Rescuing Dopamine Transporter Deficiency Syndrome

Home , Substituted amphetamine, Trimethylamine

Charles Sutton

A thesis submitted in conformity with the requirements

for the degree of Master of Science

Department of Pharmacology & Toxicology

University of Toronto

Charles Sutton

Master of Science

Department of Pharmacology & Toxicology

University of Toronto

2018

Abstract The dopamine transporter (DAT) is a membrane protein essential to dopamine homeostasis.

Abnormal dopamine homeostasis is implicated in multiple conditions including the newly discovered dopamine transporter deficiency syndrome (DTDS). DTDS is an autosomal-recessive disorder caused by mutations that impair DAT maturation. Bupropion and ibogaine can rescue DTDS DAT mutants. We therefore examine the structure-activity relationships responsible, in hopes of discovering more efficacious and potent compounds. An SAR-based screen of bupropion and ibogaine analogs demonstrated necessity for a secondary amine and single halogen substitution on bupropion along with a flexible heterocyclic ring, chair-like substituent and hydroxyl substitution on ibogaine. Lead candidates noribogaine and PAL594 were able to rescue mature DAT. We also report that there is no link between

DAT inhibition and chaperoning, suggesting that chaperoning is mediated allosterically. Future studies should aim to validate the presence of the putative DAT allosteric site, computer-assisted drug discovery and a mouse model.

ii Acknowledgements Being brought up in a household where my parents share almost 2 decades of combined post- secondary education is surely a reason that I am still here today. Many thanks are due to my parents and sister for helping me find my way through this long path of scholastic wandering. In some regards, I feel like I’ve been here forever, in others, I feel like I’ve just got here. In reality, its been 2 years. Two years of redoing assays 4-5 times until they work and running half month-long mouse dosing regimes to find out the drug wasn’t made up properly. Did I mention I’m very allergic to mice? Graduate school is one of the most negatively portrayed academic pathways with students constantly complaining about employment opportunities, their supervisor and the hours. Given all this, I feel like I share the same mentality as the many grad students before me, and the many that will follow. That this was an arduous journey that I wouldn’t trade for anything in the world. I chose to do my MSc with Dr Ali Salahpour because of his enthusiasm and drive for research. I must thank you for showing me that there is no substitute for passion in what you do. I would like to extend this thanks to Dr Amy Ramsey always being there when I needed advice and for always acting extremely interested in my lab meeting presentations.

You either love science or are a great liar, thank you. Furthermore, my big brothers in the lab, Pieter

Beerepoot and Vincent Lam, have been essential in teaching me how to be an effective scientist “behind the scenes”. Thanks to my advisor Dr Martin Beaulieu for keeping my project on track and to Wendy

Horsfall and Marija Milenkovic for their endless help and support. I would have no results and be buried in a pile of my own clutter if it wasn’t for you two. I’d like to thank my teammates on the University of

Toronto track & field team, specifically Augi Augimeri, Jack Berkshire and Anthony Kwan for helping me stay sane and chase my dream of simultaneous academic and athletic success. I could not have asked for better partners in idiocy, teammates and friends. A special thanks Katrina Allison, if I wrote down all the things you’ve done for me I’d run out of space. Without you I wouldn’t be the man I am today, let alone be completing my master’s thesis. Thanks to Parker Arnold for taking over the VMAT2 project and doing

iii a great job at that. A final shout out, to my right-hand man, Hoomam Homsi. Lab life would be much duller if we had not handpicked you out of millions of applicants for what turned out to be an excellent partnership. Here’s to the future.

iv Table of Contents

Acknowledgements ...... iii Table of Contents ...... v Tables ...... vii Figures ...... viii Abbreviations ...... ix 1. Introduction ...... 1 1.1. Dopamine ...... 1 1.2. The Dopamine Transporter ...... 2 1.1.2 Structure ...... 2 1.1.3 Ligand Binding & Translocation ...... 3 1.1.4 Regulation of DAT ...... 5 1.1.5 Implication in Human Disease...... 8 1.2. DAT Pharmacology ...... 10 1.2.1. DAT Substrates ...... 11 1.2.2. DAT Inhibitors ...... 12 1.2.3. Non-classical Pharmacology of DAT ...... 13 1.3. Membrane Protein Trafficking ...... 15 1.3.1. Membrane protein folding...... 15 1.3.2. Molecular Chaperones ...... 16 1.3.3. ER Export & Quality Control ...... 17 1.4. Pharmacological Chaperoning ...... 20 1.4.1. Pharmacological Chaperones ...... 20 1.4.2. Pharmacological Chaperones & Disease ...... 22 1.4.3. Potential & Clinical Use ...... 26 1.5. Specific Aims and Working Hypothesis ...... 27 2. Materials & Methods ...... 28 2.1 Drugs ...... 28 2.2 Constructs ...... 28 2.3 Cell Culture ...... 29

v 2.3.1 Conditions ...... 29 2.3.2 Transfections & Generation of Stable Lines ...... 29 2.4 Immunoblotting ...... 30 2.5 ß-lactamase Surface Expression Assay ...... 31 2.6 Dopamine Uptake ...... 31 2.7 Data Analysis & Statistics ...... 32 3. Results ...... 32 3.1 Identification of Pharmacological Chaperones of DAT ...... 32 3.1.1 Effect of Bupropion Analogues on DAT Surface Expression ...... 32 3.1.2 Effect of Ibogaine Analogues on DAT Surface Expression ...... 39 3.1.3 Selectivity of DAT pharmacological chaperones ...... 43 3.2 The Effect of DAT Pharmacological Chaperones on WT & Mutant Total Protein Levels ...... 44 3.3 Dopamine Uptake Inhibition of DAT Ligands ...... 52 4. Discussion, Conclusion, Future Directions ...... 56 4.1. Identification of Putative Pharmacological Chaperones of DAT ...... 56 4.1.1. Structure-Activity Relationship – Bupropion and Ibogaine Backbones ...... 56 4.1.2. DAT Chaperones Increase Mature Protein Levels of WT and Mutants ...... 66 4.2. The Discrepancy Between Dopamine Uptake Inhibition and DAT Chaperoning Effect...... 68 4.3. Clinical Potential of Bupropion & Noribogaine ...... 69 4.4. Conclusions and Future Directions ...... 73 References ...... 77 Appendix ...... 86 Copyright ...... 90

vi Tables Table 1- B1R- bradykinin B1 receptor, CaSR- calcium-sensing receptor, D4R- dopamine 4 receptor, DP1R- D-type prostanoid 1 receptor, FSHR- follicle stimulating hormone receptor, GCGR- G-protein coupled glucagon receptor, GnRhR- Gonadotrophin releasing hormone receptor, LHR- Lutenizing hormone receptor, MC4R- melanocortin 4 receptor, RhR Rhodopsin receptor, V2R- vasopressin 2 receptor, β1/β2 AR - β1/β2 adrenergic receptor, κ/δ OR- κ/δ opioid receptor, CFTR- cystic fibrosis transmembrane regulator, DAT- dopamine transporter, GABAAR- GABAA receptor, NAchR- nicotinic acetylcholine receptor, P-gp- P-glycoprotein, SERT- serotonin transporter. Table adapted and supplemented from [98] ...... 25 Table 2- Structure and effect of bupropion analogues on DAT surface expression ...... 34 Table 3- Structure and effect of ibogaine analogues on DAT surface expression ...... 40

Table 4- Comparison of Surface expression Emax, DAT binding and Dopamine uptake inhibition values for Bupropion and RTI 2-11 in WT DAT cells. DAT binding and dopamine uptake inhibition values from [138]...... 53

vii Figures Figure 1. 2D Structure of DAT ...... 3 Figure 2. Translocation mechanism of the DAT ...... 4 Figure 3 Simple overview of protein folding pathway. Adapted from [94]...... 19 Figure 4 Overview of SCL6 Transporter Rescue by chemical and pharmacological chaperones ...... 20 Figure 5 PC mechanisms of action...... 22 Figure 6- Bupropion analogues increase YFP-HA-βLAC-DAT surface expression ...... 38 Figure 7- Effect of overnight bupropion analogue treatment on YFP-HA-βLAC-DAT surface expression .. 39 Figure 8- Effect of overnight ibogaine analogue treatment on YFP-HA-βLAC-DAT surface expression ..... 42 Figure 9- Emax comparison between βLAC-DAT and βLAC-Β2AR of hit compounds ...... 43 Figure 10- Location of the 3 DAT mutants chosen for the mature DAT expression assay. Both DTDS mutants (A314V and L368Q) are located in the extracellular side of transmembrane domain 6 and 7, respectively. K590A is located in the C-terminus, a region heavily implicated in ER export...... 45 Figure 11-Effect of bupropion analogues on WT, K590A and A314V DAT expression ...... 47 Figure 12- Effect of ibogaine and noribogaine on WT, K590A and A314V DAT expression ...... 49 Figure 13- Effect of noribogaine, THH, PAL594, PAL1219 and RTI20 on WT, K590A, A314V, L368Q DAT expression ...... 51 Figure 14- Effect of bupropion, noribogaine, and the chemical chaperone 4-PBA on the clinically relevant L368Q DAT mutant ...... 52 Figure 15- Scatter plot comparing the surface expression and dopamine uptake inhibition Emax of each hit compound...... 54 Figure 16- Comparing the impact on DAT uptake and DAT surface expression for our hit compounds.... 55 Figure 17- Necessity of a secondary amine for the chaperoning effect of bupropion in the WT DAT ΒLAC assay ...... 58 Figure 18- Impact of phenyl substitutions on the bupropion backbone ...... 60 Figure 19 - Effect of alkyl chain length on the efficacy of bupropion in the WT DAT ΒLAC assay ...... 61 Figure 20- Effect of the tertiary butyl substituent on the efficacy of bupropion in the WT DAT ΒLAC assay ...... 61 Figure 21 - Effect of the hydroxyl and methyl groups on the efficacy of 6-hydroxybupropion in the WT DAT ΒLAC assay ...... 62 Figure 22-Effect of the hydroxyl substitution on the efficacy of ibogaine in the WT DAT βLAC assay ...... 63 Figure 23- Effect of the chair-like substituent on the efficacy of ibogaine in the WT DAT ΒLAC assay ...... 63 Figure 24- Comparison of pinoline and the FDA-approved frovatriptan ...... 64 Figure 25- Effect of the heterocyclic ring on the efficacy of ibogaine and β-carboline in the WT DAT ΒLAC assay ...... 65 Figure 26- Effect of the deconstructed heterocyclic ring on the efficacy of tryptamine analogues in the WT DAT ΒLAC assay...... 66 Figure 27- PHYRE2 homology model of the human DAT created using the crystal structure of the drosophila DAT (56% homology & 100% confidence)...... 73 Figure 28- Structural comparison of PAL 1219 to bupropion and ibogaine backbones ...... 75

viii Abbreviations

4-PBA - 4-phenylbutyrate

ADHD – Attention deficit hyperactivity disorder

AMPH – Amphetamine

BSA – Bovine Serum Albumin

CaMKII - Ca2+/calmodulin-dependent protein kinase II

CF – cystic fibrosis

CFTR - cystic fibrosis transmembrane regulator

COMT - catechol-O methyltransferase

COPII – Coat complex 2

D2 – dopamine receptor 2

DAT – Dopamine Transporter

DMSO – Dimethyl Sulfoxide

DTDS – Dopamine transporter Deficiency Syndrome

ENAP - N-ethyl-naphthylaminopropane

ER – Endoplasmic Reticulum

ERAD – ER-associated degradation

FDA – Food & Drug Administration

HBSS – Hank’s Balanced Salt Solution

HVA – homovillanic acid

iDAT – immature dopamine transporter

MAO -monoamine oxidase

mDAT – mature dopamine transporter

NET- norepinephrine transporter

PBS – Phosphate Buffered Saline

PC – Pharmacological Chaperone

PD – Parkinson’s Disease

PICK1 - Protein Interacting with C Kinase 1

ix PKC – protein kinase C

PNGase F - Peptide N-glycosidase F

PPI – Pre-pulse inhibition

SAR – Structure-Activity Relationships

TH – Tyrosine hydroxylase

THH - Tetrahydroharmine

VMAT2 – vesicular monoamine transferase 2

WT – Wild Type

βLAC – β-lactamase

β2AR – β-2 adrenergic receptor

β-CFT - (–)-2-β-Carbomethoxy-3-β-(4-fluorophenyl)tropane

β-PEA – β-Polyethylamine

1. Introduction

1.1. Dopamine

Dopamine is a small organic molecule that belongs to the catecholamine family and plays many important roles in both the peripheral and central nervous systems [1]. Peripherally, dopamine is produced mainly by cells of the sympathetic nervous system and the adrenal medulla [2]. It acts primarily as a local chemical messenger to regulate blood pressure and respiration, among other things.

Since dopamine cannot cross the blood-brain barrier, its role in the periphery can be considered isolated from those in the central nervous system [1]. Centrally, dopamine is synthesized in the substantia nigra, ventral tegmental area, and hypothalamus. It functions as a neurotransmitter and is only used in specific dopaminergic circuits in the brain. These circuits help regulate voluntary locomotion, motivation, learning, and reward [3]. Dysfunctional dopamine signaling is involved in conditions such as schizophrenia [4], Attention Deficit/Hyperactivity Disorder (ADHD) [5], Tourette’s syndrome [6], substance abuse [7], bipolar disorder [8], obesity [9] and the newly characterized Infantile Parkinson’s-

Dystonia called Dopamine Transporter Deficiency Syndrome (DTDS) [10]. It is also implicated in neurodegenerative conditions such as Parkinson’s Disease (PD) [11] and Huntington’s disease [12].

Given the considerable variety of dopamine-related conditions, it makes sense that the dopaminergic system is highly complex. The amino acid tyrosine enters the dopaminergic neuron via the aromatic L- amino transporter and is subsequently hydrolysed to L-DOPA [13]. This conversion is carried out by the rate-limiting enzyme in dopamine synthesis, tyrosine hydroxylase (TH). AADC (amino acid decarboxylase) then removes a carbonyl group from the L-DOPA molecule to yield dopamine.

Cytoplasmic dopamine is packaged into secretory vesicles by the proton antiporter vesicular monoamine transferase 2 (VMAT2). The proton gradient necessary for this step is created by a proton ATPase that transports hydrogen ions into the vesicles. When an action potential stimulates dopaminergic neurons,

1 the vesicles fuse with the presynaptic membrane in a Ca+ dependent manner. Upon release into the synaptic cleft, dopamine can interact with either postsynaptic dopamine receptors or presynaptic autoreceptors [13]. The former helps propagate downstream signals while the latter sends feedback to the presynaptic neuron. Of note, dopamine can be spontaneously oxidized to reactive quinones and cause dopaminergic cellular toxicity [14]. Oxidative cellular damage has been linked to diseases including PD and schizophrenia [14]. Because of this, extracellular dopamine needs to be inactivated quickly. Dopaminergic signalling is terminated primarily by diffusion out of the synapse and into the peri-synaptic space. Here it is taken up by DAT and translocated to the cytosol. Upon recycling, dopamine is either metabolized by the enzyme monoamine oxidase (MOA) into the metabolite carboxylic acid 3,4-dihydroxyphenylacetic acid (DOPAC) or repackaged into secretory vesicles by VMAT2

[13]. The remaining extracellular dopamine is broken down into the non-reactive homovillanic acid

(HVA) by catechol-O methyltransferase (COMT). The dopaminergic system can be modulated pharmacologically at many of these processes.

1.2. The Dopamine Transporter

1.1.2 Structure

The gene for the human dopamine transporter is located on chromosome 5p15 [15]. DAT

(SLC6A3) is a member of the solute carrier family 6 which consists of over 600 members, 21 of which are found in humans. These include, but are not limited to, the serotonin, norepinephrine, dopamine, creatine, glycine and GABA transporters [16]. Currently there is no crystal structure of the human DAT, however, the Drosophila Melanogaster DAT (dDAT) has recently been crystallized and possesses 56% sequence homology with human DAT [17]. This, along with the crystal structures of the prokaryotic leucine transporter (LeuT) and the serotonin transporter (SERT), provides sufficient information to construct DAT homology models that are able to make detailed predictions about the structure and

2 function of the human DAT [16]. The DAT is comprised of 12 transmembrane domains (TM) linked by 5 intracellular and 6 extracellular alternating loops. Moreover, DAT has cytosolic amino and carboxyl- termini that modulate uptake and substrate recognition [18]. TMs 1-5 and 6-10 are arranged in two pseudo-symmetrical groups with TM 11 and TM 12 excluded from the core and contribute little to substrate translocation (Figure 1). TM 1 and TM6 have non-helical hinge regions that form numerous bonds with all translocation cargo. DAT forms quaternary interactions in the form of homologous dimers or oligomers in addition to distinct protein-protein interactions [19].

Figure 1. 2D Structure of DAT

The dopamine transporter has 12 transmembrane domains organized into a 5X5+2 structure. TM1-5 and TM6-10 comprise the 2 pseudo symmetrical groups. TM1 and TM6 have hinge regions in the middle that make up a large part of the binding pocket

(S1). Adapted from [20]

1.1.3 Ligand Binding & Translocation

DAT is a Na+/Cl- dependent symporter that takes advantage of the transmembrane Na+ gradient, established by Na+/K+ ATPase, to translocate dopamine across the cell membrane. It is further

3 characterized by the co-transport of a Cl- ion. DAT terminates dopaminergic neurotransmission by simultaneously moving 2 Na+ ions, 1 Cl- ion and a dopamine molecule from the extracellular space into the presynaptic neuron [21]. This results in a net charge of positive one, however, some evidence suggests that a potassium (K+) ion exits the cell as the transporter resumes outward-facing conformation, bringing the net charge back to neutral [22]. DAT can do this by sampling a series of large- scale structural changes to transport cargo to the inner side of the membrane (Figure 2). When DAT is in the outward-facing conformation, it is ready to bind dopamine, Na+ and Cl-. The cargo interacts with transmembrane domains including TM1 and TM6 at S1, located in the hydrophobic core of the transporter. Doing so closes the extracellular gate and immediately causes the DAT to shift conformation to the occluded state. DAT homology models have shown a second site necessary for dopamine translocation, called S2. Once in the occluded S1-bound state, a second dopamine molecule can bind the extracellular S2. Binding of dopamine to S2 is essential for the opening of the intracellular gate, the transition to the inward-facing conformation and release of the S1-bound cargo into the cytosol. The release of Na+/ Cl- ions from S1 allows the transporter to return to the outward-facing conformation and reset the translocation cycle [23]. It is important to understand that the interaction of dopamine with S2 is likely low affinity and transient when compared to that of S1 [24].

Figure 2. Translocation mechanism of the DAT

(A) With the extracellular gate open and DAT in the outward facing conformation, the substrate can bind S1. (B) The extracellular gate closes, causing a shift to the occluded conformation and (C) allowing another substrate to transiently bind S2.

(D) This opens the intracellular gate and allows the S1-bound substrate to enter the cytoplasm as the DAT shifts to the inward- facing conformation. Adapted from [23]

1.1.4 Regulation of DAT

The total amount of DAT protein in a cell is dependent on the relationship between DAT synthesis and degradation whereas the extent of functional DAT at the cell surface is dependent on the relative rates of endo and exocytosis [25]. It is important to note that dopamine translocation via DAT occurs at the plasma membrane and as a result, overall transport capacity of DAT is a function of its surface density. However, the mechanisms that underlie these principles are poorly understood thus little is known about the complex trafficking of DAT. The transport and binding properties of DAT are regulated by complex and overlapping mechanisms that provide neurons with the ability to continually control synaptic dopamine in response to constantly changing physiological demands [26]. Interestingly, there is evidence to suggest that DAT is regulated by transcriptional mechanisms. Following the chronic inhibition of DAT with cocaine, DAT protein expression is considerably upregulated [27]. This, however, does not explain how DAT is able to adapt to the dynamic changes of synaptic dopamine real-time.

Therefore, DAT must be regulated in the short term to accommodate the rapidly changing synaptic environment.

Expression levels of DAT localized to the plasma membrane are mediated by changes in protein- protein interactions and posttranslational modifications [26]. To this end, DAT is constitutively internalized into vesicles and is either sorted to a late endosomal/lysosomal degradative pathway or an early endosomal recycling pathway, in a process known as post-endocytic sorting [6]. Many factors can increase the rates of internalization such as the activation of protein kinase C (PKC) by phorbol 12- myristate 13-acetate (PMA) [28, 29], treatment with DAT substrates like dopamine or amphetamine [28]

5 and membrane depolarization [30]. Interestingly, PKC-dependent and substrate-induced internalization seem to be governed by separate regulatory mechanisms as pre-treatment with DAT inhibitors has no effect on PKC mediated internalisation, with the exception of GBR 12909 [29]. These findings further the notion that DAT internalization is managed by a complex entanglement of factors. To add to the mix, different cultured cell types appear to have varying internalization rates and intracellular pool sizes making it difficult to study true in-vivo DAT regulatory factors [31]. Regardless of these discrepancies, this demonstrates the principle of DAT regulation by the governance of internalization.

The N and C-termini of DAT are highly conserved across species and are much larger than that of the bacterial homologue LeuT. The terminal regions of DAT contain binding domains that are essential for post-translational modifications and protein-protein interactions which are heavily involved in transporter-specific regulation. Post-translation modification of DAT and other SCL6 transporters include, but are not limited to, palmitoylation [32], phosphorylation [33], and glycosylation [34].

Palmitoylation is likely the least studied modification but has been shown to be important for dopamine uptake and regulating degradation [32]. Phosphorylation can occur via many different kinases including

PKC and Ca2+/calmodulin-dependent protein kinase II (CaMKII), among others [33]. CaMKII-dependent phosphorylation of the N-terminus plays a significant role in amphetamine-induced dopamine efflux

[18]. The specific role of phosphorylation on the DAT lifecycle is still not clear. Finally, glycosylation is a modification in which carbohydrate moieties are added to the transporter during the folding trajectory.

DAT contains 3 N-linked glycosylation sites in the second extracellular loop. When glycosylation is prevented, surface and intracellular DAT levels are reduced [34]. Furthermore, stability, transporter kinetics and inhibitor sensitivity are also altered [34] showing the impact glycosylation has on DAT. For example, cocaine has a greater inhibitory effect on non-glycosylated DAT than when fully glycosylated

[34] and reduced DAT glycosylation in human striatum and midbrain strongly correlates with greater

Parkinson’s disease rates [35]. It is also important to note that balance between types of modifications

6 dictates transport capability, therein suggesting a more complex regulatory framework instilled by post- translational modifications [32]. In addition to post-translation modifications, DAT can form interactions and complexes with proteins. DAT can interact with other DAT monomers to create oligomeric complexes, usually tetramers, before being exported from the ER [19]. The exact role of quaternary interactions of DAT is still relatively unknown, however, it is likely that dimerization affects trafficking, surface stability, ligand binding, and dopamine uptake kinetics [36]. Additionally, many other proteins are found to interact with either the N or C-terminus of DAT and seem to impart their own effects on surface expression. Syntaxin1A associates with the N-terminus of DAT in a CaMKII dependent manner to promote dopamine efflux [37]. At approximately the same location on the DAT N-terminus, the dopamine receptor 2 (D2) can also bind to enhance DAT trafficking to the plasma membrane and thus reduce synaptic dopamine levels [38]. The N-terminus also houses a domain in which the synaptic vesicle protein synaptogyrin-3 interacts in a VMAT2 dependent way to potentially form the link between synaptic and vesicular dopamine [39]. In addition to interacting with heat shock proteins and coat complex 2 (COPII) machinery which help SCL6 transporters reach their native state and the plasma membrane, the C-terminus is heavily implicated in protein-protein interactions that regulate DAT function [40]. The DAT C-terminus contains the PDZ-domain binding site which is essential for enabling synaptic expression of DAT. It is not completely understood how it achieves this, but studies suggest a role for protein interacting with C Kinase 1 (PICK1) as deletion of the PDZ binding site nullifies DAT’s interaction with PICK1 and impairs trafficking to the plasma membrane [41]. DAT, again through its C- terminus, can interact with the protein α-synuclein. This interaction promotes synaptic DAT and α- synuclein clustering that accelerate dopamine uptake and apoptosis [42]. Moreover, α-synuclein may act as a negative regulator of TH and dopamine biosynthesis [43]. A reoccurring theme, however, is the many holes in understanding that accompany the molecular control of DAT. Further work on more

7 complex and relevant models is essential to piecing together an accurate structured understanding of how DAT is universally regulated.

1.1.5 Implication in Human Disease

Given the role DAT plays in regulating dopamine homeostasis, it is conceivable that alterations in the DAT gene can lead to dopamine dysregulation and potentially a mental health disorder. Many mutations have been linked to genetic irregularities in DAT, one such disorder is ADHD. ADHD is a neuropsychiatric disorder characterized by hyperactivity, short attention span, and impulsivity that are all age-inappropriate [44]. ADHD rates in America are 9% for people aged 13-18 years [45] and 4.1% in people aged ≥18 years [46]. These rates are on the rise which could be due to the semantics of the term

ADHD and an increased rate of diagnosis [47]. Although the pathogenesis of ADHD is not well understood, evidence suggests that irregular dopamine signalling may be a culprit [48, 49]. A 40 bp variable number tandem repeat (VNTR) in the 3’ untranslated region of DAT that reduces DAT RNA stability and translation efficiency [49] has been linked to ADHD [50].

Loss of function DAT mutations result in a phenotype that resembles PD, not ADHD [51] and some studies suggest that ADHD-prone mutations might be much more subtle [52]. Two point mutations in DAT, A559V [53] and R615C [54], have been identified in human patients with ADHD. In agreement with the subtle mutation hypothesis of DAT and ADHD, A559V is said to leak dopamine into the synapse, a trait that can be prevented by low doses of amphetamine and methylphenidate [53].

Furthermore, maybe the clinical efficacy of low dose DAT inhibitors in the treatment of children, adolescents, and adults with ADHD can be explained by correctable irregularities in the transport kinetics of DAT [55].

PD is a neurodegenerative disorder characterized by progressive loss of nigrostriatal dopaminergic neurons that results in bradykinesia, resting tremor, and postural instability. PD affects 3% of people aged ≥65 years [56] and is treated with L-DOPA or dopaminergic agonists [57]. PD patients show a reduction in DAT levels via human imaging studies. Although the pathogenesis of PD is poorly understood, it is widely accepted that Lewy bodies containing α-synuclein accumulate within the substantia nigra pars compacta [42]. As mentioned before, α-synuclein plays a role in the regulation of

DAT via protein-protein interactions that can cause DAT clustering at the membrane and enhanced

uptake (Vmax ) under normal conditions. In the disease state, there is potential that the overproduction of

α-synuclein and subsequent cluster-induced elevated DAT Vmax can lead to oxidative stress and terminal damage [42]. PD has a complex and poorly understood pathogenesis, but it would not be surprising if

DAT was implicated given its critical role in maintaining dopamine homeostasis.

ADHD and PD both have debate surrounding their etiology and the role that DAT could play in them is insufficiently understood. There is, however, a condition that is most definitely linked to dysfunction in DAT. Hereditary Dopamine Transporter Deficiency Syndrome [51] is a recently discovered rare pediatric disorder that is caused by autosomal recessive loss of function mutations in DAT [51].

Parkinsonism-dystonia such as hypotonia, feeding difficulties, dyskinesia, dystonia, and tremors as well as raised levels of dopamine metabolites in the CSF are the main features of the condition and make the diagnosis a difficult one. The prevalence of the condition worldwide is unknown, however, many cases are initially misdiagnosed as Cerebral Palsy [58]. This suggests that there may be many other cases in the world right now that are currently misdiagnosed. This should reverse itself as awareness for DTDS among the medical profession increases. A major issue with the condition is the lack of treatment and eventual death in childhood or adolescence [58]. Initially, the syndrome presents as hyperkinetic but then transitions to an akinetic state followed by eventual death due to cardiovascular or pulmonary complications [10]. The syndrome progression can be explained in two steps. First, loss of DAT function

9 allows for increased synaptic dopamine and thus the hyperkinetic state. Second, dopamine stores are not replenished by DAT mediated dopamine recycling and lead to the akinetic state. Due to the heightened dopamine levels, there may also be dopamine-mediated cellular toxicity which could damage dopamine neurons and also lead to an akinetic state. A similar state is observed in DAT-

Knockout mice [59]. Physicians attempted to treat the akinetic state with L-DOPA or dopamine agonists to no avail [10]. As one would expect, the severity of the syndrome is related to the degree of dysfunction of DAT. Patients that have 10% DAT surface expression have a much less severe condition compared to those who have none. As mentioned earlier, the specific effect that each mutation has on

DAT may be the determining factor in what condition the patient presents as. For example, a loss of function mutation may present as DTDS whereas a transporter kinetics altering mutation may present as

ADHD [51, 52]. Dopamine and binding properties of several DTDS mutants show a heavily compromised

3 or nullified Vmax but differential effects on H-CFT binding affinity (KD) [10, 51, 60]. This suggests that the mutated DAT, in most cases, is functional but is being retained in the ER due to folding deficits. It seems that mutation-mediated structural deficits will affect the folding trajectory before function, leaving a large pool of DTDS mutants functional yet ER-retained. Taken together, these observations suggest that some DTDS mutants could be functional at the plasma membrane if folding deficits can be corrected by pharmacological intervention, thus providing a potential mechanism for the treatment of DTDS [61].

1.2. DAT Pharmacology

DAT is a target of many therapeutic and recreational psychostimulants, nootropics, and antidepressant drugs [62]. Drugs of abuse, such as cocaine and methamphetamine, act through the DAT.

The reinforcing effects of these recreational drugs are well documented and stem from their ability to enhance synaptic dopamine. Clinically, drugs that target DAT either do so selectively or as part of a

10 diffuse array of neurotransmitter proteins, which is reflected in the pharmacodynamic profile of the drug. Classically, DAT ligands have been divided into two categories, inhibitors and substrates [63]. Most exogenous substrates and inhibitors increase synaptic dopamine and are therefore classified by whether they are translocated by DAT or not. As the understanding of DAT pharmacology increases, this dichotomy has been challenged and other categories such as atypical inhibitors, allosteric modulators, and partial substrates have arisen.

1.2.1. DAT Substrates

The prototypical DAT substrate is dopamine. Other exogenous compounds such as amphetamine and methamphetamine are transported inside the cell similar to dopamine [44]. These compounds present an interesting biphasic dose-dependent behaviour. At low doses, they act as DAT inhibitors by simple substrate competition with dopamine for the S1 site [64]. Conversely, at high doses, amphetamine causes reverse transport of dopamine from intracellular vesicles into the extracellular space [64, 65]. Amphetamine may achieve this by putting DAT in a conformation that acts more like a channel than a transporter [66]. It is unclear how exactly amphetamine and other DAT substrates do this. Phosphorylation of the N-terminus [67] through interaction with phosphatidylinositol (4,5)- bisphosphate [68] and C-terminal-mediated CaMKII interactions [18] have both been implicated.

Additionally, increased intracellular Na+ has also been linked to reverse transport rates of dopamine

[69]. The biphasic behaviour of amphetamine and DAT is apparent in amphetamine’s usage. Clinically, it is used at low doses to relax and grant focus to ADHD patients [55]. Recreationally, it is used at much higher doses, due to its potent reinforcing properties and euphoric effects [62]. The clinical and recreational dose discrepancy may be explained by the biphasic mechanism of amphetamine.

1.2.2. DAT Inhibitors

DAT reuptake inhibitors, like cocaine or methylphenidate, increase extracellular dopamine by binding to the DAT and inhibiting its translocation cycle [70]. This mechanism contributes to the powerful reinforcing effects of DAT-targeting drugs of abuse. However, DAT inhibitors display a variety of behavioural profiles, something one wouldn’t expect if all DAT inhibitors acted through the same mechanism [71]. Furthermore, different classes of DAT inhibitors stabilize distinct conformations of DAT which have been linked to the reinforcing effects of each compound [71]. For example, extracellular Na+ depletion drives DAT into predominantly the inward-facing conformation and drastically reduces cocaine-like inhibitors’ affinity for the transporter, suggesting cocaine needs the outward-facing DAT to bind. Conversely, Na+ depletion has partial to no effect on the affinity of modafinil and GBR12909 binding, suggesting these inhibitors do not require the outward-facing conformation to bind and may instead bind to the occluded or inward-facing transporter [72]. This can be further corroborated by mutational binding studies, which insert a mutation that favours inward or outward-facing conformations. In doing this, two groups of compounds emerge. A group consisting of cocaine, methylphenidate and β-CFT, which bind the outward-facing conformation; and a group consisting of bupropion, modafinil, benztropine and GBR12909, which bind a more inward-facing conformation [20,

72, 73]. These are termed typical and atypical inhibitors, respectively and are grouped based on the DAT conformation needed for binding. Interestingly, typical inhibitors have strong reinforcing effects whereas atypical do not, most likely due to the difference in the conformational state they stabilize [62].

It has been shown that typical inhibitors stabilize the outward-facing state by sterically blocking the extracellular gate from closing [74]. Atypicals, however, allow the extracellular gate to close, allowing for the occluded conformation [72]. Additionally, the Tabernanthe iboga-derived alkaloid, Ibogaine, is proposed to stabilize an even more inward-facing conformation by binding the extracellular side of DAT

[75].

1.2.3. Non-classical Pharmacology of DAT

The facilitated exchange model of dopamine efflux dictates that when extracellular substrate is translocated inside a cell it provides an opportunity for a cytosolic dopamine molecule to bind the now inward-facing conformation of DAT. This postulates that the rate at which dopamine efflux occurs is dependent on the uptake rate [63]. It is now becoming clear that uptake (ie forward transport) and efflux (ie reverse transport) are not regulated by uptake kinetics and that they are in fact distinct processes. This is suggested by a set of 4-quinazolinamine compounds (SoRI-9804, SoRI-20040 & SoRI-

20041) which have shown differential effects on DAT uptake and efflux. The three compounds had similar affinities (1–5 μM) and uptake inhibition of DAT. SoRI-9804, SoRI-20040 inhibited dopamine efflux, however, SoRI-20041 had no effect [76]. This provides evidence for a separate regulation of the forward and reverse translocation mechanisms of DAT. Furthermore, it shows that it is possible to design molecules that can target different aspects of the DAT translocation cycle and differentially affect substrate uptake and efflux.

While it appears that a DAT ligand can have distinct effects on separate aspects of the DAT translocation cycle (uptake vs efflux), it is also becoming apparent that different DAT ligands can affect the same aspect of the DAT translocation cycle differently. Specifically, different DAT substrates have shown variable efficacies for inducing DAT-mediated efflux. The compound ENAP [62] and 3,4-

methylenedioxy-N-ethylamphetamine [76] were only able to reach Emax efflux values of 78% and 65%, respectively. The plateau in DAT-mediated efflux was insurmountable with increased dosage, suggesting these compounds act as partial substrates. The mechanism for this is unknown, however, it may be that they work as substrates with a slower translocation exchange rate.

In addition to having differentially selective control of DAT, the 4-quinazolinamine compounds were shown to inhibit radioligand binding in a non-competitive manner. The compounds were only able

13 to inhibit radioligand binding by approximately 40-60% before reaching saturation [76]. Classical competitive inhibitors, such as cocaine, are able to inhibit 100% of radioligand binding [77]. Moreover,

the compounds increased the KD value, decreased the Bmax value and slowed the dissociation rate of the radioactive tracer [125I]RTI-55 in radioligand binding assays [77]. It is possible that SoRI-20041 induces a subtle conformational change in DAT that slightly impairs the forward direction of transport but not the reverse. Another example similar to the 4-quinazolinamine compounds can be found in a group of rigid adenine nucleoside derivatives which can either enhance or block binding of DAT inhibitors [78]. These molecules show a similar atypical pharmacological profile to the 4-quinazolinamine compounds. These results, combined with the differential DAT regulation, suggest that these ligands do not compete for S1, the same binding site as dopamine and other classical substrates and inhibitors. Instead, they may allosterically modulate DAT from a novel site somewhere else on the transporter. Another compound acting as a potential allosteric modulator is ibogaine. Ibogaine shows non-competitive inhibition of SERT

[75]. An allosteric site on SERT has recently been discovered through X-ray crystallography [79], which given the structural similarity to DAT, could provide insight into the non-classical pharmacology of DAT.

Altogether, the 4-quinazolinamine ligands, ENAP, 3,4-methylenedioxy-N-ethylamphetamine and the adenine nucleoside derivatives demonstrate that DAT activity can be partially modulated in a non- competitive, saturable, and functionally selective manner. These putative partial and allosteric mechanisms could be similar to biased G-protein–coupled receptor signalling, in which ligands differentially affect different signalling pathways through binding to the same receptor. Further studies are needed to fully elucidate the novel mechanism behind these findings and demonstrate the in-vivo potential of non-classical pharmacological regulation of DAT.

1.3. Membrane Protein Trafficking

1.3.1. Membrane protein folding

Membrane protein folding is a highly complex, poorly understood and inefficient process [80].

The amount of years needed for a protein to reach its mature and native folded state by random interaction is in the billions [81]. In reality, this process takes seconds or less to be achieved therefore there must be other mechanisms involved to guide protein folding [81]. The disparity between theoretical and actual folding times is termed Levinthal’s paradox [81] and can, in short, be explained by the environmental constraints imposed on the peptide during its folding trajectory [82]. Simply put, protein folding occurs in a step-by-step manner where the completion of each step limits the conformational flexibility of the next, thus drastically reducing the overall number of conformations to be visited before maturity is reached. Moreover, as more interactions are made, the protein collapses and further increases the overall number of interactions, perpetuating the cycle [83]. This behaviour of protein folding is called the energic funnel and allows proteins to achieve their thermodynamically stable, native state much faster than if the completely unfolded protein was simply allowed to fold by random interaction [84].

Membrane proteins are synthesised by ER-bound ribosomes and are then or simultaneously transferred to the ER in a process known as translocation [82]. The first folding occurs inside the Sec61 translocation complex as local secondary interactions are formed [85]. The initial tertiary structure starts to assemble as the peptide chain enters the ER, however, complete tertiary folding requires interactions between domains that are far apart and can only be achieved after sufficient peptide has emerged [82].

Luminal and cytosolic molecular chaperones are proteins that associate with the nascent peptide, reduce energy barriers involved in establishing native conformations and guide the protein’s progression through the folding trajectory [82]. Concomitantly, post-translational modifications such as disulfide

15 bonds and glycosylation are added, resulting in the completion of the native conformation. Pending approval by cellular quality control and successful (COPII) mediated vesicular packaging, the protein will travel to the Golgi where the final post-translation modifications will occur. Following this, the protein is sent to the plasma membrane via vesicular trafficking [86]. Folding is now complete; however, the native state is not a finish line. In reality, most native states lie somewhere in a range of being not stable enough or too stable [87]. This range is tightly conserved in evolution and allows proteins to be stable enough to retain their native state but flexible enough to function and be susceptible to degradation.

Stability is inversely proportional to conformational flexibility and thus the range of conformations a native protein must go through to be functional will have an impact on its stability [88]. Any alterations to this tightly regulated system can have detrimental effects on cell homeostasis. With such complexity involved in protein folding pathways, it is not surprising that this is an error-prone process. These errors can arise at many different stages of folding, from mutations in the genetic code to faulty post- translation modifications [82]. Any error can cause alterations in the properties of the protein that lead to non-native stable conformations, which can have detrimental effects on the cell. These non-native stable conformations can either reduce functional protein levels or form toxic aggregates [89].

Accordingly, living systems have evolved ways to reduce folding errors and the detrimental impact of the resultant proteins.

1.3.2. Molecular Chaperones

Peptides sample many different conformations along their folding trajectory which inescapably expose hydrophobic domains susceptible to inter and intramolecular interactions [83]. These undesired interactions may promote the stability of reaction intermediates or protein-protein complexes that likely have little functionality and may accumulate inside the ER [88]. To prevent this, cells have evolved proteins called molecular chaperones which recognize these uncharacteristic hydrophobic domains and protect them from unwanted interactions throughout the folding trajectory [83]. This means that

16 molecular chaperones are present in both the cytosol and the ER, representing distinct regulation of folding and quality control mechanisms. In the cytosol, a group of molecular chaperones called heat shock proteins (HSP) are upregulated in periods of cellular stress [88]. HSC70 and HSP70, members of the HSP70 family, and HSP90 are among some of the major heat shock protein groups that help protect against unwanted aggregation in the cytosol [83]. In the ER, there is a separate set of molecular chaperones that consist of BIP, another member of the HSP70 family, in addition to the lectin chaperones, entitled calnexin and calreticulin. Lectin chaperones are part of a unique chaperone system to the ER; they act as quality control for post-translation modifications such as glycoproteins [82].

1.3.3. ER Export & Quality Control

Once a protein is properly folded and clears all quality control mechanisms, it is ready for ER export. Most ER export is dependent on COPII, specifically its subunit Sec 24 [90]. Sec24 contains cargo binding sites that recognize distinct C-terminal motifs on native proteins ready for ER export [91]. These

ER export motifs can be concealed by improper folding or protein-protein interactions. Incomplete or improperly folded proteins are retained in the ER by continued interaction with molecular chaperones to either allow complete folding or targeting for degradation [90]. Although it is energetically more efficient to refold proteins rather than degrade them, chronically misfolded proteins will collect in the

ER and will not be degraded or sent to the membrane. Proteins caught in this cycle must be degraded.

This is in-part signalled by prolonged interaction with molecular chaperones which leads to poly- ubiquitination of the retained peptides by ubiquitin ligases [82]. Once an ER-retained protein has been ubiquitinated, it must be retro-translocated via the Sec61 translocation complex into the cytosol as the

ER does not contain the required enzymes for degradation. Here the ubiquitinated protein is recognized by the proteasome and degraded [82]. This process is termed ER-associated degradation (ERAD). As for cytosolic proteins in need of degradation, it is much simpler. Cytosolic proteins are poly-ubiquitinated and subsequently degraded via the proteasome or autophagy [82]. Altogether, cellular quality control

17 works in conjunction with molecular chaperones to ensure that non-native proteins are properly dealt with (Figure 3). However, sometimes the quality control mechanisms are too harsh and end up retaining and degrading proteins that may otherwise be partially or fully functional [92]. Presents an opportunity for pharmacological intervention as modulating cellular quality control may be useful in genetic conditions where an essential protein is semi-functional but ER-retained (Figure 4). Pharmacological modulation can be achieved by chemical chaperones, such as sodium 4-phenylbutyrate, trimethylamine

N-oxide and bile acids which directly inhibit quality control proteins [93]. This will, however, induce a universal upregulation of proteins and may cause toxicity in some cases [92]. For selective rescue, pharmacological intervention can also target the misfolded protein itself, stabilizing a conformation that passes the quality control mechanisms. This approach is known as pharmacological chaperoning [92].

Figure 3 Simple overview of protein folding pathway. Adapted from [94].

Figure 4 Overview of SCL6 Transporter Rescue by chemical and pharmacological chaperones

Adapted from [95]

1.4. Pharmacological Chaperoning

1.4.1. Pharmacological Chaperones

The process of folding newly translated polypeptides is a complex and inefficient process. In some cases, WT protein folding efficiency is no greater than 50% [80]. Mutations that reduce the folding efficacy of a protein can further exacerbate folding deficiency and result in clinically significant

20 phenotypes [96]. By taking advantage of the quality control mechanisms of a cell, we can pharmacologically increase expression, and potentially, the function of mutated proteins [97].

Pharmacological chaperones (PC) present a unique opportunity to selectively target the folding and quality control processes of a specific peptide in order to rescue it [92]. A PC, also known as a pharmacochaperone or pharmacoperone, is a small molecule that binds selectively to a misfolded protein, increases folding efficiency by stabilizing a conformation that passes the quality control mechanisms and enables improved cell membrane expression [97]. PCs stabilize conformations in a variety of circumstances that lead to increased cell surface expression (Figure 5). Moreover, PCs have been able to rescue cytosolic and membrane proteins, including GPCRs and transporters [98]. Recently, a PC has been approved by the FDA and is used clinically for the treatment of cystic fibrosis [99] which demonstrates that PCs have the potential to be a widespread treatment for many health concerns.

Figure 5 PC mechanisms of action.

Adapted from [98]

1.4.2. Pharmacological Chaperones & Disease

PCs have a relatively short history, as the first demonstration of their mechanism and efficacy was made approximately 20 years ago. Mutants of the multidrug resistance transporter P-gp were rescued by capsaicin, cyclosporin A, verapamil, and vinblastine. [100]. Although the clinical potential for this case is low, it served as proof of concept for PCs and since then roughly 20 proteins have been pharmacologically chaperoned [98]. The most important PC-related discovery to date is that involving

22 the genetic pulmonary condition, cystic fibrosis (CF). CF is the most common autosomal recessive disease among Caucasians and affects over 70000 people worldwide [101]. A mutation in the cystic fibrosis transmembrane regulator (CFTR), called ΔF508, accounts for 70% of cystic fibrosis. This mutation causes CFTR misfolding and leads to ER retention [102]. The only FDA-approved PC in clinical use goes by the name of Orkambi. It is a combination of the CFTR PC VX-809 and ivacaftor, a potentiator for ΔF508-

CFTR [99]. VX-809 is effective at rescuing ΔF508-CFTR and some of the clinical endpoints, however, it has drawn criticism since symptomatic improvement is marginal given the yearly treatment price of $259

000 USD [103]. VX-661, another ΔF508-CFTR PC is currently in clinical trials and thus far has outperformed both VX-809 alone and Orkambi [104]. Furthermore, RDR1 is another ΔF508-CFTR PC that has shown promise in cell and rodent models [105].

Many proteins can be rescued by PCs (Table 1). The concept of PCs, therefore, has a broad application for rescuing mutations in membrane proteins. Furthermore, this effect is not restricted to mutants as PCs can often increase expression of WT proteins, excitingly demonstrating a possible new therapeutic avenue [61]. SLC6 family transporters have been recently rescued by pharmacological chaperones. Ibogaine and its metabolite noribogaine, were able to rescue C-terminal mutants of SERT

[106]. Furthermore, noribogaine was able to restore serotonin uptake in a dose-dependent manner, both demonstrating functionality of SERT and the potential for PCs to evoke functional change in neurotransmitter systems [40]. Excitingly, a recent study shows that a set of SERT partial substrates with a napthylpropane-2-amine backbone are able to rescue SERT, suggesting the search for PCs can take advantage of the atypical pharmacology of SLC6 transporters to fine-tune the desired actions of the drug and reduce side-effects [107]. Given the structural similarity between SLC6 family transporters, it is not surprising that DAT is also able to be rescued by similar drugs. The clinical impact of SERT chaperones is unclear, however, DAT chaperones provide an approach to potentially treat DTDS. DAT was rescued in cells and Drosophila melanogaster by noribogaine [108]. In another study, the compounds that

23 promoted rescue were all atypical inhibitors, including the FDA-approved drug bupropion. Typical inhibitors did not have an effect [109]. This suggests that the folding trajectory of DAT progresses through the inward-facing conformation and that atypical inhibitor-mediated stabilization of this conformation helps promote proper folding. Furthermore, Ibogaine may stabilize a more inward-facing conformation of DAT [75], which may explain its strong efficacy as a DAT chaperone.

Table 1- B1R- bradykinin B1 receptor, CaSR- calcium-sensing receptor, D4R- dopamine 4 receptor, DP1R- D-type prostanoid 1 receptor, FSHR- follicle stimulating hormone receptor, GCGR- G-protein coupled glucagon receptor, GnRhR- Gonadotrophin releasing hormone receptor, LHR- Lutenizing hormone receptor, MC4R- melanocortin 4 receptor, RhR Rhodopsin receptor, V2R- vasopressin 2 receptor, β1/β2 AR - β1/β2 adrenergic receptor, κ/δ OR- κ/δ opioid receptor, CFTR- cystic fibrosis transmembrane regulator, DAT- dopamine transporter, GABAAR- GABAA receptor, NAchR- nicotinic acetylcholine receptor, P-gp- P- glycoprotein, SERT- serotonin transporter. Table adapted and supplemented from [98]

Pharmacological chaperones of membrane proteins System GPCRs Compound Clinical application in vitro Rodent Human Source Quinoxalinone- derived B1R Compound 11 Inflammation + [110] CaSR Cinacalcet Hypercalcemia + [111] D4R Quinpirole - + [112] DP1R MK-0524 Bone and joint health + [113] FSHR Org41841 Ovarian failure + [114-116] GCGR L-168,049 Mahvash disease + [117] GnRhR IN3 Hypogonadism + + [115, 116] Reproductive LHR Org 42599 dysfunction + [118] MCR4 ML00253764 Obesity + + [119] 11-cis-7-ring- RhR retinal Retinitis pigmentosa + [120] Nephrogenic diabetes V2R SR121463A insipidus + + [121] Alprenolol & [113, 122, β1/β2 AR prazosin - + 123] Naltrexone & [121, 124, κ/δ OR etorphine Pain + 125]

Other Membrane Proteins VX-809, VX-661 CFTR & RDR1 Cystic fibrosis + + + [104],[105] Bupropion & DAT ibogaine DTDS + [61, 108] GABAAR GABA Epilepsy + [126] hERG Channel E4031 Long QT syndrome + [127] Parkinson's Disease & NAchR Nicotine Epilespy + [128] Capsaicin, cyclosporin A, verapamil, & P-gp vinblastine - + [100] Ibogiane & SERT noribogaine - + [40, 75]

1.4.3. Potential & Clinical Use

DTDS mutations cause DAT to be expressed at very low levels or not at all (Table 2). The expression level is tightly linked with the severity of the disease as it appears that DTDS mutations affect folding of the transporter, more than its function [10, 51, 60]. For example, the mutation Alanine-314-

Valine (A314V) causes DAT to be expressed at 9% of normal levels [61]. Mice with 10% DAT expression present a hyper-locomotion phenotype that is much less severe than DAT-Knockout mice, which express no DAT protein [129, 130]. This again shows that DAT is tightly regulated, and small changes are enough to alter behavioural phenotype. If a patient were to present with a mutation such as A314V, a small increase in DAT expression might be enough to significantly reduce symptoms and delay disease progression. Mice that are heterozygous for DAT, which express 50% of WT DAT levels, appear to have a normal phenotype [131]. Bupropion and ibogaine are able to rescue A314V DAT total mature protein by

300% in cells, which would translate to approximately 30% of normal DAT levels [109]. Based on these findings, it is understandable the restoring mutant DAT levels to the 30-50% range could be enough to restore DAT function sufficiently and reduce DTDS symptoms in patients.

Ibogaine and noribogaine seem to have the largest positive impact on DAT surface expression, however, it is unlikely ibogaine could be used in a clinical setting due to its cardiotoxic effects [132].

Noribogaine, however, is currently being tested for tolerability issues and the results are mixed. Some clinical studies suggest it is well tolerated [133, 134] yet some mechanistic studies suggest it is just as toxic as ibogaine [132]. Conversely, bupropion is FDA-approved could be used off label in patients with

DTDS. Of note, bupropion is metabolized to 6-hydroxybupropion by CYP2B6 and its steady-state plasma concentrations are 400-700% higher than the parent drug [135]. Preliminary data from our lab suggests that 6-hydroxybupropion performs to the same extent as bupropion, in respect to its pharmacological

26 chaperone effect on DAT. Given the increased steady-state concentration and half-life of 6- hydroxybupropion [135] and noribogaine [133] over their parent compounds, it may be an interest of future studies to address these compounds themselves as potential PCs of DAT. Regardless of what compound is the best PC of DAT, it is vital to note that DTDS clearly presents early in life and slowly progresses towards death [10]. Disease progression might be due to excessive extracellular dopamine- mediated cellular damage, therefore, highlighting the importance of early intervention [136]. Early DAT rescue, even marginally, may prevent this damage and thus the progression towards death.

1.5. Specific Aims and Working Hypothesis

Once dopamine has been released into the synapse it is mostly regulated by DAT. Therefore, it is not surprising that issues in DAT trafficking and function can perturb the tightly balanced dopamine homeostasis. Antagonists and partial substrates are useful in reducing DAT activity, however, there are currently no clinical strategies to increase DAT function. We believe that pharmacological chaperones offer a legitimate strategy to selectively increase functional DAT at the cell surface and ultimately rectify abnormal dopamine homeostasis.

We hypothesise that an ideal DAT PC can be formulated from the bupropion and ibogaine backbones using structure-activity relationship strategies. We aim to identify the key molecular features that are necessary and complimentary for the pharmacological chaperoning effect of these compounds.

In pursuit of this, we aim to identify bupropion and ibogaine backbone analogues that selectively augment DAT surface expression. Furthermore, we strive to determine the effects of these compounds on WT and mutant DAT protein levels and function. The aim of this thesis is to identify the ideal DAT PC, based on structure-activity relationships.

2. Materials & Methods

2.1 Drugs

Bupropion Hydrochloride and 6-hydroxybupropion were obtained from Toronto Research

Chemicals (Toronto, Canada), ibogaine from Ibogaworld, cocaine from Medisca (Montreal, Canada), β-

PEA, Pinoline, Indole, Tryptamine, Ephedrine, Frovatriptan, Alprenolol, Isoproterenol, and Tyramine from Sigma (Oakville, Canada), fluoxetine and amphetamine were from Tocris Bioscience (Bristol, United

Kingdom). PAL and RTI analogues were provided by Dr Bruce Blough. Noribogaine was a gift from Dr

Deborah Mash and Ibogamine was obtained from Specs (Zoetermeer, The Netherlands). Phenylbutyrate

Sodium was purchased from Enzo Life Sciences Inc (Farmingdale, NY).

2.2 Constructs

The βLAC sequence was cloned from the ampicillin resistance gene within the pcDNA3.1 plasmid

[137], using Spe I and an Asc I restriction sites. An HA epitope was added to the N terminus of the βLAC, yielding HA- βLAC [137]. To insert HA-βLAC into YFP-HA-DAT, Quikchange II site-directed mutagenesis

(Agilent, Mississauga, Canada) was used to insert Spe I and Asc I restriction sites on either side of the HA sequence of YFP-HA-DAT. The HA sequence in YFP-HA-DAT was removed by cuts with the Spe 1 and Asc1 restriction enzymes, prior to ligation of the HA-βLAC sequence. The Spe1 and Asc1 sites were then removed using site-directed mutagenesis, yielding YFP-HA-βLAC-DAT (referred to as βLAFC-DAT). The human β2AR (β 2 adrenergic receptor) cDNA expression vector was a gift from Dr Michel Bouvier. HA-

βLAC construct was subsequently inserted into this vector via Asc1 and Not1 restriction sites. Ligation of the HA-βLAC construct and into the β2AR cDNA expression vector yielded HA-βLAC-β2AR. To study the effects of alterations in the DAT, single point mutations were introduced into YFP-HA-DAT using

Quikchange II site-directed mutagenesis. Leucine-368-Glutamine (L368Q), Alanine-314-Valine (A314V),

Lysine-590-Alanine (K590A) in the human DAT and Alanine-313-Valine (A313V) in the mouse DAT were all confirmed by DNA sequencing.

2.3 Cell Culture

2.3.1 Conditions

HEK293 cells were obtained from ATCC (Manassas, VA) and maintained in Dulbecco’s Modified

Eagle’s Medium (DMEM) (Sigma) supplemented with 10% FBS (Sigma), 100 U/ml penicillin and 100

µg/ml streptomycin. Cells were kept in 5% atmospheric CO2 and 37°C. Cells expressing SS-HA- βLAC- β

2AR were further supplemented with 1µg/ml puromycin. All cells were treated with plasmocyin (250

µg/ml) for 2 weeks to ensure they were not infected.

2.3.2 Transfections & Generation of Stable Lines

HEK293 cells were used to create stable transfections. On day 1, cells (2 × 106) were seeded in a

10cm tissue culture plate and incubated for 24 hours. Day 2, seeded cells were transfected with 1-2 µg of plasmid DNA and 3 µl of polyethylenimine (1mg/ml) (Polyscience Inc, Warminster, PA) per µg of DNA.

They were then incubated in DMEM and transfection agent for 24 hours. On day 3, to create stable lines, the media was replaced with media containing the selection agent G418 (500 µg/ml) (Bioshop,

Burlington, Canada). In 5-7 days, most cells died, and media was replaced with fresh G418 selection media. Cells remaining on the plate were expanded until it was approximately half confluent. 96 well plates were seeded with a solution (5 cells/ml) with the goal of obtaining 1 cells per well. Plates were then incubated for 1-2 weeks. Clonal cell lines were the generated by choosing wells with only 1 visible colony. These were then grown up and protein expression was confirmed by western blot or the β- lactamase surface expression assay.

2.4 Immunoblotting

Cells (1 × 106 cells/well) were seeded into a 6-well plate. Following 24 hours, cells were treated with drugs and incubated for 16 hours. After the incubation period, cells were lysed in RIPA buffer supplemented with protease inhibitors (working concentrations: pepstatin A, 5 μg/mL; leupeptin, 10

μg/mL; aprotinin, 1.5 μg/mL; benzamidine, 0.1 μg/mL; PMSF, 0.1 mM) on ice. At 4°C, cell lysates were gently shaken for 15 minutes followed by centrifugation at 15000 rpm for 15 minutes to pellet debris. A

BCA protein assay kit (Thermo Scientific, Canada) was used to determine the protein concentration of the supernatant. To reach a concentration of 40 µg of protein per 20 µL, samples were diluted in fresh

RIPA buffer with 12.5% Laemmli sample buffer and 2.5% β-Mercaptoethanol prior to being heated to

55°C. They were then loaded on a 7.5% or 10% SDS-PAGE gel and run at 100V for approximately an hour or until the dye front reached the bottom of the gel. Next, the protein was transferred to a PVDF membrane at 22V for 16 hours or 100V for 1 hour. Blots were blocked with LI-COR blocking buffer for 1 hour and stained for 1 hour at room temperature with rabbit anti-GFP (1:1000 dilution; Life

Technologies, cat#A11122). After washing three times with TBST, blots were incubated with goat anti- rabbit IRDye-680RD secondary antibody (1:15000; LI-COR, cat#926-68071). As a loading control, blots were incubated with mouse anti-GAPDH (1:5000, Sigma-Aldrich, Canada, cat#G8795,) prior to secondary antibody goat anti-mouse IRDye-800GR (1:5000 dilution, Rockland, Limerick, PA, cat#610-132-007). All antibodies were diluted in LI-COR blocking buffer. Additionally, REVERT total protein staining (LI-COR,

Lincoln, NE) was also used as a loading control. No drug treatments showed an effect on GAPDH levels as determined by comparing GAPDH to total protein staining. Protein bands were visualized using the

Odyssey Imaging System (LI-COR, Lincoln, NE) and Image Studio Version 5.2 software.

2.5 ß-lactamase Surface Expression Assay

βLAC (β-lactamase) surface expression assay was performed by following the method outlined in [137]. The assay substrate nitrocefin (BD Biosciences) was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mM and frozen at -80°C. Immediately before use, it was thawed and diluted to a final concentration of 100 µM in phosphate buffered saline (PBS). Cells (1 × 105 /well) were seeded into a poly-D-lysine-coated 48-well plate and incubated for 24 hours, prior to drug treatment. Drugs were added to individual wells at varying concentrations and incubated for 16 hours. Following this, the media was aspirated, and the cells were washed twice with PBS. After removal of the final PBS wash,

200µL of the nitrocefin solution was added to each well and the absorbance was immediately read. The

EPOCH microplate spectrophotometer (Biotek) was used to measure absorbance (486 nm) for each well every minute for 30 minutes. The rate of reaction (slope of the curve) was used as the readout for the assay [109]. To determine suitable concentration ranges for each drug, they were dissolved in PBS or

DMSO at the highest concentration possible. From here, the solutions were serial diluted 1/10 to yield 8 different concentrations. βLAC cells were then dosed with drug concentrations and cell viability was recorded using light microscopy. For additional measure, nitrocefin was added and absorbance read. For certain cases, Trypan Blue was used to quantitatively differentiate increased cell death from reduced cell division. Drug doses that resulted in significant cell death and zero or heavily impaired reaction rates were not used in further experiments. Five or six doses were generally used in subsequent dosing experiments, starting at the highest non-toxic concentration.

2.6 Dopamine Uptake

Dopamine uptake inhibition was conducted using the Neurotransmitter Transporter Uptake

Assay Kit (Molecular Devices, prod#R8173). Cells (1 × 105 /well) were seeded into 96-well plate and incubated for 24 hours. The following day, wells were washed and incubated for 30 minutes with a

31 mixture of 1X HBSS (Hank’s Balanced Salt Solution) and 0.1% BSA (Bovine Serum Albumin) Buffer. Drugs being tested for dopamine uptake inhibition were added for 30 minutes. Following this incubation, the fluorescent dye is added, and the plate is immediately transferred to the bottom-read fluorescence microplate reader (Spectramax M3, Molecular Devices) for kinetic read-mode. The assay reads the amount of fluorescent activity which increases as a fluorescent dye is taken up by the DAT into the cytoplasm where it fluoresces. Therefore, a decrease in fluorescence relative to the vehicle-treated well can be taken as a reduction in the function of DAT. The read-out for this assay is the slope which equals the fluorescent measurements/ time, ultimately denoting the uptake rate.

2.7 Data Analysis & Statistics

Data analysis was performed using GraphPad Prism (GraphPad Software, La Jolla, CA). Linear regression analysis was performed on βLAC time points to determine the slope, thus the rate of reaction, for comparisons. Furthermore, it was also used to determine the degree of dopamine uptake in the dopamine uptake assays. Non-linear regression, fitting to the log(agonist) vs response and log(inhibitor) vs response model curve, was used to determine dose-response relationships of surface expression and uptake assays, respectively. Two-tailed t-tests or one-way ANOVA with Bonferroni correction were used to determine the differences between treatment levels.

3. Results

3.1 Identification of Pharmacological Chaperones of DAT

3.1.1 Effect of Bupropion Analogues on DAT Surface Expression

Bupropion, 6-hydroxybupropion, 3-dechloro-3-bromo bupropion, ephedrine and bupropion analogues from two different libraries, PAL and RTI, were selected to test for activity in the microplate

LAC surface expression assay. Analogues from PAL and RTI libraries were obtained through

32 collaboration with Dr Bruce Blough of RTI. Analogues were selected based on small desired derivations on the bupropion backbone from a library of compounds that was created to find more potent DAT inhibitors.

Table 2- Structure and effect of bupropion analogues on DAT surface expression

Bupropion RTI 17 RTI 2 Emax =144% Emax =78% Emax =127%

RTI 3 RTI 4 RTI 5 Emax =112% Emax =110% Emax =140%

RTI 6 RTI 7 RTI 8 Emax =134% Emax=102% Emax =107%

RTI 9 RTI 10 RTI 11 Emax =87% Emax =34% Emax =131%

6-hydroxybupropion 3-Deschloro-3-Bromobupropion RTI 21 Emax =148% Emax =160% Emax=103%

RTI 18 PAL 594 PAL 1411 Emax=122% Emax=166% Emax=110%

PAL 1007 PAL 945 PAL 1101 Emax=136% Emax=83.4% Emax=105%

RTI 12 RTI 20 Ephedrine Emax=113% Emax=147% Emax=128%

RTI 13 PAL 1270 PAL 1461 Emax=99% Emax=104% Emax=54%

PAL 1219 Emax= 149%

HEK cells expressing YFP-HA-βLAC-DAT were incubated overnight with different concentrations of these drugs. The βLAC assay has been validated and used for this exact purpose in the past by [61, 109]. Drugs were dosed in log and half-log dilutions starting at 100µM final concentration, with the exception of ephedrine, which was started at 1mM final concentration. Bupropion has been previously shown to increase WT DAT surface expression with an Emax of 137% [61]. After some slight assay optimization, we found bupropion had an Emax of 144%, similar to what was published before. Bupropion was not alone in its effect on DAT as many other analogues were able to increase WT DAT surface expression as well

(Figure 6). Compounds with small derivations on the bupropion backbone such as 3-Deschloro-3-

Bromobupropion (160%), RTI 2 (127%), RTI 5 (140%), RTI 6 (134%), RTI 11 (131%), RTI 20 (147%) and PAL

1007 (136%) caused significant improvements in WT DAT surface expression. Furthermore, bupropion’s main metabolite 6-hydroxybupropion (148%) and its analogue PAL 594 (160%) seemed to have similar or better maximum surface expression than bupropion. Other compounds with larger bupropion backbone

37 derivations had varied success with ephedrine (128%) having marginal improvements and PAL 1219

(148%) having considerable success (Table 2). Dose-response curves were conducted to assess potency of these hit compounds (Figure 7). There were no significant changes in EC50 values between bupropion and any of the hit compounds, however, it is important to note that the potency of bupropion in the

βLAC surface expression assay (39µM) was nearly 100 fold right-shifted compared to the reported IC50 of bupropion for dopamine uptake inhibition (0.6µM) [138]. To experimentally test this in our conditions, a dopamine uptake inhibition experiment was done to determine whether the insertion of

YFP-HA-βLAC into the DAT caused a reduction in bupropion uptake inhibition potency. Our results show that the potency of bupropion as a dopamine uptake inhibitor is 1.3µM demonstrating that bupropion’s

IC50 for dopamine uptake inhibition is similar to the YFP-HA-βLAC construct (0.84µM) (Supplemental

Figure 1).

180 * * * * 160 * * * * * 140 * * * 120

100

80 Surface Expression Surface (% untreated well) of 60

RTI 2RTI 3RTI 4RTI 5RTI 6RTI 7RTI 8RTI 9 RTI 11RTI 17RTI 18RTI 20RTI 21 PAL 594PAL PAL539 945 PAL 1007 PALPAL 1270 1101PAL PAL 1411 PAL1461 1219 Bupropion

Ephedrine (10-3 M) 6-Hydroxybupropion

3-Deschloro-3-Bromobupropion Figure 6- Bupropion analogues increase YFP-HA-βLAC-DAT surface expression

Summary of the maximum observed effects of bupropion analogues on WT DAT surface expression. HEK293 cells stably transfected with βLAC-WT-DAT were treated with drugs at 100µM except in the case of ephedrine, where it was used at 1mM. Drugs were dissolved in DMSO to a final concentration of 1%.

Data are means ± S.E. *, p < 0.05; negative hits due to solubility issues were not included in significance calculations; n=3-6.

Figure 7- Effect of overnight bupropion analogue treatment on YFP-HA-βLAC-DAT surface expression

Concentration-effect curves for most hit compounds of the bupropion backbone. Doses ranged between 10nM to 1mM and were dissolved in DMSO to a final concentration of 1%. Data were fitted to one-site dose-response non-linear regression curves using GraphPad Prism. n=3-6. 3.1.2 Effect of Ibogaine Analogues on DAT Surface Expression

Analogues of ibogaine and the structurally similar tryptamine were tested in the βLAC-DAT surface expression assay. Ibogaine analogues, ibogamine and noribogaine, were the first drugs chosen.

These were then followed by a set of β-carboline analogues: harmine, harmane, tetrahydroharmine, the endogenous pinoline and the FDA-approved frovatriptan. Finally, tryptamine and a set of analogues from the PAL library, courtesy of Dr Bruce Blough, were tested for effects on βLAC-DAT surface expression (Table 3).

Table 3- Structure and effect of ibogaine analogues on DAT surface expression

Ibogaine Ibogamine Noribogaine Emax=194% Emax=202% Emax=251%

Frovatriptan Pinoline Harmol Emax=103% Emax=141% Emax=54%

Harmine Tetrahydroharmine PAL 709 Emax=76% Emax=137% Emax=97%

PAL 1407 PAL 1416 Tryptamine Emax=98% Emax=127% Emax=104%

Experiments were conducted in the same fashion as those pertaining to the bupropion analogues. HEK cells expressing YFP-HA-βLAC-DAT were incubated overnight with different concentrations of drugs.

Compounds were dosed in log and half-log dilutions starting at 100µM final concentration. It was of particular interest to ascertain whether 1) any of the ibogaine analogues could display a higher Emax and/or EC50 2) if the β-carboline analogues could also elicit an effect on surface expression considering their departure from the DAT ligand classification. As the backbone of interest in this experiment, ibogaine has already been shown to increase WT DAT with an Emax of 187% [61] which served as a tool in selecting ligands that aren’t conventionally known as DAT ligands [139]. Furthermore, after assay optimization in our hands, Ibogaine achieved an Emax of 194% (Figure 8A). Its analogues, ibogamine

(202%) and noribogaine (251%), were also very effective at increasing WT DAT surface expression with the latter being our most effective compound. β-carboline analogues pinoline (141%), frovatriptan

(103%), harmol (54%), harmine (76%) and tetrahydroharmine (137%) had varied success. Tryptamine analogues were mostly ineffective except PAL 1416 (127%) (Table 3). Dose-response curves were conducted to assess potency of these hit compounds (Figure 8B). Similar to the bupropion analogues, there were no significant changes in EC50 values between ibogaine and any of the hit compounds.

Figure 8- Effect of overnight ibogaine analogue treatment on YFP-HA-βLAC-DAT surface expression

Figure (A) Summary of the maximum observed effects of ibogaine analogues on WT DAT surface expression. HEK293 cells stably transfected with βLAC-WT-DAT were treated with drugs at a final concentration of 100µM. Drugs were dissolved in DMSO to a final concentration of 1%.Data are means ± S.E. *, p < 0.05; n=3-6. (B) Concentration-effect curves for compounds of interest. Data were fitted to one-site dose-response non-linear regression curves using GraphPad Prism. n=3-6

3.1.3 Selectivity of DAT pharmacological chaperones

Given the variety of chemical compounds that can increase DAT surface expression (Table 2 &

Table 3), it is of interest to assess whether the effects are selective for DAT or not. Pharmacological chaperones rescue in a selective manner whereas chemical chaperones have widespread effects on many different proteins. To address this, we tested the set of hit compounds from the bupropion and ibogaine backbones on the β2-adrenergic receptor (β2AR), an unrelated membrane protein. β-LAC-β2AR and β-LAC-DAT constructs were used in parallel to assess surface expression. None of the DAT hits, except ephedrine, were able to induce a significant increase in β2AR surface expression at corresponding DAT chaperone doses (Figure 9). Ephedrine might act as a general chemical chaperone or may cause ER stress and induce the unfolded protein response, both of which could explain its dual effect on β2AR and DAT surface expression. Furthermore, the effective dose of ephedrine (1mM) is likely high enough to induce ER stress. We used alprenolol [122] and isoproterenol [140] as positive controls of compounds that increase or decrease β2AR levels, respectively .

300 * DAT 2AR * * 200 * * * * * * * * * * * * 100 *

Surface Expression Surface wells) treated (% vehicle of

Vehicle Pinoline PAL 594 Ibogaine PAL 1416PAL 1007PAL 1219RTI 144BRTI 101A IbogamineBupropionEphedrine Alprenolol Noribogaine Isoproterenol

Figure 9- Emax comparison between βLAC-DAT and βLAC-Β2AR of hit compounds

Summary of the maximum observed effects of hit compounds on WT DAT and β2AR surface expression. HEK293 cells stably transfected with βLAC-WT-DAT were treated with drugs at a final concentration of 100µM, except for ephedrine (1mM). Drugs were dissolved in DMSO to a final concentration of 1%. Data are means ± S.E. *, p < 0.05; n=3-6. 3.2 The Effect of DAT Pharmacological Chaperones on WT & Mutant Total Protein Levels 3.2 The Effect of DAT Pharmacological Chaperones on WT & Mutant Total Protein Levels Next, we assessed whether the changes in surface expression were also present in total protein levels. This was done to see if the observed changes in DAT surface expression were linked to an effect on protein trafficking or stability. Cells expressing YFP-HA-human DAT were treated with hit compounds for 16 hours and then lysed. Total DAT protein levels were examined using western blotting with a selective DAT antibody. Immunoblots of YFP-HA-DAT expressing cells yield 2 bands not seen on non- transfected mock cells. These bands run at 110kDa and 75kDa and represent mature and immature DAT

(mDAT & iDAT), respectively (Supplemental Figure 2). Mature DAT represents the fully glycosylated form whereas immature DAT represents the core-glycosylated form that has not yet been processed by the Golgi and thus resides in the ER. A mutation that causes DAT to be marked and degraded by the cellular quality control mechanisms would reduce the total amount of fully glycosylated DAT. The expression of mature DAT is, therefore, a readout for a compound’s ability to rescue expression levels of fully glycosylated, plasma membrane-directed WT and mutant DAT.

Effects of compounds on mature protein were assessed on four different DAT proteins. In addition to WT DAT, we assessed the effects of the compounds on A314V, L368Q and K590A mutant. In our lab, A314V has previously been shown to be rescued by bupropion and ibogaine indicating it is part of the group of DTDS mutations that are indeed rescuable [61]. L368Q was selected as there is currently a clinicalDTDS patient with this mutation which if rescued could provide evidence towards potential clinical treatment [51, 60]. K590A is a well characterised artificial, ER-retained, a trafficking-deficient mutant which could provide additional support for the mechanism of action of these drugs [61, 98].

A314V is reduced to 9% of WT DAT surface expression and L368Q/ K590A have little to none [10, 60].

These mutations are represented schematically in (Figure 10). The experimental compounds were

44 received in three different batches; Batch 1 - RTI 2-11; Batch 2- Noribogaine & Ibogamine; batch 3- tetrahydroharmine, PALs and the remaining RTIs. All compounds were initially tested in the βLAC surface expression assay and some hit compound of each batch were tested in western blots.

Figure 10- Location of the 3 DAT mutants chosen for the mature DAT expression assay. Both DTDS mutants (A314V and L368Q) are located in the extracellular side of transmembrane domain 6 and 7, respectively. K590A is located in the C-terminus, a region heavily implicated in ER export.

Altered from [141]

Bupropion, RTI 2, RTI 5, RTI 6, and RTI 11 were the hits from our initial batch of RTI bupropion analogues. First, they were tested on HEK cells expressing YFP-HA-DAT to corroborate their effects on

WT DAT which had previously been assessed in the βLAC surface expression assay. These results backed up what we had originally seen, however, it did highlight a drawback of the western blot approach. Since the magnitude of difference that certain compounds display on WT DAT is small, the western blot may not be sensitive enough to pick up the minute changes without significance-annulling error.

Nevertheless, western blots were carried out with success. As seen with the βLAC assays (Figure 6), WT

DAT protein was increased to a similar extent by bupropion, RTI 2, RTI 5, RTI 6 and RTI 11 (Figure 11).

When tested on K590A and A314V expressing cells, the compounds performed similarly. The two exceptions were the lack of significance of RTI 6 and the significantly stronger effect of bupropion over

RTI 6 and RTI 11 in the K590A DAT cells. This discrepancy may be because bupropion structure is better at rescuing the specific structural flaws of the K590A mutant or that more bupropion is able to get to the

ER, where the majority of K590A DAT is located.

Figure 11-Effect of bupropion analogues on WT, K590A and A314V DAT expression

A) Quantification of mDAT protein levels in WT DAT, K590A DAT, A314V DAT cells treated overnight with 100 µM of bupropion, RTI 2, RTI 5, RTI 6 and RTI 11. B) Representative blot of WT DAT cells. C) Representative blot of K590A DAT cells. D) Representative blot of A314V DAT cells. Drugs were dissolved in DMSO to a final concentration of 1%. mDAT = Mature DAT. Data are the means ± S.E.; *=P<0.05; n=4- 5.

Upon the arrival of noribogaine and the results that it vastly out-performs ibogaine as the best hit compound in the βLAC surface expression assay, we wanted to see if these results were consistent with respect to mature DAT total protein as well. As shown in (Figure 12), WT DAT results were consistent with the βLAC assay demonstrating that noribogaine is significantly better than ibogaine when it comes to increasing mature DAT levels. The same is observed in the K590A and A314V DAT with the former having a much larger magnitude of rescue. This is expected as there is so little K590A DAT reaching maturity that only a slight rescue is needed to outperform vehicle by 2 or 3-fold. As previously mentioned, K590A DAT was used as a tool to learn more about the mechanism of action of our hits. The enhancing effect on mature K590A protein levels demonstrates that the effect is not merely due to protein trafficking, but our drugs are able to stabilize mature DAT as well. It also suggests that the drugs act within the ER, as the rescue of K590A can be blocked by inhibiting ER export to the Golgi using the brefeldin A, a compound that inhibits protein transport from the endoplasmic reticulum to the Golgi apparatus [142]. This rescue has also been shown to be blocked by knockdown of the COPII component of SEC24D [61]. This suggests that our hits are acting as pharmacological chaperones in the ER, prior to

SEC24D-mediated ER-exit. It is important to note that bupropion and ibogaine had no effect on transcription showing that the effects seen are happening at the protein level (unpublished observation by Beerepoot et al).

Figure 12- Effect of ibogaine and noribogaine on WT, K590A and A314V DAT expression

A) Quantification of mature DAT (mDAT) protein levels in WT DAT, K590A DAT and A314V DAT cells treated overnight with 100 µM Ibogaine and Noribogaine. B) Representative blot of WT DAT cells. C) Representative blot of K590A DAT cells. D) Representative blot of A314V DAT cells. mDAT=110kDa, iDAT=80kDa. Drugs were dissolved in DMSO to a final concentration of 1%. Data are the means ± S.E.; *=P<0.05; n=3.

Similar to the first mDAT screen (Figure 13AB) WT DAT results are in agreement with the βLAC surface expression data when assessing noribogaine, PAL 594, PAL 1219, RTI 20 (Figure 7 & Figure 8).

However, the western blot results are not significant with tetrahydroharmine which did show an effect in the βLAC-DAT assay (Figure 7 & Figure 8). It is likely that the discrepancy in sensitivity between the

βLAC-DAT assay and the western blot can explain the finding of a significant result for tetrahydroharmine in one but not the other. We predict that this is going to be a trend for compounds that have a relatively small Emax in the βLAC-DAT assay. Indeed, a similar situation is observed with RTI

20. In the mutants, we also observed that noribogaine was by far the best chaperone, followed by PAL

594. This was not seen in the first round of bupropion analogues as there were discrepancies across

49 mutants (Figure 11). That being said, there was still a discrepancy in the magnitude of the difference between certain compounds across the three mutants. In K590A DAT, noribogaine was by far the most efficacious, being significantly better than vehicle and all other treatments. PAL 594, although significantly less effective than noribogaine, has a statistically higher Emax then the other 2 hits, PAL 1219 and RTI 20 (Figure 13AC). In the A314V DAT, the results were very similar to WT DAT, both in distribution and magnitude, however, RTI 20 lost its significance (Figure 13AD) which may be attributed to the same reason tetrahydroharmine lost significance (reduced sensitivity of western blot vs βLAC-DAT). Finally, the L368Q DAT cells were similar to the results observed in A314V, however, noribogaine was only significantly different from the hit PAL 1219. PAL 594 seemed to have a larger impact on L368Q DAT, in respect to the magnitude of effect relative to others, such as noribogaine (Figure 13). Overall, this data provides us with the knowledge that noribogaine and PAL 594 may be our best chaperones, not only in

WT DAT but in the mutants as well.

Figure 13- Effect of noribogaine, THH, PAL594, PAL1219 and RTI20 on WT, K590A, A314V, L368Q DAT expression

(A) Quantification of mDAT protein levels in WT DAT, K590A DAT, A314V DAT and L368Q DAT cells treated overnight with 100 µM Noribogaine, Tetrahydroharmine, PAL 94, PAL 1219 and RTI 20. B) Representative blot of WT DAT cells. C) Representative blot of K590A DAT cells. D) Representative blot of A314V DAT cells. E) Representative blot of L368Q DAT cells. mDAT=110kDa, iDAT=80kDa. Drugs were dissolved in DMSO to a final concentration of 1%. Data are the means ± S.E.; *=P<0.05; n=3-4.

Considering there is a patient with the L368Q mutation [51] we wanted to see if our only FDA- approved compound, bupropion, was able to enhance the expression of this mutant. Furthermore, we also tested our top candidate compound, noribogaine, as we know it is the best for rescuing all DAT mutations tested. In addition, we tested these conditions with and without the chemical chaperone 4- phenylbutyrate (4-PBA). 4-PBA has been shown to be a potentiator of chaperones in the past [143-146].

It is important to note that bupropion is not effective at chaperoning the L368Q mutant. This is continuous with the findings that RTI 20, a methylated bupropion, is also unable to rescue it (Figure 14).

Interestingly, PAL 594, a 6-hydroxybupropion analogue, is able to rescue L368Q DAT (Figure 14).

Noribogaine with and without 4-PBA is especially effective, as seen before.

Figure 14- Effect of bupropion, noribogaine, and the chemical chaperone 4-PBA on the clinically relevant L368Q DAT mutant

(A) Quantification of mDAT protein levels in L368Q DAT cells treated overnight with 100 µM Noribogaine & Bupropion and 10 4-PBA B) Bupropion representative blot of L368Q DAT cells. C) Noribogaine representative blot of L368Q DAT cells. mDAT=110kDa, iDAT=80kDa. Drugs were dissolved in DMSO to a final concentration of 1%. Data are the means ± S.E.; *=P<0.05; n=3. 3.3 Dopamine Uptake Inhibition of DAT Ligands

Upon testing bupropion and analogues RTI 2 to RTI11 for DAT surface expression effect, we noticed that the previously reported DAT binding and DAT uptake inhibition values of these compounds

52 had no predictive value for their chaperoning effects on surface expression. The results have been summarized in (Table 4) which is comprised of our surface expression data in addition to DAT binding and dopamine uptake inhibition data from [138, 147]. The five hits, bupropion, RTI2, RTI 5, RTI 6, and RTI

11 help highlight the major trends observed in this table. Specifically, RTI 5 and RTI 11 display similar surface expression efficacy of 140% and 131%, however, with a Ki of >10000nM and an IC50 of 6840 nM,

RTI 5 has relatively no DAT binding and poor inhibition in relation to RTI 11 with a Ki of 459nM and IC50 of 31nM. Furthermore, RTI 4, which has almost no effect on surface expression (Emax of 109%), has strong DAT binding and dopamine uptake inhibition with a Ki of 472nM and an IC50 of 271nM. This preliminary data shows that there may not be a correlation between the chaperoning efficacy of a compound and its classical DAT pharmacology.

WT DAT Surface DAT Binding (Ki - nM) Dopamine Uptake Expression Inhibition (IC50 - nM) Bupropion 144% 871 945 RTI 2 127% 1918 1295 RTI 3 112% 2195 2319 RTI 4 110% 472 271 RTI 5 140% >10000 6840 RTI 6 134% >10000 NA RTI 7 102% 1148 1033 RTI 8 107% 4133 1534 RTI 9 87% 6400 206 RTI 10 34% >10000 NA RTI 11 131% 459 31 Table 4- Comparison of Surface expression Emax, DAT binding and Dopamine uptake inhibition values for Bupropion and RTI 2-11 in WT DAT cells. DAT binding and dopamine uptake inhibition values from [138].

We then wanted to see if this trend continued with our more efficacious hits. It is important to note that dopamine uptake inhibition and DAT binding reported in (Table 4) are from reference [138]

However the results presented hereafter were conducted in our lab. Our findings for the new compounds did not differ much from those previously mentioned. This is simply exemplified in Figure

15, which is a scatter plot comparing the surface expression Emax of each hit with dopamine uptake

53 inhibition efficacy. To be more relatable to surface expression assay results, we have expressed uptake inhibition values as the percentage of vehicle-treated uptake that has been inhibited. For example, the poor DAT antagonist tetrahydroharmine only inhibits dopamine uptake by approximately 18% compared to the efficacious DAT antagonist PAL 1007 which inhibits uptake by 97%. Figure 15 is a representation of the trend that a compound’s inhibitory or binding effect on DAT is not an indication of its chaperoning capabilities, demonstrated by an R2 value of 0.07. Figure 16 shows dose responses for each hit compound and the comparison of uptake inhibition and surface expression efficacies on the left and right Y axes. Two compound curves that further depict the lack of correlation are noribogaine and RTI

12. Noribogaine has an IC50 of 29µM and an inhibition Emax of 76% compared to RTI 12 which has an

IC50 of 110nM and an inhibition Emax of 96%. The roles are reversed when the surface expression is concerned as noribogaine has an Emax of 251% vs 113% of RTI 12 (Table 2 & Table 3). This data serves as the first evidence that chaperoning and inhibiting DAT are distinct processes.

Figure 15- Scatter plot comparing the surface expression and dopamine uptake inhibition Emax of each hit compound.

Cells were treated with compounds for R2= 0.07, no significant correlation between surface expression and dopamine uptake inhibition (Emax) of each hit compound.

Figure 16- Comparing the impact on DAT uptake and DAT surface expression for our hit compounds.

Dose responses of 15 hit compounds are represented with uptake inhibition represented on the left Y axis and surface expression on the right Y axis. Doses used are half-log dilutions between 100µM and 100nM, with 2 full log dilutions yielding 10nM and 1nM. Data were fitted to one-site dose-response non- linear regression curves using GraphPad Prism.

4. Discussion, Conclusion, Future Directions

4.1. Identification of Putative Pharmacological Chaperones of DAT

Given the specific requirements of our work, the β-lactamase assay is best suited. This assay is based on the insertion of a ß-lactamase enzyme in the second extracellular loop of DAT. This is an ideal site for insertion as it is a large unstructured extracellular region of the protein. However, the second extracellular loop does play a role in substrate translocation [23] and has key glycosylation sites required for DAT stability [34]. It is therefore plausible that inserting the ß-lactamase could compromise protein function. We performed a test to determine the validity of a β-lactamase model for our studies and the

β-lactamase had no deleterious effect on dopamine uptake kinetics (Supplemental Figure 1) and is, therefore, a reliable tool for screening compounds that affect DAT surface expression.

4.1.1. Structure-Activity Relationship – Bupropion and Ibogaine Backbones

It is known that bupropion has an affinity for the norepinephrine transporter (NET) and nicotinic acetylcholine receptors (nAChR)s in addition to DAT [148]. In contrast, ibogaine and most likely noribogaine, have a diverse range of pharmacological targets including DAT and SERT [149]. We observed that analogues of bupropion and ibogaine vary in their ability to increase DAT levels. Ibogaine has also been shown to rescue levels of SERT [75]. It is important to know whether these effects are selective for these transporters or are driven by more widespread effects on protein expression. We report that all the hits that will be discussed, except for ephedrine, are selective for at least the SLC6 transporter family and are not general chaperones (Figure 9).

Certain structural features appear to be necessary for the positive effect bupropion and ibogaine demonstrate on WT DAT surface expression. Other traits seem to only modulate the already present effect. These structure-activity relationships are important in understanding why these two structurally distinct compounds are able to increase WT DAT surface expression and what an ideal WT

DAT pharmacological chaperone should look like. Furthermore, elucidating the structural features that make an ideal WT DAT pharmacological chaperone can help guide future drug discovery and may be important in understanding how to recuse DTDS mutants.

4.1.1.1. Bupropion Backbone

One structural feature that appears to be essential to the DAT chaperoning effect of bupropion is the presence of a secondary amine. For example, RTI 9 is identical to bupropion except a methyl group has been added to the secondary amine changing it to a secondary amine group. This single change effectively abolished the Emax of RTI 9 for increasing surface expression (Figure 17A). This is further exemplified by the effectiveness of RTI 20 (147%) and the loss of Emax in RTI 8 (107%) (Figure 17A). It is important to note that changing the secondary amine group to a primary amine is also deleterious as shown by the single change from RTI 5 (140%) to PAL 1270 (104%) (Figure 17B).

Figure 17- Necessity of a secondary amine for the chaperoning effect of bupropion in the WT DAT ΒLAC assay

There are many things about the phenyl ring of bupropion that can influence the efficacy of the drug. The most robust trait observed is the number of halogens present on the phenyl ring (Figure 18A).

When bupropion (144%) has another chloride added to its phenyl ring, as seen in RTI 17 (78%) and RTI 4

(110%), the chaperoning efficacy is abolished. This trend is also observed in bupropion’s main metabolite, 6-hydroxybupropion (148%). When a second chloride is added to form RTI 21 (103%) the

Emax is again reduced to no better than the vehicle. The addition of a second halogen may make binding to WT DAT energetically unfavourable and therefore reduce the efficacy. Interestingly, the second halogen substituent has a favourable effect on DAT binding and uptake properties (Table 4).

If the molecule can only be effective with one halogen, it raises the question as to where on the phenyl ring that halogen should be placed? It appears that the meta position is favourable over the para

(Figure 18B). Bupropion (144%) and 3-deschloro-3-bromobupropion (160%) are more efficacious than

RTI 3 (112%) and RTI 2 (127%), respectively where the halogen is in the para position.

A third observable trend is that the substitution of a bromine for the chloride makes the compound more efficacious while methyl substitution does the opposite (Figure 18C). A complete example of this is seen in 3-deschloro-3-bromobupropion (160%), bupropion (144%) and RTI 6 (134%).

To provide additional support, PAL 1007 (136%) and PAL 1411 (110%) show the impact of chloride over methyl and RTI 2 (127%) and RTI 3 (112%) show the impact of bromide over chloride. This improvement in Emax of Br>Cl>CH3 may potentially be explained by an increase in a positive logP value of bupropion.

Increasing the lipophilicity of a potential chaperone would enhance its ability to pass through lipid membranes and act to alter protein homeostasis in the ER. Theoretically, any substitution that would enhance lipophilicity would then improve the efficiency of the pharmacological chaperone, provided that the substitution did not alter the ligand-transporter interaction. Whether or not this is the case remains to be tested as for most of these compounds the logP values are unknown.

Figure 18- Impact of phenyl substitutions on the bupropion backbone

Moving away from the phenyl ring, we wanted to determine the necessity or effect of the alkyl chain that lies between the carbonyl group and the secondary amine on the backbone of bupropion.

Lengthening the chain seems to have a negative effect on bupropion’s ability to increase WT DAT surface expression (Figure 19). Bupropion (144%), RTI 11 (131%) and RTI 12 (113%) have methyl, ethyl and propyl groups, respectively. As discussed earlier, adding extra carbons at any position may add steric hindrance that can mask other interacting groups such as the carbonyl, primary amine and the tertiary butyl group from forming the ideal ligand-transporter interaction thus reducing its ability to properly chaperone WT DAT.

Figure 19 - Effect of alkyl chain length on the efficacy of bupropion in the WT DAT ΒLAC assay

Keeping with the idea of steric hindrance and substituents on the bupropion backbone, we then looked at the impact of the tertiary butyl group (Figure 20). Changing it to a cyclobutyl group had a dampening effect on Emax, as seen by the change in bupropion (144%) and RTI 6 (134%) to PAL 1007

(136%) and PAL 1411 (110%). Substitution of the cyclobutyl group from PAL 1411 (110%) by a methyl to form PAL 1101 (105%) further decreases the Emax. It is important to note that the change from PAL 1411 to PAL 1101 is minuscule and not significantly different. It does, however, seem that the tertiary butyl is a complementary feature of the bupropion backbone that confers some steric benefit towards ideal ligand-transporter binding.

Figure 20- Effect of the tertiary butyl substituent on the efficacy of bupropion in the WT DAT ΒLAC assay

The final structure-activity relationship that we aim to highlight is one on the 6- hydroxybupropion backbone. Although not expressly planned, this association is interesting to point out.

PAL 594 (166%), our most efficacious bupropion compound, has a significantly higher Emax than 6- hydroxybupropion (148%) (Figure 21). This can either be attributed to the removal of the hydroxyl group, the two methyl groups or all three. Potentially the removal of the hydroxyl group increases the

LogP and enhances efficacy. Future experiments need to be done to elucidate the mechanisms behind this and as our strongest bupropion compound, PAL 594 warrants further attention.

Figure 21 - Effect of the hydroxyl and methyl groups on the efficacy of 6-hydroxybupropion in the WT DAT ΒLAC assay

4.1.1.2. Ibogaine Backbone

The structure-activity relationship study thus far of the ibogaine backbone is not as thorough and extensive since commercial ibogaine analogues were not available. Ibogamine was the only commercially available analogue since it is a synthesis precursor with its empty phenyl ring. Noribogaine was a gift from Dr Deborah Mash.

The most obvious and simple structural change that confers an efficacy benefit is the substitution of a hydroxyl group for the methyl on the aromatic ring of ibogaine (Figure 22). This substitution results in noribogaine (251%) having an enhanced Emax over ibogaine (194%). Additionally, the substituent free aromatic ring of ibogamine (202%) is very similar to ibogaine (194%) suggesting that the lack of polarity conferred by the naked or methylated ring is the reason the hydroxyl substitution increases efficacy. It is likely that the polar hydroxyl group of noribogaine aids the compound in binding to DAT and promotes more efficient ligand-transporter interactions.

Figure 22-Effect of the hydroxyl substitution on the efficacy of ibogaine in the WT DAT βLAC assay

One component of the ibogaine structure that seems to explain part of its Emax is the chair-like substituent on the heterocyclic ring. Removing this group from ibogaine (194%) to form pinoline (141%) greatly reduces the chaperoning ability of ibogaine (Figure 23). It is also important to note that ibogaine has a 7-membered heterocyclic ring where pinoline has only 6. This could potentially contribute to some of the effect we see in this comparison, however, the steric impact of an extra carbon in the heterocyclic ring would be minimal compared to the bulky chair-like substituent and thus we believe the major impact of this change is due to the latter. That being said, it would be of interest to test analogues with a

7-membered heterocyclic ring to truly make this distinction. This could prove to be difficult as commercial analogues with such specific requirements are unavailable and custom synthesis by a medicinal chemist might be necessary.

Figure 23- Effect of the chair-like substituent on the efficacy of ibogaine in the WT DAT ΒLAC assay

Given that ibogaine has safety concerns and is not a very suitable drug for human consumption

[150], we searched the FDA library for compounds that contained similar structural features. The search yielded one reasonably similar drug called frovatriptan which is recommended for the perimenstrual

63 prophylaxis of menstrual attacks in women with pure menstrual migraine or menstrually related migraine [151]. Unfortunately, this compound was no better than vehicle in the βLAC surface expression assay. Structurally it most resembles pinoline which is depicted in Figure 24. It seems placing the secondary amine outside the previously heterocyclic ring in addition to substituting an amide for the methoxy group on the benzene ring abolishes all efficacy.

Figure 24- Comparison of pinoline and the FDA-approved frovatriptan

Interestingly, the β-carbolines seems to have a varied effect on WT DAT surface expression. The endogenously expressed pinoline [152] was able to increase WT DAT surface expression by 141%. This finding sparked our interest because pinoline has been reported as a selective monoamine oxidase inhibitor [153] and not a ligand of DAT [154]. In addition, it is a strong antioxidant with few deleterious effects [155, 156]. Another interesting finding was the discrepancy of effects between tetrahydroharmine (137%), which is very similar to pinoline, and its analogues harmine (76%) and harmol (54%). Harmine is well documented to inhibit cellular replication [157-159]. This could pose a problem in our βLAC assay as cells are incubated overnight with drugs and a reduction in cellular replication over this period would present as a reduction in DAT surface expression, when in reality there are simply fewer cells. This is similar to the reduction in WT DAT readout exhibited by some bupropion analogues that are toxic due to insolubility. In this case, the readout is lower not because the cells have not replicated but because they have died. A subsequent βLAC assay was done to determine the cellular viability and count with B-carboline treatment. Cells were counted before and after the

64 overnight incubation (Supplemental Figure 3). Harmine and harmol did have a significant reduction in cell count, while frovatriptan and pinoline were unaffected. Interesting, although not significant, tetrahydroharmine showed a trend towards a reduction in cell count. It is therefore possible that the structural differences between tetrahydroharmine, harmol and harmine contribute to varying degrees of cell cycle arrest. It is possible that all 3 compounds can increase WT DAT surface expression, however, this is compromised and masked by their effect on cellular replication. HEK cells replicate every 24 hours so it is possible that with an overnight incubation a reduction in cell count by up to 2 fold could be observed [160]. Given the marginal reduction in tetrahydroharmine cell count, it is possible this compound increased DAT surface expression by slightly more than its reported Emax of 137%.

Structurally, this variation may be represented by the aromaticity of the harmol (54%) and harmine

(76%) rings. (Figure 25). Pinoline (141%) and tetrahydroharmine (137%) have flexible, non-aromatic rings which may contribute to their improved efficacy over harmine and harmol or their lack of cell cycle inhibition. It is important to note that Pinoline (141%) and tetrahydroharmine (137%) have secondary amines embedded in the flexible ring, whereas harmol (54%) and harmine (76%) do not. This is likely not the driving factor between the compounds as Ibogaine, Noribogaine and Ibogamine have flexible, non- aromatic rings but no secondary amines (Figure 22). These compounds share a very strong Emax and no effect on cellular replication.

Figure 25- Effect of the heterocyclic ring on the efficacy of ibogaine and β-carboline in the WT DAT ΒLAC assay

A final structure-activity relationship observed in this study is a preliminary observation that tryptamine analogues generally don’t have efficacy in the βLAC surface expression assay for WT DAT.

Tryptamine (104%) was chosen since it has a slight resemblance to ibogaine analogues and that the major change is the deconstruction of the heterocyclic ring and chair-like substituent. PAL 709 (97%) resembles a deconstructed ibogaine with similar features such as a benzene mounted methoxy group, a

5-membered central heterocyclic ring with a secondary amine and the deconstructed heterocyclic ring with a tertiary amine and chair-like substituent. PAL 1416 (127%), under the same structural assumptions as PAL 709, resembles noribogaine and performs 30% better than its congener (Figure 26).

It is possible that PAL 1416 and PAL 709 could achieve a similar shape to noribogaine in an active site that favours this conformation due to its highly flexible carbon chain and tertiary amine. The difference observed could be explained by the hydroxyl group of PAL 1416 (127%) replacing the methoxy group of

PAL 709 (97%). This change could provide the enhancement needed to make a noticeable impact on WT

DAT surface expression, as it has been shown to improve the efficacy of noribogaine (251%) over ibogaine (194%) (Figure 22). The negative effect of the deconstructed heterocyclic ring of tryptamine is obvious and the fact that the one enhancing structure-activity relationship we have found for the ibogaine backbone (methoxy → hydroxyl) has already been incorporated into the low efficacy, tryptamine analog PAL 1416 (127%), suggests that further work on the tryptamine backbone might be futile.

Figure 26- Effect of the deconstructed heterocyclic ring on the efficacy of tryptamine analogues in the WT DAT ΒLAC assay

4.1.2. DAT Chaperones Increase Mature Protein Levels of WT and Mutants

With respect to the major structure-activity relationship trends that are essential for chaperone efficacy, such as the secondary amine of bupropion or the hydroxyl substitution and chair-like

66 substituent of noribogaine, it is very probable that they are conserved among most rescuable DAT mutants. They are defining features of the chaperoning efficacy of these drugs and would, therefore, contribute to Emax values of most rescuable mutants. This is exemplified by the evidence that noribogaine is the most efficacious compound for all tested mutants. However, the subtler structure- activity relationships observed in the WT DAT βLAC assay may not hold true for other mutations. For example, bupropion being of equal Emax to RTI hits in WT and A314V DAT but significantly better in

K590A (Figure 11). Moreover, the fact that bupropion is unable to significantly rescue L368Q but can rescue other DAT mutations may be a testament to the individual structural deficits each mutation imparts on the DAT. The structural features that contribute to the chaperone promoting qualities of the ligand-transporter interaction between bupropion and WT, A314V and K590A DAT may not be present in that of L368Q. Furthermore, RTI 20, a methylated bupropion, is also unable to rescue L368Q just like its parent drug. However, PAL 594, a 6-hydroxybupropion analogue is effective, further demonstrating the importance of the specific ligand-transporter interactions involved in the chaperoning of each DAT mutant. A mutant that deviates too far from the structure-activity relationship features of WT DAT may be too far gone and dysfunctional at the plasma membrane. It is important to understand that most of our compounds are DAT blockers, meaning they will inhibit DAT to a certain extent when present. This means that the drug needs to be washed off before the rescued transporter can function efficiently. The transporter will, therefore, need to be somewhat stable on its own after the drug is washed off. If the mutation has compromised it heavily, it may be dysfunctional and immediately degraded. Assuming this to be true, WT DAT chaperones should be useful for most mutations that can indeed be rescued to a functional state. Further studies are needed and should be aimed at developing a sensitive enough valid high-throughput method for testing the effects of drug libraries on specific DAT mutants. After all, DTDS is a very rare condition that could be the result of any given DAT mutant. Therefore, testing drugs on

67 one single mutation is unlikely to make a huge impact. A method for screening 20-30 compounds in many mutant models would be the next big step.

4.2. The Discrepancy Between Dopamine Uptake Inhibition and DAT Chaperoning Effect

The IC50 of bupropion is 0.6µM when it comes to dopamine uptake inhibition of the DAT [138].

Conversely, its EC50 for increasing DAT surface expression is 50µM [61], almost two orders of magnitude larger. It has been shown in our lab that bupropion is able to increase SERT total mature protein levels despite having no appreciable inhibitory effect [61, 98]. It is important to note that this effect is one of a pharmacological chaperone and not a general widespread effect on protein expression. The recent crystal structure of SERT has helped identify an allosteric site within the protein [79] which may already be the site of SERT allosteric modulation [161]. This, instead of the orthosteric site, could be the target for pharmacological chaperones. Recent studies have identified a set of partial agonists and allosteric modulators of DAT [76, 78, 162] and considering DAT shares a significant sequence homology with SERT, it is plausible that DAT also contains an allosteric site [16]. The strong DAT chaperone, ibogaine, seems to weakly inhibit SERT and DAT in a non-competitive manner, which could be mediated by another binding site [75]. A two-site model would explain these results and those seen in (Table 4, Figure 15,

Figure 16), the discrepancy between chaperoning and inhibitory qualities of a given compound.

Noribogaine has a strong chaperone effect of 251% and an un-potent inhibitory effect with an IC50 of

29µM. Conversely, PAL 1007 has an IC50 of 150nM, two orders of magnitude more potent than noribogaine, yet its chaperoning effect is less than half of noribogaine’s. Conventionally, this discrepancy could be explained by the differences in binding affinities between the extracellular transporter and the

ER-resident intermediates. Intracellular changes in K+ and Na+ concentrations alter the binding affinity of inhibitors[163, 164]. Furthermore, the immature intermediates that are the target for pharmacological chaperones are likely to have different affinity for inhibitors due to incomplete folding, unprocessed glycosylation, and interaction with chaperone proteins. Generally, pharmacological

68 chaperones have lower potency than expected based on their binding affinities [96]. This can explain the dosing discrepancy of inhibitors and chaperones but not the lack of correlation between the features.

An alternative or supplementary explanation is that of a two-site model, in which each compound is more suited for one of the two sites. It is likely that the chaperoning site is relatively unstructured and creates weak transient bonds with ligands which explains why so much more drug is needed to see a chaperoning effect. This could explain the discrepancy between IC50 and the chaperoning Emax of a given compound as well as the lack of correlation between effects. If true, this would be a major finding in the field of monoamine transporters, not only further proving the existence of an allosteric site on DAT but confirming the importance of it for pharmacological chaperoning.

4.3. Clinical Potential of Bupropion & Noribogaine

Bupropion is an FDA-approved compound that has a long history of clinical use and is indicated for depression as well as smoking cessation [148]. It has been used in pediatric populations with no serious safety issues. The only major side effect of bupropion treatment is a risk of seizures and this is only at higher doses [165]. Bupropion is able to reach a human serum concentration of approximately

0.5-1 µM after a single oral dose of 150mg [166-168]. Animal studies have shown that bupropion can reach 15-20 times serum concentrations in the brain, resulting in potential concentrations of 15/20 µM, which is within the pharmacological chaperoning range we have observed. Furthermore, the major metabolite 6-hydroxybupropion accumulates at levels 10-100-fold of bupropion and is responsible for some of bupropion’s pharmacological effects [169, 170]. 6-hydroxybupropion has similar efficacy to bupropion in our surface expression assay (Figure 7). Bupropion is definitely the compound with the shortest road to clinical use as it is already approved by the FDA for off-label use and many more harmful substances have already been tried in patients with DTDS [58]. However, this does not mean it would be the most effective in treating DTDS. The efficacy of bupropion and 6-hydroxybupropion is low at 144% and 148%, respectively. Furthermore, at the doses achievable in the brain it may be lower. The

6-hydroxybupropion analogue, PAL 594, exhibits a stronger efficacy at 166% and warrants more attention, even though it is a long way from clinical use. Drugs like ibogaine (194%) and its O-desmethyl analogue noribogaine (251%) have double and triple bupropion’s surface expression efficacy, respectively. These compounds thus spark our interest greatly and justify further investigation.

Ibogaine was once a highly touted anti-addictive substance but has recently fallen out of favour due to cardiotoxic effects [150], however, it is still being used on a large scale for treatment for drug addiction in private treatment centres [171]. Ibogaine blocks hERG K+ channels in the heart which are vital for repolarization of myocardial tissue after contraction. This blockade causes a prolongation in the

QT interval (time interval between the start of the Q wave and the end of the T wave in the electrical cycle of the heart) which leads to arrhythmias, Torsade to Pointes and sudden cardiac arrest [150].

Twenty-seven fatalities have been reported following the ingestion of ibogaine, 8 can be attributed to cardiotoxic effects in which most patients had pre-existing cardiac conditions [150]. The clinical potential for ibogaine is thus low and for chaperoning of DAT, potentially non-existent as doses needed for improved surface expression are high (100-10µM). Noribogaine, the O-desmethyl metabolite of ibogaine and the most efficacious DAT chaperone discovered in this study, displays greatly improved Emax over ibogaine. Furthermore, it may be much safer than its parent compound [150]. Many recent studies have forayed into the safety and pharmacokinetics of noribogaine with intriguing results. Peak plasma noribogaine concentrations can greatly exceed those of ibogaine in rats without causing adverse events

[172]. Doses of 10mg/kg in unconditioned rats were well-tolerated compared to ibogaine. Noribogaine appears less likely to produce adverse effects associated with ibogaine with no elevation in tremors and stress axis activation [172]. In another study, done on cynomolgus monkeys, EEG patterns were within normal limits following administration of noribogaine at doses up to 320mg/kg [173].This is especially important for our purposes as it demonstrates that a chaperoning dose is well within no observed adverse effect level (NOAEL) for EEG in conscious freely moving cynomolgus monkeys [173]. Considering

70 its biological activity as an anti-addictive alternative to ibogaine and its considerably upgraded safety in animals, it is no surprise that studies involving noribogaine treatment in humans have been conducted.

In two different ascending single-dose, placebo-controlled, randomized, double-blind studies 36 healthy drug-free males and 27 patients looking to discontinue methadone treatment received oral doses of vehicle or noribogaine at 3, 10, 30, or 60 mg and 60, 120 or 180 mg, respectively [133] [134]. These studies demonstrated the rapid absorption of noribogaine, with peak plasma concentrations occurring in 2-3 hours post-administration and slow elimination with a half-life of 24-49 hours [133] [134]. It is obvious but worth noting that noribogaine is able to cross the blood-brain barrier [174]. A large volume of distribution, 1417-3086L [133], and a brain/blood ratio of 7±1 [174] shows that noribogaine can concentrate in the brain. This is important as it makes reaching a chaperone appropriate dose of 100-

10µM more achievable. In fact, rats treated with one dose of 40 mg/kg reached brain concentrations of

20µM, 10-20 times higher than their plasma concentration [175]. Furthermore, single doses of 60, 120 and 180mg can yield plasma concentrations of 0.27, 0.55 and 0.9µM, which can accumulate to as high as

5.5, 11 and 18µM in the brain [134]. These are well within the chaperoning range. No safety or tolerability issues were identified in the clinical study [133] where doses ranged from 3-60mg. In doses of 60, 120 and 180mg there was a dose-dependent increase in QTc of 0.17 ms/ng/ml, which resulted in mean QTc prolongation of 16, 28 and 42ms, respectively [134]. According to the FDA, drugs that cause a

QTc prolongation of <10ms are not clearly associated with the effect, 10-20ms prolongation are cause for caution but are approved if there is evidence for therapeutic effect, and >20ms prolongation have a substantially larger chance of being proarrhythmic (Drug-induced QT interval prolongation: mechanisms and clinical management). Drugs that fall in the latter category need to have a comprehensive dose- response data through the expansion of ECG data in phase II and III trials. It is important to note that the patients in the 60, 120, and 180mg dose study were already using methadone, a known prolonger of the

QTc interval [176]. This could potentially have contributed to the QTc prolongation observed that was

71 attributed to noribogaine treatment as methadone can prolong QTc more than noribogaine and is considered “safe” by the FDA. Even with this prolongation at higher doses, noribogaine was well tolerated [134]. Other minor adverse events were non-euphoric changes in light perception such as headache and nausea beginning roughly 1-hour post-treatment [134]. Considering DTDS is a life- threatening condition that has no available treatment, it is logical to accept some side effects if a treatment can seriously improve the clinical prognosis. Many drugs currently on the market have worse safety concerns than noribogaine and still remain in circulation. It would be advised, however, that serious ECG monitoring be implemented as part of the treatment, should noribogaine ever be used clinically for DTDS.

It is interesting to observe that removing the methoxy group of ibogaine and adding a hydroxyl group to form noribogaine (Figure 22) seems to have a positive relation with chaperoning but a negative effect on QTc. It is, therefore, reasonable to postulate that further changes to the noribogaine backbone that can increase chaperoning efficacy may decrease QTc and result in a compound with improved efficacy and safety. A preliminary structure-activity relationship study was done on a hand full of ibogaine analogues to determine their inhibition and binding properties for the hERG channel [177]. It yielded the following IC50 values: manufactured ibogaine (4.09 ± 0.69 µM); extracted ibogaine from T. iboga (3.53 ± 0.16 µM); noribogaine (2.86 ± 0.68 µM); voacangine (2.25 ± 0.34 µM); and 18- methoxycoronaridine (18-MC) (>50 µM). hERG channel binding affinities for all compounds ranged from 0.71 to 3.89 µM, suggesting that 18-MC binds to the hERG channel with similar affinity but creates a substantially smaller hERG blockade [177]. This data makes 18-MC an interesting candidate compound for DAT pharmacological chaperone studies. Furthermore, 18-MC is currently in clinical trials for addiction treatment by the companies DemeRX and Savant HWP, respectively [133]. FDA approval of 18-MC would drastically improve the clinical potential of the compound, should it show chaperoning efficacy.

4.4. Conclusions and Future Directions

DAT plays an integral role in the regulation of dopamine neurotransmission. Perturbation of dopamine homeostasis is implicated in several mental and neurological disorders, including DTDS. DTDS mutations severely compromise the structure and function of DAT, resulting in a lethal pediatric movement phenotype that currently has no treatment. Pharmacological chaperones of mutated membrane proteins have been successfully transitioned to clinical use and provide proof of principle that this approach could also be applicable to DAT and DTDS. The evidence outlined in this thesis elaborates on the proof of concept of DAT pharmacological chaperones and strives to identify the structural features of bupropion and ibogaine important for chaperoning efficacy. Our structure-activity relationship studies demonstrate many structural features that impact DAT surface expression. These include the secondary amine, single high molecular weight halogen substitution, short carbon chain and

tertiary butyl group on bupropion and

the flexible heterocyclic ring, chair-

like substituent and hydroxyl

substitution on ibogaine. Many

compounds that rescued WT DAT

surface expression were also able to

increase mature DAT protein

expression including lead candidates

noribogaine and PAL594. Additionally,

our work is also the first to provide Figure 27- PHYRE2 homology model of the human DAT created using the crystal structure of the drosophila DAT (56% homology & 100% confidence). evidence that there is no link between

DAT inhibition and chaperoning, suggesting that chaperoning could be mediated via an allosteric site

Due to the recent discovery of an active allosteric site on the SLC6 transporter SERT [79], demonstration of allosteric modulators of DAT [76, 77, 162, 178], and the observed discrepancy between DAT chaperone and inhibition efficacy (Table 4, Figure 15, Figure 16), we hypothesise that DAT pharmacological chaperones are acting through an allosteric site distinct from the conventional orthosteric site that regulates the translocation cycle. Future studies should be aimed at determining the validity of this claim as well as finding the specific site. To directly test the contribution the orthosteric site makes to chaperoning efficacy of any compound we aim to create and test a DAT construct with the following mutations: V152M, N157C, V328I and S422A. These residues are essential in the orthosteric site for cocaine binding [179]. Furthermore, mutation of specific residues in the newly crystalized SERT allosteric site may shed light on the intriguing finding that bupropion can chaperone

SERT but has no known inhibitory effect on it [79].

Besides being inefficient, testing libraries of drugs by hand is not optimized for drug discovery since the major search criteria used was a structural similarity to bupropion and ibogaine backbones. For example, PAL 1219 demonstrates considerable chaperone efficacy and bears a poor resemblance to bupropion at best (Figure 28). Given this, we may be missing out on the ideal DAT chaperone as it may not have a structural resemblance to either bupropion or ibogaine. A DAT structure-based approach may be more appropriate in this situation. A method, that would help open the horizons of our compound search, is that of computer-assisted drug design via molecular docking. This method is used to predict the predominant binding mode of a ligand with a protein given its 3D structure [180]. Using these techniques, it would be ideal to test our lead chaperone candidates on the homology model in

(Figure 27) which uses the Drosophila DAT structure. This model would be especially useful for elucidating where on the molecule the compounds interact as it is of particular interest in the debate of orthosteric or allosteric mediated chaperoning efficacy. Furthermore, molecular docking of bupropion on the crystal structure of SERT could shed light on the chaperoning site considering the paradox we

74 reported earlier where bupropion is not able to inhibit SERT.

Figure 28- Structural comparison of PAL 1219 to bupropion and ibogaine backbones

There seem to be three elusive features of an ideal DAT chaperone that could have a significant clinical impact. One, finding a compound with high efficacy that does not have serious side effects. The two most efficacious compounds in this study, noribogaine and ibogaine, have potentially cardiotoxic effects at higher doses [134]. This is the major cause for concern with prolonged noribogaine treatment and is the leading reason for pushback on the idea of trying noribogaine in patients with DTDS.

Furthermore, these patients range from infancy to adolescence, creating a larger need for caution [58].

Further studies should be done to expand the efficacy and safety of the 6-hydroxybupropion analogue

PAL 594 as it displays strong efficacy without the ibogaine history of cardiotoxicity. Two, improving potency would be an excellent fix for safety concerns. In this study, we were able to discover compounds that greatly improved chaperoning efficacy for both bupropion and ibogaine, however, improved potency remains elusive. The chaperoning EC50 remains an order of magnitude higher than the IC50 [61]. It is possible, assuming our hypothesis of an allosteric site governing chaperone-induced rescue, that improved potency might be difficult to come by. The site may itself be relatively unstructured and prevent the strong interactions needed for high potency compounds. Regardless, reducing the amount of drug needed for a significant chaperone effect would certainly reduce the safety concerns surrounding this concept. Three, the dissociation of chaperoning and inhibitory efficacy. Simply put, rescuing DAT won’t be clinically effective if the compound doing so is also a DAT blocker. We have

75 shown that there is no correlation between these features (Figure 15), which suggests that a compound with a high chaperone and low inhibitory efficacy is out there. Furthermore, a large-scale screen was also able to dissociate pharmacological chaperoning activity from antagonist activity for the GPCR V2R

[181]. One issue with our study in terms of finding a DAT chaperone that also doesn’t inhibit DAT is our sourcing. Apart from our individually collected ibogaine analogues, the rest of our compounds have come from our collaborator Dr Blough who has sent us compounds that are part of a library designed to inhibit DAT. Therefore, it is likely that most compounds we received had some inhibitory effect on DAT and would reduce our chances of finding a DAT chaperone with no inhibition properties. If stabilizing the inward-facing conformation of DAT is integral to the chaperoning effect, then an ideal chaperone may be more elusive. A drug that stabilizes any state of a translocation cycle may have an impact on the rate of transport. It is with great interest that a set of 4-quinazolinamine compounds demonstrated that differential regulation of the DAT translocation cycle was possible [76, 77, 162, 178]. This finding improves the outlook for discovering a safe and potent DAT chaperone with low inhibitory properties.

Certain future experiments may prove useful in finding this ideal compound.

A final future direction is the inevitable next step in the DAT chaperoning field, a mouse model.

Our lab has constructed a mouse model of the human DTDS mutant A314V. The corresponding mutant, mDAT A313V, provides an excellent medium through which we can translate our findings into HEK293 cells to a potential therapeutic intervention. Another group has demonstrated that noribogaine can rescue DAT L368Q in Drosophila [108, 182]. We are currently characterizing our DTDS mouse model. It appears they have many interesting behavioural and molecular traits (Supplemental Figure 4) some of which resemble the well-characterized DAT-KO and DAT-knockdown animals (10% DAT) [59, 130].

Interestingly, the model does not display the expected reduced locomotor activity with cocaine administration. Overall, the DTDS mouse model has high similarity with human aspects of the condition.

Furthermore, the locomotor phenotype is a great tool to determine the degree of transporter rescue.

The one caveat with the model is that rescue would not necessarily predict rescue of all other mutants.

Our data suggest that a strong enough chaperone such as noribogaine or PAL594 will be able to rescue all rescuable mutants. It is of note that many mutations cannot be rescued as their structural deficits are far too great to allow them past the cellular quality control mechanisms. None the less, if mouse model pharmacological chaperoning of DTDS mutants can be demonstrated, it would be a great step forward on the path to potentially helping the young patients who suffer from the horrible condition that is

DTDS.

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Appendix

Bupropion Inhibition Curve 120 BLAC IC50- 0.84µM 100 WT IC50- 1.3µM 80

40 BLAC DAT WT DAT

Dopamine Uptake Dopamine 20

(% of vehicle treated(% well) vehicle of 0 -8 -7 -6 -5 -4

Supplemental Figure 1- Saturation dopamine uptake experiment of YFP-HA-βLAC-DAT after 30 minutes of incubation with vehicle or bupropion.

Supplemental Figure 2- Glycosidase Digestion of YFP-HA-DAT immunoblot bands

Lane 2 represents mock non-transfected HEK293 cells. Lane 3 represents undigested YFP-HA-DAT cells. Lane 4 is digested with Endo H and lane 5 is digested with PNGase F. The upper band (110kDa) represents the mature, fully glycosylated mature DAT whereas the lower band (75kDa) represents the core glycosylated, ER-retained, immature DAT. Adapted from ([109]).

By digesting the protein lysate with glycosidases peptide N-glycosidase F (PNGase F) and EndoH. PNGase F removes all glycosylation whereas Endo H only removes glycosylation not yet processed by the Golgi. When protein lysates were digested with PNGase F, they collapsed into one band, suggesting the two bands are two separate glycosylated isoforms of DAT. Endo H treatment caused no change in the upper (110kDa) band indicating that it represents the mature, Golgi processed DAT. Conversely, the size of the lower (75kDa) band was reduced, suggesting it is immature, has not undergone Golgi processing, and resides within the ER.

150

100 * *

Cell Count Cell 50 (% vehicle treated(% well) vehicle 0

Harmol Pinoline Harmine Frovatriptan

Tetrahydroharmine

Supplemental Figure 3- YFP-HA-ΒLAC-DAT cell count following 16 treatments with B-carboline analogs. Data are means ± S.E. *, p < 0.05; n=3.

Supplemental Figure 4- DTDS A313V mouse model A) Basal locomotor activity of WT and A313V mice for 60 minutes followed by a saline injection and subsequent locomotor for 90 minutes. B) Quantification of total distance in the 90 minutes following saline injection showing basal hyperactivity in A313V mice. C) Basal locomotor activity of WT and A313V mice for 60 minutes followed by the effect of an amphetamine (AMPH)(3mg/kg) injection on locomotor activity. D) Quantification of total distance for 90 minutes after amphetamine injection, showing enhanced locomotor activity in WT animals and inhibition of motor activity in the A313V mice. E) Basal locomotor activity of WT and A313V mice for 60 minutes followed by the effect of a cocaine (20mg/kg) injection on locomotor activity. F) Quantification of total distance for 90 minutes after cocaine injection, showing enhanced

88 locomotor activity both WT and A313V animals. G) Pre-pulse inhibition (PPI) data for WT and A313V mice, showing no different at 4, 8 and 16dB. Data are means ± S.E. ***, p < 0.001; n=6-12.

Figure 2

The copyrighted material in this figure was available under Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/),according to the copyright statement provided by the journal (included below). The material was adapted without modification from Figure 8 in Shan J, Javitch JA, Shi L and Weinstein H (2011) The substrate-driven transition to an inward-facing conformation in the functional mechanism of the dopamine transporter. PLoS One 6:e16350.

At the time of writing it was available at the following URL: http://dx.doi.org/10.1371/journal.pone.0016350

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