Inhibition of Glycine Transporter 2: an Emerging Target for Chronic Pain

University of Texas at El Paso ScholarWorks@UTEP

Open Access Theses & Dissertations

2020-01-01

Inhibition Of Glycine Transporter 2: An Emerging Target For Chronic Pain

Elvia Lilia Padilla University of Texas at El Paso

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Recommended Citation Padilla, Elvia Lilia, "Inhibition Of Glycine Transporter 2: An Emerging Target For Chronic Pain" (2020). Open Access Theses & Dissertations. 3186. https://scholarworks.utep.edu/open_etd/3186

This is brought to you for free and open access by ScholarWorks@UTEP. It has been accepted for inclusion in Open Access Theses & Dissertations by an authorized administrator of ScholarWorks@UTEP. For more information, please contact [email protected]. INHIBITION OF GLYCINE TRANSPORTER 2: AN EMERGING TARGET

FOR CHRONIC PAIN

ELVIA LILIA PADILLA MENDEZ

Master’s Program in Biological Sciences

APPROVED:

Manuel Miranda-Arango, Ph.D., Chair

Sukla Roychowdhury, Ph.D.

Suman Sirimulla, Ph.D.

Stephen L. Crites, Jr., Ph.D. Dean of the Graduate School DEDICATION

Dedicated to my family, who gave me everything I have and supporting me in every new

decision in my life. INHIBITION OF GLYCINE TRANSPORTER 2: AN EMERGING TARGET

FOR CHRONIC PAIN

ELVIA LILIA PADILLA MENDEZ, B.S.

THESIS

Presented to the Faculty of the Graduate School of

The University of Texas at El Paso

In Partial Fulfillment

of the Requirements

for the Degree of

MASTER OF SCIENCE

Department of Biological Sciences

THE UNIVERSITY OF TEXAS AT EL PASO

December 2020 ACKNOWLEDGMENTS

The support of several professionals from several disciples fields made possible the development of the research presented herein. First and foremost, I would like to thank an essential person in this road of knowledge, Dr. Manuel Miranda. I met him taking his class on

Neurobiology, and it is impressive how a person can express in that way the passion of his field. The help and support of Dr. Miranda helps me to learn and grow in the research field.

His dedication and discipline encouraged me to be part of his lab three years ago. At that time,

I started as a volunteer, but through the time, I decided to be a master student from Dr.

Miranda's lab one year later; and it has been one of the most critical decisions in my life.

Dr. Miranda taught me that hard work and dedication could give you enormous satisfaction at the moment of finalizing your project. Every technique that Dr. Miranda taught me, it was a new and exciting step in these exhausting years. I appreciate extraordinary every moment that Dr. Miranda took from his time to help me and teach me during this long time.

Then, I would like to thank Dr. Sirimula and Dr. Fillip from the department of Pharmacy for all the collaboration in the process of this project and their support helping me to understand better certain concepts about virtual screening techniques. Also, I want to be grateful for every time and the full support that Dr. A. Bryan Lopez spent helping me. He was like a second mentor for me. He helped me from the beginning of the process of learning several techniques and his unconditional assistance during the whole process of this project.

Unforgivable to say thank you to my thesis committee members: Dr. Miranda, Dr.

Sirimula, and Dr. Roychowdhury. Your counseling has advised me to realize how my work

iv and process can always be better and look for the best way to express my project's significance.

Finally, I want to thank those members of our lab, several other labs, or in general, from the department that, in any way, helped me during these two years. Thank you to Zulema Uresti for her impressive help in understanding immunofluorescence techniques or how to work with animals. Thank you to Dr. Varela that taught me how to analyze the cytotoxicity on cells.

Thank you for the CORE of the fifth floor, which always facilitates the analysis of my samples. Last but not least, I would like to thank my parents, without their support, help, and unconditional love during my whole life, I would never be where I am now. Their sacrifices these years looking for better education, it develops in me the research of more knowledge.

v ABSTRACT

Chronic pain is a complicated, completely unknown condition that has been studied based on its severe impact on the population. According to the American Academy of Pain

Medicine, 1.5 billion people suffer from tension-type headaches, one of the most common symptomatic chronic conditions. Christopher L. Cioffi, 2019; mentioned approximately in

US 116 million adults are explicitly afflicted by chronic pain. Chronic pain is described as dysfunction in nerves that have been continuous during a long period (Mills, 2019).

Treatments for chronic pain are different types of common analgesics that relieve the pain for short periods such as aspirin, ibuprofen, paracetamol, etc. (Hunging, 2017) Accord to the level of it, such as opioids, anticonvulsants drugs, antidepressants, and anti-inflammatory drugs are some of the treatments used for severe pain. One of the most common therapies is opioids, as relievers of pain available legally by prescription. Opioids are considered one of the most effective drugs for the treatment of several levels of pain (Wood, 2019). In most of the world, opioids have been the solution to controlling severe or constant pain, such as chronic pain.

The several side effects that opioids produce create several concerns related to patient safety and abuse addiction. (Rosenblum, Marsch, Joseph, & Portenoy, 2008) Secondary side effects that do not let opioids be a correct option for chronic pain treatment are diverse; for example, the high rate of addiction or possible drug abuse (Edlund, 2007). "Injury to the somatosensory system (neuropathic pain), degenerative processes and chronic inflammation

(i.e., osteoarthritis and rheumatoid arthritis), disease (i.e., cancer pain)" (Cioffi, 2019) are

vi some of the suggested risks or suffer chronic pain and the analgesic resistance that patients development through their fight with the problem makes harder to avoid the use of opioids.

Therefore, several research types are interested in finding new therapies that avoid the use of opioids—looking for better options to find relief for those patients who continuously are suffering from pain. The virtual screening method developed achieved a high hit rate of

75%. By analyzing more and more new compounds and looking for possible chronic pain treatment targets, the discovery of a group of new compounds with good inhibitory properties and selectivity it seems to be possible. Hit1 is considered to be the first and main candidate for further optimization of potential customers and detailed, more specific analysis of the ligand. We observed that some of the factors that make Hit1 the main ligand, such as the stable hydrogen bond that interacts with glycine transporter 2, showed a development similar to ALX1393 activity. Through several MD simulations. The results provided a structural basis for the Hit1 selectivity observed experimentally in GlyT2. The result displayed by Hit2 is closest to Hit1. The inhibition of glycine transporters, especially glycine transporter 2

(GlyT2), has been shown promise for new treatments for chronic pain based on their role in the neurotransmission of pain signals. They are improving the knowledge in better ways to find cures for chronic pain with fewer consequences in the process.

vii TABLE OF CONTENTS

PAGE AKNOWLEDGEMENTS...... iv

ABSTRACT………………………...... ………………...………….…..…...... …vi

TABLE OF CONTENTS…………………………………….…...... …...... ….…viii

LIST OF FIGURES………………………………..……………..…...... ……...... ix

CHAPTER I: Introduction ...... 1

1.1. Solute Carrier 6 Family (SLC6) ...... 4

1.2. Glycine Transporters difference and characteristics...... 5

CHAPTER II: Glycine’s relevance ...... 7

2.1. The importance of glycine transporters in neurological disorders...... 7

2.2.Glycine over opioids use on treatments for chronic pain...... 9

AIMS...... 10

Methods and procedure...... 12

CHAPTER III: Results ...... 14

CONCLUSION...... 31

REFERENCES...... 34

GLOSSARY...... 39

VITA...... 40

viii LIST OF FIGURES

Fig. 1 Synaptic interaction of glycine…………...…………...... ……...... ….……... 3

Fig. 2 Glycine Transporters and Chronic Pain …………...... ……...... …….....8

Fig. 3. Chemical structure of the four ligands...... 16

Fig. 4 Inhibition of GlyT2 activity by ALX1393 inhibitor...... 18

Fig. 5 Inhibition of GlyT2 activity by HIT1/Lead1 compound...... 19

Fig. 6 Inhibition of GlyT2 activity by HIT2/Lead2 compound...... 20

Fig. 7 Inhibition of GlyT2 activity by HIT3/Lead3 compound...... 21

Fig. 8 GlyT2 activity with HIT4/Lead4 compound...... 22

Fig. 9 Inhibition of GlyT1 activity by ALX5407 inhibitor...... 24

Fig. 10 Inhibition of GlyT1 activity by HIT4/Lead4 compound...... 25

Fig. 11 Inhibition of GlyT1 activity by HIT1/Lead1 compound...... 26

Fig. 12 Inhibition of GlyT1 activity by HIT2/Lea2 compound...... 27

Fig. 13 Inhibition of GlyT1 activity by HIT4/Lead4 compound...... 28

Fig. 14 Cytotoxicity of HIT compounds and ALX inhibitor on PAE cell...... 30

ix CHAPTER I- INTRODUCTION

Chronic pain has been mysterious and complicated to understand the condition, and through the years, the rate of people suffering from this condition have been increasing (Seth,

2016). Nerve damage due to injury or disease, causing deep and intense pain, with a time prolongation higher than 12 weeks is considered chronic pain (Weatherspoon, 2017). There are various treatments for chronic pain, some more efficient than others, such as opioids, antidepressants, or anti-inflammatory drugs. All these previous therapies have side effects such as short time of pain relief, high addiction levels, etc. (Edlund, 2007). Scientists are interested in developing new therapies that avoid the use of opioids and other analgesics, preventing their dangerous side effects.

The opioids in the central nervous system have a fascinating interaction with its close interaction with the limbic areas such as the amygdala or some glands like the pituitary and adrenal glands(Proehlich, 2017). Showing the problematic division between scientists on its administration as a treatment for some chronic diseases, that every year more frequently overdoses cases related to opiates intake are present in society(Kopec et al., 2009).

Then, glycine is a major inhibitory neurotransmitter in the vertebrate central nervous system. It is known to act as an inhibitory transmitter via its interaction with strychnine-sensitive glycine receptors (Hashimoto, 2010). Two glycine transporters control its extracellular levels, glycine transporter 1 and glycine transporter 2 (GlyT1 and GlyT2, respectively).

Glycine is involved in the synapse based on the termination by its rapid reuptake into the

1 nerve terminal and adjacent glial cells. Glycine transporters have been analyzed for several reasons, having essential importance in the pharmacology field because of its emerging relevance in relationship with some neurological diseases such as chronic pain.

2 SYNAPTIC INTERACTION OF GLYCINE

Figure 1. Glycine neurotransmission at excitatory and inhibitory synapses. After glycine release into the synapse, it binds to glycine receptors (GlyRs) located in the postsynaptic terminals. The glycine levels at the glycinergic synapses returned to normal levels by specific glycinergic transporters (GlyT1 and GlyT2) in the presynaptic terminal for GlyT2 and GlyT1 in glial cells and astrocytes.

3 The inhibition of glycine transporters has been established several neuronal damages shown by several animal experiments, whereby the inhibition of GlyT, mice die due to respiratory arrest (Schwarcz, 2016). The transmission of pain signals in the brain presents a co-relation with GlyT2; the inhibition of GlyT2 causes an increase in glycine levels in the synaptic cleft that results in prolonged activation of glycine receptors onto pain fibers and decreases the pain signal in the central nervous system (CNS). Chronic pain has been studied for its development through years around the population of different ages affecting up to 30% of those aged 18-39 yr (Mills, 2019). As a persistent pain caused by damage in a nerve on the peripheral or central nervous system and without efficient treatments (Vandenberg et al.,

2014), GlyT2 inhibition is a promising effective treatment against it.

1.1 Solute Carrier 6 Family (SLC6)

GlyT1 and GlyT2 are secondary active transporters and belongs to the Solute Carrier 6

Family (SLC6). The SLC6 family characterized themselves as secondary active co-transporters that depend on the Na+/Cl- concentration gradient. Akula Bala et al., 2013 analyzed the SLC6 family and mentioned four different subgroups. The neurotransmitter transporters are formed by the glycine transporters, the amino acid transporters, the osmolyte transporters, and creatine transporters, and the orphan transporters in which no substrates have been identified (Thimgan, 2006). This SLC6 family has significant relevance in the clinical aspect by its localization around the body, such as, "nervous system, kidney, intestine, pancreas, adrenal gland, and testis" (Akula et al., 2013). As can be analyzed, the SLC6 family is fundamental in understanding how glycine's development in the central nervous system works at the molecular level. The analysis of which family comes from glycine transporters

4 helps research and analyze how possible it can do. Having the antecedent of how the structure of glycine transporters is, it proposes a better idea of how the interaction of those transporters works around several amino acids, useful in better knowledge of them. In order to find a connection between glycine transport and treatment for neurological diseases and understand the physiology of glycine transporters being very similar, everything makes more challenging the development of new therapies.

1.2 Glycine Transporters difference and characteristics

The glycine transporters share several similitudes that complicate the development of specific treatments for each one in pharmacology. The creation of specific inhibitors for each glycine transporter without damaging the molecular development of the opposite one is the next step on emerging treatments with high specificity and better results against diseases.

GlyT1 needs two sodium ions; on the other hand, GlyT2 requires three sodium ions to transport one molecule of glycine. Also, glycine plays an essential role in excitatory neurotransmission with its interaction in the glycine sites located on the

N-methyl-D-aspartate (NMDA) receptors, the three ionic glutamate receptors (VanDongen,

2009). GlyT-1 and GlyT-2 share 12 putative transmembrane spanning domains and approximately 50% amino acid sequence identity. Those transporters differ in several aspects.

For example, they are different thermodynamically coupled to the sodium electrochemical gradient, GlyT1 needs two sodium ions, and GlyT2 requires three sodium ions for the transport of their residues.

Although their structures have shared several similitudes (Zafra, 2008), the plasma membrane distribution on glycine transporters differs, finding a specific interaction of GlyT2

5 with glycinergic neurons in the spinal cord, the brainstem, and the cerebellum, where is possible to locate the protein enriched in the presynaptic terminals (Zafra, 2008). Contrarily, in glial cells (the most abundant cell types in the CNS and responsible for homeostasis maintenance), GlyT1 is mainly concentrated in glutamatergic terminals at lower levels (Zafra,

2008). The analysis of GlyT2 relevant functions during the neurotransmission is an iconic beginning in the search about its appropriate utilization or manipulation of its tasks in several science and health fields, getting significant discoveries about treatment for certain diseases.

6 CHAPTER II - GLYCINE’S RELEVANCE

2.1 The importance of glycine transporters in neurological disorders

As mentioned, it is essential to understand the function and development of the glycine transporters in the CNS. Scientists have been looking for new ways to inhibit the activity of

GlyTs in order to understand their importance in the central nervous system. In the case of

GlyT1, its inhibition has been studied for its relationship against several diseases such as schizophrenia (Hashimoto, 2010), which in the inhibition of GlyT1, the extracellular glycine concentration increased and the NMDA response increased too, showing an essential modulation of NMDA receptors by glycine transporters (Lim et al., 2004).

On the other hand, the inhibition of GlyT2 has been studied concerning its high co-relation with neurological diseases such as chronic pain (Cioffi, 2017). Vandenberg,

Robert J., et al., 2014 showed how the population that suffers from chronic pain increases in the last years. It has a powerful influence on other diseases such as depression, anxiety, and cognitive dysfunction. GlyT2 is involved in glycinergic inhibitory neurotransmission, an essential pathway in the maintaining of pain control.

The importance and understanding of glycine transporters in the mammalian central nervous system are indispensable in search of new treatments against certain neurological diseases such as hyperekplexia (Schwarcz, 2016). In this project, we focus primarily on the development of GlyT2 inhibitors. Our goal is to produce new therapies for the treatment of chronic pain, which helps decrease the use of several pharmaceutic treatments such as anticonvulsants, antidepressants, NMDA receptor antagonists, and opioids.

7 GLYCINE TRANSPORTERS AND CHRONIC PAIN

Figure 2. Inhibition of GlyT2 suppressing pain signals. A) Glycine receptors regulate pain signals transmitted by the projection neurons located in the superficial layers of the dorsal horn. B) Inhibition of glycine transporter 2 (GlyT2) potentiates the activation of glycinergic inhibitory neurotransmission by reducing the reuptake of glycine from glycinergic synapses, which further inhibits the firing of projection neurons and thereby suppresses pain. C)

Reuptake of glycine by GlyT2 from glycinergic synapses.

8 The inserts depict local glycinergic transmission in the absence (top panel) and presence

(bottom panel) of glycine reuptake inhibition. Blockade of GlyT2, as shown in the top panel of the insert, does not seem to impair vesicular refilling of glycine at the presynaptic terminals and therefore does not limit presynaptic glycine release. Nociceptive C‐ and Aβ fibers stimulate the pain projection neuron and activate glycinergic interneurons that inhibit the pain projection neuron. The activation of glycinergic interneurons via NMDA

(N‐methyl‐d‐aspartate) receptors may be strengthened by a suggested GlyT1 inhibition, thereby providing additional inhibition of the pain signals destined for the brain.

2.2 Glycine over opioids use on treatments for chronic pain

Opioids have been one of the most controversial topics discussed between scientists; their use divides still this time every debate about functional and efficient treatments for chronic pain. Considered as the regular treatment for severe pain or chronic pain, opioids are not well thought out to been used for long-term administration (Rosenblum, Marsch, Joseph,

& Portenoy, 2008). Opioids' side effects are affiliated to safety or addiction from the consumers. It is has been the principal factor in order to provide a detailed analysis of the mechanism of them. The side effects of opioids in long-term treatments such as chronic pain or diseases like cancer have been a severe problem. It made scientists look forward to new treatments avoiding the use of opioids. Glycine inhibition has not been involved in the activation of neuronal mechanisms that affect the emotional stability of improving human addiction. Opioids showed to be involved in specific mechanisms in the central nervous system, specifically the amygdala (Proehlich, 2017), and altered the modulation of feelings on the brain, producing an artificial sensation of euphoria on patients.

9 AIMS

AIM 1) The Inhibition of glycine transporter 2 with Zinc molecules.

One of the primary purposes of this project is to understand the interaction and the inhibition of glycine transporters (especially glycine transporter type 2) in contact with several inhibitors; to analyze the efficiency of the inhibitors to obtain a possible target in the exploration of new treatments for chronic pain relief. We will examine four promising compounds (selected by screening over 3.5 million lead like compounds from the ZINC15 library) to hit that may inhibit the glycine transporter two and possible selectivity over

GlyT1. a) ZINC6620309 (HIT 1) b) ZINC6865169 (HIT2) c) ZINC30678404 (HIT3) d) ZINC1606495 (HIT4)

AIM 2) Glycine transporter 1 inhibition to test selectivity for glycine transporter 2.

To obtain accurate data is necessary for the analysis of several factors. It is essential the analyze how the four promising compounds interact with glycine transporter type 1 to get a more specific view of the specificity of those compounds in the interaction with both glycine transporters.

10 AIM 3 ) Cytotoxicity analysis on HIT 1-3 compounds.

Following the process of satisfaction in those possible treatment targets, it is essential to analyze cytotoxicity, showing acceptable results of not side effects, looking for the agreement of safety in the use of Zinc molecules. Subsequently, studying how the compounds interacted in the glycine transporter inhibition process, it was also necessary to analyze the compounds' toxicity to be considered possible factors for chronic pain treatments.

11 METHODS AND PROCEDURE

Virtual screening. To find the best inhibitors for GlyT2, the virtual screening technique was used. The computational method is used to discover drugs from libraries of molecules, helping identify the most likely to bind to our target. The process starts at the begging of a free database (ZINC15 library) of available and commercial compounds for virtual screening.

As a cone, the system begins to get rid of those compounds that do not have the affinity necessary in the methodology of selection and specification for an inhibitor of GlyT2.

Molecular docking. For a good characterization of new GlyT2 inhibitors, we will combine the use of computational methodologies such as Molecular docking and biochemical assays to obtain a full characterization and interaction of 4 potential inhibitors for GlyT2.

Molecular docking will help us in the understanding of the molecular interaction of the inhibitors with the glycine transporter 2, and with the use of induced-fit docking (IFD) can be possible to evaluate the stability of those four ZINC inhibitors.

In vitro assay. To follow a correct improvement in the experiments, it is necessary to start with several in vitro assays with selective GlyT1 inhibitor ALX5407 and GlyT2 inhibitor ALX1393. Human GlyT2 gene will be cloned into the expression vector pcDNA3.1 and used it for transfection and selection of porcine endothelial aortic (PAE) cells. PAE cells were selected as our preferred model cells based on their plastic stability and low over-expression of ectopic proteins. The reaction buffer will be a mix of 4uci[H3] glycine/ml

12 isotope and cold glycine stock solution. The [3H] glycine is extracted from the cells by adding 250µL of NaOH. Glycine uptake will be determined by a scintillation spectroscopy machine analyzing the samples with the help of 100µL lysate in 4ml of scintillation cocktail.

Cytotoxicity. Differential nuclear staining (DNS) assay to quantify cytotoxicity. It is necessary to gently wash the PAE cells that will be used on the experiment with a fresh medium. The mid-exponential phase of the cells for the experiment with a 60 to 70% confluence will be sufficient. The wash is necessary to remove any debris and floating cells to obtain a better analysis (Lema et al. 2011). Following the protocol trypsinization, harvest, and count the cells using the Neubauer chamber. Then, into a 96-well tissue culture plate at a density of 10,000 cells/well, add 100 μl of complete medium. The culture plate should be incubated overnight. The next day, the cells should be exposed to a concentration gradient of the HIT compounds and ALX inhibitor 24 hours. It was necessary to add Hoechst and propidium iodide (PI) at a final concentration of 1 ug/ml to each well two hours before the plate reading. (Gutierrez et al., 2019). Next, using a bioimager (BD Pathway 855 system; BD

Biosciences) equipped with a 10x objective will be used to capture 3x3 montages in two fluorescence channels, the fluorophores excitation, and emission wavelength. Finally, the

Attovison software (BD Biosciences) is used in this case to get both the image capture and their analysis to find out the percentage of cytotoxicity per individual well (Robles-Escajeda et al. 2016).

13 CHAPTER III - RESULTS

The virtual screening technique gave the beginning of finding the possible compounds to use as inhibitors of the glycine transporter 2. In the free database (ZINC15 library) is found around 3.5 million potential compatible compounds. From there, the compounds are analyzed based on docking simulation, and 35,000 compounds were possible options for inhibitors.

Just 100 scoring ligands were selected for better development, and finally, for those 100, only

20 ligands were obtained from the induced fit-docking looking for the best specification from the compounds. Yet, four compounds (2 different classes of ligands) were selected with the most possible specificity as a GlyT2 inhibitor.

The use of molecular docking was necessary to confirm the way of how the compounds possibly would interact with our target (GlyT2) based on several aspects, such as their chemical structure (figure 3)—confirming that our selected compounds would cancel the glycine binding site affinity. After MD simulations, it was necessary to prove the in-vitro assays' results, starting with the known inhibitor of the glycine transporter 2, ALX1393.

Using the control (ALX1393) on our experiments was possible to obtain more accurate results, for the assays were used epithelial cells, which expressed the human GlyT2. It was observed an IC50 of 25.9nM, value approved by literature data(Mingorance-Le Meur et al.,

2013).

First, it was necessary to analyze in detail the glycine transporter 2, seeking to verify the position and interaction of the third sodium binding site, which is one of the main factors that differentiate it from the glycine transporter 1. As a result of several MD studies, the third Na+

14 ion position could be observed, and it was guided mainly by GLU248, Met276, Trp263, and

Glu648 (Fratev, Miranda-Arango, Lopez, Padilla, & Sirimulla, 2019). An exhaustive analysis of this third sodium ion was sought since the glycine transporter 1 does not present it. It was essential to support our hypothesis about how this third sodium binding site could be more stable for the four compounds to be treated. Data that could be possible thanks to several molecular docking simulations on the GlyT1 that does not have that third Na+ ion (Fratev,

Miranda-Arango, Lopez, Padilla, & Sirimulla, 2019).

15 CHEMICAL STRUCTURE OF LIGANDS

ZINC6620309-HIT1 ZINC6865169-HIT2

ZINC30678404-HIT3 ZINC1606495-HIT4

Figure 3. Chemical structural of the four Hits (1-4) by VS. Visual screening method was used in order to identify the chemical structure of each compound.

16 It was acquiring the chemical structures of the compounds, as shown in figure 3. It was an indispensable aid in the respective MD, and virtual screening analyzes were carried out to verify the interactions between the four compounds and the glycine transporter 2. The first class of ligands was tested; the Hit1/Lead1 or ZINC6620309 showed an average of IC50 =

0.48μM, then, Hit2/Lead2, or ZINC6865169 showed an average of IC50 = 0.52μM; one of the possible reasons should be the difference in its structure comparing to Hit1; having two carbons atoms extra after the propionic acid segment (Figure 3). Then, the next two compounds were analyzed; Hit3/Lead3 or ZINC30678404 showed an average of IC50 = 33.2

μM higher than the first two ligands because of its closed interaction with the first Na+ atom of the GlyT2. Finally, HIT4/Lead4 or ZINC1606495 did not show good interaction as an inhibitor for GlyT2. After several experiments, we observed, based on the experiments, that

HIT4 did not inhibit GlyT2.

17 GlyT2 ACTIVITY WITH ALX 1393 INHIBITOR

Figure 4. Inhibition of GlyT2 activity by ALX1393 inhibitor. The graph shown is representative of 3 independent experiments. Porcine endothelial aortic cells (PAE cells) were used for the expression of the human glycine transporter two at several concentrations

(0.0001 μM - 100 μM). Based on in-vitro experiments calculating IC50 based on the sigmoidal curves obtained.

18 GlyT2 ACTIVITY WITH HIT1/LEAD1 COMPOUND

Figure 5. GlyT2 activity by HIT1/LEAD1 compound. Porcine endothelial aortic cells

(PAE cells) were used for the expression of the human glycine transporter 2 at several concentrations (0.5 μM - 500 μM). Based on in-vitro experiments calculating IC50 based on the sigmoidal curves obtained. The graph shown is representative of 3 independent experiments.

19 GlyT2 ACTIVITY WITH HIT2/LEAD2 COMPOUND

Figure 6. Inhibition of GlyT2 activity by HIT2/LEAD2 compound. The graph shown is representative of 3 independent experiments. Inhibition of GlyT2 activity by HIT2/LEAD2 compound with porcine endothelial aortic cells (PAE cells) used for the expression of the human glycine transporter 2 at several concentrations (0.5 μM - 500 μM). Based on in-vitro experiments calculating IC50 based on the sigmoidal curves obtained.

20 GlyT2 ACTIVITY WITH HIT3/LEAD3 COMPOUND

Figure 7. Inhibition of GlyT2 activity by HIT3/LEAD3 compound. The graph shown is representative of 3 independent experiments. Inhibition of GlyT2 activity by HIT3/LEAD3 compound with porcine endothelial aortic cells (PAE cells) used to express the human glycine transporter 2 at several concentrations (0.5 μM - 500 μM). Based on in-vitro experiments calculating IC50 based on the sigmoidal curves obtained.

21 GlyT2 ACTIVITY WITH HIT4/LEAD4COMPOUND

Figure 8. GlyT2 activity with HIT4/LEAD4 compound. Porcine endothelial aortic cells

(PAE cells) were used for the expression of the human glycine transporter 2 at several concentrations (0.5 μM - 500 μM). Based on in-vitro experiments was confirmed no inhibition from the HIT4 compound. The graph shown is representative of 3 independent experiments.

22 Continuing to analyze the specificity of these compounds, we tried the same compounds on the GlyT1. Based on the very close similarities between both transporters, the specificity of a compound is one of the must address essential concerns. It is known that when seeking to confirm the specificity of an inhibitor of a specific glycine transporter, it is fundamental and indispensable to analyze its interaction with the glycine transporter opposite to be treated.

That is why the next step on this project was based on test the four HIT compounds as on previews experiments but now with an inhibitor known as specific for GlyT1; ALX5407

(Kopec et al., 2009).

As we expected, the ALX5407 was tested on a mammalian cell line. It worked at low concentration (IC50:14 nM) as it is known in the literature that it is normal (Kopec et al.,

2009). Looking for our compounds' specificity, we tested the four compounds at the same concentrations from the last experiments for GlyT2, but on cells expressing glycine transporter 1. The results were favorable to the project; Hits 1-3 did not show any inhibition activity, as expected. Furthermore, Hit4 showed inhibition activity for glycine transporter 1.

23 GlyT1 ACTIVITY WITH ALX5407 INHIBITOR

Figure 9. Inhibition of GlyT1 activity by ALX5407 inhibitor. The graph shown is representative of 3 independent experiments. Porcine endothelial aortic cells (PAE cells) were used for the expression of the human glycine transporter 1 at several concentrations (0.1 nM - 200 nM). Based on in-vitro experiments calculating IC50 based on the sigmoidal curves obtained.

24 GlyT1 ACTIVITY WITH HIT1/LEAD1 COMPOUND

Figure 11. GlyT1 activity with HIT1/LEAD1 compound. Porcine aortic endothelial cells

(PAE) cells are used for the expression of the human glycine transporter 1 at several concentrations (0.5 μM - 500 μM). Based on in-vitro experiments, no presence of inhibition activity from the HIT1 compound on glycine transporter 1. The graph shown is representative of 3 independent experiments.

25 GlyT1 ACTIVITY WITH HIT2/LEAD2 COMPOUND

Figure 12. GlyT1 activity with HIT2/LEAD2 compound. Porcine aortic endothelial cells

(PAE) cells are used for the expression of the human glycine transporter 1 at several concentrations (0.5 μM - 500 μM). Based on in-vitro experiments, no presence of inhibition activity from the HIT2 compound on glycine transporter 1. The graph shown is representative of 3 independent experiments.

26 GlyT1 ACTIVITY WITH HIT3/LEAD3 COMPOUND

Figure 13. GlyT1 activity with HIT3/LEAD3 compound. Porcine aortic endothelial cells

(PAE) cells are used for the expression of the human glycine transporter 1 at several concentrations (0.5 μM - 500 μM). Based on in-vitro experiments, no presence of inhibition activity from the HIT3 compound on glycine transporter 1. The graph shown is representative of 3 independent experiments.

27 GlyT1 ACTIVITY WITH HIT4/LEAD4 COMPOUND

Figure 10. GlyT1 activity with HIT4/LEAD4 compound. Porcine aortic endothelial cells

(PAE) cells are used for the expression of the human glycine transporter 1 at several concentrations (0.5 μM - 500 μM). Based on in-vitro experiments was confirmed an inhibition activity from the HIT4 compound on glycine transporter 1.

28 Continuing with the exhaustive analysis on the search for the specification of each compound towards glycine transporters. The next step to be considered in this project was the cytotoxicity analysis of the treated compounds (HIT1, HIT2, HIT3). It is well known that in order to carry out a complete process in the pharmacological area of acceptance of any treatment, the pertinent analyzes must be carried out regarding the toxicological effects that they would have on humans. Furthermore, if said would be considered acceptable for said treatment to be considered for possible use in the medicinal field.

Following the process, only the HIT1-3 compounds showed favorable inhibition results to the glycine transporter 2 and followed by the fact that the results favor a selectivity only for the glycine transporter 2. A series of treatments on cytotoxicity was performed on the compounds to know how feasible the compounds' possible use is later in chronic pain treatments.

29 CYTOTOXICITY OF HIT COMPOUNDS ON PAE CELLS

Figure 14. Three HIT compounds and ALX inhibitor were tested for their capacity to inflict cytotoxicity on PAE cells. Expose the cells to the concentration gradient (in µM) of each inhibitor and incubate for 24 hours. Differential nuclear staining and bioimaging system

(BD Pathway 855) are used to determine the percentage of cytotoxicity (y-axis). When the experimental samples were compared with 1% v/v PBS solvent (*) and untreated (Unt.) (‡) control cells, the P values were <0.001 and <0.001, respectively. Cells treated with 1 mM

H2O2 were used as a positive control for cytotoxicity. Each experimental point represents the average of four replicates, and the error bar represents its corresponding standard deviation—experiments and analysis by a collaboration with Dr. Armando Varela.

30 CONCLUSION

All in all, these data together prove that the structure of the project has high quality. The first four compounds' development showed the efficient possible work made by using homology/MD-derived structures. Our visual screening method can still result in a high hit rate of 75%. Allowing us to discover a set of new compounds with good inhibitory properties and selectivity can be further optimized in the analysis of more and more new compounds, looking for possible targets for chronic pain treatments. Furthermore, Hit1 was considered the first and principal candidate for further optimizing potential customers and making a detailed and more specific analysis on that ligand.

We observed some factors that made Hit1 a primary ligand, such as the stable hydrogen bonds interacting with glycine transporter 2, showing a similar development to ALX1393 activity. Through several MD simulations. Exciting interactions between the ligand and several residues can be observed. The carbonyl group of Hit1 interacted with three residues

Leu211, Gly212, Tyr287, and the first sodium of the receptor, too; interactions showed similarities with those of ALX1393 activity.

These results provide a structural basis for the Hit1 selectivity observed experimentally in GlyT2. Hit2 showed the closest results to Hit1. We considered some factors, such as the carbonyl group's orientation and the missing of one oxygen in the ring structure. Moreover, the lack of interaction with one of the compounds (HIT4) could be observed. In the interest-based on their stoichiometry similarities, you can make the mistake of assuming that if it works with a glycine transporter when in fact, it would be inhibiting both transporters, bringing severe damage to the subject to whom it was supplied.

31 Situation for which, we made the decision to try four HIT compounds in interaction with the glycine transporter 1 to check if said compound ultimately had no interaction with the two glycine transporters or indeed demonstrated an inhibiting interaction with the glycine transporter 2 and the glycine transporter 1 too. As could be observed in the beneficial pertinent experiments, it was shown that the HIT4 compound was more refined to an interaction with the glycine transporter 1 and not as we assumed with the transporter of glycine 2 the three compounds before that interacted. Besides, it could be shown that the other three compounds did not lead to any interaction with glycine transporter 1.

Finally, our research shows that the combination of structure-based pharmacophores and docking technology can help identify new biologically active and selective compounds. This combination allows us to identify new and selective GlyT2 inhibitors. The FEP+ method provides detailed insights into the structural basis of selectivity and proposes ideas for further optimization of leads.

The in-vitro assays were very useful in the analysis of the inhibition interaction from the different compounds to the glycine transporters. Confirming the interaction efficiency of the compounds showed on the docking technology and the FEP+ method. Corroborative the idea that it is possible to find new treatment targets for chronic pain and decrease the uncontrolled use of opioids as the first conventional option to treat chronic pain. Although there is high sequence similarity between the binding sites of GlyT2 and GlyT1, on our project, one of the main aspects to be considered was the specificity of the compounds tested.

The experimental data of the Hit1 compound that we found shows that it is a highly selective GlyT2 inhibitor. The difference between the binding sites of these transporters

32 (GlyT1 and GlyT2) subclasses is only five residues. Therefore, Hit1 is an ideal compound to reveal selectivity. Thanks to the experiments on cytotoxicity that were carried out, the HIT compounds obtained favorable results, which bring us closer to good possibilities of using the compounds as treatment—showing data in low concentrations a good development of the three compounds interacted favorably with the glycine transporter 2. The graph shows as in comparison with the positive, negative controls and the untreated cells.

The interaction of these compounds show similarities with ALX1393. This series of virtual analyzes on molecules, as well as in-vitro experiments and cytotoxicity, help to consolidate further the idea that there are more possibilities to achieve new possible alternatives for chronic pain treatments. Avoiding a relapse into the use of opioids, improving patients' opportunities to lead a life free of secondary complications, and more stable lifestyles. If it is true that there is still much to know about new alternatives for the treatment of chronic pain, this project has been another step in that search.

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38 GLOSSARY

CNS: Central nervous system is the segment of the nervous system that involves the brain stem, spinal cord and cerebellum.

GlyT: Glycine transporter require on the transport of glycine.

GlyT1: Glycine transporter 1 is located on the glial cells, astrocytes and post-synaptic terminals, the excitatory synapses of glycine on the central nervous system are controlled by this transporter.

GlyT2: Glycine transporter 2 is located on the presynaptic terminals, the inhibitory synapses of glycine on the central nervous system are controlled by this transporter.

IFD: known as the Induced-fit docking a computational technique of the virtual docking studies about the interaction between molecules.

MD: Molecular docking, computational technique used on the analysis finding the most stable complex between a molecules interaction.

NMDA: N-methyl-D-aspartate is a glutamate receptor found the nerve cells.

PAE: the Porcine endothelial aortic cells are considered as a stable line of cells used on the study of several systems

PI: the propidium iodide is a red fluorescent dye, not permeant to live cells, that detects damage membrane cells.

SLC6: Solute carrier 6 family is one of the extended family of homologous transporters that includes many prokaroytic transporters.

39 CURRICULUM VITA

Elvia Lilia Padilla Mendez was born in Cd. Juarez, Chihuahua. I graduated from the high school El Colegio de Bachilleres del Estado de Chihuahua in June, 2011. On same year (2011)

I entered to the University of Texas at El Paso, where I received the Bachelor of Science degree based on Biochemistry in 2016. From 2017-2018, I entered to the OPT (optional practical training) program, which helps international students to develop their knowledge obtained during their studies on the United States, during this period I obtained an opportunity as a coordinator I laboratory/research. From 2018-2020 I was teacher assistant, developing my aptitudes preparing classes and helping students to understand topics on the biology and anatomy fields.

The project in which the master’s degree is based on, was published on the ACS

Medicinal Chemistry Letters website as “Discovery of GlyT2 inhibitor using structure-based pharmacophore screening and selectivity studies by FEP+ calculations”. Also, it was possible to participate on two different conference; the Society of Neuroscience conference in Chicago,

Illinois, and the Southwest and rocky Mountain Regional Meeting in El Paso, Texas.

It is possible to contact me by [email protected]