Download - DRAFT DISSERTATION4
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
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Contents
Abstract..................................................................................................2
Introduction..........................................................................................2
Materials and Methods....................................................................14
Results..................................................................................................17
Discussion...........................................................................................26
Acknowledgements..........................................................................33
References..............................................................................................34
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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
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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
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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).
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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
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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.
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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).
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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
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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
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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
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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).
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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.
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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
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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.
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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.
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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
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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
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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.
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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
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