molecules

Review
Challenges in Chagas Disease Drug Development
Amanda F. Francisco 1 , Shiromani Jayawardhana 1 , Francisco Olmo 1 , Michael D. Lewis 1 ,
Shane R. Wilkinson 2 , Martin C. Taylor 1 and John M. Kelly 1, *
1 Department of Infection Biology, London School of Hygiene and Tropical Medicine Keppel Street,
London WC1E 7HT, UK; amanda.francisco@lshtm.ac.uk (A.F.F.);
shiromani.jayawardhana@lshtm.ac.uk (S.J.); francisco.olmo@lshtm.ac.uk (F.O.);
Michael.lewis@lshtm.ac.uk (M.D.L.); martin.taylor@lshtm.ac.uk (M.C.T.)
2 School of Biological and Chemical Sciences, Queen Mary University of London Mile End Road,
London E1 4NS, UK; s.r.wilkinson@qmul.ac.uk
* Correspondence: john.kelly@lshtm.ac.uk

Academic Editors: Andrew Tsotinis and Ioannis Papanastasiou




Received: 22 May 2020; Accepted: 12 June 2020; Published: 17 June 2020

Abstract: The protozoan parasite Trypanosoma cruzi causes Chagas disease, an important public health
problem throughout Latin America. Current therapeutic options are characterised by limited efficacy,
long treatment regimens and frequent toxic side-effects. Advances in this area have been compromised
by gaps in our knowledge of disease pathogenesis, parasite biology and drug activity. Nevertheless,
several factors have come together to create a more optimistic scenario. Drug-based research has
become more systematic, with increased collaborations between the academic and commercial sectors,
often within the framework of not-for-profit consortia. High-throughput screening of compound
libraries is being widely applied, and new technical advances are helping to streamline the drug
development pipeline. In addition, drug repurposing and optimisation of current treatment regimens,
informed by laboratory research, are providing a basis for new clinical trials. Here, we will provide
an overview of the current status of Chagas disease drug development, highlight those areas where
progress can be expected, and describe how fundamental research is helping to underpin the process.

Keywords: Trypanosoma cruzi; Chagas disease; drug development

1. Introduction
Chagas disease is endemic in many areas of Latin America, and is a particular problem
amongst the rural poor, where an estimated 6–7 million people are infected with the causative
agent, the protozoan parasite Trypanosoma cruzi [1]. The disease has also become a global health
problem, with several hundred thousand infected individuals within migrant populations, mainly in
the USA and Europe [2,3]. The principal route of T. cruzi infection is via hematophagous triatomine
bugs, although oral (contaminated food and drink) and congenital transmission are also important,
along with blood transfusion and organ transplantation. Encouragingly, public health measures,
specifically insecticide spraying of poor-quality housing, have had a significant impact in breaking
transmission cycles in some areas [4,5], but the infection is a zoonosis, and these measures will have to
be sustained to maintain this improved situation. Eradication by this route is unlikely to be feasible.
There are no vaccines against Chagas disease, and given the immunological complexity and long-term
nature of the infection, progress in this area is uncertain. Therefore, new drugs, which avoid the
drawbacks associated with the current therapeutic options, will have the potential to play a significant
role in eliminating the massive disease burden that results from this infection, and in reducing the
resulting constraints on the socio-economic development of many rural communities. The economic
impact associated with Chagas disease is more than $7 billion per annum, a figure exceeding the total

Molecules 2020, 25, 2799; doi:10.3390/molecules25122799 www.mdpi.com/journal/molecules

Molecules 2020, 25, 2799 2 of 14

global costs linked to uterine, cervical and oral cancers [6,7]. For further comparison, the estimated
productivity gains from elimination of lymphatic filariasis, schistosomiasis and soil-transmitted
helminths from the Americas would amount to approximately $0.5 billion per annum [8].
T. cruzi is an obligate intracellular parasite with an extremely wide mammalian host range, and an
ability to infect the vast majority of nucleated cells. Typically, transmission occurs when infected faeces
from the insect vector are deposited near the bite wound after a blood-meal. Flagellated metacyclic
trypomastigote forms of the parasite are then introduced via the wound or mucous membranes, by
scratching or rubbing. Following host cell invasion, trypomastigotes escape from the parasitophorous
vacuole into the cytoplasm, differentiate into the small rounded amastigote form, and replicate by binary
fission. Four to five days later, after several rounds of division, and differentiation into bloodstream
trypomastigotes, lysis of the host cell results in parasite release and dissemination of the infection.
In humans, the initial acute stage of the disease lasts 4–6 weeks, and is associated with patent
parasitemia and infection of most tissues and organs. However, symptoms are usually mild and
non-specific, with transient fever and muscle pain; the majority of individuals are unaware that they
have been infected. In some cases, the disease can be more serious, particularly in children, where death
can result from myocarditis or encephalopathy. Control of the infection is mediated predominantly by
a strong CD8 + T cell response, which reduces the parasite burden by 2–3 orders of magnitude [9,10],
although sterile immunity is rarely achieved. The disease transitions to an asymptomatic chronic stage,
where the parasite burden is low and focal. Despite the life-long nature of the infection, the majority
of individuals do not develop overt pathology, although a significant minority (~30%) progress to
a symptomatic chronic state characterised by progressive cardiac and/or digestive disease. In most
cases, this takes decades to become apparent. Cardiomyopathy is the most serious outcome of T. cruzi
infection [11,12], and in many areas of South America it is a major cause of heart disease. The digestive
symptoms, which include megaoesophagus and megacolon, also have serious consequences and can
require surgery to alleviate the symptoms [13].

2. The Current Status of Chagas Disease Chemotherapy

For the last 50 years, the orally-administered compounds benznidazole and nifurtimox have
remained the only drugs available to treat T. cruzi infections [14,15]. However, they require long-term
periods of administration (typically 60 days), are often noncurative (generally in the range 10–30% of
cases), toxicity is a significant problem, and use during pregnancy is contraindicated [16–18]. Side
effects are reported in up to 90% of patients, with cutaneous, digestive and neurological complications
being the most common [19]. As a result, patient compliance can be a major issue. Furthermore,
because only a minority of cases are diagnosed in the acute or asymptomatic chronic stages, the number
of T. cruzi-infected individuals offered antiparasitic drug treatment is relatively small [20,21].
Benznidazole and nifurtimox are nitroheterocyclic compounds, containing a nitro group attached
to imidazole and furan rings, respectively (Figure 1A,B). They function as prodrugs and are
bioactivated within the parasite by the same mitochondrial-localised flavin-dependent enzyme,
the type 1 nitroreductase TcNTR-1 [22,23]. It is the substrate-specificity of TcNTR-1 and the lack
of a corresponding enzyme in the mammalian host that accounts for the selectivity of drug action.
Reductive drug metabolism generates a series of reactive intermediates that have trypanocidal
activity, most notably glyoxal in the case of benznidazole [24], and an unsaturated open-chain
nitrile with nifurtimox [25] (Figure 1A,B). For benznidazole, drug-induced mutagenesis has been
identified as a possible mode of action, resulting, for example, in disruption to DNA-repair mechanisms,
and chromosome instability [26–30]. Possible inducers of mutagenesis include reactive drug metabolites,
enhanced oxidative stress resulting from drug adduct interactions with trypanothione, and the
production of 8-oxo-guanine and other oxidised nucleotides.
Molecules 2020, 25, 2799 3 of 14
Molecules 2020, 25, x FOR PEER REVIEW 3 of 14

Figure Nitroaromatic
1. 1.
Figure Nitroaromaticdrugsdrugsused
used to treat Trypanosoma
to treat cruziinfections,
Trypanosoma cruzi infections,ororundergoing
undergoing clinical
clinical trial.
trial.
(A)(A) Reductive
Reductive metabolism
metabolism of benznidazole,
of benznidazole, initiated
initiated by TcNTR-1,
by TcNTR-1, leadsleads
to thetoproduction
the production of an
of an unstable
unstable hydroxylamine
hydroxylamine derivative.derivative. This is
This is readily readily converted
converted to a hydroxyto a intermediate
hydroxy intermediate
(possibly(possibly
through a
through aion
nitrenium nitrenium ion form),
form), which thenwhich
reactsthen
withreacts
waterwith
towater to generate
generate a dihydro-dihydrooxy.
a dihydro-dihydrooxy. This
This slowly
slowly
breaks breaks
down down the
to release to release the highly
highly reactive reactive dialdehyde,
dialdehyde, glyoxal
glyoxal (circled (circled
in red) [24]. in red)
The [24]. The
intermediaries
andintermediaries
final productand canfinal
formproduct
adductscanwith
form proteins,
adducts withDNA,proteins, DNA,molecules
and small and small such
molecules such as
as glutathione
glutathione and trypanothione. (B) Nifurtimox is reduced by TcNTR-1, leading
and trypanothione. (B) Nifurtimox is reduced by TcNTR-1, leading to the generation of an unstable to the generation of
an unstable hydroxylamine. This decomposes, potentially via a ketoxime intermediate,
hydroxylamine. This decomposes, potentially via a ketoxime intermediate, to form unsaturated (circled to form
unsaturated
in red) and then(circled
saturated in open-chain
red) and then saturated
nitriles [25]. Theopen-chain
unsaturated nitriles
form [25]. The trypanocidal
mediates unsaturated activity.
form
mediates trypanocidal activity. (C) Fexinidazole, an NTR-1-activated 5-nitroimidazole
(C) Fexinidazole, an NTR-1-activated 5-nitroimidazole prodrug [31], has recently been approved prodrug [31], as
has recently been approved as an oral treatment for African trypanosomiasis [32,33]. It outperforms
an oral treatment for African trypanosomiasis [32,33]. It outperforms other nitroaromatic drugs as
other nitroaromatic drugs as a curative treatment for experimental T. cruzi infections [34], and is
a curative treatment for experimental T. cruzi infections [34], and is undergoing clinical trial against
undergoing clinical trial against Chagas disease [35].
Chagas disease [35].

Cross-resistance to both benznidazole and nifurtimox can be readily generated in vitro, often
Cross-resistance to both benznidazole and nifurtimox can be readily generated in vitro, often
resulting from reduced expression of functional TcNTR-1 that impacts on the ability of the parasite
resulting from reduced expression of functional TcNTR-1 that impacts on the ability of the parasite
to reduce nitro-drugs [22,36,37]. Given the relatively small numbers of patients who are actually
to reduce nitro-drugs [22,36,37]. Given the relatively small numbers of patients who are actually
drug-treated [20,21], the infrequency with which genetic exchange occurs in T. cruzi, and the
drug-treated [20,21], the infrequency with which genetic exchange occurs in T. cruzi, and the extensive
extensive animal reservoir of the parasite, it is unlikely that acquired drug-resistance will develop or
animal reservoir of the parasite, it is unlikely that acquired drug-resistance will develop or spread
spread widely at a population level. However, natural parasite populations do display a broad range
widely at a population
of susceptibility level. However,
to benznidazole natural
[36,38,39], a factorparasite
that couldpopulations
have a roledo display
in some a broadfailures.
treatment range of
susceptibility to benznidazole [36,38,39], a factor that could have a role in some
The level of sensitivity in natural strains is not obviously associated with specific parasite lineages,treatment failures.
The level of sensitivity in natural strains is not obviously associated with
and not linked to polymorphisms in the TcNTR-1 gene, suggesting that other processes could be specific parasite lineages,
and not linked
involved. Theseto might
polymorphisms in the TcNTR-1
include alternative gene, suggesting
drug-activation enzymes [40],that other processes
enhancement could be
of oxidative
involved. These might include alternative drug-activation enzymes [40], enhancement
defence [41] or DNA repair pathways [29], increased drug efflux [42] or decreased drug uptake. In of oxidative
defence [41]it or
addition, hasDNA repair pathways
been observed that within[29], increased
infected drug numbers
cells, small efflux [42] or decreased
of the amastigote drug uptake.
population
In can
addition,
enter it has been
a state observeddormancy
of apparent that withinin infected
which they cells, small increased
display numbers of the tolerance
drug amastigote population
[43]. In the
canabsence
enter aofstate
newof treatments,
apparentitdormancy
is importantin that
whichthese
theyalternative mechanisms
display increased are tolerance
drug explored further
[43]. Intothe
optimise the usage of the current drugs.
absence of new treatments, it is important that these alternative mechanisms are explored further to
optimise Chronic
the usagechagasic
of theheart
currentdisease
drugs.is characterised by inflammation, fibrosis, blood clots and
arrhythmias, which lead
Chronic chagasic to progressive
heart disease is cardiac failure, and
characterised in some cases sudden
by inflammation, fibrosis,death [44,45].
blood clots It is
and
now generally accepted that the presence of the parasite is a prerequisite for driving
arrhythmias, which lead to progressive cardiac failure, and in some cases sudden death [44,45]. It is the development
nowof generally
this pathology [46,47],
accepted thatalthough
the presence persistent
of the infection
parasite isofa the target organs
prerequisite may not
for driving thebedevelopment
essential.
Rather, cumulative collateral damage could result from localised inflammatory immune
of this pathology [46,47], although persistent infection of the target organs may not be essential. Rather, responses
generated against continuous rounds of reinfection by parasites trafficked from other more
cumulative collateral damage could result from localised inflammatory immune responses generated
immunotolerant sites of persistence. Importantly, the central role played by the parasite in disease
against continuous rounds of reinfection by parasites trafficked from other more immunotolerant
pathogenesis strongly implies that effective therapy should block or reduce the development of
sites of persistence. Importantly, the central role played by the parasite in disease pathogenesis
Molecules 2020, 25, 2799 4 of 14

strongly implies that effective therapy should block or reduce the development of pathology. Evidence
supporting this has come from several reports on the beneficial effects of curative treatment of acute
stage infections in experimental models [48–52]. Studies, using highly sensitive in vivo imaging, further
suggest that the beneficial outcomes, in terms of protection against cardiac pathology, may be lessened
if treatment is withheld until chronic stage symptoms develop [53]. In humans, the evidence suggests
that curative treatment of acute-stage infections also provides long-term therapeutic efficacy [54], with
a gradual diminution of benefit if treatment is delayed. Much of the cardiac damage in chronic patients
seems to be irreversible once it has developed. For example, in the BENEFIT study, no significant
improvements in cardiac function were observed in chronic chagasic patients 5 years after benznidazole
treatment [55]. In this clinical trial, volunteers had been preselected on the basis that they already
displayed cardiac disease, a decision that has provoked some discussion [56–58]. Taken together,
the current evidence supports the premise that early curative treatment of T. cruzi infections will have
optimal benefit in preventing the development of symptomatic cardiac pathology and other outcomes.
More recently, the BENDITA phase II clinical trial, carried out in Bolivia under the auspices
of the Drugs for Neglected Diseases initiative (DNDi), has investigated the impact of reducing the
benznidazole treatment period from 8 weeks to 2 weeks, and the dose from 300 mg/day to 150 mg [59].
In both cases, the curative rate (~80%) was similar to that in those who received the standard regimen,
and was accompanied by a reduction in the number of patients who discontinued treatment because
of drug toxicity. Although these findings are preliminary, they highlight the potential for minimising
the side effects of benznidazole, maintaining antiparasitic efficacy and removing some of the barriers
to successful treatment.

3. Progress in Chagas Disease Drug Development

Both target-based and phenotypic screening approaches have been widely applied to Chagas
disease drug-discovery. In the case of the former, there are an increasing number of enzymes
and metabolic pathways that have been genetically and/or chemically validated, and these have
been the focus of much research. For example, in T. cruzi, as in fungi, ergosterol is a major and
essential component of cell membranes, and inhibitors of its biosynthetic pathway have been prime
candidates for drug development [60,61]. Azoles, which act through inhibition of lanosterol 14-α
demethylase (CYP51), are effective antifungal agents [62], and attempts to repurpose several of
these for use against T. cruzi infections have generated considerable interest. Most prominently,
the antifungal drug posaconazole, a highly efficient inhibitor T. cruzi CYP51, showed great promise
in preliminary studies and was advanced into clinical trial. However, it was found to have limited
curative potential as a monotherapy, and provided no added benefit when used in combination with
benznidazole [63,64]. Similarly, the related triazole ravuconazole also performed sub-optimally in
clinical trial [65]. These failures were a great disappointment to the Chagas disease research community.
More recent studies have demonstrated that although posaconazole has an in vitro EC50 in the low
nanomolar range, a subpopulation of parasites seem refractory to drug activity, a phenomenon that
shows great variability between strains [66]. Furthermore, in vivo experiments have revealed that
posaconazole has limited ability to confer sterile cure on murine models, despite an initial pronounced
knockdown in the parasite burden [67,68]. Understanding why cytostatic drugs like posanconazole
struggle to eliminate T. cruzi infections will be critical to inform the drug development process. An
unexpectedly high number of “hits” obtained from phenotypic screens have been found to be inhibitors
of CYP51, perhaps reflecting features of the active site of the enzyme [69]. Given the failure of
posaconazole in clinical trials and a desire to focus on alternative targets, small molecule ligands with
CYP51 inhibitory properties are now actively excluded from further progress along drug development
pipelines [70].
Proteases have been successful drug targets in several pathogens, and members of the
cruzipain family of T. cruzi cysteine proteases have been intensively studied in this context [71,72].
Cruzipain functions in a wide variety of roles throughout the parasite life-cycle, including host cell
Molecules 2020, 25, 2799 5 of 14

invasion, differentiation, evasion of the immune response, and in several aspects of host–parasite
interaction [73–76]. The enzyme family has been subject to intense biochemical and structural scrutiny,
with inhibitors from several chemical classes having been shown to have potent trypanocidal properties
and promising in vivo activity, with treatment outcomes that include parasitological cure and reduction
of cardiac pathology [77–79]. Although none of these have yet progressed to clinical trial, parasite
cysteine proteases remain a research area with some promise. The parasite oxidative defence pathway
has also been an important focus of drug-related research. Many components and pathways display
parasite-specific features and have been genetically validated [80,81]. For example, the unique thiol
trypanothione (a glutathione:spermidine conjugate) plays a central role in maintaining redox balance
within the parasite [82–84]. The key enzyme trypanothione reductase (TR) has been widely targeted for
drug design, a process that has benefitted considerably from the availability of a crystal structure [85].
Other potential drug targets that have been investigated in T. cruzi and related parasites include
methionyl-tRNA synthetase [86] and enzymes involved in glycoconjugate biosynthesis [87].
Despite the widespread application of rational drug design, few new compounds identified
by this approach have yet advanced far along the Chagas disease drug development pipeline.
This has renewed interest in exploring the potential of other nitroaromatics that might have superior
properties to benznidazole and nifurtimox, even though they may share a common mechanism
of bioactivation, mediated by TcNTR-1. The recent approval of fexinidazole (Figure 1C) by the
European Medicines Agency as an oral treatment for African trypanosomiasis caused by Trypanosoma
brucei gambiense [32,33] has further strengthened this interest. Fexinidazole, an NTR-1-activated
5-nitroimidazole pro-drug [31], has been shown to outperform both benznidazole and nifurtimox
as a curative treatment for experimental T. cruzi infections [34] (Figure 2A,B, as example), and a
clinical trial to assess efficacy and tolerability against patients with asymptomatic chronic infections
has been undertaken [35]. In addition, there have also been a number of recent studies reporting
encouraging preliminary data on the antiparasite activities of other novel nitroaromatics [88], and of a
new generation of diverse organometallic compounds [89–92].
Probably the most prominent recent development in Chagas disease drug research has been the
increased use of high-throughput phenotypic screening [93–95]. The requirement for large compound
libraries, robotic sample handling equipment, expertise in parasite biology, and over-arching funding
mechanisms has brought together both the academic and commercial sectors, with not-for-profit drug
development agencies, such as the DNDi. These consortia are international in make-up and encompass
large research teams and networks with expertise in medicinal chemistry, pharmacology, toxicology,
molecular biology, biochemistry, and clinical sciences. Examples of such collaborative partnerships
include the GSK and Tres Cantos Open Lab Foundation; initiatives such as the kinetoplastid boxes, each
with ~ 200 chemical hits, which have been made freely available to the research community [96]; and the
Novartis-led project that identified the parasite proteasome inhibitor GNF6702, which has broad
spectrum anti-kinetoplastid activity [97]. This breakdown of drug-development into its component
parts has brought a more systematic approach to the process and has offered economies of scale that
are particularly important when the research area is drugs for neglected diseases.
The failure of posaconazole in clinical trial identified the urgent need for improved preclinical
methodologies with greater predictive power. One response has been the development of long-term
washout experiments, which demonstrated that even with prolonged posaconazole exposure
in vitro [98], there is an inability to kill all parasites. This is indicative of phenotypic heterogeneity
within the population, and perhaps the presence of a metabolically quiescent sub-group that is more
resistant to drug activity [43]. These findings further highlight where gaps in our understanding of
parasite biology can impact negatively on drug development. For the remainder of this review, we
will discuss how improvements in imaging procedures applied to predictive murine models have
allowed some of these issues to be addressed, and have helped to streamline aspects of the drug
screening process.
Molecules 2020, 25, 2799 6 of 14
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Figure
Figure 2. 2. Fexinidazoleoutperforms
Fexinidazole outperforms benznidazole
benznidazole andand nifurtimox
nifurtimoxas asaatreatment
treatmentfor forexperimental
experimental
Trypanosoma
Trypanosoma cruzi
cruzi infections.(A)
infections. (A)BALB/c
BALB/cmicemice were
were infected
infected with
with bioluminescent
bioluminescentT.T.cruzicruziCL
CLBrener
Brener
strain
strain [99].
[99]. AtAtthethechronic
chronicstage
stage of infection
infection(124
(124days),
days),they were
they treated
were with
treated benznidazole
with benznidazole(BNZ) or
(BNZ)
fexinidazole (FXN)
or fexinidazole (FXN)(30 (30mg/kg,
mg/kg, orally, once
orally, oncedaily) for 5for
daily) days (marked
5 days by redby
(marked arrow). TreatedTreated
red arrow). mice weremice
immunosuppressed
were immunosuppressed on days 138, 142
on days 138,and
142146
andusing cyclophosphamide
146 using (200 mg/kg,
cyclophosphamide (200 i.p) (red i.p)
mg/kg, lines).
(red
Images were acquired using the Lumina II IVIS system (Caliper Life Science) [100].
lines). Images were acquired using the Lumina II IVIS system (Caliper Life Science) [100]. (B) BALB/c (B) BALB/c mice
at the
mice acute
at the stage
acute of infection
stage (15 days)
of infection were treated
(15 days) with nifurtimox
were treated (NFX) or
with nifurtimox FXN (100
(NFX) mg/kg,
or FXN (100orally,
mg/kg,
once daily) for 10 days (red arrow), and then immunosuppressed on
orally, once daily) for 10 days (red arrow), and then immunosuppressed on days 35, 39 and days 35, 39 and 4343using
using
cyclophosphamide
cyclophosphamide (red
(red line).UT,
line). UT,untreated
untreatedcontrol
controlmice.
mice. Heat-maps
Heat-mapsare areon
onlog10
log10scales
scalesand
and indicate
indicate
intensity of bioluminescence from low (blue) to high (red). Full data set available in reference [34].
intensity of bioluminescence from low (blue) to high (red). Full data set available in reference [34].

4. Highly Sensitive
4. Highly InIn
Sensitive Vivo Bioluminescence
Vivo BioluminescenceImaging
Imagingand
and Its
Its Application to the
Application to the Chagas
ChagasDisease
Disease
Drug
DrugDevelopment
Development Pipeline
Pipeline
Mice have
Mice have been
been widely
widelyusedusedas asexperimental
experimental models in in Chagas
Chagasdisease
diseasedrug
drugresearch.
research. However,
However,
during
during thethe
chronic
chronicstage stageofofinfection,
infection,which
which is
is the
the main
main target for new
target for new therapeutics,
therapeutics,the theparasite
parasite
burden
burden is extremely
is extremely low,
low, thesites
the sitesofofinfection
infectionare
arehighly
highly localised,
localised, and
andimaging
imagingsuggests
suggeststhat these
that these
focifoci
areare oftentransient
often transient[100].
[100]. As
Asaaresult,
result,neither
neitherlight microscopy
light microscopy nor nor
PCR-based
PCR-basedmethodologies can
methodologies
canbebeused
usedto reliably
to reliably monitor
monitorin vivoininfections in real in
vivo infections time, and
real evenand
time, end-point assays can assays
even end-point be uncertain
can be
uncertain [67]. However, it is now clear that non-invasive bioluminescence imaging of micewith
[67]. However, it is now clear that non-invasive bioluminescence imaging of mice infected T.
infected
cruzi
with that that
T. cruzi stably express
stably a firefly
express luciferase
a firefly gene
luciferase cancan
gene provide
providea method
a method forfor
following
following chronic
chronic
infections, if sufficient sensitivity can be achieved [99–101]. The major determinants
infections, if sufficient sensitivity can be achieved [99–101]. The major determinants of sensitivity of sensitivity in in
these situations are the level of expression of the luciferase enzyme, and the wavelength of light
these situations are the level of expression of the luciferase enzyme, and the wavelength of light emitted
emitted by oxidation of the luciferin substrate. Since this is an ATP-dependent reaction, only live
by oxidation of the luciferin substrate. Since this is an ATP-dependent reaction, only live parasites
parasites are detected, which is not necessarily the case with PCR. Insertion of a luciferase gene into
are detected, which is not necessarily the case with PCR. Insertion of a luciferase gene into highly
highly expressed ribosomal loci of the parasite solved the first of these issues, and the use of a
expressed ribosomal loci of the parasite solved the first of these issues, and the use of a genetically
genetically engineered codon-optimised red-shifted luciferase [102] addressed the second. Visible
engineered codon-optimised red-shifted luciferase [102] addressed the second. Visible light towards
light towards the red end of the spectrum (617 nm in this case) has greater tissue penetration due to
thereductions
red end ofinthe thespectrum
absorbance (617
andnm in this case)
scattering of thehas greater
emitted tissue
light. penetration
Using duechronic
these systems, to reductions
T. cruzi in
theinfections
absorbance in mice can be monitored for more than one year, with a limit of detection close to 100 in
and scattering of the emitted light. Using these systems, chronic T. cruzi infections
mice can be monitored for more than one year, with a limit of detection close to 100 parasites using
Molecules 2020, 25, 2799 7 of 14
Molecules 2020, 25, x FOR PEER REVIEW 7 of 14

in vivo imaging
parasites [100],
using andimaging
in vivo less than 20 parasites
[100], and less thanwhen 20ex vivo imaging
parasites when ex is subsequently
vivo imaging isused to examine
subsequently
mouse
usedtissue
to examine With in
[103].mouse vivo [103].
tissue imaging,
Withthere is imaging,
in vivo a linear relationship
there is a linearbetween the parasite
relationship between burden
the
(above 1000burden
parasite parasites) and1000
(above whole-body
parasites)bioluminescence
and whole-bodyintensity [99]. In addition
bioluminescence intensity to a central
[99]. role in
In addition
to a drug
in vivo central role in in(Figure
screening vivo drug screening
2), these (Figure
imaging 2), these have
procedures imaging procedures
provided have provided
new insights new
into infection
insightsand
dynamics intotissue
infection dynamics
tropism and tissue
[100,101,104], tropism
disease [100,101,104],
pathogenesis [53] disease pathogenesis
and vaccine development [53] and
[105].
vaccine development [105].
The standard experimental model used for drug testing is the BALB/c mouse infected with
The standard T.
the bioluminescent experimental
cruzi CL Brener model strain,
used fora drug testing
parasite is the
that BALB/c
belongs to mouse infected
the discrete with the
typing unit
bioluminescent T. cruzi CL Brener strain, a parasite that belongs to the
(DTU) VI lineage. In terms of replication rate and virulence, the genetically modified strain is discrete typing unit (DTU) VI
lineage. In termsfrom
indistinguishable of replication rate and
the parental linevirulence,
[100]. The theacute
genetically
stagemodified
infectionstrain is indistinguishable
follows a regular profile
from the parental line [100]. The acute stage infection follows a regular profile in which the
in which the bioluminescence-inferred parasite burden reaches a peak at approximately 14 days
bioluminescence-inferred parasite burden reaches a peak at approximately 14 days post-infection
post-infection (Figure 2B). At this time-point, it is brought under control by the adaptive immune
(Figure 2B). At this time-point, it is brought under control by the adaptive immune system, resulting
system, resulting in a drop of up to 1000-fold in parasite numbers as the infection transitions to the
in a drop of up to 1000-fold in parasite numbers as the infection transitions to the chronic stage,
chronic stage, approximately 50–60 days post-infection [100]. During the acute stage, parasites infect
approximately 50–60 days post-infection [100]. During the acute stage, parasites infect all organs and
all organs and tissues, whereas in the chronic stage, they are mainly confined to the colon, stomach
tissues, whereas in the chronic stage, they are mainly confined to the colon, stomach and skin (Figure
and3A).skinIn(Figure
C3H/HeN 3A).mice,
In C3H/HeN
infection mice, infection
of skeletal muscleof skeletal
is also amuscle
regularisfeature
also a regular featurestage
of the chronic of the
chronic stageAs
[101,103]. [101,103].
in humans, AsT. incruzi
humans, T. cruzi
infections are infections are generally
generally life-long, and inlife-long, and has
vivo imaging in vivo imaging
revealed a
hashighly
revealed a highly dynamic profile during the chronic stage in which transient
dynamic profile during the chronic stage in which transient bioluminescence foci appear and bioluminescence
focidisappear
appear and overdisappear
a time period overof a time
hours.period of hours.
The precise Theofprecise
nature nature
these foci of these foci
is unknown, but is unknown,
they could
butrepresent
they could represent
infected infectedinphagocytes
phagocytes the processin ofthe process
being of being
trafficked fromtrafficked from sitesinofthe
sites of persistence persistence
mouse
in the mouse to sites
to peripheral peripheral
[47]. sites [47].

Figure 3. Monitoring
Figure 3. Monitoring parasite
parasitetropism
tropismand
anddrugdrug activity Trypanosomacruzi
chronicTrypanosoma
activity in chronic cruziinfections
infections using
using
ex vivo imaging. (A) BALB/c mice were
ex vivo imaging. (A) BALB/c mice were infected infected with the bioluminescent T. cruzi CL Brener
the bioluminescent T. cruzi CL Brener strain. strain.
Animals
Animals were sacrificed
were at various
sacrificed at various points
pointsthereafter, and
thereafter, and organs
organsand andtissue
tissuewere
wereremoved,
removed,arranged
arrangedin
in a Petri
a Petri dish dish as indicated,
as indicated, andand immersed
immersed in in luciferin[100].
luciferin [100].Bioluminescence
Bioluminescence imagingimagingrevealed
revealedwide
wide
dissemination
dissemination and and
highhigh parasite
parasite burden
burden at at
thethe peak
peak of of
thethe acute
acute stage
stage (14(14 days
days post-infection),
post-infection), and
and the
theofeffect
effect of immune-mediated
immune-mediated controlcontrol of the infection
of the infection during the during the transition
transition to the phase
to the chronic chronic(typically
phase
day(typically
40–60). In daythis
40–60). In thismodel,
infection infection
themodel, the predominant
predominant long-term long-term
sites of sites of parasite
parasite persistence
persistence are the
are the colon and/or stomach. Infection of other organs/tissues is more
colon and/or stomach. Infection of other organs/tissues is more sporadic, although parasites sporadic, although parasites
are often
are often
located located
in the in the skin
skin [103]. [103]. (B) Exploiting
(B) Exploiting ex vivo imaging
ex vivo imaging to assesstodrug
assess drug efficacy
efficacy againstagainst chronic
chronic T. cruzi
T. cruzi infection. Detailed information on parasite tropism and drug susceptibility
infection. Detailed information on parasite tropism and drug susceptibility can be established by can be established
by including
including the entire
the entire carcass
carcass andandheadhead of the
of the mouse
mouse ininthe
theimaging
imagingprocess.
process. InInthe
theexample
example shown,
shown,
benznidazole (BZN) treatment has eliminated detectable parasites. In the nontreated mouse, infection
benznidazole (BZN) treatment has eliminated detectable parasites. In the nontreated mouse, infection
of the head region was observed (parietal, frontal, zygomatic and lacrimal bones; red arrows).
of the head region was observed (parietal, frontal, zygomatic and lacrimal bones; red arrows).
Molecules 2020, 25, 2799 8 of 14

This bioluminescence imaging system has now been incorporated into several Chagas disease
drug development programmes [34,52,106]. It can be used to monitor drug efficacy against both acute
and chronic infections (Figure 2A,B, Figure 3B) [34,67], enables several different dosing regimens to be
assessed in parallel, allows tissue-specific differences in drug sensitivity to be investigated, and can be
adapted to study the correlation between drug activity and disease pathology [53]. Typically, when
treatment is complete, the mice are followed for another 10–20 days to determine the extent of any
relapse. They are then immunosuppressed by cyclophosphamide (3 injections, 200 mg/kg i.p., at 3-day
intervals) to facilitate the expansion of any residual parasites to detectable levels (Figure 2A,B) [101].
As a final step, mice are designated as cured/non-cured on the basis of both in vivo and ex vivo imaging.
The sensitivity of this system seems to circumvent many of the issues related to the high false-cure
rate that can be a problem with PCR-based diagnosis, as well as providing valuable insights into
the dynamics of drug activity. In addition, the ability to continuously monitor individual mice in a
non-invasive manner provides more comprehensive data sets, and reduces the number of animals
required for experimentation.
The crucial factor with any experimental model is the reliability with which findings are directly
translatable to human patients. In mice, the infection profile revealed by bioluminescence imaging
closely mirrors that in humans, with a clearly defined acute stage, that transitions to a life-long
chronic infection characterised by an extremely low parasite burden (Figure 2) [100]. This general
trend holds true in a range of mouse models, although there can be some minor variation in the
precise timing of events [101], a situation that also seems to be the case in humans. In terms of drug
efficacy, the bioluminescence mouse model was predictive of the failure of posaconazole to cure human
infections, despite impressive transient reductions in the parasite load [67]. Similar murine experiments
have also demonstrated that benznidazole and nifurtimox have greater curative efficacy in the chronic
stage than in the acute stage [34]. This may simply reflect the much larger number of parasites that
have to be eliminated during acute stage infections. If these findings can be extended to humans,
it suggests that there could be scope to decrease the treatment length and/or the drug dose without
reducing the chronic infection cure rate. As outlined above, preliminary data from the BENDITA
clinical trial [59] suggest that this may be the case, a finding that could have an enormous impact
on patient compliance. Likewise, an on-going clinical trial [35] will provide further information on
predictive value of the bioluminescence model, which was used to demonstrate that fexinidazole has
superior curative properties compared to the current front-line drugs [34]. Murine models coupled with
bioluminescent parasites will also provide a flexible platform for exploring issues such as combination
therapy, so that the relative compound doses can be rapidly optimised before being advanced into
clinical trial [64,65].

5. Concluding Remarks
In recent years, technical advances in several areas of the drug development process have
contributed to a more favourable landscape for improving the therapeutic options available to
treat neglected tropical diseases. In parallel, changes in research practice, accompanied by a more
sympathetic funding environment, have contributed to the establishment of large drug discovery
consortia that take a more systematic and multidisciplinary approach. After many years in which
few therapeutic candidates advanced far along the development pipeline, the outlook for improved
treatments for Chagas disease is looking considerably more favourable.

Funding: This work was supported by the following awards: UK Medical Research Council (MRC) Grants
MR/T015969/1 to JMK and MR/R021430/1 to MDL.
Conflicts of Interest: The authors declare no conflict of interest.
Molecules 2020, 25, 2799 9 of 14

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