennoch war auf endogen exprimierten TRPA1 nur ein sehr geringer Einfluss nachweisbar. Isosakuranetin blockiert endogen exprimierten TRPM3 in sensorischen Neuronen, zeigte jedoch keinen blockierenden Einfluss auf endogen exprimierten TRPV1, TRPA1 und TRPM8. In Zelltoxizit¨ats-Tests zeigte sich, dass unter den untersuchten TRPM3-blockierenden Substanzen nur Narin- genin gravierende zellsch¨adigende Eigenschaften aufweist. Daher wurden Isosaku- ranetin und das bereits im Prim¨arscreen identifizierte Hesperetin verwendet um die Substanzen im Tierversuch auf ihre schmerzhemmende Wirkung hin zu unter- suchen. Etwa 30 Minuten nach intraperitonealer Injektion von 2 mg/kg oder 10 mg/kg Isosakuranetin zeigte sich, dass die M¨ause auf einer heißen Platte (52 ◦C; hot-plate-Assay) eine verl¨angerte Latenzzeit hatten, bevor sie ein nozifensives Ver- halten zeigten. Die Tiere, die den schw¨acher potenten TRPM3-Blocker Hesperetin gespritzt bekamen, zeigten nur nach der Injektion der h¨oheren Dosierung von 10 mg/kg eine statistisch signifikant verl¨angerte Verweildauer auf der heißen Plat- te. Des weiteren konnten wir zeigen, dass die durch PregS-Injektion ausgel¨oste nozifensive Reaktion der M¨ause durch vorherige Injektion von Isosakuranetin (2 und 10 mg/kg) signifikant geringer ausfiel. Bereits nach intraperitonealer Injekti- on von 2 mg/kg erreichte die Plasmakonzentration von Isosakuranetin Werte, die ausreichend sind, um TRPM3 in vitro zu blockieren. Im Rahmen dieser Arbeit konnte gezeigt werden, dass TRPM3 durch Flavano- ne aus Zitrusfruchten¨ selektiv und potent blockiert wird. Die neu identifizierten Blocker sind sowohl fur¨ in vitro- als auch fur¨ in vivo-Versuche anwendbar. Die neuen pharmakologischen Werkzeuge k¨onnen nun dazu genutzt werden, die Dis-

10 krepanz der Ergebnisse zwischen den in vitro-Versuchen und den Befunden in der knock-out-Maus bezuglich¨ der Bedeutung von TRPM3 bei der Insulin-Sekretion zu kl¨aren. TRPM3 zeigt in nozizeptiven Neuronen ¨ahnliche Funktionen, wie der Schmerz-relevante TRPV1. TRPV1-Blocker wurden bereits in klinischen Studi- en gepruft.¨ Allerdings bewirkte eine Blockade von TRPV1 nicht nur analgetische Effekte, sondern auch die K¨orpertemperatur wurde erh¨oht. Dies fuhrte¨ zum vor- zeitigen Abbruch einiger klinischer Studien. Im Gegensatz zu TRPV1-Blockern zeigten Isosakuranetin und Hesperetin keinen Einfluss auf die K¨orpertemperatur systemisch behandelter M¨ause. TRPM3 ist nicht nur an der Wahrnehmung von schmerzhafter Hitze, sondern auch an der inflammatorischen Hyperalgesie betei- ligt. Daher sind weitere Versuche n¨otig um zu prufen,¨ ob die neu identifizierten Blocker auch die uber¨ TRPM3 ausgel¨oste inflammatorische Hyperalgesie blockie- ren k¨onnen. Sollten weiterfuhrende¨ Experimente mit TRPM3 dessen Funktion als Schmerzrezeptor auch bei Entzundungsreaktionen¨ best¨atigen, k¨onnte diese Ziel- struktur zur Entwicklung neuer analgetischer Therapieans¨atze an Bedeutung ge- winnen.

11

3 Introduction

Ion channels are integral membrane proteins that form water-filled pores through cell membranes (plasma membranes or intracellular organelle membranes) that allow the movement of ions across the membrane along their electrochemical gra- dients. Typically, ion channels are oligomeric complexes of several subunits, ar- ranged in circular symmetry to form a central pore. They allow millions of ions per second to permeate the membrane to change the membrane potential or intra- cellular ion concentrations. Various mechanisms may lead to channel activation. They include changes in membrane potential, increases in intracellular Ca2+, bind- ing of neurotransmitters or other ligands, changes in intracellular levels of second messengers, or pH1.

3.1 Helix-loop-helix superfamily of cation channels

Cation channels can be divided into several superfamilies with strongly divergent structures and functional features: voltage-gated channels, ligand-gated channels, second messenger-operated channels and store-operated channels. In the human genome, helix-loop-helix-type ion channels form the largest superfamily, including the voltage-gated Ca2+, Na+ and K+ channels. A common feature of the voltage- gated ion channels is their four-fold symmetry, with a central pore domain that is surrounded by the voltage-sensor regions2. Each of the channel domains contains six transmembrane (TM) segments. The pore of the voltage-gated ion channels is formed by transmembrane-spanning α-helices S5 and S6, flanking a membrane- reentrant loop that forms the selectivity filter1. The transmembrane segment S4 contains several positively charged amino acids, providing the voltage-sensor domain.

13 Fig 3.1: A phylogenetic tree of the TRP family. Mammalian TRP channels are grouped into six subfamilies that comprise distinct channel properties. TRPC2 is a pseudogene in humans and the TRPN family is absent in mammals. Figure is taken from Nilius et al. 2011 4.

Apart from the voltage-gated ion channels the superfamily also comprises hyper- polarisation-activated (HCN) and weakly voltage-dependent ion channels, includ- ing cyclic nucleotide-gated (CNG) and transient receptor potential (TRP) chan- nels2,3.

3.2 Transient receptor potential channels

Most TRP channels are non-selective cation channels that can lead to cell de- polarisation or transmit a Ca2+ entry. They were first described in Drosophila melanogaster, where a mutation showed a transient rather than a sustained re- sponse, when exposed to continuous light5. This mutant was called transient receptor potential, and it took 20 years to finally identify the trp gene6. Based on their amino acid sequence similarity about 30 different mammalian TRP channels

14 have been identified so far (Fig. 3.1). The mamailian TRP channels are grouped into six subfamilies7:

1. TRPC (canonical or classical): The TRPC group contains the proteins with the highest similarity to the Drosophila TRP channels.

2. TRPM (melastatin-related): The name was given to the group because of the tumour suppressor melastatin (TRPM1), the founding member of the TRPM family.

3. TRPV (vanilloid): The group was named vanilloid because of the vanilloid compound capsaicin, the ligand of the first member, vanilloid receptor 1 (VR1, now TRPV1).

4. TRPA (ankyrin): The name of the group derived from the protein ankyrin- like with transmembrane domains 1 (ANKTM1, now TRPA1). TRPA1 is the only member of this subfamily in mammals.

5. TRPML (mucolipin): This group was named after mucolipin 1 (TRPML1), the founding member of the mucolipin subfamily.

6. TRPP (polycystin): Named after the polycystic kidney disease-related pro- tein 2 (PKD2, now TRPP2).

In invertebrates and fishes an additional TRP subfamily has appeared: the so- called TRPN (NOMPC-like) family. The name derived from the no mechanore- ceptor potential C (nompC) gene from Drosophila. For more information about the TRP channel subfamilies see Clapham 20038. Like voltage-gated K+ channels, TRP channels feature six transmembrane- spanning segments (S1-S6) and both termini are located intracellularly (Fig. 3.2A). To form a functional ion channel, four TRP subunits assemble, and S5 and S6, together with the connecting loop, form the predicted cation-conducting pore9,10. The tetrameric stoichiometry allows homomeric or heteromeric arrangements of subunits from the same subfamily (for review see Schaefer 200511). First struc- tural insight into the architecture of the TRP ion channels came from the crystal structure of the bacterial cation channel KcsA from the bacterium Streptomyces

15 lividans 12 (Fig. 3.2B). KcsA is a two TM channel with an amino acid sequence of the pore region that closely resembles that of vertebrate voltage-gated K+ chan- nels12, and bears sequence homology to the TRP channel pore region13. Point mutations in the connecting loop of TRPV channels affect the ion permeability of the respective TRP channel and foster the function of the linker as the core selectivity filter of TRP channels9,14.

Fig 3.2: Topology of TRP channels and architecture of the pore region. (A) TRP channels comprise six transmembrane-spanning segments with intracellularly located N- and C- terminal tails. The putative pore is located between S5 and S6 and the selectivity filter is formed by the connecting loop that dips into the membrane. (B) Schematic ribbon model of the typical helix-loop-helix pore structure of cation channels. Shown is the pore region of the bacterial 2 TM channel KcsA (only two subunits are shown). The connecting loop that forms the selectivity filter is shown as ball-and-stick model with K+ ions (blue balls) bound in the permeation pathway. (A) is modified from Clampham 2001 15; (B) is obtained from Owsianik 2006 13.

Carboxyl and amino termini contain structural and functional motifs or domains that differ between different TRP channel subfamilies. These cytosolic domains may be important for the assembly and regulation of the channel by intracellu- lar signalling cascades. An example for such domains are the ankyrin repeats located at the N-terminus in TRPC, TRPN, TRPV and TRPA channels and are expected to provide binding sites for interaction partners16. Further regulatory motifs include the TRP domain, coiled-coil motifs, calmodulin binding sites, lipid interaction domains, EF hands and phosphorylation sites (for more information see Owsianik 200617). Some members of the TRPM subfamily have, besides their function as an ion channel, an enzyme domain on their carboxy terminus: TRPM2

16 contains a Nudix hydroxylase domain that functions as an ADP ribose pyrophos- phatase18, TRPM6 and TRPM7 contain an atypical α-kinase domain19,20. Their naming as ”chanzymes” refers to a possible bimodal function of these large pro- teins.

3.3 TRP channels as temperature sensors

In complex organisms, thermosensation is essential to avoid noxious temperatures that may result in tissue damage. Thermosensory afferent neurones project into the dorsal horn of the spinal cord, where glutamatergic synapses contact and activate the second neuron of the pain pathway. Different TRP channels that are expressed in sensory nerve endings or in keratinocytes are involved in the detection of innocuous and noxious temperatures21. TRPV1 was the first cloned thermosensitive TRP channel. In resting cells, it features a thermal activation threshold of about 43 ◦C. TRPV1 has been iden- tified in an expression cloning approach with capsaicin, the active component of chili peppers, and resiniferatoxin, an extremely potent capsaicin analogue from Euphorbia plants22. The vanilloid moiety of these compounds was name-giving for the newly identified cDNA as VR1, for vanilloid receptor subtype 122. In the meantime, a plethora of different compounds that modulate TRPV1 has been identified, for review see Vriens 200823. TRPV1 is predominantly expressed in small and medium diameter primary sensory neurones Aδ and C fibers24. Besides TRPV1, nine other mammalian TRP ion channels are also activated by changes in temperature22,25–33. Six of them are currently classified as thermo-TRPs be- cause of their expression in primary somatosensory neurones and their ability to be activated by cold (TRPM8, TRPA1)34, warm (TRPV3, TRPV4), or hot tem- peratures (TRPV1, TRPV2) (Fig.3.3). TRPV2 has been identified by homology cloning25. In contrast to TRPV1, TRPV2 is insensitive to capsaicin, and its acti- vation threshold is 52 ◦C25. TRPV2 is expressed in a subpopulation of medium- diameter Aδ sensory neurones that respond to high-threshold noxious heat, but its contribution to the thermosensation still remains elusive: TRPV2 knock-out mice display normal thermal and mechanical nociception35. The temperature threshold for TRPV3 and TRPV4 activation is within the physiological temperature range

17 of skin temperatures 25-34 ◦C. TRPV3 is mainly expressed in keratinocytes of the skin, but not in sensory neurones of mice26. However, in studies with human and monkey tissues TRPV3 expression has been found in sensory neurones36,37. Mice lacking TRPV3 expression have an impaired thermosensation, indicating its involvement in innocuous warm sensation38. Besides warm temperatures, TRPV3 is activated by natural compounds that, in humans, exert a pleasantly warm sen- sation. Similar to TRPV3, TRPV4 expression in thermosensory neurones is still

Fig 3.3: Schematic representation of the classical thermo-TRP channel that sense temperatures from noxious cold to noxious heat. The temperature range for the activation of the respective TRP channel is indicated on the thermometer. Various natural compounds modulate the thermo-TRPs, prominent modulators are indicated in the figure. The figure is modified from Vay 2012 34.BAA = bisandrographolide A; THC = tetrahydrocannabinol controversially discussed27,39,40. Studies with TRPV4−/− mice have shown that TRPV4 participates in the detection of warmth in the sensory system41. TRPV4 is also activated by nonthermal activators, such as the endocannabinoid anan- damide, its metabolites42 and synthetic phorbol esters. The perception of cold can, similar to heat, range from mild to painful cold. TRPM8 has been identi- fied as a receptor for the cooling compound menthol and temperatures below 20 ◦C28,29. TRPM8 is expressed in non-peptidergic small-diameter sensory neurones that do not express substance P, calcitonin gene related peptide (CGRP)29, and

18 TRPV130. This, together with the fact that TRPM8−/− mice show deficits to sense moderately cold temperatures43–45, indicates an important role of TRPM8 in the detection of innocuous cold. TRPA1 is highly expressed in a subset of small to medium diameter peripheral sensory neurones that also express TRPV1 but not TRPM830. The threshold to activate TRPA1 in a heterologous expression system is in the noxious cold range (below 17 ◦C)30. However, the activation of TRPA1 by noxious cold is still under debate: a study with TRPA1−/− mice has shown that TRPA1 is not required for noxious cold sensation46,47, whereas in an- other study TRPA1−/− mice had displayed an impaired sensation of intense cold temperature48. One should note that TRPA1 is activated by a rise in intracellular Ca2+ concentrations. Thus, cooling cells down may indirectly activate TRPA1 2+ 49,50 through an increase in [Ca ]i . Further activators for TRPA1 are different plant-derived compounds and irritants that elicit pain and inflammation in hu- mans such as allyl isothiocyanate the major active ingredient of mustard oil51, cinnamaldehyde from cinnamon, eugenol and gingerol52. In 2011, TRPM3 has been identified as an ion channel that is expressed in sensory neurones and activated by temperatures above 37 ◦C31. Within the fam- ily of TRPM channels, three other members (TRPM2, TRPM4 and TRPM5) are modulated by temperature, but their relevance in thermosensation has not been firmly established so far32,33. Recently, mRNA of the newly identified ther- mosensitive TRPM (TRPM2-TRPM5) channels has been detected in dorsal root ganglia (DRG) of mice53,54, but only TRPM331 and TRPM2 currents55 have been measured. Interestingly, thermosensitive TRPM4 and TRPM5 channels are also co-expressed in pancreatic β-cells and contribute to insulin secretion (for review see Uchida 201156).

3.4 TRPM3

TRPM3 is a member of the subfamily of melastatin-related TRP channels. The TRPM3 gene encodes a plethora of different splice variants. Lee et al. have identi- fied six TRPM3 splice variants in humans (TRPM3a-f), ranging from 1544 to 1579 57 amino acids (aa) in length . An additional human TRPM31325 variant has been identified, which in contrary to the TRPM3a-f variants, consists only of 1325 aa

19 and exhibits a shorter C-terminus and 153 additional aa on its N-terminus58. The 2+ TRPM31325 and TRPM3a variants form both Ca -permeable and constitutively active channels when heterologously expressed in HEK293 cells57,58. In mice, five TRPM3 splice variants have been described by Oberwinkler et al. in 2005. These splice variants contain a previously not known sequence of 61 aa on their amino terminus59. Alternative splicing in a region that encodes the putative pore region results in TRPM3 proteins with different biophysiological properties. TRPM3α1 is permeable for monovalent cations but not for divalent cations, whereas TRPM3α2 is poorly selective and Ca2+-permeable59. Within the pore loop, TRPM3α2 re- sembles the human TRPM3 splice variants.

Fig 3.4: Schematic representation of different TRPM3 variants. The complete TRPM3 protein is shown as yellow bar, with the different spliced exons (orange), the alternative N-termini (blue), and the TM segments (grey). Different splice variants are indicated as black bars with the total size of the protein indicated in brackets. TRPM3α1 is a 12 aa longer splice variant compared to TRPM3α2, this difference results in TRPM3 channels with different ion selectivity. The aa differences are indicated as insertion. The figure is modified from Oberwinkler 2005, Fruehwald 2012 60,61.

TRPM3 is expressed in various neuronal and nonneuronal tissues including kid- ney, eye, pancreas, brain, DRG and trigeminal neurones57–59,62,63. TRPM3α2 can be activated by the neurosteroid pregnenolone sulphate (PregS), high concentra-

20 tions of nifedipine64, D-erythro-sphingosine65 or noxious heat31. The physiological role of TRPM3 is still not firmly established. TRPM3 is expressed in insulin- secreting β-cells and has been linked to insulin secretion64, and Zn2+ uptake in β-cells66. However, the importance of TRPM3 to regulate insulin secretion is still under debate, since TRPM3−/− mice were not different from wildtype mice con- cerning resting blood glucose level and body weight31. TRPM3 is expressed in different cell types in the eye, including the retinal pigment epithelium, ciliary body and M¨ullercells67. Notably, TRPM3−/− mice show a diminished pupillary response to bright light63. Besides the attenuated pupil constriction of TRPM3−/− mice, no functional data of the channel is available for TRPM3-expressing cells of the eye. Other data concerning TRPM3 reveals its involvement in the glutamater- gic transmission at cerebellar Purkinje neurones, but no functional activation of TRPM3 with PregS or other activators has been shown68. TRPM3 has been found in oligodendrocytes, and during the oligodendrocyte differentiation different splice variants of TRPM3 are expressed69. Furthermore, TRPM3 is expressed in synovial fibroblasts and activation of TRPM3 by PregS or sphingosine causes a decrease in the hyaluronan secretion70. In vascular smooth muscle cells, activation of TRPM3 has been shown to suppress cytokine secretion71. TRPM3 is expressed in DRG and trigeminal neurones and is involved in the detection of noxious heat31. Elevating the temperature above 37 ◦C strongly activates TRPM3, and TRPM3−/− mice showed impaired behavioural responses to noxious heat and in the development of inflammatory thermal hyperalgesia31.

3.5 Pharmacological modulation of TRPM3

Since the physiological significance of TRPM3-mediated effects is still not unam- biguously established, substances that potently and selectively inhibit TRPM3 in vitro and in vivo may help to clarify the physiological function of TRPM3. The advantage of pharmacological inhibition of an ion channel is, in contrary to knock-out animals, that compensatory effects of redundant proteins could not be accomplished so fast. Previously published inhibitors of TRPM3 include unspe- cific TRP blockers like 2-aminoethoxydiphenyl borate (2-APB)72 and gadolinium ions57. Other modulators of TRPM3 are the peroxisome-proliferator-activated

21 receptor-γ-agonists (PPAR-γ) rosiglitazone and troglitazone that are used for the treatment of type-2 diabetes73, and the nonsteroidal anti-inflammatory drugs (NSAIDs) mefenamic acid and tolfenamic acid74. All recently published blockers may be helpful tools to investigate the function of TRPM3 in vitro, but are not applicable for in vivo experiments because of significant off-target effects of the required modulator concentrations.

3.6 Aim of the thesis and systematic approach

Specific modulators for TRP channels help to identify activation mechanisms for the respective channels and lead to an improved understanding of the physiological and pathophysiological roles of these channels. TRPM3 is still poorly characterized and the aim of this thesis was to identify modulators of the Ca2+-permeable ion channel TRPM3α2 that can be used as pharmacological tool to help to identify the function of TRPM3 in vivo. Since TRPM3 is involved in the detection of noxious heat and inflammatory-induced hyperalgesia, the newly identified TRPM3 blockers were used to investigate the function of TRPM3 as pain and heat receptor in vivo. To identify modulators of TRPM3, we performed a medium throughput screen- ing with a compound library containing 2000 drugs, drug-like molecules, toxins and natural compounds (Chapter 4). In this primary screen four compounds were identified that completely block TRPM3 at a concentration of 20 µM. The newly identified TRPM3 blockers are plant-derived flavonoids, and three of them belong to the group of citrus fruit flavanones. Flavanoids are plant secondary metabolites that contribute to the flower coloura- tion and are, in higher plants, involved in the defence against ultraviolet radia- tion75. They chemically derive from the flavan substructure and are composed of two aromatic cycles (A ring and B ring), that are linked with a heterocycle (C ring) (Fig 3.5). Flavonoids can be divided into different subgroups depending on the oxidation state in the C ring, the hydroxylation patterns of the A and B rings and the substitution on the C3 position76. They naturally occur in fruit and vegetables and are, thus, an important part of the human diet. The daily intake of flavonoids of adults has been estimated to amount around 200 mg/d77. Major dietary sources of flavonoids are tea, citrus fruits and citrus fruit juices as well as

22 wine. Because of the large quantity of citrus fruit juice intake, approximately 7.6 % of the total amount of alimentary flavonoids are citrus fruit flavanones77. Different clinical and preclinical studies have shown that citrus fruit flavanone consumption contributes to cardiovascular protection (for review see Chanet 201278). In most of these clinical and preclinical studies, the effect of the flavonoids was elvaluated with a focus on cancer and coronary heart disease. The results of these studies have been inconclusive, since some studies report beneficial effects whereas others did find no or even potentially harmful effects79. However, the bioavailability of most flavonoids is limited and concentrations used for in vitro experiments are often very high78.

Fig 3.5: Molecular structure of the flavan backbone. The flavan backbone consists of two aromatic rings (A ring and B ring) that are linked together with a heterocycle (C ring).

Since the bioavailability of citrus fruits flavanones is relatively small and the ingested flavanones were intensively metabolised80, we searched for chemically re- lated flavanones that display favourable properties with respect to their bioavail- ability and their inhibitory potency. For this reason, flavonoids from different subgroups and flavanones that bear different substitutions on relevant chemical positions were tested for their ability to block TRPM3. Since the novel TRPM3 blockers should be used for in vivo studies, the cytotoxicity of each compound was determined. Two non-toxic TRPM3 blockers were chosen to be applied in in vivo experiments. The ability of the TRPM3 blockers to diminish PregS- and heat-induced nocifensive behaviour in vivo was investigated.

23 3.7 Relationship of the publications

The first publication summarizes the results from the primary screen (Chapter 4). Three of the four idendified TRPM3 blockers belong to the citrus fruit flavanones (naringenin, hesperetin and eriodictyol) and the fourth compound is a fabacea sec- ondary metabolite (ononetin). The IC50 for the compounds ranged from 0.3 and 0.5 µM for ononetin and naringenin to 1.0 µM and 2.2 µM for eriodictyol and hesperetin, respectively. Besides the potency of the compounds to block TRPM3, we investigated the selectivity of the TRPM3 blockers to discriminate between other TRP channels. Furthermore, the ability of naringenin and ononetin to block endogenously expressed TRPM3 was tested in DRG neurones. Based on the TRPM3-blocking compounds identified in the first screen, we tried to narrow down and optimise the phamacophore within the flavanone and flavonoid structure in a second screen (Chapter 5). Except for flavanones, no other tested flavonoid blocked TRPM3 in concentrations up to 25 µM, indicating that the flavanone substructure already represents an optimized backbone structure. Within the sec- ondary screen, we identified isosakuranetin and liquiritigenin as novel TRPM3 blockers that were, compared to the previously identified TRPM3 blockers, either more potent (isosakuranetin IC50 = 50 nM) or more selective (liquiritigenin) . Fur- thermore, two TRPM3 blockers (isosakurantin and hesperetin) diminished PregS- and noxious heat-induced nocifensive behaviour of mice. To obtain information about the bioavailability of isosakuranetin, the plasma concentrations of intrape- riotenally injected isosakuranetin was measured at different time points after the injection.

The function of TRPM3 in DRG neurones is similar to the pain-relevant TRPV1, and TRPV1 blockers have been investigated as potential analgesic drugs in clinical studies. However, besides their analgesic effect, TRPV1 blockers elevate the body temperature of the subjects in clinical studies, and thus, some clinical trials were stopped81. In contrary to TRPV1 blockers, neither isosakuranetin nor hesperetin elevated the body temperature of systemically treated mice. In conclusion, our in vivo experiments confirmed TRPM3 as a receptor for noxious heat. Furthermore, we showed that TRPM3 can be blocked by citrus fruit flavanones in vitro and in

24 vivo, and thus, flavanones can be used as a starting point for the development of novel analgesic therapies by targeting TRPM3.

25

British Journal of DOI:10.1111/bph.12076 www.brjpharmacol.org BJP Pharmacology

RESEARCH PAPER Correspondence M Schaefer, Rudolf-Boehm-Institut für Pharmakologie und Toxikologie, Citrus fruit and fabacea Universität Leipzig, Härtelstr. 16-18, 04107 Leipzig, Germany. E-mail: michael.schaefer@ secondary metabolites medizin.uni-leipzig.de ------Keywords potently and selectively TRP channels; flavonoids; DRG neurones block TRPM3 ------Received 5 July 2012 I Straub1, F Mohr2, J Stab2, M Konrad2, SE Philipp3, J Oberwinkler2 and Revised M Schaefer1 6 November 2012 Accepted 1Rudolf-Boehm-Institut für Pharmakologie und Toxikologie, Universität Leipzig, Leipzig, 19 November 2012 Germany, 2Institut für Physiologie und Pathophysiologie, Philipps-Universität Marburg, Marburg, Germany, and 3Experimentelle und Klinische Pharmakologie und Toxikologie, Universität des Saarlandes, Homburg, Germany

BACKGROUND AND PURPOSE The melastatin-related transient receptor potential TRPM3 is a calcium-permeable nonselective cation channel that can be activated by the neurosteroid pregnenolone sulphate (PregS) and heat. TRPM3-deficient mice show an impaired perception of noxious heat. Hence, drugs inhibiting TRPM3 possibly get in focus of analgesic therapy. EXPERIMENTAL APPROACH Fluorometric methods were used to identify novel TRPM3-blocking compounds and to characterize their potency and selectivity to block TRPM3 but not other sensory TRP channels. Biophysical properties of the block were assessed using electrophysiological methods. Single cell calcium measurements confirmed the block of endogenously expressed TRPM3 channels in rat and mouse dorsal root ganglion (DRG) neurones. KEY RESULTS By screening a compound library, we identified three natural compounds as potent blockers of TRPM3. Naringenin and hesperetin belong to the citrus fruit flavanones, and ononetin is a deoxybenzoin. Eriodictyol, a metabolite of naringenin and hesperetin, was still biologically active as a TRPM3 blocker. The compounds exhibited a marked specificity for recombinant 2+ TRPM3 and blocked PregS-induced [Ca ]i signals in freshly isolated DRG neurones. CONCLUSION AND IMPLICATIONS The data indicate that citrus fruit flavonoids are potent and selective blockers of TRPM3. Their potencies ranged from upper nanomolar to lower micromolar concentrations. Since physiological functions of TRPM3 channels are still poorly defined, the development and validation of potent and selective blockers is expected to contribute to clarifying the role of TRPM3 in vivo. Considering the involvement of TRPM3 in nociception, TRPM3 blockers may represent a novel concept for analgesic treatment.

Abbreviations 2+ 2+ [Ca ]i, intracellular free Ca concentration; DRG, dorsal root ganglion; HBS, HEPES-buffered solution; NSAIDs, non-steroidal anti-inflammatory drug; PregS, pregnenolone sulphate; TRPA, transient receptor potential ankyrin-like; TRPM, transient receptor potential melastatin-related; TRPV, transient receptor potential vanilloid-related

© 2012 The Authors British Journal of Pharmacology (2013) 168 1835–1850 1835 British Journal of Pharmacology © 2012 The British Pharmacological Society 27 BJP I Straub et al.

Introduction was introduced in frame after the start codon of the TRPM3a2 cDNA (GenBank accession AJ544535), ligated into pcDNA3 TRPM3, the third member of the melastatin-related TRPM and transfected into HEK293 cells. Transfected cells were -1 channel subfamily, is still poorly characterized. The TRPM3 selected in a medium containing 500 mg·mL G418 for 4 gene is transcribed into different alternative splice variants. weeks. Single cells were separated by FACS on a MoFlo cell Alternative splicing in a region that encodes the putative pore sorter (Beckmann Coulter, Krefeld, Germany) and expanded. region of TRPM3 changes the selectivity of ion permeation. Clones were tested in Western blots using monoclonal anti- Whereas the TRPM3a2 variant is a calcium-permeable, poorly TRPM3 and anti-myc antibodies, and analysed for PregS- 2+ selective cation channel, TRPM3a1 features 12 additional induced Ca signals in single cell calcium measurement amino acids within the pore domain and is selective for experiments. HEK293 cells stably expressing other sensory monovalent cations (Oberwinkler et al., 2005). In this manu- TRP channels (HEKhTRPA1, HEKhTRPM8:CFP, HEKrTRPV1:YFP) were script, we focus on the TRPM3a2 variant, which is expressed obtained by a limiting dilution method as described recently in various neuronal and non-neuronal tissues and can be (Hellwig et al., 2004; Urban et al., 2012). All cells were grown activated by the neurosteroid pregnenolone sulphate (PregS) at 37°C in a humidified atmosphere containing 5% CO2. or high concentrations of nifedipine (Wagner et al., 2008), Unless otherwise stated, cells were seeded 24 h prior to the experiments on poly-L-lysine-coated coverslips. by noxious heat (Vriens et al., 2011), and by D-erythro- sphingosine (Grimm et al., 2005). The physiological role of TRPM3 is still not firmly estab- DRG neuron preparation lished. PregS-induced activation of TRPM3 has been linked to Adult C57BL6/N mice, bred and kept at the local animal care insulin secretion in pancreatic beta-cells (Wagner et al., facility (Uniklinikum des Saarlandes, Homburg, Germany) 2008), to vascular smooth muscle cell contraction (Naylor were killed by an overdose of i.p. urethane (25% in 0.9% NaCl et al., 2010), and to potentiation of glutamatergic transmis- solution). Mice were opened from the dorsal side, and the sion in cerbellar Purkinje neurons from developing rats spinal cord was carefully exposed. The spinal cord was (Zamudio-Bulcock et al., 2011). Recently, Vriens et al. then gently removed; and DRG from all cervical, thoracic observed that TRPM3-deficient mice show an impaired per- and lumbar segments were obtained. Ganglia were digested ception of noxious heat. Hence, drugs inhibiting TRPM3 may for 15 min at 37°C in an enzyme solution containing -1 provide novel instruments for analgesic therapy. In addition, 0.1 mg·mL trypsin (Sigma-Aldrich, Deisenhofen, Germany), -1 TRPM3 blockers may help to unravel biological functions of and 44.4 mg·mL liberase DH (Roche, Mannheim, Germany), TRPM3 that might be obscured by compensatory effects in dissolved DMEM in (Gibco; Invitrogen). After repeated tritu- gene targeting or knock-down approaches. ration with a 1 mL pipette, the digestion was stopped by TRPM3 channel blockers described so far include the adding 100 mL FCS; the cell suspension was centrifuged antidiabetic PPARg-agonists rosiglitazone and troglitazone (3.5 min at 670 g) and resuspended in DMEM medium, sup- -1 (Majeed et al., 2011) and nonsteroidal anti-inflammatory plemented with 10% FCS, 100 units·mL penicillin, and -1 drugs (NSAIDs) of the fenamate group (Klose et al., 2011). 0.1 mg·mL streptomycin. Subsequently, droplets of cell sus- -1 Furthermore, TRPM3 is blocked by 2-APB (Xu et al., 2005) or pension were plated on poly-D-lysine (1 mg·mL ; Sigma- by lanthanum and gadolinium ions (Harteneck and Schultz, Aldrich)-coated coverslips, placed in a culture dish and left to 2007) that block a plethora of different TRP channels. adhere for at least 1 h in an incubator (37°C, 5% CO2). The By screening a compound library, we identified three culture dish was then filled with growth medium. All experi- natural compounds as potent TRPM3 blockers. Two of them, ments were performed within 36 h after preparation. naringenin and hesperetin, belong to the citrus fruit fla- To obtain rat DRG neurones, 8 week-old Wistar rats were vanones and ononetin is a deoxybenzoin. Based on its struc- killed by an overdose of CO2. The preparation of DRG neu- tural similarity to naringenin and hesperetin, we also rones was performed as described above. For patch clamp -1 identified eriodictyol as a potent TRPM3 blocker. Further- measurements, NGF (30 ng·mL ; Sigma-Aldrich) was added more, we confirm the biological activity of naringenin, onon- to the medium. Wistar rats (Janvier, Saint Berthevin Cedex, etin and eriodictyol in natively TRPM3-expressing dorsal root France) were housed under a 12 h light–dark cycle and ganglia (DRG) neurones. allowed access to laboratory animal feed and water ad libitum. All studies involving animals are reported in accordance with the ARRIVE guidelines for reporting experiments involving animals (Kilkenny et al., 2010; McGrath et al., 2010). All Methods experimental procedures involving animals were approved by the Committee on Animal Care and Use of the local govern- Cell culture mental body. Procedures were optimized to reduce the HEK293 cells used for transient transfection with TRPM1 number of animals and their suffering, according to the regu- were cultured in MEM medium (Invitrogen, Darmstadt, lations of the German Animal Welfare Act. A total number of

Germany) containing 10% FCS at 37°C and 5% CO2. Cells nine animals (two mice and seven rats) were used in experi- were passaged one to three times a week. HEK293 cells were ments described here. cultured in Earle’s Minimum Essential Medium supple- mented with 10% fetal calf serum, 2 mM L-glutamine, 100 Cell transfection units·mL-1 penicillin, 0.1 mg·mL-1 streptomycin. For genera- HEK293 cells were transiently transfected with a bi-cistronic tion of a HEK293 cell line stably expressing myc-tagged plasmid encoding Myc-tagged TRPM1 and enhanced green

mouse TRPM3a2 (HEKmTRPM3), the cDNA of the myc epitope fluorescent protein as previously described (Lambert et al.,

1836 British Journal of Pharmacology (2013) 168 1835–1850 28 Identification of novel TRPM3 blockers BJP

2011). The Polyfect transfection reagent (Qiagen, Hilden, tained 3 mM glucose and 7 mM mannitol instead of 10 mM Germany) was used according to the instructions of the glucose. Images were captured at alternating excitation wave- manufacturer. One day after the transfection, cells were split lengths of 340 and 380 nm with a 10¥ SFluor objective and reseeded at a reduced density. Cells were used for experi- (Nikon, Düsseldorf, Germany) and a cooled CCD camera ments 36 to 72 h after the transfection. For heat activation of (QImaging, Surrey, Canada). Background subtraction and TRPM3, HEK293 cells were transiently transfected with a ratio calculation were performed with ImageJ utilizing a plasmid encoding TRPM3a2-GFP. Transfection was per- modified version of the ‘ratio-plus’ plugin. 2+ formed with FugeneHD (Promega, Mannheim, Germany) and For single-cell [Ca ]i measurement in rat DRG neurones, 48 h after transfection, cells were split and reseeded on poly- coverslips were mounted on a monochromator-equipped L-lysine-coated coverslips. (Polychrome, TILL-Photonic) inverted epifluorescence micro- scope (Carl Zeiss, Jena; 10¥/0.5 Fluar objective), and super- Intracellular Ca2+ analysis in cell suspensions fused with HBS buffer. Detection and calibration of the All fluorometric assays in cell suspensions were performed at calcium concentration was performed with a spectral unmix- room temperature in a 384-well microtitre plate format. For ing method as described earlier (Lenz et al., 2002). All meas- detailed information, see Norenberg et al. (2011). Shortly, urements were performed at room temperature. HEK293 cells stably expressing TRPM3 were incubated with Fluo-4/AM (4 mM; Molecular Probes, Invitrogen) for 30 min Whole-cell patch clamp measurements at 37°C, washed and resuspended in HEPES-buffered solution Standard electrophysiology methods were used. All measure- (HBS) buffer, containing 135 mM NaCl, 6 mM KCl, 1 mM ments were performed at room temperature, expect for tem-

CaCl2, 1 mM MgCl2, 5.5 mM D-glucose, and 10 mM HEPES perature activation of TRPM3, using an Axopatch 200B adjusted to pH 7.4 with NaOH. For the primary screening, amplifier with a Digidata 1200 digitizer (Axon CNS, Molecu- wells were prefilled with individual compounds of a Spec- lar Devices, Sunnyvale, CA, USA) under the control of the trum Collection compound library (MicroSource Discovery Pclamp 9 software (Molecular Devices). Voltage ramps (from Systems, Gaylordsville, CT) at a final concentration of 20 mM. -113 to +87 mV, 1 mV·ms-1) were applied every second from Measurement was executed with a filter-based plate reader a holding potential of -13 mV. The intracellular solution device (Polastar Omega, BMG Labtech, Offenburg, Germany), contained 80 mM Cs-aspartate, 45 mM CsCl, 4 mM Na2ATP applying 485/10- and 520/20-nm band pass filters. After or TRIS2-ATP, 10 mM HEPES, 10 mM BAPTA, pH 7.2 adjusted recording a baseline of 160 s (10 cycles), PregS 35 mM, final with CsOH. The osmolarity was 300–315 mosm. The extra- concentration, was injected into each well; and fluorescence cellular solution used contained 145 mM NaCl, 3 mM KCl, intensities were followed for 10 min after agonist injection. 10 mM CsCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM HEPES, To obtain concentration–response functions, various concen- 10 mM D-glucose, pH 7.4 adjusted with NaOH. The liquid trations of the blocking compounds were pre-incubated for junction potential of approximately 13 mV between the bath 10–15 min, and data were subjected to nonlinear curve fitting solution and the pipette solution (calculated with Clampex, applying a four-parameter Hill equation (Emin, Emax,IC50 and Axon Instruments, Molecular Devices) was corrected. The Hill coefficient). sampling rate was 5 kHz in standard whole-cell measure- Measurements of agonistic properties of the compounds ments. During fast application of naringenin, the sampling were performed using a custom-made fluorescence plate rate was 20 kHz. Modulators were applied via a SF-77B Per- imaging device built into a robotic liquid handling station fusion Fast Step system (Warner Instruments). Current decay (Freedom Evo 150, Tecan, Switzerland). The plate imaging kinetics were calculated with Clampfit 9.2 (Molecular device comprised a 460-nm LED array, filtered with a 475 nm Devices), using a biexponential fitting. For heat activation of short-pass filter (DT-blue, Optic Balzers, Oerlikon, Switzer- TRPM3, the bath solution was cooled to 17–20°C under land) and a cooled CCD camera (Coolsnap FX, Photometrics, control conditions. To activate and block TRPM3, pre-cooled Tucson, AZ), equipped with a Xenon 0.95/25 C-mount lens solutions were heated to 37–40°C with an inline solution (Schneider Kreuznach, Bad Kreuznach, Germany), and a heater (Warner Instruments) under control of a TC-324B 515 nm long-pass filter (Y515-Di, Fujifilm, Tokyo, Japan). The Temperature Controller (Warner Instruments). To monitor imaging was controlled with the MicroManager software the actual temperature of the bath solution, a thermistor (Edelstein et al., 2010), and fluorescence intensities were electrode was situated in the vicinity of the patched cell, and quantified over single wells, corrected for background signals the temperature was logged during the measurement with the and expressed as F/F0, using ImageJ (Abramoff et al., 2004). Pclamp 9 software. For recordings in DRG neurones, the

extracellular solution contained 140 mM NaCl, 2 mM MgCl2, Single-cell calcium measurement 4 mM KCl, 0.5 mM lidocain, 10 mM TRIS (pH 7.4 with HCl), Transfected HEK293 cells attached to coverslips were loaded and the pipette solution contained 140 mM CsCl, 0.6 mM with 5 mM Fura-2/AM (Biotium, Hayward, CA, USA) for MgCl2, 1 mM EGTA, 10 mM HEPES, 5 mM TEA (pH 7.2 with 30 min at room temperature or, in the case of DRG neurons, CsOH). Although lidocain activates recombinant rTRPV1 at 37°C. Coverslips were mounted in a recording chamber with an EC50 of about 12 mM (Leffler et al., 2008), the con- (Warner Instruments, Hamden, CT, USA) and superfused with centration we used to block voltage-dependent sodium chan- the aid of a computer-controlled valve bench (ALA Scientific, nels should not strongly influence TRPV1. Farmingdale, NY, USA). For the measurements in mouse DRG neurones, we used the same extracellular solution as for Stock solutions and drugs whole-cell patch clamp measurements (see below). For meas- All modulators were dissolved in DMSO. The concentration urement of transfected HEK293 cells, the bath solution con- of stock solutions was chosen in such a way that the DMSO

British Journal of Pharmacology (2013) 168 1835–1850 1837 29 BJP I Straub et al.

concentration in the final solution never exceeded 0.14% at of 35 mM is higher than the reported EC50 of 12 and 23 mM for the highest concentration of the respective modulator and TRPM3 inward and outward currents (Wagner et al., 2008) but further reduced by serial dilution of the compounds. PregS, is not yet saturating. Naringenin and hesperetin displayed a

naringenin, hesperetin, eriodictyol and ononetin were pur- potent block with IC50 values of 0.5 Ϯ 0.07 mM (Figure 2A) and chased from Sigma-Aldrich. 2.0 Ϯ 0.1 mM (Figure 2B) respectively. Ononetin displayed an

IC50 of 0.3 Ϯ 0.03 mM (Figure 2C) and eriodictyol blocked Ϯ Statistical analysis TRPM3 with an IC50 of 1.0 0.07 mM (Figure 2D). The Hill All error bars represent the SEM. The number of cells analysed coefficients ranged between 1.4 and 2.2 for the investigated in each experiment is indicated in the figure legends. To test compounds (see Figure 2). for statistically significant differences, we used ANOVA with the To further characterize the new blockers, we performed Tukey’s post test. In the figures, one star indicates P < 0.05, whole-cell patch clamp measurements in the presence of two stars indicate P < 0.01, three stars indicate P < 0.001. naringenin (Figure 3) and ononetin (Figure 4). The onset of the naringenin-induced block was fast, but a complete block was only achieved after a few seconds (Figure 3A). Wash-out Results of naringenin allowed a reactivation of TRPM3 channels with PregS (Figure 3A). However, naringenin-blocked TRPM3 cur- rents recovered only slowly after removal of the modulator Screening for compounds that modulate (Figure 3A). Applying I/V ramp protocols, we observed that TRPM3 channel activity the naringenin-induced block is largely voltage-independent To identify compounds that exert a biological activity to (Figure 3B) and reversible. In order to more rapidly deliver the modulate TRPM3, we performed a medium throughput compound to the patched cell, we performed some experi- screen. To this end, the Spectrum Collection compound ments with a fast application system and supramaximally library, comprising 2000 drugs, drug-like molecules, natural effective concentrations of naringenin (Figure 3C). A biexpo- compounds or toxins, was used at a final concentration of nential fit of the decaying current kinetic revealed fast and 20 mM. HEK293 cells stably expressing mTRPM3 (HEKmTRPM3) slow t-values of 291 and 2370 ms. The major component of were loaded with the fluorescent calcium indicator dye the current (77%) decayed with tfast, and only 23% of the Fluo-4, dispensed into 384-well plates, and PregS-induced current was blocked with a tslow (Figure 3C). Interestingly, calcium entry was detected. As a result of the primary screen, naringenin exerted a slightly higher blocking potency in we identified three compounds that completely blocked the whole-cell measurements compared with the calcium assay. PregS-induced calcium entry (Figure 1A–C). Furthermore, we Electrophysiologically measured concentration–response confirmed the previously identified TRPM3 channel-blocking curves of naringenin showed that inward currents were more properties of the fenamates tolfenamic acid and mefenamic potently blocked (IC50 = 0.27 Ϯ 0.04 mM) compared to the acid (Klose et al., 2011). The three newly identified blocking outward currents (IC50 = 0.37 Ϯ 0.005 mM). Although the compounds belong to a class of plant secondary metabolites. potency of TRPM3 block displayed some voltage dependence, The flavonoids naringenin and hesperetin are contained in both current components were naringenin-sensitive and grapefruits and oranges respectively. Ononetin is a deoxyben- almost completely blocked at a concentration of 3 mM zoin that has originally been synthesized from Ononis (Figure S1). spinosa-derived isoflavanones, but may also be obtained from The current decay after ononetin-induced block devel- fabaceae. In grapefruit, a major fraction of naringenin is con- oped faster than the naringenin induced block of TRPM3, jugated with the disaccharide neohesperidose. This glycoside and the block was rapidly reversible (Figure 4A). Comparable is referred to as naringin, and accounts for the bitter taste of to naringenin, ononetin blocked TRPM3 in a voltage- grapefruit (Manach et al., 2004). Interestingly, neither nar- independent fashion (Figure 4B). In electrophysiological ingin nor hesperidin, the glycoside of hesperetin, blocked recordings, ononetin showed approximately the same TRPM3 (Figure 1A,B). efficiency as previously found in Fluo-4 measurements. A Hit verification in a secondary screen with freshly dis- concentration of 0.3 mM ononetin blocked 62% Ϯ 4% of solved compounds confirmed the ability of the citrus fruit PregS-induced currents at -113 mV and 53% Ϯ 4% at +87 mV, flavanones and ononetin to block TRPM3. Furthermore, we and at a concentration of 3 mM, the currents were completely found that eriodictyol, which is found in lemons or formed abolished (Figure 4C). by hepatic metabolism of naringenin and hesperetin (Brein- To exclude that the blocking effect of naringenin and 2+ holt et al., 2002), completely blocked the PregS-induced Ca ononetin is restricted to PregS activation of TRPM3, we per- entry in HEKmTRPM3 cells when pre-incubated at a concentra- formed whole-cell measurements with nifedipine as activator tion of 20 mM (Figure 1D,E). of TRPM3 (Wagner et al., 2008). As shown in supplemental Figure 2, nifedipine (50 mM)-induced TRPM3 currents were Concentration dependence and concentration-dependently (tested concentrations: 1 mM and electrophysiological properties of the 3 mM) blocked by naringenin or ononetin. flavonoid-induced TRPM3 block Since heat may be a physiological activator of TRPM3, we To evaluate the potency of the flavonoids to block TRPM3, tested if the newly identified compounds also block heat- we used a fluorometric Ca2+ assay to obtain concentration- induced TRPM3 currents. HEK293 cells transiently trans-

response curves. HEKmTRPM3 cells were incubated with different fected with TRPM3a2 showed small outward and inward concentrations of the test compounds, and PregS-induced currents after raising the bath solution to temperatures above calcium entry was measured. The applied PregS concentration 37°C (Figure 5). These currents were partially blocked by

1838 British Journal of Pharmacology (2013) 168 1835–1850 30 Identification of novel TRPM3 blockers BJP

Figure 1

Identification of TRPM3 channel-blocking compounds. Fluo4-loaded HEK293 cells stably expressing TRPM3 (HEKmTRPM3) were incubated with single compounds (20 mM) of the Spectrum Collection, and fluorescence intensities were detected during injection of the TRPM3 activator PregS. (A–D) Examples of calcium entry traces extracted from a single 384-well plate measurement. Light grey trace represents a DMSO control (A–D). Black trace: (A) hesperetin, (B) naringenin, (C) ononetin and (D) eriodictyol. Dark grey: (A) hesperidin and (B) naringin. (E) Chemical structures of verified hits naringenin, hesperetin, ononetin and eriodictyol as well as the flavonoid glycoside hesperedin and naringin.

5 mM naringenin and ononetin (Figure 5A). About 80% of the also gave rise to substantial inward and outward currents heat-dependent outward currents were blocked by narin- (Figure 5F), In contrast to transfected HEK293 cells, ononetin genin or ononetin, whereas only 40–60% of the inward cur- and naringenin did not diminish neither inward nor outward rents were blocked (Figure 5C). Hesperetin (10 mM) and currents in the untransfected parental HEK293 cells. We con- eriodictyol (20 mM) also partially blocked heat-induced acti- clude that background currents that are not sensitive to vation of TRPM3 (Figure 5D,E). Of note, raising the tempera- TRPM3 blockers may cause an underestimation of the effi- ture in measurements with untransfected parental HEK cells ciency of blocker effects on the heat-activated TRPM3.

British Journal of Pharmacology (2013) 168 1835–1850 1839 31 BJP I Straub et al.

Figure 2

Concentration–response curves of TRPM3-blocking compounds. HEKmTRPM3 cell suspensions were incubated with various inhibitor concentrations as indicated. Fluorescence intensities were measured during PregS-induced activation of TRPM3. TRPM3 activation without an inhibitor was set as 100% and fluorescence intensities containing inhibitors were normalized to this value. (A–D) Concentration–response curves of naringenin, ononetin, hesperetin and eriodictyol are shown. Each data point represents at least six independent experiments for naringenin and ononetin and

four independent experiments for hesperetin and eriodictyol with duplicates each. Shown are mean values and SEM. IC50 values and Hill coefficients (n) were obtained by non-linear curve fitting, applying a four-parameter Hill equation.

Biophysical properties of TRPM3 block by these observations prompted us to investigate whether the naringenin and ononetin blockers may interfere with the activator binding. To this To more precisely specify the blocking mechanism we tested end, TRPM3-expressing cells were incubated with naringenin from which side of the plasma membrane naringenin and or ononetin, and calcium entry was measured after activation ononetin act on TRPM3 channels by adding the substances of TRPM3 with different PregS concentrations (Figure 7). to the intracellular solution. Even at a concentration of Lowering the PregS concentration did not lead to significant 3 mM, intracellularly applied ononetin was unable to block changes of the IC50 values of ononetin and naringenin. Increasing the concentration of PregS to 100 mM, however, PregS-induced currents in HEKmTRPM3 cells, whereas addition of the TRPM3 blocker to the bath solution was still effective shifted the IC50 of ononetin to a higher concentration (Figure 6A). Naringenin showed similar results, however at (1.9 mM) (Figure 7A); whereas the IC50 of naringenin concentrations above 0.6 mM in the pipette, gigaseals were remained almost unaffected (Figure 7B). frequently lost during whole-cell patch-clamp measure- ments (Figure 6D). Although this concentration is sufficient Specificity of TRPM3 channel blockers to almost completely block PregS-induced currents when To examine the specificity of the blockers, we first investi- added from the outside (Figure 3C), there was no overt gated the ability of naringenin and ononetin to block the decrease in the current densities after PregS-induced TRPM3 most closely related channel TRPM1. Like TRPM3, TRPM1 channel activation in cells that were patched with narin- can be activated by PregS (Lambert et al., 2011). At a concen- genin in the pipette solution. These findings strongly hint tration of 10 mM, naringenin did not exert a strong inhibition at an extracellularly accessible binding site for naringenin of the PregS-mediated calcium entry in HEK293 cells that and ononetin. express TRPM1 (Figure 8A). In contrast to naringenin, 10 mM Electrophysiological measurements showed differences in ononetin partially attenuated the PregS-induced calcium the blocking behaviour of naringenin and ononetin. Whereas responses in TRPM1-expressing cells by about 50% ononetin showed a fast and rapidly reversible block, the (Figure 8B). In agreement with Lambert et al., TRPM1 chan- naringenin-induced block recovered with a slower off-rate. nels were almost completely and reversibly blocked by Together with the fact that PregS is an activator of TRPM3 100 mMZn2+ in contrast to TRPM3 channels (Wagner et al., channels from the extracellular side (Wagner et al., 2008), 2010).

1840 British Journal of Pharmacology (2013) 168 1835–1850 32 Identification of novel TRPM3 blockers BJP

Figure 3

Electrophysiological properties of naringenin-mediated TRPM3 block. (A) Representative recordings of whole-cell currents in HEKmTRPM3 cells induced by 35 mM PregS and reversibly blocked with 3 mM naringenin. Data are extracted from voltage ramps and depict the current at 87 mV

(upper trace) and -113 mV (lower trace). Dotted line: zero current level. (B) To assess the blocker activity in HEKmTRPM3 at different potentials, slow ramps (1 mV·ms-1) ranging from -100 to 50 mV were applied in the presence of PregS before (1), during (2) and after (3) addition of 3 mM naringenin. (C) Representative whole-cell recording, a HEKmTRPM3 cell clamped at -83 mV, showing currents in response to 35 mM PregS and currents blocked with 10 mM naringenin. Naringenin was acutely applied with a fast perfusion system. Insert: superposition of the decaying phase of TRPM3 currents and a biexponential fit of the data. (D) Statistical analysis of 6–12 independent experiments performed as shown in (A), but with various concentrations of naringenin. Dashed line: Level of background conductivity in the absence of PregS and naringenin. Statistically significant differences of TRPM3 activation with PregS and block with naringenin are indicated as asterisks.

Since an involvement of TRPM3 in thermal nociception genin (Figure S3B). However, further investigations are has been shown, we further investigated the ability of the needed to clarify the complex mechanism of naringenin to flavanones hesperetin, naringenin and eriodictyol as well as block and activate TRPM8. the deoxybenzoin ononetin to block sensory TRP channels Hesperetin and eriodictyol neither blocked nor activated that are expressed in DRG neurones, including TRPA1, any tested TRP ion channels other than TRPM3 when applied TRPM8 and TRPV1. Naringenin did not have an influence on in concentrations up to 50 mM (Figure 9B,D). Ononetin, the TRPA1, whereas at very high concentrations, naringenin most potent TRPM3 channel blocker, activated TRPA1 in exerted a partial inhibition of TRPV1 (Figure 9A). Naringenin fluorometric measurements (Figure 9F) but had no overt activated TRPM8 (Figure 9E) and prevented a subsequent acti- influence on TRPM8 or TRPV1 at concentrations up to vation by menthol (Figure S3A). Interestingly, menthol- 100 mM (Figure 9C). Since many compounds that promote induced currents in HEK cells expressing TRPM8 were TRPA1 activation in fluorometric Ca2+ assays act in a light- blocked after an additional perfusion of the cells with narin- dependent fashion (Hill and Schaefer, 2009), we reassessed

British Journal of Pharmacology (2013) 168 1835–1850 1841 33 BJP I Straub et al.

Figure 4

Electrophysiological properties of ononetin-induced TRPM3 block. (A) Whole-cell recording from a HEKmTRPM3 cell during stimulation with 35 mM PregS and acute addition of 3 mM ononetin. Data are extracted from voltage ramps and depict current amplitudes at 87 mV (upper trace) and -113 mV (lower trace); dotted line: zero current level. (B) I/V curve of PregS-activated TRPM3 currents before (1), during (2) and after (3) addition

of 3 mM ononetin. (C) Statistical analysis of peak current densities in resting HEKmTRPM3 cells after addition of 35 mM PregS and different concentrations of ononetin. Data represent means and SEM of at least 11–16 independent experiments.

the TRPA1 activation in electrophysiological experiments. Next, we investigated if flavonoids are able to block Whole-cell measurement of TRPA1 currents did not show TRPM3 that is endogenously expressed in rat DRG neu- significant current increases after challenging the cells with rones. First, we detected mRNA of TRPM3 in freshly isolated ononetin at concentrations up to 30 mM (Figure S4A). We rat DRG neurones via RT-PCR (data not shown). Single cell 2+ further tested if the activation of TRPA1 by ononetin involves [Ca ]i measurements revealed PregS-inducible, TRPM3-like 2+ oxidative intermediates. Indeed, [Ca ]i responses in TRPA1 signals that were blocked by naringenin (data not shown) or cells stimulated with low ononetin concentrations (6.25 mM) ononetin (Figure 11A). Of note, the KCl-triggered activation were strongly attenuated in the presence of the reducing of voltage-gated Ca2+ channels, identifying neuronal cells in agent DTT (10 mM, applied to the bath solution; Figure S4C– the cultures, was not significantly affected by eriodictyol, 2+ E). At higher ononetin concentrations of 12.5–25 mM, DTT naringenin and ononetin, reaching steady-state [Ca ]i con- exerted a lower or no inhibitory activity. By contrast, DTT centrations of 698 Ϯ 45 nM (n = 10), 626 Ϯ 130 nM (n = 6) supplementation did not attenuate the block of TRPM3 by and 700 Ϯ 76 nM (n = 6) respectively. We further showed ononetin. We conclude that oxidative intermediates of onon- that eriodictyol blocked PregS-induced calcium entry in rat etin may be involved in activating TRPA1, but not in the DRG neurones (Figure 11B). From 165 measured DRG neu- block of TRPM3 by ononetin. rones, 90 responded to PregS of which 39 also showed a Next, we tested if the TRPM3-blocking compounds inter- response to 2 mM capsaicin. These data indicate that rat fere with the endogenously expressed TRPM7 in HEK293 DRG neurones also functionally express Ca2+-permeable cells. Since external divalent cations are permanent blockers TRPM3 isoforms. of TRPM7, switching to a bath solution lacking Ca2+ and Mg2+ Eriodictyol has been proposed as a blocker of TRPV1 with gives rise to large inward currents (Nadler et al., 2001). antioxidant activity (Rossato et al., 2011). In our hands, erio- Neither TRPM7-dependent inward nor outward currents were dictyol neither blocked capsaicin induced calcium entry in influenced by ononetin, hesperetin or eriodictyol (20 mM HEK293 cells stably expressing rat TRPV1 (Figure 9D) nor in each; Figure S5A). Naringenin partially blocked TRPM7 cur- freshly isolated rat DRG neurones (Figure 11B). Overall, 114 rents at a concentration of 20 mM (see Figure S5A,B). of 165 DRG neurones (69%) were still sensitive to capsaicin, although the neurones were treated with 20 mM eriodictyol. Efficiency of naringenin and ononetin to To specify the influence of eriodictyol on TRPV1, we per- block endogenous TRPM3 channels in formed whole-cell measurements of freshly isolated rat DRG isolated DRG neurones neurones. Capsaicin-induced inward currents in DRG neu- We used mouse DRG neurones to investigate the influence rones were not diminished after perfusion with 20 mM erio- of naringenin and ononetin on endogenously expressed dictyol (Figure 11C). TRPM3. Neither ononetin nor naringenin (5 mM each) had an influence on DRG neurones without a previous activation of TRPM3 by PregS (Figure 10A,D), whereas PregS-induced Discussion calcium entry was blocked by naringenin as well as ononetin (Figure 10B,E). The block of ononetin and naringenin on In this study, we identified citrus fruit flavanone aglycones as

endogenously expressed TRPM3 was reversible, according to potent and selective TRPM3 channel blockers. With IC50

the measurements in HEKmTRPM3 cells. A significant fraction of values in the upper nanomolar (naringenin and ononetin) or calcium entry reappeared upon removal of naringenin or low micromolar range (hesperetin and eriodictyol), these 2+ ononetin in the continuous presence of PregS (Figure 10B,E). natural compounds completely and reversibly abrogate Ca

1842 British Journal of Pharmacology (2013) 168 1835–1850 34 Identification of novel TRPM3 blockers BJP

Figure 5 Heat-induced TRPM3 currents were partially blocked by TRPM3 blocker, whereas currents in non-transfected HEK293 cells were not affected by TRPM3-blocking compounds. (A) Representative whole-cell recording from a HEK293 cell transiently transfected with TRPM3a2. Elevation of the temperature of the bath solution elicited outward and inward currents (measured at 87 and -113 mV, respectively) that could be partially blocked by ononetin (10 mM) and naringenin (10 mM). (B) I/V curve of heat-activated (about 38°C) TRPM3 currents before (2) and during superfusion with 10 mM ononetin (3) or naringenin (4). (C) Aggregated data of five to six independent measurements. (D,E) Representative whole-cell measure- ments performed as shown in panel A, but with 10 mM hesperetin (D) or 20 mM eriodictyol (E). (F) Control experiments in non-transfected parental HEK293 cells reveal that heat-activated background currents are unaffected by superfusion with either naringenin or ononetin (10 mM each). In the presence of NS8593 (30 mM), a blocker of small conductance K+ channels (SK1-SK3) and TRPM7 channels, heat-activated outward currents were partially inhibited. entry and ionic currents through recombinantly expressed citrus fruits. Epidemiological and animal studies point to a TRPM3a2 and block the pregnenolone sulphate-inducible possible biological activity of citrus flavonoids that might be Ca2+ entry in primary cultures of mouse or rat DRG neurones, advantageous in diseased states, such as cardiovascular disor- indicating biological activity towards endogenously ex- ders or cancer (for review see Benavente-Garcia and Castillo, pressed TRPM3. 2008). In contrast to agonistic compounds that mimic the Sensory TRP channels are influenced by a large and still sensory quality of the respective TRP channel, such as a growing number of natural compounds, thereby mediating burning sensation induced by capsaicin via TRPV1 activation, thermal, gustatory, odorant or eventually painful sensations chemically defined blockers do not interfere with the channel (Vriens et al., 2008). Flavonoids comprise the most common activity in the absence of an activator. Thus, it is not unex- group of plant-derived polyphenolic compounds in the pected that, thus far, no specific sensation has been linked to human diet. Flavanones, like naringenin and hesperetin, are the TRPM3-blocking agents. The characteristic taste of nar- a class of polyphenols that is almost exclusively contained in ingin or hesperidin is restricted to the respective glycoside

British Journal of Pharmacology (2013) 168 1835–1850 1843 35 BJP I Straub et al.

Figure 6

Binding site of naringenin and ononetin is accessible from the extracellular side. (A) and (D), representative whole-cell recording from a HEKmTRPM3 cell internally perfused with 3 mM ononetin (A) or 0.6 mM naringenin (D). An additional extracellular application of 35 mM PregS is still able to activate TRPM3 induced currents, which were reversibly blocked by 1 mM ononetin (A) or 1 mM naringenin (D) when added to the bath solution in the continued presence of PregS. Data are extracted from voltage ramps and depict the current at 87 mV (upper trace) and -100 mV (lower trace). Dashed lines: zero current level. Slow voltage ramps (1 mV·ms-1) were applied, in the presence of 3 mM ononetin (B) or 0.6 mM naringenin

(E) in the pipette, to PregS-stimulated HEKmTRPM3 before (1), during (2) and after (3) extracellular perfusion with 1 mM ononetin (B) or 1 mM naringenin (E). (C and F) Statistical analysis of experiments indicated in panel A or D. Data represent mean values and SEM of at least seven independent experiments for panels A–C and six independent experiments for panels D and E.

forms, but absent in the cognate TRPM3-blocking aglycones, 1995). Considering that the glycosides did not exhibit thereby ruling out that TRPM3 is involved in sensing the TRPM3-blocking properties, the intestinal processing to their bitter taste of grapefruit. cognate aglycones represents a bioactivation pathway. In the intestine, the flavanone glycosides hesperidin and Until recently, naringenin has been thought to cause naringin are deglycosylated to the membrane-permeable food-drug interactions by inhibiting cytochrome P450 aglycones, and are then absorbed (Felgines et al., 2000). enzymes. However, in vitro studies have shown that naringin Specifically, the rutinose or neohesperidose moieties are and naringenin attenuate CYP3A4 activity by about 50% rapidly hydrolysed by enzymes of bacterial origin, such as only when applied at concentrations of 200 mM (Bailey et al., a-rhamnosidase and b-glucosidases (Fuhr and Kummert, 2000). Meanwhile, the inhibitory effect of grapefruit juice on

1844 British Journal of Pharmacology (2013) 168 1835–1850 36 Identification of novel TRPM3 blockers BJP

Figure 7

Naringenin and ononetin show different blocking behaviour. A HEKmTRPM3 cell suspension was loaded with Fluo-4 and exposed to different concentrations of ononetin (A) and naringenin (B). Calcium entry was stimulated with two different concentrations of PregS to obtain concentration–response curves. (A) Concentration–response curves of ononetin in the presence of 17.5 mM PregS and in the presence of 100 mM PregS. (B) Concentration–response curves of naringenin-induced TRPM3 channel block in the presence of 17.5 mM PregS and in the presence of

100 mM PregS. Shown are means and SEM of at least three to five different experiments in duplicates each. IC50 values and Hill coefficients (n) were obtained by non-linear curve fitting, applying a four-parameter Hill plot.

Figure 8 Naringenin affects Ca2+ entry through TRPM1only weakly and ononetin partially blocks TRPM1 channels. Single cell calcium imaging experiments with HEK293 cells transiently transfected with cDNA plasmids encoding TRPM1 and loaded with the fluorometric Ca2+ indicator fura 2. TRPM1 was activated with 50 mM PregS alone and in the presence of 10 mM naringenin (n = 44) (A) or 10 mM ononetin (n = 50) (B), followed by application of PregS alone and in combination with 100 mM ZnCl2. Shown are means and SEM of at least four to six independent imaging experiments.

CYP3A4 has been attributed to more potently inhibiting con- TRP ion channels TRPV1, TRPM8, TRPA1. By contrast, higher stituents, such as the furanocoumarins bergamottin and ber- concentrations of naringenin modulated HEK293 cells stably gapten (Hanley et al., 2011). Nonetheless, naringenin and expressing TRPM8 in a complex fashion that was reminiscent hesperetin are substrates of CYP-mediated hydroxylation, of a partial agonism, followed by deactivation of TRPM8. resulting in the formation of the common metabolite erio- With respect to TRPM1, the closest relative of TRPM3, we dictyol. Since both the CYP substrates as well as their product found that 10 mM ononetin, but not naringenin inhibited 2+ display a similar IC50, and since the kinetics of the block is fast PregS-induced [Ca ]i signals that are mediated by TRPM1 by and reversible, we conclude that the biological activity to about 50%. Considering that the IC50 of ononetin to block block TRPM3 is neither related to CYP activity, nor to its TRPM3 is 0.3 mM, the selectivity of ononetin is still 30-fold inhibition. and can be employed to differentiate between these channels.

With an IC50 of 0.5 mM, naringenin is the most potent With an IC50 of 300 nM, the deoxybenzoin ononetin is, to TRPM3-blocking flavanone. The additional hydroxyl group at our knowledge, the most potent TRPM3 blocker that has been the C3′ atom found in hesperetin and eriodictyol diminishes identified so far. Ononetin is a still poorly characterized com- the potency of the TRPM3 block to low micromolar concen- pound. Ononetin and other deoxybenzoins have been shown trations. Despite a slightly lower potency, however, the C3′ to confer antioxidant properties and, at concentrations above hydroxylation increased the selectivity for TRPM3. Neither 10 mM, to inhibit tyrosinases (Ng et al., 2009). Since the eriodictyol nor hesperetin discernibly influenced the sensory ononetin-induced activation of TRPA1 is partially sensitive to

British Journal of Pharmacology (2013) 168 1835–1850 1845 37 BJP I Straub et al.

Figure 9 Effects of TRPM3 channel blockers on other sensory TRP channels. HEK293 cells stably expressing the indicated TRP channels were preincubated with naringenin (A), hesperetin (B), ononetin (C) or eriodictyol (D); and activation of the respective channels was followed by measuring increases 2+ in the fluorescence intensity of intracellularly loaded Fluo-4, or in HEKrTRPV1:YFP cells, by monitoring the Ca influx-mediated intracellular acidification, causing a decrease in the fluorescence intensity of co-expressed YFP (Hellwig et al., 2004). The respective activators are 2 mM capsaicin for TRPV1, 30 mM AITC for TRPA1 and 300 mM menthol for TRPM8. Data represent mean and SEM of three to six different experiments, performed in duplicate each. Grey curves indicate the Hill fit of TRPM3 block by the indicated compounds. (E) Naringenin-induced Ca2+ entry in

HEKhTRPM8:CFP and (F) ononetin-induced calcium entry in HEKhTRPA1 cells.

the reducing agent DTT, oxidated ononetin intermediates competitive interference between ononetin and the neuros- may be involved. The block of TRPM3, however, requires very teroid PregS with regard to TRPM3 binding. Indeed, the low ononetin concentrations, develops within a few seconds blocker sensitivity was attenuated when exposing TRPM3 to and is not counteracted by 10 mM DTT, arguing against a higher PregS concentrations (see Figure 7). However, both necessity of oxidation for TRPM3 block. Ononetin can be competitive binding to the activator site or allosteric modu- produced from the isoflavone ononin (Hlasiwetz, 1855), an lation that mutually impedes on the ligand-binding proper- isoflavone glycoside that is not only found in the name- ties may explain these effects. giving plant O. spinosa but also contained in edible plants, Of note, the TRPM3 gene transcripts are spliced into such as soy bean and clover. Owing to their structural simi- several splice variants that either bear a Ca2+-permeable pore larity to estradiol and transactivation of the estrogen recep- (e.g. the a2 variant) or to an a1 variant whose pore is selective tors ERa and ERb, isoflavones are regarded as phytoestrogens for monovalent cations and not activated by PregS (Oberwin- (Cederroth and Nef, 2009). A structural relationship between kler et al., 2005). Since activators of the a1 variant are still ononetin and steroids may provide the structural basis of a unknown, and since fluorometric Ca2+ assays are inapplicable

1846 British Journal of Pharmacology (2013) 168 1835–1850 38 Identification of novel TRPM3 blockers BJP

Figure 10 PregS-evoked Ca2+ influx in mouse DRG neurones is reduced by naringenin or ononetin. (A,B) Example traces of single DRG neurones in calcium imaging measurements. Freshly dissociated DRG neurones were loaded with Fura-2/AM, and fluorescence changes at 340 and 380 nm were measured. DRG neurones were superfused with 5 mM naringenin alone (A) or simultaneously with 50 mM PregS that was applied longer than the inhibitor (B). (C) Statistical analysis of data similar to those presented in (B). A total number of 70 DRG neurones were monitored in nine microfluorometric imaging experiments to obtain these data. (D,E) Sample traces of single DRG neurones similar to panel A except 5 mMof ononetin was applied alone (D) or simultaneously with PregS (applied longer than ononetin) (E). (F) Statistical analysis of experiments performed in panel E. A total number of 163 neurones were analysed in eight imaging experiments. Bath solutions contained (20 mM) verapamil to suppress voltage-gated calcium channel activity. Intact neurones were identified at the end of the experiment by applying 75 mM KCl in the absence of verapamil (indicated by arrows).

with this isoform, we did not assess the effects of ononetin or Although the most common interpretation would be that the flavanones on TRPM3a1. In addition, fluorometric assays in binding site is accessible from the extracellular space, the DRG neurones relied on a PregS-triggered Ca2+ entry and, unrestricted membrane permeability of the aglycones may thus, most likely reflect splice variants that share the a2 pore result in a rapid intracellular dilution of the TRPM3 modulator. conformation. In addition, the insensitivity of TRPM3 to the glycosides is Since naringenin and ononetin also blocked the TRPM3 likely to result from an inability of flavanone glycosides to activation by nifedipine, an activator that is chemically unre- bind to TRPM3. An alternative explanation could be that lated to PregS, a competitive mode of action appears unlikely. the more hydrophilic compound is largely membrane- In addition, heat-mediated TRPM3 activation is also strongly impermeable and might not reach an intracellular binding site blocked by both compounds. Thus, naringenin and ononetin on TRPM3. Thus, more detailed analyses of the modulator- induced block is not restricted to PregS activation of TRPM3. binding properties and on the interference with the channel In this study, we provide evidence for a binding mode gating will be addressed in future by electrophysiological that involves the extracellularly accessible moieties of the studies. channel protein and exhibits a slight voltage dependence TRPM3 is expressed in nociceptive sensory neurones in in favour of blocking the physiologically more relevant dorsal root and trigeminal ganglia, and its activation has been inward current component and a reversible mode of action. linked to thermal pain. Specifically, TRPM3-/- mice show an A more thorough electrophysiological characterization, attenuated response to thermal nociceptive stimuli, an effect including a detailed kinetic analysis, the assessment of that is maintained after induction of inflammatory hyperal- use dependence, and profiling of single channel properties gesia (Vriens et al., 2011). Here we show that naringenin as in the presence of the blockers, may provide additional well as ononetin block the PregS-induced Ca2+ entry in freshly insights in the mechanism of TRPM3 block by these natural isolated neurones from mouse DRG. Besides DRG neurones compounds. from mice, TRPM3-like responses were also found in PregS- Another still unresolved question relates to the localiza- stimulated freshly isolated rat DRG neurones, and these tion of the binding site of the flavanones and deoxybenzoins responses were blocked by eriodictyol. Rossato et al. (2011) on TRPM3. Electrophysiological data obtained with intracel- showed that eriodictyol is a potent blocker of TRPV1 with lularly perfused modulators demonstrated that TRPM3 was analgesic effects on capsaicin-treated mice and complete Fre- still activated by PregS, and that the same modulator only und’s adjuvant-induced thermal hyperalgesia. Astonishingly, blocked this conductance when added to the bath solution. and in contrast to this report, we observed that eriodictyol is

British Journal of Pharmacology (2013) 168 1835–1850 1847 39 BJP I Straub et al.

Figure 11 Eriodictyol blocks TRPM3, but not TRPV1 in rat DRG neurones. (A) Sample traces of intracellular calcium concentration in single rat DRG neurones during addition of 50 mM PregS, 5 mM ononetin and 75 mM KCl. (B) Similar experiment as in (A), but with addition of 20 mM eriodictyol and 2 mM capsaicin. (C). Upper panel: Whole-cell currents in a freshly isolated rat DRG neuron voltage-clamped at -70 mV. Capsaicin (1 mM) alone, or in combination with eriodictyol (20 mM) was applied for 5 s intervals. Lower panel: Statistical analysis of 8 independent measurements (means and SEM) performed as shown in the upper panel. n.s., statistically not significant. ᭣

a potent blocker of TRPM3, but not of TRPV1. The inability of eriodictyol to block the capsaicin-induced activation of rat TRPV1 was tested in a recombinant expression system and confirmed in freshly isolated rat DRG neurones. Since we cannot confirm the TRPV1-blocking properties of eriodictyol, one should consider the possibility that the thermal hypoalgesia that has been observed by Rossato et al. may be related to the block of TRPM3. Due to the availability of pharmacokinetic and toxicologi- cal data, flavanones such as naringenin may be applied in

vitro as well as in vivo studies. In mice, naringenin has an LD50 of more than 5 g·kg-1 (Ortiz-Andrade et al., 2008). Therefore, a potential antinociceptive effect of citrus fruit flavanones may be tested in vivo and might reveal a therapeutic potential for analgesic treatment. After consumption of grapefruit juice and orange juice, plasma concentrations of naringenin and hesperetin may reach low micromolar concentrations (Erlund et al., 2001). Once resorbed, they undergo extensive phase 1 and phase 2 metabolization via hydroxylation, glucuronidation and sul- fatation (Fuhr and Kummert, 1995). Thus, if citrus fruit flavonoids exert promising pharmacodynamic properties, a further compound optimization should be considered to avoid a rapid modification. Alternatively, the modulators might be applied topically, or the bioavailability and stability of naringenin may be enhanced by complexation with hydroxyl-b-cyclodextrin (Shulman et al., 2011). In summary, we identified specific and potent blockers of TRPM3 that can be used to investigate its function in vivo and in vitro. Since the physiological functions of TRPM3 are still not firmly established, we expect that acute TRPM3 modula- tion with pharmacological modulators provides a useful and validated cell-biological tool to study TRPM3 in vitro and in vivo and to complement transgenic approaches. Furthermore, if the involvement of TRPM3 in nociception could be strengthened, TRPM3-blockers may offer a novel mode of action for analgesic treatment.

Acknowledgements

This work was supported by the Deutsche Forschungsgemein- schaft within the frameworks of FOR 806 and the GK 1097 to MS, the Emmy Noether-programme, GK 1326, SFB 593 and SFB 894 to JO, and the GK1326 and SFB 894 to SEP. We thank Helga Sobottka, Marion Leonhardt, Sandra Plant and Raissa Wehmeyer for excellent technical support.

1848 British Journal of Pharmacology (2013) 168 1835–1850 40 Identification of novel TRPM3 blockers BJP

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Vriens J, Owsianik G, Hofmann T, Philipp SE, Stab J, Chen X et al. current amplitudes at 87 mV (upper trace) and -113 mM (2011). TRPM3 is a nociceptor channel involved in the detection of (lower trace); dotted line: zero current level (B) IV curve of noxious heat. Neuron 70: 482–494. PregS- (1) and nifedipine-activated TRPM3 currents (2) and Wagner TF, Loch S, Lambert S, Straub I, Mannebach S, Mathar I during an additional stimulation with 1 mM (3) or 3 mM et al. (2008). Transient receptor potential M3 channels are ononetin (4). (C) Statistical analysis of peak current densities

ionotropic steroid receptors in pancreatic beta cells. Nat Cell Biol in resting HEKmTRPM3 cells after addition of 50 mM nifedipine 10: 1421–1430. and different concentrations of ononetin. (D–E) Similar Wagner TF, Drews A, Loch S, Mohr F, Philipp SE, Lambert S et al. experiments as described for panels A–C, whereas ononetin (2010). TRPM3 channels provide a regulated influx pathway for was replaced by naringenin. Data represent means and SEM zinc in pancreatic beta cells. Pflugers Arch 460: 755–765. of at least 5–10 independent experiments for naringenin and Xu SZ, Zeng F, Boulay G, Grimm C, Harteneck C, Beech DJ (2005). six independent experiments for ononetin. Block of TRPC5 channels by 2-aminoethoxydiphenyl borate: a Figure S3 Naringenin has activating and blocking potency differential, extracellular and voltage-dependent effect. Br J in HEK293 cells stably expressing TRPM8. (A) Examples of Pharmacol 145: 405–414. calcium entry traces from Fluo 4-loaded HEKhTRPM8 cells activated with different concentrations of naringenin Zamudio-Bulcock PA, Everett J, Harteneck C, Valenzuela CF (2011). Activation of steroid-sensitive TRPM3 channels potentiates and menthol. Challenging the cells with naringenin glutamatergic transmission at cerebellar Purkinje neurons from concentration-dependently prevented the subsequent activa- developing rats. J Neurochem 119: 474–485. tion with 300 mM menthol. (B) Example of a whole-cell recording of a HEK293 cell stably expressing TRPM8. The cell was either perfused with 100 mM naringenin alone, simulta- neously with 300 mM menthol or after an activation of Supporting information TRPM8 with 300 mM menthol. Note the partial activation of outward currents and the block of menthol-induced currents in the presence of naringenin. Additional Supporting Information may be found in the Figure S4 Ononetin does not activate TRPA1 in whole-cell online version of this article at the publisher’s web-site: patch clamp measurements, but it activates DTT-dependent Figure S1 Concentration–response curves of naringenin- TRPA1 in fluorometric measurements. (A–B) Representative

induced TRPM3 block. Whole-cell recordings of HEKmTRPM3 whole-cell recordings from HEK293 cells stably expressing cells stimulated with 35 mM of PregS and blocked with differ- TRPA1. Cells were first perfused with 10 mM (A) or 30 mM ent concentration of naringenin were performed to obtain ononetin (B). An additional perfusion with AITC (10 mM) concentration–response curves. To avoid that the rundown of induced TRPA1 currents. (C–D) Examples of calcium entry

the channel is considered as block, an interpolated value traces from Fluo4-loaded HEKhTRPA1 cells activated with differ- between before and after block was used to calculate the ent concentrations of ononetin and 30 mM of AITC (C) and in percent of block. A time-matched activation of TRPM3 the presence of 10 mM of DTT (D). (E) Statistical analysis of without naringenin was set as 100%, and measurements in differences in ononetin-induced calcium entry in the pres- the presence of naringenin were normalized to this value. ence of 10 mM DTT. Calcium entry after 30 mM AITC without (A) Representative recordings of whole-cell currents. Data are ononetin was set as maximal activation. Data represent extracted from voltage ramps and depict the current at 87 mV means and SEM of at least three independent experiments in (upper trace) and -113 mV (lower trace). Dotted line: zero duplicate each. *P < 0.05; **P <0.01 current level. (B–C) Concentration–response curves of Figure S5 Endogenously expressed TRPM7 in HEK293 cells naringenin to block PregS-induced inward currents (B) and is partially blocked by naringin, whereas ononetin, hespere-

outward currents (C). IC50 values and Hill coefficients (n) were tin and eriodictyol did not affect TRPM7 currents. (A) obtained by non-linear curve fitting, applying a four- Representative whole-cell recordings from HEK293 cells parameter Hill equation. Each data point represents SEM of endogenously expressing TRPM7. Perfusion of the cells with 5–12 independent experiments. a solution containing no divalent cations elicited linear Figure S2 Naringenin and ononetin block nifedipine- TRPM7 currents. An additional perfusion with 20 mM onon-

induced TRPM3 currents in HEKmTRPM3 cells. (A) Whole-cell etin, 20 mM hesperetin and 20 mM eriodictyol did not signifi-

recording from a HEKmTRPM3 cell stimulated with 50 mM nifed- cantly influence the TRPM7 current, whereas 20 mM ipine and 35 mM PregS. And further nifedipine-induced naringenin partially blocked the inward and outward cur- TRPM3 currents were blocked with either 1 or 3 mM of onon- rents. (B) Statistical analysis of percentage of block of 5–10 etin. Data are extracted from voltage ramps and depict independent measurements. **P < 0.01

1850 British Journal of Pharmacology (2013) 168 1835–1850 42

Figure S1: Concentration-response curves of naringenin-induced TRPM3 block. Whole-cell recordings of HEKmTRPM3 cells stimulated with 35 µM of PregS and blocked with different concentration of naringenin were performed to obtain concentration-response curves. To avoid that the rundown of the channel is considered as block, an interpolated value between before and after block was used to calculate the percent of block. A time-matched activation of TRPM3 without naringenin was set as 100 % and measurements in the presence of naringenin were normalized to this value. (A) Representative recordings of whole cell currents. Data are extracted from voltage ramps and depict the current at 87 mV (upper trace) and -113 mV (lower trace). Dotted line: zero current level. (B) – (C) Concentration- response curves of naringenin to block PregS-induced inward currents (B) and outward currents (C). IC50 values and Hill coefficients (n) were obtained by non-linear curve fitting, applying a four parameter Hill equation. Each data point represents S.E.M of 5 - 12 independent experiments.

43

Figure S2: Naringenin and ononetin block nifedipine-induced TRPM3 currents in HEKmTRPM3 cells. (A) Whole-cell recording from a HEKmTRPM3 cell stimulated with 50 µM nifedipine and 35 µM PregS. And further nifedipine-induced TRPM3 currents were blocked with either 1 or 3 µM of ononetin. Data are extracted from voltage ramps and depict current amplitudes at 87 mV (upper trace) and -113 mM (lower trace); dotted line: zero current level (B) IV curve of PregS- (1) and nifedipine-activated TRPM3 currents (2) and during an additional stimulation with 1µM (3) or 3 µM ononetin (4). (C) Statistical analysis of peak current densities in resting HEKmTRPM3 cells (grey bars and dashed line) after addition of 50 µM nifedipine and different concentrations of ononetin. (D) – (E) Similar experiments as described for (A) – (C), whereas ononetin was replaced by naringenin. Data represent means and S.E.M. of at least 5 – 10 independent experiments for naringenin and 6 independent experiments for ononetin.

44

Figure S3: Naringenin has activating and blocking potency in HEK293 cells stably expressing TRPM8. (A) Examples of calcium entry traces from Fluo 4-loaded HEKhTRPM8 cells activated with different concentrations of naringenin and menthol. Challenging the cells with naringenin concentration-dependently prevented the subsequent activation with 300 µM menthol. (B) Example of a whole-cell recording of a HEK293 cell stably expressing TRPM8. The cell was either perfused with 100 µM naringenin alone, simultaneously with 300 µM menthol or after an activation of TRPM8 with 300 µM menthol. Note the partial activation of outward currents and the block of menthol-induced currents in the presence of naringenin.

45

Figure S4: Ononetin does not activate TRPA1 in whole cell patch clamp measurements, but it activates TRPA1 DTT-dependent in fluorometric measurements. (A) – (B), representative whole- cell recordings from HEK293 cells stably expressing TRPA1, cells were first perfused with 10 µM (A), or 30 µM ononetin (B). An additionally perfusion with AITC (10 µM) induced TRPA1 currents. (C) – (D) Examples of calcium entry traces from Fluo4-loaded HEKhTRPA1 cells activated with different concentrations of ononetin and 30 µM of AITC (D) and in the presence of 10 mM of DTT. (E) Statistical analysis of differences in ononetin-induced calcium entry in the presence of 10 mM DTT. Calcium entry after 30 µM AITC without ononetin was set as maximal activation. Data represent means and S.E.M of at least 3 independent experiments in duplicate each. * p<0.05; ** p<0.01

46

Figure S5: Endogenously expressed TRPM7 in HEK293 cells is partially blocked by naringin, whereas ononetin, hesperetin and eriodictyol did not affect TRPM7 currents. (A) Representative whole cell recordings from HEK293 cells endogenously expressing TRPM7. Perfusion of the cells with a solution containing no divalent cations elicited linear TRPM7 currents. An additional perfusion with 20 µM ononetin, 20 µM hesperetin and 20 µM eriodictyol did not significantly influence the TRPM7 current, whereas 20 µM naringenin partially blocked the inward and outward currents. (B) Statistical analysis of percentage of block of 5 – 10 independent measurements. **p<0.01

47

Nachweis über Anteile der Co-Autoren, Isabelle Straub

Identification and application of novel and selective blockers for the heat-activated cation channel TRPM3

Nachweis über Anteile der Co-Autoren:

Titel: Citrus fruit and fabacea secondary metabolites potently and selectively block TRPM3 Journal: British Journal of Pharmacology Autoren: Isabelle Straub, Florian Mohr, Julia Stab, Maik Konrad, Stephan Philip, Johannes Oberwinkler, Michael Schaefer

Anteil Isabelle Straub : - Zellkulturen - Screening der TRPM3 Zelllinie - Fluoreszenzmessungen in Zellsuspension - Elektrophysiologie der TRPM3 Zellinie mit PregS, Nifedipin und Hitze- Aktivierung - Einzelzell Fluoreszenzmessungen der DRG Neurone von Ratten - Elektrophysiologie von DRG Neuronen - Analyse und Verarbeitung der Daten - Schreiben der Publikation

Anteil Florian Mohr: - Einzelzell Fluoreszenzmessungen TRPM1

Anteil Julia Stab: - Einzelzell Fluoreszenzmessungen TRPM1 - Einzelzell Fluoreszenzmessungen der DRG Neurone von Mäusen

Anteil Maik Konrad: - Einzelzell Fluoreszenzmessungen TRPM1

Anteil Stephan Philip: - Herstellung der stabilen TRPM3 Zelllinie

Anteil Johannes Oberwinkler: - Konzeption

Anteil Michael Schaefer: - Projektidee - Konzeption - Schreiben der Publikation

49

50 1521-0111/84/5/1–15$25.00 http://dx.doi.org/10.1124/mol.113.086843 MOLECULAR PHARMACOLOGY Mol Pharmacol 84:1–15, November 2013 Copyright ª 2013 by The American Society for Pharmacology and Experimental Therapeutics

Flavanones That Selectively Inhibit TRPM3 Attenuate Thermal Q:1 Nociception In Vivo

Isabelle Straub, Ute Krügel, Florian Mohr, Jens Teichert, Oleksandr Rizun, Maik Konrad, Johannes Oberwinkler, and Michael Schaefer Rudolf-Boehm-Institut für Pharmakologie und Toxikologie, Universität Leipzig, Leipzig, Germany (I.S., U.K., J.T., M.S.); and Institut für Physiologie und Pathophysiologie, Philipps-Universität Marburg, Marburg, Germany (F.M., O.R., M.K., J.O.) Received April 16, 2013; accepted September 3, 2013

ABSTRACT Transient receptor potential melastatin 3 (TRPM3) is a calcium- with respect to their inhibitory potency and a selective mode of permeable nonselective cation channel that is expressed in action. Isosakuranetin, a flavanone whose glycoside is con- a subset of dorsal root (DRG) and trigeminal ganglia sensory tained in blood oranges and grapefruits, displayed an IC50 of 50 neurons. TRPM3 can be activated by the neurosteroid preg- nM and is to our knowledge the most potent inhibitor of TRPM3 nenolone sulfate (PregS) and heat. TRPM32/2 mice display an identified so far. Both compounds exhibited a marked specificity impaired sensation of noxious heat and thermal hyperalgesia. for TRPM3 compared with other sensory TRP channels, and We have previously shown that TRPM3 is blocked by the citrus blocked PregS-induced intracellular free Ca21 concentration fruit flavanones hesperetin, naringenin, and eriodictyol as well as signals and ionic currents in freshly isolated DRG neurons. by ononetin, a deoxybenzoin from Ononis spinosa. To further Furthermore, isosakuranetin and previously identified hesperetin improve the tolerability, potency, and selectivity of TRPM3 significantly reduced the sensitivity of mice to noxious heat and blockers, we conducted a hit optimization procedure by PregS-induced chemical pain. Because the physiologic func- rescreening a focused library that was composed of chemically tions of TRPM3 channels are still poorly defined, the de- related compounds. Within newly identified TRPM3 blockers, velopment and validation of potent and selective blockers is isosakuranetin and liquiritigenin displayed favorable properties expected to contribute to clarifying the role of TRPM3 in vivo.

Introduction 2008). Furthermore, TRPM3a2 is expressed in neurons of dorsal root ganglia (DRG) and trigeminal ganglia, where it Transient receptor potential melastatin 3 (TRPM3) is contributes to the detection of noxious heat in healthy and a member of the large superfamily of TRP ion channels. The inflamed tissue (Vriens et al., 2011). Activation of TRPM3 has TRPM3 gene encodes a number of different alternative splice also been coupled to vascular smooth muscle cell contraction variants (Lee et al., 2003; Oberwinkler et al., 2005; Frühwald (Naylor et al., 2010), to oligodendrocyte differentiation, and to et al., 2012). Alternative splicing within exon 24 affects the neuronal myelination (Hoffmann et al., 2010). putative pore region, resulting in distinct biophysical proper- Recently, we showed that TRPM3 is blocked by citrus fruit ties of the permeation pathway. The TRPM3a1 variant is flavanones and fabacea secondary metabolites (Straub et al., predominantly selective for monovalent cations whereas 2013). Flavanones, a subgroup of flavonoids, comprise the TRPM3a2, a 12-amino-acid shorter variant, forms a cal- most common group of plant-derived polyphenolic compounds cium-permeable poorly selective cation channel (Oberwinkler in the human diet (Manach et al., 2004). Flavonoids share et al., 2005). a common structure: they consist of two aromatic rings (A and TRPM3a2 channels can be activated by the neurosteroid B rings) that are linked with a heterocycle (C ring) (Fig. 1A). pregnenolone sulfate (PregS), nifedipine (Wagner et al., They can be divided into different subgroups, depending on 2008), D-erythro-sphingosine (Grimm et al., 2005), and by the oxidation of the C ring, the hydroxylation pattern of the noxious heat (Vriens et al., 2011). TRPM3 is functionally phenolic rings, and the substitution at the 3-position. expressed in insulin-secreting pancreatic beta cells, and its For a better understanding of the biologically active activation has been linked to insulin secretion (Wagner et al., flavonoid substructure that bears responsibility for TRPM3 block, we performed a rescreening of a focused library that This work was supported by the Deutsche Forschungsgemeinschaft (DFG) was composed of members of different flavonoid subgroups Q:2 within the framework of FOR 806 (to M.S.), and the SFB 593 (to J.O.). and of flavanones with different substitutions on specified dx.doi.org/10.1124/mol.113.086843.

21 21 ABBREVIATIONS: AITC, allyl isothiocyanate; BAPTA, (1,2-bis(o-aminophenoxy)ethane-N,N,N9,N9-tetraacetic acid; [Ca ]i, intracellular free Ca concentration; DMEM, Dulbecco’s modified Eagle’s medium; DMSO, dimethylsulfoxide; DRG, dorsal root ganglia; ER, estrogen receptor; HEK, human embryonic kidney; HPLC, high-performance liquid chromatography; MTT, 3-(4,5-dimethylthioazol-2-yl)-2,5-diphenyltetrazoliumbromid; NGF, nerve growth factor; P450, cytochrome P450; PregS, pregnenolone sulphate; TRPA, transient receptor potential ankyrin; TRPM, transient receptor potential melastatin; TRPV, transient receptor potential vanilloid.

1 51 2 Straub et al.

Fig. 1. Chemical structures of flavonoids and their potency to block TRPM3. Fla- vonoids can be divided into different subgroups: (A) flavanones, (B) flavones (apigenin) and flavonols (quercetin), (C) flavanols, (D) isoflavanones, and (E) dihy- drochalkones. To clarify the blocking- potency of the different flavonoid sub- groups, HEKmTRPM3 cells were incubated with the calcium indicator dye Fluo-4. Cells were dispensed into 384-well plates that were prefilled with single flavonoids (25 mM), and fluorescence intensities were measured during PregS-induced activa- tion of TRPM3.

positions. Within the flavonoids, only substances that com- HEKrTRPV1:YFP) were obtained by a limiting dilution method, as prise a flavanone backbone displayed a biologic activity to described recently elsewhere (Hellwig et al., 2004; Urban et al., 2012). block TRPM3. Within the flavanones, we identified isosakur- Stably transfected cell lines were selected and maintained in HEK293 21 anetin and liquiritigenin as novel potent and specific TRPM3 medium, containing 400 mg ml G418 (geneticin). All cells were blockers. The novel identified compounds blocked endoge- grown at 37°C in a humidified atmosphere containing 5% CO2. Unless otherwise stated, cells were seeded 24 hours before the experiments nously expressed TRPM3 in freshly isolated DRG neurons. onto poly-L-lysine–coated coverslips. Furthermore, we demonstrate that isosakuranetin and the Cell Transfection. HEK293 cells were transiently transfected previously identified hesperetin block TRPM3-related noci- with a bicistronic expression plasmid, encoding Myc-tagged TRPM1 ception in vivo. and enhanced green fluorescent protein, as previously described elsewhere (Lambert et al., 2011; Straub et al., 2013). For heat activation of TRPM3, HEK293 cells were transiently transfected with Materials and Methods a bicistronic plasmid encoding TRPM3a2 and a green fluorescent protein protein. For detailed information, see Straub et al. (2013). Cell Culture. Human embryonic kidney 293 (HEK293) cells used DRG Neuron Preparation. Eight-week-old male and female for transient transfection were cultured in Earle’s minimum essential Wistar rats were sacrificed by an overdose of CO . To expose the medium (Invitrogen/Life Technologies, Carlsbad, CA), supplemented 2 21 spinal cord, a careful single dorsal-midline incision along the entire with 10% fetal calf serum, 2 mM L-glutamine, 100 units ml length of the body was made. The spinal cord was then gently penicillin, and 0.1 mg ml21 streptomycin. HEK293 cells stably removed, and dorsal root ganglia from all cervical, thoracic, and expressing myc-tagged mouse TRPM3a2 (HEK ) were obtained mTRPM3 lumbar segments were obtained. Ganglia were digested for 35 and maintained as described elsewhere (Frühwald et al., 2012; Straub 21 minutes at 37°C in an enzyme solution containing 1.5 mg ml et al., 2013). HEK293 cells stably expressing other sensory transient 2 collagenase (Sigma-Aldrich, St. Louis, MO) and 3 mg ml 1 dispase receptor potential (TRP) channels (HEKhTRPA1, HEKhTRPM8:CFP,

52 Selective TRPM3 Inhibitors 3

(Roche Applied Science, Indianapolis, IN) in Hanks’ balanced salt TRIS (pH 7.4 with HCl), and the pipette solution contained 140 mM Q:5 solution (GIBCO/Life Technologies, Grand Island, NY). After re- CsCl, 0.6 mM MgCl2, 1 mM EGTA, 10 mM HEPES, 5 mM TEA (pH 7.2 peated trituration with a 1-ml pipette and termination of the with CsOH). digestion by adding 100 ml of fetal calf serum, the cell suspension To determine the I-V relationship of PregS-induced currents, Q:6 was centrifuged (3.5 minutes at 670g) and resuspended in Dulbecco’s measurements were performed under monovalent free extracellular modified Eagle’s medium (DMEM), supplemented with 5% fetal calf conditions to suppress other cationic conductance present in DRG 21 21 serum, 100 units ml penicillin, and 0.1 mg ml streptomycin. DRG neurons. The monovalent free solution contained 2 mM CaCl2, 2 mM neurons used for measurement of transient receptor potential MgCl2, 10 mM HEPES, 280 mM D-mannitol (pH 7.4 with N-methyl-D- ankyrin-like (TRPA)1-related Ca21 entry were resuspended in glucamine). The holding potential for measurements in DRG neurons a DMEM medium additionally supplemented with 100 ng ml21 nerve was 260 mV between voltage ramps. Voltage ramps were applied as growth factor (NGF). Subsequently, droplets of cell suspension were described for measurements in HEK293 cells. Modulators were 21 plated on coverslips coated with poly-L-lysine (1 mg ml ; Sigma- applied via a SF-77B Perfusion Fast Step system (Warner Instru- Aldrich), placed in a culture dish, and left to adhere for at least 1 hour ments, Hamden, CT). Heat activation of TRPM3 was performed as in an incubator (37°C, 5% CO2). Afterward, the culture dish was then described earlier (Straub et al., 2013). Shortly, solutions were heated filled with DMEM. All experiments were performed within 36 hours with an inline solution heater (Warner Instruments) under control of after preparation. a TC-324B heater (Warner Instruments). The temperature of the Intracellular Ca21 Analysis in Cell Suspensions. All fluoro- solutions was recorded with the pClamp 10 software (Molecular metric assays in cell suspensions were performed in 384-well plates. Devices). For detailed information, see Nörenberg et al. (2012) and Straub et al. Giant Excised Patch Electrophysiology. Experiments were (2013). Shortly, HEK293 cells stably expressing TRPM3 were performed with oocytes from Xenopus laevis obtained from EcoCyte incubated with fluorescence-indictor dye Fluo-4/AM (4 mM; Molecular Bioscience (Austin, TX). A cRNA was synthesized in vitro from Probes/Life Technologies, Eugene, OR) for 30 minutes at 37°C, a TRPM3a2-encoding cDNA subcloned in a pBF1-based plasmid Q:3 washed, and resuspended in HEPES-buffered solution, containing (kindly provided by Prof. Bernd Fakler, Freiburg, Germany) with the 135 mM NaCl, 6 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 5.5 mM D- mMESSAGE mMACHINE SP6 kit (Ambion/Life Technologies, glucose, and 10 mM HEPES adjusted to pH 7.4 with NaOH. Austin, TX) according to the manufacturer’s instructions. Oocytes Fluorescence was monitored with a filter-based plate reader device were injected with 69 ml of TRPM3a2 cRNA (1000 ng/ml) and (Polastar Omega; BMG Labtech, Offenburg, Germany) in the bottom- incubated for at least 48 hour at 20°C in an oocyte medium (EcoCyte)

read mode, applying 485\10-nm and 520\20-nm bandpass filters for containing (in mM): 90 NaCl, 2 KCl, 2 CaCl2, 1 MgCl2, 5 HEPES, 0.5 g excitation and emission, respectively. Measurements of agonistic l21 polyvinylpyrrolidone, 100 U ml21 penicillin, and 100 mg ml21 properties of the compounds were performed using a custom-made streptomycin (pH 7.2). The pipette solution contained (in mM): 115 fluorescence plate imaging device built into a robotic liquid handling NaCl, 5 KCl, 1 CaCl2, 10 HEPES, and 50 mM PregS (pH 7.3), yielding station (Freedom EVO 150; Tecan Group, Männedorf, Switzerland), pipette resistances of 200–500 kV. The bath solution contained (in as recently described elsewhere (Nörenberg et al., 2012). mM): 130 KCl, 2 MgCl2, 1 EGTA, 2 NaATP, and 10 HEPES (pH 7.5). Single-Cell Calcium Measurement. Single-cell measurement of Isosakuranetin, Mg21, or dimethylsulfoxide (DMSO) was added to the 21 [Ca ]i in rat DRG neurons and in transiently transfected HEK293 bath solution at the indicated concentrations. In all solutions, pH was cells was performed as previously described elsewhere (Straub et al., adjusted with KOH. Voltage ramps (1 mV ms21) were applied from 2013). All measurements were performed at room temperature (22– 295.4 mV to 1104.6 mV (liquid junction potential corrected by 24.6 24°C). mV) from a holding potential of 24.6 mV at a rate of 1 Hz with an Whole-Cell Patch-Clamp Measurements. All electrophysio- EPC-10 amplifier controlled by the Patchmaster software (HEKA logic measurements were performed in the whole-cell voltage-clamp Instruments, Bellmore, NY). Pipettes were coated with paraffin and mode at room temperature, using an Axopatch 200B amplifier, washed with water. Seal formation was achieved by approaching the connected to a desktop computer via a Digidata 1200 digitizer (Axon oocyte and releasing the positive pipette pressure after cell contact. CNS; Molecular Devices, Sunnyvale, CA) under the control of the After a seal had been formed, a giant patch was excised by fast back- Q:4 Pclamp 9 or Pclamp10 software (Molecular Devices). To obtain I/V and-forth movement of the pipette. The giant patch was placed in curves, Vh was set to 2113 mV for 100 milliseconds, and voltage front of a custom application system with three channels. By moving ramps from 2113 to 1 87 mV (0.4 mV ms21) were applied once per the pipette, the patch was exposed to the constantly flowing solution second. The intersweep holding potential was 213 mV. Concentration stream of the different application channels. response curves were obtained with a voltage step protocol by MTT Viability Test. To measure a possible cytotoxicity of the repeatedly applying a Vclamp of 2113 mV and 187 mV (duration of flavonoids, enzymatic conversion of MTT [3-(4,5-dimethylthioazol-2- 50 milliseconds each) every 200 milliseconds. TRPM3 currents yl)-2,5-diphenyltetrazoliumbromid] was assessed. HEK293 cells were typically reached a steady state within less than 15 milliseconds seeded onto a 96-well plate coated with poly-L-lysine (10,000 cells per and were averaged over a time window of 20–50 milliseconds after well). The next day, the cells were incubated with the compounds applying voltage steps. The intracellular solution contained 80 mM diluted in cell culture medium for at least 24 hours. For colorimetric

Cs-aspartate, 45 mM CsCl, 4 mM Na2ATP, 10 mM HEPES, 10 mM detection, the medium was exchanged to a phenol red-free medium BAPTA [(1,2-bis(o-aminophenoxy)ethane-N,N,N9,N9-tetraacetic (100 ml per well), containing 1.2 mM MTT, and the cells were placed in acid], 5 mM EGTA pH 7.2 adjusted with CsOH. The osmolarity was the incubator for 4 hours. Formazan complexes that arose were 300–315 mOsm. The extracellular solution used contained 145 mM solubilized with DMSO, and absorbance was measured at 560 nm and

NaCl, 3 mM KCl, 10 mM CsCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM at 670 nm with the transmission spectrometer head of a plate reader 1 HEPES, 10 mM D-glucose, pH 7.4 adjusted with NaOH. The Cs device (POLARstar Omega). A solvent control was set as 100%, and -based, divalent-free external solution for TRPM7 measurements measurements in presence of the indicated substances were normal- contained 130 mM CsCl, 50 mM mannitol, 10 mM HEPES, pH 7.4 ized to this value. with CsOH. The liquid junction potential between the bath and the Behavioral Analysis. All animal experiments were conducted in pipette solutions was 13 mV (calculated with Clampex; Axon accordance with the accepted standards of animal care and were Instruments/Molecular Devices) and corrected. Series resistances approved by the local authorities (regional governmental agency of were ,10 MV and were compensated by 75–85%. The sampling rate the State of Saxony, Germany; TVV 02/12). We used 3-week-old was 5 kHz in standard whole cell measurements. For whole-cell female and male C57BL/6J mice. For the hot plate test, isosakur- measurements in DRG neurons, the extracellular solution contained anetin (2 mg kg21) and hesperetin (10 mg kg21) were dissolved in 140 mM NaCl, 4 mM KCl, 2 mM MgCl2, 100 nM tetrodotoxin, 10 mM Miglyol 812 (Caesar & Loretz GmbH, Hilden, Germany) containing

53 4 Straub et al.

0.1% DMSO, and compounds or the vehicle alone were injected evaporated at 40°C under nitrogen, reconstituted in 100 ml of 30% intraperitoneally (6 ml kg21). Thirty minutes after the injection, mice acetonitrile containing 0.2% aqueous phosphoric acid (v/v), and were placed on a hot plate set to a temperature of 52°C. The delay to centrifuged. Cleared supernatant (20 ml) was injected onto a high- the first pain-related response (hindpaw shaking, lifting or licking, performance liquid chromatography (HPLC) column (5 mm, 3 mm  jumping) was measured by an investigator blind to substance 150 mm; UltraSep ES PHARM RP18E; SepServ HPLC, Berlin, application. Measurements of the body temperature were performed Germany) implemented in a HPLC system with absorbance detection with a digital ear infrared thermometer (Thermoscan IRT 4520; at 288 nm (1100 series; Agilent Technologies, Böblingen, Germany). Braun, Kronberg, Germany) in the mouse’s armpit before and 30 Separation was performed isocratically at 30°C with a mobile phase minutes after injection of the compounds or vehicle. consisting of 44% acetonitril in 0.2% aqueous phosphoric acid (vol/vol) The PregS-induced pain was assessed 30 minutes after i.p. with a flow of 0.55 ml min21. injection of isosakuranetin (2 mg kg21) and hesperetin (10 mg kg21) Stock Solutions and Drugs. All modulators were dissolved in or vehicle. PregS was solved in distilled water. A volume of 10 ml DMSO, if not indicated otherwise. The concentration of stock containing 5 nmol of PregS was injected into the midplantar right solutions was chosen in such a way that the DMSO concentration in hindpaw. The latency to, duration of, and number of nocifensive the final solution never exceeded 0.14% at the highest test responses (paw licking, shaking, or lifting) were registered. concentration of the respective modulator and further reduced by Plasma Concentrations. Plasma concentrations of isosakurane- serial dilution of the compounds. PregS, pinocembrin, 49-hydroxy- tin were determined with modifications according to (Vega-Villa et al., flavanone, liquiritigenin, phloretin, formononetin, ononetin, and 2008). Male and female mice were i.p. injected with 2 or 10 mg kg21 hesperetin were purchased from Sigma-Aldrich. Isosakuranetin was isosakuranetin, and whole blood samples were collected under purchased from Roth. Q:7 anesthesia by heart puncture. Blood samples were centrifuged to Statistical Analysis. All data are expressed as mean 6 S.E.M. obtain the plasma, and 200 ml of plasma were mixed with 25 ml of The number of cells analyzed in each experiment is indicated in the ethoxycoumarin (4 mM in acetonitrile) as internal standard and 600 ml figure legends. To test for statistically significant differences, analysis of ice-cold acetonitrile. The plasma was stirred for 1 minute and of variance with the Tukey post hoc test was used. P , 0.05 was centrifuged at 12,000g for 5 minutes. The supernatant was considered statistically significant. The pharmacochemical properties

Fig. 2. Identification of isosakuranetin and liquiritigenin as novel potent inhibitors of TRPM3. Fluo4-loaded HEK293 cells stably expressing TRPM3 were incubated with different concentrations of isosakuranetin and liquiritigenin, and the fluorescence intensities were measured during injection of the TRPM3 activator pregnenolone sulfate (PregS). (A) Examples of calcium entry traces extracted from a single 384-well plate measurement. Each trace represents a different liquiritigenin concentration. Light gray trace shows a solvent control. (B) Concentration-response curve of liquiritigenin. TRPM3 activation without an inhibitor (DMSO control) was set as 100%, and fluorescence intensities evoked by solutions containing inhibitors were normalized to this value. (C and D) Similar experiments as described for isosakuranetin. (C) Example traces of a single 384-well measurement with different concentrations of isosakuranetin. (D) Concentration-response curve of isosakuranetin. Data represent at least four independent experiments for liquiritigenin and five independent experiments for isosakuranetin with duplicates of each. Shown are mean 6 S.E.M. IC50 values and Hill coefficients Q:12 (n) were obtained by nonlinear curve fitting, applying a four parameter Hill equation.

54 Selective TRPM3 Inhibitors 5

21 of isosakuranetin and liquiritigenin were obtained from www.chemic- [Ca ]i signal when preincubated at a concentration of 25 mM. alize.org. Pinocembrin differs from the potently TRPM3-blocking naringenin by a complete lack of hydroxyl groups on the B 21 ring. It only partially inhibited the [Ca ]i signal, highlighting Results the importance of these substitutions for a TRPM3 block. Identification of Novel TRPM3-Blocking Flavanones. Conversely, isosakuranetin features a methoxy group at the C49 atom of the ring and completely blocked the PregS- The three previously described TRPM3 blockers (Straub et al., 21 2013)—naringenin, hesperetin, and eriodictyol—differ chem- induced [Ca ]i entry into HEKmTRPM3 cells at a concentration ically only in the substitutions on the B ring of the flavanone of 25 mM. structure (Fig. 1A). These different substitutions account for Further, we were interested in whether the flavanone differences in the potency to block TRPM3 and for the backbone is required to block TRPM3. To this end, we differences in the selectivity (Straub et al., 2013). We now investigated several compounds from different flavonoid tested whether flavanones with different modifications at the subgroups, including the flavonol quercetin, the flavone A or C rings are still capable of blocking TRPM3 (Fig. 1). apigenin (Fig. 1B), the flavanoles epicatechin and epigalloca- techin (Fig. 1C), the isoflavones genistein, daidzein, and HEK293 cells stably expressing TRPM3 (HEKmTRPM3) were incubated with the fluorescent calcium indicator dye Fluo-4, formononetin (Fig. 1D), and the dihydrochalcone phloretin dispensed into 384-well plates that were prefilled with (Fig. 1E). Among the tested flavonoid subgroups, only different concentrations of the flavonoids, and PregS (35 phloretin partially blocked the PregS-induced calcium entry m (Fig. 1B) whereas none of the others exerted more than 20% M)-induced calcium entry was measured. Although weakly 21 potent, 49-hydroxyflavanone, a flavanone that has no hydroxyl inhibition of PregS-induced [Ca ]i signals when applied at group on the A ring, was still a blocker (47% block at 25 mM) of a concentration of 25 mM (see Fig. 1). We conclude that the TRPM3. Liquiritigenin, a flavanone that has a single hydroxyl flavanone backbone and substitutions at the A and B rings are group on the A ring (C7 atom), abrogated the PregS-induced required to strongly block TRPM3. Within the tested

Fig. 3. Electrophysiologically measured concentration-response curves of an iso- sakuranetin (Iso)-induced TRPM3 block. Whole-cell recordings of HEKmTRPM3 cells stimulated with 20 mM of PregS and blocked with different concentrations of isosakuranetin were performed to obtain concentration-response curves. Due to the channel rundown, an interpolated value between before and after isosakuranetin- induced block was used to calculate the percentage of the block. (A and B) Repre- sentative recordings of whole-cell currents obtained from voltage steps from 2113 mV (s, lower trace) to 87 mV (d,upper trace). Dotted line: zero current level. (C) Concentration-response curves of isosa- kuranetin to block PregS-induced inward currents (s, 2113 mV) and outward currents (d, +87 mV). IC50 values and Hill coefficients (n) were obtained by nonlinear curve fitting, applying a four parameter Hill equation. Each data point represents the mean 6 S.E.M of 5 to 12 independent experiments. (D) Repre- sentative whole-cell currents from a HEKmTRPM3 cell activated with 35 mM of PregS and blocked with 3 mM of isosakur- anetin. Data are extracted from voltage ramps and depict the current at 87 mV (upper trace) and 2113 mV (lower trace). Arrows show the respective data point that was used to obtain the I/V curves shown in E. (E) Current-voltage relation- ship of PregS-induced TRPM3 currents 1) before and 2) after block with 3 mM of isosakuranetin.

55 6 Straub et al. flavanones, isosakuranetin and liquiritigenin almost com- 21 pletely abrogated the PregS-induced elevations of [Ca ]i in HEKmTRPM3 cells. Concentration-Dependence of Liquiritigenin- and Isosakuranetin-Induced Block of TRPM3-Dependent Ca21 Entry and Ionic Currents. To assess the potency of the newly identified blockers, we first performed fluorometric 21 Ca assays in HEKmTRPM3 cell suspensions that were exposed to serially diluted compounds and then stimulated with 35 mM PregS. Liquiritigenin elicited a half-maximal block with an IC50 of 500 6 70 nM (Fig. 2, A and B). 21 Isosakuranetin blocked PregS-induced [Ca ]i with an IC50 of 50 6 6 nM (Fig. 2, C and D). The Hill coefficients were 0.8 for liquiritigenin and 1.3 for isosakuranetin. To characterize the impact of the more potently acting isosakuranetin on TRPM3 currents, we performed whole-cell patch-clamp measurements. To reassess the concentration- response curves with the more quantitative electrophysiologic method, PregS-induced currents were recorded in HEKmTRPM3 cells, and isosakuranetin was acutely added via the bath solution at various concentrations (Fig. 3). The isosakuranetin- induced TRPM3 block was slowly developing and recovering after removal of the modulator (Fig. 3A). Isosakuranetin blocked the inward currents at 2113 mV slightly more potently (IC50 5 80 6 13 nM) compared with the outward currents measured at 187 mV (IC50 5 120 6 24 nM) (Fig. 3, B and C). The Hill coefficients were 1.0 for both liquiritigenin and isosakuranetin. Although the potency of TRPM3 block displayed some voltage dependence, both currents were isosakuranetin sensitive and almost completely blocked at a concentration of 3 mM (Fig. 3D). Moreover, the current- Fig. 4. Isosakuranetin (Iso) blocks heat-induced TRPM3 currents in voltage relationship showed no prominent voltage depen- transiently transfected HEK293 cells. (A) Representative whole-cell dence of the isosakuranetin-induced TRPM3 block (Fig. 3E). recording from a HEK293 cell transiently transfected with a TRPM3- encoding plasmid. During the recording, the superfused bath solution To test the biologic activity of the flavanones in the heat- was heated to 37–40°C, and outward (+87 mV) and inward currents activated mode of TRPM3, we performed whole-cell measure- (2113 mV) were recorded. At 40 seconds after the onset of heating, 3 mM ments with bath solutions heated to 37–40°C. Under these isosakuranetin was added to the bath solution. Dotted line: zero current level. (B) Statistical analysis of peak current densities in six independent conditions, inward and outward currents in TRPM3- measurements performed as shown in A. (C) Representative whole-cell expressing cells were partially blocked by 3 mM of isosakur- currents in a nontransfected HEK293 cell recorded as described in A. (D) anetin (Fig. 4, A and B). Raising the temperature in Statistical analysis of peak current densities of four independent measurements with untransfected parental HEK293 cells measurements as shown in C. also yielded small inward and outward currents that remained constant in the presence of 3 mM isosakuranetin side (Wagner et al., 2008), whereas isosakuranetin gets access (Fig. 4, C and D). Thus, isosakuranetin presumably blocks the to its binding side from the intracellular side of the TRPM3-mediated component of heat-induced currents. membrane, a competitive mechanism is unlikely. From the To assess the accessibility of the binding site and to exclude inside-out patch experiments we also conclude that isosakur- a requirement of cellular signaling cascades, we performed anetin blocks TRPM3 in a membrane-confined and pre- giant patch inside-out measurements on X. laevis oocytes that sumably direct fashion. heterologously express TRPM3 (Fig. 5). The pipette solution Specificity of Flavanones to Modulate Sensory TRP contained 50 mM PregS to activate TRPM3. The PregS- Channels. To evaluate the specificity of the novel TRPM3 induced currents were strongly blocked by 1 or 10 mM blockers, we first examined the impact of these flavanones on isosakuranetin applied from the intracellular side (Fig. 5). the most closely related TRPM1. TRPM1 can also be activated 21 Because high intracellular concentrations of Mg are known by PregS. Unlike TRPM3, TRPM1 is strongly blocked by to inhibit TRPM3-induced currents (Oberwinkler et al., 2005), extracellularly applied ZnCl2 (Lambert et al., 2011). When the inside-out configuration of the patch was confirmed by tested at a concentration of 10 mM, liquiritigenin had almost applying 10 mM Mg21. Because 10 mM Mg21 is expected to 21 no inhibitory effect on TRPM1-mediated [Ca ]i signals (Fig. eliminate TRPM3 currents, the remaining inward and out- 6A). The more potent TRPM3 blocker isosakuranetin reduced ward currents are likely to reflect TRPM3-unrelated leak 21 PregS-induced [Ca ]i responses by about 36% (Fig. 6B). In currents that could not be subtracted because TRPM3- both experiments, ZnCl2 (100 mM) was applied as a positive activating PregS in the pipette was present throughout the control for the TRPM1 block. whole experiment. Notably, inhibition by 10 mM isosakur- TRPM3 is expressed in DRG neurons, and its activation is anetin was equally effective as the TRPM3 block by 10 mM linked to thermosensation. We thus tested the ability of Mg21. Because PregS binds to TRPM3 from the extracellular

56 Selective TRPM3 Inhibitors 7

Fig. 5. Isosakuranetin (Iso) applied to the cytosolic side of oocyte giant patches blocks PregS-induced TRPM3 currents. (A) Example recording of an inside-out giant patch excised from a X. leavis oocyte expressing TRPM3. PregS (50 mM), ap- plied via the pipette to the extracellular side of the patch, activated TRPM3 cur- rents that were reversibly blocked with 10 mM isosakuranetin or with 10 mM Mg2+. Data are extracted from voltage ramps and depict the current at +80 mV (upper trace) and 280 mV (lower trace). Arrows indicate the time points of I/V curves shown in B. (B) I/V curve of PregS- activated TRPM3 currents 1) before and during superfusion 2) with 10 mM isosa- kuranetin or 3) with 10 mM Mg2+ (3). (C) Similar experiment as in A, but with 1 mM isosakuranetin. (D) I/V curve of PregS- activated TRPM3 currents measured in (C) 1) before and during superfusion 2) with 1 mM isosakuranetin or 3) with 10 mM Mg2+. (E and F) Statistical analysis of currents at (E) +80 mV and (F) 280 mV recorded in PregS-stimulated giant patches treated with solvent (0.02% DMSO), 1 or 10 mM isosakuranetin, or 10 mM Mg2+. Data were obtained from 5–10 indepen- dent measurements each. Q:13

isosakuranetin and liquiritigenin to modulate other thermo- concentrations .20 mM (Fig. 7C). Isosakuranetin also sensitive or sensory TRP channels, including transient activated TRPA1 when added at concentrations .5 mM, and receptor potential vanilloid-related (TRPV)1, TRPM8, and activation of TRPA1 by 50 mM isosakuranetin reached levels TRPA1. HEK293 cell lines stably expressing the respective comparable to those elicited by AITC itself (Fig. 7D). TRP channel were loaded with Fluo-4/AM, preincubated with To further evaluate the specificity of isosakuranetin and different concentrations of liquiritigenin or isosakuranetin, liquiritigenin among other members of the TRPM family, we and agonist-induced increases in the Fluo-4 fluorescence were investigated the blocking potency of the TRPM3-blocking measured. Neither liquiritigenin nor isosakuranetin had an compounds on endogenously expressed TRPM7 in HEK293 impact on the capsaicin (2 mM)-induced TRPV1 activation or cells. TRPM7 is permanently blocked by divalent cations, but the menthol (300 mM)-triggered activity of TRPM8 (Fig. 7, A when patched with Mg21-free pipette solution, switching to and B). a bath solution lacking Ca21 and Mg21 ions gives rise to large The TRPA1 activation induced by allyl isothiocyanate inward and outward currents (Nadler et al., 2001). Because (AITC) was, however, concentration dependently (2–50 mM) TRPM7-like currents exhibit a fast rundown when using diminished by preincubation with either flavanone (Fig. 7, A a Na1-based bath solution, we used a Cs1-based bath and B). Because the irritant sensor TRPA1 is activated by solution, as described elsewhere (Chubanov et al., 2012). 21 many chemical compounds, we performed additional [Ca ]i Neither isosakuranetin nor liquiritigenin (20 mM each) measurements with acutely added liquiritigenin and isosa- affected TRPM7-dependent inward or outward currents in kuranetin and compared the effects to those elicited by the HEK293 cells (Fig. 8, A and B). known TRPA1 activator AITC (30 mM). Indeed, liquiritigenin Effects of TRPM3-Blocking Compounds on Cell partially activated TRPA1 when acutely added at Viability. Because we aimed at testing biologic effects of

57 8 Straub et al.

stimulation with 2 mM capsaicin or to 50 mM KCl, we conclude that TRPM3 is expressed in a subpopulation of DRG neurons that also express voltage-gated Ca21 channels and very often TRPV1. Likewise, PregS-induced currents in rat DRG neurons were partially blocked upon addition of 1 mM isosakuranetin. Similar to heterologously expressed TRPM3, the isosakuranetin- induced TRPM3 block was reversible (Fig. 10C) and showed a TRPM3-characteristic, outwardly rectifying current-voltage relationship (Fig. 10, D and E). PregS-induced outward currents were concentration dependently blocked by isosa- kuranetin (Fig. 10F), and the potency and efficacy of the block was comparable to that seen in HEK293 cells heterologously expressing TRPM3 (see Fig. 3B). Because isosakuranetin blocked TRPM3 in DRG neurons, we retested the selectivity of isosakuranetin on other sensory TRP channels within their native context. To this end, we investigated the effect of isosakuranetin (5 mM and 20 mM) on 21 menthol- and capsaicin-induced increases in [Ca ]i in freshly isolated DRG neurons (Fig. 11, A–D). Isosakuranetin itself elicited a calcium entry in a small number of DRG neurons (Fig. 11, B and C), whereas no obvious effect was observed on Fig. 6. Effects of liquiritigenin and isosakuranetin on Ca2+ entry through menthol- and capsaicin-sensitive DRG neurons (Fig. 11D). TRPM1. Single-cell calcium imaging experiments were performed with Because overexpressed TRPA1 was partially activated by 20 HEK293 cells transiently transfected with cDNA plasmids encoding mM isosakuranetin (Fig. 7D), we performed measurements in TRPM1 and loaded with the fluorometric Ca2+ indicator Fura-2/AM. TRPM1 was activated with 50 mM PregS alone and in the presence of (A) NGF-treated, TRPA1-expressing DRG neurons (Fig. 11, E– 10 mM isosakuranetin (Iso; n = 55) or (B) 10 mM liquiritigenin (Liqu; n = H). Again, 3 of 150 and 5 of 136 DRG neurons were activated 48), followed by application of PregS alone or in combination with 100 mM by 5 mM and 20 mM isosakuranetin, respectively. Of note, all ZnCl2. Data shown are the mean 6 S.E.M. of at least four to six independent imaging experiments. of them also responded to the subsequent stimulation with Q:14 21 the TRPA1 activator AITC. AITC elicited increases in [Ca ]i in 26–30% of NGF-treated DRG neurons. The previous the TRPM3 blockers in vivo, we next assessed the cytotoxicity exposure to isosakuranetin did not cause a decrease in 21 of the TRPM3-blocking substances in the parental HEK cell AITC-induced [Ca ]i responses in all DRG neurons nor in line by applying MTT tests. Within the tested compounds, the AITC-sensitive subpopulation (Fig. 11H). There was also 21 liquiritigenin and ononetin did not significantly impair the no inhibition of KCl-induced [Ca ]i signals in DRG neurons cell proliferation and viability (Fig. 9). Hesperetin and cultured either in the absence or in the presence of NGF. isosakuranetin significantly reduced the MTT-converting Isosakuranetin and Hesperetin Attenuate the Sensi- enzymatic activity only partially and at the highest test tivity of Mice to Noxious Heat and PregS-Induced Pain concentration of 50 mM. Eriodictyol diminished the cell Behavior. TRPM3 has previously been shown to be involved proliferation in concentrations of 50 and 25 mM, respectively. in the perception of noxious heat in vivo (Vriens et al., 2011). With an almost complete abrogation of the MTT conversion, We therefore investigated nocifensive responses in mice that naringenin was the most cytotoxic compound among the were intraperitoneally injected with TRPM3-blocking flava- tested substances. All other flavanones were well tolerated by nones 30 minutes before a hot plate assay. HEK cells at concentrations that would completely block the Naringenin and hesperetin are citrus fruit flavanones that activation of TRPM3. were often used for in vivo studies in mice and even in humans Effects of Flavanones on TRPM3 in Freshly Isolated (Kanaze et al., 2007). We therefore decided to perform the in Rat DRG Neurons. To test the biologic activity of novel vivo experiments with the newly identified and poorly TRPM3-blocking flavanones on endogenously expressed described isosakuranetin and with hesperetin, which has TRPM3, single-cell calcium measurements were performed already been published and tested in in vivo studies. We using isolated rat DRG neurons that were loaded with the preferred hesperetin over naringenin because naringenin Ca21 indicator dye Fura-2. Functional expression of TRPM3 strongly impaired cell viability of HEK293 cells in concen- has previously been described in small to medium diameter trations up to 12.5 mM (Fig. 9). neurons (Vriens et al., 2011). Therefore, all neurons that met The latency of nocifensive behavior assessed on a hot plate this criterion were selected by defining regions of interest over (52°C) was significantly prolonged in mice after hesperetin single cells, and all cells that were sensitive to KCl were (10 mg kg21 i.p.) or isosakuranetin (2 or 10 mg kg21 i.p.) considered as neurones. About 60–70% of the neurons showed treatment compared with vehicle-treated animals (Fig. 12A). a PregS-induced calcium entry. A majority of but not all Note that 2 mg/kg hesperetin were not sufficient to signifi- PregS-positive neurons were also sensitive to stimulation cantly increase the pain latency. with capsaicin (Fig. 10, A and B). The PregS-induced calcium Because antagonists of the thermosensitive TRPV1 elicited entry was strongly counteracted either by 5 mM isosakur- hyperthermia in different species such as rat and mouse anetin (Fig. 10A) or by 10 mM liquiritigenin (Fig. 10B). (Gavva et al., 2008; Gunthorpe and Chizh, 2009), we Because most PregS-sensitive DRG neurons also responded to measured the temperature of mice before and 30 minutes

58 Selective TRPM3 Inhibitors 9

Fig. 7. Effects of liquiritigenin and isosakuranetin on other sensory TRP channels. HEK293 Fluo-4 loaded cells stably expressing the indicated TRP channels were dispensed into 384-well plates and preincubated with either liquiritigenin (A) or isosakuranetin (B). Activation of the respective channel 2+ was followed by measuring increases in the fluorescence intensity or in HEKrTRPV1:CFP cells by monitoring the Ca influx-mediated intracellular Q:15 acidification, causing a decrease in the fluorescence intensity of coexpressed yellow fluorescent protein (Hellwig et al., 2004). The respective activators are 2 mM capsaicin for TRPV1, 30 mMAITCforTRPA1,and300mM menthol for TRPM8. Acute stimulation of HEK293 cells stably expressing TRPA1 with liquiritigenin (C) and isosakuranetin (D) led to an activation of TRPA1. Data represent the mean 6 S.E.M. of four to six different experiments, performed in duplicate each.

after injection of hesperetin and isosakuranetin. As shown in number of nocifensive events after blocking TRPM3 by Fig. 12B, the newly identified TRPM3 blocker isosakuranetin isosakuranetin and hesperetin were measured at a PregD Q:8 or hesperetin attenuated the response to noxious heat in mice dose of 5 nmol per injected paw. Isosakuranetin at dosages of without altering their body temperature at either dosage. 2 and 10 mg kg21 significantly diminished the duration and The endogenous neurosteroid PregS acts as a chemical the number of nocifensive movements (Fig. 12, C and D). activator of TRPM3 (Wagner et al., 2008) and evokes pain Hesperetin injected in the same doses did not significantly (Ueda et al., 2001). Intraplantar injection of PregS (2.5, 5.0, reduce nocifensive responses. No differences in the latency to and 10.0 nmol) dose-dependently elicited prolonged nocifen- the first response to PregS were observed (data not shown). sive responses, consisting of vigorous licking and lifting of the The antinociceptive effects of both isosakuranetin doses hindpaw, whereas the injection of the vehicle did not cause were similar and more pronounced than those observed by a nocifensive behavior (data not shown). The duration and applying 10 mg kg21 hesperetin, indicating that

Fig. 8. Endogenously expressed TRPM7 in HEK293 cells is not affected by isosakuranetin (Iso) or liquiritigenin (Liqu). (A) Representative whole- cell recordings from HEK293 cells endogenously expressing TRPM7. Perfusion of the cells with a solution containing no divalent cations elicited linear TRPM7-like currents. An additional perfusion with 20 mM isosakuranetin or 20 mM liquiritigenin did not significantly influence the TRPM7 current. (B) Descriptive statistics of 5–10 independent measure- ments performed as shown in A.

59 10 Straub et al.

Fig. 9. Effect of TRPM3-blocking flava- nones on cell viability. HEK293 cells were incubated with different concentrations of TRPM3-blocking flavanones, and cell via- bility was detected by performing a MTT viability test. Data represent the mean values 6 S.E.M. of at least 5–8 indepen- dent experiments. To test for statistically significant differences, analysis of vari- ance with Tukey’s post-hoc test was per- formed with the normalized data (*P , 0.05; **P , 0.01).

isosakuranetin effects may be already saturated at a dose of 2 therefore assessed the plasma concentrations that were mg kg21. Saturating effects may be caused by its higher achieved after intraperitoneal application of 10 mg kg21 potency but also by a lower percentage of conjugation. We isosakuranetin. Peak concentrations were achieved about 10

Fig. 10. PregS-evoked Ca2+ influx in rat DRG neurons is reduced by liquiritigenin or isosakuranetin (Iso). (A and B) Freshly dissociated DRG neurons 2+ were loaded with Fura-2/AM, and the [Ca ]i was measured in individual neurons. Cells were superfused with 50 mM PregS and simultaneously with (A) 5 mM of isosakuranetin or (B) 10 mM liquiritigenin, followed by stimulation with 2 mM capsaicin and addition of 50 mM KCl to select neuronal cells that express voltage-gated Ca2+ channels. (C) Example trace of whole-cell currents from a freshly isolated rat DRG neuron measured in a Na+-free extracellular solution. Data are extracted from voltage ramps and depict the current at +100 mV (upper trace) and 2100 mV (lower trace). The neuron was perfused with 50 mM PregS alone or in combination with 1 mM isosakuranetin. (D) Current-voltage relationship 1) before stimulation and 2) after stimulation with PregS, and 3) after addition of 1 mm isosakuranetin. (E) Current-voltage relationship of the PregS-induced current depicted as the difference between trace 2 and 1 in D. (F) Example of a whole-cell measurement of a DRG neuron as described in D during perfusion with PregS and various concentrations of isosakuranetin. Shown are currents at +100 mV (dotted line: zero current level).

60 Selective TRPM3 Inhibitors 11

Fig. 11. Isosakuranetin (Iso) does not inhibit TRPM8, TRPV1, or TRPA1 channels in rat DRG neurons. Single-cell calcium imaging experiments were performed with freshly isolated DRG neurons. (A–C) DRG neurons were superfused with solutions containing no isosakuranetin (A) or with solutions containing (B) 5 mMand 2+ (C) 20 mMisosakuranetin.DRGneuronsweresequentiallychallengedwith300mMmenthol,1mMcapsaicin,and50mMKCl.[Ca ]i traces of cells that responded to isosakuranetin, menthol, and capsaicin were grouped and highlighted by green, blue, and red colors, respectively. Numbers of responsive cells aregiveninthe 2+ application bars. (D) Statistical analysis of [Ca ]i determined in DRG neurons treated with buffer (open bars), with 5 mMisosakuranetin(hatched bars), or with 20 mMisosakuranetin(filledbars).Colored bars reflect results determined only in menthol-positive (blue) or in capsaicin-responding (red) subpopulations. (E–G) Experiments were performed essentially as in A–C, but in NGF-treated DRG neurones that were stimulated with 20 mMAITC(yellowlines).(H)Statisticalanalysis of all DRG neurons measured in E–GandintheAITC-positivesubpopulation(yellowbars).Datarepresentfour(A–C) or three (E–G) independent experiments for each condition performed with DRG neurons from different animals.

61 12 Straub et al.

Fig. 12. Isosakuranetin (Iso) and hesperetin (Hes) diminish the sensitivity of mice to noxious heat and attenuate the PregS-induced pain behavior. (A) Mice were intraperitoneally injected with solvent (control), with 2 mg kg21 and 10 mg kg21 isosakuranetin, or with 2 mg kg21 and 10 mg kg21 hesperetin and placed onto a hot plate (52°C) 30 minutes after the injection. The latency to the first nocifensive response was recorded. (B) Body temperature was measured before (gray bars) and 30–35 minutes after (black bars) injection of hesperetin or isosakuranetin, respectively. (C and D) Chemical pain was evoked 30 minutes after i.p. application of the flavanones (performed as described in A) by intraplantar injection of PregS (5 nmol in 10 ml) into the right hindpaw. The duration (C) and number (D) of nocifensive behavioral events (paw licking, shaking, or lifting) was recorded and counted. Data represent the mean 6 S.E.M. of 9–12 mice per group; *P , 0.05. (E) Blood was obtained from mice sacrificed without receiving isosakuranetin (0 minutes) or at the indicated time points after injection of 10 mg kg21 isosakuranetin (i.p.). Concentrations of unconjugated isosakuranetin were determined by HPLC and ultraviolet light spectroscopy.

minutes after the injection, but the individual variability was dropped rapidly, presumably due to fast formation of high (Fig. 12E). A mean plasma concentration of 5.0 6 1.8 mg metabolites. Thirty minutes after application of 2 mg ml21, ml21 (17.5 6 6.3 mM) unconjugated isosakuranetin was the mean plasma concentration of unconjugated isosakur- measured 30 minutes after intraperitoneal application and anetin was 1.8 mM and may be sufficient to block TRPM3. with a lower individual variability. Plasma levels then

62 Selective TRPM3 Inhibitors 13

Discussion more complex substituents at the C49 position may provide a promising target for further investigation. Owing to their low potency and to their known actions on When compared with other biologic activities that have other biologic effectors, previously published TRPM3 blockers been reported for flavanones, inhibition of TRPM3 by various Q:9 such as the peroxisome proliferator-activated receptor-g- flavanones displays a distinct and unique preference. Liquir- agonistic antidiabetic drug rosiglitazone (Majeed et al., 2011) itigenin, a flavanone found in many plants including licorice, or the nonsteroidal anti-inflammatory drug mefenamic acid is a selective and potent estrogen receptor b (ER-b) agonist (IC 5 6.6 mM) (Klose et al., 2011) have not been used to 50 (EC 5 36.5 nM) (Mersereau et al., 2008), which may relate investigate TRPM3 functions in vivo. Starting from the recent 50 to the beneficial effects observed after applying a liquiritige- finding that citrus fruit flavanones block TRPM3, we sought nin-containing herbal drug (MF101) used in traditional Q:10 to obtain nontoxic, selective, and potent TRPM3 inhibitors Chinese medicine to treat menopausal symptoms (Mersereau that may be used in animal models of pain perception. et al., 2008). Isoflavones from soybeans, such as genistein and Hereby, the pharmacophore was confirmed, and substituent daizein, also act as phytoestrogens that bind to both ER-b and variation led to the identification of isosakuranetin and ER-a (An et al., 2001; Mersereau et al., 2008), but are poorly liquiritigenin as noncytotoxic, more potent, and selective effective in blocking TRPM3. TRPM3 inhibitors. Both recombinant TRPM3 and TRPM3- Compared with our results with a higher potency of related [Ca21] signals and ionic currents in DRG neurons i isosakuranetin to block TRPM3, the structure relationship were strongly and reversibly inhibited. Other sensory TRP for binding ER receptors differs from the structure needed to channels or TRPM1, the closest relative of TRPM3, remained block TRPM3. Furthermore, glycosylation of naringenin, largely unaffected at modulator concentrations that fully found in naringenin-6-C-glucoside or in naringin enhanced blocked TRPM3. In vivo application of isosakuranetin and the estrogenic effect (Swarnkar et al., 2012), whereas we hesperetin confirmed an antinociceptive effect on heat or previously demonstrated that glycosylation in naringin and PregS-induced pain responses. Interestingly, the intraperito- hesperedin prevents the inhibition of TRPM3 (Straub et al., neal administration of both flavanones did not affect the body 2013). temperature, an effect that would be expected for compounds Besides their property to bind to ER receptors, flavonoids with TRPV1-antagonistic properties. modulated phase I metabolizing enzymes via binding to Besides a hit optimization strategy, we intended to more proteins of the cytochrome P450 family (P450). Naringenin Q:11 clearly assign the pharmacophore of flavonoid that is required and eriodictyol have been shown to block CYP1 activity, with to block TRPM3 and to assess the impact of the hydroxylation an IC . 4 mM. Naringenin also inhibits CYP3A4 and CYP19 pattern at the A and C rings. In plants, flavonoids are usually 50 (Moon et al., 2006). With an IC of 0.34 mM, liquiritigenin has present as glycosides. Upon enteral uptake, flavonoid glyco- 50 been identified as a potent inhibitor of CYP19 (Paoletta et al., sides are metabolized in a complex fashion. After consump- 2008). Inhibitory effects of isosakuranetin on CYP1 enzyme tion of naringin, the glycoside moiety is hydrolyzed by isoforms require concentrations in the range of 1–3 mM bacteria in the distal part of the intestine (Fuhr and (Takemura et al., 2010), and inhibition of other P450 enzymes Kummert, 1995), and the corresponding aglycones are has, to our knowledge, not been demonstrated. We conclude absorbed. In addition to phase I modification, flavanone that liquiritigenin may be the TRPM3 inhibitor with the aglycones undergo three types of phase II conjugation highest selectivity compared with other TRP channels. Thus, reactions: methylation, sulfation, and glucuronidation (Man- when used as a cell biologic tool to profile TRP channel-related ach et al., 2004). Accordingly, Silberberg et al. (2006) found responses, liquiritigenin may be favorable. However, its plasma concentrations of 10–17 mM naringenin but also 100– effects on P450 enzymes and its ER-b–agonistic properties 120 nM of isosakuranetin and 230 nM hesperetin after may interfere with its TRPM3-blocking properties in vivo. feeding rats with a diet containing 0.5% naringin. Unlike Isosakuranetin, by contrast, with respect to non-TRP channel naringenin, which is extensively metabolized to its glucuro- off-target effects is a preferable compound for use in in vivo nide or sulfate conjugates, 52% of isosakuranetin and 94% of pharmacologic studies. hesperetin remain unconjugated; therefore, they may exert The physiologic and pathophysiologic functions of TRPM3 biologic activities in vivo. Conjugated flavanone aglycones are are still poorly understood. Different splice variants that typically eliminated, with half-times in the range of about 4 to impede on the Ca21 permeability of the pore (e.g., the a1 8 hours (Erlund et al., 2001). To avoid possible uncertainties splicing event) (Oberwinkler et al., 2005) or even eliminate that may result from enteral resorption or hepatic first-pass a functional pore (Frühwald et al., 2012) further complicate elimination, we applied hesperetin or isosakuranetin by the situation. In TRPM3-deficient transgenic mice, pheno- intraperitoneal injection. typic alterations hint at a role of TRPM3 in thermal pain Although the flavanone backbone appeared optimal for perception (Vriens et al., 2011), an effect that is closely TRPM3 block, the hydroxylation pattern strongly impacted mimicked by a pharmacologic block of the channel, as the potency to block TRPM3. The C79 hydroxylation at the A described in the present study. Considering the expression ring is favorable. The missing C59 hydroxylation at the A ring of TRPV1 and TRPM3 in DRG neurons, the contribution of in liquiritigenin did not improve the potency to block TRPM3 each of the heat-sensing sensory channels to thermal pain compared with naringenin, but further increased the selec- perception is intriguingly complex and seems redundant. tivity of the effect and lowered the cytotoxicity in HEK293 However, pharmacologic inhibition of either TRPV1 or cells. Concerning the B ring, hydroxylation at the C39 atom TRPM3 exerts similar antinociceptive effects. At present, appeared unfavorable, but 49-hydroxylation or 49-O- a cross-talk between both channels has not been described. An methylation improved the potency to block TRPM3 without efferent function of heat-sensitive, CGRP-releasing nocicep- strongly compromising the blocker selectivity. Thus, adding tive neurons has been demonstrated in neurogenic

63 14 Straub et al. inflammation, but may also contribute to the pyrexia- effects that are related to the anti-inflammatory or antioxi- inducing effects of TRPV1 blockers. In contrast to TRPV1 dant properties of flavanones. Within this context, a pain- blockers, the systemic administration of isosakuranetin did alleviating effect of eriodictyol, another flavanone with not cause a discernible increase in the body temperature. This antioxidant properties, has been described and attributed to observation hints to a promising feature of TRPM3 as TRPV1 inhibition (Rossato et al., 2011). As outlined earlier, a potential analgesic target molecule and may provide we were unable to reproduce inhibition of TRPV1, but a strategy to avoid the adverse effects of TRPV1 blockers. demonstrated that eriodictyol efficiently blocks TRPM3. The exact reason why a TRPM3 block is not associated with Interestingly, the eriodictyol-induced antinociceptive effect similar side effects than inhibitors of TRPV1 is not known. was not associated with hyperthermia. Likewise, the TRPM3- Possibly, the thermal threshold of TRPM3 activation is blocking compounds hesperetin and isosakuranetin did not sufficiently high to avoid regulation by physiologic body or increase the body temperature. Hence, flavanones with TRP skin temperatures. Likewise, the marked shift of threshold channel-inhibiting properties and/or inhibition of TRPM3 temperatures described for TRPV1 in acidic or inflamed may provide a promising starting point to develop novel and tissues (Gavva et al., 2008) may not apply to TRPM3. Finally, tolerable analgesic therapies. the population of DRG neurons that express TRPV1 or TRPM3 is not completely identical (Vriens et al., 2011). Thus, Acknowledgments TRPM3 activation may not exactly mimic TRPV1-induced The authors thank Helga Sobottka, Marion Leonhardt, and Raissa responses. Wehmeyer for excellent technical assistance. Another open question relates to possible consequences of Authorship Contributions TRPM3 inhibition other than attenuation of thermal pain Participated in research design: Straub, Krügel, Mohr, Rizun, sensation. Obviously, we cannot exclude that such on- or off- Konrad, Oberwinkler, Schaefer, Teichert. target effects may limit the therapeutic potential of TRPM3 Conducted experiments: Straub, Krügel, Mohr, Rizun, Konrad, inhibition. On the other hand, two lines of evidence support Teichert. the assumption that TRPM3 inhibition may be tolerated: 1) Performed data analysis: Straub, Krügel, Mohr, Rizun, Konrad, 2/2 the phenotype of TRPM3 mice is relatively mild, and 2) Teichert. TRPM3-blocking compounds identified in this study are Wrote or contributed to the writing of the manuscript: Straub, contained predominantly in citrus fruit that are part of our Krügel, Mohr, Rizun, Konrad, Oberwinkler, Schaefer, Teichert. 2/2 daily diet. In TRPM3 mice, a partially reduced pupillary References response to light has been described (Hughes et al., 2012). In An J, Tzagarakis-Foster C, Scharschmidt TC, Lomri N, and Leitman DC (2001) contrast to a loss of TRPM1 that directly affects the function of Estrogen receptor b-selective transcriptional activity and recruitment of cor- egulators by phytoestrogens. JBiolChem276:17808–17814. bipolar cells in the retina, thereby causing congenital night Chubanov V, Mederos y Schnitzler M, Meißner M, Schäfer S, Abstiens K, Hofmann T, blindness (Koike et al., 2010), the effect of TRPM3 deficiency and Gudermann T (2012) Natural and synthetic modulators of SK (K(ca)2) po- on pupillary responses is less pronounced (Hughes et al., tassium channels inhibit magnesium-dependent activity of the kinase-coupled cation channel TRPM7. Br J Pharmacol 166:1357–1376. 2012). Future work will have to clarify whether acute Erlund I, Meririnne E, Alfthan G, and Aro A (2001) Plasma kinetics and urinary inhibition of TRPM3 interferes with retinal functions. TRPM3 excretion of the flavanones naringenin and hesperetin in humans after ingestion of orange juice and grapefruit juice. JNutr131:235–241. is functionally expressed in insulin-secreting beta cells. Frühwald J, Camacho Londoño J, Dembla S, Mannebach S, Lis A, Drews A, Wis- Although no obvious effects were observed in the resting senbach U, Oberwinkler J, and Philipp SE (2012) Alternative splicing of a protein 2/2 domain indispensable for function of transient receptor potential melastatin 3 blood glucose level in TRPM3 mice that exhibit no obvious (TRPM3) ion channels. JBiolChem287:36663–36672. developmental or metabolic deficits (Vriens et al., 2011), the Fuhr U and Kummert AL (1995) The fate of naringin in humans: a key to grapefruit juice-drug interactions? Clin Pharmacol Ther 58:365–373. acute application of TRPM3 blockers may cause alterations of Gavva NR, Treanor JJ, Garami A, Fang L, Surapaneni S, Akrami A, Alvarez F, Bak glucose homeostasis. Using the TRPM3-blocking flavanones, A, Darling M, and Gore A et al. (2008) Pharmacological blockade of the vanilloid receptor TRPV1 elicits marked hyperthermia in humans. Pain 136:202–210. these questions may be addressed and help to clarify possible Grimm C, Kraft R, Schultz G, and Harteneck C (2005) Activation of the melastatin- endocrine functions of TRPM3 in the pancreas. related cation channel TRPM3 by D-erythro-sphingosine [corrected]. Mol Phar- macol 67:798–805. As an off-target effect of flavanones, anti-inflammatory and/ Gunthorpe MJ and Chizh BA (2009) Clinical development of TRPV1 antagonists: or antioxidant properties may add to effects on TRPM3 targeting a pivotal point in the pain pathway. Drug Discov Today 14:56–67. Hellwig N, Plant TD, Janson W, Schäfer M, Schultz G, and Schaefer M (2004) TRPV1 specifically, if pain regimes are being tested in models of acts as proton channel to induce acidification in nociceptive neurons. JBiolChem inflammation. Such properties may thus overlap with the 279:34553–34561. pain-alleviating effects of the TRPM3 inhibitors, such as in Hoffmann A, Grimm C, Kraft R, Goldbaum O, Wrede A, Nolte C, Hanisch UK, Richter-Landsberg C, Brück W, and Kettenmann H et al. (2010) TRPM3 is pain threshold tests with complete Freund’s adjuvant– expressed in sphingosine-responsive myelinating oligodendrocytes. JNeurochem induced paw inflammation. Experimentally, the inhibition 114:654–665. Hughes S, Pothecary CA, Jagannath A, Foster RG, Hankins MW, and Peirson SN of carrageenan-induced paw edema formation by orally (2012) Profound defects in pupillary responses to light in TRPM-channel null mice: applied (50 mg kg21 day21) or intravenously injected (15 mg a role for TRPM channels in non-image-forming photoreception. Eur J Neurosci 35: 21 21 34–43. kg day ) liquiritigenin has been demonstrated (Kim et al., Kanaze FI, Bounartzi MI, Georgarakis M, and Niopas I (2007) Pharmacokinetics of 2008). In a cell culture model using a lipopolysaccharide- the citrus flavanone aglycones hesperetin and naringenin after single oral ad- ministration in human subjects. Eur J Clin Nutr 61:472–477. primed macrophagelike cell line, Kim et al. (2008) observed an Kim YW, Zhao RJ, Park SJ, Lee JR, Cho IJ, Yang CH, Kim SG, and Kim SC (2008) inhibition of cytokine production by 3–30 mM liquiritigenin. Anti-inflammatory effects of liquiritigenin as a consequence of the inhibition of NF- kB-dependent iNOS and proinflammatory cytokines production. Br J Pharmacol However, the TRPM3 block by liquiritigenin requires lower 154:165–173. concentrations of the flavanone. In addition, systemically Klose C, Straub I, Riehle M, Ranta F, Krautwurst D, Ullrich S, Meyerhof W, and Harteneck C (2011) Fenamates as TRP channel blockers: mefenamic acid se- administered isosakuranetin was effective to elicit antinoci- lectively blocks TRPM3. 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64 Selective TRPM3 Inhibitors 15

(TRPM1) is an ion-conducting plasma membrane channel inhibited by zinc ions. J antagonist of the TRPV1 receptor with antioxidant activity. Biochem Pharmacol Biol Chem 286:12221–12233. 81:544–551. Lee N, Chen J, Sun L, Wu S, Gray KR, Rich A, Huang M, Lin JH, Feder JN, Silberberg M, Gil-Izquierdo A, Combaret L, Remesy C, Scalbert A, and Morand C and Janovitz EB et al. (2003) Expression and characterization of human transient (2006) Flavanone metabolism in healthy and tumor-bearing rats. Biomed Phar- receptor potential melastatin 3 (hTRPM3). JBiolChem278:20890–20897. macother 60:529–535. Majeed Y, Bahnasi Y, Seymour VA, Wilson LA, Milligan CJ, Agarwal AK, Sukumar Straub I, Mohr F, Stab J, Konrad M, Philipp SE, Oberwinkler J, and Schaefer M P, Naylor J, and Beech DJ (2011) Rapid and contrasting effects of rosiglitazone on (2013) Citrus fruit and fabacea secondary metabolites potently and selectively transient receptor potential TRPM3 and TRPC5 channels. Mol Pharmacol 79: block TRPM3. Br J Pharmacol 168:1835–1850. 1023–1030. Swarnkar G, Sharan K, Siddiqui JA, Mishra JS, Khan K, Khan MP, Gupta V, Rawat Manach C, Scalbert A, Morand C, Rémésy C, and Jiménez L (2004) Polyphenols: food P, Maurya R, and Dwivedi AK et al. (2012) A naturally occurring naringenin de- sources and bioavailability. Am J Clin Nutr 79:727–747. rivative exerts potent bone anabolic effects by mimicking oestrogen action on Mersereau JE, Levy N, Staub RE, Baggett S, Zogovic T, Chow S, Ricke WA, osteoblasts. Br J Pharmacol 165:1526–1542. Tagliaferri M, Cohen I, and Bjeldanes LF et al. (2008) Liquiritigenin is a plant- Takemura H, Itoh T, Yamamoto K, Sakakibara H, and Shimoi K (2010) Selective derived highly selective estrogen receptor beta agonist. Mol Cell Endocrinol 283: inhibition of methoxyflavonoids on human CYP1B1 activity. Bioorg Med Chem 18: 49–57. 6310–6315. Moon YJ, Wang X, and Morris ME (2006) Dietary flavonoids: effects on xenobiotic Ueda H, Inoue M, Yoshida A, Mizuno K, Yamamoto H, Maruo J, Matsuno K, and carcinogen metabolism. Toxicol In Vitro 20:187–210. and Mita S (2001) Metabotropic neurosteroid/sigma-receptor involved in stimula- Nadler MJS, Hermosura MC, Inabe K, Perraud AL, Zhu Q, Stokes AJ, Kurosaki T, tion of nociceptor endings of mice. JPharmacolExpTher298:703–710. Kinet JP, Penner R, and Scharenberg AM et al. (2001) LTRPC7 is a Mg.ATP- Urban N, Hill K, Wang L, Kuebler WM, and Schaefer M (2012) Novel pharmaco- regulated divalent cation channel required for cell viability. Nature 411:590–595. logical TRPC inhibitors block hypoxia-induced vasoconstriction. Cell Calcium 51: Naylor J, Li J, Milligan CJ, Zeng F, Sukumar P, Hou B, Sedo A, Yuldasheva N, 194–206. Majeed Y, and Beri D et al. (2010) Pregnenolone sulphate- and cholesterol- Vega-Villa KR, Remsberg CM, Podelnyk KL, and Davies NM (2008) Stereospecific regulated TRPM3 channels coupled to vascular smooth muscle secretion and high-performance liquid chromatographic assay of isosakuranetin in rat urine. J contraction. Circ Res 106:1507–1515. Chromatogr B Analyt Technol Biomed Life Sci 875:142–147. Nörenberg W, Hempel C, Urban N, Sobottka H, Illes P, and Schaefer M (2011) Vriens J, Owsianik G, Hofmann T, Philipp SE, Stab J, Chen X, Benoit M, Xue F, Clemastine potentiates the human P2X7 receptor by sensitizing it to lower ATP Janssens A, and Kerselaers S et al. (2011) TRPM3 is a nociceptor channel involved concentrations. JBiolChem286:11067–11081. in the detection of noxious heat. Neuron 70:482–494. Nörenberg W, Sobottka H, Hempel C, Plötz T, Fischer W, Schmalzing G, Wagner TF, Loch S, Lambert S, Straub I, Mannebach S, Mathar I, Düfer M, Lis A, and Schaefer M (2012) Positive allosteric modulation by ivermectin of human but Flockerzi V, and Philipp SE et al. (2008) Transient receptor potential M3 channels not murine P2X7 receptors. Br J Pharmacol 167:48–66. are ionotropic steroid receptors in pancreatic beta cells. Nat Cell Biol 10: Oberwinkler J, Lis A, Giehl KM, Flockerzi V, and Philipp SE (2005) Alternative 1421–1430. splicing switches the divalent cation selectivity of TRPM3 channels. JBiolChem 280:22540–22548. Paoletta S, Steventon GB, Wildeboer D, Ehrman TM, Hylands PJ, and Barlow DJ Address correspondence to: Michael Schaefer, Rudolf-Boehm-Institut für (2008) Screening of herbal constituents for aromatase inhibitory activity. Bioorg Pharmakologie und Toxikologie, Härtelstr. 16-18, 04107 Leipzig, Germany. Med Chem 16:8466–8470. E-mail: [email protected] Rossato MF, Trevisan G, Walker CI, Klafke JZ, de Oliveira AP, Villarinho JG, Zanon RB, Royes LF, Athayde ML, and Gomez MV et al. (2011) Eriodictyol: a flavonoid

65

Nachweis über Anteile der Co-Autoren, Isabelle Straub

Identification and application of novel and selective blockers for the heat-activated cation channel TRPM3

Nachweis über Anteile der Co-Autoren:

Titel: Flavanones that selectively inhibit TRPM3 attenuate thermal nociception in vivo Journal: Molecular Pharmacology Autoren: Isabelle Straub, Ute Krügel, Florian Mohr, Jens Teichert, Oleksandr Rizun, Maik Konrad, Johannes Oberwinkler, Michael Schaefer

Anteil Isabelle Straub : - Schreiben des Tierversuchsantrag - Zellkulturen - Zusammenstellen der Screening Componenten - Screening der TRPM3 Zelllinie - Fluoreszenzmessungen in Zellsuspension - Elektrophysiologie der TRPM3 Zellinie mit PregS und Hitze-Aktivierung - Einzelzell Fluoreszenzmessungen der DRG Neurone von Ratten - Elektrophysiologie von DRG Neuronen - Analyse und Verarbeitung der Daten - Schreiben der Publikation - Zellcytoxizitätstest

Anteil Ute Krügel: - Verhaltenstests Mäuse

Anteil Florian Mohr: - Einzelzell Fluoreszenzmessungen TRPM1 - Giant excised patch Elektrophysiologie

Anteil Jens Teichert: - Bestimmung der Plasmakonzentration

Anteil Oleksandr Rizun: - Einzelzell Fluoreszenzmessungen TRPM1

Anteil Maik Konrad: - Einzelzell Fluoreszenzmessungen TRPM1

Anteil Johannes Oberwinkler: - Konzeption

Anteil Michael Schaefer: - Projektidee - Konzeption - Schreiben der Publikation 67

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Lebenslauf

Pers¨onliche Daten: Name: Isabelle Straub geboren am: 18.01.1980 Geburtsort: Neunkirchen Staatsangeh¨origkeit: Deutsch Familienstand: ledig, 1 Kind

Schulausbildung: 1986 – 1990 Grundschule G¨ottelborn 1990 – 1999 Illtalgymnasium Illingen 1999 Schulabschluss: Abitur

Akademische Ausbildung/ Beruflicher Werdegang: 10/1999 – 10/2000 Studium der Komparatistik, Philosophie und Geschichte Universit¨atdes Saarlandes 10/2000 – 11/2006 Studium der Biologie am Zentrum f¨urMolekular- und Hu- mangenetik Universit¨atdes Saarlandes 2/2007 - 09/2008 wissenschaftliche Mitarbeiterin Molekulare Pharmakologie und Zellbiologie Charite, Berlin 10/2008 - 08/2013 wissenschaftliche Mitarbeiterin Rudolf-Boehm-Institut f¨urPharmakologie und Toxikologie, Medizinische Fakult¨at,Universit¨atLeipzig 01/2010 - 10/2010 Elternzeit

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Wissenschaftliche Publikationen:

Transient receptor potential M3 channels are ionotropic steroid receptors in pan-creatic beta cells. Wagner TF, Loch S, Lambert S, Straub I, Mannebach S, Mathar I, Dfer M, Lis A, Flockerzi V, Philipp SE, Oberwinkler J Nat Cell Biol. 2008 Dec; 10(12):1421-30 Fenamates as TRP channel blockers: mefenamic acid selectively blocks TRPM3. Klose C*, Straub I*, Riehle M, Ranza F, Krautwurst D, Ullrich S, Meyerhof W, Harteneck C Br J Pharmacol. 2011 Apr; 162(8): 1757-69 The first authors contributed equally to the work Citrus fruit and fabacea secondary metabolites potently and selectively block TRPM3. Straub I, Mohr F, Stab J, Konrad M, Philipp SE, Oberwinkler J, Schaefer M Br J Pharmacol. 2013 Apr; 168(8): 1835-50 P2X7 receptors at adult neural progenitor cells of the mouse subventricular zone. Messemer N, Kunert C, Grohmann M, Sobottka H, Nieber K, Zimmermann H, Franke H, Nrenberg W, Straub I, Schaefer M, Riedel T, Illes P, Rubini P Neuropharmacology 2013; May 28; 73C:122-137 Flavanone that selectively inhibit TRPM3 attenuate thermal nociception in vivo. Straub I, Krgel U, Mohr F, Teichert J, Rizun O, Konrad M, Oberwinkler J, Schaefer M Mol Pharmacol. 2013 Sep 4. [Epub ahead of print]

Tagungsbeitr¨age:

Straub, I., Hill, K., Schaefer M., Transmembrane channel like (TMC) proteins and their role in calcium homeostasis. 9th Leipzig Research Festival for Life Sciences 2010. Leipzig, Deutschland 12/2010 Straub, I., Oberwinkler, J., Schaefer M., Citrus fruit flavonoids naringenin and hesperetin are potent TRPM3 blockers. 10th Leipzig Research Festival for Life Sciences 2011. Leipzig, Deutschland 12/2011 Straub, I., Mohr, F., Konrad M., Oberwinkler, J., Schaefer M., Citrus fruit flavonoids as potent TRPM3 blockers. Posterpr¨asentation. 78.

81 Jahrestagung Deutsche Gesellschaft f¨urexperimentelle und klinische Pharmakologie und Toxikologie, Deutschland 03/2012 Straub, I., Oberwinler, J., Schaefer M., Citrus fruit flavonoids as potent and selec-tive TRPM3 blockers. 11th Leipzig Research Festival for Life Sciences 2012. Leipzig, Deutschland 12/2012 Straub, I., Krgel, U., Mohr, F., Konrad M., Oberwinkler, J., Schaefer M., Optimization of citrus fruit flavanones as TRPM3 blockers. Vortrag. 79. Jahrestagung Deutsche Gesellschaft f¨urexperimentelle und klinische Pharmakologie und Toxikologie, Deutschland 03/2013

82 Selbstst¨andigkeitserl¨arung

Hiermit versichere ich, dass die vorliegende Dissertation ohne unzul¨assigeHilfe und ohne Benutzung anderer als der angegebenen Hilfsmittel angefertigt wurde. Direkt oder indirekt ¨ubernommene Gedanken aus fremden Quellen wurden in dieser Arbeit als solche kenntlich gemacht.

Leipzig, den 30. September 2013

Isabelle Straub

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”To absent friends, lost loves, old gods, and the season of mists; and may each and every one of us give the devil his due”

Neil Gaiman, The Sandman, Vol.4: Season of Mists

Danke, an alle die mir geholfen haben....

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