Characterization of Novel Synthetic Analgesics Selectively Targeting Α3 Glycine Receptors

Characterization of novel synthetic analgesics selectively targeting α3 Glycine receptors

Maleeha Waqar

B.S. Bioinformatics

A thesis submitted for the degree of Master of Philosophy at

The University of Queensland in 2019

Queensland Brain Institute

i Abstract

Glycine receptors (GlyRs) are ligand-gated ion channel proteins that mediate fast inhibitory neurotransmission by selectively permitting passage of Cl- ions through its pore, which is activated by the binding of the neurotransmitter glycine. The major inhibitory receptors in the spinal cord are the GlyRs, and are therefore physiologically very important. Mutations in the genes that code for the GlyRs result in several disorders such as hyperekplexia or startle disease. However, disruption of the glycinergic currents that are mediated by GlyRs, particularly in the dorsal horn of the spinal cord that receives sensory input from the superficial laminae, has implications in other disorders too, for example chronic inflammatory pain. Potentiating these GlyRs and their activity can reduce the chronic pain sensitization, which is why potent and selective modulators for the GlyRs are strong candidates for analgesic development. Considering the importance of α3 GlyRs as targets for potentiators to alleviate pain, the aim of this project was to model and predict the binding site of a previously developed GlyR potentiator designated as HO1, and the screening of novel cannabinoids for potentiation in GlyRs, with the use of patch-clamp electrophysiological techniques. HO1 selectively potentiates α3 over α1 GlyRs, expressing in both HEK293 cells and artificial inhibitory synapses. The binding site of HO1 on the α3 GlyR was modelled via molecular docking and was verified by performing site directed mutagenesis of the modelled binding residues in the α3 GlyR and then subjecting these mutants to electrophysiological studies. The mutations retained the functionality of the α3 GlyRs but shifted the receptor’s glycine EC50 and showed no potentiation on binding to HO1. The site for HO1 binding modelled and studied here may have properties of a site involved in allosteric regulation, considering the drastic effects of its mutation on both glycine sensitivity and HO1 potentiation. Recent studies have also shown that certain cannabinoids and their derivatives have a strong effect on glycine receptors, producing analgesic effect in animal models. Two novel cannabinoid derivatives, 5-Desoxy-CBD (1803) and 1,2-dimethylheptyl-desoxy CBD (1816) were subjected to electrophysiological studies in artificial inhibitory synapses expressing α1, α3, α1β and α3β GlyRs to measure potentiating effects. The cannabinoids significantly potentiated α3 GlyR, with 1803 also potentiating α1 and α1β, and 1816 showing more selectivity for GlyRs containing α3 subunits over α1. Both drugs showed a stronger affinity for homomeric channels in comparison to heteromeric, suggesting that their possible site of action is present on the α subunits. Based on the experiments in this study, we

ii conclude that HO1 and the cannabinoids are potential leads in development of strong analgesic drugs with GlyR isoform selectivity for the treatment of inflammatory pain.

iii Declaration by author

This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis.

I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, financial support and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my higher degree by research candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award.

I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School.

I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis and have sought permission from co- authors for any jointly authored works included in the thesis.

iv Publications during candidature

No publications included.

Submitted manuscripts included in this thesis

No manuscripts submitted for publication.

Other publications during candidature

No other publications.

Contributions by others to the thesis

Significant contribution was made by Lynch JW in conception of the architecture of the study and in designing the experimental methods, in particular the electrophysiological assays. Talwar S had done the preliminary work of screening and identifying the lead compound CMB-C28-HO1 that is further characterized in this study. Other than that no significant contributions were made to the design, research work and writing of this thesis by any other individuals or parties.

Statement of parts of the thesis submitted to qualify for the award of another degree

None

v Acknowledgements

First and foremost, I would like to thank my supervisor Joe, for his unlimited support, guidance, critique and humour. These past 2 years have been an incredible privilege, to have had the opportunity to learn from him. Most admirably, Joe has never taken a day off from being a diligent supervisor, not even when he is rock climbing or recovering from a serious health problem. I also want to thank my co-supervisor Angelo, for always answering any and all questions I had, even when I would be afraid to ask them. More than that, I have truly found the greatest inspiration in Angelo’s commitment to this lab and in being a dedicated teacher.

I cannot thank enough Justine Haddrill, the greatest lab manager that any lab could have. It is safe to say that Lynch lab could not run as smoothly as it does without her, and most of my experiments would not have worked if she wasn’t there to guide me properly on where everything is and how it all works. Chen, Nela, Atif and Parnayan, I owe you greatly for always offering great advice, feedback and support wherever and whenever I needed it. A special thanks to Dr. Helen Cooper and Dr. Bruno Van Swinderen for being very supportive in this program and always having their doors and emails open for any problems.

I am greatly thankful to my amazing friends Drew, Chai Chee and Consuelo, who have been my family here in Brisbane, have constantly made sure that I not only survive but enjoy my time here, and have also been the smartest and funniest people to be around. I owe my life and all the privileges to my parents, who raised me to be a strong and independent Muslim woman, understand and acknowledge my roots and always raise my head high in the face of any challenge. To my siblings, I am thankful for the constant fights and laughs, for them always reminding me where my true strength comes from, and for always looking up to me. I have relied greatly on the support of all my friends back home, and am truly thankful to all of them.

Lastly, but mostly, I am thankful to my wonderful husband, Qasim. Thank you for always being the voice of reason and support, for loving me unconditionally from seven thousand miles away, for always meeting me halfway in every adventure, and for being my best friend.

vi Financial support

This research was supported by an Australian Government Research Training Program Scholarship

Keywords

Glycine receptor, Chronic Inflammatory pain, Electrophysiology, Analgesics, Drug Discovery

Australian and New Zealand Standard Research Classifications (ANZSRC)

ANZSRC code: 060110, Receptors and Membrane Biology, 30%

ANZSRC code: 111504, Pharmaceutical Sciences, 40%

ANZSRC code: 110903, Central Nervous System, 20%

ANZSRC code: 030402, Biomolecular Modelling and Design, 10%

Fields of Research (FoR) Classification

FoR code: 0601, Biochemistry and Cell Biology, 30%

FoR code: 1115, Pharmacology and Pharmaceutical Science, 40%

FoR code: 1109, Neuroscience, 30%

vii Table of Contents

Title page ...... i Abstract ...... ii, iii Declaration by author ...... iv Publications during candidature ...... v

Submitted manuscripts included in this thesis ...... v

Other publications during candidature ...... v

Contributions by others to the thesis ...... v

Statement of parts of the thesis submitted to qualify for the award of another degree ...... v Acknowledgements ...... vi Financial support ...... vii Keywords ...... vii Australian and New Zealand Standard Research Clasifications (ANZSRC) ...... vii Fields of Research (FoR) Classification ...... vii Table of contents ...... viii List of Figures and Tables...... x Abbreviations ...... xi

Chapter 1: Introduction 1.1. Ligand gated ion channels ...... 1 1.2. Pentameric ligand-gated ion channels (pLGICs) Cys-loop receptors ...... 2 1.3. GlyRs ...... 3 1.3.1 Ligand binding domain ...... 4 1.3.2 Transmembrane domains ...... 4 1.3.3 GlyR subunit diversity and distribution ...... 6 1.4. GlyR pharmacology ...... 7 1.4.1 Channel activity and agonists ...... 7 1.4.2 Inhibitors ...... 9 1.4.3 Modulators ...... 10 1.4.4 Cannabinoids ...... 11 1.5. GlyRs and diseases ...... 11 1.5.1 GlyR α1 and hyperekplexia ...... 12 1.5.2 GlyRs and chronic inflammatory pain ...... 12

viii 1.6. Aims of this thesis ...... 14 1.7. References ...... 15

Chapter 2: Effect of HO1 on glycine evoked currents and on spontaneous glycinergic IPSCs in Glycine receptor alpha 3 and alpha 1 2.1. Background ...... 25 2.2. Materials and methods ...... 26 2.2.1 Expressing GlyR subunit of choice in HEK293 cells ...... 26 2.2.2 Generating functional inhibitory “artificial synapses” expressing GlyR subunit of choice ...... 27 2.2.3 Patch-clamp electrophysiology ...... 29 2.2.4 Data analysis ...... 30 2.3. Results ...... 31 2.3.1 Glycine concentration-responses and HO1 potentiation at GlyRs α1 and α3 ...... 31 2.3.2 HO1 potentiation on α1 and α3 GlyRs in inhibitory artificial synapses ...... 32 2.3.3 Discussion ...... 34 2.4. References ...... 37

Chapter 3: Effect of HO1 on glycine evoked currents in wild type (WT) and mutated α3 GlyR 2.1. Background ...... 41 2.2. Materials and methods ...... 42 3.2.1 Docking studies ...... 42 3.2.2 Site directed mutagenesis ...... 43 3.2.3 Plasmid preparation ...... 43 3.2.4 HEK293 Cell Culture and transfection ...... 44 3.2.5 Electrophysiological studies ...... 44 3.2.6 Data analysis ...... 44 2.3. Results ...... 44 3.3.1 Predicted Binding site for HO1 ...... 44 3.3.2 Glycine concentration response in α3 GlyR WT vs mutants...... 46 3.3.3 HO1 concentration response in α3 GlyR WT vs mutants ...... 48 3.3.4 Discussion ...... 49 2.4. References ...... 51

Chapter 4: The potentiating effects of cannabinoid derivatives at α1 and α3 GlyRs 4.1. Background ...... 54 4.2. Materials and methods ...... 54 4.3. Results ...... 55 4.3.1 5-desoxy-CBD ...... 55 4.3.2 1,2-dimethylheptyl-desoxy CBD ...... 57 4.3.3 Discussion ...... 60 4.4. References ...... 63

Chapter 5: Conclusions 5.1. Conclusions and future directions ...... 66 5.2. References ...... 70

List of Figures and Tables Figures

Fig. 1 General structure of the pLGICs and the ligand binding subunit interface (4)

Fig. 2 pLGIC TM domains (6)

Fig. 3 Agonist activity for GlyRs (9)

Fig. 4 Potentiator AM-3607 bound to α3 GlyR (PDB ID: 5TIO) between two subunits (13)

Fig. 5 Structure of CMB-C28-H01 (26)

Fig. 6 Calcium phosphate-DNA co-precipitation method for expressing GlyR subunit of

choice (27)

Fig. 7 Generating functional inhibitory synapses expressing GlyR subunit of interest (28)

Fig. 8 Diagrammatic representation of whole cell voltage clamp (patch-clamp)

electrophysiology (30)

Fig. 9 HO1 concentration response with α1 GlyR vs α3 GlyR in HEK293 cells. (32)

Fig. 10 Glycinergic IPSCs produced by inhibitory synapses expressing α1 and α3 GlyR (33)

Fig. 11 IPSC kinetics for HO1 on inhibitory synapses expressing α1 and α3 GlyR (34)

Fig. 12 Docking protocol (43)

Fig. 13 Predicted binding site of HO1 for α3 GlyR (45)

Fig. 14 Comparison of binding site for neurotransmitter Glycine and HO1 on α3 GlyR (46)

Fig. 15 Glycine dose response and EC50 comparisons for WT and mutants (47)

Fig. 16 HO1 concentration response for WT α3 GlyR compared to that of the mutants (49)

Fig. 17 Structures for cannabidiol and its derivatives (54)

Fig. 18 Comparison of spontaneous glycinergic IPSCs mediated by α1, α3, α1β and α3β

GlyRs for the cannabinoid 1803 (56)

Fig. 19 The mean values for the peak amplitudes, 10–90% rise times and decay time (57)

Fig. 20 Comparison of spontaneous glycinergic IPSCs mediated by α1, α3, α1β and α3β

GlyRs for the cannabinoid 1816 (58)

Fig. 21 The mean values for the peak amplitudes, 10–90% rise times and decay time (59)

Tables

Table 1 The Glycine EC50 and Hill slope values observed for all α3 GlyR mutants (47)

Abbreviations pLGIC Pentameric ligand gated ion channel

GABAAR γ-aminobutyric acid receptors

GlyRs glycine receptors

ECD extracellular domain

TM Transmembrane nAChR Nicotinic acetylcholine receptor

PTX Picrotoxin

xi µM Micromolar nM Nanomolar mM Millimolar ms Millisecond

IPSC Inhibitory Postsynaptic currents cDNA Complimentary Deoxyribonucleic acid

GFP Green Fluorescent protein

DMEM Dulbecco’s modified eagle medium

THC Tetrahydrocannabinol

CBD Cannabidiol

PGE2 Prostaglandin E2

PKA Protein kinase A

PDB Protein Data Bank

HEK Human Embryonic Kidney mRNA Messenger ribonucleic acid mV millivolts

Zn2+ Zinc ion

μg microgram

EC50 half-maximal effective concentration

xii

Chapter 1 Introduction

1 1.1 Ligand gated ion channels

Ligand gated ion channels (LGICs) are a superfamily of transmembrane channel proteins that allow selective passage of ions through it, initiated on binding to a ligand, usually a neurotransmitter (Acuna et al. 2016; Lynch 2004). Different type of neurotransmitters bind and activate different type of synaptic LGICs, triggering different type of ions to influx. This allows the LGICs in the central and peripheral nervous system to mediate fast chemical transmission of nerve signals (Corringer et al. 2012). Presynaptic neuron releases neurotransmitter into the synaptic cleft on excitation, which binds to the LGICs located on the post synaptic neuron. The neurotransmitter binding induces a conformational change, opening the ion channel pore and allowing ionic influx (Betz and Laube 2006). Depending on either cationic or anionic inflow, the postsynaptic neuron will either depolarize to produce an excitatory effect, or hyperpolarize to produce an inhibitory effect.

1.2 Pentameric ligand-gated ion channels (pLGICs) Cys-loop receptors

Cys-loop receptors are pentameric LGICs (pLGICs) that have a characteristic Cysteine loop, which is composed of 13 amino acids that are highly conserved in all the receptors belonging to this family, except in prokaryotes (Bocquet et al. 2006). The loop is formed between two cysteine residues on both ends of the short amino acid sequence, that bind covalently to each other by making sulphide bridges (Betz and Laube 2006; Sine and Engel 2006). pLGIC receptors exist as homomers or heteromers of five subunits assembled in a circular arrangement around a central ion channel. Members of this subfamily of pLGICs include nicotinic acetylcholine receptors (nAChR), glycine receptors (GlyRs), γ-aminobutyric acid receptors (GABAARs), glutamate-gated ion channel receptors (GluClRs) and serotonin type-

3 receptors (5HT3Rs) (Schofield et al. 1987; Tasneem et al. 2004; Beg and Jorgensen 2003; Davies et al. 2002).

PLGICs have a wide expression in many multicellular species including invertebrates such as insects, to zebrafish and in mammals including humans (Cartaud et al. 1973; Brisson and Unwin 1985; Mitra 1989; Unwin 2005). Various pLGIC receptors have had their structures resolved using techniques such as Nuclear Magnetic Resonance (NMR) spectroscopy, X-ray crystallography or electron microscopy. Crystal structures from bacteria Erwinia chrysanthemi (ELIC) and Gloeobacter violaceus (GLIC) that have been resolved and crystallized in both the closed and open states (Hilf and Dutzler, 2008; Hilf and Dutzler, 2009), and the first eukaryotic glutamate-gated chloride channel receptor from

2 Caenorhabditis elegans (GluClR) (Hibbs and Gouaux, 2011), show structural homology to each other and with other Cys-loop receptor isoforms. Though eukaryotic and prokaryotic pLGICs have a shared sequence homology to be as low as about 20%, the presence of a common transmembrane topology and conserved residues and motifs in the extracellular and transmembrane regions ensure basic structural functional similarities (Corringer et al. 2012). These findings have suggested the possibility of a phylogenetic linkage between eukaryotic and prokarytoic pLGIC (Corringer et al. 2012). Recently, x-ray crystallographic techniques

have been used to determine high resolution structures of a GABAARs and GlyR subtype in humans (Huang et al. 2017; Miyazawa et al. 2003). pLGIC receptors bear many characteristic structural similarities, where each subunit has a large N-terminal extracellular domain (ECD), four transmembrane α helical segments (M1-M4) an intracellular loop that connects the M3 and M4 segments, and a short C terminal extracellular domain (Huang et al., 2015). The large ECD has the characteristic cysteine bonded motif or the Cys-loop, and has the essential ligand binding sites. Isolated crystal structures of the ECD of many LGICs have all shown to be folded in an immunoglobulin like highly conserved β sandwich topology which is stabilized by the hydrophobic inner residues (KatjuS̆ a et al. 2001). The M2 alpha helical segment from each subunit lines the ion channel pore (Hucho et al. 1986; Webb and Lynch, 2007). Another remarkable feature conserved among pLGICs is the ECD-TMD interface which has allowed for successful coupling of ECDs and TMDs from different pLGICs to form functional chimeric receptors, such as α7 nACh and α1 GlyR and more recently, the fully functional chimera formed with bacterial GLIC and α1 GlyR (Grutter et al. 2005; Duret et al. 2011).

1.3 GlyRs

GlyRs are present at inhibitory synapses where they are activated by pre-synaptically released glycine. This activation triggers a subsequent influx of Cl- ions into the postsynaptic cell, which leads to a rapid postsynaptic response (Betz and Laube, 2006). The major inhibitory receptors in the spinal cord are the GlyRs, and are therefore physiologically very important. Mutations in the genes that code for the GlyR subunits result in several disorders such as hyperekplexia or startle disease (Bode and Lynch 2014). However, disruption of glycinergic currents, particularly in the dorsal horn of the spinal cord that receives sensory input from the superficial laminae, has implications in other disorders too, for example inflammatory pain (Harvey et al. 2004). Potentiating the activity of these GlyRs can reduce the chronic pain

3 sensitization, which is why potent and selective modulators for the GlyR receptors are strong candidates for analgesic development.

1.3.1 Ligand binding domain

A good model of the ECD for pLGICs, has been the glial Acetylcholine binding protein (AChBP) from freshwater snail Lymnaea stagnalis, which lacks the TM domains and intracellular loops but is homologous to the ECD of nAChRs (Snit et al. 2003). The AChBP has hence served as a considerably accurate template to study the ECD of PLGICs, including GlyRs (Lynch 2004). The ligand-binding domains are present within the ECD of the receptor and involves the flexible loops in its beta sheets. The interface between two adjacent GlyR subunits serves as a binding pocket for the agonist to bind (fig. 1). Distinct regions in the adjacent subunits contribute residues that interact with the ligands. This binding site is formed by three loops (A, B and C) of the principal (+) subunit with the adjacent (-) subunit contributing complimentary beta sheets (loops C, D and F), effectively allowing the ligand to bind in between the two (Brejc et al. 2001). Successful binding of the ligand in the interface between subunits induces conformational changes that ultimately lead to channel opening and allow subsequent ion influx. Another secondary cysteine loop is present in GlyR subunits that forms the ligand binding C loop domain and is crucial for glycine binding (Rajendra et al.

1995).

Fig. 1 General structure of the pLGICs and the ligand binding subunit interface. Panel A shows the pentameric arrangement of 5 subunits with a centrally located ion channel pore, characteristic to pLGICS (PDB ID: 5TIO). Panel B shows the top down view of the central ion pore and the principal (+) and complimentary (-) subunit interfaces. Panel C shows the close up side view of a ligand binding interface between two subunits with loops A, B and C in the (+) subunit and loops D, E and F in the (- ) subunit of the interface.

1.3.2 Transmembrane domains

4 Recent high resolution crystal structures available for glycine receptors have shown that like other pLGICs receptors, GlyRs also have four α-helical transmembrane (TM) domains. The transmembrane domains provide a separation between the hydrophobic lipid membrane and the polar ion conduction pathway (Lynch 2004). While the TM2 domain lines the ion channel pore, the TM1, TM3 and TM4 domains separate it by acting as a barrier between it and the nonpolar lipid molecules in the surrounding membrane (Miyazawa et al. 2003). The TM1- TM2 and TM3-TM4 domains are connected to each other through flexible intracellular linker domains, whereas the TM2-TM3 are connected via an extracellular domain (Hibbs and Gouaux 2011; Miller and Aricescu 2014; Miyazawa et al. 2003).

While the TM2 domain of each subunit in the pentamer form the ion channel pore, other TM domains also play structurally important roles in the ion channel gating. The placement of the TM1 domain in direct connection to the N-terminal domain makes it ideal for a linkage between the ligand binding site and the channel gate (Keramidas et al. 2006). Several studies have shown the importance of residues present prior to the start of TM1 domain, termed as extra-membranous TM1 residues that have been implicated in channel gating as a result of modifications due to hydrophilic agents (Akabas and Karlin 1995). This makes the TM1 domain important in the overall LGIC function as studies have shown that mutation of residues in this domain result in changes to the channel gating (fig. 2) (Akabas and Karlin 1995; Bianchi et al. 2001; Engblom et al. 2002; Tamamizu et al. 1995).

The pore forms with 5 TM2 α-helices that are bent inwards near the centre to form a narrow constriction in the middle. 15 α-helices, contributed by the other TM domains, from neighbouring subunits that twist around each due to hydrophobic interactions and shelter the inner pore from lipids (fig. 2) (Miyazawa 2003). The TM2 domain hence not only forms the channel gate but is involved in controlling ionic selectivity and has binding sites for important pharmacological and physiological agents (Krasowski and Harrison 2000; Bormann et al 2004).

Extensive studies of the TM3 domain have revealed its contribution in the formation of a water filled pocket that is distinct from the channel pore (Miyazawa 2003). More evidence

about the structures of GABAAR and GlyR has also shown that the residues in TM3 form binding sites for ivermectin, alcohols and anaesthetics (Lynch, 2004).

The TM3 and TM4 are connected by a large intracellular loop that is poorly conserved throughout the pLGIC receptor family and has been shown to not play a role in the overall

5 function and assembly of the subunit (Jansen et al., 2008). However, the loop plays an important role in modulation due to the presence of phosphorylation sites, which can alter the functioning of the receptor (Harvey et al., 2004). The loop also has an 18 amino acid long binding site for the neuronal assembly protein, gephyrin, which is involved in synaptic clustering of GlyRs (Maric et al. 2011). This site is only present in the β GlyR subunit.

Recent studies have also shown the important role of TM4 residues in determining differential glycine efficacy between GlyR subtypes α1 and α3 by looking at TM4 orientation in both receptors (fig. 2) (Han et al. 2013; Chen et al. 2009).

Fig. 2 pLGIC TM domains. Panel A shows the amino acid sequence alignment of the human α1 and α3 GlyRs with the known residues for glycine and strychnine binding site are highlighted in blue. Residues involved in formation of TM domains are shaded as: TM1 (pink), TM2 (green), TM3 (orange) and TM4 (blue). Panel B shows the side view arrangement of the TM domains highlighted in different colours for α3 GlyR. Panel C and D show the bottom up view of strychnine bound conformation and glycine + ivermectin bound conformation for α3 GlyR (PDB ID: 5TIO).

1.3.3 GlyR subunit diversity and distribution

Five GlyR subunits have been identified, α1, α2, α3, α4 and a β subunit, with the α subunits sharing a homology of ~90% in their amino acid sequences. All α subunits can form functional homomers, however, the β subunit can only form functional heteromers with either of the α subunits in a 2α:3β (Lynch 2009; Grudzinska et al. 2005; Yang et al. 2012) arrangement, or differently in a more recently revealed stoichiometry of 3α:2β (Durisic et al. 2012).

6 Strychnine binding studies have revealed the distribution of GlyRs in the nervous system. Higher brain and somatic regions show a near absence of GlyR subunits at synapses, with them being most expressed in spinal cord and medulla, and to lesser amounts in the thalamus and hypothalamus (Lynch 2004). The spinal cord has a more diffused distribution of the GlyRs as opposed to the brain stem, where they are expressed in specific nuclei. Prenatally, α1 and the β subunit are expressed in lower amounts, which increases after birth to a maximum at 3 weeks postnatal. α2 expresses in an opposite fashion with its level being highest prenatally and then declining postnatal (Watanabe and Akagi 1995). The α3 subunit shows a similar expression to α1, by being scarce in its distribution at birth but gradually increasing with age. The dorsal horn of the spinal cord is the main site of expression for α3 subunit with lower levels outside the spinal cord (Webb and Lynch 2007). The beta subunit is well spread throughout the brain and spinal cord in its distribution. The only α subunit absent from humans is the α4, which is expressed in rodents and zebrafish among other species but is a pseudogene in humans. The β subunit is critical for the clustering of the GlyRs at the synapses due to its interaction with gephyrin, which is why, the α1β heteromers are the most widely distributed isoform in the synapses of an adult spinal cord and brainstem (Singer et al. 1998).

1.4 GlyR pharmacology

1.4.1 Channel activity and agonists

GlyRs are predominantly involved in the neural circuits by glycine mediated Cl- channel pore opening, which allows Cl- influx to cause hyperpolarization of the cell membrane potential and inhibit the neuronal firing (Du et al. 2015). The release of glycine into the synaptic cleft takes place when the synaptic vesicles containing them fuse with the pre-synaptic membrane in a calcium dependant process (Betz and Laube 2006). Glycine release results in the activation of post-synaptic GlyRs, which mediates inhibitory neurotransmission throughout the spinal cord and brain stem. GlyRs (Du et al. 2015). Gephyrin, which is a neuronal assembly protein, facilitates the clustering and anchoring of the GlyRs at inhibitory synapses. The β subunit plays the critical role of binding to gephyrin due to the presence of a gephyrin- binding amino acid sequence in its M3-M4 loop, and hence makes the synaptic clustering possible (Kneussel and Betz, 2000) (Kneussel and Betz 2000).

Glycine is the physiological agonist for the GlyRs. Hence, glycine has the highest affinity for GlyRs among all the agonists, fig. 3 shows the binding interaction of glycine with α3 GlyR.

7 Other high affinity agonists include, β alanine, taurine and GABA. Due to the similarity in structure of these ligands, all of them bind to the same orthosteric site on GlyRs, and hence act as competitive antagonists to glycine (Jan et al., 2001).

Receptor activation does not require all the subunits to be bound to the ligand, especially for glycine activation, which can happen with three of its molecules bound to the subunits in the receptor (Burzomato et al., 2004). Agonist binding loop C plays an important role in the functional state of the ion channel by its conformation in relation to the opening or closing of the channel. The loop has been shown to be open when an antagonist is bound and the channel is closed, whereas the loop takes a closed conformation when the agonist is bound and the channel is open (Brams et al. 2011; Du et al. 2015). This conformational relevance is an important marker of the correlation of loop C to the channel’s open or closed state.

A naturally occurring macrocyclic lactone, ivermectin, derived from the bacteria Streptomyces avermitilis is a non-amino acid agonist and potentiator of GlyRs (fig. 3). It also targets other pLGICs such as GABAARs and GluClRs causing enhanced inhibitory neurotransmission (Lynagh and Lynch 2012a; Lynagh and Lynch 2012b). A recent α GluClR structure from C. elegans co-crystallized with ivermectin has shown that it binds in an interface between two adjacent subunits in the transmembrane domain forming hydrogen and hydrophobic bonds (fig. 3). At higher levels, ivermectin can directly activate GlyRs in an irreversible or slowly reversible mechanism, while at lower levels it can act as an allosteric modulator by enhancing glycine-induced currents (Shan et al. 2001a).

Fig. 3 Agonist activity for GlyRs. Panel A shows glycine (red) bound in the interface between two α3 GlyR (PDB ID: 5VDH) subunits in the ECD, with binding residues highlighted. Panel B shows ivermectin (blue) bound between two subunits in the TM region.

1.4.2 Inhibitors

Another ligand that binds the orthosteric binding site is strychnine (Vandenberg et al. 1992). Strychnine is a complex alkaloid from plant origins that acts as a competitive antagonist of the GlyRs. It causes convulsions and muscle spasms due to its inhibition of glycinergic inhibitory input to target neurons (Huang et al. 2015). The binding of strychnine induces a closed channel conformation. Strychnine is a very potent and highly selective antagonist that binds to GlyRs over other pLGICs, and is an effective tool for differentiating GlyRs from other pLGICs, since the original GlyR isolated was done by using strychnine affinity chromatography in rat spinal cord (Pfieffer et al. 1982). This enables strychnine to be used in studies aimed at revealing GlyR distribution and physiological roles. Several studies have also indicated that strychnine and glycine do not compete for the same site on GlyRs, but bind to overlapping yet different sites in the loops B and C of principal domain (Vandenberg et al. 1992; Grudzinska et al. 2005).

9 Picrotoxin (PTX) is a naturally occurring macrocyclic alkaloid from the plant Anamirta cocculus composed of picrotoxinin and picrotin in equimolar fractions. It non-specifically

inhibits anion selective pLGICs and is used to differentially characterize GABAARs and GlyRs due to the difference in its mechanism of inhibition for both (Chen et al. 2006; Sivilotti

and Nistri 1991). For GABAARs, picrotoxinin is the active constituent, whereas for GlyRs,

both picrotin and picrotoxinin are equally active in inhibition. In GABAARs the effect of PTX is use dependent and non-competitive and hence classifies it as an allosteric inhibitor rather than channel blocker (Porter et al. 1992). However for GlyRs, its effect is not use dependent but shows less sensitivity for heteromeric as compared to homomeric GlyRs (Pribilla et al. 1992). This has been attributed to the TM2 domain of α subunits, which when inserted into the β subunit, restored the high sensitivity in heteromeric GlyR. This has made PTX an important pharmacological tool in identifying the presence of β subunit in native and recombinant GlyRs (Shan et al. 2001b; Pribilla et al. 1992).

Sharing common structural characteristics with picrotoxin, are a class of naturally occurring macrocyclic terpene compounds called ginkgolides, isolated from the Gingko tree (Ivic et al. 2003). Ginkgolide B is a potent and specific channel blocker for GlyRs. Application on GlyRs has shown the effect of this compound to be non-competitive, use dependent, and not affected by the binding of competitive antagonist strychnine (Hawthorne et al. 2006). As opposed to PTX, Ginkgolide B on application shows an increase in its blocking ability as the cell depolarizes, strongly suggesting the binding of this negatively charged compound in the ion pore of the channel (van Beek 2005; Hawthorne et al. 2006).

1.4.3 Modulators

Zinc ions modulate both inhibitory and excitatory synaptic function and is an important allosteric modulator for the GlyRs. Free zinc (Zn2+) is present locally at the presynaptic vesicles and has been observed with glycine vesicles in spinal neurons as well (Smart et al. 2004). Zinc affects the GlyR in a biphasic fashion multiple binding interactions for the receptor. Low concentrations of zinc (0.01-10 µM) increase the apparent glycine affinity for the receptor to potentiate the current, whereas higher concentrations greater than 10 µM reduce this glycine affinity and hence the glycinergic currents as well (Nevin et al. 2003). Mutation of important residues such as D194A, that is present on the extracellular domain, have shown a drastic decrease in sensitivity to Zn+ induced potentiation (Lynch et al. 1998).

10 Alcohols have been shown to directly potentiate GlyR mediated currents (Vengeliene et al. 2008; Celentano et al. 1988). Despite previous predictions, alcohol does not act on GlyRs by disordering the membrane lipid bilayer, and has in fact a specific binding site on the receptor (Tapia et al. 1998). This binding site has been shown to be formed by residues from the TM2 and TM3 facing each other and forming a pocket for the alcohol molecule to fit into (Mihic et al. 1997). The potentiation for GlyRs increases with the length of the side chain for the alcohol up to decanol (n=10), after which the increase in chain size decreases the effect of potentiation (Mascia et al. 1996; Wick et al. 1998; Ye et al. 1998). This is likely due to limitations imposed by the volume of the alcohol binding pocket. This site is also where most anaesthetics have been shown to bind to the GlyR (Webb and Lynch 2007; Yevenes and Zeilhofer 2011).

1.4.4 Cannabinoids

Cannabinoids are a class of compounds that primarily act on cannabinoid receptors (CB1 and CB2). However, recent evidence has shown that cannabinoids and their derivatives have strong potentiating effects on GlyRs. Endogenous cannabinoids such as anandamide significantly potentiate glycinergic currents in isolated neurons as well as Xenopus laevis oocytes expressing homomeric and heteromeric α1 GlyRs (Hejazi et al. 2005; Xiong et al. 2012a). Tetrahydrocannabinol (THC), the most active and abundant constituent of cannabis, is an exogenous cannabinoid that also directly potentiates glycinergic currents in homomeric α1 and α3 GlyRs. This potentiation results in strong analgesia, as has been shown in various animal models of pain. Xiong et al. (2011) have reported a serine residue at 296 position in the TM3 domain, via NMR and mutagenesis to be critically involved in the THC induced potentiation, as it interacts with the hydroxyl groups present on THC (Xiong et al. 2011).

Other than THC, there has been growing interest in the non-psychotropic cannabidiol (CBD), which also potentiates glycinergic currents mediated by both α1 and α3 GlyR (Ahrens et al. 2009; Xiong et al. 2012a). Due to its structural similarity to THC, CBD also seems to interact with the GlyRs at the serine on 296 position. Certain derivatives of CBD have been shown to exhibit differential potentiating and modulating effects on GlyRs subtypes and are an increasing subject of interest for designing potent and non-psychoactive cannabinoid derived analgesics (Xiong et al. 2012a; Demir et al. 2009).

1.5 GlyRs and diseases

1.5.1 GlyR α1 and hyperekplexia

11 GlyRs mediate the neurotransmission in motor reflex circuits in spinal cord, primarily via α1 GlyR. Disruption of this neurotransmission can cause human startle disease or hyperekplexia. The disease is characterized by exaggerated reflex and startle episodes to tactile and auditory stimuli, followed by temporary but acute muscle rigidity and hypertonia (Shiang et al. 1993). Heritable mutations are the cause of hyperekplexia and the symptoms start appearing from birth. The disruption of inhibitory neurotransmission mediated by α1 GlyRs results in increased excitability of motor neurons. The mutations can reduce α1 GlyR activity, decreasing the receptor’s glycine sensitivity, surface channel expression and conductance and increasing desensitization rate and spontaneous activity (Chung et al. 2010; Langosch et al. 1994; Bode and Lynch 2014). Increased level of channel opening spontaneously can cause the ionic gradient across the membrane to collapse due to tonic chloride influx and also disrupt the channel inhibitory activity. Most of the mutations occurs in the TM1-TM3 domains of the α1 GlyR subunit (Harvey et al. 2008). The only α subunit affected in hyperekplexia is the α1 subunit, but mutations of other proteins present on glycinergic synapses, such as the β GlyR subunit, glycine transporter GlyT2 and gephyrin, have also been reported in isolated cases of hyperekplexia (Rees et al. 2002; Rees et al. 2003).

1.5.2 GlyRs and chronic inflammatory pain GlyRs, particularly those containing α1 and α3 subunits are predominantly expressed in the laminae I and II of the spinal cord. Inhibition of dorsal horn neurons in the spinal cord is mediated by glycinergic currents evoked by α1 and α3 –containing GlyR channels, inhibiting the propagation of spinal nociceptive transmission (Ahmadi et al. 2001). Increased sensation of pain happens when injury induced prostaglandin overproduction takes place in both the spinal cord and the tissues. Of these prostaglandins, Prostaglandin E2 (PGE2) is the most actively involved in pain sensitization in central as well as peripheral regions, and inhibits the activity of GlyRs in the spinal cord (Harvey et al. 2004; Demir et al. 2009). Studies have shown PGE2 to inhibit α3 –containing GlyRs via a Protein Kinase A (PKA) dependent phosphorylation, although this does not occur to α1-containing GlyRs, since α1 subunit lacks the sites for PKA phosphorylation (Bregman et al. 2017). Thus, when GlyR α3 is inhibited, it is unable to prevent the transmission of the pain stimuli from peripheral nociceptors to the brain, and this results in increased sensation of pain. This involvement of the α3 subunit in pain sensitization makes it an interesting target for treatment of chronic inflammatory pain. Small molecules that can bind effectively to the α3 GlyR receptor to potentiate the transmission of its currents can help in alleviating the pain sensation and serve as potent analgesics. Many ligands that can selectively bind to α3 GlyR to enhance 12 its activity have been shown to alleviate chronic pain in animal models (Xiong et al. 2011; Bregman et al. 2017; Huang et al. 2017). These possible analgesics should effectively bind to sites that do not disrupt or inhibit the binding of glycine or other important allosteric agonists such as zinc. A recent study published by Amgen Inc., showed a novel potentiator of the α3 GlyR, called AM-1488 that successfully reversed tactile allodynia in a mouse model when administered orally. The α3 GlyR was then co-crystallized with another strong compound designed by them, called AM-3607, that binds to a novel allosteric ligand binding site (fig. 1) adjacent to the glycine binding site (Bregman et al. 2017; Huang et al. 2017). The structurally similar AM-3607 is a more potent drug than AM-1488, and was shown to bind more competitively to α3 GlyR compared to AM-1488. After glycine binds to the receptor, the loop C adopts a closed conformation, as it should in an agonist bound state (Hibbs and Gouaux 2011). The AM-3607 binds near the top of the extracellular domain at subunit interfaces (fig. 4). It was hypothesized initially in the study that since the loop B residue Tyr161 is involved in the AM-3607 binding site, while other loop B residues such as Glu157, Ser158 and Phe159 are involved in glycine binding, it is possible that the drug binding would initiate conformational changes in the α3 GlyR glycine binding site. The binding affinities were measured before and after the AM-3067 bound to the receptor. The results showed that the binding of the potentiator increased the glycine affinity for the receptor by almost 200 fold (Huang et al. 2017). The compounds, however, did not exhibit any selectivity for the α3 over the α1 GlyR.

Fig. 4 Potentiator AM-3607 bound to α3 GlyR (PDB ID: 5TIO) between two subunits. Expanded view shows the residues in α3 GlyR interacting with the drug, bound in the top of the ECD.

13 Recent studies have shown that certain cannabinoids and their derivatives have a strong effect on GlyRs, producing analgesic effects in animal models (Xiong et al. 2011; Ahrens et al. 2009; Xiong et al. 2012a). This has led to a growing interest for α1 and α3 GlyRs as potential targets for cannabinoids, with recent data showing that potentiation of both α1 and α3 GlyRs results in cannabinoid-induced analgesia. However, due to lack of subunit specific agonists or co-agonists, it has been difficult to elucidate the involvement of either α1 or α3 GlyRs solely, or together, in the resultant cannabinoid-induced analgesia (Demir et al. 2009; Foadi et al. 2010; Xiong et al. 2012b; Yang et al. 2008).

1.6 Aims of this thesis

Chronic inflammatory pain remains a challenge for developing potent treatments, since most of the strong drugs with analgesic effects also pose a variety of adverse side effects. The aim of this MPhil is to characterize the binding of a novel analgesic designated CMB-C28-HO1, or simply HO1 to α3 GlyR, to identify novel synthetic cannabinoids targeting α1 and α3 GlyRs, and to determine their subtype selectivity for GlyRs containing the α3 subunit. The project was designed to achieve the following objectives:

1. To elucidate the effect of HO1 on glycinergic inhibitory post-synaptic currents (IPSCs) in wild-type (WT) α3 and α1 GlyRs, to further validate the compound’s selectivity. 2. Determine the binding site of HO1 for α3 GlyR and the site’s role in the potentiation of glycinergic currents in α3 GlyR. 3. To determine the effects of various cannabinoid derivatives on potentiating glycinergic currents in α1 and α3 GlyRs, and their subtype selectivity for particular subunit containing GlyRs.

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Chapter 2 Effect of HO1 on glycine evoked currents and on spontaneous glycinergic IPSCs in α3 and α1 GlyRs

24 2.1 Background GlyRs, particularly α1 and α3, are predominantly expressed in the synaptic puncta present in the laminae I and II of the spinal cord. Inhibition of dorsal horn neurons in the spinal cord is mediated by glycinergic currents evoked by α1 and α3 –containing GlyR channels, inhibiting

the propagation of spinal nociceptive transmission (Ahmadi et al. 2001). PGE2 is realeased

during inflammation and inhibits α3 GlyRs in the spinal cord via a PKA dependent mechanism (Harvey et al., 2004; Acuna et al., 2016). Inhibition of the GlyRs allows the spinal neurons to facilitate the transmission of nociceptive stimuli such as pain from spinal cord to higher brain regions (Zeilhofer, 2005). Small molecules that can bind effectively to α3 GlyR to potentiate its glycinergic currents, without having any effects on other GlyRs can help in alleviating the pain sensation (Lynch and Callister 2006). Many ligands that have selectively bound to α3 to enhance its activity have been shown to produce analgesia in animal models (Huang et al., 2016; Bregman et al. 2017, Xiong et al. 2011, Xiong et al. 2012).

Over the past 10 years, our lab has focused on designing and screening novel small molecular drugs that selectively potentiate the effects of GlyRs (Balansa et al., 2010; Balansa et al., 2013a; Balansa et al., 2013b), particularly the GlyR α3. Professor Robert J. Capon at IMB, University of Queensland, designed a library of 120 compounds using pharmacophores that had been constructed based on the naturally occurring marine sponge metabolites. The drug designated CMB-C28-HO1, or simply HO1, was selected from this library after rigorous screening against GlyR α1 and α3 for strong potentiating effects. Structurally, the compound comprises an aliphatic linker chain between two different moieties, a tetronic acid and a glycinyl lactam ring (fig. 5). The compound showed strong potentiation of both glycine-induced currents and spontaneously evoked inhibitory post synaptic currents (IPSCs) in α3 GlyR as compared to α1 GlyR. However, the site and mechanism by which HO1 binds to the α3 GlyR remains to be determined.

Fig. 5 Structure of CMB-C28-H01. Compound is shown with the lactam ring on the left, aliphatic carbon chain linker in the middle, and tetronic acid moiety on the right.

2.2 Materials and methods

2.2.1 Expressing GlyR subunit of choice in HEK293 cells

Expression of neuronal membrane proteins like pLGICs has been successfully achieved for years in the Human Embryonic Kidney (HEK293) cell line. This is attributed to a number of factors, one of which is the more recently proven neuronal lineage of HEK293 cells due to the expression of certain proteins and mRNAs found primarily in neurons (Graham et al. 1977). Many signalling pathways present in native neurons have also been found in the intracellular environment of HEK293 cells (Thomas and Smart 2005). Keeping this in mind, HEK293 cells were considered the most effective method of transient transfection of GlyR subunit of choice, and for subsequent use in electrophysiological experiments.

HEK 293 Cell Culture and transfection

HEK293 cells were cultured in T75 flasks in Dulbecco’s modified Eagle Medium (DMEM) with L-glutamine (Invitrogen), supplemented with 30 U/ml Penicillin and 30 µg/ml Streptomycin (Invitrogen) and 10% Serum Supreme Foetal Bovine Alternative (Lonza), aseptically. The cell culture dishes were maintained in a 5% CO2 incubator at 37 and were trypsinized and resuspended weekly to maintain optimum confluency. HEK293 cells at 50% confluency were transiently transfected with 300 ng of cDNA for rat α3L GlyR (PcDNA 3.1) and human α1 GlyR (pCIS) in separate culture dishes using calcium phosphate-DNA co- precipitation method, and co-transfected with green fluorescent protein (GFP) as a reporter of expression in 1:3 ratio with the receptor DNA (fig. 6) (Phillips 2001). The calcium phosphate method involved a mixture of calcium chloride (CaCl2) with Bis(2-hydroxyethyl)taurine

26 (BES) buffer (Kingston et al. 2003). The transfected dishes were incubated in 3% CO2 at 37 ̊C for 16 hours, then washed with phosphate buffered saline (PBS) and DMEM to terminate transfection. After overnight incubation, the cells were checked under a fluorescence microscope to confirm successful transfection and GFP expression.

Fig. 6 Calcium phosphate-DNA co-precipitation method for expressing GlyR subunit of choice.

2.2.2 Generating functional inhibitory “artificial synapses” expressing GlyR subunit of choice

It is often difficult to determine the physiological activity of individual isoforms of ion channels in their native neuronal environment and to pharmacologically or genetically isolate them, because of the presence of other channel isoforms. However, transient expressions in non-neuronal cells not only require exogenous agonist application to evoke currents but also do not simulate the dynamics of synaptic neurotransmission. A previously developed neuron- HEK293 co-culture technique has been recently optimized in our lab to efficiently generate recombinant glycinergic synapses with the expression of GlyR of interest (Dixon et al. 2015; Zhang et al. 2015). The GlyR of interest is expressed in HEK293 cells, along with a trans- synaptic protein, neuroligin 2A that mediates synapse formation between the HEK293 cells and surrounding neurons.

Preparation of spinal neurons

Timed-pregnant rats were euthanized by CO2 inhalation, with the procedures approved in accordance by the University of Queensland Animal Ethics Committee. For the generation of glycinergic inhibitory synapses, E15 embryos were dissected out and placed into chilled Ca- Mg-free Hank’s Balanced Salt Solution (CMF-HBSS). The spinal cord from each embryo was then removed after carefully removing the meninges. This minimised the possibility of too many glial cells causing contamination in the culture. The dissected spinal cords were then trypsinized to purify the neurons from the rest of the tissue. Centrifugation of the

27 neuronal concentrate in DMEM-FBS and subsequent resuspension ensured the purification of spinal cord neurons from the rest of the spinal tissue and debris. The neuronal suspension was then plated onto sterilized and poly-D lysine (PDL) coated 12 mm glass coverslips at a density of 80,000-100,000 cells per coverslip in four-well dishes (fig. 7). The neuronal

cultures were maintained in a 5% CO2 incubator at 37 ̊C. After 24 hours the DMEM-FBS was replaced with 0.5 ml of Neurobasal medium supplemented with 2% B27 and 1% GlutaMAX. Half of this medium was replaced after 1 week. After 1-4 weeks of incubation transfected HEK293 cells were introduced to the neurons for the formation of neuronal- HEK293 co-cultures.

HEK293 cell transfection and seeding

HEK293 cells plated in 35 mm dishes with DMEM-FBS were transfected using the calcium phosphate-DNA co-precipitation protocol. For homomeric α1 and α3 GlyR expression, the total cDNA comprised of 0.5 μg of GlyR subunit of interest, 0.2 μg of Neuroligin 2A, 0.1 μg of gephyrin and 0.1 of μg GFP. After overnight incubation for 16-20 hours at 3% CO2, 37 ̊C, the cells were washed with PBS to terminate transfection, trypsinized and centrifuged, and then resuspended in 50 μl of the neurobasal medium from each well of the neuronal cultures. 50 μl of the transfected HEK293 cells suspension was then seeded onto the spinal neurons

and incubated overnight at 5% CO2, 37 ̊C for the formation of synapses (fig. 7). The co- cultures were then used for experiment for the next 1-3 days.

Fig. 7 Generating functional inhibitory synapses expressing GlyR subunit of interest. Section

28 highlighted in green shows the steps for primary neuronal culture preparation from E15 rat spinal cords. Section highlighted in blue shows the steps for HEK293 cells transfection and seeding onto neurons to form inhibitory synapses.

2.2.3 Patch-clamp electrophysiology

Patch-clamp electrophysiology is an effective tool to study ionic currents and channel activity in small mammalian cells (Hamill et al. 1981). All HEK293 cells expressing α1 and α3 GlyRs, and HEK293-neuronal co-cultures expressing homomeric and heteromeric GlyRs were subjected to patch-clamp electrophysiology in the whole cell configuration. Two types of experiments were carried out. Firstly, glycine concentration-responses and drug testing was done on isolated HEK293 cells. In these experiments glycine at different concentrations and test drugs were applied directly onto the recorded cell via gravity-fed plastic tubing. In a second set of experiments, spontaneously evoked IPSC events were recorded from HEK293 cells in co-culture with neurons. The effects of test drugs on IPSCs was determined by applying the drug directly onto the recorded cell. The extracellular solution used to perfuse the cells was composed of: 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, and 1 mM MgCl2, 10 mM HEPES / NaOH and 10 mM glucose (pH adjusted to 7.4 with NaOH). Harvard apparatus borosilicate standard wall capillaries with filament, 1.0 mm OD, 0.50 mm ID, 75 mm L were used as patch pipettes for experiments with both HEK293 cells and HEK293- neuronal co-cultures. The patch pipettes were fabricated using a Sutter Instruments P-97 Flaming/Brown type micropipette puller, giving pipettes with a tip resistance of 2-5 MΩ when filled with a CsCl intracellular pipette solution consisting of: 145 mM CsCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM HEPES and 10 mM EGTA (pH 7.4 adjusted with NaOH). The patch pipette was placed onto the cell membrane using a micromanipulator, and the parallel perfusion tubes were also controlled by a micromanipulator for solution application to the cell. Membrane currents were recorded while voltage was clamped at -40 mV to -70 mV using pCLAMP 10 software and analysed on Clampfit (Molecular Devices).

Whole cell patch-clamping

To record evoked or spontaneous macroscopic currents from ion channels present on the entire cell membrane, the whole cell voltage clamp electrophysiology was used (Fernandez et al. 1984). Glass pipettes with a 2-5 MΩ tip resistance were placed on top of the HEK293 cell surface and a high resistance seal, ideally of 1-10 Gigaohms referred to as a ‘gigaseal’, or typically a 200-500 MΩ seal, is formed after providing gentle suction through the pipette tip. 29 To break the seal and acquire the whole cell configuration, a current pulse of 10mV/100-200 μs was applied. Once the seal breaks the pipette’s intracellular solution become contiguous with the cell’s internal cytosol (fig. 8). Whole-cell currents were then measured at a clamped voltage of -40 mV for HEK293 cell experiments, and at -70mV for the HEK293-neuronal co- cultures.

Fig. 8 Diagrammatic representation of whole cell voltage clamp (patch-clamp) electrophysiology technique used for HEK293 cells expressing GlyR of interest.

Whole cell patch experiments for measuring glycine and drug dose response on GlyR of interest expressed in HEK293 cells is done by applying different concentrations to measure the glycine evoked deflection, and subsequent potentiations due to drug application. In case of the latter, the experiment is done on the HEK293 cell, which is expressing the GlyRs of interest and neuroligin 2A for synapse formation. The neighbouring spinal neurons with glycinergic presynaptic vesicles form synapses with the HEK293 cells and hence the whole cell patch facilitates the measurement of spontaneous IPCSs. No glycine application is required to evoke the synaptic events, and the drug can be applied for a duration of time to observe and record changes to the IPSC kinetics.

2.2.4 Data analysis

Glycine and HO1 concentration-responses

All results are expressed as mean ± SEM of six or more independent experiments. The

saturating current magnitude (Imax), half-maximal concentration (EC50) and Hill coefficient (nH) values were obtained using SigmaPlot (SigmaPlot 13.0) by fitting concentration- response data to the Hill equation, as follows:

30 1 =

1 + 𝐼𝐼 [ ] 𝑛𝑛 𝐼𝐼 𝑚𝑚𝑚𝑚𝑚𝑚 𝐸𝐸𝐸𝐸50 � � 𝐴𝐴 Where A is the concentration of the drug or ligand, and n is the Hill coefficient. One-way or two-way, as appropriate, ANOVA was performed to observe significance in differences

between the HO1 concentration- responses for both GlyRs α1 and α3 and to establish statistical validity of the data. Significance was represented with the value of p < 0.05.

IPSC kinetics

Spontaneous IPSCs develop rapidly (< 10 ms) and decay back to baseline with an exponential waveform (Legendre 2001). In addition to the current peak amplitude, it is important to analyse at the rise time and decay time of the IPSC event. For each GlyR subtype close to 100 events were recorded after applying extracellular solution (control) and then the drug, to 4 to 6 cells. For each cell, the events were averaged to get mean for peak amplitudes, 10-90% rise times and decay times. For multiple subtype comparisons, one-way and two-way ANOVAs, as appropriate, were employed. Significance was represented with the value of p < 0.05.

2.3 Results

2.3.1 Glycine concentration-responses and HO1 potentiation at GlyRs α1 and α3

The glycine concentration-response relationship was measured determined for the WT

homomeric α1 and α3 GlyRs in the whole-cell configuration, voltage clamped at -40mV. Peak

currents were recorded at 1 mM and 3 mM glycine for both α1 and α3 GlyRs, respectively.

The EC50 values of 320 ± 4 μM (nH = 2.2 ± 0.1, n = 5) and 32 ± 2 μM (nH = 1.9 ± 0.8, n = 5)

were determined for WT α3 and α1 GlyRs, respectively. The EC50 values for both receptors

were similar to previously reported for homomeric α1 and α3 GlyRs (Islam and Lynch 2012).

An EC20 concentration of glycine was used determine the potentiating effects of HO1 at

varying concentrations at homomeric α1 and α3 GlyRs. The previously calculated EC50

values for each receptor rendered their subsequent EC20 values using the equation:

1 F H EC = . EC F 100-F 50 � �

31 The EC20 for the homomeric α1 GlyR was taken as 15 μM, whereas for α3 GlyR it was taken as

150 μM (Fig. 9A). The response was plotted as the percentage of potentiation, where the EC20 response was set to 100% (Fig. 9B). For WT α3 GlyR, there was potentiation observed between 20-70%, whereas for α1 GlyR only about 10-20% potentiation was observed.

Fig. 9 HO1 concentration response with α1 GlyR vs α3 GlyR in HEK293 cells. Panel A shows EC20 glycine evoked current traces with different concentrations of HO1, on α1 and α3 GlyRs. Panel B shows percentage of potentiation averaged for increasing HO1 concentrations on both α1 and α3 GlyRs (n=6), fitted to the Hill equation.

2.3.2 HO1 potentiation on α1 and α3 GlyRs in inhibitory artificial synapses

The HEK293-neuronal co-cultures expressing homomeric α1 and α3 GlyRs showed IPSC characteristics similar to those that have been previously observed in native synapses, expressing similar GlyR subunits. The 10-90% rise time and decay time constants observed for both GlyR subtypes were similar to those previously observed using the same co-culture technique (Zhang et al. 2015; Dixon et al. 2015). The recorded cells were perfused with HO1 at 10 nm concentration for 5-10 minutes to observe changes in the IPSC kinetics and frequency of events.

Fig. 10 Glycinergic IPSCs produced by inhibitory synapses expressing α1 and α3 GlyR,, with current traces at different timescales during control (black) and 10 nM HO1 application for α3 GlyR (red), and for α1 GlyR (green).

For α3 GlyR, the IPSC traces observed under HO1 application had significantly higher peak amplitudes and slower decay time constants than control, suggesting that HO1 potentiates

IPSCs mediated by α3 GlyR, as observed in experiments with HEK293 cells expressing GlyR

α3. Similar to experiments in HEK293 cells, no significant changes were observed in the peak amplitudes, 10-90% rise time and decay time of IPSCs from cells expressing α1 GlyR.

Fig. 11 IPSC kinetics for HO1 application on inhibitory synapses expressing α1 and α3 GlyR. A- C show peak amplitude, rise time and decay time for α3 GlyR. D-F show peak amplitude, rise time and decay time for α1 GlyR. HO1 significantly potentiated peak amplitude, rise time and decay time for α3 GlyR selectively while showing no potentiation for α1 GlyR (n=6, paired student’s t test, *p < 0.05, **p < 0.01, ***p < 0.001).

2.3.3 Discussion

Recent studies have shown the direct involvement of the α3 subunit containing GlyRs in inflammatory pain. GlyRs, particularly α3 GlyR, are involved in mediating inhibitory neurotransmission in the nociceptive sensory neurons of the spinal cord dorsal horn (Harvey et al. 2004). This glycinergic neurotransmission is effectively disrupted when Prostaglandin

E2 (PGE2) mediated postsynaptic phosphorylation of α3 GlyR disinhibits the nociceptive

sensory neurotransmission and causes increased pain sensitization. This PGE2 mediated phosphorylation is, however, only specific for α3 GlyR, and does not affect α1 GlyR as it lacks the sites for phosphorylation. In addition to that, the localisation of the α3 GlyR specifically in the dorsal horn of spinal cord also makes it an effective target for potentiation

34 by small therapeutic drugs and analgesics. Therefore, drugs that target GlyRs which contain the α3 subunit are of key interest. Previously characterized small drugs have efficaciously potentiated currents in α3 GlyR and show successful analgesia in animal models too (Xiong et al. 2011; Huang et al. 2017). However, lack of subtype selectivity can produce off target effects on other GlyR isoforms. In addition to this, achieving strong potentiation at low concentrations is also of interest in terms of determining the drug’s overall potency.

After rigorous screening of a library of 120 compounds, a lead hit was identified, designated as HO1, which strongly potentiated α3 GlyR without producing any significant potentiation for α1 GlyR. In experiments with separate HEK293 cell cultures expressing α3 GlyR and α1 GlyR transfected using the same protocols, cells were washed with different concentrations of HO1 (0.01 to 100 nM) + glycine EC20, to observe any potentiation of the glycine-induced current. With 100 % potentiation being the glycine EC20 current α3 GlyR were potentiated by 70-80%, whereas up to a 20% of potentiation was observed for α1 GlyR. Peak potentiation was observed at 1 nM and 10 nM concentrations with no increasing effect seen at higher concentrations. The drug potentiated reversibly as the effect diminished as soon as the cell

was washed with control solution and reapplication of EC20 glycine produced the original

EC20 response. The drug does potentiate in a concentration-dependent manner, starting at a concentration of 0.1 nM and saturating at 10-100 nM. The dose response curves also confirm the drug’s dose dependent activity for both α3 GlyR and α1 GlyR. For α1 GlyR, HO1 potentiates at a concentration of 0.1 nM and the highest potentiation is observed at 10 nM, much similar to α3 GlyR though significantly less.

Considering the limitations of ion channel expression in HEK293 cells, since most GlyRs are present on inhibitory synapses, makes it important to see the effect of the drug on GlyRs in functional synapses and their native neuronal environment. An HEK293 cell and spinal neuronal co-culture technique was recently developed in our lab that allows synapse formation between HEK293 cells expressing GlyR subunit of interest, and neighboring neurons (Dixon et al. 2015). This is aided by neuroligin 2A, which is a trans-synaptic protein that facilitates the formation of synapses. The effect of a drug at potentiating IPSCs is different to what we observe in HEK293 cell experiments. A drug may potentiate the IPSCs by potentiating individual kinetic parameters such as peak amplitudes, 10-90% rise times and decay times. 10 nM HO1 application at α1 GlyR and α3 GlyR potentiated IPSC kinetics as predicted from the HEK293 experiments. For α3 GlyR there was significant potentiation of the IPSC peak amplitudes, which increased almost 1.5-fold much like the ~60-80%

35 potentiation of glycine EC20 induced currents in HEK293 cells. No significant potentiation of the peak amplitudes for IPSCs mediated by α1 GlyR were observed on application of 10 nM HO1. This shows that while HO1 might bind and mildly potentiate evoked currents from individually expressing α1 GlyR in HEK293 cells, it does not seem to have a similar effect on synaptic α1 GlyR, suggesting that physiologically, HO1 might not potentiate α1 GlyR currents significantly. However, there are other IPSC kinetic parameters to consider, such as IPSC 10-90% rise and decay times. Keeping this in mind, the effect of HO1 on increasing the 10-90% rise times and decay times were observed for both α1 GlyR and α3 GlyR. The rise time was significantly increased for α3 GlyR on application of 10 nM HO1, suggesting that the drug may enhance agonist activated opening of the channel. No significant increase in rise time was observed for α1 GlyR on HO1 application. As for decay times, HO1 application significantly slowed down the decay of the glycinergic IPSCs in α3 GlyR, as compared to no significant potentiation in α1 GlyR, again suggesting the selective activity of the drug.

HO1 was also applied to GlyR α1 and GlyR α3 expressing in HEK293 cells without glycine or any primary ligands, to observe any direct agonist activity by the drug. No direct agonist activity was observed at any concentration of HO1 from 0.01 nM to 100 nM, suggesting that HO1 is an allosteric modulator, only.

36 2.4 References

Acuna, MA et al. 2016, ‘Phosphorylation state–dependent modulation of spinal glycine receptors alleviates inflammatory pain’. The Journal of Clinical Investigation, vol. 126, pp. 2547–2560.

Balansa, W., et al. Ircinialactams: subunit-selective glycine receptor modulators from Australian sponges of the family Irciniidae. Bioorganic and Medicinal Chemistry 18, 2912-9 (2010).

Balansa, W., et al. Sesterterpene glycinyl-lactams: a new class of glycine receptor modulator from Australian marine sponges of the genus Psammocinia. Organic and Biomolecular Chemistry 11, 4695-701 (2013)

Balansa, W., et al. Australian marine sponge alkaloids as a new class of glycine-gated chloride channel receptor modulator. Bioorganic and Medicinal Chemistry 21, 4420-5 (2013).

Dixon, CL, Zhang, Y, Lynch, JW 2005, ‘Generation of Functional Inhibitory Synapses Incorporating Defined Combinations of GABA(A) or Glycine Receptor Subunits’, Frontiers in Molecular Neuroscience, vol. 8, no. 80.

Fernandez, JM, Fox, AP, Krasne, S, ‘Membrane Patches and Whole-Cell Membranes: a Comparison of Electrical Properties in Rat Clonal Pituitary (GH3) Cells’, The Journal of Physiology, vol. 356, no. 1, 1984, pp. 565–585.

Graham, F, Smiley, J, Russell, W, Nairn, RJ 1977, ‘Characteristics of a Human Cell Line Transformed by DNA from Human Adenovirus Type 5’, Journal of General Virology, vol. 36, no. 1, pp. 59-72.

Hamill, OP, Marty, A, Neher, E, Sakmann, B, Sigworth FJ, ‘Improved Patch-Clamp Techniques for High-Resolution Current Recording from Cells and Cell-Free Membrane Patches’, Pflügers Archiv - European Journal of Physiology, vol. 391, no. 2, 1981, pp. 85– 100.

37 Harvey, RJ et al 2004, ‘GlyR α3: An Essential Target for Spinal PGE2-Mediated Inflammatory Pain Sensitization’, Science, vol. 304, pp. 884-887.

Huang, X et al. 2017, ‘Crystal structures of human glycine receptor α3 bound to a novel class of analgesic potentiators’, Nature, vol. 24, pp. 108-116.

Islam, R and Lynch, JW 2012, ‘Mechanism of action of the insecticides, lindane and fipronil, on glycine receptor chloride channels’, British Journal of Pharmacology, vol. 165, pp. 2707- 20.

Kingston, RE, Chen, CA, Okayama, H 2003, ‘Calcium Phosphate Transfection’, Current Protocols in Cell Biology, vol. 19, no. 1.

Legendre, P 2001, ‘The glycinergic inhibitory synapse. Cellular and Molecular Life Sciences’, vol. 58, pp. 560–593.

Lynch, JW and Callister, RJ 2006, ‘Glycine receptors: a new therapeutic target in pain pathways’, Current Opinion in Investigational Drugs, vol. 7, pp. 48-53.

Phillips, GJ 2001, ‘Green Fluorescent Protein-- a Bright Idea for the Study of Bacterial Protein Localization’, FEMS Microbiology Letters, vol. 204, no. 1, pp. 9–18.

Thomas, P, and Smart, TG 2005, ‘HEK293 Cell Line: A Vehicle for the Expression of Recombinant Proteins’, Journal of Pharmacological and Toxicological Methods, vol. 51, no. 3, pp. 187–200.

Zhang, Y, Dixon CL, Keramidas, A, Lynch, JW 2015, ‘Functional Reconstitution of Glycinergic Synapses Incorporating Defined Glycine Receptor Subunit Combinations’, Neuropharmacology, vol. 89, pp. 391–397.

38 Zeilhofer, HU 2005, ‘The Glycinergic Control of Spinal Pain Processing’, Cellular and Molecular Life Sciences, vol. 62, no. 18, pp. 2027-2035.

Chapter 3 Effect of HO1 on glycine evoked currents in wild type (WT) and mutated α3 GlyR

40 3.1 Background Elucidating the binding mechanism and site of a drug on a receptor is crucial to understanding its functional effects. Recent identification of GlyR as a target for clinically relevant compounds has also helped understand the overall pharmacology of the ion channels. Newly available crystal structures of GlyRs have also been aided with the understanding of critical binding sites for compounds, such as the strychnine (Huang et al. 2015). Co-crystallization of ligands such as agonists, antagonists and even co-agonists have also allowed for the structures to be studied in their closed and open conformations and understand their molecular determinants as well (Huang et al. 2017; Du et al. 2015). The structure of the ligand is another important determinant of ligand-receptor binding characteristics, due to side chain or chemical group interactions with the amino acids constituting the receptor binding site. Changes in such amino acid side chains or polarity have had direct effects on the binding and efficacy of important chemical compounds, such as alcohols (Mascia et al. 1998; Wick et al; 1998). Other amino acids that bear close structural similarity to glycine have been shown to act as full or partial agonists for GlyRs though with less potency as compared to glycine (Bormann et al. 1996; Lynch et al. 1997). However, this affirms that certain chemical constituents of a ligand are crucial for its effect on structural conformation and subsequent change in the function of the ion channel. In addition, amino acid mutations in GlyRs are attributed as causes of severe diseases such as hyperekplexia (Harvey et al. 2008; Langosch et al. 1994; Shiang et al. 1993), whereas other biochemical changes to amino acids are also shown to be disease causing, such as the phosphorylation of a critical serine residue in α3 GlyR is the cause of chronic inflammatory pain (Harvey et al. 2004). α1 GlyR mutations not only increase the potency of regular agonists but also enable the channel to fully activate due to partial agonists alone (Rajendra et al.1995). With the help of such evidence, allosteric modulation and hence the design and synthesis of subtype selective potentiators has significantly improved in the last decade.

In order to map the binding site of HO1 on α3 GlyR, its binding site was initially modelled via molecular docking. The subtype selectivity of the HO1 molecule for α3 GlyR over α1 GlyR was a strong reason for its selection as a lead molecule for investigating its potentiating properties. The current chapter details the experiments designed to provide insight into the binding of the HO1 molecule at α3 GlyR. Predicting the preferred binding site of HO1 for α3 GlyR and mutating it should help determine the functional importance of the site and its residues. A clearer idea of the drug’s selectivity for α3 GlyR over α1 GlyR can be deduced

41 from electrophysiological analysis of the mutants and their impact on the channel function as well.

3.2 Materials and Methods

3.2.1 Docking studies

To model the binding site of the HO1 molecule with α3 GlyR, in silico docking studies were performed using the AutoDock docking suite. The crystal structure for the human α3 GlyR was used (pdb no. 5TIO), which was obtained from the Protein Data Bank (Berman et al. 2000). The structure for HO1 molecule was available as it had been previously generated in the lab after its characterization. The docking protocol involved the introduction of partial charges in the protein crystal structure of α3 GlyR for it to facilitate binding to HO1 using two docking programs, Autodock 4.2 (Morris et al. 2009) and Autodock Vina (Trott and Olson 2010). The binding pose count was set to 100 and the search space for the drug to find a suitable binding site on the receptor was restricted to 3 different controls: just the known ligand binding domain in the first docking, to the whole receptor in the second docking to validate first docking results, and then lastly, to entire receptor for the ligand to bind anywhere, with subunits A, B and C co-crystallized with other ligands in pdb structure 5TIO to further validate HO1’s preferred site of binding to the receptor (fig. 6). Due to presence of rotatable carbon-carbon bonds, the ligand can acquire many poses in the binding conformation, and each pose would suggest a different interaction between the amino acids of the receptor and the atoms of the drug. The resulting complexes with the drug docked into its preferred binding site were analysed further using MGLTools GUI (Morris et al. 2009) from the Autodock suite, which ranked each protein-ligand complex run based on its binding affinity, free energy and ligand efficacy. This allowed for the best model with the least free energies and the highest ligand efficacy and binding affinity to be selected for further analysis. The residues in the receptor’s binding pocket that formed conventional hydrogen bonds, water aided hydrogen bonds, electrostatic and hydrophobic interactions with the HO1 molecule and its moieties were further analysed and visualised using PyMOL (The PyMOL Molecular Graphics System, Version 1.8 Schrödinger, LLC) and Discovery Studio molecular visualization systems (Dassault Systèmes BIOVIA, Discovery Studio Modelling Environment, 2017).

Fig. 12 Docking protocol included 3 controls for ligand binding to the receptor; the known ligand binding domain in the first docking (green), to the whole receptor in the second docking (blue), to entire receptor for the ligand to bind anywhere (pink), with subunits A, B and C co-crystallized with other ligands (yellow dots), PDB ID: 5TIO.

3.2.2 Site Directed Mutagenesis

The site directed mutagenesis was performed using the Phusion High-Fidelity DNA polymerase protocol (New England BioLabs) for five different mutations. The double stranded DNA template used was the plasmid DNA for rat α3L GlyR subunits provided by Prof. Robert J Harvey (University College, London) to our lab previously. The α3L construct was used in 4 mutagenesis experiments to introduce mutations, R27A, R29A, F32A and D86A, numbered according to the original α3L sequence including the signal peptide. The mutagenesis reaction for each mutant was then transformed on Lysogeny Broth agar (LB- agar) plates with ampicillin and left to incubate for 16 hours at 37 ̊C.

3.2.3 Plasmid Preparation

Plasmids are usually purified using liquid bacterial cultures, such as E. coli, since almost all bacterial plasmid vectors contain single or multiple antibiotic resistance genes that also serve as selectable markers in their growth (Hershfield et al. 1974; Sutcliffe 1978). After successful colony formations on the LB-agar plates, single colonies were selected and cultured in separate tubes in LB broth with ampicillin and again left to incubate for 16 hours at 37 ̊C. DNA extraction and purification for mutant positive colonies was done using the NucleoSpin® Plasmid (NoLid) high copy miniprep plasmid kit and protocol (Macherey- Nagel) for a DNA yield of about 10-50 μg. After the DNA was purified, the samples were

43 prepared for sequencing using the BigDye® Terminator v1.1 Cycle Sequencing Kit (ThermoFisher) to check for successful mutations. The mutant DNA samples were then labelled accordingly and stored at -20 .

3.2.4 HEK293 Cell Culture and transfection

HEK293 cell culture was maintained as described in detail in the previous chapter and the α3

GlyR WT and mutant DNA were transfected in separate cultures using the CaCl2 protocol, as detailed previously.

3.2.5 Electrophysiological studies

All HEK293 cells expressing WT α3 GlyRs and mutants were subjected to patch-clamp in the whole cell configuration to measure glycine evoked current activity at different concentrations and HO1 dose response studies as described in the previous chapter. Voltage was clamped at -40 mV for all the dose responses. All recordings were done using pCLAMP 10 software and analysed on Clampfit (Molecular Devices).

3.2.6 Data Analysis

All results are expressed in the form of mean ± SEM of three or more independent experiments. The saturating current magnitude (Imax), half-maximal concentration (EC50) and Hill coefficient (nH) values were calculated using the Hill equation for activation by glycine using SigmaPlot (SigmaPlot 13.0). A one way or two way ANOVA, as appropriate, was performed to compare the EC50 calculated for α3 GlyR WT and mutants. Statistical significance was set at p < 0.05.

3.3 Results 3.3.1 Predicted Binding site for HO1

In previous experiments done in our lab, it was believed that HO1 possibly binds to the glycine binding site due to the presence of a glycinyl lactam ring on one of its ends. However, newer docking studies using Autodock 4.2 and Autodock Vina detailed in this chapter revealed that the HO1 molecule binds to a novel binding site closely adjacent to the orthosteric glycine binding site.

This binding site was consistently observed in all 3 docking experiments with their respective controls and search space restrictions described in the previous section. The first docking experiment restricted the drug’s search space on the receptor to the known ligand binding site

44 in the extracellular domain. The docking runs were analysed and the complex with least free energy and highest ligand efficacy was selected. HO1 docked to a novel site that has been shown for a previously characterized potentiator for α3 GlyR (Huang et al. 2017). This result was further validated by doing two more successive dockings; first with the drug’s search space given to the entire receptor, and second with two consecutive unbound subunits with the rest of the subunits bound to co-crystallized ligands in their ligand binding domains. Both dockings showed that the HO1 molecule occupied the same binding site on the α3 GlyR just as the first one (fig. 13A), suggesting a strong possibility that this would be the drug’s preferred binding site.

Fig. 13 Predicted binding site of HO1 for α3 GlyR. Panel A shows the HO1 drug (red) docked into the ligand binding site between principal (+) and complimentary (-) α3 subunits. Panel B represents a 2D schematic illustration of the types of interactions between the binding residues and the HO1 drug, where each type of bond is represented by a different colour according to the legend. The residues are numbered according to the sequence of the α3 GlyR structure (PDB ID: 5TIO).

Based on this analysis, a molecular model of the α3 GlyR with HO1 docked to its binding site was created, and the residues observed in this binding site were assessed as possible candidates for undergoing site directed mutagenesis (Fig. 13B). Any residue involved in the binding site that is also a critical site of action for glycine or other allosteric modulators such as Zn2+ is not ideal for mutation since it will affect the channel’s activity via mechanisms independent of HO1 binding. This is important to keep in mind since any mutation that

45 renders the ion channel non-functional is not suitable for any further electrophysiological assays. After eliminating such residues, the remaining ones that also actively formed the HO1 binding site on α3 GlyR were, R27A, R29A, F32A and D86A. The docking showed that the modelled binding site for HO1 was adjacent to the glycine binding site (fig. 14), similar to the drug AM-3607 that has been reported previously, which is shown to bind at a distance of ~10 Å between its benzodioxole moiety and the glycine molecule.

Fig. 14 Comparison of the binding site for neurotransmitter Glycine and HO1 on α3 GlyR. Panel A shows the Glycine binding site in the extracellular domain. Panel B shows the HO1 bound to a site closely adjacent to the glycine binding site (PDB ID: 5TIO).

3.3.2 Glycine concentration response in α3 GlyR WT vs mutants

The glycine concentration-response relationship was measured for the wild type homomeric

α3 and the mutant R27A, R29A, F32A and D86A α3 GlyRs. Peak currents were recorded at between 1 mM and 10 mM glycine for both WT and mutant. The EC50 values of 320 ± 4 μM

(nH = 2.2 ± 0.1, n = 3) was obtained for WT, which was close to the previously observed value for α3 GlyR (Islam and Lynch 2012). The EC50 values of the mutants (n = 3) are shown in table 1, and sample current traces of WT and each mutant GlyR in response to glycine concentrations of 3 mM are shown in Fig 15A.

Table 1 The Glycine EC50 and Hill slope values observed for all α3 GlyR mutants

Mutant Glycine EC50 (µM) Hill coefficient (nH) D86A 1264 ± 19 1.1 ± 0.1 R27A 2858 ± 27 1.3 ± 0.3 F32A 2958 ± 34 1.7 ± 0.3 R29A 3431 ± 31 2.0 ± 0.4

These data demonstrate a drastic change in the channel’s sensitivity to glycine due to the mutation. This shift in sensitivity to the neurotransmitter glycine and the subsequently evoked current at even higher concentrations than that of the WT are apparent in the rightward shift of the dose response curve for all the mutants compared to WT (fig. 15B).

Fig. 15 Glycine dose response and EC50 comparisons for WT and mutants. Panel A shows current traces evoked for glycine concentration of 3mM for α3 GlyR WT and all mutants. Panel B shows the Glycine dose response for WT and mutants R27A, R29A, F32A and D86A stably expressed in HEK293 cells. The rightward shift of the dose response for each mutant (n = 3) shows a reduction in glycine

sensitivity. Panel B shows the EC50 for WT GlyR α3 (left, 320 ± 4 μM) compared to EC50 for each mutant. The asterisk denotes a statistically significant difference compared to WT (one way ANOVA, *p < 0.05, **p<0.01, ***p<0.001)

R29A exhibited the highest EC50 value and the smallest glycine evoked current, with no saturating peaks observed even at a10 mM concentration (table. 1). F32A and R27A had

slightly similar EC50 values and gave peak currents at 10 mM, but still a much smaller response to concentrations between 1 mM and 3 mM. D86A had a glycine dose response closest to the WT, with peak currents observed at 3 mM and 10 mM. Three out of four 47 mutants exhibited significantly different glycine evoked currents from the WT (fig 15B). Panel A shows the glycine dose response for WT and mutants R27A, R29A, F32A and D86A using HEK293 stable cell lines. The rightward shift of the dose response for each mutant

demonstrates a decrease in glycine potency for the mutants. Panel B shows the EC50 for WT

(left, 320 ± 4 µM) compared to EC50 for each mutant.

3.3.3 HO1 concentration response in α3 GlyR WT vs mutants

The glycine concentration response for the mutants revealed that mutation did not abolish function of the receptor, and hence makes the mutant receptors suitable candidates to study the effect of HO1. The WT and mutant R27A, R29A, F32A and D86A α3 GlyR were again

subjected to patch-clamp recordings. An EC20 concentration of glycine was used to administer to the cell along with the different concentrations of HO1, to see the effect of the drug on each mutant.

To validate the computational model of the binding site of HO1 on α3 GlyR, the HO1 dose response for each mutant was measured and compared to that of WT. No significant potentiation was observed for any of the mutant receptors (fig. 16). The response was plotted

as the percentage of potentiation, where the EC20 response was set to 100%. For WT α3 GlyRs, there was significant potentiation observed between 160-180%, which has been observed as discussed in the previous chapter. In addition, for all mutant GlyRs a decline in

EC20 current potentiation was also observed with increasing doses of HO1, which may not only suggest that each of the mutations impaired HO1 binding, but also that the mutated residues might also be important for the binding of glycine.

Fig. 16 HO1 concentration response for WT α3 GlyR compared to that of the mutants. Due to

differing values of glycine concentrations evoking EC20 currents in each mutant, % Potentiation was averaged for increasing compound concentrations across at least 6 cells, and the averaged group data

was fitted with the Hill equation. Glycine (EC20) is considered as 100 %. No significant potentiation was observed across any of the mutants above the EC20 current in comparison to the WT (one way ANOVA, *p < 0.05, **p<0.01, ***p<0.001).

The close proximity of the glycine binding site to the predicted HO1 binding site, may suggest the involvement of the site itself in potentiating the effect of glycine. The site for HO1 binding that was modelled and studied here may have properties of a site involved in allosteric regulation, considering the drastic effects of its mutation on both glycine sensitivity and HO1 potentiation.

3.3.4 Discussion

Several mutations to the GlyR have been shown to alter the sensitivity of the receptor to glycine. It was therefore important to see that the mutant α3 GlyR retained channel functionality. The resultant mutant-mediated currents reveal receptor expression and functional changes that point mutations might confer on the receptors. For instance, point

49 mutations can impair surface expression (Bode and Lynch, 2014) or assembly of subunits (Kuhse et al., 1993) or their release from the endoplasmic reticulum (Griffon et al., 1999). On application of concentrations of glycine that are saturating for the α3 GlyR WT, different sized current amplitudes were observed in the mutant receptors, suggesting that they are functional. However, the drastically reduced response to glycine in the mutants showed a change in agonist sensitivity. The binding site model showed that HO1 binds in a site relatively close to the glycine binding site. The site has also been shown to be the binding site for a previously characterized novel analgesic that was co-crystallized in the structure of α3 GlyR along with glycine. The binding site for HO1, being in close proximity to glycine binding site, might be sensitive to conformational changes that are induced upon glycine binding. However, this also means that mutations in this binding site will alter glycine affinity as well, since any structural changes brought on by the mutation will have effects on the nearby glycine binding site. Considering how every mutation did not have the same shift in sensitivity to glycine, it is important to note that due to the individual amino acid structures and their contribution to the overall protein stability, each mutation had different effects on channel’s in affinity to glycine. Substituting an aromatic and hydrophobic phenylalanine at position 32 with an aliphatic alanine may affect the channel’s pharmacological properties,

consistent with the observed 10-fold increase in glycine EC50. Though it is hard to draw any conclusions about the structural changes due to point substitutions, the channel activity and agonist response can be an indication of some conformational changes. However, for now it is important to recognize the possibility that the HO1 binding site residues may have a functionally important role in glycine activity due to their close proximity to its binding site.

Since the mutants had reduced sensitivity to glycine, HO1 was applied with their respective

glycine EC20s. For all mutants, no potentiation was observed on application of HO1 at any concentration.

Considering that HO1 forms a strong pi-alkyl bond with phenylalanine at position 32 in α3 GlyR and multiple hydrogen bonds with the arginine at position 29, it is very indicative of their importance, since these two mutations result in the highest shift in glycine sensitivity. In addition to the drastic effect on glycine sensitivity and lack of potentiation, a considerable

decline in EC20 currents was observed at increasing doses of HO1. This reinforces the idea that the two binding sites are possibly functionally coupled.

50 3.4 References

Berman, HM, Westbrook, J, Feng, Z, Gilliland, G, Bhat, TN, Weissig, H, Shindyalov, IN, Bourne, PE 2000, ‘The Protein Data Bank’, Nucleic Acids Research, vol. 28, pp. 235-242.

Bode, A and Lynch, JW 2014, ‘The impact of human hyperekplexia mutations on glycine receptor structure and function’, Molecular Brain, vol. 7, no. 2.

Bormann, J, Rundström, N, Betz, H, Langosch, D 1994, ‘Residues within Transmembrane Segment M2 Determine Chloride Conductance of Glycine Receptor Homo- and Hetero- oligomers’, The EMBO Journal, vol. 13, no. 6, pp. 1493.

Du, J et al. 2015, ‘Glycine receptor mechanism illuminated by electron cryo-microscopy’, Nature, vol. 526, pp. 224-229.

Griffon, N, Buttner, C, Nicke, A, Kuhse, J, Schmalzing, G, Betz H 1999, ‘Molecular determinants of glycine receptor subunit assembly’, EMBO Journal, vol. 18, pp. 4711–4721.

Harvey, RJ et al 2004, ‘GlyR α3: An Essential Target for Spinal PGE2-Mediated Inflammatory Pain Sensitization’, Science, vol. 304, pp. 884-887.

Harvey, RJ, Topf, M, Harvey, K, Rees, MI 2008, ‘The Genetics of Hyperekplexia: More than Startle!’, Trends in Genetics, vol. 24, no. 9, pp. 439-47.

Hershfield, V, Boyer, HW, Yanofsky, C, Lovett, MA, Helinski, DR 1974, ‘Plasmid ColE1 as a Molecular Vehicle for Cloning and Amplification of DNA’, Proceedings of the National Academy of Sciences, vol. 71 no. 9, pp. 3455-3459.

Huang, X et al. 2015, ‘Crystal structure of human glycine receptor α3 bound to antagonist strychnine’, Nature, vol. 527, pp. 277-282.

Huang, X et al. 2017, ‘Crystal structures of human glycine receptor α3 bound to a novel class of analgesic potentiators’, Nature, vol. 24, pp. 108-116.

Islam, R and Lynch, JW 2012, ‘Mechanism of action of the insecticides, lindane and fipronil, on glycine receptor chloride channels’, British Journal of Pharmacology, vol. 165, pp. 2707- 20.

51 Kuhse, J, Laube, B, Magalei, D, Betz, H 1993, ‘Assembly of the inhibitory glycine receptor: Identification of amino acid sequence motifs governing subunit stoichiometry’, Neuron, vol. 11, no. 6, pp. 1049-1056.

Langosch D, Laube B, Rundstrom N, Schmieden V, Bormann J, and Betz H. Decreased agonist affinity and chloride conductance of mutant glycine receptors associated with human hereditary hyperekplexia. EMBO Journal, vol. 13, pp. 4223–4228.

Lynch JW, Rajendra S, Pierce KD, Handford CA, Barry PH, and Schofield PR. Identification of intracellular and extracellular domains mediating signal transduction in the inhibitory glycine receptor chloride channel. EMBO Journal 16: 110–120, 1997.

Mascia, MP, Machu, TK, Harris, RA 1996, ‘Enhancement of homomeric glycine receptor function by long-chain alcohols and anaesthetics’, British Journal of Pharmacology, vol. 119, pp. 1331–1336.

Morris, GM, Huey, R, Lindstrom, W, Sanner, MF, Belew, RK, Goodsell, DS, Olson, AJ 2009, ‘Autodock4 and AutoDockTools4: automated docking with selective receptor flexibility’, Journal of Computational Chemistry, vol. 16, pp. 2785-2791.

Rajendra, S et al. 1995, ‘The unique extracellular disulfide loop of the glycine receptor is a principal ligand binding element’, The EMBO Journal, vol. 14, pp. 2987-2998.

Sutcliffe, JG 1978, ‘Nucleotide sequence of the ampicillin resistance gene of Escherichia coli plasmid pBR322’, Proceedings of the National Academy of Sciences, vol. 75, no. 8, pp. 3737- 3741.

Trott, O and Olson, AJ 2010, ‘AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization and multithreading’, Journal of Computational Chemistry, vol. 31, pp. 455-461.

Chapter 4

The potentiating effects of cannabinoid derivatives at α1 and α3 GlyRs

53 4.1 Background

Recent studies have shown that certain cannabinoids and their derivatives have a strong effect on GlyRs and produce analgesia in animal models (Xiong et al. 2011; Xiong et al. 2012; Lu et al. 2018). This has led to a growing interest for α1 and α3 GlyRs as potential targets for cannabinoids, with recent data showing that potentiation of both α1 and α3 GlyRs results in cannabinoid-induced analgesia (Hejazi et al. 2006; Yang et al,. 2008; Ahrens et al,. 2009a, b; Demir et al. 2009; Foadi et al. 2010; Xiong et al, 2011, 2012; Yevenes and Zeilhofer. 2011a, b). However, due to lack of receptor specificity, it has been difficult to elucidate the involvement of either α1 or α3 GlyRs solely, or together, in the resultant cannabinoid- induced analgesia.

Recently, our lab was provided with multiple cannabinoid derived compounds for electrophysiological studies on α1 and α3 GlyRs. Since native GlyRs are predominantly expressed as heteromers of α and β subunits, it was important to examine the effects of these compounds on homomeric and heteromeric receptors. Two of these drugs were derived from the cannabinoid “cannabidiol” or CBD, designated as 5-desoxy-CBD and 1, 2- dimethylheptyl-desoxy CBD (fig. 17). These CBD derivatives were tested at α1, α3, α1β and α3β GlyRs to measure effects on IPSC kinetics.

Fig. 17 Structures for cannabidiol and its derivatives 5-Desoxy-CBD (1803), and 1,2-dimethylheptyl- desoxy CBD (1816).

4.2 Materials and methods

Functional inhibitory “artificial synapses”

54 IPSCs were prepared and recorded as previously described for cells expressing homomeric α1 and α3 or heteromeric α1β and α3β GlyRs.

Electrophysiological studies

Whole cell patch-clamping was performed as detailed previously, to record IPSCs from homomeric and heteromeric GlyRs before and during application of drugs. Voltage was clamped at -70 mV during all the experiments, and patched cells were perfused with control (ringer) for 5 minutes and then with the drug for 10 minutes before washing again with control. All recordings were done using pCLAMP 10 software and analysed on Clampfit (Molecular Devices).

Data Analysis

All results are expressed in the form of mean ± SEM of six or more independent experiments. The peak amplitides (pA), 10-90% rise times (ms) and decay times (ms) were calculated during IPSC analysis on Clampfit for each recording. One way or two way ANOVA, as appropriate, were done for comparing the effect of the drug between multiple receptors, and to determine statistical significance.

4.3 Results

4.3.1 5-desoxy-CBD

5-desoxy-CBD, referred to as compound 1803, has an oxygen removed on the 5th carbon in the middle ring of CBD. The drug had shown strong potentiation at homomeric GlyRs α1 and

α3 in HEK293 cells in experiments conducted previously in the lab. To determine whether this structural change has an effect on glycinergic IPSCs, cells were washed with 1803 at a concentration of 10 μM, for 5-10 minutes after washing with extracellular solution (control). The resultant IPSCs recorded for each receptor (n=6), were analysed to determine changes in IPSC kinetics from those mediated by GlyRs without drug (fig. 18).

Fig. 18 Comparison of spontaneous glycinergic IPSCs mediated by α1, α3, α1β and α3β GlyRs during drug free control (black) and 1803 (blue). Panel A shows sample traces of the voltage clamp recordings done for α1, α3, α1β and α3β GlyRs at 20 second timescale. Panel B shows the mean IPSC waveforms for drug free control and 1803 for all 4 receptors, not normalized for α1 and α1β GlyR to show significant increase in amplitude, each averaged from >100 events from a single cell.

For α1 GlyR a significant increase in the peak amplitude of the current was observed after 1803 application (1153 ± 260 pA) as compared to control (939 ± 251 pA). A significantly slower 10-90% rise time (26 ± 3 ms) and slower decay rates (39 ± 8 ms), were observed compared to drug free control conditions, which were 7 ± 1 ms and 9 ± 1 ms, respectively. Similarly, α1β GlyR also showed a significant increase in the peak amplitude (855 ± 116 pA), rise time (18 ± 2 ms) and decay rate (26 ± 6 ms) compared to control (612 ± 93 pA, 6 ± 0.6 ms, 14 ± 3 ms, respectively). α3 GlyR showed a significant increase in the rise time (33 ± 4 ms) and decay rate (52 ± 9 ms) compared to control (12 ± 4 ms, 26 ± 6 ms, respectively) but no effect on the overall size of the current. α3β GlyR showed no significant changes in the IPSC kinetics as compared to the drug free control (fig 19). All IPSC kinetics observed for

56 α1, α3, α1β and α3β GlyRs at drug free control were similar to previously reported (Zhang et al. 2015; Dixon et al. 2015).

Fig. 19 The mean values for the peak amplitudes,10–90% rise times and decay time constants of IPSCs recorded for cannabinoid 1803 from α1, α3, α1β and α3β GlyRs, compared to the drug free control (one way ANOVA, *p < 0.05, **p<0.01, ***p<0.001).

Overall, the analysis showed that 1803, or 5-Desoxy-CBD produces greater potentiation at α1 –containing GlyRs than at α3 –containing GlyRs.

4.3.2 1,2-dimethylheptyl-desoxy CBD

1,2-dimethylheptyl-desoxy CBD, referred to as compound 1816, also has an oxygen removed from the 5th carbon in the middle ring of its parent structure, in addition to having two methyl groups added to the carbon chain. Unlike 1803, 1816 showed stronger potentiation at homomeric α3 GlyR as compared to α1 GlyR, in HEK293 cell experiments conducted separately in the lab. The resultant IPSCs recorded for each receptor (n=6), were analysed to determine changes in IPSC kinetics from those mediated by GlyRs without drug (fig. 20).

Fig. 20 Comparison of spontaneous glycinergic IPSCs mediated by α1, α3, α1β and α3β GlyRs during drug free control (black) and 1816 (orange). Panel A shows sample traces of the voltage clamp recordings done for α1, α3, α1β and α3β GlyRs at 20 second timescale. Panel B shows the mean IPSC waveforms (all normalised) for drug free control and 1816 for all 4 receptors, each averaged from >100 events from a single cell.

No significant increase in peak amplitude was observed for any of the receptors even after washing with 1816 for 10-15 minutes, continuously. For α3 GlyR, significantly slower events with increased 10-90% rise times (38 ± 8 ms) and decay rates (72 ± 9 ms) were observed, as compared to control (13 ± 3 ms, 35 ± 11 ms, respectively). For α1 GlyRs, only a significantly slower decay rate (25 ± 4 ms) of the IPSC events was observed with 1816, as compared to control (9 ± 2 ms). No other significant changes in IPSC kinetics were observed for either of the heteromeric α1β and α3β GlyRs (fig. 21).

Fig. 21 The mean values for the peak amplitudes,10–90% rise times and decay time constants of IPSCs recorded for the cannabinoid 1816 from α1, α3, α1β and α3β GlyRs, compared to the drug free control (one way ANOVA, *p < 0.05, **p<0.01, ***p<0.001).

1816 had a greater potentiating effect on α3 GlyR than α1 GlyR, and no significant effect on either of the heteromeric channels.

The results show that both drugs were more potent potentiators at for homomeric GlyRs, suggesting that their possible site of action is present on the α subunits. In addition to this, a more detailed analysis of the structure-activity relationship of both the cannabinoids, should help to better understand their respective preferences and partial selectivity for one type of α subunit. By far, 1803, or 5-Desoxy-CBD, seems to have the strongest effect on any of the receptors, and shows less selectivity due to its potentiation of α1, α3 and α1β GlyRs. The cannabinoid significantly increases the amplitude of the current events in the homomeric channels, since they have a higher number of α subunits that possess the functional glycine binding sites required for potentiation.

4.3.3 Discussion

59 Several studies have recently shown the strong potentiating effects of cannabinoids on GlyRs. The primary psychoactive cannabinoid, THC, has been shown to induce analgesia in animal models of pain, by potentiating glycinergic currents mediated by α3 GlyR in a mechanism that is independent of the activation of cannabinoid receptors CB1 and CB2 (Zimmer et al. 1999; Costa et al. 2007; Xiong et al. 2011; Xiong et al. 2012). In addition to α3 GlyR, involvement of the α1 GlyR subunit in cannabinoid-induced analgesia has also been studied and reported (Lu et al. 2018). In addition to THC, non-psychoactive phytocannabinoid cannabidiol has also been shown to potentiate GlyRs and so have other non-psychoactive synthetic derivatives (Xiong et al. 2011; Xiong et al. 2012; Lu et al. 2018). This has generated a fair amount of interest in the design and synthesis of novel cannabinoid derivatives that are non-psychoactive and can strongly potentiate GlyRs and alleviate pain in animal models and humans. While these cannabinoids have shown analgesia by potentiating both α3 and α1 GlyRs, it is still unclear how α1 GlyR plays a role in this. Thus, α3 GlyR selective potentiation by a cannabinoid is still unreported.

Since native GlyRs are predominantly expressed as postsynaptic heteromers of α and β subunits, it is also important to see the effect of such cannabinoid based compounds on them. So far electrophysiological studies with cannabinoids have been done in HEK293 cells expressing single GlyR subunits, cultured spinal neurons and in rat spinal cord slices (Xiong et al. 2011; Xiong et al. 2012; Lu et al. 2018). However, their potentiating effects on IPSCs have not been detailed so far. Recently our lab was provided with multiple novel cannabinoid derivatives to study their effects on potentiating homomeric and heteromeric α1 and α3 GlyRs. Two of these drugs were derived from the cannabinoid “cannabidiol” or CBD, designated as 5-desoxy-CBD referred to as 1803, and 1,2-dimethylheptyl-desoxy CBD referred to as 1816. 5-desoxy CBD is structurally the same compound that has been referred to as 5-Desoxy-THC (also referred to as Dehydroxyl CBD or DH-CBD) by Xiong et. al (2012), and has been previously shown to potentiate glycinergic currents in both α1 and α3 GlyRs (Xiong et. al 2012). These CBD derivatives were then used in patch-clamp recordings of α1, α3, α1β and α3β GlyRs to measure the effect of the drugs on IPSC kinetics in neuronal-HEK293 cell co-cultures that form artificial synapses.

1803 significantly potentiates homomeric α1 and α3 GlyRs

1803 significantly potentiated IPSCs in homomeric α1 and α3 GlyRs, and potentiated heteromeric α1β, but not α3β GlyRs. The lack of a potentiation at α3β GlyRs has not been

60 previously reported. However, 1803 has been previously shown to potentiate α3, α1 and α1β GlyRs (Ahrens et al. 2009a; Xiong et al. 2011). The cannabinoid had the strongest effect on α1 and α1β since it potently increased their peak amplitudes, 10-90% rise times and decay times. For α3 GlyR, it slowed the rise and decay times but had no significant effect on increasing the peak amplitudes. No potentiation in any of the IPSC kinetics was observed for α3β GlyRs. This strongly suggests that 1803 either has a much lower affinity for α3 subunit containing GlyRs or has a reduced effect on the agonist activity of glycine. Studies by Xiong et al. (2012) have shown NMR evidence for the binding of DH-CBD in the TM3 domain in both α1 and α3 GlyRs, which is why it still potentiates both homomeric α1 and α3 GlyRs but the respective effect reduces for each subunit with the introduction of the β subunit. It has been previously shown that heteromeric α1β GlyRs have significantly lower affinity for THC as compared to homomeric α1 GlyR, suggesting that cannabinoids ma have stronger preference for the α subunit (Ahrens et al. 2009a). However, the difference in its overall effect on potentiating α1 and α3 GlyRs is indicative of certain structural differences between the two subunits.

1816, an α3 GlyR selective potentiator

Unlike 1803, 1816 selectively potentiated of α3 subunit-containing GlyRs. A significant increase the 10-90% rise or activation time was observed for homomeric α3 GlyR, and significant increase in the decay or deactivation time was observed for both α3 and α3β GlyR. No increase in peak amplitudes was observed for any receptors, suggesting that the structural difference between 1803 and 1816 makes the latter derivative an α3 GlyR selective drug. 1816, designated as 1,2-dimethylheptyl-desoxy CBD, has its carbon pentyl chain replaced with a dimethylheptyl chain (two methyl groups on position 1 and 2). The insertion of the methyl group to the existing 5-desoxy-CBD seems to significantly affect its selectivity for α3 containing GlyRs. This provides not only an interesting insight into the effect of modifications in cannabinoid structures, but also gives an indication of how side chain additions or substitutions can alter the binding mechanism of the drug to the GlyR. This might suggest that while still being structurally closer to THC and CBD, 1816 might still occupy another binding site on α3 GlyR, or the addition of the methyl groups may alter its hydrophobic interactions to close accompanying lipids.

It would ultimately be of interest to design and synthesise close structural analogues to these cannabinoids, to observe whether drug potency and subunit selectivity can be increased.

61 Another important approach would be to simulate the molecular binding interaction and mechanism of these cannabinoids at α1 and α3 GlyRs and consider their sensitivities to surrounding lipid molecules. The hydrophobic nature of the overall structure of the derivatives makes them interesting compounds for binding to and potentiating membrane proteins like ion channels, and hence makes them effective drugs in targeting GlyRs.

62 4.4 References

Ahrens, J, Demir, R, Leuwer, M, De La Roche, J, Krampfl, K, Foadi, N, Karst, M, Haeseler, G 2009a, ‘The Nonpsychotropic Cannabinoid Cannabidiol Modulates and Directly Activates Alpha-1 and Alpha-1-Beta Glycine Receptor Function’, Pharmacology, vol. 83, no. 4, pp. 217-22.

Ahrens, J, Leuwer, M, Demir, R, Krampfl, K, de la Roche, J, Foadi, N, Karst, M, Haeseler, G 2009b, ‘Positive allosteric modulatory effects of ajulemic acid at strychnine-sensitive glycine alpha1- and alpha1beta receptors’, Naunyn Schmiedeberg’s Archives of Pharmacology, vol. 379, pp. 371–378.

Costa, B, Trovato, AE, Comelli, F, Giagnoni, G, Colleoni, M 2007, ‘The non-psychoactive cannabis constituent cannabidiol is an orally effective therapeutic agent in rat chronic inflammatory and neuropathic pain’, European Journal of Pharmacology, vol. 556, pp. 75– 83.

Demir, R, Leuwer, M, de la Roche, J, Krampfl, K, Foadi, N Karst, M, Dengler, R, Haeseler, G, Ahrens, J 2009, ‘Modulation of glycine receptor function by the synthetic cannabinoid HU210’, Pharmacology, vol. 83, pp. 270–274.

Hejazi, N et al 2005, ‘9-Tetrahydrocannabinol and Endogenous Cannabinoid Anandamide Directly Potentiate the Function of Glycine Receptors’, Molecular Pharmacology, vol. 69, no. 3, pp. 991-97.

Howlett, AC, Barth, F, Bonner, TI, Cabral, G, Casellas, P, Devane, WA, Felder, CC, Herkenham, M, Mackie, K, Martin, BR, et al 2002, ‘International Union of Pharmacology. XXVII. Classification of cannabinoid receptors’, Pharmacological Reviews, vol. 54, pp. 161– 202.

63 Izzo, AA, Borrelli, F, Capasso, R, Di Marzo, V, Mechoulam, R 2009, ‘Non-psychotropic plant cannabinoids: new therapeutic opportunities from an ancient herb’, Trends in Pharmacological Sciences, vol. 30, pp. 515–527.

Xiong, W, Wu, X, Lovinger, DM, Zhang, L 2012b, ‘A common molecular basis for exogenous and endogenous cannabinoid potentiation of glycine receptors’, Journal of Neuroscience, vol. 32, pp. 5200–5208.

Yévenes, GE and Zeilhofer, HU 2011a, ‘Allosteric modulation of glycine receptors’, British Journal of Pharmacology, vol. 164, pp. 224-236.

Yévenes, GE and Zeilhofer, HU 2011b, ‘Molecular sites for the positive allosteric modulation of glycine receptors by endocannabinoids’, PLoS ONE, vol. 6, pp. e23886.

Zimmer, A, Zimmer, AM, Hohmann, AG, Herkenham, M, Bonner, TI 1999, ‘Increased mortality, hypoactivity, and hypoalgesia in cannabinoid CB1 receptor knockout mice’, Proceedings of the National Academy of Science USA, vol. 96, pp. 5780–5785

Chapter 5 Conclusion

65 5.1 Conclusions and future directions

GlyRs are involved in fast inhibitory neurotransmission in various regions of the CNS such as the spinal cord, brain stem and retina (Lynch 2004). Due to the presence of Cl- selective ion permeable pore, the passive diffusion of the ions cause a rapid hyperpolarization of the postsynaptic neuron, thereby inhibiting neuronal firing (Lynch 2004). This ionic influx occurs upon receptor activation by the primary neurotransmitter, glycine, which binds to the receptor and induces channel opening (Webb and Lynch 2007). The involvement of glycinergic inhibitory neurotransmission in motor reflex and nociceptive sensory circuits makes the GlyRs important targets for modulations in the pathophysiology of startle disease and chronic inflammatory pain.

GlyRs, particularly α1β and α3β are predominantly expressed in the laminae I and II of the spinal cord, and the nociceptors that receive pain stimuli transmit to the spinal cord via glycinergic currents mediated by these channels (Harvey 2004). PGE2 mediated phosphorylation of α3 GlyR disrupts the inhibition of the transmission of the pain stimuli from peripheral nociceptors to the brain, which results in increased sensation of pain (Harvey et al. 2004; Zeilhofer, 2005; Acuna et al. 2016). While α1 GlyR lacks this phosphorylation site and hence is not involved in chronic inflammatory pain, its co-localization with α3 GlyRs in the spinal cord dorsal horn points to its possible involvement in mediating inhibitory neurotransmission in nociceptive pathways. This has been shown by involvement of α1 GlyRs in cannabis induced analgesia in animal models and its absence in a transgenic α1 GlyR mutant model (Ahrens et al. 2009; Lu et al. 2018). However, due to lack of subunit specific agonists or co-agonists, it has been difficult to elucidate the involvement of either α1 GlyRs or α3 GlyRs solely, or together, in the resultant cannabinoid-induced analgesia. Considering the emerging interest in GlyRs, particularly α3 GlyR as potential target for small drugs as modulators, the main theme of this thesis has been identifying and characterizing these modulators and their subtype specificity.

In the past 10 years, our lab has focused on designing and screening novel small molecular drugs that selectively potentiate the effects of GlyRs (Balansa et al., 2010; Balansa et al., 2013a; Balansa et al., 2013b), particularly α3 GlyR. Previous analgesics designed by other groups, including a recent novel analgesic by Amgen Inc. (Huang et al., 2016) show significant potentiation of the glycinergic currents of α3 and α1 GlyR, but lack the subtype selectivity for α3. The drug designated CMB-C28-HO1, or simply HO1, was previously

66 identified in our lab as a strong lead for its potentiating effect on α3 over α1 GlyR in

HEK293 cells. EC20 glycine-induced currents in α3 were significantly potentiated as compared to α1, for which the potentiation observed was insignificant suggesting HO1’s strong affinity for the α3 subunit. In order to ascertain further the drug’s selectivity for α3 GlyR, functional inhibitory synapses expressing α1 and α3 GlyRs were generated and used for electrophysiological studies with application of HO1. The effect of 10 nM HO1 on IPSCs mediated by α3 GlyRs showed significant potentiation as compared to α1. HO1 increased the IPSC peak amplitude, decay time and 10-90% rise times for α3 GlyR but not α1 GlyR. Since synaptic events occur due to brief but high concentration exposure to neurotransmitter, slow application of agonist with drug directly onto transfected HEK293 cells can only provide information regarding the relative sensitivity of test drugs (Lynch, 2004). The IPSC events however can show the effect of the drug in functional synapses. The artificial inhibitory synapse system is an efficient way to physiologically recreate IPSC events in close similarity to those in native GlyRs and have been helpful in determining location and clustering of different synaptic GlyR isoforms. While experiments with α1 and α3 GlyR expression in HEK293 cells strongly suggested HO1’s selectivity, it was important to validate its efficacy for α3 GlyR under synaptic activation and deactivation conditions. Hence this approach was useful in providing insight about the pharmacological changes to the receptor on binding with HO1 that affect the channel’s kinetic properties, and can also be an efficacious in vitro experimental set up to allow comparisons between modulators to be made. The increase in decay time by HO1 for α3 GlyR is suggestive of the change in agonist affinity and its access and removal, as physiologically slower decay times are shown to be governed by these properties (Pitt et al. 2008). The slower rise times due to HO1 show a possible effect of the drug on the channel’s gating mechanism due to agonist unbinding or the closing of the receptor (Lester and Kahr, 1992). Modelling the binding site for HO1 and its association to the glycine binding site clarified some of the assumptions regarding the drug’s effect on glycine’s inherent agonist activity.

Binding of small molecules to receptor in functionally important binding sites can induce conformational changes that ultimately exert influence on the function of the receptor (Huang et al, 2015, Unwin et al. 2002). The activity of a modulator is strongly related to the site it can occupy on an ion channel and what insights that can provide in characterizing the mechanism of the drug’s action. HO1 had been established as a selective potentiator for α3 GlyR and hence it was important to elucidate its binding mechanism. Using predictive structural bioinformatics tools for molecular docking, the binding site of HO1 was modelled 67 for α3 GlyR which showed that the site was in close proximity to the glycine binding site. This closeness of the glycine binding site could mean that any conformational change induced by the HO1 binding might be affecting the glycine binding site as well. Agonist induced structural changes are important for channel activation and desensitization (Pless and Lynch 2009). Allosteric modulators can affect these gating mechanisms by introducing alterations to the ion channel in their local binding vicinity and more globally to the pore architecture of the receptor (Hibbs and Gouaux 2011). Mutations of the residues involved in the predicted binding site for HO1 showed a drastic difference in response to glycine and reduced glycine sensitivity. Considering how every mutation did not have the same shift in sensitivity to glycine, it is important to note that due to the individual amino acid structures and their contribution to the overall protein stability, each mutation had different effects on the channel’s change in affinity to glycine. Each mutation affected the ability of HO1 to bind

and potentiate the activity of EC20 glycine. This combined with the changed sensitivity to glycine, points to this site being the possible site of action for HO1, as can be best assessed from the electrophysiological data presented in Chapter 3. Considering these properties of HO1 and its subsequent selective potentiation of α3 GlyR, the drug is a strong candidate for a possible analgesic and an allosteric modulator for the receptor. Other pLGICs have been previously co-crystallized with allosteric modulators like ivermectin (Hibbs and Gouaux 2011; Huang et al. 2017; Lu et al. 2015). These studies give clear insights into structural and conformational changes induced by modulators. Such a clear idea of the physiological effect of HO1 can be validated by obtaining a crystal structure of α3 GlyR in its bound state to HO1 and glycine.

Chapter 4 of this thesis further explored the potential of novel cannabinoid derivatives as analgesics that target α1 and α3 GlyRs. Recent evidence has strongly shown certain cannabinoids to induce potent analgesia in animal models via mechanisms that are independent of the CB1 and CB2 receptors, but mediated by α1 and α3 GlyRs (Hejazi et al. 2006; Xiong et al 2011; Xiong et al. 2012). This recent data coupled with the myriad of evidence that has shown the non-psychoactive properties of cannabinoids such as cannabidiol has renewed interest in its use in designing and synthesizing novel derivatives that can induce analgesia without any psychoactive side effects (Ahrens et al. 2009; Lu et al. 2018; Xiong et al. 2011; Xiong et al. 2012). Two cannabinoids that were similar in structure to cannabidiol with slight structural changes were subjected to electrophysiological analysis on both homomeric and heteromeric GlyRs containing subunits α1 and α3 using the artificial

68 inhibitory synapses. Previous studies have shown certain cannabinoids to significantly potentiate recombinantly expressed GlyRs in HEK293 cells and in native isolated spinal cord slices that have translated into strong analgesia in animal models (Lu et al. 2018; Xiong et al. 2011; Xiong et al. 2012). However effect of cannabinoids on potentiating glycinergic IPSCs kinetics has not been addressed sufficiently. The results presented in chapter 4 of this thesis show that cannabinoid 1803 (5-desoxy-CBD) showed strong potentiation of glycinergic IPSCs mediated by both GlyRs α1 and α3. However the drug showed more potent effects on homomoeric α1 and α3 GlyRs. It had the strongest effect on α1 and α1β GlyRs, since it potently increased their peak amplitudes, 10-90% rise times and decay times. For homomeric α3 GlyR it still affected the agonist activity significantly by slowing down the rise and decay times. Though 1803 displayed stronger potentiation in α1, this still did not firmly establish its subunit specific site of action, although affinity for homomeric > heteromeric does suggest its binding site possibly on α subunit. Cannabinoid 1816 (1,2-dimethylheptyl-desoxy CBD) showed much more potentiation in α3 and α3β GlyR than α1 GlyR. Considering the close structural similarities between the two cannabinoids, this switch towards selectivity for one subunit over the other strongly hints at sidechain substitutions and their structure activity relationship (SAR). A more detailed SAR analysis with more analogues should provide more insight into the mechanisms of GlyR isoform selectivity. This makes studying cannabinoids as a new class of analgesics an interesting expedition since the possibility of designing numerous and novel structural analogues is still very high and their effects unreported. In addition, many cannabinoids have not been validated in terms of receptor selectivity and in vivo analgesic potency. SAR analyses are very helpful in determining the strongest candidates and using those as leads for designing more optimal drugs.

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Balansa, W., et al. Australian marine sponge alkaloids as a new class of glycine-gated chloride channel receptor modulator. Bioorganic and Medicinal Chemistry 21, 4420-5 (2013).

Harvey, RJ et al 2004, ‘GlyR α3: An Essential Target for Spinal PGE2-Mediated Inflammatory Pain Sensitization’, Science, vol. 304, pp. 884-887.

Hibbs, RE and Gouaux, E 2011, ‘Principles of activation and permeation in an anion- selective Cys-loop receptor’, Nature, vol. 474, no. 7349, pp. 54-60.

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