Endochin-like quinolones are highly efficacious against
acute and latent experimental toxoplasmosis
J. Stone Doggetta,b,1, Aaron Nilsenb, Isaac Forquerb, Keith W. Wegmannb, Lorraine Jones-Brandoc, Robert H. Yolkenc,
Claudia Bordónc, Susan A. Charmane, Kasiram Katnenie, Tracey Schultzd, Jeremy N. Burrowsf, David J. Hinrichsb,
Brigitte Meunierg, Vern B. Carruthersd, and Michael K. Riscoeb,h,1
aDivision of Infectious Diseases andhDepartment of Molecular Microbiology and Immunology, Oregon Health and Science University, Portland, OR 97239;
bVeterans Administration Medical Center, Portland, OR 97239;cStanley Division of Developmental Neurovirology, The Johns Hopkins University School of
Medicine, Baltimore, MD 21287;dDepartment of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109;eCentre for
Drug Candidate Optimisation, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052, Australia;fMedicines for Malaria
Venture, Geneva 15, Switzerland; andgCentre de Génétique Moléculaire, Unité Propre de Recherche 3404, Centre National de la Recherche Scientifique,
Gif-sur-Yvette 91198, France
Edited by Thomas E. Wellems, National Institutes of Health, Bethesda, MD, and approved July 30, 2012 (received for review May 13, 2012)
Toxoplasma gondii is a widely distributed protozoan pathogen
that causes devastating ocular and central nervous system disease.
We show that the endochin-like quinolone (ELQ) class of com-
pounds contains extremely potent inhibitors of T. gondii growth
in vitro and is effective against acute and latent toxoplasmosis
in mice. We screened 50 ELQs against T. gondii and selected two
lead compounds, ELQ-271 and ELQ-316, for evaluation. ELQ-271
and ELQ-316, have in vitro IC50values of 0.1 nM and 0.007 nM,
respectively. ELQ-271 and ELQ-316 have ED50values of 0.14 mg/kg
and 0.08 mg/kg when administered orally to mice with acute toxo-
plasmosis. Moreover, ELQ-271 and ELQ-316 are highly active against
the cyst form of T. gondii in mice at low doses, reducing cyst burden
by 76–88% after 16 d of treatment. To investigate the ELQ mecha-
nism of action against T. gondii, we demonstrate that endochin and
ELQ-271 inhibit cytochrome c reduction by the T. gondii cytochrome
bc1complex at 8 nM and 31 nM, respectively. We also show that
ELQ-271 inhibits the Saccharomyces cerevisiae cytochrome bc1com-
plex, and an M221Q amino acid substitution in the Qisite of the
protein leads to >100-fold resistance. We conclude that ELQ-271
and ELQ-316 are orally bioavailable drugs that are effective against
acute and latent toxoplasmosis, likely acting as inhibitors of the
Qisite of the T. gondii cytochrome bc1complex.
antiparasitic agents|electron transport|antimalarial|quinoline|
Approximately 1 billion people worldwide are seropositive for
T. gondii, including more than 10.8% of the United States pop-
ulation (1). In developing countries, seroprevalence can be as
high as 80% (2). T. gondii infection is acquired by ingesting
T. gondii oocysts or undercooked, infected meat. Frequently,
T. gondii infection is asymptomatic or causes an influenza-like ill-
ness and lymphadenopathy. However, 2–25% of people in-
fected with T. gondii develop ocular lesions, making T. gondii
the leading cause of blindness in South America and the leading
cause of posterior uveitis worldwide (2). When T. gondii infects
pregnant women, it can cross the placenta and infect the de-
veloping fetus. Fetal exposure results in up to 4,000 congenital
infections per year in the United States, causing neurocognitive
deficits, chorioretinitis, and abortion (1).
After initial infection, T. gondii establishes latent infection.
Reactivation of latent infection in immunocompromised persons
causes encephalitis, myocarditis, and pneumonitis. Most immu-
nocompromised individuals with AIDS live in the developing
world, and do not have access to first-line anti-Toxoplasma
therapy. Moreover, the impact of toxoplasmosis is expected to
increase as immunosuppression for solid-organ and stem-cell
transplant patients becomes more frequent in the developing
world, where latent T. gondii infection is common (3).
he widely distributed protozoan parasite Toxoplasma gondii
causes devastating ocular and central nervous system disease.
Because of the large global burden of disease and the short-
comings of current therapeutic options, there is an urgent need for
better anti-Toxoplasma drugs. Current therapy for toxoplasmosis
suppresses active infection but does not cure latent infection and
is poorly tolerated. In AIDS patients, therapy is continued until
6 mo after immune reconstitution with antiretroviral therapy. For
some patients, immune suppression is life-long, requiring indefinite
drug suppression. Without prolonged suppressive treatment up to
80% of cases relapse, and 20–30% of patients on suppressive
therapy relapse. Drug side effects have led to discontinuation of
therapy in up to 40% of patients (4, 5). Moreover, current drugs
do not prevent relapsing ocular disease that causes cumulative
scarring and leads to blindness. An ideal anti-Toxoplasma drug
would be potent, nontoxic, and eliminate latent infection.
The drug endochin provides a scaffold for promising anti-
Toxoplasma drugs. Endochin is a 4-(1H)-quinolone initially in-
vestigated as an antimalarial drug in an avian model of malaria
(Fig. 1) by Salzer et al. (6) in 1948. Gingrich and Darrow (7)
subsequently found endochin to be active against avian and
murine toxoplasmosis in 1951. Recent 4-(1H)-quinolone derivatives,
endochin-like quinolones (ELQ), exhibit an in vitro IC50against
Plasmodium falciparum as low as 0.1 nM (8). Although highly
active in vitro, the initial series of ELQs exhibited poor aqueous
solubility and were unstable in the presence of rat and human
microsomes (8). A library of 4(1H)-quinolone-3-diarylethers was
made to improve these properties. Of the 4(1H)-quinolone-3-
diarylethers synthesized in our laboratory, we found that ELQ-
271 and ELQ-316 have the greatest efficacy against T. gondii.
Herein, we describe the in vitro and in vivo efficacy of ELQ-271
and ELQ-316 against T. gondii and provide evidence that the
mechanism of action of ELQ-271 is inhibition of the T. gondii
cytochrome bc1complex at the Qisite.
In Vitro Inhibition of T. gondii and Host-Cell Toxicity. The growth
inhibition of T. gondii by ELQ-271 and ELQ-316 was tested
against the 2F strain of T. gondii expressing β-galactosidase, allow-
ing colorimetric quantitation of T. gondii. The results are shown
Author contributions: J.S.D., I.F., L.J.-B., R.H.Y., S.A.C., K.K., T.S., J.B., D.J.H., B.M., V.B.C.,
and M.K.R. designed research; J.S.D., I.F., K.W.W., L.J.-B., C.B., K.K., T.S., and D.J.H. per-
formed research; A.N. and B.M. contributed new reagents/analytic tools; A.N. designed
and synthesized ELQ-271 and ELQ-316; M.K.R. designed ELQ-271 and ELQ-316; J.S.D., I.F.,
L.J.-B., S.A.C., K.K., T.S., V.B.C., and M.K.R. analyzed data; and J.S.D. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Freely available online through the PNAS open access option.
1To whom correspondence may be addressed. E-mail: email@example.com or riscoem@
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| September 25, 2012
| vol. 109
| no. 39www.pnas.org/cgi/doi/10.1073/pnas.1208069109
in Table 1. ELQ-271 and ELQ-316 inhibit T. gondii at an IC50of
0.1 nM and 0.007 nM, respectively. By comparison, under these
experimental conditions, atovaquone inhibits T. gondii at an IC50
of 138 nM.
Host-cell viability was measured colorimetrically using Cell-
Titer 96Aqueous One Solution Reagent to evaluate host-cell tox-
icity that may influence antiparasite activity. This measurement
also provides an initial indication of potential human toxicity.
The TD50dose of ELQ-271 against human foreskin fibroblast
(HFF) cell culture was 9.3 μM, whereas toxicity was not observed
with ELQ-316 or endochin at >50 μM. Based on these results,
the calculated in vitro therapeutic index (TI) is 93,490 for ELQ-271
and >7.1 × 106for ELQ-316. The TI calculated for atovaquone is
274, which is 341-fold lower than ELQ-271 and at least 25,912-fold
lower than ELQ-316.
Human and Rat Microsome Metabolism of ELQ-271 and ELQ-316. No
measurable degradation of ELQ-271 and ELQ-316 was seen in
the presence of human or rat liver microsomes, with or without
cofactors for chromosome P450- and glucuronosyltransferase-
mediated metabolism, suggesting that both compounds would be
subject to low hepatic metabolic clearance in vivo.
Efficacy of ELQ-271 and ELQ-316 Against Acute Murine Toxoplasmosis.
We tested ELQ-271 and ELQ-316 in comparison with atovaquone
against acute murine toxoplasmosis in two separate experiments
(Fig. 2). The initial experiment included drug concentrations of
50, 20, 5, and 1 mg/kg or the vehicle, polyethylene glycol (PEG)
400, only. The subsequent experiment included drug concen-
trations of 5, 1, 0.2, and 0.04 mg/kg and the vehicle-only control.
In both experiments, mice were divided into groups of four and
inoculated i.p. with 20,000 RH strain T. gondii tachyzoites that
express YFP. Beginning 48 h after inoculation, drugs were ad-
ministered daily for 5 d. Twenty-four hours after the final drug
administration, mice were killed and underwent peritoneal la-
vage with PBS. The lavage fluid was stained with allophycocyanin
(APC)-conjugated anti-mouse CD45 antibody and analyzed by flow
cytometry. The anti-mouse CD45 antibody stains bone marrow-
derived cells other than red blood cells and platelets. Cells infected
with T. gondii and cell-free tachyzoites are counted by flow
cytometry as YFP-positive events. We calculated the T. gondii
burden of infection as the percentage of YFP-positive events out
of the number of all CD45-positive events and YFP-positive events.
The use of anti-CD45 antibody staining excludes from analysis red
blood cells introduced at the time of peritoneal puncture. One of
the eight mice in the control group that exhibited a very low level of
infection and one of the four mice in the 0.2-mg/kg atovaquone
group that exhibited a very high burden of disease were excluded
ELQ-271 and ELQ-316 had an ED50of 0.14 and 0.08 mg/kg
and an ED90of 1.06 and 0.33 mg/kg, respectively. By compari-
son, atovaquone was given as a positive control and had an ED50
of 0.85 mg/kg and an ED90of 3.51 mg/kg. Mice in the high-dose
groups of 50 mg/kg showed no signs of overt toxicity (i.e., weight
loss, inactivity, or ruffled fur).
Efficacy of ELQ-271 and ELQ-316 Against Latent Murine Toxoplasmosis.
We tested ELQ-271, ELQ-316, and atovaquone against latent
murine toxoplasmosis (Fig. 3). Mice were infected i.p. with the
cyst-forming ME49 strain of T. gondii. Five weeks after infection,
mice were treated once daily with 5 mg/kg of atovaquone or either
5 or 25 mg/kg of ELQ-271 or ELQ-316 for 16 d. Two weeks after
the final drug dose, mice were killed, and slides were prepared
from brain homogenate for cyst enumeration. The control group of
mice that were given vehicle alone had a mean of 2,523 cysts per
brain. Atovaquone at 5 mg/kg reduced the number of brain cysts by
44%. ELQ-271 at 5 mg/kg and 25 mg/kg reduced the number of
brain cysts by 87% and 84%, respectively. ELQ-316 at 5 mg/kg and
25 mg/kg reduced the number of brain cysts by 76% and 88%. The
number of cysts per brain was significantly fewer for each ELQ
group than for the atovaquone group (P < 0.0001).
Inhibition of T. gondii Cytochrome c Reductase. Based on our
prior published work demonstrating that ELQ analogs inhibit
Plasmodium yoelli oxygen consumption, we suspected that ELQs
target the T. gondii bc1complex (9). The cytochrome bc1complex
is a membrane-bound enzyme complex located in the inner mi-
tochondrial membrane that contributes to pyrimidine biosynthesis
and oxidative phosphorylation (10). Electron transfer through the
Fig. 1. Chemical structures of endochin, ELQ-271, and ELQ-316.
Table 1. In vitro and in vivo drug efficacy against T. gondii and host cell toxicity
In vitro IC50nM
In vitro HFF TD50nM
In vivo ED50mg/kg
In vivo ED90mg/kg
T. gondii growth inhibition and host-cell toxicity of selected drugs were tested in vitro. Efficacy against experimental toxoplasmosis
was tested in a murine acute infection model (Fig. 2). HFF, human foreskin fibroblasts; ND, not done.
Doggett et al.PNAS
| September 25, 2012
| vol. 109
| no. 39
cytochrome bc1complex proceeds via the Q-cycle, resulting in
ubiquinol oxidation at the Qosite, ubiquinone reduction at the
Qisite, and cytochrome c reduction that shuttles electrons to
cytochrome c oxidase. Atovaquone is known to bind to the Qo
site, and point mutations in cytochrome b confer resistance to
atovaquone (11, 12). We selected ELQ-271, atovaquone, and
endochin to test against cytochrome bc1function in an enriched
T. gondii mitochondrial preparation. ELQ-271, atovaquone
and endochin inhibition of the T. gondii bc1was compared to
the inhibition of the HFF cytochrome bc1(Table 2).
Cytochrome c reduction was measured spectrophotometrically
at 550–542 nm. T. gondii cytochrome bc1complex has an apparent
Kmof 7.4 μM for ubiquinol that is several-fold lower than the
50 μM decylubiquinol used in our experimental system. ELQ-271,
endochin, and atovaquone inhibited T. gondii cytochrome c re-
duction at IC50s of 30.6 nM, 8.2 nM, and 785 nM, respectively.
The IC50against HFF cytochrome c reduction was 818 nM for
ELQ-271 and >10 μM for endochin and atovaquone.
Inhibition of Saccharomyces cerevisiae Cytochrome c Reductase. To
investigate further the mechanism of action of ELQ-271, we tested
the activity of ELQ-271 against a panel of Saccharomyces cerevisiae
strains that have deletions of ATP-binding cassette transporter
genes and amino acid substitutions in the Qoand Qisite of the
cytochrome bc1complex. The S. cerevisiae cytochrome bc1complex
has been used as a model to understand the mechanism of ato-
vaquone and other cytochrome bc1complex inhibitors against
T. gondii, P. falciparum, and Pneumocystis jirovecii (13, 14). This
model is valuable, because neither crystallization of the T. gondii
cytochrome bc1complex nor direct manipulation of the T. gondii
mitochondrial genome, which includes the cytochrome b gene,
has been accomplished. Hill et al. (15) demonstrated that deleting
efflux transporter genes from S. cerevisiae resulted in susceptibility
to atovaquone. This susceptibility allows the testing of cytochrome
b inhibitors against S. cerevisiae strains with point mutations in-
troduced to the cytochrome b gene with a growth-inhibition assay.
We tested 14 S. cerevisiae strains with point mutations in the
Qior Qosite and the parental strain (AD1-9) for resistance
to ELQ-271 on glycerol medium that forces S. cerevisiae to rely
on respiration for ATP production (Table S1). Of these strains,
the mutant strain with a point mutation that results in an amino
acid substitution of methionine by glutamine at position 221 in
the Qisite shows drug resistance compared with AD1-9 (Fig. 4).
Growth of the M221Q strain was not inhibited by ELQ-271 when
grown on fermentable medium.
We then evaluated the inhibitory activity of ELQ-271 against
cytochrome c reduction of the parental strain and the M221Q
mutant. Atovaquone was included as a positive control. The
IC50of ELQ-271 against parental cytochrome c reduction and
M221Q mutant cytochrome c reduction were 61 nM and >10 μM,
respectively. The IC50of atovaquone against parental cytochrome c
reduction and M221Q mutant cytochrome c reduction was 45 nM
and 78 nM, respectively. The >100-fold ELQ-271 resistance of
the M221Q mutant, compared with the parental strain, strongly
suggests that ELQ-271 inhibits the S. cerevisiae cytochrome bc1
complex by targeting the Qisite and that replacing methionine
with glutamine at position 221 interferes with ELQ-271 binding.
Current clinically used anti-Toxoplasma drugs have limited efficacy
against ocular and congenital toxoplasmosis and leave immuno-
compromised patients susceptible to recurrent infection. Despite
advances in understanding T. gondii biology and identification
of multiple unique drug targets, current first-line anti-Toxoplasma
therapy is based on the discovery of synergy between pyrimeth-
amine and sulfadiazine by Eyles and Coleman in 1953, 14 y after
T. gondii was proven to be a pathogen (16). The need for im-
proved therapy that is safe, effective, and well tolerated should
be a global priority.
We describe the efficacy of two orally active drugs, ELQ-271
and ELQ-316, in vitro and against acute and latent murine toxo-
plasmosis. It is difficult to compare the ED50of the ELQs with
the published efficacy of other anti-Toxoplasma drugs because of
heterogeneous experimental designs and variations in T. gondii
virulence. However, the atovaquone efficacy that we found is
T. gondii infection in mice. The T. gondii burden reflects the percentage of
T. gondii infection. Treatment groups consisted of the control (n = 7), 0.04 mg/kg
(n = 4), 0.2 mg/kg (n = 4; one value from the atovaquone 0.2 mg/kg group
was excluded from analysis as an outlier because it was excessively high),
1 mg/kg (n = 8), 5 mg/kg (n = 8), 20 mg/kg (n = 4), and 50 mg/kg (n = 4) of
each drug. ED50and ED90values for each compound are listed in Table 1.
Error bars represent SEM. ATQ, atovaquone.
The efficacy of ELQ-271, ELQ-316, and atovaquone against acute
Cysts / brain
of cysts/ brain
0 44%87%84% 76%88%
T. gondii infection. The number of T. gondii cysts per brain was reduced by
atovaquone, ELQ-271, and ELQ-316 compared with the control. Five weeks
after inoculation with the T. gondii ME49 strain, mice were treated with each
drug for 16 d. Brain cysts were counted 2 wk after the final day of drug
administration. The number of cysts per brain was significantly lower for
each ELQ group than for the atovaquone group (P < 0.0001). Error bars
represent SEM. ATQ, atovaquone.
The efficacy of ELQ-271, ELQ-316, and atovaquone against latent
| www.pnas.org/cgi/doi/10.1073/pnas.1208069109 Doggett et al.
comparable to that described by Araujo et al. (17, 18), who
showed that atovaquone doses <10 mg/kg partially suppressed
T. gondii RH strain infection during the first 8 d of infection.
The observation that ELQ-271 and ELQ-316 are threefold and
10-fold more effective than atovaquone is promising and indicates
that these compounds and structurally similar 4-(1H)-quinolone-3-
diphenylethers should be evaluated further for potential clinical
use against acute toxoplasmosis. Moreover, the remarkable ef-
ficacy of ELQ-271 and ELQ-316 in decreasing T. gondii brain
cysts by up to 88% after 16 d of treatment suggests that they have
the potential to eradicate latent infection at clinically applicable
doses. A drug capable of eliminating latent infection would
revolutionize current T. gondii therapy by preventing T. gondii
reactivation in patients who are immunosuppressed or who are
anticipating immunosuppression for stem-cell or solid-organ trans-
plantation. This treatment paradigm would prevent toxoplasmosis
and supplant the need for prolonged suppressive anti-Toxoplasma
therapy that often is toxic or poorly tolerated.
We chose atovaquone for comparison in these studies because
ELQs have a similar mechanism of action. Atovaquone, a Qo-site
inhibitor and the only clinically used cytochrome bc1complex
inhibitor, provides a basis for speculation regarding the clinical
potential for bc1inhibitors. Prospective clinical studies of ato-
vaquone as monotherapy and in combination with sulfadiazine or
pyrimethamine against Toxoplasma encephalitis in AIDS patients
have established atovaquone as an alternative anti-Toxoplasma
regimen (19, 20). As for latent infection, a retrospective study of
patients with ocular toxoplasmosis suggests that atovaquone may
decrease the frequency of disease recurrence more than other
anti-Toxoplasma drugs (21). The efficacy against recurrent ocular
toxoplasmosis is likely related to atovaquone’s activity against
latent infection that has been demonstrated against T. gondii
brain cysts in mice (22, 23).
Cytochrome bc1complex inhibitors bind to the Qosite or the
Qisite of the enzyme complex. The marked resistance against
ELQ-271 inhibition caused by the M221Q substitution in the
S. cerevisiae Qisite suggests that ELQ-271 targets the Qisite. The
M221Q substitution is a reversion mutation from a respiratory-
deficient M221K substitution, and its effects on enzyme kinetics
are well characterized (24-26). The methionine at position 221 is
thought to form a hydrophobic interaction with ubiquinone (26).
The M221Q substitution causes resistance to multiple Qisite
inhibitors (14, 25). The structure and function of the Qiand
Qosites are highly conserved across a wide range of species.
Currently, there are no clinically used Qisite inhibitors. Although
further investigation into the mechanism of action of ELQ-271
and ELQ-316 against T. gondii is needed, the present experiments
suggest that ELQ-271 and potentially other ELQs act at the
T. gondii Qisite. New ELQs may be designed to exploit the
Subsequent investigation of ELQ-271 and ELQ-316 will include
more extensive studies of pharmacokinetics, toxicology, and opti-
mization of the route of administration and dosing schedule for
eradication of latent T. gondii infection. We suspect that either
increased penetration of the blood–brain barrier or more fa-
vorable pharmacokinetics underlie ELQ-271’s excellent activity
against T. gondii brain cysts at 5 mg/kg, despite its lower intrinsic
potency and efficacy against acute infection as compared with
ELQ-316. Future studies also will evaluate synergy with other
clinically used anti-Toxoplasma agents.
Atovaquone typically is combined with other agents to prevent
resistance and for synergy. Resistance mutations have developed
in the cytochrome b gene when atovaquone is used alone to treat
Plasmodium and Pneumocystis (27, 28). Atovaquone-resistance
mutations have not been found in clinical T. gondii isolates but
have been generated experimentally (11, 29). Although it is un-
clear if a drug that acts at the Qisite will have a similar threshold
for the development of resistance, discovering synergistic com-
binations that enhance the antiparasitic effect and shorten the
duration of therapy will diminish the risk of drug toxicity. Syn-
ergy between atovaquone and clindamycin against murine
toxoplasmosis has been observed, and we expect that similar
combinations may enhance efficacy against acute and latent
The results presented demonstrate that ELQ-271 and ELQ-316
are orally available drugs that are remarkably effective against
acute and latent murine toxoplasmosis at low doses. Further
in vivo toxicology studies are warranted, but our current limited
evaluation does not prompt concerns for toxicity. Mechanistic
studies of ELQ-271 show inhibition of the T. gondii bc1complex.
Based on the high-level resistance caused by the M221Q amino
acid substitution in the S. cerevisiae cytochrome b Qisite, it is likely
that ELQ-271 and potentially other members of the ELQ series
act at the T. gondii cytochrome b Qisite. Qisite inhibition offers
a mechanism of action that differs from current clinically used
anti-Toxoplasma therapy. ELQ-271 and ELQ-316 are promising
candidates for the treatment and prevention of toxoplasmosis.
Chemicals and Biologic Strains. The chemical structures of endochin, ELQ-271,
and ELQ-316 are shown in Fig. 1. The methods for the synthesis of ELQ-271
and ELQ-316 are described in the supporting information (SI Methods).
Atovaquone was obtained from Sigma-Aldrich or DeF PharmaChemical Co.
HFF cells were obtained from American Type Culture Collection, and YFP2 RH
strain T. gondii was obtained from Boris Striepen (University of Georgia,
Athens GA). Mutations of the cytochrome b gene were introduced into the
yeast mitochondrial genome by site-directed mutagenesis and the mito-
chondrial transformation procedure previously described (15). Alternatively,
mutations resulted from the selection of respiratory-competent revertants
from respiratory-deficient mutants with a point mutation in the cyto-
chrome b gene (24). The mutated and wild-type mitochondrial genomes
were transferred by cytoduction into the nuclear background AD1-9 (Mat α
ura3 his1, yor1Δ::hisG, snq2Δ::hisG, pdr5Δ::hisG, pdr10Δ::hisG, pdr11Δ::hisG,
ycf1Δ::hisG, pdr3Δ::hisG, pdr15Δ::hisG, pdr1Δ::hisG) (kindly given by M. Ghislain,
Catholic University of Louvain, Louvain, Belgium).
Inhibition of T. gondii and host-cell cytochrome c
Drug T. gondii IC50nM HFF IC50nM
The rate of cytochrome c reduction was measured spectrophotometrically
in the presence and absence of inhibitors. HFF, human foreskin fibroblasts.
S. cerevisiae strain AD 1-9 with and without the M221Q point mutation.
ELQ-271 concentrations are listed below the disks.
Comparison of growth inhibition by disk diffusion of ELQ-271 against
Doggett et al.PNAS
| September 25, 2012
| vol. 109
| no. 39
In Vitro Inhibition of T. gondii And Host Cell Toxicity. Drugs were evaluated
for growth inhibition of T. gondii using a 96-well assay in which T. gondii
expressing β-galactosidase were cultured in an HFF cell monolayer and were
quantified colorimetrically. This assay was reported originally by Mcfadden
et al. (30) and has been modified by Jones-Brando et al. (31). Compounds
dissolved in DMSO were added to the first column of HFF cells (320 μM) and
then were diluted serially across the plate by dilutions of 0.5 log10, leaving
the final column drug free. Fifty T. gondii tachyzoites were added to
each well in six of the eight rows. After 4 d of incubation at 37 °C/5% CO2,
chlorophenol red-β-D-galactopyranoside (CPRG) was added, and on the fifth
day absorbance was measured at 570–650 nm. To measure cell viability,
CellTiter 96Aqueous One Solution Reagent (Promega Corp.) was added to
the two rows of uninfected HFF cells, and absorbance at 490–650 nm was
measured after 3 h. Color reactions were read in a Vmaxmicroplate reader
(Molecular Devices). The IC50and TD50were calculated using CalcuSyn software
(Biosoft). A therapeutic index for each compound was calculated as TD50/IC50.
Human and Rat Microsome Metabolism of ELQ-271. Human and rat liver
microsomes (BD Gentest, Discovery Labware, Inc.) were suspended in 0.1 M
phosphate buffer (pH 7.4) at a final protein concentration of 0.4 mg/mL and
incubated with compounds (0.5 or 1 μM) at 37 °C. An NADPH-regenerating
system (1 mg/mL NADP, 1 mg/mL glucose-6-phosphate, 1 U/mL glucose-6-
phosphate dehydrogenase) and MgCl2(0.67 mg/mL) was added to initiate
the metabolic reaction, and mixtures were quenched with ice-cold acetoni-
trile at time points ranging from 0–60 min. Samples also were incubated
in the absence of the NADPH-regenerating system to monitor for non–
cytochrome P450-mediated metabolism in the microsomal matrix. Samples
were centrifuged, and the amount of parent compound remaining in the
supernatant was assessed by LC-MS. The first-order rate constant for sub-
strate depletion was determined by fitting the data to an exponential decay
function. The rate constant was used to calculate the in vitro intrinsic clearance
value (CLint) and the predicted in vivo intrinsic clearance value (CLint vivo) (32).
The predicted in vivo hepatic extraction ratio (EH) was calculated using the
following relationship: EH= CLint vivo/(Q + CLint vivo) where Q is liver blood
flow (20.7 mL·min−1·kg−1, 55.2 mL·min−1·kg−1, and 90 mL·min−1·kg−1in
humans, rats, and mice, respectively) (33). Potential binding of compounds
to microsomal protein was not taken into account in these calculations.
Efficacy Against Acute Murine Toxoplasmosis. We used a model of acute
murine toxoplasmosis that is adapted from a protocol described by Bajohr
et al. (34). Twenty thousand T. gondii tachyzoites were inoculated i.p. into
4- to 5-wk-old female CF-1 mice with four mice in each experimental group.
Drugs were dissolved in PEG 400 and administered by oral gavage 48 h after
inoculation at concentrations of 0.04, 0.2, 1, 5, 25, and 50 mg/kg/d for 5 d.
The control group received PEG 400 only. On the eighth day, the mice were
killed, and 3 mL of PBS (pH 7.4) was injected into the peritoneum. Peritoneal
fluid and PBS were withdrawn and examined by flow cytometry using a BD
FACSCalibur. Then 200 μL of peritoneal fluid was stained with 2 μg of anti-
mouse APC-conjugated CD45 antibody for 20 min at 4 °C, centrifuged at
1,000 × g, and resuspended in 500 μL of PBS. CD45 staining was performed
to distinguish uninfected macrophages from red blood cells that might have
been introduced at the time of peritoneal lavage. The T. gondii burden of
disease was calculated as the percentage of FACS events that were positive
for both YFP and CD45 plus YFP alone events, out of the events that were
positive for both YFP and CD45 plus YFP alone events plus CD45 alone
events. Analysis included 1 × 105FACS events. Nonlinear regression analysis
was performed with GraphPad Prism 5.0 software (GraphPad Software). This
protocol was approved by the Institutional Animal Care and Use Committee of
the Portland Veterans Administration Research Foundation.
Treatment of Latent Toxoplasma Infections in Mice. Six groups of 7-wk-old
CBA/J strain mice (Jackson Laboratory) (23 mice per group) were injected
i.p. with 200 μL of sterile PBS containing 18 cysts of ME49 strain T. gondii
(type II genotype) from infected CBA/J donor mice. Zero to four mice per
group succumbed to the infection, leaving 19–23 mice per group available
for treatment. Beginning 5 wk postinfection, mice were injected i.p. daily for
16 consecutive days with 50 μL solvent (DMSO) or drug dissolved in DMSO.
Atovaquone was administered at 5 mg/kg; ELQ-271 and ELQ-316 were
administered at 5 mg/kg and 25 mg/kg, respectively. Mice were euthanized
humanely 2 wk after the final injection. The mouse brains were placed in
1 mL sterile PBS and individually minced with scissors, vortexed, and ho-
mogenized by three or four passages through a 22-G needle and syringe.
Three 10-μL samples (30 μL total) of each brain homogenate were mixed on
a glass microscope slide, smeared, air dried, fixed with ethanol for 15 min,
dried further, and then mounted on a coverslip in Eukitt mounting medium
(Sigma-Aldrich). Penicillin and streptomycin (Gibco) (50 U/mL and 50 μg/mL
final concentrations, respectively) were added to remaining brain homog-
enate for storage at 4 °C and resampling as necessary. Cysts were enumer-
ated by phase-constrast microscopy without knowledge of the sample’s
treatment group. This protocol was approved by the Committee on the Use
and Care of Animals of the University of Michigan.
T. gondii Cytochrome c Reductase Inhibition. T. gondii tachyzoites were har-
vested from lysed HFF cell culture, filtered through a 3-μm polycarbonate
filter, and centrifuged at 8,000 × g for 5 min. The T. gondii pellet was
washed in PBS containing 1 mM PMSF and centrifuged at 8,000 × g for
10 min. The T. gondii pellet then was resuspended in ice-cold lysis buffer
(75 mM sucrose, 225 mM mannitol, 5 mM MgCl2, 5 mM KH2PO4, 1 mM EDTA,
1 mM PMSF, 5 mM Hepes, at pH 7.4). This suspension was homogenized
in a cooled glass Dounce homogenizer. Cell debris was removed after cen-
trifugation at 800 × g for 5 min, and then mitochondrial particles were
centrifuged at 20,000 × g for 30 min. The pellet was suspended in lysis buffer
and mixed to a final concentration of 30% (vol/vol) glycerol. Aliquots of mi-
tochondrial fragments were frozen at −80 °C until needed.
Cytochrome c reduction was monitored at 550 nm using 542 nm as the
baseline with an Agilent Diode Array 8453 spectrophotometer. Mitochon-
drial T. gondii protein (40 μg) solubilized in 2 mg/mL dodecyl maltoside was
added to a cuvettete containing 50 μM oxidized cytochrome c (horse heart;
Sigma-Aldrich), 50 μM decylubiquinol, 2 mM KCN, 100 mM KCl, 50 mM Tricine,
pH 8.0. Drugs were dissolved in DMSO. The initial rate of cytochrome c re-
duction in the presence and absence of inhibitors was measured after the
addition of mitochondrial protein and after sufficient time to measure the
background reaction between decylubiquinone and cytochrome c.
S. cerevisiae Growth Inhibition. S. cerevisiae strains were grown overnight in
liquid YPG medium [1% yeast extract, 2% (wt/vol) peptone, and 3% (vol/vol)
glycerol]. Overnight cultures were diluted to an OD600of 0.05 and grown for
2 h. Cultures were combined with YPG containing 0.6% melted agar for
a total volume of 10 mL and a final OD600of 0.008 and were applied evenly
to 2% (wt/vol) agar YPG plates. Drugs were dissolved in DMSO at various
concentrations. Ten microliters of each concentration was applied to 7-mm
diameter, 3-μm-thick filter paper. The disks were placed on the YPG agar
plates. Plates were incubated at 30 °C. Images were obtained after 4 d.
S. cerevisiae Cytochrome c Reductase Inhibition. Enriched mitochondrial prep-
arations were obtained from S. cerevisiae strains grown overnight in 250 mL
of YPD liquid medium [1% yeast extract, 2% (wt/vol) peptone, 2% (wt/
vol) dextrose]. Cells were washed with distilled water and then were
resuspended in buffer [100 mM Tris·H2SO (pH 9.4), 10 mM DTT] and in-
cubated for 20 min at 30 °C. Cells were resuspended in buffer [2.4 M sorbitol,
1 M Hepes·KOH (pH 7), 1 M NaN3, 0.5 M EDTA] containing 5 mg Zymolase
(Sigma-Aldrich) / g cells and incubated at 30°C for 30 min with shaking. Cells
were resuspended in ice-cold lysis buffer and homogenized in a cooled glass
Dounce homogenizer. The suspension was centrifuged at 1,500 × g for 5
min. The supernatant was centrifuged at 4,000 × g for 5 min, and the
resulting supernatant was centrifuged at 12,000 × g for 15 min. The pellet
was suspended in lysis buffer and mixed to a final concentration of 30% (vol/
vol) glycerol. Aliquots of mitochondrial fragments were frozen at −80 °C
until needed. Cytochrome c reductase activity was measured as described
above for T. gondii.
ACKNOWLEDGMENTS. This project was supported by funds from the Stanley
Medical Research Institute and the Medicines for Malaria Venture, by National
Institutes of Health National Institute of Allergy and Infectious Diseases
Grant 1-R01-AI079182, and by the US Department of Veterans Affairs Merit
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| vol. 109
| no. 39