An Investigation into the Efficacy of

Aureobasidin A

Analogues to Treat

Trypansoma brucei

through In Vitro

Fluorometric Cell Viability

Assays

Name: Alexander Rowan Armit Project Supervisor: Dr Helen PriceStudent ID: 13005483 LSC-300035Word Count: 8,355

1

Contents

Abstract..................................................................................................2

Introduction..........................................................................................2

Materials and Methods....................................................................14

Results..................................................................................................17

Discussion...........................................................................................26

Acknowledgements..........................................................................33

References..............................................................................................34

2

AbstractAureobasidin A is an antifungal compound which inhibits the activity

of the enzyme inositol phosphorylceramide in yeast and protozoa systems,

a key enzyme used to produce certain sphingolipids, essential lipid

components of eukaryotic cell membranes. As this drug inhibits

sphingolipid synthases, it could be used as a possible candidate to treat

Trypanosoma brucei brucei, a sub-species of T. brucei that causes fatal

Animal Trypanosomiasis (Nagana) in Sub-Saharan Africa. Analogues of

Aureobasidin A were experimented with bloodstream forms of

Trypanosoma brucei brucei in vitro in fluorometric cell viability assays with

various concentrations of each analogue (log10 -5µM – 1.5µM) to determine

the IC50 of each drug, to determine the potency of each analogue in killing

trypanosomes. AUGC-9 and 26 were concluded as being the most effective

at killing trypanosomes with IC50 values of 0.635µM and 0.63µM,

suggesting these analogues had the highest affinity to bind to and inhibit

sphingolipid synthases. In contrast AUGC-10 & 30 produced the highest

IC50 values of the 13 analogues tested, suggesting these drugs had the

weakest affinity and were the least effective at inhibiting sphingolipid

synthases in bloodstream forms of the trypanosomes.

Introduction

Trypanosoma brucei (T. brucei) is a unicellular eukaryotic parasite

found in the bloodstream of infected individuals, and is known to cause

fatal African Trypanosomiasis (sleeping sickness) in those afflicted. T.

brucei is a flagellated parasite and is classified as a kinetoplastid parasite

due to the parasite containing a unique organelle found only in this class

3

of parasites called a kinetoplast, DNA found outside the nucleus enclosed

in its own mitochondrial membrane and is associated with the flagellar

pocket of the parasite (Smith et al. 1998). The kinetoplastid in T. brucei is

essential to its motility via the flagellum as this organelle is linked directly

to the cytoskeleton and the flagellum via a filament system called the

tripartite attachment complex, which is essential for T. brucei to complete

its cell cycle and produce daughter cells (Zhao et al. 2008).

T. brucei consists of three zoonotic sub species known as T. brucei

gambiense, T. brucei rhodiesiense, and T. brucei brucei. However only T.

brucei gambiense and T. brucei rhodiesiense infect humans and cause

chronic and acute forms of Human African Trypanosomiasis respectively,

whereas T. brucei brucei infects mainly animals and causes Animal

Trypanosomiasis/nagana (Deborggraeve et al. 2008). The T. brucei sub

species are all transmitted by insect vectors in the form of

hematophagous Tsetse fly Glossina spp, such as the species G. morsitans,

G. palpalis and G. fuscipes which are all infected and used as vectors for

the three sub species of T. brucei (Krafsur, 2009). Due to the parasite’s

choice of vector, it is restricted to sub-Saharan Africa where each sub-

species is restricted to a particular geographic area such as Gambia in

West Africa, where (T. b. gambiense), Eastern/Southern Africa (T. b.

rhodiesiense) and can also be prevalent across the sub-Sahara such as in

the case of T. b. brucei (Balmer et al. 2011).

The life cycle of T. brucei begins when the metacyclic form of the

trypanosome is inoculated into the human host from an infected tsetse fly

(Glossina spp.) takes a blood meal; this causes the metacyclic

trypanosomes to become established in the skin of the host (Sternberg.

2004). The metacyclic trypanosomes move to the blood and differentiate

into trypomastigotes which then travel in the bloodstream to various

4

bodily fluids such as the circulatory/lymphatic system and the spinal cord

where the trypomastigotes divide via binary fission (Stuart et al. 2008).

Eventually the trypomastigotes differentiate again into stumpy forms

which have a reduced size in flagella; this form is then taken up into a

tsetse fly when it feeds on an infected human host, as this form is pre-

adapted to survive the change in environment from human to tsetse

(Barrett et al. 2007). In the fly’s mid-gut, the stumpy form transforms into

a procyclic trypomastigote and continues cell multiplication (via binary

fission), before then migrating to the salivary glands of the tsetse fly and

undergoes restructuring where they transform into another form of T.

brucei called the epimastigote (Langousis and Hill. 2014). The

epimastigotes attach via their flagellum to the microvilli found in the

salivary glands and proliferate before transforming into the infective

metacyclic stages and completing the parasite’s life cycle, this attachment

has been found to be an essential step in metacyclic trypanosome

transformation (Vickerman. 1985).

In addition, the many transformations and switching from proliferative

stages (procyclic, epimastigote, trypmastigote) and non-proliferative

stages (metacyclic) has been suggested in Vickerman’s article (1985) to

be necessary as the non-proliferative stages are associated with a change

in environmental conditions (human – tsetse fly) whereas the proliferative

stages are associated with establishing the parasite in the new

environment, all shown in Figure

1.

Figure 1. Simplified diagram showing the life cycle of T. brucei in the vector Tsetse fly and the Mammalian/Human host. Diagram from Duque et al. 2013).

5

Human African Trypanosomiasis (HAT) is mainly prevalent in the rural

populations of sub-Saharan Africa, where there isn’t a clear number on the

amount of infected individuals per annum (pa), as some journals report

cases less than 12,000pa with a total of 50,000-70,000 infected worldwide

(Brun et al. 2010) whereas another journal states an annual prevalence of

300,000 between 1986-2004 but corroborates with the worldwide infected

total to be around 70,000 (Kennedy. 2008), mainly caused by the chronic

T. b. gambiense.

This overall number is fairly small in comparison to parasites that infect a

larger amount of people (e.g. Plasmodium spp.) however it is still a very

serious disease with limited control and treatment. Since the discovery of

the parasite T. brucei and the disease it causes nearly 100 years ago,

control of T. brucei in colonial Africa considerably reduced the number of

infected cases from as high as over 60,000pa to as low as less than 1000

by 1960 (Simarro et al. 2008). Due to the almost elimination of any new T.

brucei infections in the 1960s, awareness and control of this parasite

diminished due to it being near non-existent, allowing for a gradual

6

resurgence in HAT across sub-Saharan Africa with a general trend of

increasing infections per annum (Figure 2)(Simarro et al. 2008). At

present, Trypanosomiasis is regarded as part of a group of 13 parasitic

and bacterial infections known as the neglected tropical diseases, which

are the most common chronic infections that affect mainly humans that

inhabit the world’s poorest places and survive off less than $2 a day

(Hotez et al. 2007).

As aforementioned, the chronic form of HAT, caused by T. b. gambiense is

the main subspecies of T. brucei which infects humans and as this

subspecies is restricted to west and central Africa by its Glossina spp

vector, the highest numbers of infected cases are found in a string of

countries in central Africa such as in Angola, Democratic Republic of the

Congo, South Sudan and Sudan where there are more than 1000 cases

reported per year in the local population (Brun et al. 2010). While these

countries have an increased prevalence of T. brucei infections mainly by T.

b. gambiense, the acute form of HAT caused by T. b. rhodiesiense causes

a large amount of infections in eastern Africa, with between 101-500 cases

Figure 2. Bar graph to show number of T. brucei infected cases each year since 1927, note a massive decrease in 1960s and then a resurgence post 1967. Graph taken from Simarro et al. 2008.

7

measured in the United Republic of Tanzania and more interestingly in

Uganda, where there is an overlap in both T. b. gambiense and T. b.

rhodiesiense (Simarro et al. 2010).

In Human African Trypanosomiasis, there are two observable clinical

features and symptoms which occur in infected individuals known as the

haemolymphatic stage and the meningoencephalitic late stage. The

haemolymphatic stage occurs first where the parasite is confined in the

blood, and the meningoencephalitic stage which occurs as the parasitic

infection progresses and the trypanosomes invade the central nervous

system (Brun et al. 2010). The first symptom of infection with T. brucei

sub-species is the formation of a painful ulcer (chancre, 2-5cm diameter)

around the site of the infected Tsetse fly bite; this usually occurs 1-2

weeks after the fly bite as part of the human host inflammatory response

to the parasite (Ezzedine et al. 2007). However, chancres are rare in the

chronic T. b. gambiense and are primarily seen in the acute T. b.

rhodiesiense, as studies have shown that chancres do not seem to be

associated with infected African populations in West Africa where T. b.

gambiense is prevalent (Iborra et al. 1999). The parasites multiply in the

bloodstream during the first stage of infection and are transported to

various organs in the human body such as the spleen, liver, heart and

endocrine system. With many parasitic infections, initial symptoms are

non-specific to T. brucei and include intermittent fever, anaemia, joint

pains as well as inflammation in the organs they are found in, such as

myocarditis, lymphadenopathy and keratitis (Kennedy. 2006). Eventually

the trypanosomes in the bloodstream will enter into the second stage of

HAT, where they will invade the CNS, with some studies suggesting this is

due to the glucose rich environment of the brain as the parasite requires

glucose for ATP production (Wang. 1995).

8

The meningoencephalitic stage of infection is where the most severe

symptoms occur, as this is where the parasite severely affects the sleep

pattern/circadian rhythms of the infected, owing to its name ‘sleeping

sickness’. It has been postulated that the trypanosomes of T. brucei

penetrate the central nervous system and invade the brain/cerebrospinal

fluid by first localising to the barrier between the blood and cerebrospinal

fluid (choroid plexus) during early stages of infection (haemolymphatic

stage) and then eventually penetrate this barrier in later stages of

infection, as seen by T.b brucei in Masocha et al.’s (2007) study. Clinical

symptoms in this stage of infection can vary a great deal in terms of

neuropathology and may not be seen in all patients such as inhibition of

the motor system (muscle tremors, cerebellar ataxia), sensory system

(painful hypersensitivity known as hyperaesthesia) and also

behavioural/psychiatric changes (violence, hallucinations, mania)

(Kennedy. 2004). Moreover, the symptom that perhaps defines this

disease is the uncontrollable urge for late stage infected individuals to

sleep; this is caused by the trypanosomes as they convert the α-amino

acid tryptophan into the metabolites indole-3-acetic acid and tryptophol

(Cornford et al. 1979), the latter molecule causing haemolysis in

erythrocytes, changes in body temperature and induces a sleep like state

(Seed et al. 1978). Without rapid treatment, irreversible neurological

damage can occur, including continual mental deterioration, cerebral

oedema (inflammation), incontinence and ultimately a coma which may

regress into death of the infected (Kennedy. 2006). In addition, the

timeframe between the point of infection by Glossina spp and death varies

between the two main human infected T. b. gambiense and T. b.

rhodiesiense as the chronic T. b. gambiense may last from months to

years before death whereas acute T. b. rhodiesiense has a much smaller

9

timeframe with death occurring mainly between weeks or months (Blum et

al. 2006).

For the treatment of HAT, there is a very limited range of drugs and like

other protozoan parasites (Leishmaniasis), the drugs are old as there have

been no new advances in 40 years, highly toxic and, like with many

parasites there is a possibility of resistance building (Legros et al. 2002).

In addition, the drugs used to treat HAT differ from each stage, as drugs

used for the meningoencephalitic stage will need to be able to cross the

choroid plexus/ blood-brain barrier to attack the trypanosomes residing in

the CNS. In the haemolymphatic stage of T. brucei infection,

intravenous/intramuscular drugs such as Pentamidine are used to treat T.

b. gambiense and Suramin mainly used to treat T. b. rhodesiense (Brun et

al. 2011). The mode of mechanism for Pentamidine is poorly understood,

however it may act by binding and inhibiting components of kinetoplast

modification and RNA editing (Bouteille et al. 2003). Suramin is known to

inhibit at least 3 glycosomal enzymes involved in glycolysis (Wilson et al.

1993), blood-brain barrier permeation is minimal thus it can only be used

for stage 1 of HAT, and the side effects of this drug are vary, ranging from

pyrexia to nephrotoxicity (Brun et al. 2011).

In stage 2 of HAT, there are 3 main drugs called Eflornithine, Nifurtimox

which can be used in combination therapy and Melarsoprol, as well as a

number of drugs currently in development. Eflornithine is currently the

latest drug developed to treat stage 2 of HAT, even though it is nearly 50

years old and operates by inhibiting the enzyme ornithine decarboxylase

which is used to recycle ornithine, a by-product of the urea cycle (Burri

and Brun. 2002). Eflornithine has shown to reduce the numbers of

trypanosomes in the cerebral spinal fluid rapidly, with tolerable side

10

effects such as diarrhoea, hair loss and anaemia which worsen depending

on the length of treatment, which is dependent on the severity of stage 2

HAT in the patient (Pepin et al. 1987). However this drug’s activity is

greater in T. b. gambiense than in T. b. rhodesiense, the reason for this is

that T. b. rhodesiense has an innate tolerance of the drug as it has a

higher ornithine decarboxylase turnover (Burri and Brun. 2002).

Nifurtimox is an orally administered drug used to treat Chagas’ disease (T.

cruzi)/HAT and kills the trypanosomes by intracellular autoxidation to

produce free radicals which accumulate in the parasite to toxic levels

(Docampo and Moreno. 1986) and leads to neurological side effects such

as agitation, confusion and headaches. While in combination therapy with

Eflornithine, each drug has a lower dose and has been shown to reduce

toxicity in patients while efficiently killing the trypanosomes, while also

reducing the risk of resistance occurring in the parasite (Priotto et al.

2009).

Melarsoprol is an arsenic-based compound that is the most widely used

drug to treat stage 2 HAT, despite the fact it is also the most toxic as it

contains a toxic metal and thus produces severe side effects, the worst

being encephalopathy where ~40% of patients die from (Balasegaram et

al. 2006). While exact mode of mechanism for Melarsoprol on

trypanosomes is unknown, it has been shown to actively cause lysis in

trypanosomes exposed to the arsenical compound (Barrett et al. 2007), as

well as increasing the susceptibility of the parasite to free-radical damage

(Meshnick et al. 1978).

Unfortunately, parasitic resistance to these drugs is already occurring,

where Eflornithine has replaced Melarsoprol as the front line drug in some

areas of Africa, as well as how easy it was for Eflornithine resistance to be

11

created in a laboratory (Barrett et al. 2011). This shows an urgent need for

new drugs to be developed for treatment of HAT, as well as having

tolerable side effects.

The drug used in this project, Aureobasidin A, is a cyclic depsipeptide

antifungal compound produced by the fungus Aureobasidium pullulans

that can inhibit a wide range of pathogens such as fungi (Tan and Tay.

2013), and intracellular parasites such as Leishmania amazonensis &

Toxiplasma gondii (Tanaka et al. 2007) as well as extracellular parasites

such as Trypanosoma brucei. Aureobasidin A can be used to inhibit the

production of certain sphingolipids, which are essential lipid compounds of

eukaryotic cell membranes as they can be involved in signalling

mechanisms as a secondary messenger, such as the mammalian

sphingolipid Ceramide which is a complex sphingolipid as it contains a

backbone N-acylated with a long fatty acid (Pratt et al. 2013). There is a

contrast however, in the production of sphingolipids between mammalian

and yeast, plant and some protozoan systems (e.g. kinetoplastids) as

mammalian systems use a complex called sphingomyelin to produce

sphingolipids (Pratt et al. 2013), whereas fungi/some protozoa use inositol

phosphorylceramide (IPC) synthase to produce sphingolipids such as

inositolphosphoceramide and glycoinositolphospholipids (Figure 3).

12

Aureobasidin A (Figure 4) inhibits the enzyme inositol phosphorylceramide

(IPC) synthase found in the yeast/protozoa systems through non-

competitive binding and does not affect mammalian sphingolipid synthesis

(Salto et al. 2003), it can be considered a candidate for development to

treat some protozoa parasites such as trypanosomes. This is possible in

trypanosomes such as Trypanosoma cruzi (T. cruzi), which possesses on

its extracellular membrane glycoinositiolphospholipids (GIPL) that are

present on all life stages of this parasite (procyclic, metacyclic etc.) and

thus when inhibiting the enzyme IPC synthase responsible for the GIPL

synthesis, it impairs differentiation of the trypomastigotes in acidic pH and

could be used in therapies (Salto et al. 2003). In T. brucei, the parasite’s

genome contains 4 similar genes encoding for sphingolipid synthases,

which are essential for parasite viability during the bloodstream stage of

its lifecycle in the human host, thus it is stated as a possible drug target

(Sevova et al. 2010), however it is also stated in this journal article that

while Aureobasidin A was a potent inhibitor in yeast and other

protozoa/trypanosomes it did not significantly affect the activity of

sphingolipid synthesis in T. brucei.

13

As previously mentioned, Aureobasidin A was found to be a less potent

inhibitor in T. brucei than in yeast and surprisingly other trypanosomes

such as T. cruzi (Sevova et al. 2010). The main aim of this project was to

determine the efficacy and effectiveness of a series of new Aureobasidin

A analogues on geneticall modified T. brucei brucei (T. b. brucei) by cell

viability assays using AlamarBlue™ to measure parasite proliferation. The

Aureobasidin A analogues are overall very similar to Aureobasidin A,

however they have been chemically altered in some way (data restricted)

which may change the chemical behaviour of each molecule, thus the 13

Aureobasidin analogues that were tested will each have a slight change in

structure, for example one analogue may include a new side chain or R

group while another analogue may have another element substituted into

its structure. The change in chemical behaviour may allow the analogues

to bind to sphingolipid synthases with more affinity, with weak affinity or

may even bind preferentially to another molecule. The T. b. brucei cells

were placed in 10 varying concentrations of each analogue to determine

the IC50 of each drug. This was to determine the half maximal inhibitory

concentration which is the concentration of each analogue where 50% of

Figure 4. Simplified structure of Aureobasidin A, showing its cyclic structure and all aromatic structures and other R groups. Diagram from: http://www.guidechem.com/reference/dic-1456498.html

14

the T. b. brucei cells have been killed (Kalliokoski et al. 2013). Once the

IC50 has been calculated for each analogue, the drugs can be compared

with each other to determine which analogue has the lowest IC50 and is

thus the analogue which kills more of the parasite at lower concentrations

i.e. the most potent analogue. A negative control of Amphotericin B was

used for 0% survival of T. b. brucei and a positive control was used with no

drugs and just growth medium and drug diluent (an equivalent volume of

ethanol) as 100% cell survival.

Amphotericin B was used in this experiment as the 0% survival (Blank) as

this drug in low concentrations is able to kill all cells of T. b. brucei.

Amphotericin B is an amphipathic polyene which is known to increase the

permeability of ergosterol-containing membranes through pore formation

(Milhaud et al. 2002); this occurs as Amphotericin B forms an ion-channel

assembly in the presence of ergosterol which can lead to ion permeability

and cell death (Umegawa et al. 2011). This drug is particularly effective

against early eukaryotic cells such as Fungi and Protozoa as ergosterol is

more prevalent in their membranes as it helps preserve structural

membrane integrity in stressful environmental conditions (Dickey et al.

2009). In addition human cells do not contain the same levels of ergosterol

as Protozoa do, as ergosterol is converted into ergocaliferol in human cells

which is used in the production of Vitamin D (Bikle, 2014) and thus

Amphotericin B is not as effective so can be used as treatment in humans

for other diseases such as Leismaniasis. Due to these properties,

Amphotericin B was used as the blank to kill all the cells in the well it was

placed into, thus acting as a negative control showing that no other

factors other than those being tested have killed the T. b. brucei cells.

15

To assess the survival of T. b. brucei in the sample wells, the reagent

AlamarBlue™ was used, which quantifiably measures cell proliferation in

each well. This would determine the effectiveness of each Aureobasidin

analogue in killing the trypanosomes and the IC50. AlamarBlue™ can be

used to measure cell proliferation as its main component resazurin (Martin

et al. 2003) is a non-toxic molecule which interacts with and is catalysed

by intracellular enzymes involved in cellular metabolism such as NAD(P)H-

dependent oxidoreductases (Aleshin et al. 2015). Resazurin in

AlamarBlue™ is a blue colour and is weakly fluorescent before interacting

with metabolising cells, however when incorporated into cells, the

molecule acts as an intermediate electron acceptor for oxidative

metabolism in the mitochondria thereby becoming reduced (gains

electrons, loses oxygen) and is reduced to become resorufin which is the

colour red and is also highly fluorescent (Figure 5.) (Larson et al. 1997).

Due to the nature of the components within AlamarBlue™ that allow it to

exhibit fluorescent and colorimetric changes due to cellular metabolic

activity (Abe et al. 2002), it is a useful and accurate indicator for cell

viability as there is a direct correlation between the reduction of resazurin

and the quantity of proliferating cells (O’Brien et al. 2000). The

fluorescence of each well will increase if the T. b. brucei cell proliferation is

increasing thus any Aureobasidin analogue that does not affect T. b.

brucei’s cell viability will have a very high fluorescence when measured on

a GloMax® Multi+ Microplate Multimode Reader at Ex 530-560nm/Em

590nm.

16

Materials and Methods

Negative control setup

For the negative control a stock concentration of Amphotericin B at

271µM in 250mg/ml was required to be 50µM in 500µl thus the dilution

factor would be calculated by dividing 271 by 50, which is 5.42. The

required volume (500µl) was then divided by the dilution factor (5.42) to

calculate the volume ratio of Amphotericin B: HM19 medium (92µl:408µl),

which is then placed in a well on a 24-well plate. 250µl of this dilution is

to resorufin which is highly fluorescent and what is also the molecule that is measured in each well to determine cell viability. Note the loss of the Oxygen bonded to the Nitrogen in resazurin as the molecule is reduced. Diagram from http://gbiosciences.com/ResearchProducts/Alamar_Blue_Cell_Viability_Assay.aspx

17

extracted and placed in another well and mixed with 250µl of fresh HM19

to dilute Amphotericin B to half the previous concentration, this procedure

of extracting 250µl from the previous well into a new well with 250µl of

HM19 is continued until 10 concentrations of Amphotericin B is produced,

each well half the drug concentration as the previous well. In addition, a

control is set up in another well with a 1:250 dilution of ethanol and HM19

at 500µl (2µl ethanol: 498µl HM19).

First assay of Amphotericin B

50µl of each drug dilution and the control were placed in a 96 well

plate in triplicate prior to the addition of 50µl of the T. brucei brucei

sample at the correct concentration (discussed later).

Aureobasidin Analogue dilutions

For the cell viability assays with the Aureobasidin analogues, a

similar procedure to the negative control was used. Aureobasidin

analogues (abrev. AUGC) originally in 5mg/ml concentrations were diluted

by a ratio of 1:250 in 500µl thus 2µl of the AUGC would be mixed with

498µl of HM19 in a well. Exact procedure as the first assay where 250µl is

extracted from the 1:250 dilution well and placed in a new well with 250µl

fresh HM19, procedure repeated until 10 wells of varying concentrations of

AUGC (10µM-0.019µM) with each well half the concentration of the

previous. The negative control for the AUGC cell viability assays was

designed to have 0% survival of trypanosomes, thus consisted of a well with 2µl

Amphotericin B in 269µl HM19. The positive control well designed to have

100% survival consisted of a 1:250 dilution of ethanol and HM19 in 500µl,

the ethanol replacing the AUGC.

T.brucei brucei growth culture dilutions

18

In order to find the density of the T. brucei brucei samples used,

20µl was extracted from a 48hr growth culture and placed on a

haemocytometer under a light microscope at 20x. Live T. b. brucei cells

were then counted on the haemocytometer to determine the

concentration of the 48hr growth culture so that it can be diluted if

necessary, as the cell viability assays required a T. b. brucei concentration

of 2x105 cells per ml (2x105/ml). To determine dilution factor, the amount

of cells counted on the haemocytometer (e.g. 365 cells) was converted

into a concentration (36.5x105/ml) and then divided by the desired

concentration of 2x105/ml to determine the dilution factor needed (this

example 18.25). For 1 assay there would be 36 wells containing 50µl of

the controls and AUGC dilutions (11 drugs in triplicate) thus there needed

to be enough T. b. brucei dilution for 50µl in each well so 50 x 36 = 1800µl

of diluted cells needed, 2000µl was created as backup. The overall volume

of 2000µl was then divided by the dilution factor to calculate the volume

needed to be extracted from the original 48hr growth culture and then

diluted by HM19 to make 2000µl. 50µl of the diluted cells was then added

to each of the 36 wells before the plate was placed in an incubator at 37oC

for ~48hrs.

Alamar Blue addition

After ~48hrs incubation period, Alamar Blue was added to each of

the wells at 10% of the overall volume in the wells (100µl overall volume

in wells, thus 10µl of Alamar Blue added). Once Alamar Blue was added to

each of the 36 wells, the plate was placed back into the incubator at 37oC

for ~7hrs.

19

Fluorescence measurement and data collection

After the further 7hrs needed for the reagent Alamar Blue to be

incubated in the assays, plate is removed and placed in a Promega

Glomax Plate Reader; the plate reader @530-560nm will then measure the

fluorescence of the Alamar Blue in each well. Once been measured the

data can then be analysed and a visual representation of the data can be

produced to show the effects of the AUGC analogue concentrations of

T.brucei cell survival.

Interpretation of Results

To interpret the data, results created using GraphPad.Prism.v.6. to

calculate the % cell survival in each of the analogue concentrations. The

fluorescence in each analogue dilution is subtracted by the negative

control (Blank) and then divided by the positive control well, which has a

100% survival of cells then multiplied by 100 (Figure 6.). The percentage

values of cell viability are then plotted against the various concentrations

of the analogues to create a visual representation of results.

Figure 6. Formula used to calculate the cell survival of T. brucei in each well of the cell viability assay.

20

ResultsDeath of bloodstream forms of T. b. brucei via AUGC-3

administration resulted in a plateau and then gradual decrease of T. b.

brucei, with >90% of cells surviving at concentrations up to log10 -0.167µM

(Figure 7). After log10-0.167µM, there was a rapid decrease in cell survival

until 10% of T. b. brucei were alive at log10 1µM, where there was a return

to a continued gradual decrease until there were no cells alive at log10

1.5µM (Figure 7). IC50 of AUGC-3 was calculated to be 2.29µM with a 95%

certainty that the IC50 was located between 1.63µM and 3.22µM.

Same experiment occurred where AUGC-3 was replaced by AUGC-9,

resulted in an IC50 considerably lower than AUGC-3 at 0.635µM and a 95%

certainty between 0.282µM and 1.426µM (Figure 8). From log10 -5µM of

AUGC-9, T. b. brucei survival began to decrease gradually until ~log10 -

2µM where the survival rate of the cells began to decrease more rapidly

until 10% of cells survived at a concentration of log10 1.5µM, however the

curve does not continue past 10% survival. Large overlapping error bars

on all data points between 90% and 10% cell survival to be discussed later

(Figure 8), no repeat conducted with AUGC-9.

Experiment with AUGC-10 resulted in an IC50 of 3.586µM with a 95%

certainty the IC50 calculated is between 3.021µM and 4.257µM (Figure 9).

T. b. brucei survival plateaued initially and remained very high (above

95%) until log10 0µM, where an exponential decrease in cell survival

occurred. Rapid decrease of cell survival continued between log10 0µM and

1µM before a gradual decrease resumed until 0% cell survival at log10

1.47µM. Cell viability assay was repeated for AUGC-10 and resulted in

small errors except for one data point at ~log10 0.66µM (Figure 9).

21

AUGC-15 meta gave rise to an IC50 of 1.52µM and a 95% window of

0.932µM and 2.81µM. With previous experiments, the death of T. b. brucei

began gradually until the concentration of AUGC-15 meta reached log10 -

0.5µM, where a rapid decrease of T. b. brucei survival occurred between

log10 -0.5µM and 0.667µM (Figure 10). After log10 0.667µM cell survival

decreased at a more gradual rate until ~2% of cells were alive, where the

curve desists, no repeats of AUGC-15 meta were conducted.

Analogue AUGC-15 para which is very similar to AUGC-15 meta was

assayed with T. b. brucei and produced a IC50 of 2.174µM with 95%

confidence interval of 1.53µM to 3.51µM (Figure 11). Cell survival

plateaued at 100% until around log10 -1.8µM, where the decrease in cell

survival begins more rapidly. Gradient of the exponential slope was not as

Figure 7. Death of T. brucei bloodstream forms from AUGC-3. Experiment conducted over 72 hours and resulted in a positive correlation as the concentration of AUGC-3 increased, cell survival of T. b. brucei

Figure 8. Cell survival of bloodstream forms of T. b. brucei from AUGC-9 cell viability assay conducted over 72 hours. Positive correlation with a low IC50 of 0.635µM. Large overlapping error bars suggests ambiguity

Figure 9. Cell viability assay of brucei with AUGC-10 over 72 hours. Positive correlation with a high IC3.586µM. Only one large error bar with little overlapping of data points between the repeated assays.

22

steep as other assays (e.g. Figure 9), reaching 10% cell survival at around

log10 1.167µM. This assay was repeated and shows large overlapping error

bars at the lowest concentrations of the drug, similar to other experiments

(Figure 11).

AUGC-20 administered to T. b. brucei produced an IC50 of 2.16µM with 95%

confidence that the actual IC50 is between 1.72µM and 2.7µM for the drug

(Figure 12), a similar IC50 seen in AUGC-15 para (Figure 10). AUGC-20

caused a rapid decrease in cell survival of T. b. brucei when the

concentration of the drug reached log10 -0.32µM and continued to rapidly

decrease cell survivability until around log10 0.83µM, where the decrease

in cell survival became more gradual until 0% cell survival at log10 1.5µM

(Figure 12). AUGC-20 was not repeated and shows moderate-sized error

bars during the first few concentrations of the analogue.

Bloodstream forms of T. b. brucei administered with AUGC-21 in cell

viability assays concluded that the IC50 of the drug was approximately

1.075µM with a 95% confidence interval of 0.684µM to 1.69µM (Figure 13).

The cell survival of T. b. brucei remained at 100% until approximately log10

-3µM, where it begins to gradually decrease and then exponentially

between the concentrations log10 -1.32µM and 1µM. Cell survival never

reaches past 8% and shows the curve would’ve continued past the final

concentration of log10 1.5µM. AUGC-21 was repeated and produced small

error bars compared to other figures (Figure 13).

AUGC-25 assayed with T. b. brucei produced an IC50 of 1.734µM and 95%

confidence intervals of 1.219µM to 2.465µM (Figure 14). Cell survival

decreased fewer than 95% of cells alive at log10 -0.875µM, and then fell

exponentially between log10 -0.875µM to 1µM, and then continued to

decrease down to ~6%, where the curve desists as the maximum

23

concentration of 1.5µM is reached. AUGC-25 was repeated and large error

bars were produced, particularly at the data point at log10 0.375µM (Figure

14).

24

Analogue AUGC-26 was used in the cell viability assay and concluded an

IC50 value of 0.63µM with 95% confidence interval that the IC50 is between

0.399 and 0.997µM (Figure 14). AUGC-26 resulted in an overall more

gradual sloped curve unlike that seen in previous assays (Figure 9), and a

significantly less gradient on the slope between log10 -2µM and log10

0.5µM. in addition, the slope continued after log10 1.5µM and only reached

7% cell survival before it stopped (Figure 15). AUGC-26 was repeated and

produced large overlapping error bars around 50% survival.

AUGC-27 was calculated to have an IC50 of approximately 3.54µM with

95% confidence that the IC50 was calculated between 3µM and 4.2µM. The

cell survival of T. b. brucei remained at 100% even as the concentration of

AUGC-27 increased up to log10 ~-0.167µM before decreasing significantly

between log10 0µM and 1µM (Figure 16). After log10 1µM, the kill rate of T.

b. brucei began to decrease gradually and reached 0% cell survival at log10

1.5µM. AUGC-27 was repeated and produced overlapping error bars in

some instances, particularly at log10 0.667µM (Figure 16).

IC50 of AUGC-30 was calculated to be approximately 3.74µM with 95%

confidence the IC50 is between 2.51µM and 5.58µM (Figure 17). Cell

survival remained above 90% until approximately log10 0µM, where the

cell survival then exponentially decreases between 0µM and 1µM before

resuming a gradual decrease after 1µM of AUGC-30. The curve stops

25

before reaching 0% survival at log10 1.5µM (Figure 17), furthermore AUGC-

30 was not repeated and produced small error bars with an anomalous

point at log10 ~-0.32µM.

AUGC-36 produced an IC50 of 3µM with 95% confidence interval of 1.67µM

to 5.36µM (Figure 18). Cell survival remained above 95% until the

concentration of AUGC-36 reached log10 ~-0.32µM, after which the

exponential decrease in T. b. brucei survival occurred until log10 1µM. After

log10 1µM, kill rate of T. b. brucei decreased and became more gradual

until log10 1.5µM where the curve desists. Curve stops at 5% survival

showing the curve could continue past log10 1.5µM in AUGC-36

concentration (Figure 18). AUGC-36 was not repeated and produced small

error bars with the exception of the data point at log10 -0.5µM.

The final drug concentrations used in the assay of T. b. brucei was AUGC-

40, which produced an IC50 of 1.905µM and 95% confidence interval of

1.27µM to 2.85µM (Figure 19). T. b. brucei cell survival remained above

90% until the AUGC-40 concentration reached log10 -0.167µM, where the

expected rapid decrease in cell survival began between log10 -0.167µM

and 0.667µM. After 0.667µM, the rate of dying cells began to slow until

reaching 0% cells surviving at log10 1.5µM (Figure 19). AUGC-40 was not

repeated due to time constraints for the experiments but produced fairly

small error bars which indicate some overlapping initially, however there

are no error bars at the data points approximately around 0% cell survival.

The IC50 of each Aureobasidin A analogue has been placed in a table on

page 25 (Table 1), as well as the degrees of freedom for each drug and

the R2 value to show relationship between the curve and the actual data.

26

Figure 15. Cell viability assay of AUGC-26 produced an IC50 of 0.63µM with large overlapping error bars around 50% cell survival.

Figure 16. Kill rate of T. b. brucei from varying concentrations of AUGC-27. IC50 calculated to be 3.54µM.

Figure 17. Cell viability assay of AUGC-30 with bloodstream forms of T. b. brucei. IC50 calculated to be 3.74µM.

Figure 18. Cell viability assay of T. b. brucei and AUGC-36 of varying concentrations. IC50 calculated as 3µM.

Figure 19. Cell viability assay of varying concentrations of AUGC-40 with T. b. brucei bloodstream forms. IC50 calculated at 1.905µM.

27

AUGC-10, 15 para, 21, 25, 26 and 27 only repeated assays. IC50 values in µM with 95% confidence intervals in brackets. R2 values in table to show relationship of data points with the actual curve of the figures.

Table 1. IC50 activity of each drug for the cell viability assays with bloodstream forms of T. brucei

28

Discussion

To recap, the aim of this project was to investigate the efficacy of

Aureobasidin A derivatives to kill blood stream forms of Trypanosoma

brucei brucei through fluorescence-based cell viability assays.

Aureobasidin A was selected as this anti-fungal depsipeptide is a known

inhibitor of the enzyme phosphorylceramide synthase in Leishmania spp.

and also in T. Cruzi (Aeed et al. 2009), where it prevents the production of

extracellular membrane glycoinositiolphospholipids (GIPL). While there is

no extensive data on Aureobasidin A affecting T. brucei, procyclic forms of

T. brucei contain IPC synthase (Tsetse midgut) whereas bloodstream forms

of T. brucei contain sphingolipid synthases that are involved in the

biosynthesis of inositol phosphorylceramide (IPC) (Mina et al. 2009).

The cell viability assays of each analogue were produced on 96 well plates

with a negative control (Amphotericin B) and a positive control (growth

medium and drug diluent/ethanol) along with 10 decreasing

concentrations of each drug with the addition of AlamarBlue™ after ~72

hours. The plates were then placed GloMax®-Multi+ Microplate Multimode

Reader at Ex 530-560nm/Em 590nm then the percentage of T. brucei

survival in each well of varying concentrations was determined using the

equation (Figure 6.). The efficacy of each derivative was recorded by

calculating the IC50 of each derivative which would deduce the potency of

each derivative whereby the smaller the IC50 of the drug, the more potent

the drug is.

The first Aureobasidin A analogue assayed (AUGC-3) produced an IC50 of

2.29 which results in AUGC-3 being the 9th most potent analogue out of

29

the 13 tested. In addition, the graph produced from this cell viability assay

calculated an R2 value of 0.945, and as R2 determines how well the data

fits the statistical model, it suggests that the data points produced for this

analogue follows closely the regression curve. The IC50 of this drug

suggests that the minor change in the R group at the 3 rd carbon on

Aureobasidin A considerably altered the efficacy of AUGC-3 compared to

other analogues, suggesting that it binds to sphingolipid synthases with

less affinity and thus is a weak inhibitor. However, due to the unknown

structure of the analogue AUGC-3 through data restriction, it can only be

speculated as to what the change on carbon 3 of this analogue is, as the

unknown R group could be altering the overall properties of the drug such

as a change in polarity and hydrophobicity through methyl or amine

groups.

Compared with AUGC-3, a change in the R group on the 9 th carbon rather

than the 3rd caused an increase of inhibitor strength and AUGC-9’s affinity

for sphingolipid synthases by approximately 3.63 times, altering the IC50

from 2.29 to 0.635 (Table 1). AUGC-9 was determined by its IC50 of 0.635

to be the 2nd most potent analogue of the 13 assayed, and can be

designated as being an active drug due to it exhibiting a high potency

against sphingolipid synthases, defined by its IC50 being lower than 1µM

(Jacobs et al. 2011). However there are some discrepancies with the cell

viability assay of AUGC-9 as it produced a relatively low R2 value, along

with very large, overlapping error bars. This could have been rectified if

the assay was repeated, however due to the time restrictions of this

experiment there could be no repeats.

The IC50 of 3.586 in the cell viability assay with AUGC-10 concluded that

this analogue is one of the least potent out of the 13 tested, ranking 12th.

30

This high IC50 of this analogue suggests that the change in structure at

carbon 10 in Aureobasidin A has drastically reduced the potency and

strength of this analogue to bind to sphingolipid synthases and act as a

non-competitive inhibitor, thus would not be an applicable candidate to

pursue as a treatment for HAT. The cell viability assay of AUGC-10 was

repeated for reliability and produced consistent results with small error

bars (Figure 9) except for an anomalous point at log10 0.66µM, most likely

down to human error while conducting the experiment.

As carbon 15 of Aureobasidin A already consists of a large, 6 membered

aromatic side chain, analogues were produced by substituting the change

in structure (such as an R group) onto the aromatic ring at a particular

position, such as at the 3rd carbon on the arene (meta) and the 4th carbon

(para) which results in the analogues AUGC-15 meta and AUGC-15 para.

AUGC-15 meta and para produced IC50 values of 1.62µM and 2.174µM

respectively, suggesting that the addition of an R group to the para site of

the aromatic ring results in a decrease of affinity for analogue binding to

sphingolipid synthases. What is also remarkable about these two

analogues is how they are the most structurally similar and produced fairly

large differences in IC50, however both confidence intervals overlap thus

more repeats and refinement of these two analogues may produce more

distinct IC50 values and confidence levels. In addition, it appears that in

terms of overall chemistry that aromatic substitutions at the para position

generally produce less reactive derivatives whereas those with an arene

substitution at the meta position generally creates more active

derivatives (Troisi et al. 2009). Furthermore, AUGC-15 meta has a similar

IC50 to a derivative of an acyl hydrazide (1.66µM) which has also been

known to kill bloodstream forms of T. b. brucei (Troeberg et al. 2000).

31

AUGC-20 produced a very similar IC50 to AUGC-15 para, with only

0.0014µM between each IC50 (2.16µM and 2.174µM respectively)

suggesting a minor change in structure at carbon 20 had a similar effect

as AUGC-15 para, in terms of affinity of the inhibitor to bind non-

competitively to sphingolipid synthases. This analogue produced results

with a very good relationship to the statistical regression model used;

producing the highest R2 value, suggesting the data produced shows

accuracy. However this would be improved if the results were repeated

but due to the length of time each assay required (>72 hours), there were

restrictions into how many repeats could be conducted.

AUGC-21 and AUGC-25 produced IC50 values of 1.075µM and 1.734µM

respectively with no overlapping of the 95% confidence intervals (Figures

13 & 14, Table 1). The IC50 increase from AUGC-21 to 25 by 0.7µM

suggests that a structural change at carbon 25 has decreased the

efficiency and the affinity of AUGC-25 to bind and inhibit the sphingolipid

synthases in T. brucei. These two results were able to be reproduced and

showed consistent results; however both would need to be repeated to

improve reliability as the cell viability assay with AUGC-21 produced a low

R2 value, suggesting the data does not clearly match the curve of

inhibition. AUGC-25 produced large overlapping error bars from the 2

experiments (Figure 14) with one data point’s error bars overlapping 2

other data points, thus a 3rd repeat would eliminate any anomalous data

from the assay with this analogue.

AUGC-26, along with AUGC-9 (aforementioned) were found to be the most

potent analogues found out of the 13 tested, with AUGC-26 producing an

IC50 value of 0.63µM, and is also the only analogue tested to have 95%

confidence that its IC50 < 1µM (0.399µM – 0.997µM) indicating that this

32

analogue can be classified as active. This is in contrast to AUGC-9 which

has large 95% confidence intervals that exceed 1µM but also can range

lower than AUGC-26 (0.282µM – 1.426µM) which is understandable as

from Figure 8; AUGC-9 shows a large degree of the potential for errors,

due to its large overlapping error bars. Strong comparison between the

two most potent analogues would be more reliable should AUGC-9 be

repeated as AUGC-26 was.

Analogues AUGC-27, 30 and 36 (along with AUGC-10) produced the largest

IC50 values out of the Aureobasidin A derivatives assayed, produced IC50

values of 3.54µM, 3.74µM and 3µM respectively (Table 1) which is over 5

times the IC50 value of the lowest calculated for an analogue (AUGC- 26).

The results suggest that these analogues are the weakest in terms of

inhibition in T. brucei, as a change in structure at carbons 27, 30 & 36

causes a large decrease in affinity for sphingolipid synthase binding.

AUGC-30 and 36 were not repeated due to time restrictions and produced

very large 95% confidence levels that overlapped many of the IC50 values

of other analogues, particularly AUGC-36 which had a confidence interval

range from 1.67µM -5.36µM (Table 1). From speculation, these two

analogues suggest a large degree of error from the cell viability assays

which can also be seen in their figures (17 & 18) as the data points at the

weakest concentrations (approx. log10-1µM) begin to increase in cell

survival rather than the predicted decrease in cell survival when the

concentration increases. Unfortunately, like with other cell viability assays

produced they could not be reproduced due to the length of time of the

experiments and the length of time for the whole experiment, as repeats

would have increased reliability and eliminated anomalies within these

two sets of data. AUGC-40 produced an IC50 value of 1.905µM, a value

fairly similar to AUGC- 25 (1.734µM) which suggests that changes at these

33

carbons, even though separated by 15 carbons produced very similar

affects in terms of strength of inhibition of sphingolipid synthases.

While not included in the cell viability assays in this experiment, the IC50 of

Aureobasidin A without any chemical/structural modifications was 0.42nM

(0.00042µM) which is very significantly lower than any IC50 values of any

of the analogues tested in this experiment (Mina et al. 2009). This

illustrates that none of the analogues produced from the original

Aureobasidin A had the same inhibition strength or potency as the parent

drug, but rather showed various degrees of loss of such inhibition.

Interestingly what the analogues show however, are which carbons and

their associated R groups may be important for binding to sphingolipid

synthases. For example AUGC-30 produced the highest IC50 of 3.74µM thus

showed it was the least potent drug against T. b. brucei sphingolipid

synthases, and because the parent drug has an IC50 of 0.00042µM this

shows a staggering 8,900 decrease in AUGC-30’s affinity for the enzyme.

It is plausible to suggest then, that the 30th carbon and its associated R

groups (if any) has a key role in binding to sphingolipid synthases in

Aureobasidin A, along with other carbons which were highlighted as

having high IC50 values in analogues (AUGC-10). This can also be reversed

with analogues that produced low IC50 values such as AUGC-9 & 26, which

suggests that while there was a 1,500 fold decrease in binding affinity to

the enzyme, this is clearly significantly different to the much larger

decrease in inhibition strength found in AUGC-30 and suggests the 9 th and

26th carbons are not as important for binding. However due to data

restriction on the actual structures of the analogues tested and no

published data on the interaction of Aureobasidin A with the enzyme, this

theory is only speculation and requires further investigation.

34

In addition to Aureobasidin A used to inhibit aspects of lipid metabolism in

T. brucei, other pathways involved in lipid synthesis could be exploited

and drugs could be used to inhibit these pathways if they are essential to

T. brucei survival, such as the Kennedy and Mevalonate pathways (Smith

and Bűtikofer, 2010). The Kennedy pathway is process which is used to

produce phosphatidylcholine and phosphatidylethanolamine (PE) in T.

brucei through two separate branches of the pathway (Gibellini and Smith,

2010). The ethanolamine branch of the pathway used to produce

phosphatidylethanolamine has been discovered to be essential in

bloodstream forms of T. brucei as PE is a major component of the

trypanosome membranes (Gibellini et al. 2009). A particular enzyme in the

PE formation branch of the Kennedy pathway known as ethanolamine-

phosphate cytidylyltransferase (a cytosolic enzyme) is essential for growth

and survival of bloodstream forms of T. brucei thus could be considered a

possible drug target to inhibit (Gibellini et al. 2009). Mevalonate is a key

precursor for the production of isoprenoid groups which are incorporated

into the structures of many molecules such as sterols, ubiquinone and

dolichol (Coppens et al. 1994). This pathway can be considered for

inhibition by drugs because of the rate limiting enzyme in this pathway,

hydroxymethylglutaryl-coenzyme A reductase which is a regulatory

enzyme that controls the production of mevalonate and can be inhibited

by lipid lowering drugs already used in medicine such as simvastatin,

mevastatin and lovastatin (Field et al. 1996).

When discovering drugs to be used to treat the sub-species of T. brucei

(gambiense, rhodiesiense, brucei), important factors need to be

considered such as drug entry, toxicology and also follow (to some extent)

Lipinski's rule of five to evaluate drug activity. Drug entry for treatment of

35

T. brucei is particularly important due to the fact that the trypanosomes

will eventually penetrate the choroid plexus and enter the blood brain

barrier, thus the drug will need to be potent enough to kill the

trypanosomes during the haemolymphatic stage or must be able to cross

the blood brain barrier. The blood brain barrier is difficult for drugs to

cross due to the presence of efflux systems, cerebral blood flow and

plasma protein binding which can all alter the amount of substance the

crosses the barrier (Nau et al. 2010). Unfortunately data on drug delivery

across the blood brain barrier is limited due to the vast complexity of the

brain; however there have been approaches such as coupling drugs with

substances that can cross the blood brain barrier but this has the issue

that the barrier transporters no longer recognise the coupled molecule and

is targeted to be destroyed by lysosomes (Banks. 2009). Thus more

research will be required to understand the pharmacokinetics between the

specific drug and the blood brain barrier, as a drug that is able

simultaneously kill both haemolymphatic and meningoencephalitic stages

of T. brucei would remove the trypanosomes in the entire host rather than

just in the blood or brain.

Another factor that should be determined when underlining candidates for

drug development is Lipinski’s rule of five which describes the molecular

properties of the drug in terms of its pharmacokinetics. Lipinski’s five

states that if the drug has >5 H-bond donors, a molecular weight over 500

Daltons, >10 H-bond acceptors and has a lipophobicity LogP value over 5,

that the drug will have very poor absorption, metabolism, distribution and

excretion in the host (Lipinski et al. 2001) consequently not being a viable

drug candidate for treatment.

36

To conclude, the cell viability assays conducted in this experiment

deduced that AUGC-9 and AUGC-26 could be possible candidates for

further development for treatment of bloodstream forms of T. brucei, as

they produced the lowest IC50 values (0.635µM & 0.63µM). As

aforementioned, the IC50 values were significantly larger than the parent

drug, Aureobasidin A (0.00042µM) however they are both under 1µM and

can thus be classed as active drugs. Further experimentation would be

required such as testing the same analogues on T.b. gambiense and

T.b.rhodiesiense to determine whether any of the sub species of T. brucei

have a change in sensitivity to the derivatives. Moreover, further tests

would be needed to determine the structures of the analogues though

NMR and Fast Atom Bombardment and determine the actual changes of

each derivative which change the potency of each analogue. Drug

cytotoxicity assays can also be conducted with bloodstream forms of T. b.

brucei and the analogues; this would indicate whether AUGC-9 and AUGC-

26 are still as effective against T. brucei in the presence of mammalian

cells, which may be possible as Aureobasidin A inhibits an enzyme not

present/involved with mammalian sphingolipid synthesis (Salto et al.

2003). Should the two successful analogues be successful in the

cytotoxicity assays, the safety profile of the drug can be produced,

including the therapeutic index before the drug may enter preclinical trials

on animals and should the drug be effective against T.b. gambiense and

T.b. rhodiesiense as well, clinical trials could also be conducted to

determine suitability for treatment of Animal and Human African

Trypanosomiasis.

37

Acknowledgements I would like to express my gratitude to my project supervisor, Dr. Helen

Price for allowing me use of her laboratory to conduce the experiments, as

well as providing technical guidance and for also assisting with any

problems I encountered while writing the report. I would also like to

recognise the postgraduate student Imran Ullah for assisting me in the use

of Graphpad Prism and in producing the graphs vital for this report.

38

References

ABE, T., TAKAHASHI, S. and FUKUUCHI, Y., 2002. Reduction of Alamar Blue, a novel redox indicator, is dependent on both the glycolytic and oxidative metabolism of glucose in rat cultured neurons. Neuroscience Letters, 326(3), pp. 179-182.

AEED, P., YOUNG, C., NAGIEC, M. and ELHAMMER, A., 2009. Inhibition of Inositol Phosphorylceramide Synthase by the Cyclic Peptide Aureobasidin A. Antimicrobial Agents and Chemotherapy, 53(2), pp. 496-504.

ALESHIN, V.A., ARTIUKHOV, A.V., OPPERMANN, H., KAZANTSEV, A.V., LUKASHEV, N.V. and BUNIK, V.I., 2015. Mitochondria Impairment May Increase Cellular NAD(P)H: Resazurin Oxidoreductase Activity, Perturning the NAD(P)H-Based Viability Assays. Cells, 4(3), pp. 427-451.

BALASEGARAM, M., HARRIS, S., CHECCHI, F., GHORASHIAN, S., HAMEL, C. and KARUNAKARA, U., 2006. Melarsoprol versus eflornithine for treating late-stage Gambian trypanosomiasis in the Republic of the Congo. Bulletin of the World Health Organization, 84(10), pp. 783-791.

BALMER, O., BEADELL, J., GIBSON, W. and CACCONE, A., 2011. Phylogeography and Taxonomy of Trypanosoma brucei. PLOS, 5(2), pp. 1-11.

BANKS, W., 2009. Characteristics of compounds that cross the blood-brain barrier. BMC Neurology, 9(1), pp. 1-5.

BARRETT, M., BOYKIN, D., BRUN, R. and TIDWELL, R., 2007. Human African trpanosomiasis: pharmacological re-engagement with a neglected disease. British Journal of Pharmacology, 152, pp. 1155-1171.

BARRETT, M., VINCENT, I., BURCHMORE, R., KAZIBWE, A. and MATOVU, E., 2011. Drug resistance in human African trypanosomiasis. Future Microbiology, 6(9), pp. 1037-1047.

BARRY, J. and MCCULLOCH, R., 2001. Antigenic variation in trypanosomes: Enhanced phenotypic variation in a eukaryotic parasite. Advances in Parasitology, 49, pp. 1-70.

BIKLE, D., 2014. Vitamin D Metabolism, Mechanism of Action, and Clinical Applications. Chemistry & Biology, 21(3), pp. 319-329.

BLUM, J., SCHMID, C. and BURRI, C., 2006. Clinical aspects of 2541 patients with second stage human African trypanosomiasis`. Acta Tropica, 97(1), pp. 55-64.

BOUTEILLE, B., OUKEM, O., BISSER, S. and DUMAS, M., 2003. Treatment perspectives for human African trypanosomiasis. Fundamental & Clinical Pharmacology, 17, pp. 171-181.

39

BRUN, R., BLUM, J., CHAPPUIS, F. and BURRI, C., 2010. Human African trypanosomiasis. The Lancet, 375(9709), pp. 148-159.

BRUN, R., BLUM, J., CHAPPUIS, F. and BURRI, C., 2010. Human African trypanosomiasis. The Lancet, 375(9709), pp. 148-159.

BRUN, R., DON, R., JACOBS, R., ZHUO WANG, M. and BARRETT, M., 2011. Development of novel drugs for human African trypanosomiasis. Future Microbiology, 6(6), pp. 677-691.

BURRI, C. and BRUN, R., 2002. Eflornithine for the treatment of human African trypanosomiasis. Parasitology Research, 90, pp. S49-S52.

COPPENS, I., BASTIN, P., LEVADE, T. and COURTOY, P., 1995. Activity, pharmacological inhibition and biological regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in Trypanosoma brucei. Molecular and Biochemical Parasitology, 69(1), pp. 29-40.

CORNFORD, E., BOCASH, W., BRAUN, L., CRANE, P. and OLDENDORF, W., 1979. Rapid Distribution of Tryptophol (3-Indole Ethanol) to the Brain and Other Tissues. The Journal of Clinical Investigation, 63, pp. 1241-1248.

DEBBORGGRAEVE, S., KOFFI, M., JAMONNEAU, V., BONSU, F., QUEYSON, R., SIMARRO, P., HARDEWIJN, P. and BUSCHER, P., 2008. Molecular analysis of archived blood slides reveals an atypical human Trypanosoma infection. Diagnostic Microbiology and Infectious Disease, 61(4), pp. 428-433.

DICKEY, A., YIM, W. and FALLER, R., 2009. Using Ergosterol To Mitigate the Deleterious Effects of Ethanol on Bilayer Structure. Journal of Physical Chemistry, 113(8), pp. 2388-2397.

DOCAMPO, R. and MORENO, S., 1986. Free radical metabolism of antiparasitic agents. Federation Proceedings, 45(10), pp. 2471-2476.

DUQUE, T., SOUTO, X., ANDRADE-NETO, V., ENNES-VIDAL, V. and MENNA-BARRETO, R., 2013. Autophagic Balance Between Mammals and Protozoa: A Molecular, Biochemical and Morphological Review of Apicomplexa and Trypanosomatidae Infections. Dr. Yannick Bailly.

EZZEDINE, K., DARIE, H., LE BRAS, M. and MALVY, D., 2007. Skin Features Accompanying Imported Human African Trypanosomiasis: Hemolymphatic Trypanosoma gambiense Infection Among Two French Expatriates With Dermatologic Manifestations. International Society of Travel Medicine, 14(3), pp. 192-196.

FIELD, H., BLENCH, I., CROFT, S. and FIELD, M., 1996. Characteriasation of protein isoprenylation in procyclic form Trypanosoma brucei. Molecular and Biochemical Parasitology, 82(1), pp. 67-80.

GIBELLINI, F., HUNTER, W. and SMITH, T., 2009. The ethanolamine branch of the Kennedy pathway is essential in the bloodstream form of Trypanosoma brucei. Molecular Microbiology, 73(5), pp. 826-843.

40

GIBELLINI, F. and SMITH, T., 2010. The Kennedy pathway - De novo synthesis of phospatidylethanolamine and phosphatidylcholine. IUBMB Life, 62(6), pp. 414-428.

HOTEZ, P., MOLYNEUX, D., FENWICK, A., KUMARESAN, J., SACHS, S., SACHS, J. and SAVIOLI, L., 2007. Control of Neglected Tropical Diseases. The New England Journal of Medicine, 357, pp. 1018-1027.

IBORRA, C., DANIS, M., BRICAIRE, F. and CAUMES, E., 1999. A Traveler Returning from Central Africa with Fever and a Skin Lesion. Clinical Infectious Diseases, 28, pp. 679-680.

JACOBS, R., NARE, B. and PHILLIPS, M., 2011. State of the Art in African Trypanosome Drug Discovery. Current Topics in Medicinal Chemistry, 11(10), pp. 1255-1274.

KALLIOKOSKI, T., KRAMER, C., VULPETTI, A. and GEDECK, P., 2013. Comparibility of Mixed IC50 Data - A Statistical Analysis. PLOS ONE, 8(4), pp. N/A.

KENNEDY, P., 2008. The continuing problem of human African trypanosomiasis (sleeping sickness). Annals of Nerology, 64(2), pp. 116-126.

KENNEDY, P., 2006. Diagnostic and neuropathogenesis issues in human African trypanosomiasis. International Journal for Parasitology, 36(5), pp. 505-512.

KENNEDY, P., 2006. Human African trypanosomiasis: neurological aspects. Journal of Neurology, 253, pp. 411-416.

KENNEDY, P., 2004. Human African trypanosomiasis of the CNS: current issues and challenges. Science in Medicine, 113, pp. 496-504.

KRAFSUR, E., 2009. Tsetse flies: Genetics, evolution, and role as vectors. Infection, Genetics and Evolution, 9(1), pp. 124-141.

LANGOUSIS, G. and HILL, K., 2014. Motility and more: the flagellum of Trypanosoma brucei. Nature Reviews Microbiology, 12, pp. 505-518.

LARSON, E.M., DOUGHMAN, D.J., GREGERSON, D.S. and OBRITSCH, W.F., 1997. a new, nonradioactive, nontoxic in vitro assay to monitor corneal endothelial cell viability. Investigative Ophthalmology & Visual Science, 38(10),.

LEGROS, D., OLLIVIER, G., GASTELLU-ETCHEGORRY, M., PAQUET, C., BURRI, C., JANNIN, J. and BUSCHER, P., 2002. Treatment of human African trypanosomiasis-present situation and needs for research and development. The Lancet Infectious Diseases, 2(7), pp. 437-440.

LIPINSKI, C., LOMBARDO, F., DOMINY, B. and FEENEY, P., 2001. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Advanced Drug Delivery Reviews, 46(1-3), pp. 3-26.

41

MARTIN, A., CAMACHO, M., PORTAELS, F. and PALOMINO, J., 2003. Resazurin Microtiter Assay Plate Testing of Mycobacterium tuberculosis Susceptibilities to Second-Line Drugs: Rapid, Simple, and Inexpensive Method. Antimicrobial agents and Chemotherapy, 47(11), pp. 3616-3619.

MASOCHA, W., ROTTENBERG, M. and KRISTENSSON, K., 2007. Migration of African trypanosomes across the blood-brain barrier. Physiology & Behaviour, 92(1-2), pp. 110-114.

MESHNICK, S., BLOBSTEIN, S., GRADY, R. and CERAMI, A., 1978. An approach to the development of new drugs for African trypanosomiasis. The Journal of Experimental Medicine, 148(2), pp. 569-579.

MILHAUD, J., PONSINET, V., TAKASHI, M. and MICHELS, B., 2002. Interactions of the drug amphotericin B with phospholipid membranes containing or not ergosterol: new insight, into the role of ergosterol. Biochimica et Biophysica Acta, 1558(2), pp. 95-108.

MINA, J., PAN, S., WANSADHIPATHI, N., BRUCE, C., SHAMS-ELDIN, H., SCHWARZ, P., STEEL, P. and DENNY, P., 2009. The Trypanosoma brucei sphingolipid synthase, an essential enzyme and drug target. Molecular and Biochemical Parasitology, 168(1), pp. 16-23.

NAU, R., SORGEL, F. and EIFFERT, H., 2010. Penetration of Drugs through the Blood-cerebrospinal Fluid/ Blood-Brain Barrier for Treatment of Central Nervous System Infections. Clinical Microbiology Reviews, 23(4), pp. 858`-883.

O'BRIEN, J., WILSON, I., ORTON, T. and POZNAN, F., 2000. Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytoxicity. Eur. J. Biochem, 267, pp. 5421-5426.

PEPIN, J., GUERN, C., MILORD, F. and SCHECHTER, P., 1987. Difluoromethylornithine for arseno-resistant Trypanosoma brucei gambiense sleeping sickness. The Lancet, 330(8573), pp. 1431-1433.

PRATT, S., WANSADHIPATHI-KANNANGARA, N.K., BRUCE, C.R., MINA, J.G., SHAMS-ELDIN, H., CASAS, J., CANADA, K., SCHWARZ, R.T., SONDRA, S. and DENNY, P.W., 2013. Sphingolipid synthesis and scavenging in the intracellular apicomplexan parasite, Toxoplasma gondii. Molecular and Biochemical Parasitology, 187(1), pp. 43-51.

PRIOTTO, G., KASPARIAN, S., MUTOMBO, W., NGOUAMA, D., GHORASHIAN, S., ARNOLD, U., GHABRI, S., BAUDIN, E., BUARD, V., KAZADI-KYANZA, S., ILUNGA, M., MUTANGALA, W., POHLIG, V., SCHMID, C., KARUNAKARA, U., TORREELE, E. and KANDE, V., 2009. Nifurtimox-eflornithine combination therapy for second-stage African Trypanosoma brucei gambiense trypanosomiasis: a multicentre, randomised, phase III, non-inferiority trial. The Lancet, 374(9683), pp. 56-64.

42

SALTO, M.L., BERTELLO, L.E., VIEIRA, M., DOCAMPO, R., MORENO, S.N.J. and LEDERKREMER, R.M., 2003. Formation and Remodelling of Inositiolphosphoceramide during Differentiation of Trypanosoma cruzi from Trypomastigote to Amastigote. Eukaryotic Cell, 2(4), pp. 756-768.

SEED, J., SEED, T. and SECHELSKI, J., 1978. The Biological effects of tryptophol (indole-3-ethanol): Hemolytic, biochemical and behaviour modifying activity. Comparative Biochemistry and Physiology Part C: Comparative Pharmacology, 60(2), pp. 175-185.

SEVOVA, E.S., GOREN, M.A., SCHWARTZ, K.J., HAU, F., TURK, J., FOX, B.G. and BANGS, J.D., 2010. Cell-free Synthesis and Functional Characterization of Sphingolipid Synthases from Parasitic Trypanosomatid Protozoa. Journal of Biological Chemistry, 285, pp. 20580-20587.

SIMARRO, P., CECCHI, G., PAONE, M., FRANCO, J., DIARRA, A., RUIZ, J., FEVRE, E., COURTIN, F., MATTIOLI, R. and JANNIN, J., 2010. The Atlas of human African trpanosomiasis: a contribution to global mapping of neglected tropical diseases. International Journal of Health Geographics, 9(57), pp. 1-18.

SIMARRO, P., JANNIN, J. and CATTAND, P., 2008. Eliminating Human African Trypanosomiasis: Where Do We Stand and What Comes Next? PLOS, 5(2), pp. 174-180.

SMITH, D., PEPIN, J. and STICH, A., 1998. Human African trypanosomiasis: an emerging public health crisis. British Medical Bulletin, 54(2), pp. 341-355.

SMITH, T. and BUTIKOFER, P., 2010. Lipid metabolism in Trypanosoma brucei. Molecular and Biochemical Parasitology, 172(2), pp. 66-79.

STERNBERG, J., 2004. Human African trypanosomiasis: clinical presentation and immune response. Parasite Immunology, 26, pp. 469-476.

STUART, B., BRUN, R., CROFT, S., FAIRLAMB, A., GURTLER, R.E., MCKERROW, J., REED, S. and TARLETON, R., 2008. Kinetoplastids: related protozoan pathogens, different diseases. The Journal of Clinical Investigation, 118(4), pp. 1301-1310.

TAN, H.W. and TAY, S.T., 2013. The inhibitory effects of aureobasidin A on Candida planktonic and biofilm cells. Mycoses, 56(2), pp. 150-156.

TANAKA, A.K., VALERO, V.B., TAKAHASHI, H.K. and STRAUS, A.H., 2007. Inhibition of Leishmania (Leishmania) amazonensis growth and infectivity by aureobasidin A. Journal of Antimicrobial Chemotherapy, 59(3), pp. 487-492.

TROEBERG, L., CHEN, X., FLAHERTY, T., MORTY, R., CHENG, M., HUA, H., SPRINGER, C., MCKERROW, J., KENYON, G., LONSDALE-ECCLES, J.,

43

COETZER, T. and COHEN, F., 2000. Chalcone, Acyl Hydrazide, and Related Amides Kill Cultured Trypanosoma brucei brucei. Molecular Medicine, 6(8), pp. 660-669.

TROISI, F., PIERRO, T., GAETA, C., CARRATU, M. and NERI, P., 2009. Appending aromatic moieties at the para- and meta- position of calixarene phenol rings via p-bromodienone route. Tetrahedron Letters, 50(31), pp. 4416-4419.

UMEGAWA, Y., NAKAGAWA, Y., TAHARA, K., TSUCHIKAWA, H., MATSUMORI, N., OISHI, T. and MURATA, M., 2012. Head-to-tail Interaction between Amphotericin B and Ergosterol Occurs in Hydrated Phospholipid Membrane. ACS Publications, 51(1), pp. 83-89.

VICKERMAN, K., 1985. Developmental Cycles and Biology of Pathogenic Trypanosomes. British Medical Bulletin, 42(2), pp. 105-114.

WANG, C., 1995. Molecular mechanism and therapeutic approaches to the treatment of African trypanosomiasis. Annual Review of Pharmacology and Toxicology, 35, pp. 93-127.

WILSON, M., CALLENS, M., KUNTZ, D., PERIE, J. and OPPERDOES, F., 1993. Synthesis and activity of inhibitors highly specific for the glycolytic enzymes from Trypanosoma brucei. Molecular and Biochemical Parasitology, 59(2), pp. 201-210.

ZHAO, Z., LINDSAY, M.E., CHOWDHURY, A., ROBINSON, D. and ENGLUND, P., 2008. p166, a link between the typanosome mitochondriak DNA and flagellum, mediates genome segregation. The EMBO Journal, 27(1), pp. 143-154.

ZHONG, W., MURPHY, D.J. and GEORGOPAPADAKOU, N.H., 1999. Inhibition of yeast inositol phosphorylceramide synthase by aureobasidin A measured by a fluorometric assay. FEBS Letters, 463(3), pp. 241-244.