Pharmacological Study of KM822 Analogs---A Novel Class of Dopamine Transporter

Pharmacological Study of KM822 Analogs---a Novel Class of Dopamine Transporter

Inhibitors

Xiaonan Liu

June 2018

A Dissertation Presented to the Faculty of

Drexel University College of Medicine

in partial fulfillment of the Requirements for the Degree of Master

iii

DEDICATIONS

I dedicate this thesis to my parents.

ACKNOWLEDGEMENTS

I would like to thank several people for supporting me and helping me in my thesis project. First,

I would like to give utmost thanks to Dr. Ole Mortensen for accepting me in the lab, and offering me opportunities, and giving me advices in the project. He created a very nice and warm environment in the lab for me to explore in the science world.

I would like to thank, Dr. Paul McGonigle, Dr. Joanne Mathiasen, Dr. Andreia Mortensen, and

Dr. Ole Mortensen for agreeing to be a part of my thesis committee. Their guidance and feedback are very valuable for me not only for my thesis, but also for my career in the future.

I would like to extend special thanks to Dr. Paul McGonigle, for being such a supportive director of Drug Discovery and Development program (DDD). I appreciate every advice and suggestion you had for me, and those are invaluable. And special thanks to Dr. Joanne Mathiasen, she is the co-director of DDD program, and the one making great effort to bridge China Pharmaceutical

University with Pharmacology and Physiology department.

I would also like to give special thanks to Dr. Andreia Mortensen, she provided me precious advices. I would like to thank Dr. Shaili Aggrawal for being such a patient teacher, she taught me techniques in detail, as well as providing advices in my data analysis. I would like to thank my friends in the lab, especially Stacia Lewandowski, Apeksha K, Caitlyn Rice, Stwarsti Sarks for willing to share my happiness and sadness. I would also like to thank my classmate and my friends in the department, especially for their help in and outside class. v

Finally, I would like to thank my parents. They support my ideas and my endeavors both emotionally and financially. Without their encouragement and care, I would never go this far in my life.

LIST OF ABBREVIATIONS

BZT Benztropine

CNS Central Nervous System

DAT Dopamine Transporter dDAT Drosophila Dopamine Transporter

DMSO Dimethyl Sulfoxide

HSB Hybrid Structure Based

LeuT Leucine Transporter

MATs Monoamine Transporters

MDCK Madin-Darby Canine Kidney

NAc Nucleus Accumbens

NET Norepinephrine Transporter

NSS Neurotransmitter-sodium Symporter

NTT Neurotransmitter Transporter

PNS Peripheral Nervous System

SAR Structure-activity Relationship

SERT Serotonin Transporter

SmDAT Schistosoma mansoni Dopamine Transporter

SmNET Schistosoma mansoni Norepinephrine Transporter

SLC6 Solute Carrier 6

TCA Tricyclic Antidepressant

VMAT Vesicular Monoamine Transporter

VTA Ventral Tegmental Area vii

WT Wild Type

5-HT 5-hydroxytryptamine or Serotonin

viii

TABLE OF CONTENTS

LIST OF FIGURES

Figure 1. Schematic Depiction of Monoamine Transporters………………………….4

Figure 2. Model of the Conformation Cycle for Substrate Transport by the DAT...7

Figure 3. Chemical Structure, Chemical Formula and Molecular Weight of

KM822…………………………………………………………………………………..13

Figure 4. Model of Human Dopamine Transporter (hDAT) highlighting the

Allosteric Binding Pocket in the Complex with KM822 …………………………….14

Figure 5. Interaction between KM822 and W84 in the allosteric pocket…………...15

Figure 6. Dopamine Uptake Kinetics on Stable transfected MDCK hDAT Cells.…23

Figure 7. KM822 inhibits dopamine uptake in MDCK-hDAT and -hNET, and

inhibits serotonin uptake in MDCK-hSERT cells…………………………………....25

Figure 8. Comparison of IC50 of KM822 in MATs…………………………………...25

Figure 9. KM822 Affects Cocaine Dose-response in hDAT, hNET and hSERT…...28

Figure 10. DA Uptake Kinetics in presence of several concentrations of KM822….29

Figure 11. Substitutions in KM822 Structure…………………….….……………….30

Figure 12. NP-1-145 Dose-response Curve……………………………………………31

Figure 13. NP-1-146 Dose-response Curve……………………………………………32

Figure 14. Dose-response Curves of NP-1-151 Series………………………………...34

Figure 15. Dose-response Curves of NP-1-154 Series………………………………...36

Figure 16. Dose-response Curves of NP-1-161 Series………………………………...39

Figure 17. NP-1-152 Affects Cocaine Dose-response in hDAT………………………42

Figure 18. NP-1-155 Affects Cocaine Dose-response in hDAT………………………43 ix

Figure 19. NP-1-163 Affects Cocaine Dose-response in hDAT………………………44

Figure 20. NP-1-154 Affects Cocaine Dose-response in hDAT………………………45

Figure 21. NP-1-152 Affects Biotin Accessibility at W84…………………………….47

Figure 22. NP-1-155 Affects Biotin Accessibility at W84…………………………….49

Figure 23. NP-1-163 Affects Biotin Accessibility at W84…………………………….51

Figure 24. NP-1-154 Affects Biotin Accessibility at W84…………………………….53

LIST OF TABLES

Table 1. Structures of KM822 and its analogs with substitutions indicated………..33

ABSTRACT………………………………………………………………………………………7

INTRODUCTION…………………………………………………………………………….…8

Monoamine Transporters……………………………………………………………….8

Dopamine Rewarding System and Cocaine Abuse…………………………………….9

Dopamine Transporter Structural Studies and Dopamine Uptake Cycle…………..11

Allosteric Modulation on Dopamine Transporter……………………………………13

Discovery of A Novel Allosteric Modulation Site on Dopamine Transporter………14

KM822—A Newly Identified Allosteric Modula for the Allosteric modulation.…...15

Studies on Amino Acid Residues Outlining the Novel Allosteric Modulation Site…16

Aims of Chemical Synthesis and Study of KM822 Analogs………………………….17

MATERIALS AND METHODS………………………………………………………………15

Generation of Cell Lines Stably Expressing Human DAT, NET and SERT……….19

Expression of DAT in Human Embryonic Kidney 293 (HEK293) Cells……………19

Dose-response Assays on analogs of KM822 Compounds …………………………..19

[3H]-Dopamine Uptake Kinetics Assays………………………………………………20 x

Cocaine Dose-response Assays…………………………………………………………21

Biotinylation and Western Blot…………………………………………………….….22

RESULTS……………………………………………………………………………………….23

1. Optimization of Cell Number in 96-well Plates…………………………………….23

2. KM822 effect on dopamine transporter with or without cocaine………………...24

2.1. KM822 Inhibits All Three MATs…………………………………………24

2.2. KM822 Inhibits Cocaine’s Effect on hDAT, but not on hNET or

hSERT…………………………………………………………………………...25

3. KM822 Decreases hDAT Dopamine Uptake Kinetics ……………….……………26

4. KM822 Analogs Display Varying Potencies in Inhibiting MATs ……….…….….29

4.1. NP-1-145 and NP-1-146 Dose-response Assays…………………………..31

4.2. NP-1-150, NP-1-151 and NP-1-152 Dose-response Assays………………32

4.3. NP-1-154 and NP-1-155 Dose-response Assays…………………………..35

4.4. NP-1-161, NP-1-162, NP-1-163 Dose-response Assays…………………..36

5. Selected compounds were screened in cocaine dose-response assays…………….40

5.1. NP-1-152 Affects Cocaine IC50 in hDAT..………………………………...41

5.2. NP-1-155 Affects Cocaine IC50 in hDAT………………………………….42

5.3. NP-1-163 Affects Cocaine IC50 in hDAT..………………………………...43

5.4. NP-1-154’s Effect on Cocaine IC50………………………………………..44

6. Selected Compounds Were Tested in Biotinylation Assays……………………….45

6.1. NP-1-152 Biotinylation Assays…………………………………………….46

6.2. NP-1-155 Biotinylation Assays…………………………………………….48

6.3. NP-1-163 Biotinylation Assays…………………………………………….50 xi

6.4. NP-1-154 Biotinylation Assays…………………………………………….52

DISCUSSION…………………………………………………………………………………...53

LIST OF REFERENCES…………...………………………………………………………….60

ABSTRACT

The dopamine transporter (DAT) is a membrane protein that is responsible for the reuptake of dopamine back into the presynaptic neurons. DAT has been investigated intensively as a therapeutic target for substance abuse, depression, and attention deficit hyperactivity disorder. In the category of drugs of abuse, cocaine and other psychostimulants including amphetamine competitively inhibit all monoamine transporters, including dopamine transporter, norepinephrine transporter (NET) and serotonin transporter (SERT). By this mechanism, cocaine gives a sense of euphoria, and leads to dependency in the users. Chronic cocaine users have difficulty ceasing their cocaine abuse, and this leave cocaine addiction as a severe problem to the society. DAT has stood out to be a very promising target treating cocaine addiction.

In a previous study, a region within the DAT has been found to modulate the transporter allosterically by comparing human dopamine transporter (hDAT) to a catecholamine transporter from Schistosoma mansoni. An allosteric site within hDAT was found based on the S. mansoni study using virtual screening, a small compound, called KM822, was identified. This compound was later shown to be binding at the predicted allosteric binding site in hDAT. Additionally,

KM822 was shown to inhibit dopamine uptake and to inhibit cocaine binding to the transporter.

Herein, we tested KM822 and KM822 analogs to investigate the structure-activity relationship between the transporter and these compounds. The overall goal of this project was to study the inhibitory properties of the KM822 class of compounds on dopamine uptake, as well as their effect on cocaine inhibition of DAT using cell-based functional assays. Also, we pursued to identify the structural characteristic that favors the interaction of these analogs with hDAT by 2 conducting biotinylation assays. Future in vivo studies will be performed by colleagues to investigate the effect of these compounds on cocaine mediated behaviors in rodent models.

INTRODUCTION

1. Monoamine Transporters

Monoamine transporters (MATs) are members of the solute carrier 6 (SLC6)

neurotransmitter transporter (NTT) family, which is also designated as the

neurotransmitter sodium symporters (NSS) family because they use sodium and chloride

electrochemical gradients to induce uphill movements of substrates [1, 2].

MATs are comprised of dopamine transporter (DAT), norepinephrine transporter (NET)

and serotonin transporter (SERT). They reside on the membrane of the presynaptic

neurons. They are widely distributed in the central nervous system, and are located in

both dendrites and axons, and they are correlated with the distribution of the respective

neurotransmitter systems. They regulate extracellular monoamine homeostasis by

mediating reuptake of respective monoamines, including dopamine, serotonin [5-

hydroxytrytamine (5-HT)], and norepinephrine. Upon uptake, the accumulated dopamine,

norepinephrine, or serotonin are recycled and stored in synaptic vesicles through the

vesicular monoamine transporters (VMATs) or degraded (figure 1). Since the MATs are

essential in modulating neurotransmitter concentration, pharmacological methods that

target these transporters would be expected to modulate physiological or pathological

conditions that are mediated by these neurotransmitters. For instance, monoamine

transporters inhibitors are used for treating depression, and CNS stimulants for treating

attention deficit hyperactivity disorder. 4

Figure 1. Schematic Depiction of Monoamine Transporters. [3]. The plasma membrane monoamine transporters belonging to the SLC6 family include the transporters of DAT, NET and SERT; they are expressed in the dendrites and axons of their respective monoaminergic neurons using the gradient of sodium as their primary driving force. These transporters mediate the rapid uptake of synaptically released neurotransmitters from the extracellular space. The VMATs are members of the SLC18 family and include VMAT1 and VMAT2, which use the low pH in the vesicular lumen, driven by the proton (H+) vesicular adenosine triphosphatase, as their driving force to sequester cytoplasmic DA, NE, or 5-HT into the synaptic vesicle. These two groups of transporters have similar, but distinct, structure, with a membrane topology of 12 transmembrane a-helical domains and intracellular amino (N)- and carboxy (C)-termini.

Breakthroughs in understanding the structure and function of SLC6 have been made, which largely facilitate the understanding of the structural biology and molecular 5

pharmacology of the SLC6 NTTs, and further help to translate preclinical molecules into

the clinic (Kristensen, Andersen et al. 2011). This will be discussed in more detail in the

introduction of DAT structure (section 3 and 4).

2. Dopamine Rewarding System and Cocaine Abuse

The mesolimbic-mesocortical dopaminergic system is the most well established brain

rewarding system. This system projects from the ventral tegmental area (VTA) to the

nucleus accumbens (NAc), which is part of the ventral striatum. VTA dopaminergic

neurons also innervate several areas in the prefrontal cortex (PFC), amygdala, and the

hippocampus. These regions comprise the ‘brain reward region’, and they are inter-

connected [4]. There are a lot of studies that support that the dopaminergic system is

mediating the feeling of happiness in brain, both to natural reward (such as food,

complement) and drugs (such as cocaine and amphetamine) [5].

Cocaine is a natural alkaloid originated from leaf of coca plant. It is a drug of abuse.

National Survey on Drug Use and Health found that 900,000 American adults (over age

11) struggled with a cocaine use disorder in 2014, and amongst 39,000 were adolescents

aged between 12 and 17 that used cocaine and crack cocaine at the time of the survey.

Cocaine functions by binding to DAT, NET and SERT, and inhibit binding and reuptake

of endogenous substrates in a nonselective, competitive manner [6]. The inhibitory

effects lead to accumulation of neurotransmitters in the extracellular synaptic space.

Thereby, it results in prolonged neurotransmitter action at the receptors on the

postsynaptic neurons. For dopamine neurotransmitter, specifically, cocaine impedes 6

dopamine uptake through inhibiting DAT and NET, thus dopamine activation at

dopamine receptors cannot be terminated, causing continuous downstream signaling,

resulting in the euphoric feeling in the drug user. Beside its primary effect in blocking

MATs, cocaine has the potential to cause glutamate release in the VTA through neuronal

and glial cells in experienced users, causing a central and peripheral response, moreover,

this glutamate release could feed back to dopaminergic transmission and regulate

dopamine release [7, 8]. Cocaine also affects the peripheral nervous system (PNS),

blocking voltage-gated sodium and calcium channels and calcium activated potassium

channel, therefore, the drug can be used as a local anesthetic. However, PNS effects come

faster than CNS effect by systemic administration, despite its rapid entry into brain [9].

Given its hazardous effect on individual and the society, it is urgent to find a therapeutic

approach to treat cocaine abuse. Efforts has been made in identifying both antagonist

therapy and agonist therapy for treating cocaine addiction [10, 11]. Several drugs have

shown promising results in pre-clinical and clinical studies, such as d-amphetamine,

which is also a psychostimulant, but has a slower onset and longer duration of action than

cocaine, that can be used in agonist therapy [12]. Another drug that has shown potential

is modafinil. It is a drug that provides wakefulness with a mechanism that is not fully

understood. However, evidence show modafinil could blunt cocaine-induced euphoria in

clinical studies [13]. These clinical findings could be beneficial in alleviating cocaine

addiction, and retrospective studies would also facilitate us to understand this disease

better.

3. Dopamine Transporter Structural Studies and Dopamine Uptake Cycle 7

DAT has been studied intensively since it is a major player in the dopaminergic reward system, thus a therapeutic target for psychostimulants, antidepressants, and for substance abuse like cocaine addiction. Many studies focused on identifying the structure of DAT are based on computational homology modeling, molecular docking, and molecular dynamics simulations. In 2013, the crystal structure of the Drosophila melanogaster

DAT (dDAT) was identified that provided tools to expand our understanding of NTTs

[14]. The dDAT has shown homology with hDAT, and the hDAT uptake cycle was modelled based on dDAT using steered molecular dynamics. As demonstrated in figure

2, DAT undergoes different conformation when taking up dopamine.

The uptake cycle begins with the transporter in the ion/substrate –free (apo, i.e. free from substrate) state. In this state, the DAT is opening to the extracellular space (the outward- facing state). Na+ binding stabilizes the transporter in the fully outward-facing conformation where the extracellular gate is completely open (Na+ and Cl- bound, with a stoichiometry of 2 Na+: 1 substrate molecule per transporter), then DA binds to the primary binding site (S1 site, comprised of transmembrane domain 1, 3, 6, 8). Then substrate (DA) binding to the S1 site, shifts the structure to the occluded conformation.

Based on an equivocal/controversial model, a second DA molecule is depicted to bind to the S2 site in the extracellular vestibule, which may facilitate the translocation of the transporter to a conformation open to the cytosol, that is the full inward-facing state [15,

16]. This allows dissociation of ions and substrate from the S1 site, and leads to the apo inward-facing state. In the final rate-limiting step, the DAT shifts to the outward-facing apo state allowing the initiation of another translocation cycle. 8

Figure 2. Model of the Conformation Cycle for Substrate Transport by the DAT

[11].

Mutagenesis studies have shown that cocaine interacts with different residues on DAT, and combined with computer modeling give us some insights into the conformational change when cocaine is present at its binding site. Binding of cocaine is at the S1 site, adjacent to the sodium binding site, bordered by phenylalanine 43, alanine 44, aspartic acid 46, alanine 48, phenylalaine 319, and serine 421[17]. Furthermore, it appears that cocaine can stabilize the DAT to the outward-facing conformation, therefore impeding substrate uptake [18, 19].

Some atypical DAT ligands, such as benztropine (BZT) derivatives, were found to bind

to the S1 site of DAT, but had an opposite effect to cocaine. Instead of stabilizing DAT in

the outward-facing state, BZTs could keep DAT in the occluded state [20]. Interestingly,

though dopamine uptake is inhibited by BZTs, less rewarding effects were seen in rodent

models at effective doses of BZTs [21]. The reason for this phenomenon is still under

investigation.

4. Allosteric Modulation on Dopamine Transporter

An allosteric binding site is a region in the protein that differs from the region where

endogenous substrate binds. An allosteric modulator can interact with the allosteric site,

and change the conformation of the target, thereby hindering the interaction of

endogenous substrate or exogenous inhibitors with the target.

In DAT, several allosteric sites have been discovered. The secondary binding site (S2), as

is briefly introduced above, is a substrate binding site other than the S1 site. The S2 site

sits at a site 11Å above the S1site, between transmembrane domain 1, 3, 6, 8, and 10.

Although the mechanism is not completely clear, it was found that leucine binds to the S2

site, and triggers a transporter conformation shifting to inward-facing, and intracellular

release of Na+ using the LeuT model [22]. Intriguingly, the tricyclic antidepressant

(TCA) clomipramine inhibits DAT in a noncompetitive manner. The TCA was found to

bind to a very similar site as S2 in the extracellular vestibule, stabilizing DAT in the

inward-facing state, and slows down substrate dissociation without affecting uptake of

the substrate and sodium ions [23]. 10

Another type of allosteric modulation happens when zinc ions bind to the zinc binding

site in DAT. The zinc-binding site is between the transmembrane helices 7, 8 and

extracellular loop 2. The following amino acid side chains have been validated to co-

ordinate zinc: H193 in extracellular loop 2, H374 in the first helical part of extracellular

loop 4, and E396 in the second helix of extracellular loop 4 [24]. Extracellular zinc

inhibits dopamine uptake by altering the conformational equilibrium between the inward-

and outward-facing state upon binding to zinc binding site, and constrains the movement

of DAT [25]. The zinc binding site has been shown to be between DAT transmembrane

domain 7, 8 (H375, H396) and extracellular loop 2 (H193). Moreover, extracellular zinc

interaction can allosterically modulate cocaine effects on DAT by binding to the zinc

binding site [26].

The concept of allosteric modulation encourages us to develop compounds that can

stabilize the DAT in a conformation non-favorable for cocaine binding. We hypothesize

that people addicted to cocaine would no longer feel the high or will feel a partial high

when taking cocaine, if they also take a compound with allosteric modulation properties.

This could therefore be developed as therapy for cocaine addiction.

5. Discovery of A Novel Allosteric Modulation Site on Dopamine Transporter

The trematode Schistosoma mansoni is a human parasite that cause schistosomiasis.

Previous studies for treating the parasite infection found that DA and NE are essential

inhibitory neurotransmitters in Schistosoma mansoni. They can cause muscular relaxation 11

and lengthening in the parasite, and therefore hinder movement. Followed by DA and NE

release and receptor activation, the signaling is terminated by uptake through

Schistomsoma mansoni DAT (SmDAT) and NET (SmNET) [27].

Previous studies in our lab have cloned the gene from Schistosoma mansoni that

expresses DAT of the parasite, called SmDAT. SmDAT has a typical structure of SLC6

family of transporters, and comparing with hDAT, it has a long 120 residue intracellular

N-terminal. Functional assays with SmDAT expressed in mammalian cells showed levels

of dopamine uptake affinity is similar as hDAT. Interestingly, cocaine dose-response

results showed increased IC50 for SmDAT comparing to that for hDAT with a hundred-

fold increase (IC50 on hDAT is 0.292±0.036 µM, whereas IC50 on SmDAT 29.4±3.90

µM) [28]. This difference in IC50 suggest that the differences in structure lead to changes

in interaction between the transporters and cocaine. These findings led our group to

hypothesize that hDAT could be stabilized into a conformation similar to SmDAT, using

allosteric modulation. Furthermore, this could result in diminishing or blocking of hDAT-

cocaine interaction.

6. KM822—A Newly Identified Allosteric Modulator of hDAT

The bacteria Aquifex aeolicus leucine transporter (LeuT) model is a model that has been

utilized commonly in structural studies for dopamine transporter when drosophila and

human dopamine transporter models were not available [2, 29]. Our group used the LeuT

model to simulate the allosteric binding as they hypothesized, and screened for

compounds that fit in the proposed allosteric binding site using the hybrid structure based 12

(HSB) method. This method is a computer-based technique. It can virtually screen small- molecules with the designed pharmacophore on the target protein to identify hit compounds (hits), dock the hits to the target protein and rank the protein-ligand complex using a customized scoring method. The score gives electrostatic and hydrophobic information for the complex to assess how strong the protein-ligand interaction is. The top-ranking hits can be tested in in vitro studies [30].

Based on the preliminary findings, the group docked 462 molecules that were obtained from HSB screening to the allosteric binding site formed by residues E215, E218, D385,

V464, D476, R544, Y548, L566. Compounds KM822 and KM571 were chosen to be further investigated, as they interact with the allosteric site on hDAT with favorable electrostatic and hydrophobic interactions.

To confirm their prediction, cell-based functional assays were conducted with KM571 and KM822 (figure 3). KM571 was found to enhance hDAT affinity to amphetamine (a

DAT inhibitor and dopamine releaser) (unpublished data). KM822 also showed modulatory activities toward hDAT. However, KM822 reduced cocaine and other psychostimulants’ affinities for hDAT. Furthermore, a planarian in vivo model was used to test whether KM822 produces psychostimulant-related behaviors. Specifically, the worms can show elevated locomotion and withdrawal symptoms, behaviors that are indicative of psychostimulant properties [31]. In this in vivo model, KM822 specifically inhibits locomotion elicited by stimulants that target on DAT (amphetamine and cocaine), but not stimulants that target nicotinic acetylcholine receptors. Overall, these results led 13 to the suggestion that KM571 and KM822 are both allosteric modulators of hDAT, but their functions differ dramatically. Our current interest is to study compound that can block cocaine binding on DAT, thus we focused our attention on KM822.

Figure 3. Chemical structure, chemical formula and molecular weight of KM822

Figure 4 gives a brief depiction of hDAT-KM822 interaction. The protein is in blue, with the transmembrane domain labelled (only those shown at this view). The allosteric pocket resides between transmembrane domain 1, 12 and extracellular loop 4, 5, 6, which is away from the orthosteric binding site (dopamine and cocaine binding site). Our lab is the first to identify this allosteric pocket.

Figure 4. Model of Human Dopamine Transporter (hDAT) highlighting the

Allosteric Binding Pocket in the Complex with KM822 (grey), with surrounding

amino acid residues indicated. This picture was done using ICM-Brower (Molsoft

L.L.C, CA, USA).

7. Studies on Amino Acid Residues Lining the Novel Allosteric Modulation Site

Previous studies in our lab focused on characterizing this newly identified allosteric site

on hDAT. Using mutagenesis, selected amino acid residues within the allosteric pocket

can be mutated, and the mutated hDAT can then be studied using; 1) pharmacological

methods including uptake assays and efflux assays, for assessing the importance of each

amino acid residues by observing dopamine uptake Vmax and Km; 2) biotin labeling

assays (amino acid residues substituted by cysteine) to study whether an amino acid

residue is occupied by a compound; 3) and ligand binding assays using the mutated 15

DATs and WT DAT, to determine whether the binding property is modulated by the original amino acid residue by comparing IC50 or EC50 value. This method is used to identify the residues that display significant movement during conformational changes and therefore are likely mediators of allosteric modulation. Computer modeling results show that, the amino acid tryptophan at position 84 (W84) in hDAT sequence interacts with KM822 by p-p stacking interaction, a type of non-covalent bond, that contribute to interactions between small molecules and proteins (figure 5). Also, in cell-based assays,

KM822 reduces biotin accessibility to W84, indicating that KM822 is occupying this spot. Therefore, W84 is likely to be one of the amino acids that play a role in the interaction of KM822 and the allosteric site.

KM822

Figure 5. Interaction between KM822 and W84 in the allosteric pocket. p-p stacking interaction is formed between the benzene ring on KM822 chemical structure, and the benzene ring on the 84-tyrosine.

Other amino acids have also been studied by other people in our group, including Y88,

D385, T473, as well as some residues reported by other laboratories that are located in sites of dramatic conformational change when DAT binds to cocaine or other 16

compounds, using the same methods. Together, these studies will help us to understand

the transporter biochemical structure more clearly, and if successful, provide more

information for researchers when designing compounds in the future.

8. Aims of Chemical Synthesis and Study of KM822 Analogs

As discussed above, KM822 interacts with the allosteric site with favorable electrostatic

and hydrophobic properties, and displays inhibition in cocaine-hDAT interaction, both in

in vitro and in vivo. While the interaction of KM822 with hDAT is under investigation in

the lab, we would also like to gain a better understanding of the structure-activity

relationship (SAR) on hDAT. Therefore, with the aid of a medicinal chemistry

collaborator (Dr. Joseph Salvino, Wistar Institute), several novel analogs of KM822 were

designed and synthesized. Chemical modifications were made with substitutions

commonly used for CNS penetration, such as substitution of halogen groups and amine

groups [32].

In these studies, we focused on examining these KM822 analogs with pharmacological

methods. These results are expected to give us information on the binding properties and

enable us to evaluate their functional ability to modulate hDAT and provide structural

information on what residues in DAT and what chemical moieties on the compounds are

important and necessary for the allosteric activity.

In this thesis, we are primarily focusing on measuring the IC50 values of the analogs on

DAT, NET and SERT, as well as the interaction of several selected analogs with amino acid residue W84 in the dopamine transporter.

MATERIALS AND METHODS

Generation of Cell Lines Stably Expressing Human DAT, NET and SERT

Madin-Darby canine kidney (MDCK) naïve cells and MDCK hDAT, hNET, hSERT espressing cells were used in the cell-based functional assays. To make stable MDCK blast cell lines, DAT,

SERT or NET were subcloned into an IRES DNA vector expressing the respective transporter together with the blasticidin resistance gene. These were transfected into the MDCK naïve cells and stable clones were selected in the presence of 5 mg/ml blasticidin.

MDCK naïve cells were maintained in Dulbecco modified Eagle medium (4.5 g/l glucose, L- glutamine & sodium) supplemented with FBS (10% each) at 37° C with 5% CO2. MDCK blast

DAT, NET and SERT cells were maintained in Dulbecco modified Eagle blast medium supplemented with FBS (10%) at 37° C with 5% CO2, and with addition of 0.05% of blasticidin

(10mg/ml) (InvivoGen, CA, USA).

Expression of DAT in Human Embryonic Kidney 293 (HEK293) Cells

The coding sequence of human DAT cDNA was subcloned into pcDNA3.1(+) (Invitrogen, CA,

USA) using KpnI and XbaI sites. Plasmid DNA was linearized with PvuI and transiently transfected into HEK293 cells with Lipojet Transfection Reagent (SignaGen Laboratories, MD,

USA).

Site-directed mutants of DAT

Mutants of DAT were generated using the primer-based QuikChange method. Mutations were made on cysteine 306 to alanine (C306A), and tryptophan 84 to cysteine (W84C) (Results 19 section 6 for more details). Mutations were verified by standard DNA sequencing procedures.

Mutant constructs were cloned into pcDNA3.1(+) and expressed in HEK293 cells by transient transfection. A hemagglutinin epitope tag had been added to the N-terminal of the mutant DAT gene expression vectors, to allow specific detection of the additional DAT to the cells by probing with Hemagglutinin antibody (primary antibody) and then IgG antibody (secondary antibody).

Dose-response Assays on Analogs of KM822 Compounds

Solubility of the analogs in water were tested by dissolving 20 mM of compounds in water with various percentage of DMSO (0%, 1%, 5%, 10%). According to the solubility tests, for dissolving KM822, NP-1-145, and NP-1-46, 5% DMSO was added into RO-buffer to allow for proper dissolving (both NP-1-145 and NP-1-146 were not completely dissolved, but a higher percentage of DMSO percentage would damage cell, so no additional DMSO% was add to achieve one hundred percent solubility). Other compounds were dissolved in RO-buffer without

DMSO. MDCK hDAT, hNET and hSERT cells were plated on 96-well plates (50,000 cells per well, this number was determined in result 1). 24 h later, the medium was removed and cells were washed with phosphate buttered saline [(137 mM NaCl, 2.7 mM KCL, 4.3 mM Na2HPO4,

1.4 mM KH2PO4, pH was adjusted to 7.4) containing 0.1 mM CaCl2, 1 mM MgCl2 (PBS-CM buffer)]. Dose-response assays were performed in the presence of several concentration of the compounds (NP-1-145 and NP-1-146 concentration ranged from 2 nM to 2 mM, NP-1-150, NP-

1-151, NP-1-152, NP-1-161, NP-1-162 and NP-1-163 concentrations ranged from 1nM to 1 mM). After a 10 min pre-incubation with the compounds, the uptake assays were initiated by the addition of 50 nM [3H]-dopamine for cells expressing hDAT and hNET. For cells expressing hSERT, 50 nM [3H]-serotonin was added. After a 10 min period, cells were washed and 20 solubilized in scintillation cocktail. Plates were counted using a Wallac 1450 MicroBeta liquid scintillation counter (PerkinElmer, UK). To determine IC50 values of the inhibition by the compounds, using Log[analog concentration] numbers as X axis and radioactive response as Y axis (Log 0 value in all the experiments were taken as -6.7), data were fitted to a Hill equation by nonlinear regression analysis using GraphPad Prism, version 6 (GraphPad Software, CA, USA).

IC50 values are given as mean ± S.E.M. of at least three independent experiments.

[3H]-Dopamine Uptake Kinetics Assays

Uptake assays were performed using both MDCK naïve cells and MDCK hDAT cells. The cells were plated in 96-well plates at a density of 10,000, 20,000 or 50,000 cells per well. One day later, the medium was removed and the cells were washed with PBS-CM buffer. To determine the optimal cell number per well, uptake experiments were initiated by adding a mixture of [3H]-

Dopamine and non-radiolabeled dopamine (final concentration is 0.1 µM of [3H]-Dopamine and

9.9 µM of non-radiolabeled dopamine) in RO-buffer (PBS-CM solution containing 50µM ascorbic acid and 5µM catechol-O-methyl transferase inhibitor RO 41-0960). Cells were incubated for 10 min at room temperature. Uptake reactions were terminated by washing twice with PBS-CM. Then cells were solubilized in scintillation cocktail and counted on a Wallac 1450

MicroBeta liquid scintillation counter. For studying the KM822 effects on DA uptake by hDAT,

4 different concentrations of KM822 solutions (0 µM, 1 µM, 5 µM, 25 µM respectively) were added 10 min before the dopamine mixture, and the rest of the procedures were the same.

Assuming Michaelis-Menten kinetics, the data were analyzed using nonlinear regression with

GraphPad Prism. The Michaelis constant [33] and the maximum velocity (Vmax) values were calculated by Graphpad Prism 6 simultaneously. The averaged results for 3 different experiments 21 were calculated with Microsoft Excel (Microsoft, WA, USA) and were fitted into Miclaelis-

Menten kinetics in Prism, version 6. Vmax and Km values are given as mean ± S.E.M. of at least three independent experiments.

Cocaine Dose-response Assays

For these assays, selected KM822 analogs and cocaine were diluted in RO-buffer. Cocaine was diluted to a concentration range from 1 nM to 1 mM (10-fold dilution each time) and 0 nM, and the analogs were diluted to their respective IC50 and twice IC50 concentrations. MDCK hDAT cells were plated on 96-well plates at density of 50,000 cells per well. 24h later, the medium was removed, and cells were washed with PBS-CM. The experiments were initiated by addition of either KM822 analogs or cocaine, and the cells were incubated for 10 min at room temperature.

Afterwards, [3H]-dopamine was added at a final concentration of 25 nM, and the cells were incubated for 10 min at room temperature. The reactions were terminated by removal of the solutions and washed twice with PBS-CM. Cells were solubilized by adding scintillation cocktail and counted on Wallac 1450 MicroBeta liquid scintillation counter. Data were fitted to a Hill equation by nonlinear regression analysis using GraphPad Prism, version 6, using Log [cocaine concentration] as the X axis and amount of radioactivity as Y axis (Log 0 value in all the experiments were taken as -6.7). IC50 values are given as mean ± S.E.M. of at least three independent experiments. Statistical significance between groups was assessed using unpaired T- test.

Biotinylation and Western Blot 22

Biotinylation is an assay that utilizes the strong bound formed between biotin and thiol group on cysteine to isolate the protein of interest. Biotinylation was performed on HEK293 cells transiently transfected with cDNAs of wild type hDAT or C306A and W84C/C306A mutants.

The cells were seeded into 6-well plates and cultured to 80% to 90% confluence. To improve cell attachment, plates were coated with poly-d-lysine solution (final coating concentration was 2

µg/ml) for one hour, and washed with PBS before plating. 24 hours after plating, HEK293 cells were transiently transfected with different mutants (see above). On the third day after plating

(around 72 hours), the cells were ready for biotinylation assays. Cells were cooled at 4°C for 5 to

6 minutes, and washed twice with cold PBS-CM, and incubated with either vehicle or compounds (NP-1-152, NP-1-154, NP-1-155, NP-1-163, concentrations are indicated in the results figures) for 10 minutes, then incubated with 625µM biotin MTSEA (Biotium, CA, USA) in PBS-CM for 10 min at 4°C. After incubation, the solution was completely removed from the wells, and then cells were incubated in 1mM dithiothreitol solution for 2 to 3 min. After a final wash with PBS-CM, cells were harvested and lysed in 600 µl TNE lysis buffer (10mM Tris, 150 mM NaCl, 1mM EDTA, pH 7.5) with protease inhibitor at 4°C for 40 min, followed by a 10-min centrifugation of 12,000 ´ g. The supernatants were divided into two portions: a 30-µl (5% of total lysed cell) aliquot was saved as lysate input; 450-µl (75% of the total lysed cell) supernatant was incubated with 50 µl of a 50% slurry of NeutrAvidin-agarose beads (Thermo Fisher

Scientific, PA, USA) overnight at 4°C. The beads were washed three times with 1 ml of TNE lysis buffer, with a final wash with 1 ml of PBS-CM. The biotinylated proteins were eluted with

20 µl NuPage SDS sample buffer (Thermo Fisher Scientific, PA, USA) at 65°C for 10 min, then

10 µl of each protein sample was loaded into the gel, where they were separated by LDS-PAGE sample buffer (Thermo Fisher Scientific, PA, USA) according to standard procedures, and 23 transferred to PVDF membranes. Membranes were then probed with anti-hemagglutinin (HA)

Epitope Tag, and anti-mouse IgG antibody (Cell Signaling Technology, MA, USA).

Chemiluminescent images were analyzed with LI-COR Image Studio software (LI-COR, NE,

USE) to measure integrated density values of hDAT bands. The biotinylated samples were normalized to percentage of total samples. At least two experiments were done for each compound. The densities of the western blot bands were used to conduct unpaired T-test for testing significance.

RESULTS

1. Optimization of Cell Number in 96-well Plates

In order to get the best results in our uptake assays, we first performed experiments to optimize the number of cells plated in 96-well plates with MDCK cells stably transfected with human

DAT. The cells were seeded in the 96-well plates at densities of 10,000, 20,000 and 50,000 cells/well. Then we performed the uptake kinetic assays and plotted Michaelis-Menten curves against the transporter reuptake rates at different concentrations of substrate. The results in

Figure 5 show that, at 50,000 cells/well, dopamine uptake in hDAT has a relatively large uptake velocity (Vmax=54.79±1.60 pmol/min/well) comparing to Vmax resulting from uptake in cells plated at 10,000/well, which is 25.55±4.62 pmol/min/well, but similar with Vmax of 20,000 cells/well (Vmax=59.29±3.83 pmol/min/well). Km is the concentration of dopamine at which the reaction rate reaches half of Vmax. At 10,000 cells/well, Km equaled to 2.03±0.53 µM, and at

20,000 cells/well, Km was 2.36±0.18 µM, at 50,000 cells/well, Km was 1.52±0.34 µM. The Km values indicated how much dopamine were needed to get the maximal uptake velocity at different cell number. At 50,000 cells/well, a high Vmax was reached by relatively efficient amount of dopamine. Although Vmax at 20,000 cells/well was as efficient, its Km was larger than

Km at 50,000 cells/well. In addition, it took 2 days for 20,000 cells to reach 80% confluency in the 96-well plates, whereas it only took 1 day for 50,000 cells to reach 80% confluency.

Therefore, I decided to perform further experiments using the density of 50,000 cells/well.

40 10,000 cells/well mol/min/well)

p 20,000 cells/well 50,000 cells/well 20 H-[DA] uptake (

3 0 100 102 104 106 DA concentration (µM)

10K/well 20K/well 50K/well

V max (pmol/min/well) 25.55±4.62 59.29±3.83 54.79±1.60 ! Km ( M) 2.03±0.53 2.36±0.18 1.52±0.34

Figure 6. Dopamine Uptake Kinetics on Stable transfected MDCK hDAT Cells. MDCK hDAT cells plated in 96-well plates at different cell densities one day before the uptake assays.

Cells were incubated with 3H-dopamine and dopamine mixture at the following concentrations:

0.0049 µM, 0.0195 µM, 0.0781 µM, 0.3125 µM, 1.25 µM, 5 µM, and for 10 min. Results are fitted Michaelis-Menten curves with the average results of three different experiments, and the unit was converted from cpm to pmol/min/well. Vmax and Km are presented as mean±SEM, calculated using results from the same three experiments.

2. KM822 effect on dopamine transporter with or without cocaine

2.1. KM822 Inhibits All Three MATs

To study the effect of KM822 on dopamine uptake mediated by hDAT and hNET, and on serotonin uptake mediated by hSERT, we performed cell-based functional assays on three MATs using MDCK cells stably transfected with appropriate cDNAs (hDAT, hNET, hSERT 26 respectively). We found that the compound inhibits all three transporters in dopamine/serotonin uptake to different degrees (figure 7). KM822 is most potent in inhibiting hDAT

(IC50=3.69±0.65 µM), while less potent in inhibiting hNET (IC50=119±11.41 µM), and hSERT

(IC50=191.7±17.22 µM). The t-test results showed significant differences between DAT and

NET inhibition, as well as between DAT and SERT inhibition, as shown by the IC50 values in figure 6. This indicates that the compound is more selective to inhibit hDAT than hNET or hSERT (figure 8).

100

hDAT hNET 50 hSERT % 3H-DA or 5HT% 3H-DA uptake 0 0.0001 0.01 1 100 10000 KM822 concentration (µM)

DAT NET SERT

IC50 (mean±SEM) (!M) 3.69±0.65 119.00±11.41 191.65±17.22 LogIC (mean±SEM) ( M) 0.54±0.086 2.07±0.043 2.28±0.040 50 !

Figure 7. KM822 inhibits dopamine uptake in MDCK-hDAT and -hNET, and inhibits serotonin uptake in MDCK-hSERT cells. Cells were incubated with several concentrations of

KM822 (1 mM, 102 µM, 10 µM, 1 µM, 0.1 µM, 10-2 µM, 1 nM) for 10 min, and 50 nM of [3H]- dopamine or [3H]-serotonin was added followed by 10-min incubation. The figure was plotted using average of three independent experiments, and IC50 means and SEM was calculated using 27 the same three experiments. Results are normalized to percent of the highest [3H]-DA response in each group.

****

2.5 ****

2.0

1.5

1.0

0.5 Values of KM822 Values Inihibition 50 0.0 LogIC DAT NET hDAT

Figure 8. Comparison of IC50 of KM822 in MATs. Unpaired T-test were performed between hDAT and hNET group, as well as hDAT and hSERT group, using IC50 values from three independent experiments. LogIC50 of KM822 on hDAT was significantly lower comparing to both hNET (**** denotes p<0.0001, n=4, unpaired T-test) and hSERT (p<0.0001, n=4, unpaired

T-test).

2.2. KM822 Inhibits Cocaine’s Effect on hDAT, but not on hNET or hSERT

To study the effect of KM822 on cocaine, we performed cocaine dose-response assays in MDCK expressing hDAT, hNET and hSERT cells, in the presence/absence of different concentrations of

KM822. In Figure 8a, KM822 shifted the cocaine dose-response curve to the right dose- 28

dependently. There was a significant increase in cocaine IC50 values for inhibiting DA uptake in hDAT at both 1 µM (3.6-fold, p<0.01) and 5 µM (9.3-fold, p<0.05). However, the compound did not show a similar shift of the curve, with IC50 values remained similar in both NET and SERT cocaine dose-response assays (figures 9b and 9c, respectively). The results suggest that KM822 reduced cocaine potency or affinity at the original IC50 (original cocaine IC50 was 1.17±0.66

µM). Based on the computational modeling and the dose-response curves, we propose that

KM822 modulates the function of hDAT in an allosteric mode, but not of hNET and hSERT.

a. 150

100 KM822=0 µM KM822=1 µM KM822=5 µM H]-DA uptake H]-DA

3 50 % [

0 0.0001 0.01 1 100 10000 Cocaine (µM) [KM822]=1 !M [KM822]=5 !M Vehicle (n=3) (n=3) (n=3) Cocaine IC 50 1.17±0.66 4.20±0.60 10.87±2.92 (mean±SEM) Cocaine LogIC 50 −0.0047±0.19 0.62±0.042* 0.87±0.14 * (mean±SEM) 29

b. 150

100 KM822=0 µM KM822=1 µM KM822=5 µM H]-DA uptake H]-DA

3 50 % [

0 0.0001 0.01 1 100 10000 Cocaine (µM) [KM822]=1 !M [KM822]=5 !M Vehicle (n=3) (n=3) (n=3) Cocaine IC 50 3.41±0.45 3.16±0.29 4.36±1.36 (mean±SEM) Cocaine LogIC 50 0.53±0.042 0.50±0.029 0.62±0.092 (mean±SEM)

150

100 KM822=0 µM KM822=1 µM KM822=5 µM 50 H]-SERT uptake H]-SERT 3 % [

0 0.0001 0.01 1 100 10000 Cocaine (µM) [KM822]=1 !M [KM822]=5 !M Vehicle (n=3) (n=3) (n=3) Cocaine IC 50 18.74±4.10 16.49±8.19 16.23±12.13 (mean±SEM) Cocaine LogIC 50 1.26±0.064 1.16±0.16 1.04±0.30 (mean±SEM)

Figure 9. (a) KM822 Affects Cocaine Dose-response in hDAT. MDCK- hDAT cells were incubated in KM822 solutions for 10 min, then incubated with varying concentrations of cocaine

(2 mM, 200 µM, 20 µM, 2µM, 0.2 µM, 0.02 µM, 0.002 µM). (b) KM822 and cocaine effect on

NET. MDCK-hNET cells were incubated in KM822 solutions for 10 min, then incubated in presence of varying concentrations of cocaine (2 M, 200 µM, 20 µM, 2µM, 0 µM, 0.2 µM, 0.02

µM, 0.002 µM, 0 µM). (c) KM822 and cocaine effect on SERT. MDCK-hSERT cells were incubated in KM822 solutions for 10 min, then incubated in presence of several concentrations of cocaine (2 M, 200 µM, 20 µM, 2µM, 0 µM, 0.2 µM, 0.02 µM, 0.002 µM, 0 µM). All the curves were plotted using results from three independent experiments, and cocaine IC50 value were presented as mean±SEM, and were calculated using results from the same three 31

independent experiments. Unpaired t-test were done using logIC50 values from three different experiments (* denotes p<0.05).

3. KM822 Decreases hDAT Dopamine Uptake Kinetics

Compound KM822 was previously predicted to bind to the allosteric pocket of a model of DAT based on LeuT with a favorable electrostatic and hydrophobic property in the protein-ligand interaction modeling. We provided additional suggest for these predictions by performing dopamine uptake kinetics studies in the presence of KM822.

Dopamine uptake assays showed that KM822 reduced Vmax values in a dose-dependent manner

(Figure 10). The Vmax value showed a dose-dependent reduction at higher KM822 concentration.

Km values were consistent when KM822 concentration was between 0 to 5 µM, but dramatically increased at 25 µM. Changes in Vmax indicated that KM822 inhibited dopamine uptake in a noncompetitive inhibiting manner (Figure 10). The results were consistent with our hypothesis that the compound bind allosterically in hDAT when KM822 concentration is around its IC50.

40 mol/min/well)

µ 30 Vehicle KM822=1 µM 20 KM822=5 µM KM822=25 µM 10 H]-DA response ( response H]-DA

3 0 [ 0 5 10 15 DA concentration (µM)

Vehicle [KM822]=1 !M [KM822]=5 !M [KM822]=25 !M

Vmax (!mol/min/well) 71.55±6.12 65.76±8.71 57.90±4.15 34.98±6.03 Km (!M ) 6.22±0.69 6.97±0.58 10.54±0.78 16.53±2.78

Figure 10. DA Uptake Kinetics in presence of several concentrations of KM822. Cells were incubated in different doses of KM822 for 10 min, and then [3H]-dopamine and cold dopamine mixture (final concentration was 0.1 µM of [3H]-dopamine, and 9.9 µM of cold dopamine) was added in each well in a concentration gradient (0.3125 µM, 0.625 µM, 1.25 µM, 2.5 µM, 5 µM,

10 µM) followed by a 10-min incubation. Data were fitted to a Michaelis-Menten equation using the nonlinear regression algorithm in Graphpad Prism 6. The figure was generated using an average of four independent experiments, the unit was converted from cpm to µM/min/well.

Vmax and Km were calculated based on the same four experiments.

4. KM822 Analogs Display Varying Potencies in Inhibiting MATs

KM822 has good potency and selectivity towards hDAT (figure 8), however, its water

solubility is quite low. Low solubility of the compound could cause inconsistencies in

concentration among experiments, thus introducing random error. With goals of 33 improving solubility and understanding structure/activity relationships (SAR) of the analogs, chemical modifications of the structure of KM822 were designed and synthesized by the Salvino lab. We then screened these compounds for potency and selectivity. Importantly, the chemical structure of these analogs enabled us to study the structure-activity relationship between the allosteric binding site and ligands.

In the solubility tests, NP-1-145 and NP-1-146 need to be dissolved in at least 5% DMSO

(described in method). Analogs from NP-1-150 to NP-1-163 had better solubility, and were used in RO-buffer without DMSO in the dose-response experiments.

We classified the analogs based on the structural modification on R2 group (see figure

10). NP-1-145 and NP-1-146 had the same R2 sidechain as KM822, and differed in the

R1 group, where NP-1-145 has fluorine R1 group, and NP-1-146 has chlorine R1 group.

NP-1-150, NP-1-151, NP-1-152 all had R2 sidechain changed into an ammonic group, while they differed in the R1 group. NP-1-150 has methyl group, NP-1-151 has fluorine group, NP-1-152 has chlorine group at R1 site. NP-1-154, NP-1-155 both have dimethylamine group at R2 site, but they differs in the R1 site, where NP-1-154 has fluorine group, and NP-1-155 has chlorine group. NP-1-161, NP-1-162, NP-1-163 all have morpholine ring substituted for the R2 sidechain, and they differed in the R1 group.

NP-1-161 has methyl at R1 site, NP-1-162 has fluorine at R1 site, and NP-1-163 has chlorine at R1 site. Thus, I classified the compounds as NP-1-145, NP-1-146, NP-1-150 series, NP-1-154 series, and NP-1-161 series. 34

R1 substitution R2 substitution -CH -NH 3 2 -F -N(CH ) 3 2 -Cl -Morpholine

Figure 11. Substitutions in KM822 Structure. This figure summaries the substitutions made in the chemical structure of KM822. To the left are the substitutions for R1 group, to the right are the substitutions for R2 group. Analogs structures were permutations of those listed R1, R2 substitutions (the structure is from unpublished data)

Table 1. Structures of KM822 and its analogs with substitutions indicated. The left column

contains the name, chemical structure, chemical formula, and molecular weight of the analogs.

The right columns show the substitutions at R1 and R2 group.

In the dose-response assays, MDCK- hDAT, -hNET and -hSERT cells were incubated in

presence of varying concentrations of the compounds (1 nM to 1mM, concentration

gradient for each compound was adjusted by respective solubility and response),

followed by [3H]-dopamine or [3H]-serotonin uptake. We then plotted the dose response

curve, and obtained the IC50 values for each compound.

4.1. NP-1-145 and NP-1-146 Dose-response Assays

NP-1-145 and NP-1-146 had their R1 group changed to a fluorine or chlorine group in their

chemical structure, respectively. The changes decreased the potency of both compounds on

all three transporters (increased IC50 values), when compared to KM822. However, NP-1-145

was more selective towards hNET than hDAT or hSERT (IC50 in hDAT=1442.67±205.77

µM; IC50 in hNET=56.70±18.77 µM; IC50 in hSERT=635.53±125.65 µM) (figure 12). NP-1-

146 was selective towards hDAT and hNET, yet dramatically lost potencies on all the

transporters (IC50 in hDAT=226.23±21.77 µM; IC50 in hNET=117.98±36.89 µM; IC50 in

hSERT=1342.27±271.65 µM) (figure 13). The dramatic change of property suggests that the

R1 group might play a role in interacting with specific region in the transporters. Fluorine

and chlorine are halogens, that might form a hydrogen bond with some residues in the

transporter, also they are more nucleophilic than carbon, and this might help to explain the

results.

150

DAT 100 NE SERTT

50 H]-DA or 5HT response 5HT or H]-DA 3

% [ 0 10-10 10-5 100 105 [NP-1-145] (µM)

DAT NET SERT

IC50 (mean±SEM) (!M) 1442.67±205.77 56.36±18.77 635.53±125.65 LogIC (mean±SEM) (!M) 3.15±0.066 1.70±0.16 ** 2.79±0.081 * 50

Figure 12. NP-1-145 Dose-response Curve. MDCK- hDAT, -hNET and -hSERT cells were incubated in NP-1-145 solution with the concentration gradient (2 nM, 0.02 µM, 0.2 µM, 2 µM,

20 µM, 200 µM, 2 mM) for 10 min, then incubated in 50 nM [3H]-DA or [3H]-serotonin for 10 min. The curves were plotted using average of three independent experiments, IC50 mean±SEM were calculated using the same three experiments. Unpaired t-test were done using logIC50 values from three different experiments (* denotes p<0.05, ** denotes p<0.01).

150

DAT 100 NET SERT

50 H]-DA or 5HT response response 5HT or H]-DA 3

% [ 0 10-4 10-2 100 102 104 [NP-1-146] (µM) DAT NET SERT

IC50 (mean±SEM) (!M) 226.23±21.77 117.98±36.89 1342.27±271.65 LogIC (mean±SEM) ( M) 2.35±0.044 2.02±0.15 3.06±0.039 *** 50 !

Figure 13. NP-1-146 Dose-response Curve. MDCK- hDAT, -hNET and -hSERT cells were incubated in presence of several concentrations of NP-1-145 (2 nM, 0.02 µM, 0.2 µM, 2 µM, 20

µM, 200 µM, 2 mM) for 10 min, then incubated in [3H]-DA or [3H]-serotonin for 10 min. The curves were plotted using average of three independent experiments, IC50 mean±SEM were calculated using the same three experiments. Unpaired t-test were done using logIC50 values from three different experiments (*** denotes p<0.005).

4.2. NP-1-150, NP-1-151 and NP-1-152 Dose-response Assays

With these compounds, the R2 sidechain had been modified, thus the molecular weight was

reduced. As discussed above, removing the sidechain improved water solubility of these

compounds. 38

NP-1-150 had the sidechain removed and substituted with a primary amine group, and the R1 ethyl group had been substituted to a methyl group. This change dramatically improved water solubility, so the compound was dissolved directly in RO-buffer without addition of DMSO.

NP-1-150 displayed selectivity towards hDAT (IC50=30.88 ± 20.16 µM) compared to NET

(IC50=91.45±32.16 µM) and SERT (IC50=83.81±25.34 µM) (figure 13a).

NP-1-151 had the R1 ethyl group modified into a fluorine group. The dose-response curves showed a similar pattern as NP-1-150. The compound was more selective towards DAT

(IC50=33.59±1.42 µM), than NET (IC50=62.85±31.01 µM) and SERT (IC50=84.75±15.56

µM) (figure 13b).

NP-1-152 has a chlorine group substituted for the ethyl group at R1. The compound had greater potency as the IC50 was lower than NP-1-150 and NP-1-151. However, it showed to be more selective to DAT (IC50=20.23±10.43 µM), 2.36-times more potent than NET

(IC50=47.80±10.43 µM) and 1.6-times more potent than SERT (IC50=32.47±6.54 µM) (figure

13c).

Overall, this series of compounds displays a similar pattern of activity on the transporters, with a slight selectivity toward DAT, and less selective toward NET and SERT. IC50 values all increased compared with KM822. The results demonstrate that removal of the R2 sidechain did not abolish the inhibitory effect of the compounds on all three transporters, and partly preserved the selectivity toward DAT.

a. 150 DAT NET 100 SERT

50 H]-DA or 5HT uptake uptake 5HT or H]-DA 3 % [ 0 10-10 10-5 100 105 [NP-1-150] (µM)

DAT NET SERT

IC50 (mean±SEM) (!M) 30.88±20.16 91.45±32.16 83.81±25.34 LogIC (mean±SEM) ( M) 1.35±0.22 1.93±0.99 * 2.02±0.14 * 50 !

b. 150

DAT 100 NET SERT

50 H]-DA or 5HT uptake 5HT or H]-DA 3 % [ 0 10-10 10-5 100 105 [NP-1-151] (µM)

DAT NET SERT

IC50 (mean±SEM) (!M) 33.59±1.00 62.85±17.90 84.75±8.99 LogIC (mean±SEM) ( M) 1.65±0.12 1.86±0.16 1.92±0.045 50 !

c. 150 DAT NET 100 SERT

50 H]-DA or 5HT uptake 5HT or H]-DA 3 % [ 0 10-10 10-5 100 105 [NP-1-152] (µM)

DAT NET SERT

IC50 (mean±SEM) (!M) 20.23±5.21 47.80±13.62 32.47±3.27 LogIC (mean±SEM) ( M) 1.24±0.12 1.61±0.12 1.50±0.46 50 !

Figure 13. Dose-response Curves of NP-1-150, NP-1-151, NP-1-152. (a) NP-1-150 dose response curve. MDCK- hDAT, -hNET and -hSERT cells were incubated in several concentrations of NP-1-150 (1 nM, 0.01 µM, 0.1 µM, 1 µM, 10 µM, 100µM, 1 mM) for 10 min, then incubated in [3H]-DA or [3H]-serotonin for 10 min. (b) NP-1-151 dose response curve. MDCK- DAT, -NET and -SERT cells were incubated in presence of several different concentrations of NP-1-151 (1 mM, 100 µM, 10 µM, 1 µM, 0.1 µM, 0.01 µM, 1 nM) for 10 min, then incubated in [3H]-DA or [3H]-serotonin for 10 min. (c) NP-1-152 dose response curve. MDCK- DAT, -NET and -SERT cells were incubated in presence of varying concentrations of NP-1-152 (1 mM, 100 µM, 10 µM, 1 µM, 0.1 µM, 0.01 µM, 1 nM) for 10 min, then incubated in [3H]-DA or [3H]-serotonin for 10 min. All the curves were plotted using average of three independent experiments, IC50 mean±SEM were calculated using the 41

same three experiments. Unpaired t-test were done using logIC50 values from three different

experiments (* denotes p<0.05).

4.3. NP-1-154 and NP-1-155 Dose-response Assays

NP-1-154 and NP-1-155 had modification of the R2 sidechain in the chemical structure,

which resulted in better water solubility and smaller molecular weight. The R2 had been

changed into the dimethylamine group, and the R1 ethyl group had been substituted into

halogens.

NP-1-154 has fluorine substitution at R1. The results indicated the compound had lost

potency on hDAT, with a dramatic increase in IC50 (IC50=253.33±69.62 µM), and also lost

selectivity (figure 14a).

NP-1-155 has a chlorine substitution at R1. The dose-response results showed similar IC50

values in all three transporters (IC50 in DAT=38.53±8.56, IC50 in NET= 30.00±10.26, IC50 in

SERT= 28.27±6.44). Therefore, this compound is not selective for any of the MAT

transporters examined. 42

a. 150 DAT NET 100 SERT

50 H]-DA or 5HT uptake 5HT or H]-DA 3 % [ 0 10-10 10-5 100 105 [NP-1-154] (µM) DAT NET SERT

IC50 (mean±SEM) (!M) 253.32±40.20 144.21±33.76 96.96±21.94 LogIC (mean±SEM) ( M) 2.38±0.084 2.12±0.10 1.96±0.087 * 50 !

b. 150 DAT NET 100 SERT

50 H]-DA or 5HT uptake 5HT or H]-DA 3 % [ 0 10-10 10-5 100 105 [NP-1-155] (µM)

DAT NET SERT

IC50 (mean±SEM) (!M) 38.53±8.56 30.00±10.26 28.27±6.44 LogIC (mean±SEM) (!M) 1.58±-0.064 1.45±0.10 1.44±0.066 50

Figure 14. Dose-response Curves of NP-1-154 Series. (a) NP-1-154 dose-response curve.

MDCK- hDAT, -hNET and -hSERT cells were incubated in NP-1-154 solution with the

concentration gradient (1 nM, 0.01 µM, 0.1 µM, 1 µM, 10 µM, 100µM, 1 mM) for 10 min,

then incubated in [3H]-DA or [3H]-serotonin for 10 min. (b) NP-1-155 dose response curve.

MDCK- DAT, -NET and -SERT cells were incubated in NP-1-155 solution with the

concentration gradient (1 nM, 0.01 µM, 0.1 µM, 1 µM, 10 µM, 100µM, 1 mM) for 10 min,

then incubated in [3H]-DA or [3H]-serotonin for 10 min. All the curves were plotted using

respective average of three independent experiments, IC50 mean±SEM were calculated using

the same three experiments. Unpaired t-test were done using logIC50 values from three

different experiments (* denotes p<0.05).

4.4.NP-1-161, NP-1-162, NP-1-163 Dose-response Assays

NP-1-161, NP-1-162 and NP-1-163 also had modification at the R1 ethyl group and the R2

sidechain. The sidechain has been removed and changed into a morpholine ring. Morpholine

ring is a feature that is commonly added to improve water solubility in medicinal chemistry.

NP-1-161 had a methyl group substituted for the R1 ethyl group. The dose-response assays

showed that the compound was slightly selective towards DAT (IC50=75.49±12.95 µM), and

the potency had a dramatic decrease to DAT compared to KM822. IC50s for NET and SERT

are reduced, indicating the compound gained some potency in NET and SERT with the 44

modifications (IC50 for hNET= 90.43±12.95, IC50 for hSERT= 137.68±16.68 µM) (figure

15.a).

NP-1-162 had a fluorine group substituted for the ethyl group at R1. The dose-response results showed a similar pattern of dopamine/serotonin uptake in three transporters as NP-1-

161, but with less potency (IC50 for hDAT=97.92±14.76 µM, IC50 for hNET= 140.98 ±35.99

µM, IC50 for hSERT=168.73 ±42.22 µM) (figure 15b).

NP-1-163 had a chlorine group substituted for the ethyl group at R1. The dose-response results showed that this compound was slightly selective towards hDAT (IC50=31.82±10.07

µM), however it lost some selectivity compared to KM822 (figure 15c).

150 DAT NET SERT 100

50 H]-DA or 5HT uptake 5HT or H]-DA 3 % [ 0 10-10 10-5 100 105 [NP-1-161] (µM)

DAT NET SERT

IC50 (mean±SEM) (!M) 75.49±7.48 90.43±16.72 137.68±9.63 LogIC (mean±SEM) ( M) 1.87±0.047 1.94±0.074 2.14±0.030 ** 50 ! 45

b. 150 DAT NET SERT 100

% 3H-[DA] or 5HT% 3H-[DA] uptake 0 10-10 10-5 100 105 [NP-1-162] (µM)

DAT NET SERT

IC50 (mean±SEM) (!M) 97.92±8.52 140.98±20.78 168.73±24.38

LogIC50 (mean±SEM) (!M) 1.96±0.38 2.13±0.072 2.21±0.065 *

c. 150 DAT NET SERT 100

50 H]-DA or 5HT uptake 5HT or H]-DA 3 % [ 0 10-10 10-5 100 105 [NP-1-163] (µM)

DAT NET SERT

IC50 (mean±SEM) (!M) 31.82±5.81 77.93±5.04 110.46±14.67

LogIC50 (mean±SEM) (!M) 1.48±0.077 1.89±0.028 * 2.03±0.059 *

Figure 15. Dose-response Curves of NP-1-161 Series. (a) NP-1-161 dose-response curve.

MDCK- hDAT, -hNET and -hSERT cells were incubated in presence of several concentrations of NP-1-161 (0 nM, 1 nM, 0.01 µM, 0.1 µM, 1 µM, 10 µM, 100µM, 1 mM) for 10 min, then incubated in [3H]-DA or [3H]-serotonin for 10 min. (b) NP-1-162 dose- response Curve. MDCK- hDAT, -hNET and -hSERT cells were incubated in presence of several concentrations of NP-1-162 (0 nM, 1 nM, 0.01 µM, 0.1 µM, 1 µM, 10 µM, 100µM, 1 mM) for 10 min, then incubated in [3H]-DA or [3H]-serotonin for 10 min. (c) NP-1-163 dose-response curve. MDCK- hDAT, -hNET and -hSERT cells were incubated in presence of several concentrations of NP-1-163 (0 nM, 1 nM, 0.01 µM, 0.1 µM, 1 µM, 10 µM,

100µM, 1 mM) for 10 min, then incubated in [3H]-DA or [3H]-serotonin for 10 min. All the curves were plotted using the average of three independent experiments, IC50 mean±SEM were calculated using the same three experiments. Unpaired t-test were done using logIC50 values from three different experiments (* denotes p<0.05, ** denotes p<0.01).

As shown above, compounds NP-1-161, NP-1-162, NP-1-163 displayed improved water solubility, but had lost selectivity (NP-1-161 and NP-1-162 are selective towards DAT and

NET, NP-1-163 is selective towards DAT), with decreased potency in hDAT, and increased potency in hNET and hSERT.

Taking all the dose-response results together, we found that all compounds lost the DAT selectivity that KM822 has. Compounds with chlorine group substituted at the R1 site showed lower IC50 values, and were more selective toward DAT. NP-1-152, NP-1-155 and

NP-1-163 displayed the lowest IC50 values among their respective series. NP-1-151, NP-1- 47

154, NP-1-162 showed higher IC50 values for DAT. NP-1-154 only differs with NP-1-155 in

the fluorine group at the R1 site, but showed a great difference in IC50 in DAT than NP-1-155

(6.6-times higher at IC50).

5. Selected Compounds Were Screened in Cocaine Dose-response Assays

As shown above, KM822 could target at the allosteric site in hDAT, change the conformation

of DAT, and obstruct cocaine interaction at S1 site. Since the analogs displayed inhibition in

dopamine uptake in hDAT similar to KM822, we tested whether the analogs can also affect

cocaine interaction.

Based on the results from the dose-response assays, we found compounds with a chlorine R1

group were more potent at DAT in their respect group (the group was classified by R2

substitution, see point 4 in the Results section). We selected one compound from each group

(3 compounds in total), and tested them using cocaine dose-response assays. The compounds

are NP-1-152 (IC50 in DAT=20.23±10.43 µM), NP-1-155 (IC50 in hDAT=38.53±8.56 µM),

and NP-1-163 (IC50 in hDAT=31.82±10.07 µM). And we also selected NP-1-154 (IC50 in

hDAT=253.33±69.62 µM) for a cocaine dose-response assay as a negative control, because

this compound has a fluorine R1 group, and low in potency towards DAT.

In these experiments, we used concentrations of the compounds at both their IC50 value and

twice the IC50 value to test the compounds’ effect in modulating cocaine’s interaction with

DAT. We used two concentrations in order to study the influence of dose of the compounds

on cocaine-DAT interaction. If a higher dose has more influence on cocaine IC50, then the 48

compound effect on cocaine-DAT interaction is likely to be dose-dependent. And dose-

dependency is important in predicting compound effects at high doses.

5.1. NP-1-152 Affects Cocaine IC50 in DAT

NP-1-152 displayed reduced selectivity for hDAT (still slightly selective), but was the most

potent in inhibiting dopamine uptake in hDAT among all the analogs. IC50 value of NP-1-152

is 20.23±10.43 µM in hDAT. In the cocaine dose-response assay, we used two different

concentrations of NP-1-152, which were 20 µM and 40 µM, as well as a vehicle control. The

results show that NP-1-152 shifts the cocaine dose-response curve rightward, indicating the

compound was inhibiting cocaine interaction with hDAT. Cocaine IC50 values were obtained

from the curves. At 20 µM, the compound caused a significant increase in cocaine IC50

(p<0.01). And at 40 µM, the compound caused a 4.5-times increase in cocaine IC50, which

was also statistically significant (p<0.01). The results are consistent with what we found with

KM822, suggesting that NP-1-152 is impeding cocaine interaction, through binding to the

allosteric site. 49

150

100 Vehicle NP-1-152 20µM NP-1-152 40µM H]-DA uptake (%) uptake H]-DA 3 50

2×IC50 0 Normalized [ 10-5 100 105 Cocaine (µM)

Vehicle [NP-1-152]=20 µM [NP-1-152]=40 µM

Cocaine IC50 (mean±SEM) !M 0.17±0.04 0.49±0.04 0.76±0.08 LogCocaine IC (mean±SEM) M −0.78±0.096 −0.13±0.046 −0.32±0.037 50 ! ** *

Figure 16. NP-1-152 Affects Cocaine Dose-response in DAT. MDCK- hDAT cells were

incubated in presence of 2 concentrations of NP-1-152 or vehicle for 10 min, then cocaine

was added (at final concentrations of 0 µM, 0.001 µM, 0.01 µM, 0.1 µM, 1µM, 10 µM, 100

µM, 1 mM), then the cells were incubated in 25 nM [3H]-DA for 10 min. The curves were

plotted using average of three independent experiments, and IC50 mean±SEM were

calculated using the same three experiments. Unpaired t-tests comparing vehicle group and

two compound treated groups were done using LogIC50 of cocaine (* denotes p<0.05, **

denotes p<0.01).

5.2. NP-1-155 Dose Not Affect Cocaine IC50 in hDAT

NP-1-155 was chosen to represent its class. In dose-response assays, NP-1-155 completely

lost the selectivity on hDAT. However, the compound is much more potent than NP-1-154, 50 but they only differ at the R1 site in the chemical structure, so it would be interesting to study why such a small change could result in such a big difference for SAR purpose.

The cocaine dose-response results were conducted using two concentrations of NP-1-155 (40

µM, 80 µM), and a vehicle control group. At 40 µM, the compound had little effect on cocaine IC50. At 80 µM, the compound slightly shifted the curve to right, and increased the cocaine IC50, but it was not statistically significant (p=0.42). Therefore, we could conclude that, though the compound inhibits DA uptake through hDAT, it has little effect on cocaine binding (figure 17).

150

100 Vehicle NP-1-155 40µM

H]DA uptake (%) uptake H]DA NP-1-155 80µM 3 50

0 Normalized [ 10-10 10-5 100 105 Cocaine (µM)

Vehicle [NP-1-155]=40!M [NP-1-155]=80!M

Cocaine IC50 (mean±SEM) !M 0.19±0.05 0.19±0.07 0.27±0.06

LogCocaine IC50 (mean±SEM) !M −0.75±0.10 −0.60±0.093 −0.87±0.21

Figure 17. NP-1-155 Dose Not Affect Cocaine Dose-response in hDAT. MDCK- DAT cells were incubated in NP-1-155 for 10 min, then cocaine was added (at final concentrations of 0 µM, 0.001 µM, 0.01 µM, 0.1 µM, 1µM, 10 µM, 100 µM, 1 mM), then the cells were incubated with 25 nM [3H]-DA for 10 min. The curves were plotted using average of three 51

independent experiments, and IC50 mean±SEM were calculated using the same three

experiments. Significance was calculated using unpaired t-test with Log Cocaine IC50 values

(not significant showing as no asterisk in the figure).

5.3. NP-1-163 Dose Not Affect Cocaine IC50 in DAT

NP-1-163 had a morpholine ring on the R2 site (same structure at R2 site), and chlorine at R1 site. The results in figure 17 illustrate that this series compounds displayed lower potencies toward hDAT, and similar potencies for the other transporters, therefore lower selectivity.

In the cocaine dose-response assays, we used. The compound increased cocaine IC50 at both 60

µM and 30 µM, but both changes are not statistically significant (figure 18).

150

100 Vehicle NP-1-163 30µM NP-1-163 60µM H]-DA uptake (%) uptake H]-DA 3 50

0 Normalized [ 10-5 100 105 Cocaine (µM)

Vehicle [NP-1-163]=30!M [NP-1-163]=60!M

Cocaine IC50 (mean±SEM) !M 0.13±0.01 0.15±0.01 0.19±0.01 LogCocaine IC (mean±SEM) M −0.90±0.054 −074.±0.082 −0.85±0.085 50 ! 52

Figure 18. NP-1-163 Dose Not Affect Cocaine Dose-response in DAT. MDCK- hDAT

cells were incubated with three concentrations of NP-1-163 (0 µM, 30 µM, 60 µM) for 10

min, then cocaine was added (at final concentrations of 0 µM, 0.001 µM, 0.01 µM, 0.1 µM,

1µM, 10 µM, 100 µM, 1 mM), then the cells were incubated with 25 nM [3H]-DA for 10

min. The curves were plotted using average of three independent experiments, and IC50

mean±SEM were calculated using the same three experiments. Significance was calculated

using unpaired t-test with Log Cocaine IC50 (not significance showing as no asterisk in the

figure).

5.4. NP-1-154 Dose Not Affect Cocaine IC50

As mentioned above, NP-1-154 was used as a negative control. NP-1-154 has a

dimethylamine group at the R2 site, and fluorine substitution at R1 site. The compound

displayed IC50 of 253.33 µM in inhibiting dopamine uptake. Here we used NP-1-154 at

concentration 250 µM (IC50), 500 µM (IC100=twice IC50), as well as at 0 µM in the cocaine

dose-response assay. The results in figure 19 showed that at 500 µM and 250 µM, the

compound slightly increased cocaine IC50 in DA uptake (p=0.26 when NP-1-154

concentration was 500 µM, p=0.49 when NP-1-154 concentration was 250 µM).

150

100 Vehicle NP-1-154 250µM H]DA uptake (%) uptake H]DA

3 NP-1-154 500µM 50

0 Normalized [ 10-10 10-5 100 105 Cocaine (µM)

Vehicle [NP-1-154]=250!M [NP-1-154]=500!M

Cocaine IC50 (mean±SEM) !M 0.10±0.03 0.19±0.06 0.18±0.02 LogCocaine IC50 (mean±SEM) !M −1.02±0.20 0.74±0.091 −0.76±0.24

Figure 19. NP-1-154 Dose Not Affect Cocaine Dose-response in hDAT. MDCK- hDAT cells were incubated in NP-1-154 for 10 min, then cocaine was added (at final concentrations of 0 µM, 0.001 µM, 0.01 µM, 0.1 µM, 1µM, 10 µM, 100 µM, 1 mM), then the cells were incubated with 25 nM [3H]-DA for 10 min. The curves were plotted using average of three independent experiments, and IC50 mean±SEM were calculated using the same three experiments. Significance was calculated using unpaired t-test with Log Cocaine IC50 values

(not significant showing as no asterisk in the figure).

In conclusion, NP-1-152 displayed the most influence on cocaine in DA uptake. NP-1-155 and NP-1-163 affected cocaine dose-response to a small degree, but had a trend to be dose- dependent (not statistically significant). NP-1-154 was not a potent inhibitor for DAT dopamine uptake, and did not antagonize cocaine in the similar way as KM822.

6. Selected Compounds Were Tested in Biotinylation Assays

To study the compounds’ binding to the allosteric site in DAT, we performed biotinylation

assays using selected analogs. In the biotinylation assays, MTSEA-biotin labels cysteine

residue located in an accessible region in a protein. In our case, there are 13 cysteines in

hDAT, two of them are extracellular (C90 and C306), but C306 is the only cysteine resides in

DAT that is exposed and can be labelled by MTSEA-biotin [34]. To ensure that the intrinsic

cysteine does not get labelled and contributes to variability in our experiments, we mutated

cysteine to alanine, and used a DAT single mutation of C306A as negative control, as well as

in the targeted mutants (double mutation). As we have introduced, in our previous study,

W84 had been confirmed to interact with KM822. Herein, we tested the selected analogs

using the same experimental system. The results would help us to determine the interaction

of those analogs with W84.

The hDAT mutants C306A and W84C/C306A were previously generated in the lab and their

function is intact (the mutation did not impair the dopamine uptake function). HEK 293 cells

were transfected with wild type hDAT DNA, and the mutant DNAs. The selected compounds

were the same as those tested in cocaine dose-response assays (NP-1-152, NP-1-155, NP-1-

163 and NP-1-154). Compound solutions were made in twice IC50 value of respective

compound (40 µM for NP-1-152, 80 µM for NP-1-155, 60 µM for NP-1-163, 500 µM for

NP-1-154), which is in line with the cocaine dose response assays, so that we would be able

to study the compounds and directly compare the two assays.

6.1.NP-1-152 Biotinylation Assay

In dose-response experiments, NP-1-152 was more selective to hDAT, with an IC50 of 20.23

µM. In biotinylation experiments, a solution of 40 µM (twice IC50) was used to ensure the

inhibitory effect could be detected by biotinylation. Figure 20 (a) shows that at presence of

NP-1-152, biotin accessibility to W84C in DAT was reduced (with NP-1-152, biotinylated

DAT Western Blot density was 1.59; without NP-1-152, biotinylated DAT Western Blot

density was 3.07). To have a more direct view of the reduction, unpaired t-test was used to

compare the densities. Figure 20b shows the result of the t-test. To the left, the two columns

represent the density of biotinylated WT-DAT density in the absence/presence of NP-1-152,

and to the right, the two columns represent the density of biotinylated W84C/C306A-DAT

density at the absence/presence of NP-1-152. Biotinylated WT-DAT density was not changed

by NP-1-152, while biotinylated W84C/C306A-DAT density was significantly reduced in the

presence of NP-1-152 (p<0.05). The results indicate NP-1-152 is occupying the W84C spot

in DAT, which supports the hypothesis that NP-1-152 binds in the allosteric pocket.

b. 2.5

2.0 *

1.5

1.0

0.5 Normalized band density 0.0

WT vehicle

W84C/C306A veh WT compound treated

W84C/C306A compound treated Figure 20. NP-1-152 Affects Biotin Accessibility at W84. (a) Representative western blot of biotinylated and total cell lysates. Western blot was done using the protein sample from biotinylation assay; all the rectangles shown are from the same gel. The image is representative of 3 blots from 3 separate in vitro experiments. The number on each band represents the relative density of each band on the same gel, and is used as quantification for protein quantity. (b) 57

Quantification of western blot results. Each column was calculated with biotinylated samples normalized to respective total cell samples. Each bar is represented as a mean ± SEM on three separate measurements (i.e., each measurement was done on cells prepared on a different day).

6.2.NP-1-155 Biotinylation Assays

NP-1-155 has showed a trend of decreasing cocaine potency in the cocaine dose-response experiment (as shown in figure 17), therefore possibly inhibiting cocaine interaction by binding to the allosteric pocket and shifting the conformation of human dopamine transporter. NP-1-155 dose-response results showed that its IC50 was 38.53 µM (figure 14). In the biotinylation experiment, we tested 80 µM NP-1-155 solution, which is twice the IC50 of this compound, so that the inhibitory effect can be detected by biotinylation. Figure 21a shows that in the presence of NP-1-155, biotin accessibility to W84C in DAT was reduced with NP-1-155. The density was

0.684 without NP-1-155, with NP-1-155 the biotinylated DAT Western Blot density was 2.49).

To have a more direct comparison of the reduction, unpaired t-test was conducted. Figure 21 (b) shows the result of the t-test. The two columns on the left represent the density of biotinylated

WT-DAT density at the absence/presence of NP-1-155, and the two columns to the right, represent the density of biotinylated W84C/C306A-DAT in the absence or presence of NP-1-

155. Biotinylated WT-DAT density was significantly increased by NP-1-155 (p<0.05), while biotinylated W84C/C306A-DAT density was significantly reduced at presence of NP-1-155

(p<0.001). The increased WT-DAT Western blot density suggests that NP-1-155 might have affected the position of C306, made it more accessible for biotin to interact with. The decreased

C306A/W84C-DAT density in the treatment group indicates NP-1-155 is occupying the W84C site in DAT, and therefore consistent with the hypothesis that NP-1-155 is binding to the 58 allosteric pocket. An increase in biotinylated WT-DAT density might indicate NP-1-155 presence caused a conformation changed to a state where C306 is more available for biotin to bind. 59

b. * 5

3 *** 2

1 Normalized band density 0

WT vehicel

W84C/C306A vehicle WT compound treated

W84C/C306A compound treated 60

Figure 21. NP-1-155 Affects Biotin Accessibility at W84. (a) Representative western blot.

Western blot was done using the protein sample from biotinylation assay; all the rectangles shown are from the same gel. The image is representative of 3 blots from 3 separate in vitro experiments. The number on each band represents the relative density of each band on the same gel, and is used as quantification for protein quantity. (b) Quantification of western blot results. Each column was calculated with biotinylated samples normalized to respective total cell samples. Each column is represented as a mean ± SEM on three separate measurements (i.e., each measurement was done on cells prepared on a different day).

6.3.NP-1-163 Biotinylation Assays

In the cocaine dose-response assays, NP-1-163 had little effect on the cocaine dose-response in inhibiting human dopamine transporter (as shown in figure 18). NP-1-163 dose-response in inhibiting dopamine uptake showed its IC50 was 31.82 µM (see figure 15 (c)). We tested 60 µM

NP-1-163 solution in biotinylation assays, so that the inhibitory effect can be clearly detected by biotinylation. Figure 22 (a) shows that in the presence of NP-1-163, biotin accessibility to W84C in DAT was reduced slightly (NP-1-163 (+) biotinylated DAT density was 2.19, NP-1-163 (-) biotinylated DAT density was 2.62). To have a more direct comparison of the reduction, unpaired t-test was conducted. Figure 22b shows the result of the t-test. To the left, the two columns represent the density of biotinylated WT-DAT density in the absence/presence of NP-1-

163, and to the right, the two columns represent the density of biotinylated W84C/C306A-DAT in the absence or presence of NP-1-163. Biotinylated WT-DAT density was not changed by NP-

1-163, nor was biotinylated W84C/C306A-DAT density significantly reduced at presence of NP-

1-163 (p=0.2870). The results suggest NP-1-163 is occupying the W84C spot in DAT, but the 61 results did not reach statistical significance, so that the compound might be partially impeding biotin’s access to W84C at the 60 µM concentration

b. 2.0

1.5 ns

1.0

0.5 Normalized band density 0.0

WT vehicle

W84C/C306A vehicle WT compound treated

W84C/C306A compound treated 62

Figure 22. NP-1-163’s Effect On Biotin Accessibility at W84. (a) Representative western blot. Western blot was done using the protein sample from biotinylation assay; all the rectangles shown are from the same gel. The image is representative of 3 blots from 3 separate in vitro experiments. The number on each band represents the relative density of each band on the same gel, and is used as quantification for protein quantity. (b) Quantification of western blot results. Each column was calculated with biotinylated samples normalized to respective total cell samples. Each column is represented as a mean ± SEM of three separate measurements (i.e., each measurement was done on cells prepared on a different day) (ns denotes not significant).

6.4. NP-1-154 Biotinylation Assays

NP-1-154 showed little effect on cocaine dose-response in the cocaine dose-response experiments, and it differed from the other three compounds, it did not show a dose-dependent trend (as seen in figure 19). The IC50 of NP-1-154 was 253.33 µM, which was much higher than

NP-1-155, by only differed in the R1 site in their chemical structures (see figure 14a). NP-1-154 was tested in biotinylation assay because we were curious about the difference we had observed in the previous results with the dose-response assays. In the biotinylation experiments, Figure 23

(a) shows that in the presence of NP-1-154, biotin accessibility to W84C in DAT was reduced

(with NP-1-154 biotinylated, DAT Western Blot density was 2.22; without NP-1-154, biotinylated DAT Western Blot density was 4.33). To gain a more direct comparason of the reduction, unpaired t-test was conducted. Figure 23 (b) shows the result of the t-test. The two columns on the left represent the density of biotinylated WT-DAT density at the absence/presence of NP-1-154. The two columns on the right represent the density of biotinylated W84C/C306A-DAT at the absence/presence of NP-1-154. Biotinylated WT-DAT 63 density was not changed by NP-1-154, while biotinylated W84C/C306A-DAT density was reduced at presence of NP-1-154, but not statistically significant (p=0.1574). The results indicate

NP-1-154 is occupying the W84C spot in DAT, but did not reach a statistical significance, so that the compound might partially impeding biotin to access W84C at 500 µM concentration.

b. 4

3 ns

1 Normalized band density 0

WT vehicle

W84C/C306A vehicle WT compound treated

W84C/C306A compound treated

Figure 23. NP-1-154’s Effect On Biotin Accessibility at W84. (a) Representative western blot. The biotinylated transporter density was reduced in presence of compound NP-1-154.

Western blot images are representative of 3 blots from 3 separate in vitro experiments. The number on each band represents the relative density of each band on the same gel, and is used as quantification for protein quantity. (b) Quantification of western blot results. Each column was calculated with biotinylated samples normalized to respective total cell samples. Each column is represented by mean ± SEM of three separate measurements (i.e., each measurement was done on cells prepared on a different day) (ns denotes not significant).

In conclusion, the above biotinylation results suggest that NP-1-152 and NP-1-155 significantly reduced biotin labeling at W84C, whereas NP-1-154 and NP-1-163 showed only a trend of 65 diminishing biotinylation at this site. These findings indicate that all of the compounds tested might obstruct biotin from attaching to the sulfhydryl on cysteine 84. On the other hand, NP-1-

152 and NP-1-155 might be able to bind more tightly than NP-1-163 and NP-1-154, and resulted in more reduction in the quantity of pulled-down transporter. We are looking for other amino acid residues lying around the allosteric pocket in hDAT, that could have reduced biotinylation signal when treated with KM822, and we would employ those amino acid mutants in our study of KM822 analogs.

DISCUSSION

In this study, we have screened eleven KM822 analogs for their effects on substrate uptake in dopamine transporter, serotonin transporter, and norepinephrine transporter. Results showed that

KM822 inhibits dopamine uptake by hDAT non-competitively, as it decreased Vmax dose- dependently at lower KM822 concentration (0 to 5 µM in our experiments). Km were constant at lower KM822 concentration, but increased when KM822 concentration was much higher than

IC50 (25 µM in our experiments). Therefore, at higher doses, KM822 might mimic a competitive inhibitor to hDAT, while at lower doses, KM822 inhibits hDAT non-competitively.

Selectivity of KM822 and analogs was tested because DAT, NET, SERT are homologous, and most inhibitors on the market inhibit at least two of them instead of just one specific. Thus, having an inhibitor that only inhibits DAT would be beneficial for medical use, because target specificity reduces side-effect. KM822 is selective to hDAT, but not hNET or hSERT. KM822 analogs all have changed binding properties to the three transporters. The strong DAT selectivity of KM822 is diminished in all the analogs. Comparing the structure of KM822 with NP-1-145 and NP-1-146, we find that substitution of R1 group immediately resulted in change of potency in DAT, and selectivity. This might suggest that ethyl group at R1 interacts with hDAT at a specific site, which is different in the other transporters, hSERT and hNET. Compared to

KM822, NP-1-150 to NP-1-163 have improved water solubility by changing the R2 sidechain to several ammonic groups (primary amine group, dimethylamine group, morpholine group), therefore I suspect that the R2 sidechain of KM822 might be hydrophobic, so that it prevents the compound from dissolving in water. Compounds NP-1-150 to NP-1-163 also showed higher potency than NP-1-145 and NP-1-146. Although this increased potency might be simply due to 67 bad solubility of NP-1-145 and NP-1-146 in our assay buffer (not completely dissolved in 5%

DMSO), it is more likely that substitutions by these ammonic group helped the compounds to interact with hDAT. Another suggestion might be that the decreased molecular mass helped NP-

1-150 and following compounds to get to the target site more easily because of less steric hindrance, therefore become more potent compared with NP-1-145 and NP-1-146. However,

KM822 is still the most potent compound for the dopamine transporter amongst all the tested compounds.

KM822 displayed an inhibitory effect on the cocaine hDAT interaction (see figure 8a). This result validates our hypothesis that the conformational change caused by KM822 binding shifted the availability of cocaine binding site. However, except for NP-1-152, the other tested compounds had little inhibitory effect on cocaine, and some even increased cocaine potency at the compounds’ IC50. This phenomenon may be because that affinity of these compounds for the allosteric site are low. In addition, cocaine could shift the conformation of hDAT, and change the affinity of the compounds to the allosteric binding site. Evidence shows that, upon cocaine binding, the intracellular loop 3 region of hDAT undergoes significant structural shift, as well as

TM7 in MTSET-biotin labeling studies [18, 36]. The effect of hDAT-conformation change caused by cocaine might influence the binding of the analogs to the allosteric site to a different degree. Some compounds might be more vulnerable to the conformation change and get removed more readily, thus showing no effect on the cocaine dose-response curve (for example NP-1-155 and NP-1-163, shown in figures 17 and 18), while some compounds’ binding are less influenced by conformation change caused by cocaine (for example NP-1-152, as seen in figure 16).

Overall, these compounds’ profiles have facilitated our understanding of the structure-activity relationship, and provided clues for helping to design compounds with additional modifications.

Substituting the R2 sidechain to ammonic group (primary ammonium, dimethylamine, morpholine) could improve water solubility, therefore helping us to test the compounds both in in vitro and in vivo studies in the future (water solubility is needed for compounds to cross blood-brain barrier), and facilitate the drug development process in the future. Modifications at

R1 group seem more likely to affect potency. Compounds with chlorine group substitution at R1 site are more potent than fluorine substitution on dopamine transporter, when they have the same

R2 group. It is difficult to make conclusions on structure and hDAT-selectivity relationship, because the compounds lost selectivity to various degrees, and do not show a common pattern or trend, meaning that the SAR was not very tractable. We do not have an analog that has the original ethyl group at R1 site, while with the change on R2 simultaneously. This could lead us to suspect that maybe methyl-R1 is important for hDAT-selectivity, and it would be necessary to test compound with ethyl group at R1 using the same methods.

The amino acid Tryptophan 84 resides in transmembrane domain 1 in dopamine transporter

(leucine 84 in LeuT). As it was shown in the computer modeling, W84 interacts with KM822 by a face-to-face p-p stacking (figure 4 and point 7 in Introduction), which is a relatively stable non-covalent interaction. To study W84 interaction with KM822, cysteine substituted DAT mutant was made, enabling us to use the biotinylation method. Substitution of W84 by cysteine increased KM822 IC50 in dopamine uptake, indicating W84 plays a role in the compound-hDAT binding, and hDATW84C is more readily binds to KM822. Also, the affinity of hDATW84C to 69

KM822 does not seem to be related to the increase in biotin-labeled hDAT (unpublished data by a colleague in our lab).

We tested 4 compounds, NP-1-152, NP-1-155, NP-1-163 and NP-1-154 at twice their IC50 concentration in biotinylation assays. Twice IC50 concentration instead of IC50 concentration was used because twice IC50 would produce more of an effect on DAT conformational change, and cause more inhibition on biotin labeling. Since our aim in doing biotinylation assay was to determine if the compound is interacting with the allosteric binding site (qualitative), but not to study their affinity (quantitative), it is fine for us to use twice IC50 as test concentration. If the presence of the analog decreases biotinylated dopamine transporter, then the analog is possibly interacting at the allosteric pocket similar to KM822. Amongst the compounds, NP-1-152 and

NP-1-155 significantly decreasing biotinylated DATW84C, while NP-1-163 and NP-1-154 also showed a trend of decreased (not statistically significant, figures 23 and 24). In the computer modeling, which was done by the Kortagere lab, the phenyl group of tryptophan interacts with the three rings at the backbone of KM822 (not shown). All our analogs preserved the backbone structure, which means conceptually they should all occupy W84, and reduce the accessibility of biotin to W84C. The unchanged three-ring structure enables us to study SAR of those changed parts in the structure (i.e. R1 group and R2 group, see figure 10). For example, when we compare the structure of NP-1-154 and NP-1-155, we found the only difference in the structure was the halogen group at R1 site (noting fluorine is more nucleophile than chlorine, and smaller in size), but this single change caused dramatic difference in potency (figure 15). Designing a KM822 analog with modification on the three ring structure and have it synthesized would be also 70 beneficial, because it could serve as a negative control in an hDATW84C biotinylation study, and help us to find out the role the three-ring structure plays.

To gain a better understanding of the structure-activity relationship between these compounds and the transporter, we need methods that are more specific. A combination of computer modeling with a cell-based binding assay has been widely used in protein structural studies [37].

More direct in vitro studies including the use of a radio labelled compounds in cell-based functional assays and modifying the chemical structure of the compound into an irreversible ligand, these studies are expected to give more specific signal in biotinylation assays.

Another goal of ours is to understand whether the allosteric modulator KM822 will stimulate or decrease cocaine-like behavior. Stimulation of dopaminergic system is usually related to mental alertness in short-term, and dependency if administered chronically. These can be assessed in several animal behavior tests, for example stimulated locomotor test for immediate effect of cocaine, and conditioned place preference test for chronic use of cocaine [38]. Although the underlying mechanism is not clear, some dopamine transporter inhibitors showed diminished behavior stimulation, or slower onset than cocaine, such as benztropine analogs (JHW-007, AHN

2005), piperazine derivative GBR-12909, modifinil for treating narcolepsy, and several cocaine analogs [39-41]. These findings shed light on dopamine uptake inhibitors, that not all of them are likely to be addictive, but also make the mechanism of drug abuse more vague, highlighting the need for extensive SAR studies. For example, evidence suggests that some atypical DAT inhibitors, such as GBR-12909, modafinil and benztropine analogs, are most likely to favor a more inward-facing occluded conformation, instead of the outward-open conformation that 71 cocaine favors. The DAT inward-facing structure stabilized by these atypical inhibitors might contribute to the lack of cocaine-like behavior effect [42].

In future studies, our lab plans to examine the in vivo effect of several KM822 class compounds, including KM822 itself and selected analogs on wild type rats, by intraventricular administration

(due to unclear pharmacokinetic properties, we are not sure the compound is able to cross the blood-brain barrier, so intraventricular injection would be more favorable). Upon compound administration, a locomotion test will be done. Cocaine systemic administration increases distance of traveling in the locomotion test [43]. If KM822 or analog gives same levels of increase in locomotion as cocaine in the locomotion test, then the compound is predicted to give cocaine-like behavior; if not, then the compound is predicted to have reduced stimulation in behavior. KM822 or an analog can also be given before cocaine administration. If the cocaine- stimulated increase in locomotion were inhibited, then it would indicate that the compound is blocking cocaine interaction, possibly due to the allosteric modulation at DAT. This way, these results would help my colleagues to determine whether the compounds display cocaine-like behavioral effect on rats, as well as whether the compounds can block cocaine effects.

LIST OF REFERENCES

1. Zhou, Z., et al., LeuT-desipramine structure reveals how antidepressants block

neurotransmitter reuptake. Science, 2007. 317(5843): p. 1390-1393.

2. Kristensen, A.S., et al., SLC6 Neurotransmitter Transporters: Structure, Function, and

Regulation. Pharmacological Reviews, 2011. 63(3): p. 585-640.

3. Benarroch, E.E., Monoamine transporters. Structure, regulation, and clinical

implications, 2013. 81(8): p. 761-768.

4. Russo, S.J. and E.J. Nestler, The brain reward circuitry in mood disorders. Nat Rev

Neurosci, 2013. 14(9): p. 609-25.

5. Ikemoto, S., Brain reward circuitry beyond the mesolimbic dopamine system: A

neurobiological theory. Neuroscience and biobehavioral reviews, 2010. 35(2): p. 129-

150.

6. Han, D.D. and H.H. Gu, Comparison of the monoamine transporters from human and

mouse in their sensitivities to psychostimulant drugs. BMC Pharmacology, 2006. 6: p. 6-

7. Kiyatkin, E.A., et al., Critical role of peripheral vasoconstriction in fatal brain

hyperthermia induced by MDMA (Ecstasy) under conditions that mimic human drug use.

J Neurosci, 2014. 34(23): p. 7754-62.

8. Whitton, P.S., Glutamatergic control over brain dopamine release in vivo and in vitro.

Neuroscience & Biobehavioral Reviews, 1997. 21(4): p. 481-488.

9. Wise, R.A., B. Wang, and Z.B. You, Cocaine serves as a peripheral interoceptive

conditioned stimulus for central glutamate and dopamine release. PLoS One, 2008. 3(8):

p. e2846. 73

10. Kitayama, S., et al., Dopamine transporter site-directed mutations differentially alter

substrate transport and cocaine binding. Proc Natl Acad Sci U S A, 1992. 89(16): p.

7782-5.

11. Reith, M.E., et al., Behavioral, biological, and chemical perspectives on atypical agents

targeting the dopamine transporter. Drug Alcohol Depend, 2015. 147: p. 1-19.

12. Czoty, P.W., J.L. Martelle, and M.A. Nader, Effects of chronic d-amphetamine

administration on the reinforcing strength of cocaine in rhesus monkeys.

Psychopharmacology, 2010. 209(4): p. 375-382.

13. Dackis, C.A., et al., A Double-Blind, Placebo-Controlled Trial of Modafinil for Cocaine

Dependence. Neuropsychopharmacology, 2004. 30: p. 205.

14. Penmatsa, A., K.H. Wang, and E. Gouaux, X-ray structure of the dopamine transporter

in complex with tricyclic antidepressant. Nature, 2013. 503(7474): p. 85-90.

15. Zhen, J. and M.E. Reith, Impact of disruption of secondary binding site S2 on dopamine

transporter function. J Neurochem, 2016. 138(5): p. 694-9.

16. Quick, M., et al., Experimental conditions can obscure the second high-affinity site in

LeuT. Nature structural & molecular biology, 2012. 19(2): p. 207-211.

17. Wang, K.H., A. Penmatsa, and E. Gouaux, Neurotransmitter and psychostimulant

recognition by the dopamine transporter. Nature, 2015. 521: p. 322.

18. Dehnes, Y., et al., Conformational changes in dopamine transporter intracellular regions

upon cocaine binding and dopamine translocation. Neurochemistry international, 2014.

0: p. 4-15.

19. Beuming, T., et al., The binding sites for cocaine and dopamine in the dopamine

transporter overlap. Nature neuroscience, 2008. 11(7): p. 780-789. 74

20. Wasko, M.J., et al., In Silico Molecular Dynamics Analysis of Dopamine Transporter

Conformational Preference as a Function of Occupancy with Inhibitors of Low Abuse

Potential. The FASEB Journal, 2017. 31(1_supplement): p. 1062.7-1062.7.

21. Katz, J., et al., Dopamine transporter binding without cocaine-like behavioral effects:

synthesis and evaluation of benztropine analogs alone and in combination with cocaine

in rodents. Vol. 154. 2001. 362-74.

22. Shi, L., et al., The mechanism of a neurotransmitter:sodium symporter – inward release

of Na(+) and substrate is triggered by substrate in a second binding site. Molecular cell,

2008. 30(6): p. 667-677.

23. Singh, S.K., A. Yamashita, and E. Gouaux, Antidepressant binding site in a bacterial

homologue of neurotransmitter transporters. Nature, 2007. 448: p. 952.

24. Stockner, T., et al., Mutational Analysis of the High-Affinity Zinc Binding Site Validates a

Refined Human Dopamine Transporter Homology Model. PLOS Computational Biology,

2013. 9(2): p. e1002909.

25. Richfield, E.K., Zinc modulation of drug binding, cocaine affinity states, and dopamine

uptake on the dopamine uptake complex. Molecular Pharmacology, 1993. 43(1): p. 100.

26. Bjorklund, N.L., T.J. Volz, and J.O. Schenk, Differential effects of Zn2+ on the kinetics

and cocaine inhibition of dopamine transport by the human and rat dopamine

transporters. European Journal of Pharmacology, 2007. 565(1): p. 17-25.

27. El-Shehabi, F., et al., A Novel G Protein-Coupled Receptor of Schistosoma mansoni

(SmGPR-3) Is Activated by Dopamine and Is Widely Expressed in the Nervous System.

PLOS Neglected Tropical Diseases, 2012. 6(2): p. e1523. 75

28. Larsen, M.B., et al., A catecholamine transporter from the human parasite Schistosoma

mansoni with low affinity for psychostimulants. Mol Biochem Parasitol, 2011. 177(1): p.

35-41.

29. Yamashita, A., et al., Crystal structure of a bacterial homologue of Na+/Cl--dependent

neurotransmitter transporters. Nature, 2005. 437: p. 215.

30. Kortagere, S., et al., Structure-based Design of Novel Small-Molecule Inhibitors of

Plasmodium falciparum. Journal of chemical information and modeling, 2010. 50(5): p.

840-849.

31. Raffa, R.B. and A.F. Martley, Amphetamine-induced increase in planarian locomotor

activity and block by UV light. Brain Research, 2005. 1031(1): p. 138-140.

32. Zaldini Hernandes, M., et al., Halogen Atoms in the Modern Medicinal Chemistry: Hints

for the Drug Design. Vol. 11. 2010. 303-14.

33. Schambach, S.J., et al., Application of micro-CT in small animal imaging. Methods,

2010. 50(1): p. 2-13.

34. Ferrer, J.V. and J.A. Javitch, Cocaine alters the accessibility of endogenous cysteines in

putative extracellular and intracellular loops of the human dopamine transporter.

Proceedings of the National Academy of Sciences of the United States of America, 1998.

95(16): p. 9238-9243.

35. Udi Vazana, 2,3* Ronel Veksler,1,2* Gaby S. Pell,4,5* Ofer Prager,1,2 Michael

Fassler,1,2,3 Yoash Chassidim,1,2, et al., Glutamate-Mediated Blood-Brain Barrier

Opening: Implications for Neuroprotection and Drug Discovery. Neurobiology of

Disease, 2016. 7728 • J. Neurosci. 76

36. Norregaard, L., C.J. Loland, and U. Gether, Evidence for Distinct Sodium-, Dopamine-,

and Cocaine-dependent Conformational Changes in Transmembrane Segments 7 and 8

of the Dopamine Transporter. Journal of Biological Chemistry, 2003. 278(33): p. 30587-

30596.

37. Kortagere, S., M. Lill, and J. Kerrigan, Role of Computational Methods in

Pharmaceutical Sciences, in Computational Toxicology: Volume I, B. Reisfeld and A.N.

Mayeno, Editors. 2012, Humana Press: Totowa, NJ. p. 21-48.

38. Huskinson, S.L., et al., PREDICTING ABUSE POTENTIAL OF STIMULANTS AND

OTHER DOPAMINERGIC DRUGS: OVERVIEW AND RECOMMENDATIONS.

Neuropharmacology, 2014. 0: p. 66-80.

39. Rothman, R.B., et al., Dopamine Transport Inhibitors Based on GBR12909 and

Benztropine as Potential Medications to Treat Cocaine Addiction. Biochemical

pharmacology, 2008. 75(1): p. 2-16.

40. Newman, J.L., et al., Behavioral evaluation of modafinil and the abuse-related effects of

cocaine in rhesus monkeys. Experimental and Clinical Psychopharmacology, 2010. 18(5):

p. 395-408.

41. Czoty, P.W., et al., Lower reinforcing strength of the phenyltropane cocaine analogs

RTI-336 and RTI-177 compared to cocaine in nonhuman primates. Pharmacology

Biochemistry and Behavior, 2010. 96(3): p. 274-278.

42. Loland, C.J., et al., Relationship between conformational changes in the dopamine

transporter and cocaine-like subjective effects of uptake inhibitors. Molecular

pharmacology, 2008. 73(3): p. 813-823. 77

43. Au - Smith, L.N., et al., Assessment of Cocaine-induced Behavioral Sensitization and

Conditioned Place Preference in Mice. JoVE, 2016(108): p. e53107.