Vanilloid Receptor Trpv1 in Drug Discovery

VANILLOID RECEPTOR TRPV1 IN DRUG DISCOVERY

Targeting Pain and Other Pathological Disorders

Edited by

ARTHUR GOMTSYAN CONNIE R. FALTYNEK Abbott Laboratories

A JOHN WILEY & SONS, INC., PUBLICATION

VANILLOID RECEPTOR TRPV1 IN DRUG DISCOVERY

Targeting Pain and Other Pathological Disorders

Edited by

ARTHUR GOMTSYAN CONNIE R. FALTYNEK Abbott Laboratories

Published by John Wiley & Sons, Inc., Hoboken, New Jersey

Published simultaneously in Canada

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Library of Congress Cataloging-in-Publication Data

ISBN: 978-0-470-17557-6

Printed in the United States of America

10 9 8 7 6 5 4 3 2 1 Arthur wishes to dedicate this book to his wife Natalia, daughters Anahit, Lusine, Ani, and parents, especially father, with love and gratitude.

Connie dedicates this book with love to her husband Robert, who is her lifelong dearest friend and soul mate.

CONTENTS

PREFACE AND ACKNOWLEDGMENTS ix FOREWORD xiii CONTRIBUTORS xv

PART I INTRODUCTION TO THE TRP CHANNELS 1

1 TRP Channels and Human Diseases 3 Bernd Nilius and Rudi Vennekens 2 Role of TRP Channels in Pain: An Overview 68 Arpad Szallasi 3 Biochemical Pharmacology of TRPV1: Molecular Integrator of Pain Signals 101 Carol S. Surowy, Philip R. Kym, and Regina M. Reilly 4 TRPV1 Genetics 134 Ruslan Dorfman, Hubert Tsui, Michael W. Salter, and H.-Michael Dosch

PART II ROLE FOR TRPV1 IN PAIN STATES 151

5 TRPV1 and Inﬂ ammatory Pain 153 Anindya Bhattacharya, Sonya G. Lehto, and Narender R. Gavva 6 Role of TRPV1 Receptors in Osteoarthritic Pain 175 Shailen K. Joshi and Prisca Honore 7 TRPV1 and Bone Cancer Pain 191 Juan Miguel Jimenez-Andrade and Patrick Mantyh 8 TRPV1 in Visceral Pain and Other Visceral Disorders 206 António Avelino and Francisco Cruz

vii viii CONTENTS

9 TRPV1 Receptors and Migraine 239 Philip R. Holland and Peter J. Goadsby 10 TRPV1 in Neuropathic Pain and Neurological and Neuropsychiatric Disorders 260 Enza Palazzo, Katarzyna Starowicz, Sabatino Maione, and Vincenzo Di Marzo

PART III TRPV1 ANTAGONISTS AND AGONISTS AS NOVEL ANALGESIC DRUGS 293

11 Aryl-Urea Class and Related TRPV1 Antagonists 295 Arthur Gomtsyan 12 2-Pyridinylpiperazine Carboxamide Class and Related TRPV1 Antagonists 311 Natalie A. Hawryluk and Nicholas I. Carruthers 13 TRPV1 Agonist Approaches for Pain Management 325 Keith R. Bley

PART IV ROLE FOR TRPV1 IN OTHER PHYSIOLOGICAL PROCESSES BESIDES PAIN TRANSMISSION 349

14 The TRPV1 Channel in Normal Thermoregulation: What Have We Learned from Experiments Using Different Tools? 351 Andras Garami, Maria C. Almeida, Tatiane B. Nucci, Tamara Hew-Butler, Renato N. Soriano, Eszter Pakai, Kazuhiro Nakamura, Shaun F. Morrison, and Andrej A. Romanovsky 15 The Role of TRPV1 in Respiratory Diseases 403 Serena Materazzi, Alain Tchoimou, Romina Nassini, Marcello Trevisani, and Pierangelo Geppetti 16 The Role of TRPV1 in Diabetes 423 Hubert Tsui, Ruslan Dorfman, Michael W. Salter, and H.-Michael Dosch

AFTERWORD 449 INDEX 451 PREFACE AND ACKNOWLEDGMENTS

The intended purpose of this book is to summarize available data related to the therapeutic utility for transient receptor potential vanilloid 1 (TRPV1) ligands. Many of the world’ s leading experts on TRPV1 contributed to this book, not only by reviewing the most relevant information on the subject but also by providing personal opinions on the critical issues. The history of the TRPV1 receptor is, for the most part, the history of chili pepper, a popular hot spice that has been cultivated in South America for over 6000 years and in the rest of the world since the sixteenth century. However, food fl avoring has not been the only benefi t chili peppers have offered. Chili preparations have been used as treatments for ulcers, backaches, and coughs, among other maladies. The ingredient that is responsible for the initial burning effect of chili pepper, as well as for its medicinal properties, is a compound called capsaicin, which constitutes approximately 0.14% w/w of chili peppers. Capsaicin was isolated in 1876, and its structure was determined shortly there- after. The homovanillyl fragment in the structure of capsaicin was the basis for naming the putative cell surface receptor vanilloid receptor 1 (VR1). An amazing breakthrough occurred in 1997, when VR1 was molecularly cloned by David Julius’ team. VR1 later was renamed the TRPV1 to under- score its membership in a larger family of transient receptor potential (TRP) ion channels. The Foreword by Professor Julius presents an illuminating perspective on the entire fi eld. The two introductory chapters in this volume (Nilius and Vennekens; Szallasi) are designed to familiarize the reader with the multifunctional TRP family of ion channels and, at the same time, to lay the foundation for the remaining chapters, which focus on one member of that family, TRPV1. Members of the TRP superfamily have been shown to be polymodal molecular ix x PREFACE AND ACKNOWLEDGMENTS sensors, with the important roles of detecting a variety of thermal, mechanical, and chemical stimuli. These ion channels have been shown to contribute to sensory processes such as pain transmission, vision, taste, and hearing. The complex nature of TRPV1 modulation is described in the chapter by Surowy et al., which provides basic information about the structure of the receptor, numerous regulators (activators and sensitizers), and their modes and sites of action. The chapter on TRPV1 genetics (Dorfman et al.) reviews the impact of TRPV1 genetic variability on the pharmacological properties of TRPV1 in different species and summarizes available data on identifi cation of the binding sites for biological substrates. Several key studies with TRPV1 knockout mice and with TRPV1 antagonists, support the conclusion that TRPV1 plays an important role in pain pathways. Indeed, most of the advances with TRPV1 as a new molecular target for novel medications have been in the development of new analgesics. The next chapters discuss in detail the connection between TRPV1 and various types of pain. Preclinical studies provide a strong case for the benefi cial effects of TRPV1 blockade for the treatment of infl ammatory, osteoarthritic, and bone cancer pain. The effi cacy of TRPV1 antagonists in animal models of infl ammatory pain is discussed in the chapter by Bhattacharya et al. Several preclinical studies have shown the benefi t of TRPV1 antagonists in treating the pain associated with osteoarthritis (Joshi and Honore). It has also been shown that bone cancer pain, which is often poorly controlled by existing analgesics, can be successfully attenuated in mice by treatment with TRPV1 antagonists (Jimenez - Andrade and Mantyh). TRPV1 has been actively inves- tigated as a potential target for conditions such as visceral pain and the treatment of lower urinary tract symptoms, including urinary frequency, urgency, and incontinence (Avelino and Cruz). Evidence for the role of TRPV1 in migraine (Holland and Goadsby) and in neuropathic pain (Palazzo et al.) is limited and inconclusive. To conclude the theme of TRPV1 antagonists and pain, two medicinal chemistry chapters summarize structural and structure – activity rela- tionship information on the two major classes of TRPV1 antagonists: aryl- ureas (Gomtsyan) and pyridinylpiperazine carboxamides (Hawryluk and Carruthers). While the blockade of receptor activation by TRPV1 antagonists attenuates pain transmission, TRPV1 agonists have also been shown to be effective in pain management, especially for peripheral neuropathies (Bley). Antinociception induced by TRPV1 agonists is the result of receptor desen- sitization/defunctionalization after prolonged administration of the agonist. In fact, capsaicin cream has been used for many years to treat peripheral neuropathies, and new topical and injectable formulations of capsaicin with better safety margins are undergoing late - stage clinical trials. This volume also presents experts’ opinions on the links between TRPV1 and disease states other than pain. Some of these links are well established. For example, high expression of TRPV1 on sensory nerves in airways led PREFACE AND ACKNOWLEDGMENTS xi scientists to explore the potential for TRPV1 antagonists in treating chronic cough, asthma, and other respiratory diseases (Materazzi et al.). A number of such compounds have shown potent antitussive activity in preclinical models of cough, and at least one compound (MK- 2295) has entered clinical testing. The role of TRPV1 in some other diseases is less well validated. Expression of functional TRPV1 receptors in the brain suggests the possibility of novel strategies for the treatment of neurological and perhaps neuropsychiatric disorders (Palazzo et al.), while recent observations with non- obese diabetic mice suggest a potentially important role for TRPV1 in the development of diabetes (Tsui et al.). One of the properties of TRPV1 and other members of the TRP family is the ability to detect thermal stimuli within a specifi c intensity range. The chapter by Garami et al. discusses the role of TRPV1 in thermoregulation, presenting both a mechanistic point of view and the implications for the development of safe TRPV1 antagonist - based therapies. Recent setbacks in the clinical development of TRPV1 antagonists due to the induction of transient hyperthermia and a defi cit in perception of potentially injurious heat make this chapter especially relevant. Our hope is that this book successfully highlights the role of TRPV1 as one of the best- known integrators of multiple painful stimuli and serves as a good reference regarding current and potential future applications of TRPV1 ligands in treating pain as well as other pathophysiological conditions.

The editors are very grateful to the authors for their excellent contributions and their understanding of efforts to keep the delicate balance between appre- ciation of the busy schedules of the authors and the deadlines set by the publisher. We also express our indebtedness to Jonathan Rose at John Wiley & Sons for inviting us to serve as editors for this book and for patiently providing guidance throughout the process.

A rthur Gomtsyan C onnie R. Faltynek

FOREWORD

The use of plant extracts in folk medicine can be traced back thousands of years to geographically and ethnically diverse cultures. Such accumulated wisdom has served as the starting point for the development of important experimental and therapeutic drugs. This process is perhaps best exemplifi ed by pain medications, so many of which — particularly those of the opiate and NSAID class— were inspired by the active ingredients in opium poppies and in willow bark, respectively. While some natural plant products (such as opiates) suppress pain, others produce it. This is certainly the case for capsaicin, the pungent agent in “ hot ” chili peppers that elicits that familiar and intense sensation of burning pain. Just as morphine and aspirin have served as chemical keys for unlocking cellular mechanisms of pain suppression, capsaicin and other pungent natural products have helped to defi ne the neurons, molecules, and signaling pathways that initiate or enhance pain under normal (acute) and pathological (chronic) circumstances. The culinary and medicinal wonders of capsaicin and related vanilloid compounds can be traced to Central and South America, where Capsicum plants have their indigenous roots. Thousands of years later, Christopher Columbus and other explorers introduced chili peppers to European, Asian, and African continents, where they have been similarly and widely exploited for sensorial and medicinal applications ranging from food preservation to hypothermic cooling. In more recent times, seminal work from Jansco and colleagues in Hungary showed that capsaicin functions as a highly selective excitatory agent for a subset of primary afferent sensory neurons, making sensitivity to capsaicin a defi ning functional hallmark of the nociceptor. Consequently, the goal of delineating a mechanism of capsaicin action, and in particular, identifying a “ vanilloid receptor,” became a holy grail of pain xiii xiv FOREWORD research, much in the same way that molecular identifi cation of the T- cell receptor was seen as a watershed in the genetic and functional characterization of the lymphocyte. Leading up to this goal were the fundamental contributions of Rang, Bevan, Wood, and colleagues at the Sandoz Institute, as well as those of Blumberg and coworkers at the U.S. National Cancer Institute, whose early electrophysi- ological and pharmacological studies helped to better defi ne and to validate a bona fi de “ vanilloid site. ” Still, this putative receptor defi ed genetic identifi cation for quite some time, and thus its existence and molecular nature remained enigmatic. The cloning and molecular characterization of TRPV1 (n é VR1) by Michael Caterina and by other members of my group at once resolved these issues while providing a defi nitive molecular explanation for the highly selective nature of capsaicin action. In doing so, this accomplish- ment gave molecular validation to the so- called specifi city theory of nociception fi rst espoused by Sherrington over a century ago. From a more general perspective, cloning of the vanilloid receptor put a spotlight on TRP channels as important new players in vertebrate sensory systems. In retrospect, this should not have come as a great surprise given the well - established role of TRP channels in fl y phototransduction, but for whatever reason, their relevance to sensory signaling in higher systems had not been fully appreciated. The study of TRPV1 has established several important new paradigms that are of general relevance to sensory signaling but also hold special signifi cance for nociception and pain. Of course, one of these relates to the discovery that TRPV1 functions as a thermosensor, which has provided a molecular and cellular framework for understanding how ion channels sense heat or cold, and how primary afferent neurons detect changes in ambient temperature. Additionally, these studies set the stage for understanding how TRP channels function as polymodal signal detectors that can integrate information from both physical and chemical stimuli to modulate neuronal excitability in the face of changing environmental or physiological conditions. In the context of the primary afferent nociceptor, this has great signifi cance for understanding how tissue injury and infl ammation produce pain hypersensitivity. The molecular analysis of TRPV1 has opened up exciting new vistas in the study of somatosensation, nociception, and pain. Of course, the full therapeutic impact of this work will come with the development of analgesic agents that target TRP channels on primary afferent nociceptors. Should this come to pass — as I expect it will — then we can add capsaicin to the list of natural products that have inspired novel classes of pain medicines.

D avid Julius San Francisco CONTRIBUTORS

Maria C. Almeida, Systemic Inﬂ ammation Laboratory, Trauma Research, St. Joseph ’ s Hospital and Medical Center, Phoenix, AZ

Ant ó nio Avelino, Institute of Histology and Embryology, Faculty of Medi- cine of Porto, Alameda Hernani Monteiro, Porto, Portugal; IBMC, University of Porto, R. Campo Alegre, Porto, Portugal; Email: aavelino@ med.up.pt

Anindya Bhattacharya, Department of Pain and Related Disorders, Johnson & Johnson Pharmaceutical Research and Development, L.L.C., 3210 Merryﬁ eld Row, San Diego, CA 92121

Keith R. Bley, NeurogesX, Inc., 2201 Bridgepointe Parkway, San Mateo, CA 94404; Email: [email protected]

Nicholas I. Carruthers, Johnson & Johnson Pharmaceutical Research & De- velopment, L.L.C. San Diego, CA; Email: [email protected]

Francisco Cruz, Institute of Histology and Embryology, Faculty of Medicine of Porto, Alameda Hernani Monteiro, Porto, Portugal; IBMC, University of Porto, R. Campo Alegre, Porto, Portugal; Department of Urology, Hospital Sã o Joã o and Faculty of Medicine of Porto, Porto, Portugal; Email: [email protected]

Vincenzo Di Marzo, Endocannabinoid Research Group, Institute of Biomolecular Chemistry, Consiglio Nazionale delle Ricerche, Naples, Italy; Email: [email protected] xv xvi CONTRIBUTORS

Ruslan Dorfman, University of Toronto, The Hospital For Sick Children, Research Institute, Genetics & Genome Biology Program, Toronto, Ontario, Canada

H. - Michael Dosch, University of Toronto, The Hospital For Sick Children, Research Institute, Neurosciences & Mental Health Programs, Toronto, Ontario, Canada; Email: [email protected]

Connie R. Faltynek, Neuroscience Research, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, IL; Email: [email protected]

Andras Garami, Systemic Inﬂ ammation Laboratory, Trauma Research, St. Joseph ’ s Hospital and Medical Center, Phoenix, AZ

Narender R. Gavva, Department of Neuroscience Amgen Inc., Thousand Oaks, CA; Email: [email protected]

Pierangelo Geppetti, Department of Preclinical and Clinical Pharmacology, University of Florence, Florence, Italy; Center of Excellence for the Study of Inﬂ ammation, University of Ferrara, Ferrara, Italy; Email: pierangelo. geppetti@uniﬁ .it

Peter J. Goadsby, Headache Group Department of Neurology, University of California, San Francisco, CA; Email: [email protected]

Arthur Gomtsyan, Neuroscience Research, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, IL; Email: [email protected]

Natalie A. Hawryluk, Johnson & Johnson Pharmaceutical Research & Development, L.L.C. San Diego, CA

Tamara Hew - Butler, Systemic Inﬂ ammation Laboratory, Trauma Research, St. Joseph ’ s Hospital and Medical Center, Phoenix, AZ

Philip R. Holland, Headache Group Department of Neurology University of California, San Francisco, CA

Prisca Honore, Neuroscience Research, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, IL; Email: [email protected]

Juan Miguel Jimenez - Andrade, Department of Pharmacology, University of Arizona, Tucson, AZ

Shailen K. Joshi, Neuroscience Research, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, IL; Email: shailen. [email protected] CONTRIBUTORS xvii

David Julius, Department of Physiology and Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA Email: [email protected] Philip R. Kym, Neuroscience Research, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, IL Sonya G. Lehto, Department of Neuroscience Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA Sabatino Maione, Endocannabinoid Research Group, Department of Experimental Medicine, Section of Pharmacology “ L. Donatelli, ” Faculty of Medicine and Surgery, Second University of Naples, Naples, Italy; Email: [email protected] Patrick Mantyh, Department of Pharmacology, University of Arizona, Tucson, AZ; Research Service, VA Medical Center, Minneapolis, MN Email: [email protected] Serena Materazzi, Department of Preclinical and Clinical Pharmacology, University of Florence, Florence, Italy Shaun F. Morrison, Division of Neuroscience, Oregon National Primate Research Center, Oregon Health & Science University, Beaverton, OR Kazuhiro Nakamura, Division of Neuroscience, Oregon National Primate Research Center, Oregon Health & Science University, Beaverton, OR Romina Nassini, Department of Preclinical and Clinical Pharmacology, University of Florence, Florence, Italy. Bernd Nilius, KU Leuven, Department of Molecular Cell Biology, Laboratory Ion Channel Research, Campus Gasthuisberg, Leuven, Belgium; Email: [email protected] Tatiane B. Nucci, Systemic Infl ammation Laboratory, Trauma Research, St. Joseph ’ s Hospital and Medical Center, Phoenix, AZ Eszter Pakai, Systemic Infl ammation Laboratory, Trauma Research, St. Joseph ’ s Hospital and Medical Center, Phoenix, AZ Enza Palazzo, Endocannabinoid Research Group, Department of Experimental Medicine, Section of Pharmacology “ L. Donatelli, ” Faculty of Medicine and Surgery, Second University of Naples, Naples, Italy Regina M. Reilly, Neuroscience Research, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, IL Andrej A. Romanovsky, Systemic Infl ammation Laboratory, Trauma Research, St. Joseph’ s Hospital and Medical Center, Phoenix, AZ; Email: [email protected] xviii CONTRIBUTORS

Michael W. Salter, University of Toronto, The Hospital For Sick Children, Research Institute, Neurosciences & Mental Health Programs, Toronto, Ontario, Canada Renato N. Soriano, Systemic Inﬂ ammation Laboratory, Trauma Research, St. Joseph ’ s Hospital and Medical Center, Phoenix, AZ Katarzyna Starowicz, Department of Pain Pharmacology, Institute of Pharmacology PAS, 12 Smetna str 31343 Cracow Poland; Endocannabinoid Research Group, Institute of Biomolecular Chemistry, Consiglio Nazionale delle Ricerche, Naples, Italy Carol S. Surowy, Neuroscience Research, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, IL; Email: [email protected] Arpad Szallasi, Departments of Pathology and Laboratory Medicine, Monmouth Medical Center, Long Branch, NJ; Drexel University College of Medicine, Philadelphia, PA; Email: [email protected] Alain Tchoimou, Department of Preclinical and Clinical Pharmacology, University of Florence, Florence, Italy Marcello Trevisani, Center of Excellence for the Study of Inﬂ ammation, University of Ferrara, Ferrara, Italy; present address: PharmEste srl, Ferrara, Italy Hubert Tsui, University of Toronto, The Hospital For Sick Children, Research Institute, Neurosciences & Mental Health Programs, Toronto, Ontario, Canada Rudi Vennekens, KU Leuven, Department of Molecular Cell Biology, Laboratory Ion Channel Research, Campus Gasthuisberg, Leuven, Belgium PART I

INTRODUCTION TO THE TRP CHANNELS

1 TRP CHANNELS AND HUMAN DISEASES

Bernd Nilius and Rudi Vennekens

1.1 INTRODUCTION

Transient receptor potential (TRP) cation channels have been described in detail as polymodal cell sensors in many reviews (Nilius and Voets, 2005 ; Pedersen et al., 2005 ; Voets et al., 2005 ; Nilius and Mahieu, 2006 ; Ramsey et al., 2006 ; Nilius, 2007 ; Nilius et al., 2007 ; Venkatachalam and Montell, 2007 ). The mammalian TRP superfamily consists of 28 mammalian TRP cation channels, which can be subdivided into six main subfamilies: the TRPC (“ Canonical ” ), TRPV ( “ Vanilloid ” ), TRPM ( “ Melastatin ” ), TRPP ( “ Polycystin ” ), TRPML ( “ MucoLipin ” ), and the TRPA ( “ Ankyrin ” ) groups. In the current review, we attempt to create an overview of the currently available data on the functional role of TRP channels and their possible signifi cance for human disease. We will extend and complement the very comprehensive reviews on this topic published just recently (Jordt and Ehrlich, 2007 ; Kiselyov et al., 2007 ; Nilius, 2007 ; Nilius et al., 2007 ). For that reason, and due to space limitations, it is inevitable that some topics will not be covered at this occasion (Fig. 1.1 ). Several TRP channelopathies are known, that is, diseases with identifi ed defects in the gene encoding the channels. Channel dysfunctions caused by regulatory proteins can also contribute to the genesis of several diseases and may result in changes in channel abundance, channel gating or modulation, and inadequate responses to various stimuli. Abnormal endogenous produc- tion of various agents during the development of a disease (e.g., in infl ammation conditions) can affect channel function and may determine the progression

3 4 TRP CHANNELS AND HUMAN DISEASES

TRPM3 TRPM6 “M, melastatin” “C, canonical” TRPM1 TRPM7 TRPM2 TRPC7 TRPM8 TRPC3 TRPM4 TRPC6 TRPM5 TRPC5 “P, polycystin” TRPP3 TRPC4 (PKD2L1) TRPC1 TRPP2 (PKD2) TRPV6 TRPP5 (PKD2L2) TRPV5 TRPML1 TRPV2 TRPML2 “V, vanilloid” TRPV1 TRPML3 TRPV4 TRPV3 “ML, mucolipin” Figure 1.1 A phylogenetic tree of the mammalian members of the TRP family. of the disease. This review will list all known mammalian TRP channels and will indicate links to possible diseases based on data from human patients, genetically manipulated organisms, and pharmacological studies. The increasing evidence that TRP channels are involved in human diseases has created a huge interest for these channels as novel drug targets. Comprehensive reviews have been published on the modulation of TRP channels by novel modulators and the general role of TRPs as pharmaceutical targets (see Okuhara et al., 2007 ; Szallasi et al., 2007 ; Landry and Gies, 2008 ). This topic will only sporadically be discussed.

1.2 THE “ CANONICAL” TRPC S

The mammalian TRPC (canonical) channels can be subdivided into four sub- classes: TRPC1, TRPC2, TRPC3/6/7, and TRPC4/5 (Vazquez et al., 2004 ). All TRPC channels are nonselective Ca 2+ - permeable cation channels. TRPC1, TRPC4, and TRPC5 can form heteromers, and current properties are signiﬁ - cantly different between TRPC5 and TRPC1/TRPC5 - expressing cells. Similarly, TRPC3, TRPC6, and TRPC7 also can form heteromers (Strubing et al., 2001, 2003; Goel et al., 2002 ; Hofmann et al., 2002 ). TRPC activation occurs mainly via different isoforms of phospholipases (PLCs) (Venkatachalam et al., 2002 ). TRPC1, TRPC4, and TRPC5 are activated by receptor - induced PLC, but, in contrast to TRPC3, TRPC6, and TRPC7, are completely unre- sponsive to diacylglycerol (DAG) and show a quite complicated activation pattern including changes in membrane potential (voltage dependence), binding of multivalent cations to the pore region, and interaction with phosphatidylinositol phosphates (Hofmann et al., 1999 ; Venkatachalam et al., 2003 ; THE “CANONICAL” TRPCS 5

Chromosomal location of Homo sapiens TRP channel genes

85.20 (ML3) p p MCOLN3 q p p MCOLN2 p q p q p q q (ML2) q p P2 135.58 q 85.11 q A1 C3 C7 137.26 142.09 C1 PKD2L2 V6 M8 (P5) V5 Chr 1 Chr 2 Chr 3 Chr 4 Chr 5 Chr 6 Chr 7142.13 Chr 8 M5 P1 p p p p p q q q q p q M1 p M3 q C4 q M6 M7 Mg PKDL (P3) C6 V4

Chr 9 Chr 10 Chr 11 Chr 12 Chr 13 Chr 14 Chr 15 Chr 16 V3 36.67 V1 34.19 MCOLN1 V2 p (ML1) p p p q p q p p q q p q q q q C5 M4 PKDREJ M2 (P4) Chr 17 Chr 18 Chr 19 Chr 20 Chr 21 Chr 22 Chr X Chr Y Figure 1.2 Schematic overview of the chromosomal location of human TRP genes (reproduced from Abramowitz and Birnbaumer [ 2006 ] with the permission of Springer - Verlag and the authors) . (See color insert.)

Otsuguro et al., 2008 ). TRPC channels have been linked to cardiovascular disease (Yao and Garland, 2005 ; Dietrich et al., 2007 ; Firth et al., 2007 ; Kwan et al., 2007 ; Ohba et al., 2007 ), pulmonary disease (Meng et al., 2008 ), inﬂ ammation (Brechard et al., 2008 ), cancer (Fraser and Pardo, 2008 ), skin disease (Pani and Singh, 2008 ), and proliferative diseases via dysregulation of the cell cycle (Quadid - Ahidouch and Ahidouch, 2007 ) (Fig. 1.2 ).

1.2.1 TRPC1 1.2.1.1 Cardiovascular Cardiac hypertrophy is coupled to an increased Ca2+ entry and a dysfunction of Ca 2+ signaling (Bers and Guo, 2005 ) (for a review including the role of TRPs, see Watanabe et al. [2008 ]). Two lines of evidence suggest that TRPC1 may be involved in the development of cardiac hypertrophy. First, TRPC1 expression is signiﬁ cantly increased in the hearts of abdominal aortic - banded (AAB) rats, which develop pressure - induced hypertrophy, compared to sham - operated rats. It is also increased in cultured myocardial cells pretreated with hypertrophic factors such as endothelin -1 (ET - 1), brain natriuretic peptide (BNP), and atrial natriuretic factor (ANF). Silencing of the TRPC1 gene via small interfering RNA (siRNA) prevents 6 TRP CHANNELS AND HUMAN DISEASES

Hypertrophic agonists: ATII ET-1 TRPC1 PE TRPC3 2+ Ca L-type Ca2+ channel TRPC6

2+ Δϕ Gαq DAG Ca 2+ PLC Ca2+ Ca IP3

Calcineurin ER Ca2+ store P NFAT

Positive feedback system NFAT

MEF2 NFAT GATA TRPC Cardiac growth and remodeling genes

Figure 1.3 Hypertrophic agents such as angiotensin II, endothelin- 1, and phenyleph- rin promote hypertrophy of the cardiomyocyte by causing excess Ca infl ux, which stimulates NFAT- driven gene expression of specifi c genes. Ca infl ux either occurs directly through TRP channels or the membrane potential is set by these channels, infl uencing Ca infl ux through L- type Ca channels. Expression of several TRPC channels is stimulated by NFAT, generating a positive feedback loop. See text for more details and references . ER: endo/sarcoplasmic reticulum; GATA, MEF2, and NFAT: nuclear transcription factors; IP3: inositol- 1,4,5 - trisphosphate. (See color insert.)

ET - 1 - , angiotensin II (ATII) - , and phenylephrine (PE) - induced cardiac hypertrophy. Second, ATII also induces hypertrophy of vascular smooth muscle cells (VSMCs). TRPC1 is overexpressed in these cells, and TRPC1 siRNA prevents the development of ATII- induced hypertrophy (Takahashi et al., 2007 ). The induction of TRPC1 expression is probably mediated through the nuclear factor of activated T cell (NFAT). The general mechanism for hypertrophy development might be that proliferative stimuli activate TRPC1, inducing depolarization and activation of L- type Ca 2+ channels, which subse- 2+ quently increase cardiac [Ca ]i , activate calcineurin, and trigger NFAT trans- location to the nucleus, which in turn results in an increased transcription of TRPC1 channels (Bush et al., 2006 ) (Fig. 1.3 ). Occlusive vascular disease is often a lethal complication in myocardial infarction, stroke, atherosclerosis, and clinical procedures such as angioplasty and grafting in bypass surgery. It can be caused by a switch in smooth muscle cell phenotype to an invasive and proliferative mode, leading to neointimal THE “CANONICAL” TRPCS 7 hyperplasia. TRPC1 might be involved in this switching of cell fate, which is associated with enhanced calcium entry and cell cycle activity. A speciﬁ c E3 - targeted (pore blocking) antibody to TRPC1 reduced neointimal growth in human veins, indicating that this might be a new avenue in the treatment of occlusive vascular diseases (Kumar et al., 2006 ; van Breemen et al., 2006 ).

1.2.1.2 Skeletal Muscle A role for TRPC1 in Duchenne muscular dystro- phy (DMD) has been suggested extensively (Gailly, 2002 ; Hopf et al., 2007 ). In dystrophic myocytes, an increased Ca2+ infl ux is apparent, which can be suppressed by antisense TRPC1, TRPC4, and TRPC6 oligonucleotides. Furthermore, a direct interaction between α 1 - syntrophin and TRPC1 was recently shown. Apparently, normal regulation of calcium infl ux in skeletal muscle depends on the association between TRPC1 channels and α 1 - syntrophin that may anchor the store - operated channels (SOCs) to the dystrophin - associated protein complex (DAPC). The loss of this molecular association could contribute to the calcium alterations observed in DMD cells (Vandebrouck et al., 2007 ). A further indication for the role of TRPC1 in a myopathy comes from homer 1− / − mice. Mice lacking homer 1 show decreased muscle fi ber cross - sectional area and decreased skeletal muscle force generation. Homer 1− / − myotubes displayed altered cation infl ux, which can be blocked by TRPC1 downregulation. Diminished Homer 1 expression in mouse models of DMD suggests that loss of Homer 1 scaffolding of TRP channels may contribute to the increased stretch - activated channel activity observed in mdx (dystrophin - defi cient) myofi bers (Stiber et al., 2008 ). Also, reactive oxygen species (ROS), which are increased in DMD, activate an src kinase, which in turn can activate TRPC1 (Gervasio et al., 2008 ).

1.2.1.3 Skin Darier ’ s disease (DD) is caused by mutations of SERCA2. Keratinocytes from DD patients show increased TRPC1 expression levels and increased Ca inﬂ ux. The latter might be involved in reinforcement of NFkB- mediated upregulation of antiapoptotic genes, for example, BclxL. TRPC1 therefore could increase cell proliferation, a hallmark of lesional keratinocytes (Beck et al., 2008 ; Pani and Singh, 2008 ).

1.2.1.4 Immunodefense TRPC1 is involved in antibody recognition in B lymphocytes. Suppression of TRPC1 results in reduced B- cell antigen receptor 2+ (BCR) - mediated oscillations in [Ca ]i and consequently depressed activation of the NFAT (Mori et al., 2002 ).

1.2.1.5 Neurological Disorders TRPC channels seem to be crucially involved in the signaling via metabotropic glutamate receptors (Kim et al., 2003 ) and in the actions of brain- derived neurotrophic factor (BDNF) (Li et al., 1999 ) that include netrin - 1 and BDNF - mediated growth cone guidance (Greka et al., 2003 ; Li et al., 2005c ; Shim et al., 2005 ). Recent studies indicate 8 TRP CHANNELS AND HUMAN DISEASES a potential link between TRPC1 and neurotoxicity induced by the exogenous agent 1 - methyl - 4 - phenylpyridium (MPP). The latter causes selective nigral dopaminergic lesions and induces Parkinson ’ s disease - like syndromes. (Bollimuntha et al., 2005 ). Apparently, TRPC1 may execute a neuroprotective role in dopaminergic neurons. TRPC1 (and probably also TRPC3) are activated by the peptide hormone orexin A via the GPCR OX 1 (Larsson et al., 2005 ). This activation mode may link TRP channels to important physiological functions, because orexin regu- lates sleep/wakefulness states, alertness, and food intake (appetite). In addi- tion, the orexin knockout mouse is a very useful model of human narcolepsy, a disorder that is characterized primarily by rapid eye movement (REM) sleep dysregulation.

1.2.2 TRPC2 The TRPC2 gene trpc2 is a pseudogene in humans and will not be discussed further in this review.

1.2.3 TRPC3 1.2.3.1 Cardiovascular TRPC3 might be critically involved in heart hypertrophy. RNAi- mediated knockdown of TRPC3 decreases expression of hypertrophy -associated genes such as the A- and B- type natriuretic peptides (ANP and BNP) in response to numerous hypertrophic stimuli. Overexpres- sion of TRPC3 increases BNP expression (Brenner and Dolmetsch, 2007 ). Increased Ca2+ causes cell death in response to ischemia – reperfusion (I/R) . After I/R, apoptosis was signiﬁ cantly increased in TRPC3 overexpressing cardiomyocytes compared to control. TRPC3 overexpression increased not only apoptosis but also calpain - mediated proteolysis resulting from I/R injury, as well as sensitivity to Ca2+ overload in cardiomyocytes (Shan et al., 2008 ). It is noteworthy to mention that Epo activates TRPC3. TRPC3 is expressed in human erythroid progenitor cells and may regulate Ca 2+ inﬂ ux during erythroid differentiation (Tong et al., 2008 ). Importantly, Epo secretion is critically regulated by the hypoxia - induced factor HIF - 1 , which in turn upregulates two other TRPCs, TRPC1 and TRPC6 (Wang et al., 2006 ).

1.2.3.2 Immunodefense Interestingly, mutations in TRPC3 induce a reduc- tion of T - cell receptor activation - induced Ca2+ entry in T lymphocytes, which may parallel the defective immune responses from patients with severe com- bined immunodeﬁ ciency (SCID) (Philipp et al., 2003 ).

1.2.3.3 Neurological Disorders TRPC3 seems to be critically involved in brain development. BDNF shapes synapses in hippocampal neurons. It also activates a nonselective cationic current (IBDNF ) in CA1 pyramidal neurons. Activation requires phospholipase C, IP3 receptors, and the respective stores THE “CANONICAL” TRPCS 9

2+ and depends on extracellular Ca . CA1 neurons express TRPC3, and IBDNF is absent after silencing with TRPC3 siRNA. A sustained phase of IBDNF depends on phosphatidylinositol 3 - kinase, which is required for plasma membrane insertion of TRPC3. TRPC3 channels are necessary for BDNF to increase dendritic spine density(Amaral and Pozzo - Miller, 2007 ). Furthermore, the Δ202 mouse line, which has defective TRPC3 expression, develops severe neurological problems, including paralysis and atrophy starting at week 4. Mice die around week 12. The neurologic syndrome of the Δ 202 mice appears to be a monogenic recessive neuromotor disease caused by the interruption of the trpc3 gene, leading to a failure in the postnatal development of the central nervous system (CNS) (Rodriguez - Santiago et al., 2007 ). There may be a connection between TRPC3 and Parkinson ’ s disease : Parkinsonian movement disorders are often associated with abnormalities in the GABA neuron fi ring pattern in the substantia nigra pars reticulata. These neurons express TRPC3 channels, which are tonically active and mediate an inward Na + current, leading to a substantial depolarization in these neurons. Inhibition of TRPC3 channels induces hyperpolarization, decreases fi ring frequency, and increases fi ring irregularity, all of which may contribute to movement disorders (Zhou et al., 2008a ). Spinocerebellar ataxia type 14 (SCA14) is an autosomal dominant neuro- degenerative disease caused by mutations in protein kinase PKCg . This kinase normally inhibits TRPC3. The mutant PKCγ cannot phosphorylate TRPC3, resulting in sustained Ca2+ entry. This alteration in Ca2+ homeostasis in Purkinje cells may contribute to neurodegeneration in SCA14 (Adachi et al., 2008 ).

1.2.4 TRPC4 1.2.4.1 Cardiovascular TRPC4 has been associated with regulation of vascular tone (Freichel et al., 2001 ) and endothelial permeability (Tiruppathi et al., 2002 ). Recently, TRPC4 has also been implicated in angiogenesis. The angiogenesis inhibitor thrombospondin -1 (TSP1) controls the switch in renal cell carcinoma (RCC) toward an angiogenic phenotype. This factor is highly secreted in normal kidney cells, whereas RCC cells secrete little TSP1. Ca 2+ inﬂ ux through TRPC4 may be critical for TSP1 secretion since expression of TRPC4 is very low in RCC cells, and TRPC4 silencing in normal kidney cells causes TSP1 retention and impaired secretion (Veliceasa et al., 2007 ).

1.2.4.2 Neurological Disorders Alzheimer ’ s disease has multiple genetic variants. A genome- wide screen of two extended pedigrees identifi ed a gene related to TRPC4, TRPC4 - associated protein (TRPC4AP), on chromosome 20q11.22, as relevant for the disease. Multiple signifi cant single - nucleotide polymorphisms (SNPs) in this gene were found with an initial genome scan and were confi rmed by haplotype analysis. TRPC4AP might be involved 10 TRP CHANNELS AND HUMAN DISEASES with the disease in these late- onset Alzheimer’ s families, but the mechanism is unclear (Poduslo et al., 2009 ). TRPC4 is also present in F2 - synaptic terminals of the thalamic network. These terminals provide a GABAergic input into the dorsal lateral geniculate nucleus. GABA release from such F2 terminals depends on Ca 2+ infl ux initi- ated by metabotropic receptors. In trpc4 − / − mice, the 5 - hydroxytryptamine (5- HT) - induced release of GABA from the thalamic interneurons is dramati- cally reduced (Munsch et al., 2003 ). This GABAergic component is critical for the control of the sleep/wake cycle, processing of visual information, and may also be important for the control of retinogeniculate transmission.

1.2.4.3 Skin Aberrant keratinocyte differentiation causes hyperprolifera- tive skin diseases, including basal cell carcinoma (BCC). Ca2+ inﬂ ux plays a crucial role in this differentiation process. Knocking down of both TRPC4 and TRPC1 expression prevents Ca2+ - dependent differentiation of keratinocytes, and the expression of both channels is signiﬁ cantly decreased in BCC, sug- gesting a critical role for both channels in the process controlling normal keratinocyte differentiation (Beck et al., 2008 ).

1.2.5 TRPC5 1.2.5.1 Gastrointestinal Quite recently, TRPC5 and TRPC6 have been mentioned in infantile hypertrophic pyloric stenosis (IHPS) (OMIM #179010). This disease has an incidence of one to eight per 1000 live births and is inher- ited as a complex sex- modiﬁ ed multifactorial trait with a striking male pre- ponderance. The disease is characterized by projectile vomiting due to gastric outlet obstruction caused by hypertrophy of the smooth muscle of the pylorus. In a genome - wide SNP - based high - density linkage scan, two genomic loci were identiﬁ ed, which harbor TRPC6 (11q21- 22) and TRPC5 (Xq23) (Everett et al., 2008 ).

1.2.5.2 Joints The identifi cation of endogenous agents interacting with TRP channels is a major priority in the fi eld. Recently, it has been shown that activation of TRPC5 homomultimers and TRPC5 – TRPC1 heteromultimers can be mediated by extracellular reduced thioredoxin (Xu et al., 2008 ). Thioredoxin is an endogenous redox protein, which has several intracellular functions in cancer, ischeamic reperfusion injury, infl ammation, and aging. Also, thioredoxin is highly secreted in rheumatoid arthritis . TRPC5 and TRPC1 are expressed in secretory fi broblast- like synoviocytes (FLSs) (FLS cells). FLS cells secrete matrix metalloproteinases (MMPs), which promote the progression of arthritis. Thioredoxin decreases MMP secretion in these cells. (Q: is this OK?) Block of TRPC5- containing channels enhances secretion of MMPs and prevents suppression of MMP secretion by thioredoxin. Thus, TRPC5/TRPC1– TRPC5 activation could represent a novel strategy in the treatment of rheumatoid arthritis (Xu et al., 2008 ).