Synthesis, in Vitro Characterization and Applications of Novel 8-Aminoquinoline Fluorescent Probes Adonis Mcqueen University of South Florida, [email protected]

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October 2017 Synthesis, in vitro Characterization and Applications of Novel 8-Aminoquinoline Fluorescent Probes Adonis McQueen University of South Florida, [email protected]

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Synthesis, in vitro Characterization and Applications of Novel 8-Aminoquinoline Fluorescent Probes

Adonis McQueen

A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Molecular Medicine Department of Molecular Medicine College of Medicine University of South Florida

Major Professor: Dennis E. Kyle, Ph.D. Burt Anderson, Ph.D. Yu Chen, Ph.D. Lynn Wecker, Ph.D.

Date of Approval: October 13, 2017

Keywords: Malaria, Primaquine, Drug Discovery, Mechanism of Action, Parasitology, Medicinal Chemistry

ACKNOWLEDGMENTS

This research is a culmination of everyone I’ve interacted with, everyone who’s taught me and everyone I’ve ever taught. I thank my Creator for allowing me to make it this far. As far as people go, I owe my first thanks to Dr. Dennis Kyle. Without your mentorship, patience and guidance, I wouldn’t have been able to complete this project. You are an amazing scientist and an even greater man. I am privileged to have known you and to work in your lab. My time in your lab has provided me with the skills necessary to pursue any era of research I wish. For that, and for your countless lab meeting jabs, I will always be grateful.

To Dr. Roman Manetsch, you too have been an excellent mentor and professor. Our interactions have taught me to not let setbacks weigh me down, and that any progress is good progress. I am grateful for your attentiveness, honesty, wisdom, and light hearted nature. Working in your lab made me re-discover why I enjoy chemistry so much.

To my scholarship and fellowship mentors Ms. Edna Cofield and Mr. Bernard Batson. You both have made sure that my life stayed balanced and informed during my professional transitions. Both of you have helped me develop my professional network in ways I still cannot even imagine. I am blessed to know the both of you. The progress made by thousands of students can be directly or indirectly attributed to your commitment to your work. I only hope I can have the same impact on students as the two of you.

To my family: Gloria McQueen (Mom), Monica, Lisa, Jasper and Ocie. You all have been a huge help not just in graduate school, but throughout my entire education as I reached for this goal. Now that I’ve attained it, each of your individual inputs over time have all had a great impact on my life and success. I couldn’t have done this without any of you, especially Mom. You always encouraged me, helped me when I needed it, and told me your truth even when I didn’t want to hear it. I am truly blessed and privileged to have such loving people in my family.

To my lab mates and friends, there are so many of you to thank I couldn’t even begin to list you all (but

I’m going to try!!!). Sasha, Lynn, Marvin, Debbie, Tina, Miles, TJ, Beatrice, Samantha, Steve and

Amanda from the Kyle lab; all of you have helped me in one way or another to complete my goal. I literally couldn’t have done this without any of you. Niranjan, Kurt, Cindy, Iredia, Jordainy, David and

Andrii from the Manetsch lab; each of you have been instrumental in helping me refine my rough organic chemistry skills. I hope that we all can continue to learn from each other in the years to come. To my close friends Jamarr, Liz, Jennifer, Kyle, Ahmad, Chris, Geoff, Krishna, Jessica, Stephanie, Brian, Jerome and Kali, thank you for keeping me sane in between the rough shores of endless experiments and information dense classes. You all have literally helped me maintain my sanity, and each of you has been vital in my physical and mental transformation. I couldn’t have asked for a better group of people to associate myself with.

I love and appreciate every single person listed in this acknowledgement. “Alone we can accomplish so little, but together we can accomplish so much”. This quote means so much more to me now than it ever did before, and I know it’s because of the impact you all have had on my life. I pray that I am able to exceed all of your expectations as well as my own. Let us all create the future together.

TABLE OF CONTENTS

List of Tables ...... iii

List of Figures ...... iv

Abstract ...... vi vi

Chapter 1: Introduction ...... 1 1 Malaria History ...... 1 Life Cycle ...... 1 1 Pathology ...... 3 3 Immunization and Human Host Protection Mechanisms...... 5 Immunity and Immunization...... 5 Human Host Protection Mechanisms ...... 7 Glucose 6-Phosphate Dehydrogenase Deficiency ...... 7 Thalassemias ...... 8 Sickle Cell Anemia ...... 9 Malaria Chemotherapeutics: Mechanisms of Action and Resistance ...... 10 Arylamino Alcohols ...... 10 Quinine ...... 10 Mefloquine ...... 11 Antifolates and Atovaquone ...... 11 Sulfadoxine and Pyrimethamine ...... 11 Proguanil ...... 12 Atovaquone ...... 13 Antibiotics ...... 14 Clindamycin ...... 14 Doxycycline ...... 14 4-Aminoquinolines ...... 15 Chloroquine ...... 15 Amodiaquine ...... 17 Piperaquine ...... 18 Endoperoxides ...... 18 Methylene Blue and 8-Aminoquinolines ...... 20 Methylene Blue ...... 20 8-Aminoquinlines: Primaquine and Tafenoquine ...... 21 Project Specific Background ...... 25 Focus of Study ...... 26 27

Chapter 2: Synthesis and Characterization of Novel 8-Aminoquinoline Fluorescent Probes ...... 31 Introduction ...... 31 32 Materials and Methods ...... 33 PQCP synthesis ...... 33 TQCP synthesis ...... 34 Absorbance and Steady State Emission studies ...... 35 Results ...... 35 36

Conclusion ...... 38 39

Chapter 3: In vitro Characterization of 8-Aminoquinoline Fluorescent Probes ...... 50 Introduction ...... 50 Materials and Methods ...... 51 Culturing of P. falciparum parasites and in vitro cultures ...... 51 Characterization of anti-malarial activity in asexual stages ...... 52 L. donovani axenic amastigotes assay ...... 52 L. donovani cytotoxicity assay ...... 53 Hepatocyte Culture ...... 53 Animals and Parasites ...... 53 Cytotoxicity of PQCP and TQCP in HepG2 Cells ...... 54 Fluorescent microscopy of sexual and asexual stage parasites ...... 54 Statistical analysis ...... 55 Results ...... 55 57 Coumarin does not affect 8-AQ anti-leishmaniasis or anti-malarial efficacy ...... 55 TQCP and PQCP localize in the cytosol of P. falciparum parasites ...... 56 TQCP and PQCP are not toxic to HepG2 cells ...... 56 Discussion ...... 56 58

Chapter 4: In vivo assessment of the mechanisms of action, synergy and resistance of anti-malarials ...... 68 Introduction ...... 68 69 Materials and methods ...... 69 Mosquito infections, sporozoite production and harvesting ...... 69 Animals and parasites ...... 70 Drug screening assay ...... 70 Synergy screening of primaquine with chloroquine or quinine ...... 71 Microscopy of P. berghei infected HepG2 cells with PQCP ...... 71 Generation of menoctone resistance in P. berghei and mutation detection ...... 71 In vivo antimalarial efficacy against blood stages of P. berghei...... 71 Elucidation of mutation selection by atovaquone or menoctone ...... 72 P. berghei MEN (MRA414) assessment of atovaquone cross resistance ...... 72 Results ...... 72 73 Discussion ...... 73 74

Chapter 5: Summary ...... 78 79

Chapter 6: References ...... 81

Appendix A: Copyright Permissions ...... 90

LIST OF TABLES

Table 2.1: Various screening conditions for EDCI/Dmap coupling of PQCP ...... 36

Table 3.1: IC50s of PQ, PQCP, TQ, and TQCP against P. falciparum strains ...... 61

iii

LIST OF FIGURES

Figure 1.1: Life Cycle of the malaria parasite...... 3

Figure 1.2: Structures of selected 8-AQs ...... 22

Figure 1.3: Proposed PQ metabolite redox cycling due to CYP2D6 metabolism ...... 24

Figure 1.4: Hydroxylated PQ metabolites ...... 27

Figure 1.5: Sources of ROS in plasmodium ...... 28

Figure 1.6: Thioredoxin system in plasmodium ...... 29

Figure 1.7: The glutathione system in plasmodium ...... 30

Figure 2.1: Examples of the Perkin and Pechmann reactions ...... 32

Figure 2.2: First generation synthesis of PQ-linker ...... 40

Figure 2.3: Synthesis of bromo-coumarin moiety...... 41

Figure 2.4: PQ-linker and bromo-coumarin reaction ...... 42

Figure 2.5: Second generation synthesis of PQCP and TQCP ...... 43

Figure 2.6: ESI-HRMS of PQCP ...... 44

Figure 2.7: ESI-HRMS of TQCP ...... 45

Figure 2.8: 1H-NMR of PQCP ...... 46

Figure 2.9: 1H-NMR of TQCP ...... 47

Figure 2.10: Normalized optical absorption spectra ...... 48

Figure 2.11: Normalized steady state emission spectra ...... 49

Figure 3.1: 3H-Hypoxanthine Dose response for TQ/TQCP...... 59

Figure 3.2: 3H-Hypoxanthine dose response for PQ/PQCP ...... 60

Figure 3.3: PQ/PQCP activity against L. donovani...... 62

Figure 3.4: Cytotoxicity response of PQ/PQCP and TQ/TQCP against HepG2 ...... 63

Figure 3.5: PQ/PQCP and TQ/TQCP activity against P. berghei ...... 64

Figure 3.6: Images of PQCP in asexual D10 P. falciparum parasites ...... 65

Figure 3.7: PQ saturation of D10 P. falciparum parasites ...... 66

Figure 3.8: Images of PQCP in NF54 strain of P. falciparum parasites ...... 67

Figure 4.1: Survival curves of sensitive vs menoctone resistant P. berghei ...... 76

Figure 4.2: Assessment of ATQ cross resistance with menoctone ...... 77

ABSTRACT

Malaria is a parasitic disease that is caused by the plasmodium parasite. Plasmodium infection has affected man for thousands of years. With advances in drug discovery over the past century, malaria has evolved to possess resistance to most mainline therapeutics. This war of drug discovery vs plasmodium evolution continues to be fought to this very day, with attempts to eradicate malaria worldwide. Frontline treatments such as chloroquine, artemisinin, and atovaquone/proguanil have all seen parasitic resistance in strains of P. vivax as well as P. falciparum. While plasmodium possesses resistance to most classes of anti-malarials, the 8-aminoquinoline (8-AQ) class has seen minimal resistance development. 8-AQs have been shown to be effective against erythrocytic and exo-erythrocytic forms of plasmodium, and are often given in combination with a blood schizonticide such as chloroquine or artemisinin. These combinations clear all forms of plasmodium infection. With 8-AQs unique set of anti-malarial properties and the advent of increased drug resistance to other drugs, much research is being done to understand 8-AQs mechanism of action and toxicity. 8-AQ use is limited due to inducing extreme hemolytic anemia in those with glucose-6-phosphate dehydrogenase (G6PD) deficiency. Primaquine is the only 8-AQ molecule available on the market with tafenoquine, an analog primaquine, currently in phase III clinical trials. It is believed that if the mechanism of action and toxicity of the 8-AQs are understood, then we can create new generation anti-malarials that will maintain the unique action of 8-AQs while reducing their toxicity.

Studies have shown that 8-AQ mechanism of action has been attributed to the generation of unstable metabolites that induce ROS production in the parasite, as well as mitochondrial swelling. While there is some evidence suggesting molecular targets of 8-AQs, the actual target is still unknown. When 8-AQs is given in combination with chloroquine, a synergistic effect is observed. While chloroquine has no activity against liver stages, it still somehow potentiates primaquine’s activity in those stages. This mechanism of synergy in liver stages is not well understood, and its understanding can give us increased understanding of basic plasmodium biology in the liver. Additionally, more information about the mechanisms of action

vi of both chloroquine and primaquine could be elucidated. Tagging drugs with fluorescent probes is a technique that can give much information about the drug’s pharmacological activity in vitro, and sometimes in vivo as well. Such an approach has been used for various disease states such as HIV and cancer. Malaria is no exception; fluorescent probes of artemisinin and chloroquine have been used to examine resistance mechanisms to both molecules. In addition to 8-AQs, there are other older anti- malarials that have received attention recently due to increases in resistance. Menoctone, a hydroxynapthoquinone that subsequently lead to the discovery of atovaquone, has recently gained increased attention because of its similarities to atovaquone. Research surrounding menoctone was abandoned due to the discovery of more efficacious compounds. Similar to 8-AQs, understanding the mechanisms of action and resistance to menoctone could give us much more information about plasmodium responses to this class of compounds. This understanding could potentially lead to the discovery of novel therapeutics. To understand mechanisms of action and synergy of 8-AQs, we report the creation of novel fluorescent probes of the 8-AQ molecules primaquine and tafenoquine. The organic synthesis was designed and characterization was confirmed by NMR and high resolution mass spectra, and the fluorescent properties were examined using absorbance and steady-state emission experiments.

We found that the anti-malarial, anti-leishmaniasis, and cytotoxic properties of these novel probes were similar to the parent compounds. These probes localized in the cytoplasm of infected parasites in vitro.

We also attempted to view their localization in liver stage infection, and investigated the synergistic combination of 8-AQs with chloroquine and quinine. Menoctone resistance was induced in vivo to determine mechanisms of resistance. Cross resistance to atovaquone was observed, and the mutation responsible for resistance was also found.

vii

CHAPTER 1: INTRODUCTION

MALARIA HISTORY

Malaria, the disease caused by the Plasmodium parasite, originally got its name from medieval Italian, meaning “bad air” (mal-bad, aria-air) (1). When malaria was first described, it was believed to be an airborne disease. It wasn’t until 2000 years later that it was discovered that malaria was transmitted by the anopheles mosquito via a very delicate and complicated system of infection. Over the next several millennia, Plasmodium infection would continue to ravage communities all over the world. In China, the

Artemisia annua plant was used to treat fever and was one of the first antimalarial treatments to be used before the parasite would be identified. In the late 19th century quinine would be discovered as a treatment, and Plasmodium parasites would first be observed by geimsa stain. There are several species that infect man, each with varying severity. Plasmodium falciparum, vivax, malaria, ovale, and knowlsei infect humans, with falciparum and vivax being the most deadly. Plasmodium has evolved to hundreds of different species that infect birds, pigs, primates, rodents, and other mammalian species. The definitive host is the anopheles mosquito, while the intermediate hosts are often mammalian vertebrates.

LIFE CYCLE

The life cycle of Plasmodium begins when an infected mosquito takes a blood meal from an animal. The infected mosquito injects sporozoites from its salivary glands, which migrate to the liver within 30 minutes. Once in the liver, the sporozoites infect hepatic cells. Sporozoites tend to pass through several hepatic cells killing them in the process. They select for certain hepatocytes, but how this selection occurs is unknown. Once the appropriate hepatocyte is selected, the sporozoites begin asexual reproduction, forming hepatic schizont forms filled with merozoites. These merozoite forms, which are responsible for blood stage infection, rupture from the hepatic schizonts, circulate via the blood stream, and begin

1 infecting erythrocytes. In vivax and ovale infections, it is believed that dormant forms of the parasite known as hypnozoites form a reservoir in hepatic cells and are responsible for relapsing infection. This theory however, remains a source of debate (2).

The invasion of erythrocytes by merozoites is mediated by several signaling processes and events.

Merozoites invaginate into the erythrocyte to begin infection. Merozoites then turn into the first asexual form known as rings, or young trophozoite forms. These forms are described as rings due to their structure resembling a jewelry ring, characterized by a round cytoplasmic form, and the nucleus resembling the ‘jewel’. Depending on the species of infection, these forms last between 22-24 hours. The next form is the mature trophozoite stage, characterized by the food vacuole (responsible for heme digestion), nucleus, and active heme metabolism and digestion. It is active for 6-8 hours. The final form is the schizont form, where multiple merozoites have been created, each with its own nucleus. The schizont subsequently ruptures the erythrocyte, where the merozoites are released and are free to infect other erythrocytes to continue the cycle. In P. falciparum, vivax, and ovale the cycle takes 48 hours. For malariae however, the cycle can take up to 72 hours(3).

It is believed that the ring stage form of Plasmodium differentiates into the male and female sexual forms of malaria known as gametocytes. Gametocyte growth, known as gametogenesis, occurs in five stages each with their own characteristic structure and molecular functions. The later stages of gametocytes (IV-

V) are responsible for transmission to the anopheles mosquito. This is known as the sexual stage of replication; stage V gametocytes differentiate into male and female forms. When an uninfected mosquito takes a blood meal from an infected person, the mosquito ingests the mature gametocytes. The gametocytes then turn into gametes which mate inside the mosquito, then forms a zygote, which progresses to an ookinete form. This ookinete then forms an oocyst in the midgut of the mosquito. The oocyst then develops over 10-14 days until mature sporozoites form. Once the oocyst ruptures, the sporozoites migrate from the midgut to the salivary glands, where they are injected into an uninfected

2 person during a blood meal, beginning the cycle all over again. Figure 1.1 shows a graphical representation of the life cycle.

Figure 1.1- Life cycle of the malaria parasite. Open source document from the NIAID, 2012

PATHOLOGY

Severity of infection differs greatly between parasite strains. Falciparum malaria is much more deadly than vivax malaria due to falciparum causing severe and cerebral malaria symptoms that often lead to death. Vivax infection is usually described as benign tertian malaria, and doesn’t cause severe disease unless other factors are present that exacerbate the infection such as malnutrition, co-infections, or mixed malaria infection (4).

Typically, malaria infection is seen as uncomplicated or severe, and treatment options differ depending on the type. The symptoms of malaria are primarily due to asexual replication within erythrocytes, as well as morphological changes and protein expression that occur within these stages. Each replication cycle over a period of 48 hours produces symptoms that are congruent with the various asexual stages. The set of symptoms usually associated with uncomplicated malaria infection include fever, chills, headache, nausea, fatigue, and vomiting. Physical presentations may include sweating, enlarged spleen and liver, and increased breathing rate. In severe malaria, symptom presentation may include coma, severe anemia, acute kidney failure, metabolic acidosis, bacterial meningitis and convulsions. These symptoms are often the result of extremely high parasite burdens, metabolic disorders or organ failure when infected with

Plasmodium (5). Parasite rupture of erythrocytes as well as complement mediated lysis is associated with fever/chills, increased inflammation and anemia. Sequestration of infected erythrocytes results in reduced blood flow throughout the body and is thought to be an essential event leading to cerebral malaria.

Sequestration of parasites is exacerbated by cytoadherence of infected erythrocytes. The expression of the

PfEMP1 protein on the surface of infected erythrocytes adheres to the endothelial lining of capillaries, resulting in a ‘rolling’ phenomenon that inhibits proper circulation of infected and non-infected erythrocytes alike. Symptom presentation of severe/cerebral malaria can differ between adults, children, and pregnant women (4).

Children can sometimes carry parasite burdens while being asymptomatic, though in endemic areas, severe disease can occur quickly. One life threatening symptom is usually present as opposed to many symptoms in non-immune adults. When children present multiple life threatening symptoms, the prognosis is usually much worse, often leading to death. While cerebral malaria is more deadly than malarial induced anemia, anemia results in more deaths due to its increased prevalence. Severe malarial anemia causes more healthy erythrocytes to be ruptured as the disease progresses.

In non-immune adults, severe malaria can be common when they are infected with P.falciparum. Clinical manifestations of severe falciparum malaria in adults differ from the pediatric version of the disease, and include cerebral malaria, jaundice, pulmonary edema, and kidney failure. Additionally, travelers from non-endemic regions are much more susceptible than those who reside in endemic regions who may have acquired partial immunity over time.

Women in endemic areas acquire some immunity to malaria after years of exposure. However, once becoming pregnant, their susceptibility increases. This greatly affects the newborn with low birth weight, and significantly contributes to infant mortality in malaria endemic regions. Severe malaria is also more likely to occur, with parasites sequestering around the placenta. This sequestration affects the development of the fetus, leading to birth complications, developmental defects in the newborn and even maternal mortality (4).

IMMUNIZATION AND HUMAN HOST PROTECTION MECHANISMS

Immunity and Immunization

Natural and/or acquired immunity to malaria can affect a number of factors in Plasmodium infection.

From initial transmission to the production of gametocytes to chemotherapeutic outcomes, the effects of immunity on Plasmodium biology is vast.

In areas endemic with malaria, immunity increases with age, which explains why most malaria related deaths are due to severe malaria in children under the age of 5. After this age malaria related deaths sharply decline, but susceptibility to non-life threatening episodes continues. Evidence for this age related trend was observed in a study of naive adults traveling to malaria endemic regions; the adults acquired immunity more rapidly than children from the area (6). Further evidence for this phenomenon was observed when children and adults were treated with chloroquine against parasites that were chloroquine

5 resistant. As the age of the afflicted increased, their ability to clear parasites after treatment with chloroquine also increased (7).

Immune responses could target any stage to which humans are exposed. Immune responses to exo- erythrocytic and sexual stages have been observed, but the effects on transmission are unclear.

Furthermore, there is evidence that naturally acquired immunity is predominantly against erythrocytic stages and is antibody mediated. Antibodies may act by clearing infected erythrocytes or by acting against merozoites before erythrocytic infection (8, 9). Antibody and spleen clearance of infected erythrocytes results in the lysis of erythrocytes, leading to the characteristic anemia symptom of malaria pathology.

The relative importance of each antibody clearance mechanism is still a source of debate. The most likely scenario is that the various mechanisms act in concert to offer protection against Plasmodium infection.

One of the main goals for malaria eradication is the production of a safe vaccine with long term protection and efficacy. This vaccine would need to have the ability to focus on eliminating the parasitic infection prior to clinical manifestations associated with blood stage infection. It is believed that the portion of the life cycle most vulnerable to vaccine protection is the point of mosquito to host transmission, or transfer from the definitive (Anopheles) host to the intermediate host. Despite millions of sporozoites being injected upon mosquito bite, very few make it to the liver to begin infection. No host immune protection is ever elicited against these forms despite repeated infection (10). The majority of research efforts have surrounded either the RTS,S/AS01 vaccine or the PfSPZ vaccine. RTS,S/AS01 is currently considered the gold standard to which all future vaccines will be compared, due to it being the most successful vaccine candidate until very recently. The RTS,S/AS01 is an anti-sporozoite vaccine which is currently in phase

III clinical trials. RTS,S/AS01 is comprised of a liposome-based adjuvant (AS01) and a hepatitis B virus surface antigen (HBsAg) virus-like particles which incorporates a portion of the P. falciparum derived circumsporozoite protein (CSP) fused to HBsAg (11). CSP is a protein that covers the surface of

Plasmodium spp. sporozoites. RTS,S/AS01 confers protection against malaria parasites, but this

6 protection has a short duration and is mildly successful in reducing parasite burdens in children and infants (8, 12).

The second vaccine candidate is the PfSPZ vaccine, which is a radiation-attenuated, whole-cell sporozoite vaccine. This vaccine has recently entered phase III clinical trials as well, and was found to achieve >80% sterile protection against controlled human malaria infection (CHMI) with homologous falciparum parasites. Further studies showed that the PfSPZ vaccine also confers protection against heterologous

CHMI for up to 33 weeks after the final immunization, and that protection against heterologous vs homologous CHMI is a dose dependent phenomenon (13). Both vaccines offer great promise, but both have been limited in that they’ve only been screened against CHMI rather than heterogeneous natural exposure. Age specific immunization, screening in pregnant women and immunocompromised subjects and responses in malaria endemic populations must all be addressed before these vaccine candidates can be used in widespread eradication efforts (14).

Human Host Protection Mechanisms

Glucose 6 Phosphate Dehydrogenase Deficiency. Glucose-6-Phosphate Dehydrogenase (G6PD)

Deficiency is an X-linked genetic disorder that results in mutations in the G6PD gene. G6PD is an enzyme that is present in the pentose phosphate pathway, which is responsible for supplying energy in the form of ATP to cells and also maintaining levels of nicotinamide adenine dinucleotide phosphate

(NADP+). NADP+ then maintains levels of glutathione which protect the cell from oxidative damage from ROS. G6PD catalyzes the rate limiting step of the pentose phosphate pathway, which is the reduction of NADP+ to NADPH:

D-glucose 6-phosphate + NADP+ 6-phospho-D-glucono-1,5-lactone + NADPH + H+

Due to G6PD’s importance in the pentose phosphate pathway, its inhibition or lack of function would have significant downstream effects. G6PD deficiency affects millions worldwide, and is associated with

7 many clinical presentations. Neonatal jaundice, cardiovascular disease, and hemolytic anemia are common clinical presentations of the deficiency. 8-aminoquinolines, a class of anti-malarial drugs, are known for causing severe hemolytic anemia in those with G6PD deficiency. However, G6PD deficiency has been positively correlated with increased resistance to malaria infection, as well as areas that are endemic with malaria. One study in African children with G6PD deficiency displayed a 46-58% reduction in severe malaria risk among them (15). Other studies suggest that parasite loads are lower in cells with deficient enzyme activity rather than normal levels (16). Taken together, these studies show that parasite proliferation is impaired in individuals with G6PD deficiency. How G6PD deficiency confers anti- malarial resistance is currently unknown. It is hypothesized that red cells in G6PD deficient individuals may be under increased oxidative stress, which is a toxic environment for P. falciparum parasites.

Additionally, those G6PD deficient cells that are infected by ring stage parasites have impaired anti- oxidant defenses and maybe more likely to undergo phagocytosis, leading to reduced numbers of mature parasites (16).

Thalassemia. Thalassemia is a genetic blood disorder that results in abnormal hemoglobin formation. It is an autosomal recessive trait that affects individuals in the Middle East, parts of Europe, northern Africa and Asia. This abnormal hemoglobin formation is due to the type of thalassemia present, and there are two types of thalassemia; alpha and beta. In normal adult hemoglobin, there are four protein chains (2 alpha and 2 beta) arranged in a heterotetramer structure. Alpha thalassemia affects the duplicated alpha-chain genes and causes underproduction of alpha-globin chains. Beta-thalassemia affects the single beta chain and causes abrogation or underproduction of beta chains (16). Both versions of thalassemia confer partial protection against malaria infection. In -thalassemia, erythrocytes infected with Plasmodium parasites bind high levels of antibodies from malaria endemic sera. This causes a downstream effect which ultimately results in the clearance of infected erythrocytes by immunity mechanisms such as compliment mediated lysis, opsonization, and inhibition of sequestration of infected erythrocytes. In -thalassemia, protection is conferred by reduced rates of severe malaria infection, or by 8 increasing phagocytosis of infected erythrocytes (17). Whether or not the individual is homozygous or heterozygous for either  or  thalassemia also seems to affect the degree of protection, but the extent and significance of this protection is still a matter of debate (16, 17).

Sickle Cell Anemia. Sickle-cell disease (SCD) is a group of autosomal recessive genetic blood disorders. It is one of the most prevalent erythrocytic alterations in malaria endemic regions. The most common presentation is known as sickle-cell anemia. It results in a mutation in the hemoglobin B (HBB) gene which changes the form of the hemoglobin S. Homozygous SCD is often fatal in early life without proper treatment. Even with proper treatment, homozygous SCD individuals still experience a shorter life span (16). Malaria attacks and deaths are increased in homozygous SCD individuals, and it is suggested that they consider life-long malaria chemoprophylaxis in endemic areas. This however, comes with its own risks, as life-long chemotherapy could increase side effects, impair natural immunity tolerance to malaria, and increase parasitic resistance to the drugs (18). Those who are heterozygous for SCD have less clinical crises and milder symptoms. Additionally, they enjoy a degree of protection from

Plasmodium infection. Studies have shown that these heterozygous individuals are protected against severe malaria episodes, and even have lower parasitemia burdens in uncomplicated malaria infection

(16, 17). Two main theories surround the mechanisms of protection conferred by heterozygous mutation.

The first is that intra-erythrocytic parasite growth is inhibited by hemoglobin S polymerization when oxygen levels drop below 5%. The second is that parasite-infected sickle erythrocyte phagocytosis by host immune cells is done at a much higher rate compared to infected normal erythrocytes. It has also been shown that hemoglobin S erythrocytes infected by P. falciparum lower the expression of PfEMP-1, which results in a reduction of cytoadherence and subsequent protection from severe malaria (17, 19).

MALARIA CHEMOTHERAPEUTICS: MECHANISMS OF ACTION AND RESISTANCE

Arylamino Alcohols

Quinine (QN). Quinine is an aromatic alkaloid that was discovered in the mid-1800s. It was initially isolated from the bark of the Peruvian Cinchona tree and became one of the first mainstay treatments as an anti-malarial (20). It is fast acting with a short half-life, and has been recommended for the treatment of uncomplicated malaria in pregnant women and even for drug resistant parasites (21, 22).

Despite its efficacy as an anti-malarial, its use alone is limited due to long treatment regimens as well as side effects such as blurred vision, tinnitus, headache and nausea. Therefore, QN is often given in combination with other anti-malarials such as doxycycline, or in artemisinin combination therapy (ACT), which enhances its anti-malarial activity and efficacy. It even has fluorescent activity, and is sometimes used for fluorescence studies in photochemistry (23). QN anti-malarial mechanism of action has been investigated by various studies. One study links its mechanism to the digestive vacuole via fluorescence localization, showing that it localizes to a non-acidic compartment within the digestive vacuole (24).

Other structural studies have shown that it forms complexes with ferriprotoporphin IX (FP). These complexes are -oxo dimers which are stabilized by  stacking interactions. These interactions are important for the stability of the complex and QN anti-malarial activity, as this complex is toxic to the parasite. These complexes have been studied in detail, as they occur with other anti-malarials containing the quinoline pharmacophore such as chloroquine and quinidine (25, 26). QN resistance in isolates is rare, most likely due to QN short half-life in vivo. In vitro studies suggest that decreased susceptibility to QN is due to mutations in the PfNHE-1 gene, which codes for a Na+/H+ exchanger. There is a microsatellite polymorphism consisting of DNNND repeat units, which has been defined as ms4760. Variant ms4760 with two copies of DNNND repeat units was associated with reduced QN sensitivity in vitro (27, 28).

However, this evidence was found to possibly be dependent on the genetic background of the parasites and/or geographic origin (28).

Mefloquine (Mfq). Mefloquine is a synthetic anti-malarial that was synthesized as an analog of quinine. Mfq has found much success as an anti-malarial, and is effective against all species of

Plasmodium that infect humans. Additionally, it is one of the few anti-malarials that is effective against all the asexual forms of infection (25). It is therapeutically dosed once per week, allowing for good compliance among users. It is often prescribed to travelers going to malaria endemic regions as a prophylactic. Its pharmacokinetics is characterized by low oral clearance, a large apparent volume of distribution and a long half-life, though individual differences have been observed (29). Mfq mechanism of action is a matter of debate. As an analog of QN, it possesses a shared mechanism according to various studies. These studies indicate that Mfq, like QN, inhibits the ingestion of host cell hemoglobin by potentially interfering with the endocytic process. Another study found that Mfq could treat drug resistant cancer cells in vitro by inhibiting p-glycoprotein (30). This specific inhibition has been implicated as a mechanism for anti-malarial activity; the homolog PfPgh1 is expressed as an integral digestive vacuole membrane protein. This is critical, since QN and Mfq share similar mechanisms via action in the digestive vacuole, inhibition of this protein could be a significant contributor to Mfq mechanism of action (25).

Further evidence of PfPgh1 contribution to Mfq mechanism of action is also implicated in parasites resistant to Mfq and QN. Plasmodium resistance to Mfq has been found to be linked to PfMDR1 as well as Pgh1 expression. Clinical isolates that are resistant to Mfq display an increased copy number of the

PfMDR1 gene (31), as well as increased expression of Pgh1 (25).

Antifolates and Atovaquone

Sulfadoxine and Pyrimethamine (SP). The treatment combination of sulfadoxine and pyrimethamine is known as fansidar, and is used to treat Plasmodium infection. Pyrimethamine was found to have anti-malarial activity in the early 1950s, and was used for prophylaxis and as a

11 monotherapy against P. falciparum infection. Sulfadoxine was found to be a slow acting blood schizonticide in clinical trials almost a decade later. Shortly after sulfadoxine’s anti-malarial activity was found, more studies revealed that faster action against malaria was found when the two drugs were combined (32, 33). SP is often used as a first line anti-malarial treatment for several reasons. It is a cheaper alternative to chloroquine, can be used to treat chloroquine resistant parasites, and it is safe for use in pregnant women, children and even infants (33). The mechanism of action differs slightly for each drug, yet they both act as anti-folate drugs due to their ability to competitively inhibit folate biosynthesis required for pyrimidine biosynthesis in Plasmodium. Sulfadoxine inhibits dihydropteroate synthase

(DHPS), while pyrimethamine inhibits dihydrofolate reductase (DHFR). Both enzymes are essential for parasite folate pathways, and thus, pyrimidine biosynthesis. This combination was found to be synergistic in vitro against P. falciparum, and was used as an anti-malarial treatment and prophylaxis for pregnant women and children (33). Unfortunately, resistance to SP quickly emerged after its use. The mechanism of resistance to SP has been correlated with various mutations in the DHPS and DHFR genes. These mutations subsequently result in clinical treatment failure. Additionally, it is worth noting that folic acid supplements at standard therapeutic doses can reduce the antimalarial efficacy of these drugs (34), and should be avoided if SP is to be prescribed for antimalarial treatment.

Proguanil. Proguanil is a synthetic anti-malarial prophylactic agent discovered in 1945. It is a biguanide compound, a class which was found to have anti-malarial activity due to metabolism by cytochrome enzymes. It gained popularity as a first line anti-malarial treatment when used with

Atovaquone. This Atovaquone/Proguanil combination (Malarone) treatment was found to have a synergistic effect against Plasmodium parasites. It is well tolerated, administered orally, and can even be used safely against uncomplicated malaria in pregnant women (25, 35). Proguanil’s mechanism of action has been characterized by a number of studies. The consensus indicates that proguanil’s activity is due to metabolism by CYP2C19 to the active metabolite Cycloguanil. Cycloguanil then acts as a potent inhibitor of P.falciparum Dihydrofolate Reductase (PfDHFR) (36-38). Resistance in Plasmodium against

12 proguanil/cycloguanil is primarily caused by mutations in the PfDHFR gene. Proguanil is thought to have a different target than cycloguanil, which may explain why malarone is such an effective treatment for both sensitive and resistant parasites (37).

Atovaquone (ATQ). Atovaquone is a synthetic 2-hydroxynapthoquinone that was made in the

1980s. Investigations into using hydroxynapthoquinones as potential anti-malarials began during World

War II, a time when quinine supplies were incredibly short. ATQ is effective against diseases such as

Pneumocystis carinii pneumonia; when used in combination with other drugs such as proguanil or azithromycin, it becomes a powerful treatment against malaria or babesiosis respectively. ATQ synthesis results in a racemic mixture, with the trans isomer being more active than the cis isomer. Optimized synthesis of ATQ was just recently described. This synthesis effectively separates the active trans isomer, is higher yielding than previous synthetic routes, and doesn’t involve the use of heavy metals (39, 40).

ATQ/proguanil is prescribed as malarone, a frontline anti-malarial treatment used for uncomplicated and drug resistant Plasmodium infection as well as prophylaxis. Malarone is well tolerated, administered orally, and has few detrimental side effects. Due to ATQ being highly protein bound and more lipophilic, it has an unusually long half-life (50-84 hours) when compared to other anti-malarials, and it is recommended that ATQ be taken with a meal that is high in fat in order to increase ATQ bioavailability.

It is not extensively metabolized, nor is metabolism required for elimination, which primarily occurs in the liver (41). ATQ mechanism of action has been extensively characterized and debated. It has been found that ATQ is a competitive inhibitor of ubiquinol; specifically inhibiting the Plasmodium mitochondrial electron transport chain by binding the cytochrome bc1 complex at or around the ubiquinol oxidation (QO) site. This binding results in a collapse of the mitochondrial membrane potential (41-43).

ATQ is also capable of inhibiting dihydroorotate dehydrogenase (DHOD), which is required by

Plasmodium for pyrimidine biosynthesis. Therefore, the mechanism of action of ATQ can be attributed to the combination of DNA synthesis inhibition as well as mitochondrial membrane collapse. When used in combination with proguanil as malarone, parasite clearance is rapid and effective. It is even capable of

13 acting against hepatic schizonts, though it isn’t capable of clearing hypnozoite forms. Using ATQ to clear hepatic schizonts has been exploited to develop in vitro culturing methods to investigate hypnozoite biology and detection (44). ATQ resistance in Plasmodium has been associated with a missense mutation at position 268 in cytochrome b. These mutations exchange tyrosine for serine (Y268S) or sometimes even asparagine (Y268N) or cysteine (Y268C). These mutations result in 1000-fold increased IC50 values in vitro for ATQ and have been associated with Malarone drug failure, but these mutations come at a fitness cost to the parasite (41, 45, 46).

Antibiotics

Clindamycin. Clindamycin is a licosamide antibiotic that also possesses anti-malarial properties.

It can be used as a monotherapy against P. falciparum infection, and is used to treat severe malaria. It has minimal side effects, is orally efficacious, and is often given with quinine as a combination therapy.

However, it is slow acting and can take up to a week to clear parasites. Additionally, it isn’t capable of acting against exo-erythrocytic forms responsible for relapsing infection in vivax malaria (21). Its mechanism of action in bacteria targets protein synthesis by inhibiting the peptidyl transferase reaction on the 50S ribosomal subunit (47). It affects malaria parasites by acting against the circular apicoplast genome which harbors bacterial ancestry and biochemical function. Clindamycin combination with quinine is often used to treat chloroquine resistant and sensitive strains of malaria infection due to its oral efficacy as well as effective clearance of parasites. Clindamycin resistance in Plasmodium is still being investigated, but studies have shown that resistance is conferred by a point mutation in the apicoplast genome encoding 23S rRNA, which leads to a greater than 100 fold increase in the EC50 values for clindamycin (48).

Doxycycline. Doxycycline is another antibiotic that is also effective against Plasmodium infection. Like clindamycin, it is often given in combination with quinine. It is rapidly absorbed orally, but activity can be varied due to the age of the patient and any other treatments provided along with it

(49). It can be used as chemoprophylaxis for falciparum malaria, but there is evidence showing that prophylaxis fails at times due to inadequate dosing and poor compliance (50). Nevertheless, when the regimen is adequately followed, prophylactic failure rarely occurs. Its mechanism of action in

Plasmodium has not been well characterized, but studies suggest that it may directly inhibit mitochondrial protein synthesis and decrease the activity of dihydroorotate dehydrogenase (DHOD) involved in de novo pyrimidine synthesis. Further research suggests it may inhibit nucleotide and deoxynucleotide synthesis in

P. falciparum, but the concentrations needed for said inhibition are much higher than those given therapeutically (50, 51). Characterizing resistance to doxycycline in Plasmodium parasites is not as straightforward as with other anti-malarials. Doxycycline is given in combination with quinine, and treatment failures with this combination are rare. Due to this phenomenon, quantifying resistance in

Plasmodium proves difficult. In order to attempt to elucidate a mechanism of resistance, studies have been done to characterize bacterial resistance, and then attempt to correlate bacterial genetic changes to their P. falciparum genetic homologs. While most theories point to plasmodial apicoplast genes playing a role in resistance, there is little evidence for any definitive target or mechanism for doxycycline resistance in Plasmodium (50).

4-aminoquinolines

Chloroquine (CQ). Chloroquine (originally called Resochin) was synthesized in the 1930s by

Hans Andersag. It was made as an analog of methylene blue in order to treat vivax malaria (52). Since its discovery, CQ has been used as a frontline anti-malarial treatment all over the world. CQ has many advantages to its use, including low toxicity, effective potency, and a long half-life. It is distributed throughout the body, is 60% bound to plasma proteins, and is primarily cleared by the kidney and liver. It is primarily metabolized by P450 enzymes (CYP2D6 and 3A) to its active forms desethylchloroquine and bisdesethylchloroquine (53). While it was initially used as a treatment against malaria, it is also employed as an adjuvant in cancer therapy.

Many studies have been done to elucidate CQ’s mechanism of action and resistance. CQ mechanism of action has been attributed to the inhibition of hemazoin biocrystallization. In Plasmodium parasites, hemoglobin metabolism occurs in the acidic digestive vacuole of the parasite. After hemoglobin metabolism, the parasite forms free heme which is crystallized to hemazoin. CQ inhibits this heme crystallization process by binding to free heme, forming CQ-heme complexes. These complexes prevent the formation of hemazoin, and are responsible for permeabilizing parasite cell membranes, leading to parasite death (54, 55). This theory has been supported by localization studies using fluorescent analogs of CQ to determine localization (56), cell fractionation studies using transmission electron microscopy

(55), and other studies quantifying amounts of free heme and hemazoin upon CQ treatment (7, 25, 57).

How CQ accumulates within the DV is a matter of some debate. Several theories attempt to explain this phenomenon. In the weak base model, unprotonated CQ is the only form permeable in the membrane that can diffuse into the erythrocyte up to the digestive vacuole. These forms become protonated then trapped, as protonated forms cannot cross the membrane. The next theory is the carrier mediated mechanism, in which a Na/H exchanger on the parasite plasma membrane is involved in CQ uptake with sodium ions.

The last theory is the “ferriprotoporphin IX receptor” model, in which free heme acts as a receptor for

CQ, and CQ entry into the digestive vacuole is regulated by the amount of free heme (58). In cancer, it has various modes of action, including inhibition of multidrug resistance pumps and autophagy

(chemotherapy resistance mechanisms). While these effects were observed at higher doses, clinically acceptable doses of CQ were found to increase the survival prospects of certain cancers when co- administered with chemo and radiation therapy (59).

Since the mechanism of action involves a host-derived target, resistance to CQ took over 20 years to develop within Plasmodium parasites (52). The mechanism of resistance to CQ has also been extensively studied, due to CQs widespread use and efficacy against malaria. Resistance to CQ by parasites is characterized by decreased uptake of CQ in the parasite digestive vacuole. Several genes have been implicated in this decreased uptake. One of them is the PfCRT gene (Plasmodium falciparum chloroquine

16 resistance transporter). This gene codes the PfCRT protein which is localized in the parasite’s digestive vacuole. In CQ resistant parasites, a consistent mutation is found at position 76 in the first transmembrane domain. This K76T mutation has been found in multiple studies describing CQ resistance (25, 54, 58).

Another gene is the PfMdr1 (Plasmodium falciparum multidrug resistant transporter 1), which is has been implicated in resistance to piperaquine as well as mefloquine. Studies have shown that partial CQ resistance is conferred by mutations in the PfMdr gene, but the mutations alone are not enough to give complete protection. It has been suggested that the increased frequency of pfmdr1 polymorphisms in resistant parasites could be due to physiological compensation for the altered function of mutated pfcrt

(58).

Amodiaquine (AQ). Amodiaquine is a 4-aminoquinoline that is similar to CQ. It is often used to treat uncomplicated falciparum malaria, and is a cheaper alternative to CQ, making it more widely used and available. It is taken orally and is given to children for treatment due to it being more palatable than

CQ (60). AQ has mild and severe side effects, which has limited its use over time. The WHO even went so far as to withdraw its use when travelers had fatal side effects when it was used as prophylaxis. While it is no longer used as a first line treatment, it is still used to combat Plasmodium infection, as the WHO determined it should be used if the risk of infection outweighs adverse drug reactions (60). Its mechanism of action is similar to that of CQ as well. It is metabolized to its active metabolite, accumulates in the digestive vacuole of the parasite and inhibits heme polymerization to hemazoin by complexing with heme, preventing its degradation by glutathione (61). This causes a toxic buildup of heme inside the parasite, leading to parasite death. AQ resistance has been attributed to mutations in the Pfcrt gene (62) and Pfmdr1, which pumps the metabolite out of the digestive vacuole of the parasite before it can inhibit heme polymerization in vitro and in vivo (62, 63).

Piperaquine (PQP). Piperaquine is a bis-4-aminoquinoline that was synthesized in 1960. It is structurally similar to chloroquine, one of the frontline treatments for malaria. PQP enjoys good in vitro activity against chloroquine sensitive and resistant parasites. When it was first discovered, it was used for prophylaxis, as well as a monotherapy to treat chloroquine resistant parasites (64). PQP is very slowly absorbed, and subsequently has a long elimination half-life of approximately 22 days (65). Its use as a monotherapy quickly resulted in the emergence of PQP resistant parasites, which resulted in limited use by the 1980s. Now, PQP is primarily administered with dihydroartemisinin (DHA) as a part of

Artemisinin combination therapy (ACT). DHA is fast acting, and being paired with PQP allows their differences in action to synergize their effects against Plasmodium parasites (65). PQP mechanism of action has not been thoroughly investigated, but it is thought that it is similar to chloroquine in that it binds heme in the digestive vacuole and prevents its detoxification by the parasite. This idea becomes even more interesting when PQP resistance is studied. Despite having a similar mechanism of action to chloroquine, PQP treatment it not hindered by CQ resistance mechanisms. This would imply that the parasite develops two distinct mechanisms of resistance to PQP and CQ, despite them having similar modes of action (64, 65). It has also been found that in P. berghei, PQP resistance is stable over multiple passages, but comes at a fitness cost to the parasite (66).

Endoperoxides

Artemisinin and other artemisinin relatives such as artelinic acid (AA), dihydroartemisinin (DHA), artesunate, and arteether are endoperoxide-containing compounds. Artemisinin was discovered as a natural product of the Artemisia annua plant, called “Qinghao” by the Chinese. This discovery was made in 1972 by Youyou Tu, a researcher working with the Chinese military force. The Artemisia plant has been used to treat fever, jaundice and toxic blood conditions. The extracts from Artemisia annua were found to clear rodent malaria parasites with a clearance rate of 95-100%. With anti-malarial resistance on the rise and no vaccination prospects in sight, novel therapeutics were desperately needed (1).

Artemisinin and its derivatives have been used successfully to treat acute P. falciparum malaria in areas where mefloquine- and quinine-resistant strains are endemic (67). In many instances artemisinin derivatives are used concurrently with mefloquine, quinine, or another antimalarial drug. Artemisinin combination therapy (ACT) uses artemisinin or its derivatives in combination with other anti-malarial drugs. Such combinations include, but are not limited to, dihydroartemisinin-piperaquine, artesunate plus sulphadoxine-pyrimethamine, as well as others. They are administered either orally or intravenously, depending upon the type of infection and drug combination. These combinations differ in their activity, but certain combinations are successful in clearing non-complicated falciparum malaria, and even severe vivax malaria. These combinations have been shown to clear severe vivax malaria faster than chloroquine alone. ACT activity against P. malariae and P. ovale is high, though ovale (as well as vivax) infections still require primaquine treatment to prevent relapsing infection. Oral ACT is efficacious against non- complicated P. knowlesi infection, but intravenous artesunate treatment is used for severe knowlesi (67).

Artemisinin can also act as an anti-cancer, anti-viral, and is effective against a variety of parasitic diseases. However, its effectiveness in those systems are several magnitudes smaller than in Plasmodium spp (68).

The mechanism of action of the artemisinins remains a source of fierce debate. It has been suggested that there are several molecular targets for artemisinin, which include PfATPase6, a sarcoplasmic/ER Ca2+-

ATPase (SERCA) homolog protein in mammals, translational controlled tumor protein and heme. While these targets have been proposed, how artemisinin acts on them is also unknown. There is evidence suggesting these artemisinin protein targets are alkylated, causing downstream affects that lead to parasite death (69). The mechanism of action is attributed to the endoperoxide bridge (1,2,4-trioxane bridge) present in the compound, which is activated by either mitochondrial or heme-mediated degradation. The trioxane bridge has also been implicated as essential for ART’s anti-cancer properties and synergy with other anti-cancer therapeutics (70, 71). The mitochondrial degradation pathway is responsible for depolarization of parasite mitochondria and plasma membranes, as well as lipid peroxidation induced

19 cytotoxicity via ROS.(72, 73) In heme-mediated degradation, two models have been proposed, but both models lead to the generation and activation of a carbon centered radical (70, 74). Artemisinin resistance was observed almost ten years after artemisinin and ACT were implemented in the field. Resistance is characterized by delayed parasite clearance due to mutations/polymorphisms in the PF3D7_1343700 kelch propeller domain (K13-propeller) on chromosome 13. These mutations lead to increases in phosphatidylinositol-3-kinase, which is required for the mediations of cell signaling and survival (70).

Further evidence indicates resistance is possibly a cell cycle arrest event, possibly due to prolonged parasite development at the ring stage where the activation of artemisinin is low, or to DHA induced dormancy mechanism that is yet to be understood (75, 76).

Methylene Blue and 8-Aminoquinolines

Methylene Blue (MB). Methylene Blue is a synthetic dye that was created in the 19th century by

Heinrich Caro. After its discovery, Paul Ehrlich proposed that it may potentially have anti-malarial activity in 1891 due to the belief that drugs and dyes might have similar activity against microorganisms

(77). MB was a safe and cheaper treatment for malaria, especially in endemic populations that couldn’t afford sufficient treatments. It was even found to be safe in G6PD deficient individuals at higher doses

(78). Its use quickly diminished due to the high rates of recrudescence after treatment, as well as the discovery of more potent anti-malarials. Additionally, MB dyed the eyes blue after treatment. However, due to the emergence of drug resistant Plasmodium parasites, older compounds like MB are seeing a revival in drug discovery. MB has many applications in medical therapy. Some of the most notable applications include visualization of tissues during surgery, treatment of methemoglobinemia, and for prophylaxis/treatment of ifosfamide-induced encephalopathy that occurs during cancer chemotherapy

(79). MB can be administered via I.V or orally. Studies have shown that the bioavailability is high with these administrations routes, and that co-treatment with CQ can increase plasma concentrations of MB

(79). The mechanism of action of MB against malaria parasites is due to its ability to act as a “subversive

20 substrate”, where it behaves as a redox cycler with glutathione reductase (GSH). One study suggests that the anti-malarial activity of MB does not inhibit the mitochondrial electron transport chain, and therefore does not target redox substrates within that system (80). Another aspect of its mechanism of action could be due to MB’s ability to form free heme/MB complexes. It has been shown that various “sandwich complexes” of MB with free heme can be formed to interfere with hemazoin biocrystallization, acting in a similar manner to CQ and other 4-aminoquinolines. This same study also supported the redox cycling theory of MB’s mechanism of action, showing that it has unique interactions with GSH (81). Other studies have shown that MB could potentially be effective against drug resistant strains of Plasmodium due to the lack of cross resistance between MB and other anti-malarials such as CQ, QN and MFQ (82).

Due to the lack of widespread treatment with MB and its lack of robust efficacy against Plasmodium strains, resistance to MB has not been effectively characterized. However, there is evidence that the

PfCRT is capable of reducing susceptibility to MB, similar to a manner that it confers resistance to CQ

(83). This theory is an especially viable one if you take into account MB’s ability to inhibit hemazoin formation in Plasmodium; it makes sense that a resistance mechanism to MB would be similar to CQ resistance.

8-Aminoquinolines: Primaquine and Tafenoquine. The discovery of the 8-AQ class of compounds as potent anti-malarials began between WW1 and WW2, where thousands of anti-malarials were being discovered and researched. The first 8-AQ to be discovered was pamaquine/plasmochin. It was synthesized in 1925, and was found to have anti-gametocyte activity when taken in combination with quinine (84). Pamaquine’s effectiveness was limited due to its poor schizontocidal activity as well as causing hemolytic anemia in those with G6PD deficiency, an issue that is consistent among almost all 8-

AQ analogs. Pamaquine however, lead to the discovery of primaquine (PQ) in 1946. Of the 8-AQs discovered at this time, PQ was the one that presented the highest efficacy against malaria with the lowest relative toxicity levels. PQ has enjoyed widespread use for its gametocytocidal activity, as well as its ability to clear hepatic schizonts and hypnozoite forms for over 70 years (84). During this time, resistance

21 to PQ and other 8-AQs has been not been observed to any degree of clinical relevance (85). However, due to 8-AQs toxicity in those with G6PD deficiency and the increasing prevalence of resistance to other anti- malarials, other analogs of PQ have been synthesized. Tafenoquine (TQ) is an 8-AQ analog that is currently in phase III clinical trials. It was synthesized by scientists at the Walter Reed Army Institute of

Research in 1978. Current development is under GlaxoSmithKline and MMV. The structures of some prominent 8-AQs can be seen in figure 1.2.

Figure 1.2- Structures of selected 8-AQs. A) Pamaquine, B) Primaquine, and C) Tafenoquine.

PQ is multifaceted, and is used for a variety of reasons. Against malaria, it can be used as primary prophylaxis against Plasmodium spp., anti-relapse therapy, or as a radical cure when co-administered with a blood schizonticide. It acts against gametocytes of Plasmodium, and therefore is a very effective anti- transmission chemotherapeutic. TQ also enjoys the same properties, but it has been shown to be less toxic and has better efficacy against erythrocytic forms than PQ (84, 86). PQ has been shown to have activity against leishmanisis and African trypanosomiasis in vitro, but has not seen any clinically relevant success against these pathologies (84, 87, 88). PQ also has activity against pneumocystic pneumonia, a fungal infection that plagues HIV infected individuals. It is given in combination with clindamycin for use as prophylaxis and treatment. It acts by generating superoxides which interfere with electron transport in vivo (89). PQ, when used in non-G6PD deficient individuals, is generally well tolerated with a low half- life. It is primarily metabolized in the liver, and is cleared by the kidneys. In those with G6PD deficiency, 22 it causes severe hemolytic anemia, which limits its therapeutic uses. G6PD deficiency affects 8% of the population in malaria endemic countries, where approximately 32.5% of the population in sub-Saharan

Africa and the Arabian peninsula possess it as well (90). Therefore, the need to elucidate the mechanisms of toxicity and action are considered paramount due to PQs many advantages against Plasmodium that are not seen in any other anti-malarial.

The mechanism of action of 8-AQ analogs is still a matter of debate. What is known is that their action is dependent upon metabolites generated in the liver. These metabolites have varied structures such as hydroxylated forms as well as dimers. Hydroxylated forms are created by the Cytochrome P 450

(CYP450) family of enzymes located in the liver. Previous work has confirmed various isoforms of

CYP450 responsible for metabolite generation, where 2D6 has been implicated in the creation of the major metabolite responsible for activity (91). However, the specific action of each metabolite and their targets is still unknown. They could potentially act via multiple mechanisms, though these mechanisms can’t be adequately studied because of the instability of the metabolites (84). One proposal that has significant support is the metabolite based generation of ROS species that lead to parasite death. Many of the reactive metabolites responsible for ROS generation can be seen in Figure 1.4.

The hydroxylated species have been implicated as responsible for generating hydrogen peroxide (H2O2) in vitro and in vivo. These species are also capable of redox cycling in the presence of an enzymatic system such as superoxide dismutase, which can go on to generate hydroxyl radicals via downstream effects (92,

93). During normal parasite development, ROS such as H2O2 are natively generated through hemoglobin metabolism in the parasitophorus vacuole, and through electron transport in the mitochondria. A diagram of these ROS sources can be seen in figure 1.5. Hydrogen peroxide is primarily responsible for the majority of ROS mediated stress in Plasmodium spp. Redox regulation is a complex process in

Plasmodium that is mediated by multiple mechanisms. In this context, the most relevant systems would be the Thioredoxin and glutathione redox cycling systems which are represented in figures 1.6 and 1.7

23 respectively. In the Thioredoxin system, Thioredoxin reductase (PfTrxR) as well as Thioredoxin like proteins (Trx1-3) are the main proteins responsible for clearance of H2O2. It has also been shown that

Trx1 can act as a backup to the glutathione system by directly reducing oxidized glutathione (94, 95). 8-

AQs are slightly active against metabolically active asexual stages of Plasmodium, namely the troph and schizont stages. It is worth noting that the expression of TrxR, as well as Trx proteins, is upregulated in the troph and schizont stages (96). Since the metabolites of 8-AQs increase hydrogen peroxide production, it is possible that the anti-oxidant defense of Plasmodium becomes overwhelmed when exposed to therapeutic concentrations of 8-AQs, resulting in parasite death.

The contention to this theory is that the concentrations required for parasite death are often far above peak therapeutic concentrations in sera (approx. 0.59 M) (97), which also explains why 8-AQs are not effective against erythrocytic forms of the parasite without a blood schizonticide. For this theory to prevail, it would be necessary to determine ROS increases via depolarization potentials in the parasite in vivo. This would help determine if 8-AQ generated ROS significantly contributes to parasite killing.

Given the fact that 8-AQs are most active against metabolically active stages of Plasmodium infection, it cannot be denied that 8-AQ mediated ROS production plays a part in 8-AQs anti-malarial mechanism. A proposed mechanism for PQ metabolite redox cycling can be seen in figure 1.3

Figure 1.3- PQ metabolite redox cycling due to CYP2D6 metabolism. The unstable quinone-imine form is quickly converted to the more stable 5,6-ortho-quinone. Redox cycling of 5-hydroxyprimaquine is suspected to be primarily responsible for mechanism of action as well as toxicity.

Another proposed mechanism is the inhibition of parasite mitochondrial metabolism via ubiquinone (84,

98). 3H-Primaquine has been shown to accumulate in parasite mitochondria, resulting in mitochondrial ultrastructure changes. It has also been theorized that metabolic dimers of primaquine are also responsible for decoupling oxidative phosphorylation in mitochondria, as this has been shown in the rat liver (99).

Furthermore, tafenoquine has been shown to target the respiratory III complex in L. donovani mitochondria, depolarizing membrane potential in promastigotes leading to apoptosis-like death of the parasite (84).

It is worth noting that inhibition of mitochondrial function with 8-AQs still leads to ROS production. The most likely explanation of 8-AQ action, given the various studies, is that it causes excessive ROS production via metabolite redox cycling and mitochondrial changes. This increase in ROS production inhibits the parasite’s anti-oxidant defenses while also affecting proper mitochondrial function. Coupled with an effective blood schizonticide, all parasitic forms are cleared, resulting in a full cure.

PROJECT SPECIFIC BACKGROUND

Discovery and development of new drugs that kill the liver or exo-erythrocytic (EE) stages of malaria, especially dormant forms (hypnozoites) of P. vivax and P. ovale, requires validated EE models. Current in vitro EE assays suffer from low throughput, poor correlation with human disease, and limited development of the liver stages of the major human malaria species. The Kyle laboratory, in collaboration with the Adams and Draper laboratories at USF, have developed microfluidic devices capable of long term growth and culture of chimeric mouse expanded human hepatocytes (100). These models are useful for our purposes in order to investigate mechanisms of action and synergy, and for the definitive detection of hypnozoite forms of P. vivax or ovale. While there is evidence for these forms in zoonotic species of

Plasmodium (like in P. cynomolgi), hypnozoites of P. vivax or ovale have yet to be validated due to the nature of hepatic infection. Sporozoites select for certain hepatocytes before beginning infection, and can kill hepatic cells they pass through on their journey. The deaths of these hepatocytes, while

25 inconsequential in vivo, have devastating consequences in in vitro models. The entire culture system can become compromised due to the death of these select hepatic cells. These microfluidic devices can allow us to culture liver stage infection in vitro, and attempt to perform various experiments determining the nature of these forms and therapeutics effective against them.

8-AQs are effective against late stage (IV-V) gametocytes as well as hepatic schizonts of Plasmodium infection, but are only mildly effective against asexual blood stages (84, 86, 101). Therefore, to fully clear

Plasmodium infection, 8-AQs are often given with a blood schizonticide such as CQ or artemisinin based compounds. Studies have found that synergy between CQ and PQ is attained in Plasmodium infection

(102), though the mechanism of this synergy isn’t adequately understood. Understanding this synergy could assist in finding novel therapeutics and combinations in order to treat drug resistant Plasmodium infection. Investigating this phenomenon isn’t possible with standard anti-malarial assays, as CQ is not effective against liver stage infection. Therefore, another approach is needed to understand CQ’s role in synergy against liver stage infection. Pharmacological studies of drugs using fluorescent tags have long been a method of determining the mechanisms of action, synergy, and resistance (103). These methods have seen use in anti-malarials as well (56, 73). Using these microfluidic devices, as well as tagging 8-

AQs with fluorescent tags, we can potentially elucidate the mechanism of synergy by observing differential accumulation of each drug in infected hepatocytes.

FOCUS OF STUDY

This study seeks to synthesize, characterize and investigate the effects of novel 8-AQ fluorescent probes on Plasmodium infection. These probes will then be used to examine accumulation of each drug in hepatocytes infected with Plasmodium. With this information, we can propose a mechanism of synergy and enhance the knowledge of the activity of 8-AQs.

Figure 1.4- Hydroxylated metabolites of PQ. These forms are suggested to be implicated in PQ mechanism of action. In addition to 5-hydroxyprimaquine (8), these metabolites are suspected of also undergoing redox cycling to facilitate parasite killing.

Figure 1.5- Sources of ROS in Plasmodium infection. Notice how redox cycling due to Thioredoxin occurs in the cytosol. Superoxide Dismutase (SOD) is primarily responsible for hydrogen peroxide production in the cytosol.

Figure 1.6- Thioredoxin system in Plasmodium. The redox cycling in this system influences the regulation and production of hydrogen peroxide, along with activating protein targets and regulating peroxiredoxins.

Figure 1.7- The glutathione system in Plasmodium. Redox cycling of GSSG to GSH also influences ROS regulation and production via the Thioredoxin system Trx-SS and Trx-(SH)2. GSH is an important redox regulator in Plasmodium.

CHAPTER 2-SYNTHESIS AND CHARACTERIZATION OF NOVEL 8-AMINOQUINOLINE

FLUORESCENT PROBES

INTRODUCTION

Current therapeutics to clear liver stage infections in ovale and vivax malaria include atovaquone and 8- aminoquinolines (primaquine, tafenoquine, pamaquine, etc.), though atovaquone does not clear hypnozoites or gametocytes in blood stages (43, 104). Primaquine and tafenoquine are within the 8- aminoquinoline class of compounds to which Plasmodium has little observed resistance. It is worth noting that despite 8-AQ resistance not being readily observed, the criterion for parasitic resistance against these molecules is still undefined (104, 105). These molecules not only exhibit some moderate asexual stage activity, but are also effective against sexual stages (Stage III-V gametocytes), hepatic schizonts, and hypnozoites (84).

Coumarin is an organic benzopyrone molecule that was discovered in 1820 by German chemist Alfred

Vogel. Vogel discovered coumarin by isolating it from Tonka beans and sweet clover. Its formal synthesis wouldn’t be described until 1868 by William Henry Perkin, who also discovered the Perkin reaction to synthesize cinnamic acids. The 7-hydroxy and 7-methylamino substituted coumarin molecules have significantly higher quantum yields than the coumarin moiety alone. They are often employed as fluorescent probes, enzyme substrates, or in bio-conjugation reactions (106). Other derivatives of coumarin have found medicinal applications as well (107). Coumarin synthesis can be accomplished multiple ways, but the most often used synthetic route is the Pechmann condensation, which uses a phenol and a carboxylic acid or ester that has a -carbonyl group. The basic reaction conditions for the

Perkin reaction and Pechmann condensations can be seen in figure 2.1. Coumarin versatility in reaction

31 synthesis, use in fluorescence studies, extensive characterization, and biomedical application made it an attractive choice for our purposes.

Figure 2.1- Examples of A) Perkin reaction and B) Pechmann condensation. Pechmann condensation is one of the many ways to synthesize coumarin molecules.

Synthesis of fluorescent analogs of chemotherapeutics has recently gained interest for various applications. These analogs can offer much information about the drug’s mode of action, resistance, and even basic science of the pathogen which it acts against. Using fluorescent drug labeling has found use in studying cancer therapy mode of action. Various methods are exploited to study fluorescence localization, and can even use these dyes as an indication of drug release and efflux (108). For malaria, probes of chloroquine and artemisinin have been synthesized, characterized and investigated to give information about parasite biology, as well as mechanisms of action and resistance for both chloroquine and artemisinin. Fluorescent chloroquine analogs have been used to study chloroquine influx and efflux kinetics to determine how chloroquine action differs in CQ sensitive vs resistant parasites (56), and to examine how P-glycoprotein inhibition affects malaria parasites in vitro (109). Both of these studies use different analogs, yet exploit the same localization methods and theory to determine both pharmacokinetics of chloroquine as well as basic biology of Plasmodium parasites. Furthermore, a novel artemisinin fluorescent analog was used to investigate artemisinin localization in live parasites, as well as provide more evidence linking artemisinin mechanism of action to the 1,2,4-trioxane bridge characteristic

32 of artemisinin analogs (73). These studies effectively demonstrate the value in using fluorescent analogs of drugs to further their characterization against infections of various types.

While probes of chloroquine and artemisinin have been synthesized, various probes of 8-AQ molecules have not. Fluorescent co-localization studies of these 8-AQ probes with chloroquine probes could provide evidence for why these various compound combinations (PQ/CQ and TQ/CQ) have synergistic activity in asexual stages (102). Additionally, because of 8-AQ effectiveness against liver stages, we can potentially use fluorescent probes of these molecules to help detect liver stage forms, and potentially hypnozoite forms. Coumarin was chosen as the probe of choice due to its extensive characterization and fluorescent intensity, as well as its limited areas of reactivity. These properties allowed us to create reaction schemes that exploited this limited reactivity in order to couple it to primaquine and tafenoquine.

In this study we demonstrate the synthesis and chemical characterization of the novel primaquine- coumarin probe (PQCP) and tafenoquine-coumarin probe (TQCP). There were two synthetic schemes proposed for the synthesis of PQCP. The first scheme will be discussed, but ultimately resulted in failure to obtain the final product. Specific experimental details will be omitted for coherence and clarity. The second scheme vastly improved upon the first, and the differences between the two will also be discussed.

The 1H-NMR, High Resolution Mass spectra, as well as quantum yields have been determined for both probes, though PQCP was used as the mainstay probe for in vitro experiments due to incomplete characterization of TQCP.

MATERIALS AND METHODS

PQCP synthesis

Compound 1 was synthesized in house by methods previously described (110, 111). PQCP [2] and

TQCP [3] were synthesized by utilizing the EDCI/Dmap coupling reaction. Primaquine bis-phosphate

(Sigma-Aldrich, St. Louis, MO) was combined with excess (3-4eq) NaOH and stirred at room

33 temperature for 30min-1hr (reaction not shown). The free base form of Primaquine was extracted twice with DCM. LC-MS analysis displayed a single chromatogram peak. The dark vicious liquid was used without further purification. A flame dried, round bottom flask filled with argon via balloon was charged with free base 8-aminquinoline (1eq), 4-(7-hydroxy-2-oxo-2H-chromen-4-yl) butanoic acid (1eq) and toluene (3ml). The toluene was evaporated under reduced pressure. EDCI (2eq) and Dmap (20mol%) was added to the flask, and the flask was recharged with argon via balloon. DCM was added via syringe through a rubber septum. The reaction was stirred at room temperature for 18 hours. The mixture was extracted twice with ethyl acetate and dried over anhydrous sodium sulfate. The mixture was then concentrated onto silica gel, and purified via flash chromatography. Effort was taken to make sure the reaction and fractions had minimal light exposure. Fractions were analyzed via LC-MS for pure product.

Pools containing pure compound were concentrated resulting in green crystals (78%). 1H-NMR PQCP

(400 MHz, Acetone-d6): δ 9.40 (s, 1H), 8.47 (q, 1H), 7.99 (dd, 1H), 7.68 (d, 1H), 7.34 (q, 1H), 7.18 (s,

1H), 6.81 (dd, 1H), 6.71 (d, 1H), 6.42 (d, 1H), 6.28 (d, 1H), 6.11 (d, 1H), 6.01 (s, 1H), 3.83 (s, 3H), 3.68

(m, 1H), 3.26 (m, 2H), 2.75 (t, 2H), 2.29 (t, 3H), 2.03 (m, 2H), 1.95 (m, 2H), 1.66 (d, 2H), 1.27 (d, 2H).

[M+H]=490.2349

TQCP synthesis

A flame dried, round bottom flask filled with argon via balloon was charged with Tafenoquine Succinate

(1eq), 4-(7-hydroxy-2-oxo-2H-chromen-4-yl) butanoic acid (1eq) and toluene (3ml). The toluene was evaporated under reduced pressure. EDCI (2eq) and Dmap (20 mol%) was added to the flask, and the flask was recharged with argon via balloon. DCM was added via syringe through a rubber septum. The reaction was stirred at room temperature for 18-24 hours. The mixture was extracted twice with ethyl acetate and dried over anhydrous sodium sulfate. The mixture was then concentrated onto silica gel, and purified via flash chromatography. Effort was taken to make sure the reaction and column chromatography fractions had minimal light exposure. Pools containing pure compound were

34 concentrated resulting in yellow/golden crystals (50%). TQCP 1H-NMR (400 MHz, Acetone-d6): Unable to determine. [M+H]=694.2761

Absorbance Spectra and Steady State Emission Studies

The absorption spectrum of each compound was obtained using a Shimadzu UV2401 spectrophotometer.

Samples were placed in a 1-cm quartz optical cuvette in ethanol and spectra were obtained form 200-500 nm. Steady state emission spectra were obtained using an ISS PC1 single photon counting spectrofluorimeter using a 600W Xe arc lamp as the excitation source.

RESULTS

The first generation scheme of synthesis can be seen in figures 2.2-2.4. In this first scheme, we had a 3 step approach. First we sought to synthesize the linker compound first then couple the linker to coumarin.

After the coumarin-linker compound was made, we would then couple it to primaquine or tafenoquine.

This scheme was inefficient for several reasons. The first is that too many groups required chemical protection, decreasing the overall efficiency of the synthetic scheme. BOC protection of the primary amine group in the linker analog had to be maintained throughout the reaction procedures, and adds two reactions to the overall scheme, leaving room for product loss. Additionally, when the final reaction initially encountered difficulty, further protective groups were added to the bromo-coumarin analog to prevent side reactivity. Despite this protection, the final reaction still didn’t proceed; LCMS detected less than 2% product formation after 24 hours of reaction time. More rigorous reaction methods could not be pursued due to increased potential of side product formation.

Secondly, some reactions had side products that couldn’t be separated from the main product without extensive, laborious purification methods. The initial synthesis of the bromo-coumarin analog via H2SO4 provided little product formation. This was due to a side product being formed that couldn’t be purified from the main product without extensive time consuming methods. To combat this issue, synthesis of the

35 bromo-coumarin analog was done via microwave synthesis, as described (107). Microwave synthesis offered many benefits, including minimal reaction times (<30min). The minimization of reaction time allowed faster screening of optimal conditions to obtain viable product. Another advantage is that side product formation with these conditions was less of a hindrance to purification than the H2SO4 route.

Despite all the advantages of microwave synthesis, including adequate yields of product, difficulties with this reaction scheme continued.

Lastly, the final alkylating step simply did not work, despite screening several different bases at varying concentrations, as well as varying temperature. The temperatures, bases used and their stoichiometric equivalents can be seen in Table 2-1. These varying methods either resulted in little to no product after

24hrs of reaction time. It is believed that this was due to the linker chain between coumarin and primaquine being too long, though no experiments were conducted to verify or refute this idea.

Table 2-1- Various screening conditions for EDCI/Dmap coupling of the primaquine-linker compound to coumarin.

Bases Stoichiometric equivalent Temperature (0C)

DIPEA 0.5, 1.0, 1.2, 2.0 0, 25

K2CO3 0.1, 0.5, 1.0, 1.2, 2.0 0, 25

Cs2CO3 0.1, 0.5, 1.0, 1.2, 2.0 0, 25

This led to the generation of the second scheme. This scheme worked much better overall, and can be seen in Figure 2.5. The amounts of steps, as well as purifications, were cut almost in half. Additionally, there was no need for protection reactions, as the coumarin-linker compound was formed in a single step.

The final alkylation reaction also proceeded easily, with a minimum 50% yield. Since the new coumarin- linker compound readily reacted with the primary amine group of primaquine, this led us to believe that the final reaction in the previous scheme failed due to a longer linker length, since that is the only difference between the final products.

For the second reaction scheme, the synthesis of the -keto ester via Meldrum’s acid was described (110,

112). The addition of the acyl chloride must be done slowly, otherwise side products are easily formed, making purification more difficult. The intermediate formed with meldrum’s acid can be isolated via purification, but is usually not necessary. In this study, methanolysis was done immediately after the detection of the product by LC-MS and removal of residual reactants and solvent. After synthesis of the

-ketoester, the coumarin moiety was synthesized as described (106). For this reaction, the coumarin synthesis and subsequent hydrolysis were also done without further purification of the intermediate.

Synthesis of the coumarin moiety and the subsequent fluorescent primaquine (and tafenoquine) molecule was done under limited light exposure to prevent photobleaching and maintain fluorescence. To accomplish this, the lights in the hood were turned off and the columns along with any fractions were wrapped with aluminum foil. Product concentration under reduced pressure was also done with the product vial wrapped in foil.

While electron spray ionization mass spec effectively characterized both probes, molecular characterization via NMR was hindered for TQCP. Due to limited amounts of product and the presence of the –CF3 group on TQ, initial 1H-NMR characterization as well as 13C-NMR characterization was inconclusive. 1H-NMR studies using between 1-5mg of TQCP were inconclusive. We attempted to use up to 10mg of TQCP to obtain spectra. While we did obtain a moderately good 1H-NMR for TQCP, 13C-

NMR for both probes was unable to be analyzed in our hands. For this reason, in addition to concerns about fluorescence shelf life, we attempted to further characterize the probes via fluorescence and absorption spectroscopy.

The absorption spectra of PQCP and TQCP together with the parent PQ, TQ and coumarin are displayed in Fig. 2.10. The spectra for PQ and TQ are dominated by bands at ~265 nm and ~360 nm originating

1 1 1 from a transition from the So to LB state and LA state, respectively. The LB state is a bond centered state

1 with excess charge density on the quinolone ring while the LA state is an atom centered excess charge

37 density state with the excess charge density localized on the heterocyclic nitrogen. The spectrum of coumarin contains a single transition centered at 320 nm that arises from a strongly allowed * transition overlapping with a weaker n-* transition. The absorption spectra of the PQCP and TQCP complexes are essentially the sum of the PQ/TQ and coumarin spectra indicating that there are no intramolecular interactions between the ring systems of the two chromophores within the complexes.

The corresponding steady state emission spectra of PQ, TQ, PQCP, TQCP and coumarin are displayed in

Fig. 2.11. Excitation of PQ at 320 nm results in an emission spectra with a maxima centered at 465 nm

1 and is due to the radiative relaxation from the LA excited state while excitation of coumarin at 320 nm

1 results in an emission maxima centered at 380 nm arising from relaxation from the S1 excited state. As the absorption spectrum of the PQCP complex is dominated by the coumarin chromophore in the region of 300 nm to 350 nm, excitation of the complex at 320 nm results in an emission spectrum that is nearly identical to that of the isolated coumarin with only a small shoulder at 450 nm arising from the PQ chromophore. The TQ molecule exhibits an emission spectrum similar to PQ upon 320 nm excitation.

However, excitation of the TQCP complex results in an emission spectrum with a much greater contribution from the TQ chromophore which is likely due to the presence of a weak absorption band associated with TQ that is not present in PQ.

CONCLUSION

The synthesis and characterization of a novel fluorescent 8-AQ probe has been described. The synthesis of these probes proceeds readily from previously synthesized coumarin analogs via -keto ester routes.

Care must be taken to ensure light protection after coumarin analogs have been created. The presence of

CF3 groups hinder effective proton and carbon NMR characterization of tafenoquine based probes; much product is required in order to obtain adequate spectra (>10mg). While its fluorescence is not as strong as coumarin alone, it is sufficient for in vitro studies. In order to ensure success of future microscopy analysis, their anti-malarial activity will be assessed to make sure it doesn’t differ from the parent

38 compound (PQ). Cytotoxicity assays against human hepatoma cell line HepG2 will be done to ensure the probes do not kill the host cells before entering the liver stage parasite. Finally, we will construct methods for PQCP co-localization studies with other commercially available probes, and then determine their localization in erythrocytic and exo-erythrocytic forms. Detection of these forms in erythrocytic stages (sexual and asexual) could even give information about mechanism of action if they localize to any specific receptors or organelles. If these probes prove to be viable in detecting exo-erythrocytic forms, it could give us information about the mechanism of action against hepatic schizonts and hypnozoite forms.

Figure 2.2- Synthesis of the PQ-linker compound. Reactions with an ‘x’ over the arrow either didn’t proceed to completion, or the product was unable to be isolated.

Figure 2.3- Synthesis of the bromo-coumarin moiety. Pechmann condensation yielded minimal product in this study and was abandoned, as indicated by the ‘x’ reaction. Microwave synthesis was employed to create the bromo-coumarin intermediate with adequate yields (50%).

Figure 2.4- Failed reaction of PQ-linker with bromo-coumarin intermediate. It is believed this reaction did not proceed due to the extended linker chain. Attempted basic conditions for this reaction are listed in Table 2.1

Figure 2.5- Second generation synthesis of PQCP (2) and TQCP (3). Compound (1) was synthesized by previously described methods. The EDCI/Dmap coupling reaction proceeded readily for both reactions (50-60% yield).

Figure 2.6- Experimental details of the electron spray ionization (ESI) high resolution mass spec of PQCP. The same procedure and instrumentation was used for TQCP.

Figure 2.7- ESI High resolution mass spectra of TQCP.

Figure 2.8- 1H-NMR of PQCP.

Figure 2.8- 1H-NMR of TQCP

Figure 2.10- Normalized optical absorption spectra of PQ, PQCP and coumarin (Panel A) and TQ, TQCP and coumarin (Panel B). The concentrations of each compound were ~15-20 µM in 2 mL of ethanol. Spectra were obtained in a 1-cm quartz optical cuvette.

Figure 2.11- Normalized steady state emission spectra of PQ, PQCP and coumarin (Panel A) and TQ, TQCP and coumarin (Panel B). The concentrations of each compound were ~5 µM in 2 mL of ethanol. Spectra were obtained in a 1-cm quartz optical cuvette. Excitation- 320 nm.

CHAPTER 3: IN VITRO CHARACTERIZATION OF 8-AMINOQUINOLINE FLUORESCENT

PROBES

INTRODUCTION

8-Aminoquinolines are a class of anti-malarial compounds that have great efficacy against sexual stages and moderate activity against asexual stages. The mechanism of action of these molecules is dependent upon their metabolites, and various investigations support this theory (91, 113). After undergoing various metabolic reactions, the reactive metabolites interact with the parasite causing death. How these reactive metabolites clear Plasmodium parasites is not completely understood, though it is believed that these metabolites induce intracellular oxidative potentials that lead to parasite death (84, 114). When combined with a blood schizonticide such as chloroquine, the combination acts as a radical cure, clearing the infection. 8-aminoquinoline use is limited due to toxicity in those with glucose 6-phosphate dehydrogenase deficiency, where hemolytic anemia occurs with varying severity. It is possible that the mechanism of action and the mechanism of toxicity are interlinked, and there is evidence suggesting that the mechanism of toxicity could be mediated by various groups substituted on the 5-position of the aminoquinoline ring system(115). Understanding these mechanisms is important, as they could indicate why Plasmodium has not developed an effective resistance to these compounds.

Fluorescent localization studies of Plasmodium parasites, as well as other diseases, can offer a host of information about microorganism biology, as well as the mechanism of action of particular therapeutics.

While much of Plasmodium biology has been characterized and readily understood, definitive detection of hypnozoite forms remains elusive. Various studies have confirmed their presence in zoonotic

Plasmodium species such as P. cynomolgi and P. simiovale (116), and this is often used as evidence for the existence of hypnozoites in human infections with vivax and ovale. The difficulty in hypnozoite

50 detection lies in hypnozoites’ limited metabolism and cell cycle functions, keeping them from being detected by methods that exploit those functions. Difficulty in detection is compounded by the fact that hypnozoites appear in very limited numbers when actually found in cynomolgi and simiovale.

Additionally, human hepatocytes are hotbeds of cytochrome P450 enzyme activity, many of which are fluorescently active. This auto-fluorescent activity adds considerable background to fluorescence microscopy, making detection of some fluorescent probes difficult when searching for hypnozoites.

Very recently, a transcriptome study of hypnozoites from P. cynomolgi was accomplished via laser capture microdissection. This study was the first to describe gene regulation in hypnozoites and effectively compare them to blood and liver stage forms. It was found that hypnozoite quiescence is possibly regulated by the ApiAP2 family of transcription factors, and translational repression by the elF2a kinase elK2 (117). While this work is a huge step forward in the characterization of hypnozoites, effective detection of the vivax and ovale forms remains elusive.

This study investigates the in vitro characterization of these novel 8-AQ fluorescent probes. Though their anti-malarial and anti-leishmaniasis activities significantly differ from their parent molecules, the IC50 concentration values are similar in terms of magnitude. They are not toxic against HepG2 carcinoma cells, and their localization in asexual and sexual stage parasites is cytosolic. Co-localization studies indicate there is no mitochondrial localization, though this is most likely due to the bulky nature of the

PQCP/TQCP molecules.

MATERIALS AND METHODS

Culturing of P. falciparum parasites and in vitro cultures

P. falciparum parasite strains 3D7, W2 (Indochina), D6 (Sierra Leone), and D10 (ATCC) were maintained in culture as previously described(118). All cultures were supplemented with 4% O+ human red blood cells (Interstate Blood Bank, Memphis, TN), 10% heat inactivated A+ human plasma (Interstate

Blood Bank), and RPMI1640 media supplemented with 25mM HEPES(Sigma-Aldrich). D10 parasite cultures were supplemented with 100 nM Pyrimethamine in order to select for GFP parasites. For fluorescent microscopy and drug susceptibility assays, cultures were synchronized to ring stage using 5%

(w/v) D-sorbitol (Sigma-Aldrich, St. Louis, MO) as described (119). Growth staging was determined by geimsa staining and light microscopy.

Characterization of Anti-malarial Activity in asexual stages

Anti-malarial activity assays performed against P. falciparum clones 3D7, W2, and D6 were performed using a modified 3H-Hypoxanthine uptake assay (120). Initial drug stocks were prepared in DMSO. All final drug dilutions were prepared using RPMI1640 media supplemented with 25 mM HEPES. Drugs were serially diluted 1:2 with a starting concentration of 100g/ml. Drug susceptibility curves and IC50s were calculated using GraphPad 6 software.

L. donovani axenic amastigotes assay

Compounds were tested against L. donovani axenic amastigotes (MHOM/SD/75/1246/130) using the

CellTiter 96 Aqueous one-solution cell proliferation assay (Promega, Madison, WI). In a 96 well drug plate (Costar, Assay Plate, 96 well with low Evaporation Lid, Flat Bottom, None-Treated, #3370) compounds were diluted in a series of 6 2-fold dilutions in media to produce a concentration range from

200μg/ml to 6.25μg/ml. Ten μL from each well was transferred to another 96 well plate (Costar, Assay

Plate, 96 well with low Evaporation Lid, Flat Bottom, Tissue culture Treated, # 3628) and then 90uL of parasites in media in a 66,000 cell per well concentration was added to produce a final concentration range from 20μg/ml to 625ng/ml. The plates were then incubated at 37oC for 72 hours. Twenty μL of 3-

(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium (MTS:

Promega, Madison, WI) solution was added to each well and incubated for an additional 4 hours. A

Spectra Max M2e (molecular Devices, Sunnyvale, Ca) was used to measure optical density (OD) at

490nm. 52

L. donovani cytotoxicity assay

Compounds were tested against J774.A1 macrophages using the CellTiter 96 Aqueous one-solution cell proliferation assay (Promega, Madison, WI). In a 96 well drug plate (Costar, Assay Plate, 96 well with low Evaporation Lid, Flat Bottom, None-Treated, #3370) compounds were diluted in a series of 6 2-fold dilutions in media to produce a concentration range from 500μg/ml to 15.625μg/ml. Ten μL from each well was transferred to another 96 well plate (Costar, Assay Plate, 96 well with low Evaporation Lid, Flat

Bottom, Tissue culture Treated, # 3628) and 90μL of macrophages in media in a 50,000 cell per well concentration was added to produce a final concentration range from 50μg/ml to 1.562 μg/ml. The plates were then incubated at 37oC, 5% CO2 for 72 hrs. Twenty μL of 3-(4,5-dimethylthiazol-2-yl)-5(3- carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium (MTS: Promega, Madison, WI) solution was added to each well and incubated for an additional 4 hours. A Spectra Max M2e (molecular Devices,

Sunnyvale, Ca) was used to measure optical density (OD) at 490nm.

Hepatocyte culture

Frozen HepG2 hepatocytes (ATCC) were cultured on rat tail-1 collagen coated 75cm2 vented flasks in media containing: Eagle’s Minimum Essential Medium with 10% fetal bovine serum, 1% L-glutamine additional supplement, and 1% Penicillin-Streptomycin. Cultures were incubated in a mixed gas incubator at 37°C. Cultures receive media change within the first 24 hours post-seeding from cryopreservation stock and media renewal every other day.

Animals and Parasites

All mice used in these experiments were female BALB/c mice obtained from Envigo (Indianapolis, IN).

The transgenic P. berghei line 1052 Cl1 (Pb 1052 Cl1) used in the liver stage assay was obtained from

C.J. Janse at Leiden University. This line was generated from the reference clone of ANKA strain c1115cyl and was designed to express a green fluorescent protein (GFP) and firefly luciferase. Pb 1052

Cl1 has two copies of the GFP-luc integrated into its genome. One copy is under the control of a constitutively expressed eef1aα promoter, and the other is under the control of an ama1 promoter. This study was conducted in compliance with the Guide for the Care and Use of Laboratory Animals of the

National Research Council for the National Academies. The protocol was approved by the University of

South Florida Institutional Animal Care and Use Committee.

Cytotoxicity of PQCP/TQCP in HepG2 cells

HepG2 cells were used for cytotoxicity assays once they reached >90% confluence. Afterwards, cells were trypsinized, counted, and seeded into collagen coated 96 well plates at 35,000-40,000 cells per well.

Cells were allowed to adhere overnight at 37 0C. Dilutions of PQ, TQ, TQCP, and PQCP were made and added to plates, with an initial concentration of 125g/ml. 1:2 serial dilutions were made, with a final concentration of 0.122g/ml. After the compounds were added the plate was incubated at 37 0C for 24 hours. After this period, cell viability was assessed by MTS [(3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)] cell proliferation assay (CellTiter96

Aqueous non-radioactive cell proliferation assay, Promega Corp., Madison, WI). Plates were read using a

SpectraMax M2e (Molecular Devices, Sunnyvale, Ca) at 490 nm wavelength. IC50 calculations and cytotoxicity curves were created using GraphPad 6 software.

Fluorescent Microscopy of sexual and asexual stage parasites

PQCP and TQCP were synthesized as described. Mitotracker Red (MTR) and SYTO11 were obtained from Thermo-Fisher. Cultures were grown and maintained in a similar manner described above, with the exception of being grown in RPMI1640 complete media without phenol red. Cultures of D10 or W2 parasites were allowed to grow to 2-6% parasitemia for screening. For primaquine saturation experiments,

150 M PQ was incubated with the parasites and washed before fluorescent probes were added.

Infected erythrocytes (0.1% HCT) were washed with phenol red free RPMI1640, then incubated with

7.5nM MTR, 1 M SYTO11, and 150 M PQCP/TQCP for 30-45min in 12 well plates. After incubation, cells were spun at 1850rpm for 2min. The supernatant was removed and erythrocytes were immersed in 200 l RPMI and allowed to incubate for another 30-45min. After this incubation, cells were re-suspended and transferred to 8 chamber slides for visualization. Pictures were taken using the

Deltavision inverted fluorescence microscope (GE Life Sciences, Pittsburgh, PA) with the 100x objective.

Statistical Analysis

All statistical analysis was carried out using Graph Pad Prism 6 software (GraphPad Software Inc., La

Jolla, CA). Data for anti-malarial, anti-leishmaniasis, and cytotoxicity assays were normalized against minimum negative control values and maximum positive control values. All values are expressed as means and standard deviations (SD). A  <0.05 was considered statistically significant. Data was analyzed using non-linear regression and a comparison of fits test to determine significant differences in drug action.

RESULTS

Coumarin does not affect 8-AQ anti-leishmanisis or anti-malarial efficacy in asexual or liver stage infection

Studies of PQCP and TQCP vs the parent compounds PQ and TQ indicate that the addition of the fluorescent coumarin moiety does not adversely affect compound efficacy. In 3H-Hypoxanthine assays with various strains of P. falciparum (3D7, W2, D6), no significant differences in IC50 values were observed between PQ/PQCP and TQ/TQCP. A similar phenomenon occurs in L. donovani as well.

Figures 3.1-3.3 and 3.5 shows dose response curves of their activities vs the respective strains and parasites.

PQCP localizes in the cytosol of P. falciparum asexual parasites

In our evaluation of localization, figure 3.6 displays PQCP localization in the various asexual stages

(Ring, Troph and Schizont) while fig 3.7 displays stage IV and V gametocytes. The pictures display localization of PQCP as mainly cytosolic. Co-localization of these probes with GFP and mitotracker also suggests that primaquine does not directly act upon the apicoplast or the mitochondria, due to lack of overlapping fluorescent signals. Parasites pre-loaded with primaquine support cytosolic localization, as

PQCP fluorescence is not observed when parasites are saturated with PQ.

TQCP and PQCP are not toxic to HepG2 cells

In order to make sure the probes do not adversely affect hepatic viability, we performed cytotoxicity assays using HepG2 human carcinoma cells. The probes were again screened against the parent compounds of PQ and TQ. There were significant differences in IC50 values between PQ/PQCP pair and the TQ/TQCP pair. The fluorescent probes were significantly less toxic than the parent compounds. We believe this is because the bulky probes have a more difficult time being taken up by uninfected hepatocytes.

DISCUSSION

Addition of the coumarin moiety to primaquine and tafenoquine had interesting effects on their in vitro activity. Despite significantly increasing the size of the molecules, the anti-malarial and anti-leishmaniasis activity didn’t greatly differ from the parent molecules, and the differences in IC50 were not statistically significant in most cases. It is possible that adding the coumarin moiety to TQ, along with the meta- trifluoromethyl-phenoxy group at the 5 position, affects trafficking and metabolism respectively. These two factors could be the reason why such a drastic difference is seen in the TQ/TQCP pair vs the

PQ/PQCP pair in cytotoxicity, as well as anti-malarial activity in the W2 strain of P. falciparum. It is expected that trafficking of the molecule would be impacted given such a drastic change to the parent

56 molecule. Regardless, the activities remain similar for the PQ/PQCP pair throughout the anti-malarial and anti-leishmaniasis assays. This could potentially mean that metabolism at the terminal amine in the PQ chain isn’t necessary for anti-malarial activity. It has even been theorized that blocking the primary terminal amine in PQ could help its bioavailability by blocking the conversion to carboxyprimaquine (84,

121). However, whether or not the coumarin-linker modifier has this effect has not been investigated. The difference in cytotoxicity against HepG2 cells is more affected by the addition of the coumarin moiety.

The probes are less toxic than their parent molecules, though they are still somewhat cytotoxic. Again, it is possible that the addition of the coumarin moiety affects molecule transport into and out of HepG2 cells. We attempted to determine localization of the probes in HepG2 cells, but background fluorescence of cytochrome enzymes prevented effective characterization of PQCP localization in uninfected HepG2 cells.

Despite the decrease in fluorescent intensity compared to coumarin, fluorescence is still maintained in in vitro culture and experiments. The concentrations needed for effective fluorescence are much higher when compared to commercialized probes such as mitotracker red (100 M vs 1 M respectively). This will perhaps limit the use of the probe in localization and trafficking studies, as over-exposure can increase parasite stress leading to lysis and death. Regardless, fluorescent localization can be effectively observed in both P. falciparum and L. donovani parasites. In falciparum, localization of PQCP appears to be limited to the cytosol of the parasite. This specific localization was further confirmed by saturation experiments with PQ, as well as co-localization studies with mitotracker red. Previous work with 3H- primaquine indicates that PQ localization also occurs in the mitochondria (99). The lack of localization of

PQCP in mitochondria is most likely due to the presence of the coumarin moiety on the probe itself.

We also investigated the co-localization of the nuclear stain SYTO11 with PQCP. Coumarin and therefore PQCP/TQCP fluoresce as blue, so traditional nuclear stains such as Hoechst couldn’t be used. In order to determine localization with a probe that stains nucleic acids, we employed SYTO11 as a control

57 probe to elucidate nuclear localization. Unfortunately, SYTO11 localization is primarily cytosolic, and overlapped with PQCP. The use of SYTO11 didn’t adequately detect the nucleus, so we decided to use the D10 strain of P. falciparum, which has a GFP tag on the ACL protein which localizes to the apicoplast. This proved to be especially effective in our co-localization studies, as PQ and TQ have not been implicated as having any apicoplastic targets.

Again, addition of the coumarin moiety would consequently affect trafficking. It is possible that the channel responsible for PQ efflux into parasite mitochondria isn’t able to transport PQCP as effectively and localization isn’t observed as a result. Nevertheless, cytosolic localization with no co-localization in the apicoplast or mitochondria could indicate PQ has a cytosolic target which affects its mechanism of action. Kimura et al have proposed PfPdxK as a target for PQ (97). Given the potential of PfPdxK as a target, as well as previous work indicating PQ affects SOD in Plasmodium due to increases in H2O2 production (84, 92, 93), cytosolic localization appears consistent with a mechanism that has PfPdxK as well as SOD as molecular targets. Similar localization phenomenon is observed in stage IV-V gametocytes, though more co-localization studies are needed in order to definitively define PQCP localization.

Figure 3.1- 3H-Hypoxanthine concentration response curves for TQ and TQCP against 3D7, W2, and D6 strains of P. falciparum. Similar curves indicate similar activity between compounds.

Figure 3.2- 3H-hypoxanthine concentration response curves of PQ/PQCP against 3D7, W2, and D6 strains of P. falciparum. Similar curves and statistical analysis of IC50s indicate no significant differences in drug action.

Table 3.1-IC50s of PQ, PQCP, TQ, and TQCP against various strains of P. falciparum

Figure 3.3- Activity of PQ/PQCP against L. donovani axenic amastigotes. Similar curves and statistical analysis suggest no significant differences in drug action in this system.

Figure 3.4- Cytotoxicity assay of PQ/PQCP and TQ/TQCP against HepG2 Carcinoma cells. No significant differences in cytotoxicity were observed in the dosing pairs.

Figure 3.5- Anti-malarial activity of PQ/PQCP and TQ/TQCP against P. berghei in the liver model. TQ/TQCP variability in one of the experiments causes the curves to appear to be different, but statistical tests suggest there are no significant differences in IC50s between the two pairs.

Ring Schizont Troph

DIC

GFP

TRITC

DAPI

MERGE

Figure 3.6- Images of D10 P. falciparum parasites treated with 150 M PQCP and 1 M MTR. Green channel is Green fluorescent protein tagged to the ACL carrier protein targeted to the apicoplast.

DIC GFP MTR PQCP MERGE

Figure 3.7- Pictures of D10 strain of P. falciparum parasites saturated with PQ, washed, and then treated with PQ. Localization of PQCP in the cytosol is greatly diminished, suggesting that PQ takes up space in the cytosol when interacting with plasmodium parasites. GFP-Green Fluorescent Protein (ACL-GFP). MTR-Mitotracker Red. PQCP-Primaquine-coumarin probe.

Stage IV Stage V

DIC

PQCP

MTR

MERGE

Figure 3.8- Images of Stage IV and V NF54 strain P. falciparum gametocytes treated with PQCP and MTR. Localization of PQCP in gametocytes is primarily cytosolic.

CHAPTER 4: IN VIVO ASSESSMENT OF THE MECHANISMS OF ACTION, SYNERGY AND

RESISTANCE OF ANTI-MALARIALS

INTRODUCTION

The emergence of drug resistant malaria parasites hinders eradication efforts by MMV and other organizations. Therefore, the need for novel therapeutics to combat these resistant parasites has increased efforts to understand mechanisms of action, resistance, and structure-activity relationships (SAR). Many old anti-malarials such as primaquine and menoctone have been used to combat malaria infection, but their modes of action and mechanisms of resistance are not fully understood.

Primaquine (PQ), despite being used for anti-malarial prophylaxis and anti-transmission for over 70 years, has not seen any clinically relevant resistance and treatment failure is rare (84). This phenomenon is extremely rare for anti-malarials of all classes. Primaquine’s use is limited due to inducing life- threatening hemolytic anemia in those with glucose 6-phosphate dehydrogenase (G6PD) deficiency.

Despite this limitation, it enjoys synergistic activity when administered with chloroquine (CQ) (102). This results in clearance of liver and blood stage parasites, resulting in a radical cure. The mechanism of synergy has been investigated in blood stages, but not in liver stages. Additionally, it is known that CQ potentiates the liver stage activity of PQ, but whether it is by direct parasite action or by another mechanism is still unknown (122). Fluorescent probes of PQ and CQ have been synthesized and found to be effective in localization studies for the erythrocytic stages of Plasmodium (56, 109). Using these probes, we can determine accumulation of each drug in infected hepatocytes. We hypothesize that synergism is achieved by differential accumulation of each drug in infected hepatocytes. The differential accumulation of these parasites could give us information on individual mechanisms in liver stages, as well as a mechanism of synergy.

Menoctone is a hydroxynapthoquinone compound, similar to atovaquone. It was assessed as an anti- malarial, but studies surrounding it were abandoned due to other more promising leads at the time. Due to the increase in rates of drug resistance, many older anti-malarials are being investigated to optimize their effectiveness. Additionally, information about their pharmacology may yield increased understanding about Plasmodium biology. It has been shown that menoctone is capable of anti-transmission activity due to its sporozontocidal activity, but its mechanism of action against sporozoites remains to be elucidated

(123).

These studies examine the role of PQ/CQ synergy in liver stages, and in vivo induction of menoctone resistance in P. berghei parasites. Resistance to menoctone in P. berghei was effectively characterized in vivo, and was found to confer resistance to atovaquone. The studies surrounding PQ/CQ synergy were unfortunately inconclusive. Ideas surrounding the reasons for these inconclusive experiments will also be discussed.

MATERIALS AND METHODS

Mosquito infections, sporozoite production and harvesting

P. berghei ANKA luciferase parasites were obtained from heart puncture from infected BALB/c mice and injected intraperitoneally into non-infected young female BALB/c mice. Parasitemia was monitored through tail vein blood smear bi-weekly. Once the parasitemia reached 3-5%, the mice were anesthetized with a mixture of xylene (10mg/kg), ketamine (100mg/kg), and placed on cartons of 100 3-4 day old, native female Anopheles stephensi mosquitoes. The mosquitoes were allowed to feed for approximately

30 minutes. After feeding, the mosquitoes were maintained on 10% sucrose inside environmental chambers at 26°C. On day 21 post-exposure (PE), infected salivary glands were dissected and sporozoites were isolated and counted. Mice were humanely euthanized following approved IUCAC protocols.

Animals and Parasites

All mice used in these experiments were female BALB/c mice (the average weight was approximately

18g) obtained from Envigo (Indianapolis, IN). The transgenic P. berghei line 1052 Cl1 (Pb 1052 Cl1) used in the liver stage assay was obtained from C.J. Janse at Leiden University. This line was generated from the reference clone of ANKA strain c1115cyl and was designed to express a green fluorescent protein (GFP) and firefly luciferase. Pb 1052 Cl1 has two copies of the GFP-luc integrated into its genome. One copy is under the control of a constitutively expressed eef1aα promoter, and the other is under the control of an ama1 promoter. This study was conducted in compliance with the Guide for the

Care and Use of Laboratory Animals of the National Research Council for the National Academies. The protocol was approved by the University of South Florida Institutional Animal Care and Use Committee.

Drug screening assay

Hepatocytes were seeded at an optimized density of 17,500 cells per well in a 96-well collagen coated plate (Becton Dickson, Franklin Lakes, NJ). Sporozoites harvested were added at an optimized 4,000 sporozoites per well (Derbyshire, Prudencio et al. 2012). After 3 hour incubation at 37°C, media was removed and drugs were added to their respective wells. A 48-hour incubation period yields our end point where we will lyse the cells with Promega Cell Lysis Reagent Buffer and add Luciferase Assay Reagent.

This catalyzes the firefly luciferase reaction by reacting luciferin with ATP, creating luciferyl adenylate and PPi. Luciferyl adenylate and oxygen then react to yield oxyluciferin, AMP and light. The amount of light emitted, which is read by the Perkin Elmer EnVision system, indicates parasite viability. If the parasite is affected by the drug and is no longer viable, then the luciferase signal will no longer work, thus determining the viability of the sporozoites after drug treatment. A growth curve was generated from this data to determine if the compound has liver stage activity.

Synergy screening of PQ with CQ or QN in P. berghei

To investigate synergy, various drug combinations of PQ with CQ and QN were investigated. We used a modified protocol from a previous study using 1:1, 3:1, and 1:3 concentration ratios (124). Drug screening assays were performed as described.

Microscopy of P. berghei infected HepG2 cells with PQCP

In order to evaluate localization of PQCP and the CQ-NBD probe (synthesized in house by the Manetsch lab) in P. berghei, we used HepG2 cells infected with P. berghei sporozoites as described in Ch. 3, but using microfluidic devices designed by Dr. Steve Maher at USF (100). After infection, liver stages forms were allowed to develop for 4 days. 150 M PQCP, 5nM Draq5 stain (Thermo-fisher, Waltham, MA) and

100 M CQ probe were added to P. berghei infected HepG2 cells for 1 hour. Media was removed from cells, washed with PBS and visualized on the Deltavision microscope using the 10x and 40x objectives.

Generation of menoctone resistance in P. berghei

We used similar methods as used by W. Peters to generate a new menoctone resistant parasite from the well-characterized P. berghei luc ANKA (125). Infected mice were treated with increasing levels of menoctone at doses of 3 mg/kg, 30 mg/kg, and 300 mg/kg. Recrudescent parasites were inoculated into new mice, and subsequent treatment with higher doses of drug led to resistance at a dose of 300 mg/kg.

Resistance was confirmed by the lack of response to subsequent inoculation of high doses of menoctone.

A maintenance dose of 300 mg/kg was used to maintain a high level of parasite resistance.

In vivo antimalarial efficacy against blood stages of P. berghei life cycle (modified Thompson test)

Concisely, five mice were randomly assigned to each dosage group. P. berghei luc ANKA, as well as, Pb

MEN-R were inoculated at 2x106 parasites per mouse. Menoctone was administered orally at 3 mg/kg, 30 mg/kg and 300 mg/kg and atovaquone was administered at 1 mg/kg, 3 mg/kg and 10 mg/kg. Both drugs were suspended in polyethylene glycol 400 (PEG 400) (Sigma Aldrich, St. Louis, MO) and administered 71 via oral gavage at 100 μl per mouse. Dosing by oral gavage occurred once daily for 3 days on days 6 to 8.

Blood films were made on days 3, 5, 6, 9, 12, 15, 18, 21, 24, 27, and 30 post-infection from tail vein blood to monitor parasite recrudescence.

Elucidation of mutation selection by atovaquone or menoctone

Sensitive and resistant parasites were inoculated into mice and challenged with either atovaquone or menoctone at different concentrations. Atovaquone was administered to mice at dosages of 1 mg/kg, 3 mg/kg, and 10 mg/kg, and menoctone was given at 3 mg/kg, 30 mg/kg, and 300 mg/kg respectively. Parasitized blood was then removed and sequenced for cytb mutations.

P. berghei MEN (MRA-414) assessment of atovaquone cross-resistance

Mice were infected with P. berghei MEN and allowed to grow to >4% parasitemia at which point were challenged with atovaquone at 3 mg/kg, 30 mg/kg and 100 mg/kg. Subsequent administration of atovaquone at 100 mg/kg was used as confirmation of atovaquone resistance.

RESULTS

Synergy of PQ with CQ and QN. Results of this experiment were inconclusive. Positive and negative control values were not consistent and varied greatly. In vivo concentrations where PQ has been shown to be active displayed no activity. Experiment was not repeated due to time constraints and lack of trained personnel.

Microscopy of P. berghei parasites in HepG2 cells with PQCP. We attempted to determine the localization of PQCP in HepG2 cells infected with P. berghei parasites in a microfluidic culture model.

These experiments yielded inconclusive results, as characteristic parasite structures could not be located and analyzed.

Menoctone resistance selection in P. berghei. This study attempted to select for resistance to menoctone in P. berghei in vivo by using a stepwise selection method. When mice were infected with asexual

72 erythrocytic stages and treated with a single dose of menoctone at 3 mg/kg, no decrease in parasitemia was observed. Blood from these mice was then inoculated into malaria naïve mice and allowed to develop. After reaching detectable levels of parasitemia, these mice were treated with a single dose of menoctone at 30 mg/kg. While this treatment resulted in a decrease of parasitemia to less than 1%, parasites recrudesced to 30% six days later. This procedure was repeated once more, but the dose of menoctone was increased to 300 mg/kg. Parasitemia was reduced to undetectable levels, but recrudescence still occurred. This process was once again repeated, taking blood from the recrudescent mice into malaria naïve mice. In this second passing, recrudescence occurred more rapidly despite treatment with 300 mg/kg of menoctone. This indicated that parasites were indeed resistant to menoctone treatment and is notated as MEN-R. Graphical representations of survival groups can be seen in figure 4.1

Assessment of atovaquone cross-resistance in P. berghei MEN resistant parasites. PbMEN-R infected mice were treated with 3mg/kg ATQ, and saw a decrease in parasitemia to <1%. However, parasites recrudesced by day 6 post treatment, and reached 10% parasitemia by day 9. Blood was passed into naïve mice and a dose of 30 mg/kg ATQ was given. Again, parasitemia decreased to <1%, but recrudesced much faster than the first drug exposure at 4 days post treatment vs 6. The process was repeated, but naïve mice were treated with 100 mg/kg ATQ. In this group parasites recrudesced by day 7 post treatment.

Successive treatment with 100mg/kg ATQ failed to clear erythrocytic parasites. These trends can be seen in figure 4.2.

DISCUSSION

The failure to detect PQCP and the CQ-NBD probe was not unanticipated. HepG2 cells are human hepatic carcinoma cells, which have levels of fluorescently active cytochrome enzymes. Due to the fact that high concentrations of PQCP are needed for active fluorescence, it is entirely possible that the probe was unable to be detected in parasites due to the high background of fluorescently active enzymes.

Another possibility is that the infections of the HepG2 cells themselves were unsuccessful. Parasites used

73 to investigate localization were not self-reporting due to the nature of the experiment; all available fluorescent channels were taken up by probes (PQCP, CQ-NBD, MTR). Due to limited time and materials, other hepatic cell lines were not investigated. If fluorescent detection of liver stage Plasmodium infection by tagged 8-AQ probes were to be repeated, different cell lines would need to be used or potentially use another fluorescently active probe to tag the parent 8-AQ molecules.

The synergy studies of PQ with CQ and QN had many potential issues, but two points in the assay in particular are usually responsible for confounding the experiment. The first of which is sporozoite viability, which can decrease greatly upon the death of the mosquito during dissection. Decreased viability would lead to decreased infection rates, which would cause great variability in parasite detection.

This assay in particular requires expression of the luciferase gene in P. berghei luc ANKA to interact with the luciferase reagent to generate light, which is read by the plate reader. Previous studies in the optimization of this procedure noted that the luciferase gene would sometimes be knocked out after a certain number of parasite passages in mice. Therefore, it is also possible that the luciferase gene was knocked out before the assay even began. Unfortunately, there is no way to assay the activity of this gene before feeding mice to mosquitos. While there are other ways this experiment could have been confounded (parasite death, variances in infection rates of cells, etc.), these two reasons represent the most likely scenarios of experimental failure due to previous studies by the Kyle laboratory.

Menoctone is an older anti-malarial which stopped being investigated due to more promising leads at the time. These studies were limited in their characterization of menoctone’s anti-malarial properties, leaving much to be discovered about its anti-malarial mechanisms and pharmacology. This study sought to better characterize menoctone’s mechanisms via resistance induction. Using a stepwise selection method, resistance to menoctone in P. berghei parasites was induced. Resistance to menoctone was further confirmed by cross resistance to atovaquone (ATQ). Using high doses of ATQ, parasites still recrudesced, even after parasitemia was reduced to less than 1%. This is not unexpected, as ATQ and menoctone are

74 both hydroxynapthoquinones; they should exhibit similar anti-malarial activities and potentially similar mechanisms of action. Indeed, more work surrounding menoctone’s resistance should be investigated.

Menoctone’s effect on parasite mitochondria and elucidation of molecular targets would likely give a wealth of information about mechanisms of action and resistance.

While the experiments mentioned here are specific to the author’s contributions, a full characterization of the genotype of resistant parasites, in addition to a more in depth discussion about the ramifications of this work is included in the full publication by Blake et al under the title “Menoctone resistance in malaria parasites is conferred by M133I mutations in cytochrome b that are transmissible through mosquitos" in the journal Antimicrobial Agents and Chemotherapy.

Figure 4.1- Survival curves of sensitive (A, C) versus menoctone resistant P. berghei (B, D) following treatment with menoctone or atovaquone. Menoctone offers some protection to Pb luc ANKA infected mice (A), but demonstrates a marked decrease in protection against menoctone resistant parasites, Pb MEN-R (B). Atovaquone provided significantly more efficacy than menoctone (C), however even at the highest dose of 10 mg/kg, atovaquone was not effective at preventing recrudescence of Pb MEN-R (D).

Figure 4.2- Assessment of ATQ cross-resistance with menoctone in P. berghei MEN (MRA-414). P. berghei MEN infected mice were treated with 3 (A), 30 (B), and 100 mg/kg (C) on day 0. Confirmations of ATQ cross-resistance with menoctone resistant parasites was evident by failure to significantly eliminate asexual blood stages parasites with a subsequent does of 100 mg/kg ATQ.

CHAPTER 5: SUMMARY

Malaria is a disease that affects millions of people, and billions more are at risk of infection. Due to the increase in resistance to all types of anti-malarial drugs, we need to investigate novel therapeutics, as well as older potential therapies to combat the spread of resistance. This work seeks to aid those investigations by exploring the potential of fluorescent probes made with 8-AQ molecules. Using fluorescent tagged drug molecules is an advantageous way of exploring the drug’s pharmacology and even investigating the basic science of disease states. This process has been used for cancer medications as well as for anti- malarials such as artemisinin and chloroquine. 8-AQ mechanism of action is based upon its metabolites generated by CYP450 enzymes present in the liver (84, 91, 126). However, how these mechanisms synergize activity with other anti-malarials such as chloroquine (CQ) is still unknown. By using fluorescent probes of 8-AQs we hoped to examine co-localization of 8-AQs with CQ fluorescent probes.

We hypothesized that synergism is achieved by differential accumulation of each drug in hepatocytes infected with Plasmodium.

We created two 8-AQ fluorescent probes, which were characterized by chemical and biological processes.

The synthesis of these compounds proceeds readily. While the yields of these reactions are not excellent, they are sufficient enough to perform many in vitro and in vivo experiments. Characterization of TQCP was somewhat hindered by limited research materials; tafenoquine is sold at a maximum size of 50mg.

Synthesizing a batch of TQCP takes the entire bottle of TQ and only gives a 50% yield (25-26mg).

Additionally, TQ is somewhat expensive, so making large amounts of this probe for commercial purposes will be impractical. Nevertheless, in our hands we saw very similar trends in activity for both probes when compared the parent molecules. Since PQCP is cheaper to make and easier to characterize than

TQCP, PQCP would be a more attractive choice for commercialization. Additionally, due to the 78 aforementioned similarities between PQCP and TQCP, we can suggest that localization of PQCP and

TQCP will be similar in many circumstances.

Unfortunately, localization studies to determine potential mechanisms of synergy were confounded by reasons mentioned in Ch. 4. While there is evidence for PQ/CQ synergy in vitro and in vivo, the mechanism of synergy is currently unknown. What was found was that during visualization studies with

PQCP, uptake of the probe only occurred in live samples. Slides with dead P. falciparum parasites fixed to a slide did not take up PQCP. Therefore, we can suggest that uptake of PQCP (and therefore PQ) is facilitated by an active transport process. Previous work demonstrated that transport can be investigated using fluorescent probes of CQ (56). It would be interesting to see if a similar approach could be applied to infected hepatocytes using fluorescent 8-AQ probes. However, due to confounds in using these probes in hepatocytes, different probes of 8-AQ should be created to investigate such mechanisms.

To continue this research, it would be especially interesting to tag the molecules with different fluorescent probes in the red or green absorption range. The EDCI/Dmap coupling reaction can couple any probe with an active carboxylic acid group to the primary free amine group of 8-AQs. This linkage is especially effective to preserve activity of 8-AQs, as transformations of the primary amine have been shown to not significantly impact 8-AQ anti-malarial activity (84). Furthermore, less bulky probes should be investigated, as trafficking to the mitochondria was not observed in these studies, despite already being implicated as a potential primaquine target. Another experiment that should be considered is high resolution mass spectra analysis of these probes after incubation with metabolically active hepatocytes. If the coumarin moiety is being cleaved due to CYP450 enzyme activity, it would confound experiments by presenting false positives. If such transformations occurred in vivo, rodent studies would be out of the question, as coumarin has been shown to be toxic to rodent hepatocytes (127). Finally in 1961, a few years after primaquine’s initial discovery, P. vivax parasites resistant to PQ were generated in vitro (128).

This work has not seen replication due to the lack of PQ resistance seen in wild type parasites and clinical

79 isolates. If this work were to be replicated today, we would be able to determine changes in gene regulation in the resistant parasites. Additionally, this study includes an example of such a resistance study via the generation of menoctone resistance in vivo. In order to effectively characterize such gene regulation, a combination of in vitro and in vivo studies would need to be accomplished.

With our current wealth of information surrounding 8-AQ mechanisms of action, which would include this study, the question as to why 8-AQ resistance is nearly non-existent would finally be answered.

Answering this question would lead to a new generation of anti-malarial therapeutics based in the 8-AQ class, and possibly leading to a novel class of anti-malarials. The MMV’s goal of malaria eradication may not be immediately feasible, but with this work combined with the efforts of others, it is a goal that is not farfetched.

In the author’s opinion, ROS mediated killing is a mechanism seen in multiple anti-malarial compounds.

This mechanism is a consistent weak spot in Plasmodium, as resistance to ROS generating compounds such as artemisinin come at a fitness cost to the parasite (75, 129). 8-AQ mediated ROS generation is no different, as seen by the near non-existent resistance to 8-AQ compounds despite their use for over 70 years. Future anti-malarials, as well as combination therapies, should exploit this weakness in order to further eradication efforts and reduce incidences of resistance in Plasmodium.

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