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The handle http://hdl.handle.net/1887/62061 holds various files of this Leiden University dissertation

Author: Soethoudt, Marjolein Title: Chemical tools to study the cannabinoid receptor type 2 Date: 2018-04-26

Chapter 1

General Introduction

Chapter 1 General Introduction

1.1 Drug discovery approaches and challenges In the hit‐to‐lead optimization phase, the interaction of the compound with the protein target is optimized, while reducing off‐target activities, thereby enhancing the selectivity Drug discovery and development comprises the whole process from the discovery of a and safety of the lead.10 During the hit‐to‐lead optimization a strict control of the therapeutically relevant protein to the launch of an effective and safe drug on the market physicochemical properties of a small molecule is exerted to improve the pharmacokinetic (Figure 1). This process can take more than a decade (~13.5 years per drug on average) and and toxicity profile of a drug. Binding kinetics, i.e. the association and dissociation rate of a the costs may rise above one billion dollars (~2.5 billion dollars per drug on average).1 drug to a protein target and off‐target proteins, is increasingly investigated to improve the 11‐14 Especially the development stage is very expensive, time consuming and risky.2, 3 Currently, efficacy and safety profile of a lead. market introduction rates of new drugs are low, while costs of drug development have risen substantially (the “innovation gap”),4 often due to late stage clinical failures.5, 6 Main causes 1.1.3 Target engagement in drug development for clinical trial failures are a lack of efficacy and the occurrence of adverse side effects. After optimization of a lead compound, it is essential to verify that the compound engages Possible reasons for toxicity and the lack of efficacy include 1) the protein target has not with its target at the site‐of‐action in a dose‐dependent manner in preclinical animal models been properly validated for the disease under investigation, 2) preclinical profiling of the as well as in humans. Several chemical tools and strategies have been reported to drug was not sufficient, 3) the translation between preclinical models and the human determine target engagement in living systems,15 such as radioligands,16 positron emission situation is very poor, or 4) the drug simply does not reach the protein target at the site of tomography (PET),17‐19 fluorescent or biotinylated small molecules,20 fluorescence or 21 action. bioluminescence resonance energy transfer (FRET and BRET, respectively), fluorescence 22 polarization microscopy, or activity/photoaffinity‐based protein profiling (ABPP/pAfBPP, respectively).15, 23

1.2 G protein-coupled receptors

An important class of drug targets is the 700‐membered superfamily of G protein‐coupled 24 Figure 1. Schematic representation of the drug discovery and development process. receptors (GPCRs). Currently, more than 30% of the available drugs on the market act on 25 GPCRs, which are involved in the regulation of many physiological processes, including 26‐28 1.1.1 Target identification and validation vision, behavior, mood, energy balance, immunity and inflammation. The goal of target discovery is to identify a protein (i.e. a target) which is therapeutically GPCRs are cell surface receptors consisting of seven helices spanning the cell 26, 27 relevant for a particular disease. In case of natural product‐based drugs, such as herbal membrane. They convey extracellular signals from different types of stimuli, such as mixtures, it is important to identify the protein target, because this will enable the light, (peptide) hormones and neurotransmitters, to intracellular second messenger development of synthetic and more selective lead compounds.7 Moreover, selective systems, thereby allowing cells to respond to their environment. This signaling is mediated modulators of a protein are required to establish efficacy in animal models of the disease by a G protein, which consists of three subunits α, β and γ (Figure 2). Activation of the (i.e. target validation).8 Most lead compounds and drug candidates are small molecules, receptor results in a conformational change, causing the G protein‐bound guanosine which are organic compounds with a low molecular weight (< 900 Da) and size in the order diphosphate (GDP) to be exchanged for guanosine triphosphate (GTP), leading to of 1 nm. It is essential that the small molecules used for target validation are highly selective dissociation of the βγ‐dimer. The GTP‐bound Gα‐subunit activates or inhibits (depending on and have a well‐defined molecular mode of action. Gα‐subunit subtype) several different second messenger pathways, such as cyclic adenosine 2+ monophosphate (cAMP), inositol triphosphate (IP3) and Ca ions. These second messengers 29 1.1.2 Hit discovery and optimization in turn regulate several cell signaling pathways. To date, 21 Gα‐subunits, 6 Gβ‐subunits 30 Target discovery and validation are followed by hit identification, which may involve high and 12 Gγ‐subunits have been identified, which constitute four main classes of G proteins: throughput screening of chemical libraries, identification of active ingredients from a natural Gs, Gi, G12/13 and Gq. G protein signaling is terminated when the Gα‐subunit hydrolyzes its product, and structure‐ or ligand‐based design (e.g. using previously published data and bound GTP molecule to GDP, followed by the reassociation of the Gα‐subunit with the βγ‐ three dimensional models of the protein structure).9 dimer. In addition to G protein‐dependent signaling, GPCRs can also signal through the binding and subsequent activation by G protein‐coupled receptor kinases (GRKs) and β‐ arrestins.31

8

Chapter 1 General Introduction

1.1 Drug discovery approaches and challenges In the hit‐to‐lead optimization phase, the interaction of the compound with the protein 1 Chapter target is optimized, while reducing off‐target activities, thereby enhancing the selectivity Drug discovery and development comprises the whole process from the discovery of a and safety of the lead.10 During the hit‐to‐lead optimization a strict control of the therapeutically relevant protein to the launch of an effective and safe drug on the market physicochemical properties of a small molecule is exerted to improve the pharmacokinetic (Figure 1). This process can take more than a decade (~13.5 years per drug on average) and and toxicity profile of a drug. Binding kinetics, i.e. the association and dissociation rate of a the costs may rise above one billion dollars (~2.5 billion dollars per drug on average).1 drug to a protein target and off‐target proteins, is increasingly investigated to improve the 11‐14 Especially the development stage is very expensive, time consuming and risky.2, 3 Currently, efficacy and safety profile of a lead. market introduction rates of new drugs are low, while costs of drug development have risen substantially (the “innovation gap”),4 often due to late stage clinical failures.5, 6 Main causes 1.1.3 Target engagement in drug development for clinical trial failures are a lack of efficacy and the occurrence of adverse side effects. After optimization of a lead compound, it is essential to verify that the compound engages Possible reasons for toxicity and the lack of efficacy include 1) the protein target has not with its target at the site‐of‐action in a dose‐dependent manner in preclinical animal models been properly validated for the disease under investigation, 2) preclinical profiling of the as well as in humans. Several chemical tools and strategies have been reported to drug was not sufficient, 3) the translation between preclinical models and the human determine target engagement in living systems,15 such as radioligands,16 positron emission situation is very poor, or 4) the drug simply does not reach the protein target at the site of tomography (PET),17‐19 fluorescent or biotinylated small molecules,20 fluorescence or 21 action. bioluminescence resonance energy transfer (FRET and BRET, respectively), fluorescence 22 polarization microscopy, or activity/photoaffinity‐based protein profiling (ABPP/pAfBPP, respectively).15, 23

1.2 G protein-coupled receptors

An important class of drug targets is the 700‐membered superfamily of G protein‐coupled 24 Figure 1. Schematic representation of the drug discovery and development process. receptors (GPCRs). Currently, more than 30% of the available drugs on the market act on 25 GPCRs, which are involved in the regulation of many physiological processes, including 26‐28 1.1.1 Target identification and validation vision, behavior, mood, energy balance, immunity and inflammation. The goal of target discovery is to identify a protein (i.e. a target) which is therapeutically GPCRs are cell surface receptors consisting of seven helices spanning the cell 26, 27 relevant for a particular disease. In case of natural product‐based drugs, such as herbal membrane. They convey extracellular signals from different types of stimuli, such as mixtures, it is important to identify the protein target, because this will enable the light, (peptide) hormones and neurotransmitters, to intracellular second messenger development of synthetic and more selective lead compounds.7 Moreover, selective systems, thereby allowing cells to respond to their environment. This signaling is mediated modulators of a protein are required to establish efficacy in animal models of the disease by a G protein, which consists of three subunits α, β and γ (Figure 2). Activation of the (i.e. target validation).8 Most lead compounds and drug candidates are small molecules, receptor results in a conformational change, causing the G protein‐bound guanosine which are organic compounds with a low molecular weight (< 900 Da) and size in the order diphosphate (GDP) to be exchanged for guanosine triphosphate (GTP), leading to of 1 nm. It is essential that the small molecules used for target validation are highly selective dissociation of the βγ‐dimer. The GTP‐bound Gα‐subunit activates or inhibits (depending on and have a well‐defined molecular mode of action. Gα‐subunit subtype) several different second messenger pathways, such as cyclic adenosine 2+ monophosphate (cAMP), inositol triphosphate (IP3) and Ca ions. These second messengers 29 1.1.2 Hit discovery and optimization in turn regulate several cell signaling pathways. To date, 21 Gα‐subunits, 6 Gβ‐subunits 30 Target discovery and validation are followed by hit identification, which may involve high and 12 Gγ‐subunits have been identified, which constitute four main classes of G proteins: throughput screening of chemical libraries, identification of active ingredients from a natural Gs, Gi, G12/13 and Gq. G protein signaling is terminated when the Gα‐subunit hydrolyzes its product, and structure‐ or ligand‐based design (e.g. using previously published data and bound GTP molecule to GDP, followed by the reassociation of the Gα‐subunit with the βγ‐ three dimensional models of the protein structure).9 dimer. In addition to G protein‐dependent signaling, GPCRs can also signal through the binding and subsequent activation by G protein‐coupled receptor kinases (GRKs) and β‐ arrestins.31

Chapter 1 General Introduction

Binding of these proteins to the GPCR can result in receptor desensitization and trafficking, Of note, a ligand may also preferentially activate or inactivate one or more specific signaling resulting to internalization, resensitization and/or degradation, or in the activation or pathways over others. This concept is known as “biased signaling” or “functional inhibition of G protein-independent signaling pathways, such as mitogen-activated protein selectivity”.42 Whereas biased agonism is well characterized,43, 44 biased inverse agonism is a kinases (MAPKs), tyrosine kinases or E3 ubiquitin ligases.32 relatively new concept.45, 46 The three dimensional structure of GPCRs (consisting of seven transmembrane α- helices, an extracellular N-terminus, an intracellular C-terminus, and three extra- and – intracellular loops (ECLs and ICLs, respectively)) is highly conserved (Figure 3).47 Nonetheless, the GPCR superfamily can be divided into six classes based on their functional similarity and sequence homology:48 class A rhodopsin-like receptors, class B secretin receptor family, class C metabotropic glutamate/pheromone receptors, class D fungal mating pheromone receptors, class E cyclic AMP receptors and class F frizzled/ smoothened like receptors. Class A is the largest and most studied. It is characterized by the conservation of specific amino acids (especially the DRY motif in TM349) and have relatively short extracellular domains.50, 51 An example of class A receptor GPCRs is the family of cannabinoid receptors, which is the subject of this thesis.

Figure 2. Schematic representation of GPCR signaling. Activation of a G protein-coupled receptor (GPCR) is mediated by a conformational change induced by binding of an agonist. Activated receptors couple to heterotrimeric G proteins, which consist of Gα G and G subunits. n ase α snan te α subunit activates or inhibits (after dissociation of the G subunit) the generation of second messengers, such as cyclic 2+ AMP (cAMP), inositol triphosphate (IP3) and Ca ions. Activated receptors are phosphorylated by GPCR kinases (GRKs), which then recruit -arrestins. -Arrestins can block G protein – GPCR coupling (i.e. desensitization), or mediate clathrin-dependent endocytosis of activated GPCRs (i.e. receptor trafficking, internalization, resensitization or degradation). Finally, -arrestins may mediate G protein-independent signaling pathways, such as mitogen activated protein kinases (MAPKs, e.g. extracellular signal-regulated kinases (ERKs) and c-Jun N-terminal kinases (JNKs)), tyrosine kinases or E3 ubiquitin ligases.

The activity of a GPCR is dependent on its conformational state, which varies from completely inactive to several active states.33, 34 Some GPCRs are active when no ligand is bn a enmenn tat s ae nstttve r basa atvt35 A ligand that reduces this basal activity by inducing an inactive state of the GPCR is termed an inverse agonist,36 whereas a ligand that merely binds the receptor (thereby blocking the binding of other ligands), without inducing a conformational change, is defined as an antagonist.36 A ligand that stabilizes an active conformation, which leads to modulation of cell signaling pathways, is called an agonist. Dependent on the structure of an agonist or inverse agonist, they may induce different active or inactive conformations, respectively.37 Ligands that do not modulate the signal transduction pathway to the full extent compared to a reference Figure 3. 3D structure of a GPCR. The 3D structure shown is from the cannabinoid receptor type 2 (CB2R), a ligand are classified as partial (inverse) agonists, while ligands that fully (in)activate a GPCR representative class A GPCR. The 3D structure was derived using a homology model based on a crystal 52 are classified as full (inverse) agonists, respectively.38-41 structure of the cannabinoid receptor type 1 (CB1R). which was co-crystallized with AM6538. The orthosteric 53 binding site is visualized by docking of a CB2R-selective agonist, LEI101.

1

Chapter 1 General Introduction

Binding of these proteins to the GPCR can result in receptor desensitization and trafficking, Of note, a ligand may also preferentially activate or inactivate one or more specific signaling 1 Chapter resulting to internalization, resensitization and/or degradation, or in the activation or pathways over others. This concept is known as “biased signaling” or “functional inhibition of G protein-independent signaling pathways, such as mitogen-activated protein selectivity”.42 Whereas biased agonism is well characterized,43, 44 biased inverse agonism is a kinases (MAPKs), tyrosine kinases or E3 ubiquitin ligases.32 relatively new concept.45, 46 The three dimensional structure of GPCRs (consisting of seven transmembrane α- helices, an extracellular N-terminus, an intracellular C-terminus, and three extra- and – intracellular loops (ECLs and ICLs, respectively)) is highly conserved (Figure 3).47 Nonetheless, the GPCR superfamily can be divided into six classes based on their functional similarity and sequence homology:48 class A rhodopsin-like receptors, class B secretin receptor family, class C metabotropic glutamate/pheromone receptors, class D fungal mating pheromone receptors, class E cyclic AMP receptors and class F frizzled/ smoothened like receptors. Class A is the largest and most studied. It is characterized by the conservation of specific amino acids (especially the DRY motif in TM349) and have relatively short extracellular domains.50, 51 An example of class A receptor GPCRs is the family of cannabinoid receptors, which is the subject of this thesis.

Figure 2. Schematic representation of GPCR signaling. Activation of a G protein-coupled receptor (GPCR) is mediated by a conformational change induced by binding of an agonist. Activated receptors couple to heterotrimeric G proteins, which consist of Gα G and G subunits. n ase α snan te α subunit activates or inhibits (after dissociation of the G subunit) the generation of second messengers, such as cyclic 2+ AMP (cAMP), inositol triphosphate (IP3) and Ca ions. Activated receptors are phosphorylated by GPCR kinases (GRKs), which then recruit -arrestins. -Arrestins can block G protein – GPCR coupling (i.e. desensitization), or mediate clathrin-dependent endocytosis of activated GPCRs (i.e. receptor trafficking, internalization, resensitization or degradation). Finally, -arrestins may mediate G protein-independent signaling pathways, such as mitogen activated protein kinases (MAPKs, e.g. extracellular signal-regulated kinases (ERKs) and c-Jun N-terminal kinases (JNKs)), tyrosine kinases or E3 ubiquitin ligases.

The activity of a GPCR is dependent on its conformational state, which varies from completely inactive to several active states.33, 34 Some GPCRs are active when no ligand is bn a enmenn tat s ae nstttve r basa atvt35 A ligand that reduces this basal activity by inducing an inactive state of the GPCR is termed an inverse agonist,36 whereas a ligand that merely binds the receptor (thereby blocking the binding of other ligands), without inducing a conformational change, is defined as an antagonist.36 A ligand that stabilizes an active conformation, which leads to modulation of cell signaling pathways, is called an agonist. Dependent on the structure of an agonist or inverse agonist, they may induce different active or inactive conformations, respectively.37 Ligands that do not modulate the signal transduction pathway to the full extent compared to a reference Figure 3. 3D structure of a GPCR. The 3D structure shown is from the cannabinoid receptor type 2 (CB2R), a ligand are classified as partial (inverse) agonists, while ligands that fully (in)activate a GPCR representative class A GPCR. The 3D structure was derived using a homology model based on a crystal 52 are classified as full (inverse) agonists, respectively.38-41 structure of the cannabinoid receptor type 1 (CB1R). which was co-crystallized with AM6538. The orthosteric 53 binding site is visualized by docking of a CB2R-selective agonist, LEI101.

11

ater eneral nrodcion

1.3 Cannabinoid receptors and the endocannabinoid system he is considered a heraeic arge or a arie o diseases, sch as cardioasclar, lier, kidne and nerodegeneraie diseases (menioned aboe, as e cannabinoid recetor faily consists of two subtyes te cannabinoid recetor tye ell as or he reamen o inlammaor or neroahic ain, cancer, oseoorosis, and cannabinoid recetor tye . e recetors sare an overall seuence and inlammaor diseases, sch as rhemaoid arhriis, Crohn’s disease and mlile oology of and oology in te ligandbinding doain. e is ostly sclerosis. resent in te central nervous syste wereas te is redoinantly found in cells of rrenl, he onl drgs on he marke argeing are medical cannabis, aiex te iune syste suc as acroages lyocytes and olyoronuclear (a mixture of Δ and cannabidiol ( and abilone (a analog. oeer, hese neutroils. ot cannabinoid recetors s are activated by Δtetraydrocannabinol are nonselecie cannabinoid ligands ha also aciae he , leading o naned Δ te ain sycoactive constituent of te lant Cannabis sativa ore coonly schoacie side eecs. hereore, drg discoer eors hae ocsed on he , , known as ariuana. e s are also activated by endogenous ligands called deelomen o selecie ligands, becase i is aniciaed ha hese drgs , endocannabinoids of wic Naracidonoyletanolaine anandaide and ill lack he mediaed side eecs. ince i has been reored ha no all aracidonoylglycerol are te ost wellknown. is redoinantly biosyntesied indced biological eecs are abolished in mice, research eors hae also ocsed by diacylglycerol liases and osoliase and degraded by onoacylglycerol on he ideniicaion o oher (non roein arges o . oh he ideniicaion o liase wereas is roduced by Nacylosatidyletanolaine osoliase addiional roein arges o as ell as he deelomen o selecie drgs argeing and etabolied by fatty acid aide ydrolase . e s te cold limael lead o he deelomen o noel heraeics ih eer side endocannabinoids and teir biosyntetic and etabolic enyes constitute te eecs. endocannabinoid syste . eeral selecie ligands ere acie in arios animal models o chronic and

ot s are couled to io roteins and odulate various intracellular signal inlammaor ain, diabeic nero and nehroah, lier cirrhosis, and ischemic transduction atways suc as inibition of croduction activation of itogen reersion inr,, , b onl a e ere esed in clinical rials (e.g. activated rotein kinases s also called etracellular signalregulated (1, P (annabinor, 2, (3, edalinab (4 and (5 kinases s osoliase and roteincouled inwardly rectifying (Figure 4. hese ligands hae been mosl exlored or heir analgesic ( and cannels s and recruit βarrestins to te recetor. s dislay ligand annabinor and aniinlammaor (, , edalinab and deendent biased signaling as well as intersecies differences. ifferent ceical roeries. eciicall, and annabinor ere esed as analgesics in a hird classes ave been described as ligands suc as endocannabinoids and ‘classical molar ooh exracion model, hile , and edalinab ere cannabinoids’. is latter grou is based on and is caracteried by its tricyclic core deeloed or he reamen o oseoarhriis and as esed or he reamen scaffold. ter classes include ainoalkylindoles diaryl yraoles and bicyclic ligands. ost o aoic dermaiis. of te are ‘mixed’ ligands i.e. dislay siilar affinity for bot s but also or selective ligands ave been develoed suc as yridines yriidines uinolones oouinolines triaines biaryl ydantoins and oters for reviews see akur et al. and orales et al..

1.4 The CB2 receptor as therapeutic target

e is associated wit any ysiological rocesses suc as food intake and locootor activity but also wit undesirable effects including addiction aniety and sycoactive effects. e lays an iortant role in cell igration iunosuression and ain sensation and its cellular eression is uregulated under atoysiological conditions suc as neuroinflaation and neurodegenerative diseases (e.g. Alzheimer’s disease, Parkinson’s disease, neuroatic ain ayotroic lateral sclerosis and ultile sclerosis.

Figure 4. Structures of CB2R-selective clinical trial candidates.

12

ater eneral nrodcion

1.3 Cannabinoid receptors and the endocannabinoid system he is considered a heraeic arge or a arie o diseases, sch as 1 Chapter cardioasclar, lier, kidne and nerodegeneraie diseases (menioned aboe, as e cannabinoid recetor faily consists of two subtyes te cannabinoid recetor tye ell as or he reamen o inlammaor or neroahic ain, cancer, oseoorosis, and cannabinoid recetor tye . e recetors sare an overall seuence and inlammaor diseases, sch as rhemaoid arhriis, Crohn’s disease and mlile oology of and oology in te ligandbinding doain. e is ostly sclerosis. resent in te central nervous syste wereas te is redoinantly found in cells of rrenl, he onl drgs on he marke argeing are medical cannabis, aiex te iune syste suc as acroages lyocytes and olyoronuclear (a mixture of Δ and cannabidiol ( and abilone (a analog. oeer, hese neutroils. ot cannabinoid recetors s are activated by Δtetraydrocannabinol are nonselecie cannabinoid ligands ha also aciae he , leading o naned Δ te ain sycoactive constituent of te lant Cannabis sativa ore coonly schoacie side eecs. hereore, drg discoer eors hae ocsed on he , , known as ariuana. e s are also activated by endogenous ligands called deelomen o selecie ligands, becase i is aniciaed ha hese drgs , endocannabinoids of wic Naracidonoyletanolaine anandaide and ill lack he mediaed side eecs. ince i has been reored ha no all aracidonoylglycerol are te ost wellknown. is redoinantly biosyntesied indced biological eecs are abolished in mice, research eors hae also ocsed by diacylglycerol liases and osoliase and degraded by onoacylglycerol on he ideniicaion o oher (non roein arges o . oh he ideniicaion o liase wereas is roduced by Nacylosatidyletanolaine osoliase addiional roein arges o as ell as he deelomen o selecie drgs argeing and etabolied by fatty acid aide ydrolase . e s te cold limael lead o he deelomen o noel heraeics ih eer side endocannabinoids and teir biosyntetic and etabolic enyes constitute te eecs. endocannabinoid syste . eeral selecie ligands ere acie in arios animal models o chronic and

ot s are couled to io roteins and odulate various intracellular signal inlammaor ain, diabeic nero and nehroah, lier cirrhosis, and ischemic transduction atways suc as inibition of croduction activation of itogen reersion inr,, , b onl a e ere esed in clinical rials (e.g. activated rotein kinases s also called etracellular signalregulated (1, P (annabinor, 2, (3, edalinab (4 and (5 kinases s osoliase and roteincouled inwardly rectifying (Figure 4. hese ligands hae been mosl exlored or heir analgesic ( and cannels s and recruit βarrestins to te recetor. s dislay ligand annabinor and aniinlammaor (, , edalinab and deendent biased signaling as well as intersecies differences. ifferent ceical roeries. eciicall, and annabinor ere esed as analgesics in a hird classes ave been described as ligands suc as endocannabinoids and ‘classical molar ooh exracion model, hile , and edalinab ere cannabinoids’. is latter grou is based on and is caracteried by its tricyclic core deeloed or he reamen o oseoarhriis and as esed or he reamen scaffold. ter classes include ainoalkylindoles diaryl yraoles and bicyclic ligands. ost o aoic dermaiis. of te are ‘mixed’ ligands i.e. dislay siilar affinity for bot s but also or selective ligands ave been develoed suc as yridines yriidines uinolones oouinolines triaines biaryl ydantoins and oters for reviews see akur et al. and orales et al..

1.4 The CB2 receptor as therapeutic target

e is associated wit any ysiological rocesses suc as food intake and locootor activity but also wit undesirable effects including addiction aniety and sycoactive effects. e lays an iortant role in cell igration iunosuression and ain sensation and its cellular eression is uregulated under atoysiological conditions suc as neuroinflaation and neurodegenerative diseases (e.g. Alzheimer’s disease, Parkinson’s disease, neuroatic ain ayotroic lateral sclerosis and ultile sclerosis.

Figure 4. Structures of CB2R-selective clinical trial candidates.

13

Chaer enera nrodion

esie oein eidene o eia in reinia seins hese iands aied in hase References inia rias de o a a o eia edaina has ie een disonined ease no reors ro his dr andidae hae aeared sine hoh he reasons or arin rrosih eah eeson andre en airadea ennie ie an aae eir n anasis o he ariion o dr hese aires are e near ossie eanaions a e a a o ransaiona ania andidaes ro or aor haraeia oanies Nat Rev Drug Discov 2015, odes or heraei eia ossi de o inerseies sinain dierenes no inani er oas odries aora h and ho hae dr disoer sraeies in hara haned ha are he ne indses Drug Discov Today 2016, roo o are enaeen andor he roe o C as no roer aidaed or he hes ees aindian hio rinies o ear dr disoer Br J Pharmacol aien oaion sdied ene noe heia oos and sraeies are reired o aid 2011, C are aidaion enaeen or odaion dea hese heia oos shod hae iasi ansen raosi he rie o innoaion ne esiaes o dr hih seeii and a edeined ode o aion his od hen enae he deeoen oss J Health Econ 2003, aid raonin ee haraeia he road o osiie rerns Nat Rev Drug ransaion o inoraion ro reinia sdies on nonhan seies o he aien Discov 2009, eris rodii ons he deiniion is e Science 2005, an han an C ao C ao are ideniiaion o nara rods 1.5 Aims and outline of this thesis and ioaie oonds sin ainiased roes Nat Prod Rep 2016, asha inhas oer sadah ha aes a ood dr are Drug Discov Today 2011, his hesis desries he deeoen and aiaion o heia oos and sraeies o hes ees aindian hio rinies o ear dr disoer Brit J sd he annainoid reeor e C he researh desried in his hesis a Pharmacol 2011, eser aara i disoer and hioead aroahes Drug Discov Today 2006, iae aid in he disoer and deeoen o noe Cased heraeis ha a he naned side ees assoiaed ih aiaion o he C Coeand oiano ee rare residene ie and is iiaions or ead Chapter 2 roides a orehensie oerie o he heia oos ha hae een oiiaion Nat Rev Drug Discov 2006, Coeand he drare residene ie ode a ear reroseie Nat Rev Drug Discov reored o sd C disriion eression ees oan inernaiaion and 2016, haraoo oh in vitro and in vivo odes Chapter 3 desries he deeoen o a aein an iede o anaonis dissoiaion and onasin in io reeor roeion niona assa to study βarresin rerien o C iands and a sese Trends Pharmacol Sci 2006, o ier eran eian rare esidene ie Case or rooo his hihhro sreenin assa is sed or oond haraeriaion in roeinCoed eeors Med Res Rev 2014, Chapters 4 5 and 6 Chapter 4 desries a orehensie and eensie roiin o os ion ihais Craa eerinin are enaeen in iin sses Nat Chem Biol 2013, ide sed annainoid reerene iands o ideni he os siae iand or C ioennens annoon eis risson n iro reeor indin assas are aidaion n Chapter 5 a oond irar ased on 6 Figure 5 an in enera ehods and onsideraions Q J Nucl Med Mol Im 2008, riood ari are sie oan erin eneraiaions ro inia and vivo aie Cseeie aonis is desried in order o ideni srreinei reinia sdies Pharmacol Therapeut 2009, reaionshis Chapter 6 reors on he desin snhesis and aiaion o a ased on asher rnder he oe o ain in roo o Cone or C r isoer ose hooaini roe as a oo o dee endoenos C eression and are and eeoen Neuropsychopharmacology 2009, enaeen in riar han ine es ina ose hooaini aein ih a ahes ainer asshier nn osiron eission oorah oear iain or dr deeoen Brit J Clin Pharmaco 2012, Cased roe or C roein are ideniiaion is desried in Chapter 7 Chapter 8 nnae Cheer ones are aidaion sin heia roes Nat Chem Biol saries he or desried in his hesis and resens re roses and haenes 2013, hrann annin ieer adann aoee are naeen in Ces in he ied Cell Chem Biol 2016, ah i an Carese ied eirneis ineoni C eisseder aniain drare enaeen in sine es in iro and in io Nat Chem Biol 2017, oeerin Craa o Cheoroeois Can nae r isoer and eeoen Chem Biol 2012, ao ohea ersaher een he roeinoed reeor are ai Chem Med Chem 2006, Nat asndersen en hioh rends in he eoiaion o noe dr ares Rev Drug Discov Figure 5. Structure of LEI101 2011, aersro C hioh rra diersi o roeinoed reeors and siniiane or dr disoer Nat Rev Drug Discov 2008,

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esie oein eidene o eia in reinia seins hese iands aied in hase References 1 Chapter inia rias de o a a o eia edaina has ie een disonined ease no reors ro his dr andidae hae aeared sine hoh he reasons or arin rrosih eah eeson andre en airadea ennie ie an aae eir n anasis o he ariion o dr hese aires are e near ossie eanaions a e a a o ransaiona ania andidaes ro or aor haraeia oanies Nat Rev Drug Discov 2015, odes or heraei eia ossi de o inerseies sinain dierenes no inani er oas odries aora h and ho hae dr disoer sraeies in hara haned ha are he ne indses Drug Discov Today 2016, roo o are enaeen andor he roe o C as no roer aidaed or he hes ees aindian hio rinies o ear dr disoer Br J Pharmacol aien oaion sdied ene noe heia oos and sraeies are reired o aid 2011, C are aidaion enaeen or odaion dea hese heia oos shod hae iasi ansen raosi he rie o innoaion ne esiaes o dr hih seeii and a edeined ode o aion his od hen enae he deeoen oss J Health Econ 2003, aid raonin ee haraeia he road o osiie rerns Nat Rev Drug ransaion o inoraion ro reinia sdies on nonhan seies o he aien Discov 2009, eris rodii ons he deiniion is e Science 2005, an han an C ao C ao are ideniiaion o nara rods 1.5 Aims and outline of this thesis and ioaie oonds sin ainiased roes Nat Prod Rep 2016, asha inhas oer sadah ha aes a ood dr are Drug Discov Today 2011, his hesis desries he deeoen and aiaion o heia oos and sraeies o hes ees aindian hio rinies o ear dr disoer Brit J sd he annainoid reeor e C he researh desried in his hesis a Pharmacol 2011, eser aara i disoer and hioead aroahes Drug Discov Today 2006, iae aid in he disoer and deeoen o noe Cased heraeis ha a he naned side ees assoiaed ih aiaion o he C Coeand oiano ee rare residene ie and is iiaions or ead Chapter 2 roides a orehensie oerie o he heia oos ha hae een oiiaion Nat Rev Drug Discov 2006, Coeand he drare residene ie ode a ear reroseie Nat Rev Drug Discov reored o sd C disriion eression ees oan inernaiaion and 2016, haraoo oh in vitro and in vivo odes Chapter 3 desries he deeoen o a aein an iede o anaonis dissoiaion and onasin in io reeor roeion niona assa to study βarresin rerien o C iands and a sese Trends Pharmacol Sci 2006, o ier eran eian rare esidene ie Case or rooo his hihhro sreenin assa is sed or oond haraeriaion in roeinCoed eeors Med Res Rev 2014, Chapters 4 5 and 6 Chapter 4 desries a orehensie and eensie roiin o os ion ihais Craa eerinin are enaeen in iin sses Nat Chem Biol 2013, ide sed annainoid reerene iands o ideni he os siae iand or C ioennens annoon eis risson n iro reeor indin assas are aidaion n Chapter 5 a oond irar ased on 6 Figure 5 an in enera ehods and onsideraions Q J Nucl Med Mol Im 2008, riood ari are sie oan erin eneraiaions ro inia and vivo aie Cseeie aonis is desried in order o ideni srreinei reinia sdies Pharmacol Therapeut 2009, reaionshis Chapter 6 reors on he desin snhesis and aiaion o a ased on asher rnder he oe o ain in roo o Cone or C r isoer ose hooaini roe as a oo o dee endoenos C eression and are and eeoen Neuropsychopharmacology 2009, enaeen in riar han ine es ina ose hooaini aein ih a ahes ainer asshier nn osiron eission oorah oear iain or dr deeoen Brit J Clin Pharmaco 2012, Cased roe or C roein are ideniiaion is desried in Chapter 7 Chapter 8 nnae Cheer ones are aidaion sin heia roes Nat Chem Biol saries he or desried in his hesis and resens re roses and haenes 2013, hrann annin ieer adann aoee are naeen in Ces in he ied Cell Chem Biol 2016, ah i an Carese ied eirneis ineoni C eisseder aniain drare enaeen in sine es in iro and in io Nat Chem Biol 2017, oeerin Craa o Cheoroeois Can nae r isoer and eeoen Chem Biol 2012, ao ohea ersaher een he roeinoed reeor are ai Chem Med Chem 2006, Nat asndersen en hioh rends in he eoiaion o noe dr ares Rev Drug Discov Figure 5. Structure of LEI101 2011, aersro C hioh rra diersi o roeinoed reeors and siniiane or dr disoer Nat Rev Drug Discov 2008,

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arih Chereo eens C rrenion o he roeinoed reeor a eri an hao hi i orde arairie serai Annu Rev Pharmacol Toxicol 2013, ah o ono ho area hao anson ohn arinissen ind roeinoed reeors and sinain neors eerin ariannis eens C i Crsa rre o he an Cannainoid eeor aradis Trends Pharmacol Sci 2001, C Cell 2016, e hosh ari aian ha ehodooia adanes he nsn heroes o he an der e Cas roedersosen Cone an der oeen erens de C srra reoion Nat Rev Mol Cell Bio 2015, ie in o osero osde ooi de oos an ior oe ones aa he roein sni ene aiies Genomics 1999, aer isoer and oiiaion o ridineniidaoidinedione deriaies ier a inenin o C aii reeorinerain roeins Nat Rev Mol Cell as a noe ass o seeie annainoid C reeor aoniss J Med Chem 2011, Bio 2009, eier eoi s and eaarresins roes in reeor sienin raiin and nro hoas haar oear haraeriaion o a erihera reeor or sinain Trends Endocrin Met 2006, annainoids Nature 1993, e he ae ode o eeor iaion Trends Pharmacol Sci 1995, aie isriion o annainoid reeors in he enra and erihera neros sse Handb oier nraein he srra asis o C aiaion and inaiaion Nat Struct Mol Biol Exp Pharmacol 2005 2013, Co aeea Carise Cara Cannainoid inhiiion o he roessin o Cosa Coehia Consiie reeor aii series hisoria reie eaie eia and ina soe arohaes idene or C reeor ariiaion J Pharmacol Exp Ther 1999, he onsiie aii o roeinoed reeors Trends Pharmacol Sci 2005, enain ia as a eor he reaie reaene and ai o inerse aonis Mol Caraon arhand ssosso ero io ord oaoa aiee Pharmacol 2004, ondiere enarier e r erane Caseas odaion and niona oia ei Conoraiona oei o roeinoed reeors Trends Pharmacol inoeen o C erihera annainoid reeors drin e diereniaion Blood 1998, Sci 2007, haahara a aidehi onisinded onoraiona hanes in oine rhodosin aiee ar arhand ssosso Carriere Caraon oaoa hire nsih ino aiaion o roeinoed reeors J Mol Biol 2008, e r Caseas ression o enra and erihera annainoid reeors in han ine o Chirinda res esserer a h aanoe C inenha rane isses and eoe soaions Eur J Biochem 1995, C oann C e ii orae osenis ohr nai iand indin diaes ehoa ans eree oe C ar hoannainoid heisr o aria aonis a a roeinoed reeor Nat Chem Biol 2014, endoannainoids and eond Nat Rev Neurosci 2014, aarre erorh ne aa hidhaer aadori aoni ea heier ioei seond endoenos annainoid ha odaes oner errini ran aars nerse aonis i anaoes and a oeniaion Nature 1997, narindoe anaoe Eur J Pharmacol 2000, isono oe iias inassi Casio iresi aias hianoorieo anaaa aashia hihida aae s as a aria nerse onis o eaoroi a iias anadharan os C i aro oher Conin o he irs aae eeor ih a ine ino id aion in he ranserane oain J Biol chem sn iases oins o he saia and eora reaion o endoannainoid sinain in he 2013, rain J Cell Biol 2003, enain niona eeii and iased eeor inain J Pharmacol Exp Ther 2011, anan ion Craa orehensie roie o rain enes ha hdroe he endoannainoid arahidonoero Chem Biol 2007, enain iased aonis F1000 Biol Rep 2009, in aa an aoo onai eda onin and haraeriaion o anoi rs ohn iased aonis n eerin aradi in C dr disoer han and ose Caindeenden hoshaidehanoaine aranserases Bba-Mol Cell Bioorganic Med Chem Lett 2016, Biol L 2009, ao Consiie aii in eanoorin reeor iased sinain o inerse aoniss Adv soi n aoo rai onai eda oear haraeriaion o Pharmacol 2014, aehanoainehdroin aid aidase a noe eer o he hooine hdroase ai adi C eris nro aes C aiere ane aian ar aino ih srra and niona siiari o aid eraidase J Biol chem 2005, enoee erdie ehren arine aneres arie onis aeih idorreiss e ar ehoa oe he erihera Cannainoid naonis and nerse onis ias a he hrein eeor inain J Biol Chem 2015, eeor denaeCase nhiiion and roein Coin FEBS Lett 1995, an C ao oras han i iao an i enaarishnan ei eon ae C herer a oear erins hiaaa n no iordano isani ese sinares o roeinoed reeors Nature 2013, Cin reresses ransaiaion hroh a indeenden inaseindeenden ehanis aenor eander haran ason enson onahan J Biol chem 2005, ie C ahana C onner ei in eddin arar errera Carraedo ieaera an easo is inoed in C nernaiona nion o asi and Cinia haraoo roeinCoed eeor is reeorinded aoosis o han eaeia es FEBS Lett 2005, eoendaions or e airins ih Conae iands Pharmacol Rev 2013, oaoa oinoChae C arhand Cana orrie inadiCarona Caandra Cie oddard ro rreased eene inen o he ranserane er Caseas inain aha assoiaed ih siaion o C erihera annainoid oains o an Cs hoenei rra and niona iaions Plos Comput Biol reeor noeen o oh ioenaiaed roein inase and indion o ro eression 2016, Eur J Biochem 1996, eore od ee roeinoed reeor oioeriaion and is oenia o eono aada aase i Coin o he eressed annainoid C and or dr disoer Nat Rev Drug Discov 2002, C reeors o hoshoiase C and roeinoed inard reiin hannes Receptor ood raier aie C heraei areinaiin Prog Neuro-Psychoph 2012, Channel 1999, eder C C oe rie ansori aie ond ai a ihe Coarison o he haraoo and inaransdion o he an Cannainoid C and C eeors Mol Pharmacol 1995,

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arih Chereo eens C rrenion o he roeinoed reeor a eri an hao hi i orde arairie 1 Chapter serai Annu Rev Pharmacol Toxicol 2013, ah o ono ho area hao anson ohn arinissen ind roeinoed reeors and sinain neors eerin ariannis eens C i Crsa rre o he an Cannainoid eeor aradis Trends Pharmacol Sci 2001, C Cell 2016, e hosh ari aian ha ehodooia adanes he nsn heroes o he an der e Cas roedersosen Cone an der oeen erens de C srra reoion Nat Rev Mol Cell Bio 2015, ie in o osero osde ooi de oos an ior oe ones aa he roein sni ene aiies Genomics 1999, aer isoer and oiiaion o ridineniidaoidinedione deriaies ier a inenin o C aii reeorinerain roeins Nat Rev Mol Cell as a noe ass o seeie annainoid C reeor aoniss J Med Chem 2011, Bio 2009, eier eoi s and eaarresins roes in reeor sienin raiin and nro hoas haar oear haraeriaion o a erihera reeor or sinain Trends Endocrin Met 2006, annainoids Nature 1993, e he ae ode o eeor iaion Trends Pharmacol Sci 1995, aie isriion o annainoid reeors in he enra and erihera neros sse Handb oier nraein he srra asis o C aiaion and inaiaion Nat Struct Mol Biol Exp Pharmacol 2005 2013, Co aeea Carise Cara Cannainoid inhiiion o he roessin o Cosa Coehia Consiie reeor aii series hisoria reie eaie eia and ina soe arohaes idene or C reeor ariiaion J Pharmacol Exp Ther 1999, he onsiie aii o roeinoed reeors Trends Pharmacol Sci 2005, enain ia as a eor he reaie reaene and ai o inerse aonis Mol Caraon arhand ssosso ero io ord oaoa aiee Pharmacol 2004, ondiere enarier e r erane Caseas odaion and niona oia ei Conoraiona oei o roeinoed reeors Trends Pharmacol inoeen o C erihera annainoid reeors drin e diereniaion Blood 1998, Sci 2007, haahara a aidehi onisinded onoraiona hanes in oine rhodosin aiee ar arhand ssosso Carriere Caraon oaoa hire nsih ino aiaion o roeinoed reeors J Mol Biol 2008, e r Caseas ression o enra and erihera annainoid reeors in han ine o Chirinda res esserer a h aanoe C inenha rane isses and eoe soaions Eur J Biochem 1995, C oann C e ii orae osenis ohr nai iand indin diaes ehoa ans eree oe C ar hoannainoid heisr o aria aonis a a roeinoed reeor Nat Chem Biol 2014, endoannainoids and eond Nat Rev Neurosci 2014, aarre erorh ne aa hidhaer aadori aoni ea heier ioei seond endoenos annainoid ha odaes oner errini ran aars nerse aonis i anaoes and a oeniaion Nature 1997, narindoe anaoe Eur J Pharmacol 2000, isono oe iias inassi Casio iresi aias hianoorieo anaaa aashia hihida aae s as a aria nerse onis o eaoroi a iias anadharan os C i aro oher Conin o he irs aae eeor ih a ine ino id aion in he ranserane oain J Biol chem sn iases oins o he saia and eora reaion o endoannainoid sinain in he 2013, rain J Cell Biol 2003, enain niona eeii and iased eeor inain J Pharmacol Exp Ther 2011, anan ion Craa orehensie roie o rain enes ha hdroe he endoannainoid arahidonoero Chem Biol 2007, enain iased aonis F1000 Biol Rep 2009, in aa an aoo onai eda onin and haraeriaion o anoi rs ohn iased aonis n eerin aradi in C dr disoer han and ose Caindeenden hoshaidehanoaine aranserases Bba-Mol Cell Bioorganic Med Chem Lett 2016, Biol L 2009, ao Consiie aii in eanoorin reeor iased sinain o inerse aoniss Adv soi n aoo rai onai eda oear haraeriaion o Pharmacol 2014, aehanoainehdroin aid aidase a noe eer o he hooine hdroase ai adi C eris nro aes C aiere ane aian ar aino ih srra and niona siiari o aid eraidase J Biol chem 2005, enoee erdie ehren arine aneres arie onis aeih idorreiss e ar ehoa oe he erihera Cannainoid naonis and nerse onis ias a he hrein eeor inain J Biol Chem 2015, eeor denaeCase nhiiion and roein Coin FEBS Lett 1995, an C ao oras han i iao an i enaarishnan ei eon ae C herer a oear erins hiaaa n no iordano isani ese sinares o roeinoed reeors Nature 2013, Cin reresses ransaiaion hroh a indeenden inaseindeenden ehanis aenor eander haran ason enson onahan J Biol chem 2005, ie C ahana C onner ei in eddin arar errera Carraedo ieaera an easo is inoed in C nernaiona nion o asi and Cinia haraoo roeinCoed eeor is reeorinded aoosis o han eaeia es FEBS Lett 2005, eoendaions or e airins ih Conae iands Pharmacol Rev 2013, oaoa oinoChae C arhand Cana orrie inadiCarona Caandra Cie oddard ro rreased eene inen o he ranserane er Caseas inain aha assoiaed ih siaion o C erihera annainoid oains o an Cs hoenei rra and niona iaions Plos Comput Biol reeor noeen o oh ioenaiaed roein inase and indion o ro eression 2016, Eur J Biochem 1996, eore od ee roeinoed reeor oioeriaion and is oenia o eono aada aase i Coin o he eressed annainoid C and or dr disoer Nat Rev Drug Discov 2002, C reeors o hoshoiase C and roeinoed inard reiin hannes Receptor ood raier aie C heraei areinaiin Prog Neuro-Psychoph 2012, Channel 1999, eder C C oe rie ansori aie ond ai a ihe Coarison o he haraoo and inaransdion o he an Cannainoid C and C eeors Mol Pharmacol 1995,

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Chapter 1

112. Soethoudt, M.; van Gils, N.; van der Stelt, M.; Heitman, L. H., Protocol to Study beta-Arrestin Recruitment by CB1 and CB2 Cannabinoid Receptors. Methods Mol Biol 2016, 1412, 103-111. Chapter 2

Chemical Tools and Strategies to Study the Cannabinoid Receptor Type 2