PNAS PLUS

(+)-SJ733, a clinical candidate for malaria that acts

through ATP4 to induce rapid host-mediated clearance
of Plasmodium
Mara Beln Jimnez-Daza, Daniel Ebertb, Yandira Salinasc, Anupam Pradhand, Adele M. Lehanee,
Marie-Eve Myrand-Lapierref, Kathleen G. OLoughling, David M. Shacklefordh, Mariana Justino de Almeidai,
Angela K. Carrilloc, Julie A. Clarkc, Adelaide S. M. Dennise, Jonathon Diepb, Xiaoyan Dengf, Sandra Duffyj,
Aaron N. Endsleyg, Greg Fedewab, W. Armand Guiguemdec, Mara G. Gmeza, Gloria Holbrookc, Jeremy Horstb,
Charles C. Kimk, Jian Liul, Marcus C. S. Leei, Amy Mathenyc, Mara Santos Martneza, Gregory Millerc,
Ane Rodrguez-Alejandrea, Laura Sanza, Martina Sigalc, Natalie J. Spillmane, Philip D. Steinl, Zheng Wangl, Fangyi Zhuc,
David Watersonm, Spencer Knappl, Anang Shelatc, Vicky M. Averyj, David A. Fidocki, Francisco-Javier Gamoa,
Susan A. Charmanh, Jon C. Mirsalisg, Hongshen Maf, Santiago Ferrera, Kiaran Kirke, Iigo Angulo-Barturena,
Dennis E. Kyled, Joseph L. DeRisib, David M. Floydl, and R. Kiplin Guyc,1
a
Tres Cantos Medicines Development CampusDiseases of the Developing World, GlaxoSmithKline, Tres Cantos 28760, Madrid, Spain; bDepartment of
Biochemistry and Biophysics, University of California, San Francisco, CA 94158-2330; cDepartment of Chemical Biology and Therapeutics, St. Jude Childrens
Research Hospital, Memphis, TN 38105; dDepartment of Global Health, College of Public Health, University of South Florida, Tampa, FL 33612; eResearch
School of Biology, Australian National University, Canberra, ACT, Australia 2601; fDepartments of Mechanical Engineering and Urologic Sciences, University of
British Columbia, Vancouver, BC, Canada V6T 1Z4; gToxicology and Pharmacokinetics, SRI International, Menlo Park, CA 94025; hFaculty of Pharmacy and
Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia 3052; iDepartment of Microbiology and Immunology and Division of Infectious Diseases,
Department of Medicine, Columbia University Medical Center, New York, NY 10032; jEskitis Institute, Brisbane Innovation Park, Nathan Campus, Griffith
University, QLD, Australia 4111; kDivision of Experimental Medicine, University of California, San Francisco, CA 94110; lDepartment of Chemistry and Chemical
Biology, Rutgers, The State University of New Jersey, Piscataway, NJ 08854; and mMedicines for Malaria Venture, International Center Cointrin, 1215 Geneva,
Switzerland

Edited* by Thomas E. Wellems, National Institutes of Health, Bethesda, MD, and approved October 30, 2014 (received for review July 29, 2014)

malaria

| PfATP4 | drug discovery

n 2010, there were 220 million cases of malaria, leading to

660,000 deaths. More than 400,000 of those who died were
African children (1). Recent successes in antimalarial drug discovery and development have led to renewed hope for global
eradication of malaria (2, 3). Antimalarial drugs are a critical
component of the eradication campaign, and single-dose regimens of cheap, safe, and efficacious drugs, which ideally also
reduce transmission, are required (4). The discovery of agents
that act rapidly against novel targets would greatly aid in the
development of such drugs (5). Additionally, given the ready
propensity with which the parasite develops resistance to new
www.pnas.org/cgi/doi/10.1073/pnas.1414221111

antimalarials (6), targets for which drug-resistanceconferring

mutations cause a high fitness cost are of particular interest.
In a high-throughput screen to identify molecules that block
the proliferation of Plasmodium falciparum in cocultures with
human erythrocytes in vitro, we identified three high-priority
Significance
Useful antimalarial drugs must be rapidly acting, highly efficacious, and have low potential for developing resistance. (+)-SJ733
targets a Plasmodium cation-transporting ATPase, ATP4. (+)-SJ733
cleared parasites in vivo as quickly as artesunate by specifically
inducing eryptosis/senescence in infected, treated erythrocytes.
Although in vitro selection of pfatp4 mutants with (+)-SJ733
proceeded with moderate frequency, during in vivo selection of
pbatp4 mutants, resistance emerged slowly and produced marginally resistant mutants with poor fitness. In addition, (+)-SJ733
met all other criteria for a clinical candidate, including high oral
bioavailability, a high safety margin, and transmission blocking
activity. These results demonstrate that targeting ATP4 has great
potential to deliver useful drugs for malaria eradication.
Author contributions: M.B.J.-D., A.M.L., K.G.O., A.S.M.D., W.A.G., C.C.K., L.S., N.J.S., P.D.S., F.Z.,
V.M.A., D.A.F., F.-J.G., S.A.C., J.C.M., H.M., S.F., K.K., I.A.-B., D.E.K., J.L.D., D.M.F., and R.K.G.
designed research; M.B.J.-D., D.E., Y.S., A.P., A.M.L., M.-E.M.-L., K.G.O., D.M.S., M.J.d.A.,
A.K.C., J.A.C., A.S.M.D., J.D., X.D., S.D., A.N.E., G.F., W.A.G., M.G.G., G.H., J.H., C.C.K., J.L.,
M.C.S.L., A.M., M.S.M., A.R.-A., L.S., M.S., N.J.S., P.D.S., Z.W., F.Z., and J.L.D. performed research;
J.L.D. contributed new reagents/analytic tools; M.B.J.-D., D.E., Y.S., A.P., A.M.L., K.G.O., D.M.S.,
J.A.C., A.S.M.D., A.N.E., W.A.G., M.G.G., G.H., C.C.K., J.L., M.C.S.L., M.S.M., G.M., L.S., N.J.S., P.D.S.,
F.Z., D.W., S.K., A.S., V.M.A., D.A.F., F.-J.G., S.A.C., J.C.M., H.M., S.F., K.K., I.A.-B., D.E.K., J.L.D.,
D.M.F., and R.K.G. analyzed data; and D.A.F., K.K., D.E.K., J.L.D., and R.K.G. wrote the paper.
Conflict of interest statement: M.B.J.-D., M.G.G., M.S.M., A.R.-A., L.S., F-J.G., S.F., and I.A.-B. are
employees of GlaxoSmithKline and are engaged in commercial development of antimalarial
drugs, although not this compound.
*This Direct Submission article had a prearranged editor.
Data deposition: The sequencing data have been deposited in the National Center for
Biotechnology Information BioProject database, www.ncbi.nlm.nih.gov/bioproject (project ID PRJNA253899).
1

To whom correspondence should be addressed. Email: kip.guy@stjude.org.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.

1073/pnas.1414221111/-/DCSupplemental.

PNAS | Published online December 1, 2014 | E5455E5462

PHARMACOLOGY

Drug discovery for malaria has been transformed in the last 5

years by the discovery of many new lead compounds identified by
phenotypic screening. The process of developing these compounds as drug leads and studying the cellular responses they
induce is revealing new targets that regulate key processes in the
Plasmodium parasites that cause malaria. We disclose herein that
the clinical candidate (+)-SJ733 acts upon one of these targets,
ATP4. ATP4 is thought to be a cation-transporting ATPase responsible for maintaining low intracellular Na+ levels in the parasite.
Treatment of parasitized erythrocytes with (+)-SJ733 in vitro
caused a rapid perturbation of Na+ homeostasis in the parasite.
This perturbation was followed by profound physical changes in
the infected cells, including increased membrane rigidity and externalization of phosphatidylserine, consistent with eryptosis
(erythrocyte suicide) or senescence. These changes are proposed
to underpin the rapid (+)-SJ733-induced clearance of parasites
seen in vivo. Plasmodium falciparum ATPase 4 (pfatp4) mutations
that confer resistance to (+)-SJ733 carry a high fitness cost. The
speed with which (+)-SJ733 kills parasites and the high fitness cost
associated with resistance-conferring mutations appear to slow
and suppress the selection of highly drug-resistant mutants in
vivo. Together, our data suggest that inhibitors of PfATP4 have
highly attractive features for fast-acting antimalarials to be used in
the global eradication campaign.

series (7). Pharmacological and chemical optimization of one

of these series, the dihydroisoquinolones (DHIQs), led to the
selection of a clinical candidate, (+)-SJ733, for development as
a fast clearance component (Target Candidate Profile 1; TCP1)
of a single-exposure radical cure and prophylaxis (SERCaP) drug
(4). Here, we present evidence that (+)-SJ733 acts on the recently recognized target PfATP4 (pfatp4; PFL0590c) (8) to give
rapid clearance of the parasite in vivo by inducing eryptosis or
senescence in parasite-infected erythrocytes. Mutation of pfatp4
to confer resistance to (+)-SJ733 carries a high fitness cost, and
the development of resistance in vivo is slow and produces only
low-level resistance.
Results and Discussion
(+)-SJ733 Is a Candidate for TCP1 of a SERCaP Drug. SJ733 was highly

potent in vitro against all tested strains of P. falciparum (EC50

range 1060 nM; SI Appendix, Fig. S1A and Table S1), including
those resistant to other antimalarials, and was equally potent
against all stages of the erythrocytic life cycle (SI Appendix, Fig.
S3E). Of the two enantiomers of SJ733, the (+)-enantiomer was
significantly more potent. (+)-SJ733 was, however, 10-fold less
potent against Plasmodium berghei and other rodent malarias ex
vivo (SI Appendix, Fig. S1B). (+)-SJ733 bound to a single receptor site in P. falciparum-infected erythrocytes with equivalent

affinity to its growth-inhibitory potency (kd = 50 nM; SI Appendix, Fig. S3F).

(+)-SJ733 was highly potent and efficacious against P. falciparum
3D70087/N9 in vivo when administered as four sequential daily oral
doses in the NOD-scid IL2Rnull mouse model (Fig. 1A) (9), with
a 90% effective dose, (ED90 1.9 mg/kg) and exposure [area under
the curve at ED90 (AUCED90), 1.5 Mh] superior to artesunate
(11.1 mg/kg; AUCED90 not determined), chloroquine (4.3 mg/kg;
AUCED90 3.1 Mh), and pyrimethamine (0.9 mg/kg; AUCED90
5. Mh) in the same model. When treated with the ED90 dose,
(+)-SJ733 concentrations in blood remained above the average
in vitro EC90 for 610 h after each dose (SI Appendix, Fig. S1C).
In line with ex vivo results, SJ733 was 10-fold less potent in vivo
against P. berghei when administered as four sequential daily oral
doses (racemate ED90 40 mg/kg; AUCED90 80 Mh; SI Appendix,
Fig. S1D), with (r)-SJ733 concentrations in blood remaining above
the average ex vivo EC90 for 1012 h after each dose (SI Appendix,
Fig. S1E). (+)-SJ733 had robust in vivo pharmacokinetic behavior in all preclinical species, including mouse, rat, and dog,
with excellent exposure (310 M Cmax, 540 Mh AUC, at 20
30 mg/kg doses) and reasonable clearance (Fig. 1B). The compound also had oral bioavailability of >65% in both rats (SI
Appendix, Fig. S1F) and dogs (SI Appendix, Fig. S1G).

plasma

Plasma

Fig. 1. SJ733 is an efficacious and safe orally active drug candidate. (A) SJ733 is efficacious against P. falciparum 3D70087/N9 in the nonobese diabetic Scid
interleukin-2 receptor chain null (NSG) mouse model with the (+)-enantiomer exhibiting excellent potency (1.9 mg/kg); similar to pyrimethamine (ED90
0.9 mg/kg) and superior to chloroquine (ED90 4.3 mg/kg) in this model. (+)-SJ733 achieves this efficacy from exposure (AUCED90 1.5 Mh) similar to that of
chloroquine (AUCED90 3.1 Mh) and superior to that of pyrimethamine (AUCED90 5.2 Mh) in the same model. (B) (+)-SJ733 exhibits excellent exposure after oral
administration in mouse, rat, and dog. In rodents, (+)-SJ733 reaches peak plasma concentrations of 5 M within 1 h after 2025 mg/kg doses and >20 M in dogs
following a 30 mg/kg oral dose. (C) (+)-SJ733 exhibits no significant toxicology at doses up to 200 mg/kg in rats with an exposure (AUC) 43-fold higher than that
required to produce the maximum parasitological response (fastest rate of killing) and 220-fold that required to produce the ED90 in the mouse, indicating the
potential for an excellent therapeutic ratio. (D) (+)-SJ733 potently and efficaciously blocks transmission of P. berghei from infected mice to mosquitos when the mice
are treated 1 h before feeding the mosquitos as measured by counting oocysts from dissected mosquitos after the sexual stage is allowed to mature.

E5456 | www.pnas.org/cgi/doi/10.1073/pnas.1414221111

Jimnez-Daz et al.

(+)-SJ733 Targets ATP4. To gain insight into the mechanism of action

of (+)-SJ733 and the potential for acquisition of resistance, we selected drug-resistant mutant strains of P. falciparum in vitro and
P. berghei in vivo, using (+)-SJ733 and other DHIQ analogs, and
characterized the alleles conferring resistance (6, 10). Mutant
strains were generated in vitro by pressuring erythrocytic cocultures
with (+)-SJ733 or other DHIQ analogs (SI Appendix, SI Materials
and Methods) until apparently resistant parasites emerged. Resistance, confirmed by measuring the potency of (+)-SJ733, varied
between twofold to 750-fold (SI Appendix, Table S1) and emerged
with minimum inocula of between 107 and 109 parasites (3D7
strain). In most cases, resistance was 10-fold and emerged from
108 to 109 parasites (SI Appendix, Fig. S2A). The single mutant
strain generated in vivo was produced by serial passage of parasites
in mice with multiple rounds of intermittent high-dose treatment
with (+)-SJ311, a close analog of (+)-SJ733 (SI Appendix, Fig. S2C),
with weak (twofold) stable resistance emerging only after 4 weeks
of treatment.
For six independently produced resistant P. falciparum strains,
we used whole-genome sequencing to identify mutations (SI
Appendix, Table S2). Genomic DNA was prepared for parental
and mutant strains, and libraries were sequenced (SI Appendix).
After quality filtering and alignment to the reference sequence,
the average coverage ranged from >50-fold to >700-fold for the
14 nuclear chromosomes and >200-fold to >30,000-fold for the
mitochondrial and plastid genomes. The presence of copy
number variants (CNVs) was assessed, relative to the parental
strains, by using the R-package CNV-Seq (11). Other than the
variable sequences proximal to the telomeres, no significant (P <
0.001) deletions were detected relative to the drug-sensitive parental strains (SI Appendix, Table S3). Similarly, no amplified
regions were detected for any strain. To detect genomic mutations we compared exonic sequences for both the parental and
selected strains. Because the mutant strains were not necessarily
clonal populations, we reasoned that true drivers of drug resistance would be gain-of-function mutations driven to purification as opposed to passenger mutations present at various
frequencies. Therefore, we considered only nonsynonymous
mutations that were present at 99% purity or higher, where
purity was defined as the percent of the total reads confirming
a mutation for a given position in the genome, assuming that
mutations occurred in single-copy genes.
Jimnez-Daz et al.

PNAS | Published online December 1, 2014 | E5457

PNAS PLUS

By using these criteria, only a single mutated gene, encoding the

putative Na+-ATPase PfATP4 (PFL0590c), was shared among all
six selections (SI Appendix, Table S2). In five of six selections,
100% of the mapped reads confirmed the detected mutation, all
of which were confirmed by Sanger sequencing. In five of six
strains, pfatp4 was the only mutated gene detected using these
criteria. We also investigated whether expression of the mRNA
for pfatp4, as measured by quantitative RT-PCR (qRT-PCR), was
altered in wild-type or mutated strains, with or without treatment
with (+)-SJ733. Relative to a housekeeping gene, we detected no
significant change in expression (SI Appendix, Table S4).
Two additional independently derived resistant strains were
generated by selection with SJ733 or another DHIQ. Sanger sequencing of the pfatp4 coding sequence identified nonsynonymous
mutations in both cases (SI Appendix, Table S3). For the sole
resistant P. berghei strain , described above, sequencing of the
orthologous gene pbatp4 revealed a nonsynonymous mutation
absent in parental parasites (SI Appendix, Table S5).
Finally, we examined whether cross-resistance existed for mutant
strains of malaria previously selected with other putative PfATP4
targeted compounds, including the DHIQ family, the spiroindolones,
and a series developed at GlaxoSmithKline (SI Appendix, Table S5).
In all cases there was complete and bidirectional cross-resistance for
all compounds and strains (SI Appendix, Table S1). The fold
resistance exhibited by each strain tracked with the genotype
and was consistent from compound to compound. This finding
strongly suggests that the mutation(s) in pfatp4 are the sole
determinant of resistance for each of the compounds.
Two DHIQ-resistant mutant strains (P. falciparum ATP4L350H
and ATP4P966T) were tested in competition assays and were
found to be substantially less fit than their parental strains in the
absence of drug pressure (SI Appendix, Fig. S2B). The slow
emergence of DHIQ resistance observed during our in vivo selection experiment is consistent with our finding of a fitness cost
associated with DHIQ resistance.
Together, the complete association of resistance with point
mutations in pfatp4, the absence of copy-number variations or
expression-level differences for this gene, and the cross-resistance
with spiroindolones all support the notion that mutation of
PfATP4 is both necessary and sufficient for resistance to SJ733.
A homology model for PfATP4, built from crystal structures of
the closest mammalian homolog (sarco/endoplasmic reticulum
Ca2+ -ATPase; SERCA), revealed that the majority of the
resistance-conferring mutations clustered in a small portion of
the predicted protein structure within the transmembrane, iontransporting channel of the protein (Fig. 2 A and C). All other
resistance-conferring mutations were predicted to perturb the
protein structure near this cluster by distorting the orientation of
helices adjoining the cluster. Unconstrained docking of the
structure of (+)-SJ733 to this homology model revealed one
predicted binding site, which is enveloped by the cluster of residues bearing resistance-conferring mutations (Fig. 2B).
The three rodent malaria species (P. berghei, Plasmodium
vinckei, and Plasmodium chabaudi) were all significantly less
sensitive to (+)-SJ733 than was P. falciparum both ex vivo (SI
Appendix, Fig. S1B) and in vivo (SI Appendix, Figs. S1D and S2D).
The atp4 alleles from these species carry a short variant sequence
(SI Appendix, Fig. S2E), which is predicted to encode a loop within
the (+)-SJ733 binding site (purple ribbon, Fig. 2B). Replacing the
pbatp4 gene with the pfatp4 gene fully conferred increased susceptibility to (+)-SJ733 ex vivo (SI Appendix, Fig. S1B) and partially (2-fold vs. 10-fold) restored sensitivity in vivo (SI Appendix,
Figs. S1D and S2D), thus strongly suggesting that the sequence
variations underpin the reduced susceptibility of the rodent
malarias to (+)-SJ733. The lack of a full restoration of sensitivity
in vivo may be due to differences between the humanized mouse
model used for P. falciparum and the normal mouse model used

PHARMACOLOGY

(+)-SJ733 has not exhibited either significant safety liabilities

at any dose in extensive profiling in vitro (see summary in SI
Appendix) or significant safety or tolerability liabilities in either
single- or repeat-dose studies at any dose tested in any preclinical
species (no observed adverse effect level and maximum tolerated
dose >240 mg/kg from 7-d repeat dosing study in rat; see summary in SI Appendix). Therefore, (+)-SJ733 is expected to have
a safety margin of at least 43-fold [comparing AUCmax in the rat
safety studies to AUC from the dose giving maximal parasitological response in mice (Fig. 1C)]. In addition, (+)-SJ733 blocked
transmission of P. berghei from infected mice to mosquitos (ED50
5 mg/kg; Fig. 1D), at a dose lower than that efficacious against
asexual blood stages. Finally, (+)-SJ733 was readily synthesized in
good yield from commercially available precursors in five steps
with a route that is expected to provide low cost of goods (SI
Appendix, Fig. S1H). The only potential liability noted to date was
a relatively high clearance in human microsomal models (SI Appendix). However, predicted efficacious human doses, allometrically derived from preclinical pharmacokinetics experiments, fall
into the range of 36 mg/kg, and the projected half-life is long
enough to allow (+)-SJ733 to be the fast acting component
(TCP1) of a SERCaP drug (4). Thus, (+)-SJ733 is highly potent,
efficacious against both asexual and sexual blood stages, safe, and
highly orally bioavailable. (+)-SJ733 was selected for clinical development by Medicines for Malaria Venture in March 2013.

N355Y I398F P412T,L

V415D
L350H G358S
Q172H
T418N
P437S
G223R

166

443

900

F917L
L928F
P966S,T,A
P990R

1128

Fig. 2. SJ733 targets the PfATP4 protein. (A) All SJ733-resistant strains of malaria generated worldwide contain mutations in the pfatp4 gene that cluster in
a single region of the protein. The mutations are illustrated with a homology model of the protein structure for PfATP4 (gold/blue ribbon), built from the
available SERCA crystal structures, with the location of mutations causing either high-fold resistance (red), medium-fold resistance (yellow), or low-fold
resistance (blue) shown as solid fill. The blue helices represent those in the transmembrane domain and are indicated in the gene block diagram in C. (B)
Theoretical docking studies with (+)-SJ733 reproducibly generate a single pose that places (+)-SJ733 in contact with the two residues inducing the highest fold
resistance and the one residue selected by in vivo passage with cycling drug exposure. This pose is illustrated by the docked structure of (+)-SJ733 placed into
the putative binding site on the PfATP4 structure, with residues conferring resistance color-coded as in A. A loop with sequence variability among Plasmodium
spp. that may lead to differing sensitivities to (+)-SJ733 is highlighted in magenta. (C) Block diagram of pfatp4 gene structure and positioning of mutations
detected in strains resistant to (+)-SJ733. The selected mutations are color-coded to match those in A, with the location in the coding sequence indicated. The
blue cylinders represent the helical portions of the protein shown as blue on the ribbon diagram in A. (D) SJ733 disrupted resting [Na+]i within asexual bloodstage P. falciparum as illustrated by doseresponse experiments with the active (+) and inactive () enantiomers. Resting [Na+]i was measured >60 min after
addition of the compound to sodium-binding benzofuran isophthalate-loaded, saponin-isolated parasites. Each data point represents the mean final [Na+]i
averaged from at least three independent experiments (shown SD). The relative potencies of the two SJ733 isomers in the [Na+]i assays shown here, on the
two different strains (the SJ733-resistant mutant ATP4L350H and the wild-type parent) matched those seen in parasite growth assays.

for P. berghei, but is not driven by differing pharmacokinetics,

because there were no significant exposure variations.
PfATP4 has been proposed to be a Na+-efflux ATPase that
maintains a low Na+ concentration in the parasite cytosol ([Na+]i)
(12). As with the spiroindolones (12), treatment of saponinisolated trophozoites with (+)-SJ733 resulted in a rapid increase in
[Na+]i from a resting level of 6 1 mM to a maximal level of 110
10 mM within 1.5 h of initiating treatment (Fig. 2D and SI
Appendix, Fig. S2F). As is also seen for the spiroindolones (13),
(+)-SJ733 caused a rapid increase in the transmembrane pH
gradient (0.11 0.03 pH units; mean SD n = 6) and reduced
the extent of acidification of the parasite cytosol seen following
the inhibition of the parasites plasma membrane V-type H+
ATPase by concanamycin A (SI Appendix, Fig. S2G).
E5458 | www.pnas.org/cgi/doi/10.1073/pnas.1414221111

The EC50 for the effect of (+)-SJ733 on [Na+]i in wild-type

parasites is 200 nM, within fivefold of the growth inhibition
potency in the same strain (SI Appendix, Fig. S1A) and well
within the concentrations achieved in the blood of effectively
treated animals (SI Appendix, Fig. S1C). The P. falciparum
ATP4L350H strain, which is resistant to (+)-SJ733, showed a
50-fold decrease in sensitivity (relative to the parental strain) to
the [Na+]i-disrupting effects of (+)-SJ733. Furthermore, the
relative potency of the enantiomers of SJ733 in the [Na+]i assays
for these strains was the same as in the proliferation assays (Fig.
2D and SI Appendix, Fig. S1A). The mutant strain had a significantly higher resting [Na+]i than the parent (15.8 0.8 mM vs.
5.7 0.5 mM; mean SEM from 9 or 10 independent experiments; P < 0.001 with unpaired t test), which may account for the
decreased fitness of this strain. The data are consistent with the
Jimnez-Daz et al.

Atovaquone
Pyrimethamine
Artesunate
(+)-SJ733

50

50

0
0

12

24

36 48 60 72 84
Time of Treatment (h)

96 108 120

D
100

12 h
24 h
48 h
72 h

50

0
0.0001

12

24

36

48

60

72

84

96

108 120

Time of Treatment (h)

log(Viable Parasites)

Normalized % Growth Arrest

Atovaquone, wt
Atovaquone, no spleen
Artesunate, wt
Artesunate, no spleen
(+)-SJ733, wt
(+)-SJ733, no spleen

100

Atovaquone
Pyrimethamine
Artemisinin
(+)-SJ733

0.001

0.01
0.1
[(+)-SJ733], M

10

12

24

36 48 60 72 84 96 108 120
Time of Treatment (h)

Fig. 3. SJ773 causes rapid clearance in vivo that is not dependent upon innate immunity. (A) (+)-SJ733 causes rapid clearance of parasites in vivo, with speed
equivalent to artesunate, the fastest-acting antimalarial drug, as measured by clonal dilution assays. When total parasites present in blood are measured, all
parasites are cleared systemically within 48 h of initiation of therapy. Similar pharmacodynamics are seen in P. berghei-infected animals (SI Appendix). (B) The in vivo
clearance rate of (+)-SJ733 is independent of the presence of a spleen. When total parasites in blood are measured, all parasites are cleared within 48 h of initiation
of treatment in both splenectomized and nonsplenectomized animals. Similar experiments in clodronate-treated animals infected with P. berghei indicate that
macrophages are not required for rapid pharmacodynamics (SI Appendix). (C) (+)-SJ733 arrests growth of parasites rapidly in vitro, reaching a maximal effect within
24 h as measured by proliferation of a luciferase-labeled 3D7 strain. (D) (+)-SJ733 kills parasites in vitro, but does so with modest speed, equivalent to pyrimethamine
as measured by clonal dilution assays. When viable parasites are measured, 96 h of continuous exposure above the EC99 is required for maximal effect.

Jimnez-Daz et al.

PNAS | Published online December 1, 2014 | E5459

PHARMACOLOGY

Normalized Parasitemia

100

PNAS PLUS

Inhibition of Plasmodium ATP4 Leads to Rapid Clearance in Vivo

Through Inducing Physical Changes in Infected, Treated Red Cells.

Normalized Parasitemia

Treatment of P. falciparum-infected NOD-scid IL2Rnull mice

(9) with (+)-SJ733 caused rapid clearance of parasites, which
were 80% depleted within the first 24 h and undetectable by 48 h
(Fig. 3A). Notably, (+)-SJ733 cleared parasites faster than
artesunate in the same model. The same pharmacodynamics
were observed after treatment of P. falciparum-infected splenectomized NOD-scid IL2Rnull mice (Fig. 3B) or normal mice
infected with rodent malarias, with or without liposomal clodronate depletion of Kupffer cells (SI Appendix, Fig. S3A). As
discussed above, although the rate of action remains the same,
the potency of (+)-SJ733 varied by >10-fold between these
models. Thus, the rate of clearance in vivo was independent of
both malaria species and host immune status.
When infected erythrocytes were treated with (+)-SJ733 in vitro,
growth was fully arrested within 24 h, as measured by using lucif-

erase-labeled parasites (Fig. 3C), but fully killing parasites required

96 h (Fig. 3D), as measured by using clonal dilution methods (14).
Given that a typical P. falciparum-infected mouse has 108 parasites, the same load used in the in vitro cidality assays, it appears
that (+)-SJ733 acts at least fourfold faster in vivo than it does in
vitro. These data suggest that compounds targeting ATP4 induce
physical changes in the infected erythrocyte that allow recognition
and clearance of treated, infected cells by the host.
The most obvious explanation for enhanced clearance in vivo
is that (+)-SJ733 induces an erythrocyte phenotype leading to
rapid clearance such as eryptosis (suicidal erythrocyte death) or
senescence, which can be caused by a wide range of stresses (15,
16). Eryptotic and senescent erythrocytes share several key features: an increase in exposed phosphatidylserine (PS), increased
membrane rigidity, more spherical shape, and decreased size (17,
18). The eryptotic/senescent phenotype has been correlated with
increased clearance of erythrocytes, primarily through erythrophagocytosis (1921). However, mechanical clearance in the
spleen (22) and sinusoidal clearance, independent of Kupffer
cells, in the liver (16) also both play a role and can compensate

primary growth inhibition effects of (+)-SJ733 stemming from its

ability to increase [Na+]i through inhibition of PfATP4.

for a lack of erythrophagocytosis in macrophage-depleted animals. We simultaneously measured each of these variables for
uninfected and infected erythrocytes in vitro using multimodal
fluorescence activated cell sorting measuring forward/side scatter
(size and shape), parasite infection (SYBR green binding), and
PS exposure (Annexin V binding).

Plasmodium-infected erythrocytes (iRBCs) have been reported

to possess a mild eryptotic/senescent phenotype (23) relative to
uninfected erythrocytes (uRBCs). In our studies iRBCs were
small relative to uRBCs (Fig. 4A, lower forward scatter in Top
Left vs. Bottom Left) and possessed more exposed PS [color scale
in Fig. 4A; uRBCs, green (<1,000 Annexin binding) and iRBCs,

Fig. 4. SJ733 arrests parasite development and induces eryptosis selectively in infected erythrocytes. (A) (+)-SJ733 causes a significant proportion of infected
erythrocytes to shrink and expose PS. Uninfected erythrocytes, whether treated with (+)-SJ733 or not (Bottom Left and Bottom Right), remain normally sized, do
not have exposed PS, and do not possess substantial DNA, as evidenced by examining the cell population for forward scatter, Annexin V binding, and SYBR
green binding in FACS. Cells are shown as a population contour plot that compared scatter with SYBR green binding and are color-coded to indicate Annexin
binding. After infection, magnetically purified trophozoite/schizont-infected erythrocytes can be detected in three populations (large, with little exposed PS;
small, with moderate levels of exposed PS; and intermediate-sized, with high levels of exposed PS), with the majority being in the small- and large-sized
populations. Treatment with (+)-SJ733 causes the population distribution to shift strongly toward the intermediate-sized population with high amounts of
exposed PSconsistent with strong induction of eryptosis. This shift is not seen with either control drugs or DMSO treatment. (B) When the eryptosis induction
effect is examined for (+)-SJ733 by using a concentration-response experiment, the maximal effect is seen at a concentration of 40 nM and the EC50 at 30 nM
congruent with the doses causing similar levels of response for proliferation inhibition and parasite Na+ levels. Artesunate has no such effect. (C) (+)-SJ733
causes a significant increase in rigidity of infected, treated erythrocytes. As previously reported, infection of erythrocytes (iRBC) causes a significant increase in
their rigidity (P < 0.001; JonckheereTerpstra test). After treatment with (+)-SJ733, these infected erythrocytes become significantly more rigid, with the degree
of rigidity peaking at 7 h after treatment. *P < 0.001 (JonckheereTerpstra test). There are no detectable effects on unparasitized erythrocytes at any time
point. (D) (+)-SJ733 induces a rapid arrest of parasite motility inside infected erythrocytes that is maintained for the period of treatment as shown by time-lapse
microscopy of the treated and untreated cells. The first row of images shows typical motility and growth of a ring-stage parasite over 18 h, which is in stark
contrast to the treated parasite in the second row of images that immediately arrests both motility and growth. In some cases the parasite can be observed to
lyse within the erythrocyte as shown in the third row. In additional cases the infected, treated erythrocyte itself will lyse after lysis of the parasite as shown in
the fourth row. No changes are noted to erythrocyte morphology after treatment of uninfected erythrocytes.

E5460 | www.pnas.org/cgi/doi/10.1073/pnas.1414221111

Jimnez-Daz et al.

Jimnez-Daz et al.

PNAS PLUS

hours
Treatment of iRBC
with (+)-SJ733

1.5

Na+

Na+

Inhibition of parasite
sodium efflux

Induction of host cell

changes similar to
eryptosis

24
Na
N a+

Clearance of
parasites in vitro
96

Maximum killing of
parasites in vivo

Fig. 5. Induction of eryptosis by SJ733 triggers rapid clearance in vivo.

When (+)-SJ733 is added to erythrocytes infected with Plasmodium spp., it
prevents the action of the putative Na+ ATPase PfATP4 and causes a significant increase in cytosolic Na+ within the parasite, reaching maximal effect
within 90 min after treatment. This process results in the immediate arrest of
parasite motility and blockade of intracellular parasite replication that reaches maximal potency after 24 h of exposure. Simultaneously, infected,
treated erythrocytes begin to enter eryptosis, as characterized by their
shrinking and becoming more spherical, becoming significantly more rigid,
and exposing PS on their plasma membrane. These effects maximize by 7 h
after treatment. In vitro, the drug effects lead to complete arrest of replication within 24 h of treatment, followed by slow death, which is maximal
by 96 h. In stark contrast to the in vitro setting, the induction of eryptosis
leads to rapid clearance of the infected, treated erythrocytes in vivo.

results in animals with varying phagocyte and macrophage

defects, as described above, suggest that all of these clearance
mechanisms are used in a complementary way to drive the rapid
clearance of parasites in vivo by (+)-SJ733.
The data presented here are consistent with the model outlined
in Fig. 5. Treatment of parasitized erythrocytes with (+)-SJ733
blocks the normal function of PfATP4 and leads to a rapid increase
of [Na+]i within the parasite, reaching a maximum by 1.5 h after
treatment. This immediate response triggers other cellular responses, probably including the arrest of protein synthesis (8), and these
are integrated at the level of both the parasite (manifested as a
swollen, development-arrested phenotype) and the host erythrocyte
(manifested as induction of an eryptotic/senescent phenotype).
These morphological changes reach a maximum by 78 h after
treatment. In vivo, these effects trigger rapid clearance of the infected, treated erythrocytes through host-mediated mechanisms used to
clear aging red cells. Conversely, in vitro, without the physiological
clearance mechanisms, the parasites go into a growth-arrested state,
with maximal arrest being reached by 24 h after treatment. Parasites
PNAS | Published online December 1, 2014 | E5461

PHARMACOLOGY

white to light pink (1,0005,000 Annexin binding)]. Treatment of

iRBCs with (+)-SJ733, or with another ATP4-targeted compound
(the spiroindolone NITD-246), caused the majority of the cells to
shift to the population with the most severe phenotype possessing
a 10-fold increase in exposed PS relative to uRBCs [Fig. 4A; increased pink intensity (10,000 to >1000,000 Annexin binding,
Middle Left and Middle Right)]. The potency of (+)-SJ733 in
shifting cells from the mild to more severely eryptotic/senescent
phenotype was almost identical to that observed for growth inhibition (EC50 30 nM; Fig. 4B). No other class of antimalarial
drug has this profile. For example artesunate, another rapidly
acting drug (Fig. 4A, Top Right) did not produce the more pronounced phenotype, regardless of dose. Importantly, this response is dependent upon the presence of both the parasite and
the drug, because treatment of uninfected erythrocytes did not
induce eryptosis (Fig. 4A, Bottom Right).
The biconcave cell shape of RBCs causes a multimodal distribution of forward scatter scores, whereas changing RBC morphology to a more spherical shape characteristic of eryptosis/
senescence leads to a more monomodal distribution (24) that can
be captured by regression modeling of forward and side scatter
data (25). The treatment of iRBCs with (+)-SJ733 (or NITD246)
collapsed the multimodal distribution largely to a monomodal
distribution (containing >85% of cells) of the iRBCs, which when
modeled is similar to spherical cells (MI 0.7) and not to discoid
cells (MI 0.9). This finding suggests that inhibition of ATP4 in the
parasite leads to a spherical morphology, perhaps through parasite
swelling. Individual iRBCs that are more spherical are also observed after treatment with (+)-SJ733 in video microscopy (Fig. 4D,
comparing the first row to the following three). These cell shape
changes were not observed for either uRBCs or untreated iRBCs.
The eryptotic/senescent phenotype also includes an increase in
rigidity as measured by deformation when the cell is pushed
through a capillary with fluid pressure. (+)-SJ733 treatment of
iRBCs caused them to become significantly more rigid compared
with either uRBCs or untreated iRBCs (Fig. 4C; P < 0.001;
JonckheereTerpstra test). Again, there was no effect on the
rigidity of uRBCs exposed to (+)-SJ733. This increase in rigidity
was larger in magnitude than previously reported to take place
after infection (26) and is congruent with the magnitude of loss
of deformability that is associated with rapid clearance of eryptotic/senescent erythrocytes (17, 27, 28).
Finally, treatment of infected erythrocytes led to the arrest of
parasite development (Fig. 4D, row 2, relative to row 1) and to
changes in the morphology of both the parasite (swelling) and
the erythrocyte (becoming pyknotic) as observed by serial single
cell phase contrast microscopy. All of these changes reached
maximal effect within 1 h of treatment and were maintained for
the duration of treatmentcongruent with the time scale on
which [Na+]i increases. In many cases the swelling of the parasite
was followed by parasite bursting (Fig. 4D, row 3) or by sequential bursting of the parasite and the infected, treated
erythrocyte (Fig. 4D, row 4). These effects become evident after
710 h. In all cases, the effects on erythrocyte morphology and
integrity were limited to treated, infected erythrocytes, with no
changes observed in uninfected, treated erythrocytes.
Together, these data strongly suggest that (+)-SJ733 produces
structural changes in infected erythrocytes consistent with
eryptosis/senescence, while leaving uninfected erythrocytes
unscathed. This effect is clearly not due to simple changes in
erythrocyte ion content; similar (eryptotic) changes were not
seen on treatment of uninfected erythrocytes with the Na+/K+
ATPase inhibitor ouabain under conditions shown previously to
cause major perturbation of the Na+/K+ ratio in the erythrocyte
cytosol (SI Appendix, Fig. S3G) (29). Our data suggest that
eryptosis may be induced by mechanical changes caused by
parasite swelling. Induction of the eryptosis/senescence phenotype leads to clearance by several documented mechanisms. Our

then die by a number of mechanisms, including spontaneous lysis,

with the maximal effect ultimately reached within 96 h.
In conclusion, these studies of (+)-SJ733 indicate that PfATP4
inhibitors, including both the DHIQs and the spiroindolones,
possess multiple properties that make them ideal for development
as fast-acting components of combination therapies for single dose
cure of malaria. First, the host-mediated mechanism leads to extremely rapid clearance in vivo, thus obviating the need for extended coverage. Indeed, the spiroindolones have proven faster
acting in the clinic than expected from preclinical modeling (30).
Second, they have exquisite selectivity due to a host-mediated
mechanismonly affecting infected and treated erythrocytes.
Third, there is a high fitness cost associated with resistance-conferring mutations. This trait is potentially due to the observed
higher resting [Na+]i causing cellular stress. The combination of
this fitness cost and the extremely rapid action of the compounds
make it very difficult for the parasite to acquire resistance in vivo
despite a frequency of in vitro resistance acquisition that is in line
with other targeted drugs. Therefore, we hypothesize that the
acquisition of resistance during clinical use will occur at a very low
frequency, on par with artemisinin-containing drugs. The rapid in
vivo clearance produced by compounds that target ATP4 make it
an attractive drug development target and the favorable properties
of (+)-SJ733 encourage further development.

vivo and in vitro pharmacokinetics studies in all species; in vitro and ex vivo
efficacy studies in P. falciparum and P. berghei; synthesis of (+)-SJ733; homology modeling and docking studies of (+)-SJ733 binding to PfATP4; in
vitro and in vivo selection of mutant strains of P. falciparum and P. berghei
resistant to (+)-SJ733; whole-genome and Sanger sequencing of mutant
strains; quantitative PCR of mutant strains; production of transgenic parasites; measurement of cytosolic Na + and pH; mutant fitness studies; timelapse microscopy studies; erythrocyte rigidity studies; FACS analysis of
erythrocytes; and measurement of speed of effect on P. falciparum. The
SI Appendix includes additional data related to the generation, characterization of the chemical sensitivity, and genotype of P. falciparum
strains resistant to (+)-SJ733; summaries of the in vivo efficacy and
pharmacokinetics data; and the details of safety pharmacology and
tolerability studies.

All procedures were carried out according to published methods. Details of

the methods are included in SI Appendix. The detailed methods included in
SI Appendix are the in vivo efficacy studies for blood stages in both the
P. falciparum and P. berghei models; in vivo transmission blocking studies; in

ACKNOWLEDGMENTS. We thank Dr. Alexis LaCrue for mosquito dissections;

Case McNamara, Elizabeth Winzeler, and Thierry Diagana for sharing mutant
strains of P. falciparum selected by exposure to the spiroindolones and samples of
the spiroindolones; the National Institute of Mental Health Psychoactive Drug
Screening Program for profiling (+)-SJ733 in their assay panel; Deqing Pei for
biostatistical analyses; Dr. Leonard D. Shultz and The Jackson Laboratory for providing access to nonobese diabetic SCID IL2Rc null mice through their collaboration with GlaxoSmithKline Tres Cantos Medicines Development Campus; and the
Australian and Spanish Red Cross Blood Service for the provision of blood. This
work was supported by National Institute of Allergy and Infectious Diseases Contract HHSN2722011000221; NIH Grants AI090662 and AI075517; the Medicines for
Malaria Venture; Australian National Health and Medical Research Council
(NHMRC) Project Grant 1042272; NHMRC Overseas Biomedical Fellowships
585519 and 1072217; the Howard Hughes Medical Institute; The David and
Lucille Packard Foundation; the American Lebanese Syrian Affiliated Charities;
and St. Jude Childrens Research Hospital. We acknowledge the support of the
High-Throughput Screening Center, the Biostatistics Department, and the Flow
Cytometry and Cell Sorting Shared Resource at St. Jude Childrens Research
Hospital.

1. World Health Organization (2012) World Malaria Report (WHO, Geneva).

2. Anthony MP, Burrows JN, Duparc S, Moehrle JJ, Wells TN (2012) The global pipeline of
new medicines for the control and elimination of malaria. Malar J 11:316.
3. Spangenberg T, et al. (2013) The open access malaria box: A drug discovery catalyst
for neglected diseases. PLoS ONE 8(6):e62906.
4. Burrows JN, van Huijsduijnen RH, Mhrle JJ, Oeuvray C, Wells TN (2013) Designing the
next generation of medicines for malaria control and eradication. Malar J 12:187.
5. Chatterjee AK, Yeung BK (2012) Back to the future: Lessons learned in modern targetbased and whole-cell lead optimization of antimalarials. Curr Top Med Chem 12(5):
473483.
6. Neafsey DE (2013) Genome sequencing sheds light on emerging drug resistance in
malaria parasites. Nat Genet 45(6):589590.
7. Guiguemde WA, et al. (2010) Chemical genetics of Plasmodium falciparum. Nature
465(7296):311315.
8. Rottmann M, et al. (2010) Spiroindolones, a potent compound class for the treatment
of malaria. Science 329(5996):11751180.
9. Jimnez-Daz MB, et al. (2009) Improved murine model of malaria using Plasmodium falciparum competent strains and non-myelodepleted NOD-scid IL2Rgammanull mice engrafted with human erythrocytes. Antimicrob Agents Chemother
53(10):45334536.
10. Park DJ, et al. (2012) Sequence-based association and selection scans identify drug
resistance loci in the Plasmodium falciparum malaria parasite. Proc Natl Acad Sci USA
109(32):1305213057.
11. Xie C, Tammi MT (2009) CNV-seq, a new method to detect copy number variation
using high-throughput sequencing. BMC Bioinformatics 10:80.
12. Spillman NJ, et al. (2013) Na+ regulation in the malaria parasite Plasmodium falciparum involves the cation ATPase PfATP4 and is a target of the spiroindolone antimalarials. Cell Host Microbe 13(2):227237.
13. Spillman NJ, Allen RJ, Kirk K (2013) Na+ extrusion imposes an acid load on the intraerythrocytic malaria parasite. Mol Biochem Parasitol 189(1-2):14.
14. Rathod PK, Leffers NP, Young RD (1992) Molecular targets of 5-fluoroorotate in the
human malaria parasite, Plasmodium falciparum. Antimicrob Agents Chemother
36(4):704711.
15. Lutz HU, Bogdanova A (2013) Mechanisms tagging senescent red blood cells for
clearance in healthy humans. Front Physiol 4:387.

16. Lee SJ, Park SY, Jung MY, Bae SM, Kim IS (2011) Mechanism for phosphatidylserinedependent erythrophagocytosis in mouse liver. Blood 117(19):52155223.
17. Kwan JM, Guo Q, Kyluik-Price DL, Ma H, Scott MD (2013) Microfluidic analysis of
cellular deformability of normal and oxidatively damaged red blood cells. Am J
Hematol 88(8):682689.
18. Mohandas N, Groner W (1989) Cell membrane and volume changes during red cell
development and aging. Ann N Y Acad Sci 554:217224.
19. de Back DZ, Kostova EB, van Kraaij M, van den Berg TK, van Bruggen R (2014) Of
macrophages and red blood cells: A complex love story. Front Physiol 5:9.
20. Schwartz RS, et al. (1985) Increased adherence of sickled and phosphatidylserineenriched human erythrocytes to cultured human peripheral blood monocytes. J Clin
Invest 75(6):19651972.
21. Schroit AJ, Madsen JW, Tanaka Y (1985) In vivo recognition and clearance of red
blood cells containing phosphatidylserine in their plasma membranes. J Biol Chem
260(8):51315138.
22. Klausner MA, et al. (1975) Contrasting splenic mechanisms in the blood clearance of
red blood cells and colloidal particles. Blood 46(6):965976.
23. Fller M, et al. (2009) Suicide for survivaldeath of infected erythrocytes as a host
mechanism to survive malaria. Cell Physiol Biochem 24(3-4):133140.
24. Piagnerelli M, et al. (2007) Assessment of erythrocyte shape by flow cytometry
techniques. J Clin Pathol 60(5):549554.
25. Ahlgrim C, Pottgiesser T, Sander T, Schumacher YO, Baumstark MW (2013) Flow cytometric assessment of erythrocyte shape through analysis of FSC histograms: Use of
kurtosis and implications for longitudinal evaluation. PLoS ONE 8(3):e59862.
26. Guo Q, Reiling SJ, Rohrbach P, Ma H (2012) Microfluidic biomechanical assay for red
blood cells parasitized by Plasmodium falciparum. Lab Chip 12(6):11431150.
27. Evans E, Leung A (1984) Adhesivity and rigidity of erythrocyte membrane in relation
to wheat germ agglutinin binding. J Cell Biol 98(4):12011208.
28. Williams RJ, Shaw SK (1980) The relationship between cell injury and osmotic volume
reduction: II. Red cell lysis correlates with cell volume rather than intracellular salt
concentration. Cryobiology 17(6):530539.
29. Lee P, Ye Z, Van Dyke K, Kirk RG (1988) X-ray microanalysis of Plasmodium falciparum
and infected red blood cells: Effects of qinghaosu and chloroquine on potassium,
sodium, and phosphorus composition. Am J Trop Med Hyg 39(2):157165.
30. White NJ, et al. (2014) Spiroindolone KAE609 for falciparum and vivax malaria. N Engl
J Med 371(5):403410.

Materials and Methods

E5462 | www.pnas.org/cgi/doi/10.1073/pnas.1414221111

Jimnez-Daz et al.

Supplemental Data and Methods for

(+)-SJ733: A Clinical Candidate for Malaria that Acts Through ATP4 to Induce
Rapid Host-Mediated Clearance of Plasmodium
Table of Contents
Supplemental Data ........................................................................................................ 2
Characterization of Cross-resistance of Mutant Strains of Malaria Resistant to (+)SJ733 to DHIQs and the Spiroindolones .................................................................... 2
Genetic Characterization of Mutant Strains of Malaria Resistant to (+)-SJ733 ........... 3
Sequencing Accession Numbers ................................................................................ 8
In Vivo Efficacy Summary ........................................................................................... 8
Toxicology Summary .................................................................................................. 9
Cytotoxicity (CC50) against panel of mammalian cell lines .................................... 9
In vitro human P450 inhibition (including time dependence)................................... 9
Receptor, enzyme, ion channel and kinase profile ............................................... 10
Phototoxicity risk ................................................................................................... 10
Oxidative Stress Burden ....................................................................................... 10
Reactive Metabolite Risk ...................................................................................... 10
Genotoxicity .......................................................................................................... 10
Cardiotoxicity ........................................................................................................ 11
Hemolysis ............................................................................................................. 11
In vivo rodent toxicology studies ........................................................................... 11
In vitro Microsomal Modeling of Metabolism ......................................................... 12
In vivo pharmacokinetics ...................................................................................... 13
Supplemental Methods ............................................................................................... 13
Compound Control Number ...................................................................................... 13
Animal Welfare.......................................................................................................... 13
Efficacy and in vivo clearance studies (Humanized mouse model, GSK) ................. 13
Pharmacokinetic analysis (Rat, CDCO) .................................................................... 14
Pharmacokinetic analysis (Dog, SRI) ........................................................................ 15
Transmission blocking (USF) .................................................................................... 15
In vitro P. falciparum sensitivity (SJCRH).................................................................. 16
Ex vivo efficacy of P. berghei (USF) ......................................................................... 16
Binding of (+)-SJ733 to P. falciparum infected erythrocytes (SJCRH) ...................... 17
Synthesis of (+)-SJ733 (Rutgers) .............................................................................. 17
Homology Modeling and Docking Methods ............................................................... 19
In vitro Selection of DHIQ resistant mutants (SJCRH) .............................................. 19
In vitro Selection of DHIQ resistant mutants (Columbia) ........................................... 20
In vitro Selection of DHIQ resistant mutants (UCSF) ................................................ 20
In vivo induction of resistance (USF) ........................................................................ 21
Whole-Genome Sequencing and Analysis ................................................................ 21
Quantitative PCR Analysis of Expression Levels ...................................................... 21
Production of transgenic P. berghei parasites........................................................... 22
Measurement of cytosolic [Na+] and pH (ANU) ......................................................... 23
Efficacy and parasite clearance studies (mouse models, USF) ................................ 23
1

Mutant Fitness study (UCSF) .................................................................................... 24

Time Lapse Microscopy Study (UCSF) ..................................................................... 25
Cellular Rigidity study (UCSF, UBC) ......................................................................... 25
Real Time Proliferation study (SJCRH) ..................................................................... 25
FACS analysis (SJCRH) ........................................................................................... 26
In Vitro Parasite Reduction Assay (GSK) .................................................................. 27
In Vivo Clodronate depleted rodent efficacy studies (USF) ....................................... 27
In Vitro stage specificity assay (Eskitis) .................................................................... 27
Supplemental Figure Legends ................................................................................... 29
Table of Tables
SUPPLEMENTAL TABLE 1: SENSITIVITY OF SELECTED MUTANT STRAINS TO
DHIQ'S AND SPIROINDOLONES ........................................................................... 3
SUPPLEMENTAL TABLE 2: WHOLE GENOME SEQUENCING OF SELECTED
MUTANTS ............................................................................................................... 4
SUPPLEMENTAL TABLE 3: CNV-SEQ RESULTS FOR WHOLE GENOME
SEQUENCING OF MUTANT VS. PARENTAL STRAINS, USING A 3KB WINDOW
WITH A SIGNIFICANCE THRESHOLD OF P<0.001. ............................................. 5
SUPPLEMENTAL TABLE 5. SUMMARY OF IN VIVO POTENCY OF SJ000557733 ... 9
SUPPLEMENTAL TABLE 6: IN VITRO METABOLISM .............................................. 12
SUPPLEMENTAL TABLE 8. CANINE PK PARAMETERS ......................................... 13
SUPPLEMENTAL TABLE 7. COMPARATIVE RODENT PK PARAMETERS ............. 13

Supplemental Data
Characterization of Cross-resistance of Mutant Strains of Malaria Resistant to (+)SJ733 to DHIQs and the Spiroindolones
A total of 17 strains resistant to (+)-SJ733 were either generated for this study or
tested from previously generated stocks. The strains were selected using one of three
strategies (see methods below) using drugs from three classes: the
dihydroisoquinolines (the subject of this paper), the spiroindolones(1), or a third series
of putative PfATP4 inhibitors from GlaxoSmithKline, whose structure has not yet been
disclosed. In general all strains examined exhibited cross-resistance to all putative
PfATP4 inhibitors, regardless of the drug used to select the strain, as measured by
relative fold-resistance to the parental strain (representative data in Supplemental Table
1). In our hands, historical variation of sensitivity of any given strain over time is 3-fold
from the average value. By this test, none of the strains vary significantly in their fold
sensitivity to each individual drug. This strongly suggests that the mutations are
inducing the same pharmacological sensitivity changes for each class of putative
PfATP4 inhibitors.

Supplemental Table 1: Sensitivity of Selected Mutant Strains to DHIQ's and Spiroindolones

Average Values (EC50 in uM)
UC7247

UC8279

609

3D7

K1

V1/S

camA
TP4

Dd2

ATP4I398F/P99
0R

ATP4D1247
Y

SJ72733

(r)-SJ000101247

0.33

0.20

0.60

0.06

0.07

0.08

1.06

0.05

0.45

0.13

0.34

(r)-SJ000101279

0.15

0.09

2.13

0.05

0.06

0.17

1.32

0.04

1.69

0.69

10

(+)-SJ000571311

0.10

0.04

0.31

0.02

0.02

0.06

0.18

0.01

0.14

0.07

3.0

15*

(+)-SJ000573359

0.03

0.02

0.03

0.03

0.02

0.09

0.01

0.05

0.02

1.0

15*

NITD-138

0.15

0.08

0.65

0.04

0.03

0.30

0.04

0.92

0.38

1.3

NITD-246

0.001

0.001

0.01

0.001

0.001

0.001

0.01

0.001

0.01

0.004

0.005

(+)-SJ000557733

0.19

0.07

0.44

0.03

0.04

0.06

0.42

0.02

0.18

0.10

15*

(-)-SJ000557733

12.61

3.75

9.03

0.93

1.14

5.23

12.85

0.93

10.20

3.12

15*

cmpd

SJ83
-733

15*

Curve did not provide saturated response, arbitrarily set at highest tested dose

(r)-SJ000101247

10

23

10

(r)-SJ000101279

44

31

40

16

208

(+)-SJ000571311

16

22

17

151

750

(+)-SJ000573359

10

36

517

NITD-138

18

22

36

NITD-246

12

14

(+)-SJ000557733

14

25

10

484

(+)-SJ000557733

14

10

14

11

16

484

Variation Across Compounds

mean resistance
standard deviation
resistance
fold-variation from average

19

19

16

249

447

12

11

142

326

Genetic Characterization of Mutant Strains of Malaria Resistant to (+)-SJ733

For six independent resistance selections, we used whole-genome sequencing to
discover mutations (Supplemental Table 2). For both parental parasite strains, and for
each selection, genomic DNA was prepared and libraries were sequenced on an
Illumina HiSeq-2500 using either 97 or 135 nt paired end reads (Materials and
Methods). After quality filtering and alignment to the reference sequence, the average
coverage ranged from >50-fold to >700-fold for the 14 nuclear chromosomes. Coverage
of the mitochondria and plastid genomes ranged from >200-fold to greater than 30,000fold. To detect genomic mutations, we analyzed the reads from each selection after
mapping to the reference genome, and then comparing exonic sequences for both the
parental and selected strains. These selections were not necessarily clonal populations,
and thus we reasoned that true drivers of drug resistance would be gain-of-function
mutations driven to purification as opposed to passenger mutations scattered
3

throughout the population. Therefore, we considered only non-synonymous mutations

that were present at 99% purity or higher, where purity was defined as the percent of
the total reads confirming a mutation for a given position in the genome. Using these
criteria, only a single mutated gene, the putative Na+ pump PfATP4, was shared in
common among all six selections. In all but one selection, 100% of the mapped reads
confirmed the detected mutation, all of which were subsequently confirmed by Sanger
sequencing. Notably, in five of the six selections, PfATP4 was the only mutated gene
detected.
Supplemental Table 2: Whole Genome Sequencing of Selected Mutants
Mutant
Strain

Parent
Drug
Strain Selecting

Mutation

Amino Acid
Reads
Position
Confirming
Mutation
Base Call

SJ81-733

3D7

SJ733

V415D

415

SJ82-733

3D7

SJ733

L350H

350

SJ83-733

3D7

SJ733

P412T

412

SJ72-311

3D7

SJ311

L350H

350

UC7-247

W2

SJ247

P966S

966

UC8-279

W2

SJ279

P966T

966

(32/32)
100%
(48/48)
100%
(67/67)
100%
(82/82)
100%
(479/480)
99.8%
(478/478)
100%

Parental
Reads
Confirming
Wild Type
Base Call
(613/613)
100%
(702/702)
100%
(639/639)
100%
(702/702)
100%
(585/585)
100%
(585/585)
100%

Average
Average
Parent
Mutant
Genome
Genome
Coverage Coverage
717

70

717

54

717

131

717

113

692

693

692

597

Each dataset was evaluated for the presence of copy number variants (CNVs) relative
to the parental strains using the R-package CNV-Seq [3]. Other than the variable
sequences proximal to the telomeres, no deletions of significance (p<0.001) were
detected relative to the drug sensitive parental strains. Furthermore, no amplified
regions relative to the parental strains were detected for any of the six selections
analyzed here.

Supplemental Table 3: CNV-Seq results for whole genome sequencing of mutant vs. parental strains, using a 3kb window with a significance threshold
of p<0.001.
Strain

CNV-ID

CNV-Chrm

CNV-start

CNV-end

Log2

CNV-pval

Gene

SJ81-733
SJ81-733
SJ81-733
SJ81-733
SJ81-733
SJ81-733
SJ81-733
SJ81-733
SJ81-733
SJ81-733
SJ81-733
SJ81-733
SJ81-733
SJ81-733
SJ81-733
SJ81-733
SJ81-733
SJ81-733
SJ81-733
SJ81-733
SJ81-733
SJ81-733
SJ81-733
SJ81-733
SJ81-733
SJ81-733
SJ81-733
SJ81-733
SJ81-733
SJ82-733
SJ82-733
SJ82-733
SJ82-733
SJ82-733
SJ82-733
SJ82-733
SJ82-733
SJ82-733
SJ82-733
SJ82-733

CNVR_1
CNVR_2
CNVR_3
CNVR_4
CNVR_5
CNVR_6
CNVR_7
CNVR_8
CNVR_9
CNVR_10
CNVR_11
CNVR_11
CNVR_12
CNVR_12
CNVR_12
CNVR_13
CNVR_13
CNVR_13
CNVR_14
CNVR_15
CNVR_16
CNVR_17
CNVR_18
CNVR_19
CNVR_20
CNVR_21
CNVR_21
CNVR_21
CNVR_22
CNVR_1
CNVR_1
CNVR_2
CNVR_2
CNVR_2
CNVR_3
CNVR_1
CNVR_2
CNVR_3
CNVR_3
CNVR_4

Pf3D7_09_v3
Pf3D7_08_v3
Pf3D7_08_v3
Pf3D7_08_v3
Pf3D7_07_v3
Pf3D7_07_v3
Pf3D7_11_v3
Pf3D7_11_v3
Pf3D7_11_v3
Pf3D7_13_v3
Pf3D7_13_v3
Pf3D7_13_v3
Pf3D7_13_v3
Pf3D7_13_v3
Pf3D7_13_v3
Pf3D7_01_v3
Pf3D7_01_v3
Pf3D7_01_v3
Pf3D7_06_v3
Pf3D7_06_v3
Pf3D7_06_v3
Pf3D7_12_v3
Pf3D7_12_v3
Pf3D7_05_v3
Pf3D7_04_v3
Pf3D7_02_v3
Pf3D7_02_v3
Pf3D7_02_v3
Pf3D7_10_v3
Pf3D7_13_v3
Pf3D7_13_v3
Pf3D7_14_v3
Pf3D7_14_v3
Pf3D7_14_v3
Pf3D7_10_v3
Pf3D7_11_v3
Pf3D7_11_v3
Pf3D7_13_v3
Pf3D7_13_v3
Pf3D7_13_v3

1488751
20251
39751
1437751
552751
567751
23251
33751
1923751
35251
2798251
2798251
2877751
2877751
2877751
74251
74251
74251
1302751
1359751
1407751
751
767251
21751
48751
876751
876751
876751
1646251
2798251
2798251
3272251
3272251
3272251
1574251
36751
1923751
2798251
2798251
2877751

1494750
26250
48750
1443750
558750
573750
30750
44250
1931250
42750
2805750
2805750
2894250
2894250
2894250
80250
80250
80250
1310250
1365750
1413750
9750
773250
27750
54750
882750
882750
882750
1652250
2805750
2805750
3282750
3282750
3282750
1580250
44250
1931250
2805750
2805750
2883750

-1.25
-1.15
-1.13
-1.20
-1.07
-1.20
-1.37
-1.26
-1.09
-1.12
-1.26
-1.26
-1.31
-1.31
-1.31
-1.38
-1.38
-1.38
-1.12
-1.17
-1.18
-1.39
-1.31
-1.25
-1.18
-1.12
-1.12
-1.12
-1.31
-1.34
-1.34
-1.08
-1.08
-1.08
-1.06
-1.14
-1.38
-1.36
-1.36
-1.20

1.91E-222
1.59E-205
3.05E-301
7.81E-218
4.56E-189
9.14E-221
2.03E-307
0.00E+00
4.79E-230
5.43E-244
4.41E-283
4.41E-283
0
0.00E+00
0
8.71E-241
8.71E-241
8.71E-241
9.20E-249
3.06E-211
1.23E-212
0
2.24E-243
5.05E-228
4.73E-211
9.20E-196
9.20E-196
9.20E-196
2.10E-241
6.00E-235
6.00E-235
2.45E-242
2.45E-242
2.45E-242
5.39E-141
0.00E+00
0.00E+00
0.00E+00
0.00E+00
0

PF3D7_0937700

Genestart
1492373

Geneend
1493708

PF3D7_1371000
PF3D7_1371200
PF3D7_1373300
PF3D7_1373400
PF3D7_1373500
PF3D7_0101300
PF3D7_0101400
PF3D7_0101500

2800004
2802527
2877908
2881591
2884786
74563
75982
78241

2802154
2802686
2879141
2882956
2892340
75366
76809
79891

PF3D7_0222000
PF3D7_0222100
PF3D7_0222200

876984
878468
881469

877832
879251
882267

PF3D7_1371000
PF3D7_1371200
PF3D7_1479600
PF3D7_1479700
PF3D7_1479800
PF3D7_1039200

2800004
2802527
3272578
3276230
3279500
1576441

2802154
2802686
3273848
3277501
3280662
1576991

PF3D7_1371000
PF3D7_1371200
PF3D7_1373300

2800004
2802527
2877908

2802154
2802686
2879141

Description
rifin RIF
No overlapping feature
No overlapping feature
No overlapping feature
No overlapping feature
No overlapping feature
No overlapping feature
No overlapping feature
No overlapping feature
No overlapping feature
18S ribosomal RNA
5.8S ribosomal RNA
rifin RIF
rifin RIF
PfEMP1 VAR
Pfmc-2TM
RESA-like pseudogene
PfEMP1 pseudogene
No overlapping feature
No overlapping feature
No overlapping feature
No overlapping feature
No overlapping feature
No overlapping feature
No overlapping feature
RESA-like protein
Pfmc-2TM
exported protein
No overlapping feature
18S ribosomal RNA
5.8S ribosomal RNA
rifin RIF
rifin RIF
rifin RIF
pseudogene
No overlapping feature
No overlapping feature
18S ribosomal RNA
5.8S ribosomal RNA
rifin RIF

SJ82-733
SJ72-311
SJ72-311
SJ72-311
SJ72-311
UC7-247
UC8-279_6

CNVR_4
Pf3D7_13_v3
CNVR_1
Pf3D7_09_v3
CNVR_2
Pf3D7_13_v3
CNVR_2
Pf3D7_13_v3
CNVR_3
Pf3D7_12_v3
No CNVs detected
No CNVs detected

2877751
1488751
2798251
2798251
751

2883750
1494750
2805750
2805750
8250

-1.20
-1.07
-1.20
-1.20
-1.26

0
1.15E-288
0.00E+00
0.00E+00
0.00E+00

PF3D7_1373400
PF3D7_0937700
PF3D7_1371000
PF3D7_1371200

2881591
1492373
2800004
2802527

2882956
1493708
2802154
2802686

rifin RIF
rifin RIF
18S ribosomal RNA
5.8S ribosomal RNA
No overlapping feature

We next analyzed expression levels of mutant and wild type PfATP4, with or
without treatment with (+)-SJ733. The relative expression of PfATP4 in both mutant and
wild type strains was measured with respect to time and treatment with, or without drug.
Late-ring stage synchronized parasites were mock treated, or treated with 540nM
SJ733, and time points collected. PfATP4 mRNA expression was measured by qRTPCR relative to a housekeeping gene, Pf-Chitinase, in triplicate. No significant changes
in relative expression were detected in either strain, with or without drug.
Supplemental Table 4: Relative expression of wild type or mutant Pf ATP4, with or
without treatment with SJ733.
Pf ATP4
Chitinase
Ct Ratio
Strain
Hours SJ733 Avg
Std.Dev
Avg Std.Dev
PfATP4 /
Chitinase
UC80
21.6
0.1
21.1
0.1
1.02
279_6
UC80
+
23.4
0.1
21.7
0.0
1.08
279_6
UC82
23.8
0.1
23.5
0.1
1.01
279_6
0.3
1.01
UC82
+
22.7
0.1
22.5
279_6
UC84
21.5
0.1
21.8
0.0
0.99
279_6
UC84
+
22.5
0.1
22.4
0.2
1.01
279_6
UC88
23.9
0.1
25.0
0.2
0.96
279_6
0.2
0.98
UC88
+
23.7
0.0
24.3
279_6
W2
0
22.1
0.0
21.8
0.2
1.02
W2
0
+
23.6
0.1
23.0
0.1
1.03
W2
2
22.3
0.1
23.8
0.2
0.93
W2
2
+
23.4
0.1
23.3
0.2
1.00
W2
4
23.7
0.0
24.7
0.1
0.96
W2
4
+
22.5
0.1
22.9
0.2
0.98
W2
8
22.1
0.0
25.1
0.2
0.88
W2
8
+
22.1
0.1
23.5
0.1
0.94
Finally we examined a number of other strains resistant to (+)-SJ733 by Sanger
sequencing of the ATP4 locus. These included strains selected with other DHIQs as
well as strains selected with other drugs targeting PfATP4. All of these shared
mutations in the locus.

Supplemental Table 5: Plasmodium spp. Mutations that confer resistance to

DHIQ or spiroindolones Sanger Sequencing
Parent
Strain

Drug
Selecting

Mutant
Strain

Mutation

Amino Acid Fold-change

Position

K1
K1
Dd2
K1

NITD609
NITD609
SJ733
NITD609

K1-8b
K1-9a
DD2-SJ16-D2
K1-8a

Gln->His
Asn->Tyr
Gly->Ser
Ile->Phe

172
355
358
398

6
8
5
11

Dd2
K1
P. berghei
ANKA
Dd2
Dd2
DD2
3D7
W2

SJ733
NITD609
SJ311

DD2-SJ16-B7
K1-9c
Pber

Leu->Phe
Pro->Ala
Pro->Ser

928
966
437

3
12
2

NITD678
NITD609
NITD609
GSK
GSK

NITD678-R #1
NITD609-R #2
NITD609-R #1
W2-F6-ATP4P412L
3D7-C9-ATP4F917L

Gly->Arg
Thr->Asn
Pro->Arg
Pro->Leu
Phe->Leu

223
418
990
412
917

7
7
9
100
10

Taken together with the lack of detected copy number variations, these data
support and strengthen the notion that mutation of PfATP4 was required for resistance
to SJ733 and its derivatives.
Sequencing Accession Numbers
The sequencing data discussed above has been deposited with NCBI in
Bioproject PRJNA253899 accessible at http://www.ncbi.nlm.nih.gov/bioproject/253899.
In Vivo Efficacy Summary
SJ733 has been tested in several murine models of malaria. Testing of (rac)SJ733 against P. berghei, in a standard Peters test, revealed an apparent ED90 of
roughly 40 mg/kg and that a single oral dose of 200 mg/kg appeared to reach the ED99
or higher. This result has been confirmed by three independent replicates at three other
laboratories. There is also a survival advantage in the P. berghei model of 4 to 12 days
relative to the untreated controls and a single oral dose of 200 mg/kg is as efficacious
as four daily oral doses at 100 mg/kg.
Subsequent testing of (+)-SJ733 in the Nod-SCID P. falciparum model revealed a
significant potency difference between the P. berghei model and the P. falciparum
model with (+) SJ733 being roughly 10-fold more potent against P. falciparum. As
expected, the potency of (+)-SJ000557733 was roughly 2-fold higher, giving an ED90 of
roughly 2 mg/kg in this model. This disparity between the P. berghei and P. falciparum
models is seen for all three classes of PfATP4 inhibitors. In the case of the
spiroindolones, which have progressed to human trials, the P. falciparum model has
been established as the one that correlates most closely with efficacy in humans.
8

Supplemental Table 5. Summary of In Vivo Potency of SJ000557733

Measure

(r)-SJ733

(+)-SJ733

CQ

AS

Pyr

3.8

1.9

4.3

11.1

0.9

P. falciparum
AUCED90 (Mhr)

1.5

3.1

nd

5.2

P. berghei ED90 (mg/kg)

40

--

--

--

P.
berghei
(Mhr)

80

--

--

--

P. falciparum ED90 (mg/kg)

AUCED90

Toxicology Summary
Cytotoxicity (CC50) against panel of mammalian cell lines
(+)-SJ733 has been tested against a panel of mammalian cell lines: 1) HepG2
(human hepatocellular liver carcinoma cell line), a clumping/adherent marker line for
liver toxicity that can be used for experiments involving stably and transiently
transfected reporters or microarrays; 2) HEK 293 (human embryonic kidney fibroblasts),
an adherent marker line used for kidney toxicity and experiments involving stably and
transiently transfected reporters or microarrays; 3) BJ (a fibroblast cell line), an adherent
marker line for general toxicity; and 4) Raji (human B-cell lymphoma cell line) a
suspension marker line for B-cell toxicity. No significant cytotoxicity or growth inhibition
was exhibited for any of these lines at concentrations up to 20 M; this has been the
case for all DHIQs tested. Additionally, (+)-SJ733 has been tested against Cellular
Dynamics iCell cardiomyocytes, a human induced pluripotent stem (iPS) cell-derived
cardiomyocyte. No significant cytotoxicity was noted with this line at concentrations up
to 25 M.
In vitro human P450 inhibition (including time dependence)
(rac)-SJ733 has been tested for inhibition of CYP450 isoforms 1A2, 2C9, 2C19,
2D6, and 3A4 using microsomal models, competing for turnover of established
substrates. At a fixed concentration of 10 M, there was no significant inhibition of any
isoform (< 50% inhibition of known substrate turnover). There was modest inhibition of
2C9 (25% at 10 M) and 3A4 (38% at 10 M). These studies have been repeated with
(+)-SJ733 and show no significant inhibition of any isoform. Additional studies have
examined time-dependent inhibition of p450 enzymes for both (+)-SJ733 and its Noxide, demonstrating that neither exhibits time-dependent inhibition of any p450. The
potential for induction of CYP450s was assessed by examining the effects of (+)-SJ733
on the induction of PXR or CAR responsive promoter constructs in HepG2 cells, either
in the presence or absence of rifampicin. There were no significant effects.

Receptor, enzyme, ion channel and kinase profile

(rac)-SJ733 has been profiled in a number of in vitro safety panels with no
significant signals being found. These findings are consistent with the mild toxicology
that has so far been observed during exploratory in vivo studies that will be described
below.
(rac)-SJ733 has been profiled by the NIMH Psychoactive drug screening
program (http://pdsp.med.unc.edu/) and by CEREPs preclinical safety panel that
contains many channels, transporters, and receptors found in the CNS and a small
number of other targets. To date, over 100 targets have been tested using a ligand
displacement assay with > 50% displacement of ligand by a fixed concentration of
SJ733 (10 M) being seen only with the serotonin receptor 5-HT2, histamine receptor
H1, and - and -opioid receptors. The affinity of (+)-SJ733 was evaluated by
competition with known ligand for these receptors and all affinities were 2 to > 10 M.
Follow up functional assays were carried out for the serotonin and opioid receptors
(+)-SJ733 showed no significant antagonism or agonism at any of the 5-HT2 or opioid
family receptors at doses up to 10 M. The potential for problematic interaction with
these targets was judged low and no further characterization was done.
(rac)-SJ733 has been tested for binding to the active sites of 451 human kinases
by Kinomescan. At a fixed concentration of 10 M, SJ733 showed no competition for
any substrate of any kinase tested exceeding 33%. Most kinases in the panel showed
signal. The potential for problematic interaction with human kinases was judged low
and no further characterization was done.
Phototoxicity risk
(+)-SJ733 clearly possesses a minor absorption peak at 290 nM. Therefore
there is a theoretical risk of phototoxicity. A study with (+)-SJ733 revealed minimal risk
of phototoxicity using the Oil Red method.(2)
Oxidative Stress Burden
In order to assess whether or not (+)-SJ733 was producing significant levels of
oxidative stress, its effects on the transcription of glutathione-s-transferase A1, quinone
oxidoreductase, and catalase all genes induced by NRF2 in response to oxidative
stress.(3) At a fixed dose of 20 M there were no effects on transcription of these
genes.
Reactive Metabolite Risk
The potential to produce reactive metabolites has been examined by Cyprotex
and (+)-SJ733 shows no tendency to produce such metabolites in the standard survey.
Genotoxicity
The genotoxic potential of (rac)-SJ733 was evaluated by AMES test at
concentrations of 3000, 300, 30, and 3 ug / plate against the TA100 strain of
Salmonella. Compound was tested both with and without S9 activation. The compound
was neither cytotoxic nor mutagenic at any tested concentration. A 5-strain AMES test
with (+)-SJ733 at the same concentrations with or without S9 activation has been
completed and was negative. The 3-cyano-4-fluoroaniline (SJ000568990) was tested in
a 5-strain model both with and without S9 activation over the same concentration range.
10

It was not cytotoxic or mutagenic at any concentration tested. The low risk of
genotoxicity with (+)-SJ733 was confirmed using an in vitro micronucleus test that was
negative.
Cardiotoxicity
The potential for cardiotoxicity was evaluated using three approaches: testing
against a panel of cardiac ion channels, testing against hERG in particular, and testing
for effects on contractile rate and rhythm in an -cardiomyocyte model. There was no
significant signal from any of these models.
Initial testing of cardiac ion channel activity was carried out at Chantest using a
screening concentration of 10 M of (rac)-SJ733 detecting activity using an Ionworks
instrument. The compound showed no inhibition of hNaV1.5, hERG, or hCav1.2 at this
concentration. A follow-up study was carried out for hERG using patch clamping and
monitoring potassium current with doses ranging from 100 nM to 10 M. (rac)-SJ733
showed no inhibition of hERG at any concentration tested. The N-oxide metabolite is
also without apparent risk.
Subsequent testing was carried out at Acea Biosciences where the effects of (+)SJ733 on rhythm and rate of beating of cardiomyocytes were monitored. The
compound was non-toxic to iPS-induced cardiomyocytes at concentrations up to 25 M.
When added to cells (+)-SJ733 had no effect on rate or rhythm at any dose except 23
M where it transiently and reversibly blocked beating. The candidate was judged to
have low risk for inducing changes in cardiac function.
Hemolysis
The risk for hemolysis by (+)-SJ733 in normal human blood was evaluated using
two approaches. First, the effects of the compound on erythrocyte integrity were
examined under conditions used for testing efficacy (low hematocrit, low protein). There
was no apparent effect at doses up to 10 M for up to 72 hours. Next, the effects of a
fixed concentration of (+)-SJ733 (15 M) upon fresh whole human and whole mouse
blood were examined by treating the samples with compound (or DMSO control) and
then carrying out a CBC using the clinical method. No significant changes were seen in
hematocrit, hemoglobin, red blood cell count, mean corpuscular volume, mean
corpuscular hemoglobin, or mean corpuscular hemoglobin concentration. Overall these
studies suggest there are no significant effects from (+)-SJ733 upon red blood cell
integrity per se.
In vivo rodent toxicology studies
(rac)-SJ733 was tested for toxicological responses initially in C57/B6 mice with
single doses ranged up to 200 mg/kg po and 15 mg/kg iv. Endpoints for the study
included a modified functional observational battery immediately following
administration; monitoring of body weight, signs, and symptoms for 48 h after
administration; and evaluation of hematological parameters, clinical chemistries,
pathology, and histopathology at the end of the study. There were no significant
changes to any of the measures after administration of (rac)-SJ733.
(+)-SJ733 was then tested for toxicological response in male Sprague-Dawley
rats with single oral doses ranging up to 750 mg/kg. Drug levels in plasma were
assessed at 4 and 24 hours post administration. Endpoints for the study included
11

monitoring body weight, signs, and symptoms for 72 hours post administration. No
serious adverse events were noted. The only observed sign was ruffled fur of 1 of 4
rats in the 750 mg/kg dose group. The TK satellites for this study indicated good
linearity of the relationship between dose and exposure through 200 mg/kg, with
continued but sub-proportional increases in exposure throughout the remaining dose
range.
(+)-SJ733 was also tested in a 7-day repeat dose toxicity study (with 7-day
recovery) in male rats at 0, 30, 60, 120, and 240 mg/kg po with a single doses given
each day for 7 days, followed by a 7-day recovery period. Endpoints included
monitoring of body weight, signs, and symptoms; and evaluation of hematological
parameters, clinical chemistries, pathology and histopathology at day 7; and the same
studies at day 14. Additionally, plasma drug levels were measured at 1, 4, 8, and 24
hours post dose on days 1 and 7. There were no significant changes in body weight or
signs and symptoms that were test article related. There were no significant changes in
any clinical chemistry parameters that were test article related. There were statistically
significant changes in hematocrit, hemoglobin, and red blood cell counts at all doses,
that were mildly dose related (hct reduced 9 to 15% from 30 to 120 mg/kg; hemoglobin
reduced 9 to 15% from 30 to 120 mg/kg; rbc count reduced 10 to 14% from 30 to 120
mg/kg; the 240 mg/kg group was an outlier with only 9% reductions of parameters).
However, all of these changes are within the normal ranges of historical controls and
recovered to baseline during the 7-day recovery period. They were judged to be not
significant adverse events. The only changes in histopathology were judged to be not
drug related. Based upon these considerations the consulting toxicologist set the
NOAEL and the MTD at both greater than 240 mg/kg.
In vitro Microsomal Modeling of Metabolism
Supplemental Table 6: In vitro metabolism

Mouse
t1/21

C
Clint
(
M)
20
1.2 0.1 19 2
4
0.8 0.1 29 1
0.8
0.6 0.1 37 2
1
hours; 2L/min/mg protein

t1/21
>4
>4
>4

Rat
Clint2

t1/21

<6
<6
<6

>4
>4
>4

12

Dog
Clint2
<6
<6
<6

t1/21

Human
Clint2

0.8
0.6
0.5

28 1
nd
27 1

In vivo pharmacokinetics
Supplemental Table 7. Comparative Rodent PK Parameters
Dose
(mg/Kg)

Species - route

Cmax
(
M)

AUC0-inf
(
M-hr)

Vss
(L/Kg)

Cltot
(mL/
min/kg)

T1/2
(h)

%F

Mouse i.v.
15
11 0.3 9.9 0.2 2.2 0.3
54 4
1.4
Rat i.v.2
5.5 0.5
15 1 3.6 0.3 11.4 0.3
11 2
Mouse p.o.
10
1.4 0.2 4.3 0.2
3.6
66
3
Rat p.o. solution
20 0.2
12 2
76 6
81
122 10
Rat p.o. suspension4
17 0.2
82
40 2
72
75 3
1
2
3
Racemate; mean values from 2 experiments (n=5); mean value (n=3) from PBS (pH 7.4 isotonic 50
mM phosphate buffered saline) based vehicle containing 1% (w/v) hydroxypropyl-cyclodextrin,10%
4
(v/v) ethanol, 10% (v/v) propylene glycol and 40% (v/v) PEG400; mean value (n=3) from aqueous
vehicle containing 0.5% (w/v) hydroxypropyl methylcellulose, 0.5% (v/v) benzyl alcohol and 0.4% (v/v)
Tween 80.
Supplemental Table 8. Canine PK Parameters
Dose
(mg/kg)

Cmax
(
M)

AUC0-inf
(
M-hr)

Vz
(L/kg)

Cltot
(mL/
min/kg)

T1/2
(h)

%F

Canine i.v.
3
15 5
2.8 1.7
3.4 0.9 10 5
Canine p.o.
3
3.2 0.6
41 13
2.6 0.8
2.8 0.9 11 5
115 30
Canine p.o.
30
26 2
244 53
2.1 0.8
4.6 1.1
51
74 16
1
mean value (n=3) from PBS (pH 7.4 isotonic 50 mM phosphate buffered saline) based vehicle
containing 1% (w/v) hydroxypropyl-cyclodextrin,10% (v/v) ethanol, 10% (v/v) propylene glycol and
39% (v/v) PEG400

Supplemental Methods
Compound Control Number
(+)-SJ733 is an abbreviation for the full St Jude Childrens Research Hospital control
number, which is SJ000557733.
Animal Welfare
All experimental animal work was conducted in compliance with the NRC Guide
in facilities accredited by the Association for Assessment and Accreditation of
Laboratory Animal Care International (AAALAC). The Institutional Animal Care and Use
Committee of each respective institution approved all experimental work.
Efficacy and in vivo clearance studies (Humanized mouse model, GSK)
In efficacy studies groups of n = 3 mice per dose level or n = 5 with mice treated
with different dose levels were used. The mice were non-myelodepleted, age-matched,
NODscid IL-2Rnull (Charles River, Gannat, France, obtained under license from The
Jackson Laboratory, Bar Harbor, ME). Female mice engrafted with human erythrocytes
13

were infected with 2107 P. falciparum Pf3D70087/N9 -infected erythrocytes suspended in

0.3 mL saline solution (day 0). A range of dose levels of (+)-SJ733 was administered to
mice QD from day 3 to day 6 in aqueous suspension (1% Hydroxypropyl cyclodextrin,
10% EtOH, 10% propylene glycol, 40% PEG-400, 39% PBS) by oral gavage at 10
mlkg-1. Parasitemia in peripheral blood of humanized mice was measured by flow
cytometry using SYTO-16 and anti-mouse erythrocyte phycoerythrine-conjugated Ter119 mAb (Pharmingen, San Diego, CA) in serial 2 L blood samples taken every 24
hours.(4)
Efficacy was expressed as the daily dose administered (mgkg-1) or the daily
exposure of compound in whole blood (AUC, ghml-1day-1) necessary to reduce
parasitemia at day 7 by 90 % with respect to vehicle-treated mice (ED90 and AUCED90,
respectively). The AUCED90 of (+)-SJ733 was estimated by fitting a four parameter
logistic equation for the log10 [parasitemia at day 7 for individual i] versus the AUC0-23h of
(+)-SJ733 in blood for individual i using GraphPad Prism 6.0.
The rate of parasite clearance was addressed by measuring the percentage of
parasitemia by flow cytometry, as described above, at 24, 48, 72, and 96 hours after
start of treatment of P. falciparum-infected mice. To study the effect of splenectomy on
parasite clearance, groups of n=3 splenectomized mice were treated orally qd with
Atovaquone (50 mgkg-1), Artesunate (50 mgkg-1), or SJ733 (25 mgkg-1). The results
were compared with data obtained in non-splenectomized mice treated with the same
regimen (n = 3 mice per compound, except for Atovaquone in which n = 4) in separate
experiments.
The levels of (+)-SJ733 in blood upon oral administration were measured in all
mice of the efficacy study. Serial samples of peripheral blood (25 l) were taken at 0.25,
0.5, 1, 3, 6, 8, 10 and 23 hours after the first administration, mixed with 25 l of water
containing 0.1% saponin for erythrocyte lysis, immediately frozen on dry ice and
samples stored at -80C until analysis. Protein precipitation by liquid-liquid extraction
was performed in a 96- well plate with filter system (MultiScreen Solvinert 0.45 m
Hydrophobic PTFE; Millipore). Briefly, 120 L of suitable organic solvent containing
internal standard were added to 10 L of blood/saponin lysate sample per well. The
plates were vortexed for 10 minutes filtrated and the filtrates were analyzed by
LC/MS/MS in positive ion mode with electrospray (Sciex API 4000 Triple Quadrapole
Mass Spectrometer, Sciex Division of MDS Inc., Toronto, Canada). Concentration
versus time data were analyzed by non-compartmental analysis (NCA) methods using
WinNonlin Professional Version 5.2 (Pharsight Corporation, Mountain View, CA).
Additional statistical analysis of the data was performed with GraphPad Prism Version
5.01 (GraphPad Software Inc., San Diego CA). The lower limit of quantification (LLOQ)
in these assays was 0.002 M.
All the experiments were approved by the DDW Ethical Committee on Animal
Research, performed at the DDW Laboratory Animal Science facilities accredited by
AAALAC, and conducted according to European Union legislation and GlaxoSmithKline
policy on the care and use of animals
Pharmacokinetic analysis (Rat, CDCO)
The pharmacokinetics of (+)-SJ733 were studied in overnight-fasted male
Sprague Dawley rats weighing 267 - 291 g pre-dose. Rats had access to water ad
14

libitum throughout the pre- and post-dose sampling period, and access to food was reinstated 4 h post-dose. (+)-SJ733 was administered intravenously as a 10 min constant
rate infusion (1.0 mL per rat, n = 3 rats) and orally by gavage (10 mL/kg, n = 3 rats). The
IV formulation consisted of pH 7.4 isotonic phosphate buffered saline containing 1%
(w/v) hydroxypropyl-cyclodextrin, 10% (v/v) ethanol, 10% (v/v) propylene glycol and
40% (v/v) PEG400 whereas the oral formulation was an aqueous suspension in 0.5%
(w/v) hydroxypropyl methylcellulose, 0.5% (v/v) benzyl alcohol and 0.4% (v/v) Tween
80. Aliquots of the formulations were retained for analysis of the actual dose
administered. Samples of arterial blood and total urine were collected at various time
points up to 24 h post-dose. Arterial blood was collected directly into borosilicate vials
(at 4 C) containing heparin, Complete (a protease inhibitor cocktail), potassium
fluoride, and EDTA to minimize potential for ex vivo degradation of (+)-SJ733 in
blood/plasma samples. Once collected, blood samples were centrifuged, supernatant
plasma was removed, and stored frozen (-20C) until plasma concentrations of (+)SJ733 were determined by quantitative LC-MS. The analytical LLOQ was 0.0011 M.
Plasma concentration versus time data were analyzed by non-compartmental methods.
The PK studies were conducted using established procedures in accordance with
the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes,
and the study protocols were reviewed and approved by the Monash Institute of
Pharmaceutical Sciences Animal Ethics Committee.
Pharmacokinetic analysis (Dog, SRI)
The pharmacokinetics of (+)-SJ733 were studied in overnight-fasted male beagle
dogs weighing 6 to 7.6 kg pre-dose. Dogs had access to water ad libitum throughout the
pre- and post-dose sampling period, and access to food was re-instated 4 h post-dose.
(+)-SJ733 was administered intravenously or orally by gavage. Samples of venous
blood and total urine were collected at various time points up to 72 h post-dose. Blood
was collected directly into borosilicate vials (at 4 C) containing EDTA to minimize
potential for ex vivo degradation of (+)-SJ733 in blood/plasma samples. Once collected,
blood samples were centrifuged, supernatant plasma was removed, and stored frozen (70C) until plasma concentrations of (+)-SJ733 were determined by quantitative LC-MS.
The analytical LLOQ was 0.02 M.
Transmission blocking (USF)
Transmission blocking potential for (+)-SJ733 was tested in a dose ranging study
in P. berghei infected mice. P. berghei-infected erythrocytes (1 x 106) were inoculated
IP into mice (ICR, female from Harlan) to initiate experiments. On day 4 when the blood
stage parasitemia was approximately 3%, groups of mice were treated per os with (+)SJ733, primaquine, chloroquine or vehicle control (1% HPC (w/v), 10% ethanol, 10%
propylene glycol, 40% PEG-400, and 39% PBS). For (+)-SJ733 single doses of 2.5, 5,
10, 20, 50, 100 and 200 mg/kg were given per os at 48, 24, 8, 4 and 1 hours before
mosquito feed. Then each mouse was anesthetized with IP injection of
Ketamine/Xylazine (100:10 mg/kg IP) and 50 adult female Anopheles stephensi
mosquitos were allowed to feed for 20-30 minutes. The mosquitos were then
maintained under standard laboratory conditions for 10 days to allow development of

15

oocysts. On day 11 transmission-blocking efficacy in vivo was assessed by counting the

number of oocysts in 15 mosquitos per group.
In vitro P. falciparum sensitivity (SJCRH)
Asynchronous parasites were maintained in culture based on the method of
Trager.(5) Parasite strains used were: 3D7, K1, D10, and V1/S provided by the MR4
Unit of the ATCC; 101247 resistant and 101279-6 resistant provided by Joe DeRisi;
D10yDHOD provided by Akhil Vaidya; camATP4 and Dd2attB provided by David
Fidock; C2A and C2B provided by Dennis Kyle; A6 provided by Michael Riscoe; and
609 and DD2 provided by Novartis. Parasites were grown in fresh group O-positive
erythrocytes (Key Biologics, LLC, Memphis, TN) in Petri dishes at a hematocrit of 4-6%
in RPMI-based media (RPMI 1640 supplemented with 0.5% AlbuMAX II, 25 mM
HEPES, 25 mM NaHCO3 (pH 7.3), 100g/ml hypoxanthine, and 5 g/ml gentamycin).
Cultures were incubated at 37C in a gas mixture of 90% N2, 5% O2, 5% CO2. Each
compound was tested in two fully independent experiments with each experiment using
triplicate assay plates, for a total of six replicates. 20 L of RPMI 1640 with 5 g/mL
gentamycin were dispensed into each well of an assay plate (Corning 384-well
microtiter plate, clear bottom, tissue culture treated, catalog no. 8807BC). To each well
was dispensed 40 nL of compound, previously prepared as a serial dilution series in a
separate 384-well white polypropylene plate (Corning, catalog no. 8748BC),
hydrodynamic pin transfer (FP1S50H, V&P Scientific Pin Head). Then 20 L of a
synchronized P. falciparum erythrocytic co-culture suspension (1% rings, 4%
hematocrit) was added to each well, thus giving a final hematocrit and parasitemia of
2% and 1%, respectively. Assay plates were incubated for 72 h, and the parasitemia
was determined by a previously described method(6): Briefly, 10 L of dye in PBS (10X
Sybr Green I, 0.5% v/v triton, 0.5 mg/ml saponin) was added to each well. Assay plates
were shaken for 1 min, incubated in the dark for 90 min, then read with the Envision
Multilabel Reader at Ex/Em wavelengths of 485nm/535nm. EC50s were calculated with
the proprietary Robust Investigation of Screening Experiments (RISE) software.
Ex vivo efficacy of P. berghei (USF)
ICR female mice (Harlan Laboratories) were inoculated with either P. berghei
(ANKA), PbSJ1311R (SJ1311 resistant P. berghei), transgenic-PfATP4 (PfATP4
expressing line of P. berghei), P. chabaudi chabaudi or P. vinckei vinckei. When
parasitemia exceeded 3%, blood was collected from infected mice by cardiac puncture
into CPDA anticoagulant containing 50 g/ml gentamycin. The blood was then washed
three times in RPMI 1640 media and adjusted to 1% parasitemia and 4% hematocrit in
phenol free RPMI1640 supplemented with 10% heat-inactivated mouse serum and 50
g/ml gentamycin and used for ex vivo susceptibility testing in 96 well microtiter plates.
All drugs were dissolved in DMSO (except CQ in dH2O). The drugs were serially diluted
3-fold in 96 well plates by using a Biomek 3000. All assays were initiated with drug
exposure and simultaneous addition of 3H Hypoxanthine (PerkinElmer, CA, USA). The
parasites were further grown for 24 hours under atmospheric conditions of 5% CO2, 5%
O2 and 90% N2 before plates were harvested and incorporation of 3H-hypoxanthine
assessed in a TopCount Scintillation counter. Dose response data were analyzed with a
non-linear logistic dose response function (DataAspects Plate Manager, DataAspects
16

Corporation, California, USA) to determine the 50% inhibitory concentration (IC50) for
each drug. Two biological replicates were performed for each parasite.
Binding of (+)-SJ733 to P. falciparum infected erythrocytes (SJCRH)
Parasites were grown in the presence of fresh group O-positive erythrocytes
(Lifeblood Memphis, TN) in Petri dishes at a hematocrite of 4-6% in a RPMI based
media (RPMI 1640 supplemented with 0.5% AlbuMAX II, 25mM HEPES, 25mM
NaHCO3 (pH 7.3), 100 g/mL hypoxanthine, and 5 g/mL gentamycin). Cultures were
incubated at 37C in a gas mixture of 90% N2, 5% O2, and 5% CO2. Parasites were
MACS purified (90-95%). 200 L of uninfected red blood cells (uRBCs) or infected red
blood cells (iRBCs) were seeded on a flat bottom 48-well tissue culture plate (Corning).
50 L of a solution containing 3H-SJ000557733 (SA:7 Ci/mmol) in RPMI based media
were added to each well to reach the desired concentration in triplicate (DMSO control,
1.2 nM, 12 nM, 55 nM, 285 nM, and 600 nM). Plates were incubated for 3 h at 37C in
a gas mixture of 90% N2, 5% O2, and 5% CO2. Each solution was transferred to a 1.5
mL microcentrifuge tube containing 300 L of dibutyl phthalate. The tubes were
centrifuged for 30 s at 10000 x g. 20 L of supernatant were transferred to new tubes.
The oil was discarded. 150 L of bleaching solution (EtOAc:AcOH(glacial):H2O2, 1:1:1)
was added to the pellets and the supernatant and heated at 99 C for 30 min. 50 L of
HCl(cc) was added to each hot solution and let to cool to room temperature. Each
solution was transferred to 20 mL scintillation vials followed by 6 mL scintillation cocktail
(Ultima Flo). Each vial was left at room temperature in the dark overnight and read on a
table-top counter (Beckman LS 6000TA). The evident kd was 51 nM.
Synthesis of (+)-SJ733 (Rutgers)
The key step for the preparation of (+)-SJ733 is the reaction of an aldimine with
homophthalic anhydride to yield the fully elaborated 4-carboxy DHIQ nucleus 1.(7)
Thus 2,2,2-trifluoroethylamine and 3-pyridinecarboxaldehyde (1.5:1 respectively) were
slurried in toluene, cooled in an ice bath, and treated with 1.5 equivalents of sodium
hydroxide as a 50% aqueous solution. After warming to ambient temperature and
stirring for several hours, the phases were separated, and the aqueous phase extracted
with additional toluene. The combined organic phases were dried over sodium sulfate
and concentrated to a yellow oil (solidifies at -5oC) in 94% yield based on the aldehyde
and used without further purification. The aldimine was dissolved in methylene chloride
to which 2 equivalents of N-methylimidazole was added. The solution was stirred for 30
minutes and 1 equivalent of homophthalic acid was then added. This solution was then
stirred for 24 hours, diluted with brine, and adjusted to pH 4.5 with concentrated HCl.
The resulting mixture was stirred for an additional 24 hours during which time a white
precipitate formed. The precipitate was isolated by filtration, dried and then slurried in a
4:1 mixture of cyclopentyl methyl ether and methanol (7.5 mL/gram of crude product).
The slurry was then heated to reflux for 1 hour, cooled to room temperature with stirring,
filtered and dried at 50oC in vacuuo to give a 74% yield of 1 that was 98% pure by
HPLC. (mp = 229-230oC). A solution of 1 and 1.5 equivalents of 5-amino-2fluorobenzonitrile in acetonitrile was treated with 1.1 equivalents of phosphorus(V)
oxychloride and refluxed for 15 hours. The reaction mixture was then concentrated.
The resulting material was re-dissolved in ethyl acetate and poured into a pH 9 aqueous
17

solution of sodium dihydrogenphosphate. The resulting organic layer was separated

and the aqueous layer extracted several times with additional ethyl acetate. The
combined organic phases were dried over sodium sulfate and concentrated to afford 6 g
(12.8 mmole, 90%) of highly pure racemic SJ733. Recrystallization from isopropyl
alcohol afforded gave 70% of analytically pure racemic SJ733 based on homophthalic
anhydride. (mp = 210-212oC)
The synthesis of (+)-SJ733 was accomplished through the resolution of 1 to
afford good yields of (+)-1 by the following methods. A slurry of 1 and 2 equivalents of
(1S,2S)-trans-1-amino-2-indanol was suspended in 1:1 propionitrile-heptane (30
mL/gram of 1) and heated to reflux for 3.5 hours. The suspension was filtered and the
solids washed with additional solvent mixture. The procedure was completed once
again to yield, after filtration and drying in vacuuo, an 85% yield of a 1:2 salt (acid to
amine respectively, mp = 176-178 oC). Liberation of (+)-1 was accomplished by
dissolving the 1:2 salt in aqueous 20% acetic acid (30 mL/gram of salt) followed by
exhaustive extraction with ethyl acetate. Drying and concentration of the combined
organic phases gave a 76% yield of (+)-1 based on racemic 1. (mp = 126-130 oC; []D =
+62 o, c = 1, MeOH; 99.7% ee by chiral HPLC). Material prepared in this manner was
subjected to the procedures described above to afford 95% yields of 95% pure product
by HPLC analysis. Recrystallization of this material from isopropyl alcohol afforded 6570% yields of pure (+)-SJ733 (mp = 169-170 oC, []D = +147o, c = 0.45 , MeOH). The
product as obtained directly from the reaction described above was disolved in ethanol
(3 mL/gram of material) with heating. The resulting solution was seeded with a sample
of previously obtained HCl salt and a slight excess of HCl was added as an ethereal
solution. After stirring over night at room temperature, the suspension was filtered,
washed with additional ethanol and dried to yield 93% of the desired (+)-SJ733
hydrochloride. This material was then slurried in ethanol and heated to reflux for 3
hours (10 mL/gram of product). After cooling to room temperature the suspension was
filtered, washed with additional ethanol and thoroughly dried at 62 oC in vacuuo to give
84% of (+)-SJ733 hydrochloride. (mp = 275-277 oC; []D = +137.5 o, c = 1, MeOH)
Analytical data:
Aldimine: . 1H NMR (300 MHz, DMSO-d6) 4.40 (m, 2H), 7.52 (m, 1H), 8.18 (m, 1H),
8.61 (s, 1H), 8.70 (dd, J = 1.8, 4.8 Hz, 1H), 8.92 (d, J = 1.8, 1H). MS (ESI, [M +H] +),
189.5.
Racemic Acid: 1H NMR (300 MHz, DMSO-d6) 4.03 (m, 1H), 4.29 (s, 1H), 4.66 (m,
1H), 5.59 (s, 1H), 7.22-7.32 (m, 2H), 7.40 (m, 1H), 7.42-7.54 (m, 2H), 7.98 (dd , J = 2,
6.9 Hz, 1H), 8.35 (d, J = 2 Hz, 1H), 8.41 (dd, J = 2, 4.8 Hz, 1H). MS (ESI, [M +H] +),
351.6.
Racemic SJ733: 1H NMR (300 MHz, DMSO-d6) 4.03 (m, 1H), 4.29 (s, 1H), 4.66 (m,
1H), 5.59 (s, 1H), 7.22-7.32 (m, 2H), 7.40 (m, 1H), 7.42-7.54 (m, 2H), 7.98 (dd , J = 2,
6.9 Hz, 1H), 8.35 (d, J = 2 Hz, 1H), 8.41 (dd, J = 2, 4.8 Hz, 1H). MS (ESI, [M +H] +),
351.6.
(+)-SJ773 hydrochloride: . 1H NMR (500 MHz, Methanol-d4) 4.20~4.35 (m, 1H), 4.40
(s, 1H), 4.50-4.70 (m, 1H), 5.80 (s, 1H), 7.25-7.36 (m, 2H), 7.50-7.60 (m, 2H), 7.80-7.90

18

(m, 1H), 7.90-8.10 (m, 1H), 8.10-8.16 (m, 1H), 8.13-8.16 (m, 1H), 8.41 (d, J = 8 , 1H),
8.76 (d, J = 6, 1H), 8.84 (s, 1H).
Homology Modeling and Docking Methods
A four-stage approach was used to assess the physical relationship of SJ733
with respect to PfATPase4 resistance mutations, structure, function, and interactions.
First, the Iterative Threading Assembler pipeline (I-TASSER)(8) was used to find all
relevant templates and build atomic models. The PfATPase4 protein sequence was
downloaded from UniProtKB.(9) The pipeline limiting templates were then re-run to Xray crystallographic protein structures with >0.4 TM-score structural similarity to the
most realistic initial models (PDB identifiers 3kdp, 2zxe, 2dqs, 3b8c, and 3rfu). Second,
the resistance conferring mutations were mapped to the models. Third, the docking of
(+)-SJ733 to the models was done using DOCK v6.6(10) across the entire accessible
extracellular surface. Fourth, the ligands co-crystallized with structurally similar proteins
were mapped to the docked PfATPase4 model using CO-FACTOR.(11)
The estimated accuracy of the PfATPase4 model is 0.58 0.14 by TM-score,
which signifies approximately 12.0 4.4 CRMSD, describing a mid-resolution model.
Accuracy estimates are calculated by similarity to template structures; the majority of
non-concordance for the model arises from distant extracellular domains. The
transmembrane domain and specifically the region around the mutations and putative
SJ733 binding site align well to all template structures with high resolution in this region.
Of note, the structurally resolved portion of phosphatidylethanolamine in the rabbit
sarcoplasmic/endoplasmic reticulum calcium ATPase 1 structure (PDB identifier 2dqs)
overlaps the putative (+)-SJ733 binding site, adding evidence to this pocket binding
ligands.
An open pore connects ~9 from the putative PfATPase4 (+)-SJ733 binding site
to potassium ions in the spiny dogfish Na+/K+-ATPase structure (PDB identifier 2zxe)
and the rubidium ions in the pig Na+/K+-ATPase (PDB identifier 3kdp). This occurs at
the kink in the 3rd transmembrane alpha-helix (corresponding to PfATPase4 residues
406-410). This pore and kink occur in all homologous pumps, suggesting that (+)-SJ733
blocks ion transport.
In vitro Selection of DHIQ resistant mutants (SJCRH)
10 mL of asynchronous culture suspensions (2% hematocrit), at different parasite
densities (104, 105, 106, 107, and 108 parasites), were added to each well of a 6-well
plate (Corning, clear, tissue culture treated, catalog no. 3516). (+)-SJ733 was added to
each well to make a final compound concentration of 1.8 M, corresponding to 30 x
EC50 of the compound. Three wells were used for each parasite density. Plates were
incubated at 37 C under an atmosphere of 90% N2, 5% O2, 5% CO2 for 90 days under
constant drug pressure. The media of each well was replaced 3 times a week with
freshly made media containing a compound concentration of 30 x EC50. In addition,
each well was split (1:2) once a week. Parasite outgrowth was monitored 3 times a
week by transferring quadruplicate 40 L aliquots from each well into a 384-well assay
plate (Corning 384-well microtiter plate, clear bottom, tissue culture treated, catalog no.
8807BC) and determining parasitemia by a previously described method (6): Briefly, an
10 L of the following solution in PBS (10X Sybr Green I, 0.5% v/v triton, 0.5 mg/ml
19

saponin) was added to each well. The 384-well assay plates were shaken for 1 min,
incubated in the dark for 90 min, and then read with the Envision Multilabel Reader at
Ex/Em of 485nm/535nm. Cultures from wells that showed outgrowth were transferred to
a large Petri dish and were allowed to expand under constant compound pressure (30 x
EC50) for subsequent EC50 determination and deep sequencing experiments. Cultures
from wells that did not show outgrowth were discarded after 90 days.
In vitro Selection of DHIQ resistant mutants (Columbia)
To generate mutants resistant to (+)-SJ733, the recently derived B2 clone of the
P. falciparum strain Dd2 was expanded to triplicate 100 mL flasks each with 109 infected
red blood cells (2.5% parasitemia, 4% hematocrit). Parasites were exposed to 200 nM
of compound (~5 the IC50 value of the Dd2 parent) and resistant parasites were first
detected after 1622 days. Clones were obtained by limiting dilution, and their IC50
values were determined by flow cytometry (12, 13).
Mutants resistant to NITD609 were selected from a clone of the K1 strain, using
a 5 IC50 selection procedure applied to triplicate flasks of 109 infected red blood cells,
as described above. Resistant mutant clones were obtained by limiting dilution, and
their IC50 values were determined by flow cytometry (12, 13).
In vitro Selection of DHIQ resistant mutants (UCSF)
Two cultures of W2 parasites were expanded to a total quantity of 1010 parasites
at 3% hematocrit in 500 mL RPMI (15 mL of 100% RBCs) and then each culture
inoculated into a hyperflask cell culture vessel from Corning. Upon seeding the
hyperflasks (Day 0), media was supplemented with 50 nM SJ101247 for one hyperflask
and 70 nM SJ101279 for the other hyperflask. For the next seven days, media was
changed daily with fresh drugs added at 50 nM SJ101247 and 70 nM SJ101279. Blood
smears were made each day for the first seven days of selection to ensure parasitemia
in the hyperflasks dropped to 0%. By Day 4, parasitemias were determined to be 0%
(based on microscopic inspection of 10 fields of >200 RBCs per field with no visible
infected RBCs). On Day 7, 1mL of 100% RBCs from each hyperflask was used to
inoculate a 50 mL culture with fresh blood added (final 3% hematocrit) and without drug
pressure (normal RPMI media). Subsequently, the hyperflask vessels were split 1:2 with
fresh blood added and kept under constant drug pressures. Following Day 7, both the
hyperflask cultures and 50mL no drug cultures were maintained by changing media
every three days (+ drug for hyperflask cultures, - drug for 50 mL cultures) and splitting
1:2 with fresh RBCs every six days. Blood smears were made and Giemsa stained
alongside media changes and 1:2 splits to inspect cultures for parasite recrudescence.
On Day 16, parasites were visualized in the blood smear from the 50 mL (no drug)
culture derived from the hyperflask drugged with SJ101247. On Day 28, parasites were
visualized in the blood smear from the 50 mL (no drug) culture derived from the
hyperflask drugged with SJ101279. Subsequent EC50 calculations by flow cytometry of
resistant strains confirmed approximately 10-fold resistance to respective drugs
compared to non-resistant W2 strain.

20

In vivo induction of resistance (USF)

To assess the acquisition of resistance to DHIQ analogs in vivo, we used (+)SJ311, a DHIQ analog of (+)-SJ733, and P. berghei (GFP) infected mice. Mice were
inoculated IP with 1 x 106 P. berghei infected erythrocytes and on day 3 post-infection
animals were treated with 200 mg/kg (+)-SJ311 qd for three days and monitored for
parasitemia beginning on day 6. Upon recrudescence of blood stage parasitemia, blood
from the infected animal was used to inoculate new mice for subsequent rounds of drug
exposure. Following the first two rounds of drug exposure parasite recrudescence
occurred on day 14 of the study (8 days following the last dose); in subsequent rounds
recrudescence occurred earlier and by the sixth round of treatment blood stage
parasitemia never cleared. After the 8th round of drug exposure blood was collected
from infected mice and gDNA prepared for Sanger sequencing of PbATP4 (as
described below).
Whole-Genome Sequencing and Analysis
Genomic DNA was isolated by TRIzol purification as per the manufacturers
protocol (Life Technologies, Inc.), from populations of infected RBCs selected for
resistance to SJ733 and its derivatives. Approximately 500 ng of gDNA was used as
input for the Nextera DNA Sample Prep Kit (Illumina, Inc.) to produce dual indexed
paired-end libraries for sequencing as per the manufacturers protocol. Quantified gDNA
libraries were sequenced on an Illumina HiSeq-2500. Raw reads, consisting of either
97 or 135nt paired-end sequences, were filtered for low quality reads, using the
PriceSeqFilter module, available as a component of the PRICE metagenomic
assembler
software
package
[1]
and
publicly
available
at
(derisilab.ucsf.edu/software/PRICE/). For a given read, all bases were required to have
per-base quality of 99% accuracy or better to pass filter. Reads that passed filter were
mapped to the 3D7 reference genome (PlasmoDB release 9.3) using the short read
alignment software bowtie [2], keeping reads that were uniquely mapping with no more
than a single mismatch. Following mapping, alignments were evaluated for the
presence of copy number variants (CNVs), overall coverage, and mutations. For CNVs
and coverage calculations, the R-package CNV-Seq [3] was used in conjunction with
samtools [4] to detect the presence of amplifications or deletions, using a 3kb sliding
window and a p-value threshold of <0.001, relative to the parental strain used to initiate
each drug selection.
For mutation detection, a custom written python package
(available upon request) was used to compare each alignment in the drug selected
strain relative to the parental strain and the 3D7 reference genome. For each detected
variant, the number reads featuring that variant was reported, as well as the percentage
of the total reads for that nucleotide position and variant. To account for spurious
sequencing errors, only variants confirmed by 20 reads or more were considered
reliable.
Quantitative PCR Analysis of Expression Levels
W2 parent parasites and UC8-279_6 resistant parasites, harboring theP996T
mutation in PfATP4, were synchronized grown in their respective media (RPMIc for W2;
RPMIc +90nM SJ733 for UC8-279 resistant strain, in order to prevent the allele from
21

reverting back to wild type). Late-ring stage parasites from each strain were grown with
drug (540nM SJ733) or without drug, and time points were harvested at 0, 2, 4, and 8
hours. RNA was prepared by Trizol/Phenol/Choloroform extraction and ethanol
precipitation, followed by DNAse treatment and purification using Zymo RNA Clean and
Concentrator columns (Zymo, Inc). First strand cDNA synthesis was carried out using
500ng of total RNA using SuperScript III with random primers. RNA was then removed
by treatment with RNaseH. qPCR reactions were prepared using Roche SYBR GREEN
480 master mix (Roche, Inc), using primers specific for PfATP4 and PF3D7_1252200
(Pf-chitinase), and run on a Roche LightCycler 480. All reactions were prepared in
triplicate. Primers and cycle conditions were as follows:
Pf ATP4:
5 ACCGTCTCGAGAAGTTGTTGT
5 GGTTGGATCTTTATCTGCATG
PF3D7_1252200:
5 TGTTTCCTTCAACCCTTTT
5 TAATCCAAACCCGTCTGCTC
95C(10) 56C(1m) 65C (1.5), 40 cycles
Production of transgenic P. berghei parasites
The ATP4 gene in Plasmodium berghei (PbATP4) was replaced with the ATP4
gene from Plasmodium falciparum (PfATP4) by creating a homologous recombination
construct with six different components (listed in order from the 5 to 3 end): PbATP4
5UTR, PfATP4, 3 UTR of P. berghei dihydrofolate reductase-thymidylate synthase
(PbDHFR-TS), 5 eukaryotic elongation factor 1 (eEF-1) promoter, Toxoplasma gondii
dihydrofolate reductase-thymidylate synthase (TgDHFR-TS) gene, and PbATP4 3UTR.
The PbATP4 5UTR (1,390 base pairs) and PbATP4 3UTR (997 base pairs) had exact
sequence identity to their respective sequences in the P. berghei genome, representing
the target sites for homologous recombination.
The P. berghei genome served as a template for PCR-amplification of PbATP4
5UTR and PbATP4 3UTR. PfATP4 (3,795 base pairs) was PCR-amplified from the P.
falciparum genome. Finally, a pL0017 plasmid was used to PCR amplify the 3UTR of
PbDHFR-TS, 5 eEF-1 promoter, and TgDHFR-TS (1,833 base pairs). Notably,
PbDHFR-TS 3UTR and 5 eEF-1 promoter were PCR-amplified together as a single,
contiguous fragment (1,057 base pairs) from pL0017.
We aimed to join the different fragments together in a pUC118 cloning plasmid
by ligase-independent cloning using vaccinia virus DNA polymerase, sold under the
commercial name of In-Fusion. However, to increase the probability that the fusion
reaction would be successful, we first sought to reduce the number of reaction
components from six to four. We accomplished this by using In-Fusion to join (i)
PbATP4 5UTR and PfATP4 and (ii) TgDHFR-TS and PbATP4 3UTR and to insert the
fused pieces into separate pUC118 cloning vectors between the EcoRI and BamHI
restriction sites.
22

This enabled the PCR-amplification of PbATP4 5UTR/PfATP4 and TgDHFRTS/PbATP4 3UTR as single, contiguous fragments. These two pieces, along with
PbATP4 3UTR/5 eEF-1 promoter, were then joined together by In-Fusion into pUC118
between the EcoRI and BamHI restriction sites. NheI and NcoI restriction sites were
included in the forward and reverse primers, respectively, to introduce restriction
digestion sites for the generation of a linear homologous recombination construct. sThe
homologous recombination construct (9,072 base pairs) was verified by sequencing and
single-cut restriction digestion of the plasmid.
10 g of linearized DNA in Basic Parasite Nucleofector Solution 2 (88A6) was
transfected into P. berghei ANKA using a Nucleofector set to program U33 (Lonza).(14)
On day 4, parasites had reached 3% parasitemia and were switched to drinking water
with 70 g/ml pyrimethamine. Parasites were at very low levels on days 5 and 6; on
day 7, they had reached 1% parasitemia and were harvested into Alsevers solution by
cardiac puncture. Parasites were transferred into new mice and placed immediately on
pyrimethamine selection for 5 additional days. Parasites were maintained under
selection with 35 g/ml pyrimethamine until they were used for experiments.
Measurement of cytosolic [Na+] and pH (ANU)
The P. falciparum strains were grown in culture as described previously (5), with
some modifications (15), and were synchronized by sorbitol treatment (16). Parasites
were functionally isolated from their host erythrocytes by exposing the (~ 4%
hematocrit) cultures briefly to saponin (0.05% w/v; final sapogenin concentration of ~
0.005% w/v) (17), and then washing the cells several times in bicarbonate-free
RPMI1640 supplemented with 20 mM glucose, 0.2 mM hypoxanthine, 25 mM HEPES,
and 25 mg/L gentamycin sulfate (pH 7.10). The isolated parasites were loaded with
either the Na+-sensitive dye Sodium Binding Benzofuran Isophthalate (SBFI, Molecular
Probes, Invitrogen) or the pH-sensitive indicator 2',7'-bis(2-carboxyethyl)-5,6carboxyfluorescein (BCECF, Molecular Probes, Invitrogen), as described previously (18,
19). For cytosolic [Na+] ([Na+]i) measurements suspensions of SBFI-loaded, saponinisolated trophozoites (1 107 - 3 107 cells/mL in a saline solution containing 125 mM
NaCl, 5 mM KCl, 1 mM MgCl2, 20 mM glucose, 25 mM HEPES; pH 7.10) were excited
at 340 nm and 380 nm successively, and the fluorescence measured at 515 nm, using a
PerkinElmer LS 50B Fluorescence Spectrometer or a Carey Eclipse Fluorescence
Spectrophotometer. The ratio of the two measurements (340 nm/380 nm) was used as
an indicator of parasite [Na+]i, with the relationship between the fluorescence ratio and
[Na+]i calibrated as described previously (18, 19). For cytosolic pH (pHi) measurements
suspensions of BCECF-loaded, saponin-isolated trophozoites (suspended at similar cell
numbers, and in the same media, as was used in the [Na+]i measurements) were
excited at 440 nm and 490 nm successively, and the fluorescence measured at 520 nm.
The ratio of the two measurements (490 nm/440 nm) provides an effective measure of
pHi. The relationship between this ratio and pHi. was calibrated as described previously
(18, 19).
Efficacy and parasite clearance studies (mouse models, USF)
To assess blood stage efficacy in rodent (ICR female mice, Harlan) models of malaria
we used a modified Thompson model. Infections were established by IP inoculation of
23

1 x 106 P. berghei (GFP)-infected erythrocytes. The first day of drug treatment was day
3 following infection when parasitemia typically approaches 1% of the infected
erythrocytes. (+)-SJ733, chloroquine, or vehicle control were administered per os once
daily (qd) for three days (day 3-5 PI). Chloroquine was used a standard drug for
comparison for rate of clearance and efficacy. Infected mice were followed for 30 days
PI and parasitemia was assessed on days 3, 6, 9, 12, 15, 18, 21, 24, 27, and 30.
Parasitemia was assessed by microscopic examination of Giemsa-stained blood
smears. End points for efficacy analysis were the ED50 and ED90 to determine the dose
of drug that suppressed 50% and 90% of parasitemia on day 6, respectively. Identical
experiments were conducted to compare efficacy (+)-SJ733 against P. berghei, P.
vinckei vinckei, and P. chabaudi chabaudi. Additionally, to capture parasite clearance
following each dug treatment, parasitemia was also assessed 12 and 24 hours post
treatment by microscopic observation of Giemsa-stained blood smears. These studies
were conducted in compliance with the Guide for the Care and Use of Laboratory
Animals of the National Research Council for the National Academies and the protocol
was approved by the University of South Florida Institutional Animal Care and Use
Committee.
Mutant Fitness study (UCSF)
To determine whether the mutations in PfATP4 that confer resistance to (+)SJ733 present a fitness cost, a growth competition was carried out between the
resistant mutant 7_2 (L350H) and the 3D7 parental strain in the presence and absence
of (+)-SJ733 (1.8 M). Using a hemocytometer and traditional Giemsa staining, we
attempted to mix equal numbers of each parasite line (approx. 108 of each). Cultures
were then incubated at 37C, 5% CO2, 5% O2. Aliquots from each culture were
removed on days when cultures were split. Cultures were always split 1:4 and only on
days when % parasitemia exceeded 10%.
Aliquots removed were immediately used to isolate genomic DNA by saponin
lysis and phenol/chloroform extraction. Contaminating RNA was removed by treatment
with 100ug/mL RNAse A (Qiagen). All genomic DNA samples were normalized to a
concentration of 0.5ng/uL. Using the QX 100 Droplet Digital PCR System (Bio-Rad), two
color allele specific TaqMan PCR reactions were carried out using primers designed to
amplify a 130bp region containing the variant nucleotide that differs between 7_2 and
3D7
parent.
The
primer
sequences
are
as
follows:
Forward
5
ATTGCATCCCAATTAAAAA and Reverse 5 AGCTAAGCTGATAATAACAA. Two
unique labeled TaqMan probes were as follows:
7_2 5-HEX-TAACACCTCATCAAGTAGCA-3IB
3D7 parent 5- FAM-TAACACCTCTTCAAGTAGCA-3IB
Per Bio-Rads QX 100 Droplet Digital PCR system protocol, PCR samples were
made with 900 nM final primer concentrations, and 250nM final TaqMan probe
concentrations. Droplets were prepared for all samples using Bio-Rads droplet
generator and then transferred to 96 well plates. Amplification in plates was performed
per the following program: 95C 10, 94C 45, 48C 1 , 95C 10 x 40 cycles

24

Following amplification, all plates were read on the QX 100 Droplet Digital
Reader (BioRad). Measurement of positive droplets and calculation of population ratios
was performed with QuantaSoft v1.3.2 (BioRad).
Time Lapse Microscopy Study (UCSF)
W2 parasites, cultured as described above, were pre-treated with (+)-SJ733 (540
nM, EC90) for 6 hours or with (+)-SJ733 (1.08 M, 2 x EC90) for 4 hours. Vehicle control
(DMSO) parasites were pre-treated for 4 hours. All pre-treated cultures were incubated
at 37C, 5% CO2, 5%O2. Time-lapse videos, covering a 15 hour time-course, were
performed with pre-treated parasites in 35 mm glass bottom, Poly-D-Lysine coated
dishes (MatTek Corp) and acquired using a Biostation IM (Nikon Instruments) using at
80x magnification under humidified conditions of 37C, 5% CO2, 5%O2, N2 Balance.
Images were taken approximately every 5 minutes for the duration of the time course.
Cellular Rigidity study (UCSF, UBC)
Studies of the deformability of iRBCs were performed using precisely controlled
pressure applied to single cells suspended in PDMS microchannels containing funnelshaped constrictions (20). To perform deformability measurements, trophozoite stage
iRBCs from DHIQ-treated and control samples were magnetically purified (21),
suspended at ~0.5% hematocrit in RPMI media. During testing, the samples were
resuspended at 30% hematocrit in PBS with 0.38% BSA (Invitrogen) and 0.02%
Pluronic F127 (Sigma-Aldrich) to prevent non-specific adsorption to PDMS. Measured
cells are first introduced into the microchannel while under observation using an
inverted microscope. When the cell reached the mouth of a funnel constriction, pressure
was applied across the microchannel and increased slowly until the cell transited
through the constriction. The threshold deformation pressure was recorded and used as
a measure of iRBC deformability. The funnel constrictions used in this measurement
were 2.05 0.15 m in width and 3.7 0.1 m in thickness. A minimum of 37 iRBCs
were tested in each case.
Real Time Proliferation study (SJCRH)
Transgenic 3D7-luciferase parasites were obtained from the Kirk Deitsch group.
Asynchronous 3D7-luciferase parasites were maintained in culture based on the
method of Trager (5). For EC50 determinations, 15 L of RPMI 1640 with 5 g/mL
gentamycin were dispensed to each well of an assay plate (Corning 384-well microtiter
plate, solid bottom, tissue culture treated, catalog no. 8807BC). Test compounds (60 nL
of DMSO stock), previously serial diluted in a separate 384-well white polypropylene
plate (Corning, catalog no. 8748BC), were dispensed to the assay plate by
hydrodynamic pin transfer (FP1S50H, V&P Scientific Pin Head) and then 15 L of a
synchronized culture suspension (1% rings, 4% hematocrit) was added to each well,
thus giving a final hematocrit and parasitemia of 2% and 1%, respectively. Assay plates
were incubated for 12h, 24h, 48h, 72h, and the parasitemia was determined by a
method previously described. Briefly, 30 L of Bright-Glo reagent (Promega) was added
to each assay plate well. Assay plates were shaken for 1 min, incubated in the dark for

25

2 min, and then luminescence was read using the Envision Multilabel Reader. EC50
values were calculated with Prism using a four-parameter logistic equation.
FACS analysis (SJCRH)
In order to ensure reproducibility, each drug was tested with internal triplicates in
2 independent experiments.
Human erythrocytes infected with mature parasites were purified (>90%) by
and resuspended in RPMI
magnetic cell separation (VarioMACSTM Miltenyi)
supplemented with Hypoxanthine and Albumax to a density of 106 cells/ml. 1 million
cells were dispensed per well in a 48 well microplate (Corning catalog number 3548).
For experiments with uninfected erythrocytes, the erythrocytes were resuspended in
RPMI supplemented with Hypoxanthine and Albumax to a density of 106 cells/ml. 1
million cells were dispensed per well in a 48 well microplate.
For each condition, 1 L of compound dissolved in DMSO was added to each
well at final concentration: (+)-SJ733 (10 M), Atovaquone (10 M), Chloroquine (10
M), NITD246(0.5 M) and Artesunate(0.5 M). The microplate was incubated (37 C,
95% humidity, 5% CO2, 5% O2) for 12 h or 24 h and stained thereafter. First 2 L of the
fluorescent DNA binding dye Sybr Green (S7563) were added to each well then the
resulting solution transferred to 5 ml culture tubes and incubated at room temperature
for 10 min. Staining with Annexin V APC (Pharmingen, Catalog number 556421) was
done by washing the cells in PBS and resuspending in 100 l Annexin Binding buffer
containing 2 l of Annexin V APC. Samples were incubated for 15 min at room
temperature in the dark, filtered through a 40 m nylon mesh, placed on ice and run
soon after completion.
A Becton-Dickinson LSRFortessa instrument and the Diva software version 6.2
were used to collect and analyze the data. The instrument was calibrated using beads
for standardization so that the fluorescence of each channel remained roughly constant
between independent experiments. Samples were collected and manually compensated
using appropriate controls (uninfected erythrocytes with and without dye).
FACS data were parsed by FCSExtract then further analyzed using custom R
scripts.(22) First, events with FSC-A levels between 1x102 and 3x104 and Sybr Green
levels between 1x101 and 1x105 were selected for analysis. FSC-A, Sybr Green, and
Annexin V values were then log10 transformed after assigning Annexin V levels 0 to
1x101. Contour levels representing 5% - 95% probability levels were determined from
the FSC-A and Sybr Green values by estimating the two-dimensional kernel density
using the function kde2d in the MASS package(23) with the number of points taken in
each direction set to 300. Annexin V values were summarized in the FSC-A : Sybr
Green space by first dividing the space into a 1000 by 1000 grid spanning the range of
FSC-A and Sybr Green, then computing the mean Annexin V value for events at each
grid point. The Annexin V grid was then smoothed by applying a moving window
average of size 25 points using the function focal in the raster package.(24) Grid points
lying outside of the 95% contour line were identified using the package sp,(25) and
colored as missing values.
For experiments testing the effects of ouabain in preliminary experiments
concentrations of up to 6 mM oubain were tested for their ability to inhibit growth of P.
falciparum. No dose had any effect. For further analysis, 106 RBCs were seeded per
26

well (1ml) in a 48 well microplate (Corning) and cultured with our standard conditions.
Cells were treated with ouabain at a final concentration of 300 M for 1, 2, 3 and 4 days;
control cells were treated with DMSO under the same conditions. After incubation, cells
were washed in PBS then stained with Annexin V FITC and further analyzed by FACS
using the methods described above. There was no evidence of eryptosis.
In Vitro Parasite Reduction Assay (GSK)
The assay uses limiting dilution technique to quantify number of parasites that
remain viable after drug treatment. P. falciparum strain 3D7A (MR4) was treated with
(+)-SJ733 at a concentration corresponding to 10x IC50 .Conditions of parasites exposed
to treatment are identical to the ones used in the IC50 determination (2% hematocrit,
0.5% parasitemia). Parasites were exposed to drug for 120 hours, with drug being
renewed daily over the entire treatment period. Samples of parasites were taken from
the treated culture every 24 hours (24, 48, 72, 96 and 120 hour time points), drug was
washed out and drug-free parasites were cultured in 96-well plates by adding fresh
erythrocytes and new culture media.
To quantify number of viable parasites after treatment, 3-fold serial dilutions were
used samples after removing the drug. Parasites were cultured in microtiter plates to
allow all wells with viable parasites to render detectable parasitemia. Four independent
serial dilutions were done with each sample to correct for experimental variation. After
21 days of culturing, samples were taken to examine growth. Additional sampling was
done after 28 days to confirm growth/ no growth. The number of viable parasites was
determined by counting the number of wells with growth. The assay allows to
determine parameters such as lag phase (time needed to observe the maximal killing
effects of the drug), PRR (parasite reduction ratio or number of parasites the drug can
kill in a parasite life cycle) and PCT99.9% (parasite clearance time to kill 99.9% of the
initial population).
In Vivo Clodronate depleted rodent efficacy studies (USF)
Initially we determined the efficacy of (+)-SJ733 and chloroquine for clearance
and cures of P. berghei (ANKA) or P. berghei expressing the PfATP4 transgene in
female, ICR mice. For these studies we used the Thompson test model (as described
above) and monitored parasitemia every 12 hours following the first dose of drug
through day 6. In addition we determine the ED50 and ED90 from day 6 parasitemia
data. (+)-SJ733 or chloroquine was given qd on days 3-5 PI. We also assessed the
effect of microphage depletion on parasite clearance and efficacy in a similar
experiment by using clodronate liposomes. Identical groups were established for
positive control drug vehicle control and (+)-SJ733 treated mice with or without
clodronate. Mice in the clodronate treated groups were inoculated on day -3, day 0 (just
before infection), and day 3 post-infection. There were five animals per group and
efficacy was followed up to 30 days post-infection.
In Vitro stage specificity assay (Eskitis)
Plasmodium falciparum was cultured according to the standard methods.
Schizonts were isolated from culture using a MAC column when the culture was
27

predominantly late schizonts. Blood was added to the isolated schizonts and the
parasites re-incubated for 1-2 hours. Once the culture reached more than 5% rings, the
culture was harvested and purified again using MAC column, this time collecting the
flow through. This gives a culture of parasites highly enriched for those 0-3 hours old.
The culture is adjusted to a 0.75 % hematocrit and 2 % parasitemia.
Wells of Cell Carrier imaging plates are then charged with 25l of culture. An
individual plate is made for each time point. The plates are incubated in a standard
incubator at 5 % CO2, 37 oC and 5 % humidity. At each time point, a single plate was
removed from the incubator and freshly diluted compound and controls added to the
plate, which was then re-incubated. This was performed at 5, 9, 18, 24, and 32 hours,
which were chosen to match progression of the life cycle.
A culture left in a dish was tested regularly for the identification of when the
culture had completed a single replication cycle. All the time plates were then stained
with DAPI and left overnight for the signal to reach optimum. The plates were then
measured on the OPERA and the images analyzed on a spot detection program.
Reference List
1.
2.
3.

4.

5.
6.

7.

8.
9.
10.
11.

Rottmann M, et al. (2010) Spiroindolones, a potent compound class for the

treatment of malaria. Science 329(5996):1175-1180.
Dijoux N, et al. (2006) Assessment of the phototoxic hazard of some essential
oils using modified 3T3 neutral red uptake assay. Toxicol. In Vitro 20(4):480-489.
Kraft AD, Johnson DA, & Johnson JA (2004) Nuclear factor E2-related factor 2dependent antioxidant response element activation by tert-butylhydroquinone
and sulforaphane occurring preferentially in astrocytes conditions neurons
against oxidative insult. J. Neurosci. 24(5):1101-1112.
Jimenez-Diaz MB, et al. (2009) Quantitative measurement of Plasmodiuminfected erythrocytes in murine models of malaria by flow cytometry using
bidimensional assessment of SYTO-16 fluorescence. Cytometry A 75(3):225235.
Trager W & Jensen JB (1976) Human malaria parasites in continuous culture.
Science 193(4254):673-675.
Smilkstein M, Sriwilaijaroen N, Kelly JX, Wilairat P, & Riscoe M (2004) Simple
and inexpensive fluorescence-based technique for high-throughput antimalarial
drug screening. Antimicrob. Agents Chemother. 48(5):1803-1806.
Cushman M, Gentry J, & Dekow FW (1977) Condensation of imines with
homophthalic anhydrides. A convergent synthesis of cis- and trans-13methyltetrahydroprotoberberines. J. Org. Chem. 42(7):1111-1116.
Roy A, Kucukural A, & Zhang Y (2010) I-TASSER: a unified platform for
automated protein structure and function prediction. Nat. Protoc. 5(4):725-738.
Apweiler R, et al. (2004) UniProt: the Universal Protein knowledgebase. Nucleic
Acids Res. 32(Database issue):D115-119.
Foller M, et al. (2009) Suicide for survival--death of infected erythrocytes as a
host mechanism to survive malaria. Cell. Physiol. Biochem. 24(3-4):133-140.
Narayanasamy K, et al. (2012) Malaria evolution in South Asia: knowledge for
control and elimination. Acta Trop. 121(3):256-266.

28

12.

13.

14.

15.
16.
17.

18.
19.

20.

21.
22.
23.
24.

25.
26.

Ekland EH, Schneider J, & Fidock DA (2011) Identifying apicoplast-targeting

antimalarials using high-throughput compatible approaches. FASEB J.
25(10):3583-3593.
Goodyer ID, & Taraschi TF (1997) Plasmodium falciparum: a simple, rapid
method for detecting parasite clones in microtiter plates. Exp. Parasitol. 86(2):5860.
Janse CJ, Ramesar J, & Waters AP (2006) High-efficiency transfection and drug
selection of genetically transformed blood stages of the rodent malaria parasite
Plasmodium berghei. Nat. Protoc. 1(1):346-356.
Allen RJ & Kirk K (2010) Plasmodium falciparum culture: the benefits of shaking.
Mol. Biochem. Parasitol. 169(1):63-65.
Lambros C & Vanderberg JP (1979) Synchronization of Plasmodium falciparum
erythrocytic stages in culture. J. Parasitol. 65(3):418-420.
Saliba KJ, Horner HA, & Kirk K (1998) Transport and metabolism of the essential
vitamin pantothenic acid in human erythrocytes infected with the malaria parasite
Plasmodium falciparum. J. Biol. Chem. 273(17):10190-10195.
Spillman NJ, Allen RJ, & Kirk K (2013) Na+ extrusion imposes an acid load on the
intraerythrocytic malaria parasite. Mol. Biochem. Parasitol. 189(1-2):1-4.
Spillman NJ, et al. (2013) Na+ regulation in the malaria parasite Plasmodium
falciparum involves the cation ATPase PfATP4 and is a target of the
spiroindolone antimalarials. Cell Host Microbe 13(2):227-237.
Guo Q, Reiling SJ, Rohrbach P, & Ma H (2012) Microfluidic biomechanical assay
for red blood cells parasitized by Plasmodium falciparum. Lab Chip 12(6):11431150.
Kim CC, Wilson EB, & DeRisi JL (2010) Improved methods for magnetic
purification of malaria parasites and haemozoin. Malar. J. 9:17.
Team. RDC (2013) R: A language and environment for statistical computing. . (R
Foundation for Statistical Computing, Vienna, Austria.).
Venables WN & Ripley BD (2002) Modern applied statistics with S (Springer,
New York) 4th Ed pp xi, 495 p.
Amambua-Ngwa A, et al. (2012) SNP genotyping identifies new signatures of
selection in a deep sample of West African Plasmodium falciparum malaria
parasites. Mol. Biol. Evol. 29(11):3249-3253.
Bivand R, Pebesma EJ, & Gmez-Rubio V (2008) Applied spatial data analysis
with R (Springer, New York) pp xiv, 374 p.
Lee P, Ye Z, Van Dyke K, & Kirk RG (1988) X-ray microanalysis of Plasmodium
falciparum and infected red blood cells: effects of qinghaosu and chloroquine on
potassium, sodium, and phosphorus composition. Am. J. Trop. Med. Hyg.
39(2):157-165.

Supplemental Figure Legends

Figure S1. SJ733 possesses critical characteristics of a drug candidate for
development as a rapid acting antimalarial. a. (+)-SJ733 is a potent antimalarial
agent against laboratory strains of P. falciparum possessing all known resistance
29

mechanisms. The majority of the activity resides with the (+)-isomer. b. (+)-SJ733 is
active against all rodent species of malaria tested ex vivo, but has significant variation in
potency. The wild type P. berghei is roughly 10-fold less sensitive to (+)-SJ733,
whereas P. chabaudi and P. vinckei possess intermediate sensitivities. c. There is good
correlation between in vitro potency of (+)-SJ733 against P. falciparum in vitro and in
vivo. Blood levels of (+)-SJ733 in infected mice treated with single oral doses
bracketing the ED90 reach a peak roughly 3 to 4 -fold over the in vitro EC90 and maintain
levels above the in vitro EC90 for 6 to 10 hours. d. In vivo dose response experiments
using wild-type Plasmodium berghei show that (+)-SJ733 is roughly 10-fold less potent
in vivo than it is against P. falciparum, mirroring the ex vivo results. e. There is good
correlation between ex vivo potency of (+)-SJ733 against P. berghei in vitro and in vivo.
Blood levels of (+)-SJ733 in infected mice treated with single oral doses bracketing the
ED90 reach a peak roughly 6 to 10 -fold over the in vitro EC90 and maintain levels above
the in vitro EC90 for 10 to 12 hours. f. (+)-SJ733 has high oral bioavailability in the rat,
even from suspensions. Comparison of exposure from IV dosing of a 5 mg/kg solution
to oral dosing of a 17 mg/kg suspension in hydroxypropylmethylcellulose shows oral
bioavailability exceeding 75%. Similar studies (not shown) comparing solution dosing
show oral bioavailability approaching unity. g. (+)-SJ733 has high oral bioavailability in
the dog. Comparison of exposure from IV dosing of a 3 mg/kg solution to oral dosing of
a 30 mg/kg solution shows oral bioavailability exceeding 75%. h. The synthesis of (+)SJ733 proceeds efficiently through a 4 step route from commercially available
precursors with resolution of the active enantiomer as a chiral salt in the penultimate
step. If the salt form is desired it can be formed in one step from the free base.
Figure S2. SJ733 targets Plasmodium ATP4 gene products, changes in PfATP4
sequence lead to changes in sensitivity, and mutation of ATP4, while conveying
resistance, comes at significant fitness cost. a. Relationship between parasite
inoculum, frequency of selection of mutant strains of P. falciparum (3D7 strain), and
fold-resistance. The data in this graph represent experiments carried out using the St
Jude protocol across replicate experiments carried out using inocula varying from 104 to
109 parasites (all P. falciparum 3D7 strain). Most mutations occur at relatively low
frequency (minimum inoculum of 107 to 108) and provide fairly low fold-resistance (5-15
fold) although two high fold resistance clones have been selected. No resistant strain
has ever been selected from a population smaller than 107 parasites. The low risk
bounding box represents the current MMV thinking on risk of acquisition of clinical
resistance, based on historical rates of resistance development. b. Treating a mixed
population of SJ733 resistant ATP4L350H and the 3D7 parental strain with (+)-SJ733
leads to essentially complete selection for the mutant strain within 4 days, as measured
by quantitative PCR. However, if drug pressure is relieved the population reverts
predominantly (>90%) to the parental strain within 30 days. The same trend is seen
with the ATP4P966T parasite line. c. Selection of resistant clones in vivo proceeds
slowly, requiring 3 cycles of treatment before resistant parasites emerge and an
additional two cycles before the phenotype is stable. When selective pressure is
maintained, the resistant parasites are stable. d. Using a fixed dose (50 mg/kg, the
ED75 for P. berghei) results in variable efficacy for treatment of the rodent malarias with
(+)-SJ733. When compared in vivo the sensitivity of the P. berghei genotypes still
30

follows the same trend as the ex vivo experiments with the selected mutant being the
least sensitive, the wild type intermediate, and the transgenic PfATP4 expressing strain
hypersensitive. However, the other two rodent species are less sensitive than the P.
berghei, highlighting that the situation in vivo is more complex, probably due to
variations in tropism and life cycle. e. There exists a variant sequence loop within the
ATP4 gene, believed to overlie the DHIQ binding site, which may partially explain the
variations in sensitivity to (+)-SJ733 between species. f. (+)-SJ733 causes a rise in
intracellular Na+ within the parasite that is not exhibited after DMSO treatment and
which reaches maximal effect within 90 min of treatment. The image shows a trace of
sodium levels as measured by fluorescent detection. g. (+)-SJ733 causes a rapid
cytosolic alkalinisation (shown here for isolated W2 trophozoites) as well as reducing
the extent of acidification of the parasite cytosol seen following the inhibition of the
parasites plasma membrane V-type H+ ATPase by concanamycin A. The black triangle
denotes the point of addition of (+)-SJ733 (2.5 M) or DMSO (control) and the red arrow
the point of addition of concanamycin A (200 nM).
Figure S3. SJ733 pharmacodynamic responses are similar between human and
rodent malarias, do not depend on macrophages, and do not significantly affect
normal erythrocyte populations. a. (+)-SJ733 induces rapid clearance of rodent
malarias from normal immune function mice in vivo with pharmacodynamics similar to
those seen in the P. falciparum humanized mouse model. b. (+)-SJ733
pharmacodynamics with rodent malarias do not depend on macrophages, as
demonstrated by the similarities between responses in clodronate depleted and native
immune function animals infected with the transgenic P. berghei strain expressing
PfATP4. c. (+)-SJ733 does not significantly affect erythrocyte populations in either
uninfected or infected mice. There are no significant dosage related changes in
hematocrit (hct), red blood cell counts (rbc), hemoglobin levels (Hgb), or the mean size
of red cells (RDWa). The normal range and limit of detection for all of these measures
is greater than 10% coefficient of variation, so it is not surprising that it is not possible to
detect the intravascular lysis of infected, treated erythrocytes. d. Map of the plasmid
used to generate the transgenic P. berghei strain. e. Variation in the potency (EC50) of
(+)-SJ733 when added at discrete times in the erythrocytic cycle to co-cultures of P.
falciparum. There is no significant change in potency for any stage for (+)-SJ733, or for
other DHIQs. Pyrimethamine has a similar profile. In contrast halofantrine is much
more potent on rings and equipotent across the rest of the cycle while artesunate shows
a gradual loss in potency from the ring stages to the schizonts. f. Binding isotherm for
the specific binding of tritiated (+)-SJ733 to P. falciparum infected red cells. There is a
single evident class of binding sites and the apparent affinity (kd) is roughly 50 nM,
essentially equivalent to the EC50. g. The effects of treating uninfected erythrocytes
with ouabain. On treatment of the cells with ouabain (300 M), a concentration reported
to induce marked change in erythrocyte resting cytosolic [Na+]/[K+],(26) there were no
detectable differences with respect to size (forward scatter), shape (side scatter), or
exposed phosphatidyl serine levels (green to pink color gradient) between untreated
(left panel) and treated (right panel) erythrocytes at timeframes of up to 4 days.
Therefore there is no induction of eryptosis by ouabain treatment. In parallel

31

experiments, ouabain concentrations of up to 6 mM had no effect upon proliferation of

P. falciparum, as previously reported.

32

Figure S1
a

3D7
K1

TM90C2A

A6

V1/S

TM90C2B

D10

D10-yDHODH

EC50 ( M)

10

P. berghei

P. chabaudi chabaudi

P. berghei DHIQ resistant

P. berghei PfATP4 Transgenic

P. vinckei vinckei

EC50 ( M)

0.1

0.1

ch
lo
ro
qu

in
e

io
m
er

Average P. falciparum
in vitro EC90 at 72 h

10
0

Time (h)

Average P. berghei
ex vivo EC90 at 48 h

100

C
Q

J7
33

(+
)-S

C
Q

J7
33

C
Q

(+
)-S

J7
33

C
Q

(+
)-S

P. berghei

0.1

P. berghei TgPfATP4
1.0

1.5
2.0
log (Dose (mg/kg))

10

Plasma Concentration SJ733, M

1000

0.01

10

(+)-SJ733 - 25 mg/kg
(+)-SJ733 - 50 mg/kg

10000
Blood [(+)-SJ733] (nM)

J7
33

10
log(% Parasitemia)

Blood [(+)-SJ733] (nM)

100

C
Q

(+
)-S

(+)-SJ733 - 1.6 mg/kg

(+)-SJ733 - 3.2 mg/kg

(+
)-S

c 1000

0.01
J7
33

(-)

(+
)e
na
n

en
an
t

tio
m
er

ra
ce
m
at
e

0.01

2.5

3.0

(+)-SJ733 Rat PO 17 mg/kg

(+)-SJ733 Rat IV 5 mg/kg
%F = 75 +/- 3 % at 17 mg/kg
(in suspension)

0.1

0.01

LLOQ

10
0

10

Plasma Concentration SJ733, M

12 14
Time (h)

16

18

20

22

24

12

18

30

36

42

48

(+)- SJ733 Dog PO 30 mg/kg

(+)-SJ733 Dog IV 3 mg/kg

10

24
Time (h)

%F = 78 +/- 18 % at 30 mg/kg
(in solution)

CHO
F3C

NH3Cl

a) NaOH, toluene, 94%

b) homophthalic anhydride,
N-methylimidazole,
methylene chloride, 74%

O
N

CF3
N

N
HO

O
1
NH2

2
O
N

0.1

LLOQ

CF3

HN

12

18

24
Time (h)

30

36

42

48

CN
F
(+)-SJ733

a) POCl3, 5-amino-2fluorobenzonitrile, 70%

b) HCl (84%) - optional

OH

a) PrCN, Heptane (1:1)

b) 20% HOAc,
76%, 99+% ee
O
N

CF3
N

HO

O
(+)-1

10 4

Figure S2

b
100

ATP4mut

10

3D7

Low Risk Zone

Drug Free - ATP4L350H

1

wt to

10 2

Ratio 3D7

EC50 Fold Increase

10 3

Drug Free - ATP4L350H

Drug Free - ATP4P966T

0.1

(+)-SJ733 (3 x EC90)

10 1
0.01
0

10

log(Minimum Inoculum of Resistance)

100
In Vivo Sensitivity
Fixed dose 50 mg/kg (+) SJ733 Peters Test

1 week treatment course

80

D1

D2

D3

D4

D5

D6

% Viable Parasites

(+)-SJ733
D7

Day7 P.berghei % Parasitemia

15

60

40

20

10
0

P. berghei

P. chabaudi

P. berghei PfATP4 Transgenic

P. vinckei

P.
P.
P.
P.
P.
P.
P.

10

falciparum
chabaudi
vinckei
yoelii
berghei
vivax
cynomolgi

Treatment Courses(weeks)

100

7.4

60

2.5 !M (+)-733

pHi

7.2

40

7.0

20
0

SESIENLCKEFGLESINTGLNSEQVKINRDKYGENFIEKDEVVPVWLIFLSQYCSPVVLL
VYNVEDVLRAVKV-DENRGLSENEIRKRIMQYGFNELEVEKKKGIFELILNQFDDLLVKI
IYNVEDVLRAVKV-DENRGLSENEIRKRIMQYGFNELEVEKKKGILELILNQFDDLLVKI
SESIENLCKEFDLADVNTGLNFEQVKINRERYGENHIEKDSITPIWLIFLSQYYSPVVML
SESIEKLCKEFDLADINIGLSFEQVKINRERYGENHIEKDSITPIWLIFLSQYYSPVVML
TESIENLCREFDLQDLNVGLTTEQVKINREKYGENFIEKDDAMPLWLIFLSQYCSPVVVL
----------------------------------------------------------ML
.:*.: : . : . * **. :::: . :** * :* :.
: ::*.*: . :* :

2.5 !M (+)-733

80

[Na+]i (mM)

30

Day After Initiation

10 0

20

DMSO

DMSO

6.8
0

50

Time (min)

100

50

Time (min)

100

Figure S3
a

50

50

0
0

12

24

36

48
Time (h)

60

72

84

96

HCT - no malaria
HCT - Pb->PfATP4
RBC - no malaria
RBC - Pb->PfATP4
Hgb - no malaria
Hgb - Pb->PfATP4
RDWa - no malaria
RDWa - Pb->PfATP4

60
40
20

12

24

36

48
Time (h)

60

72

84

%@B&"4&

%@3&"4&

!5=795&3&

:;#&3<'&

>?+&"4&

3<B3&"4&

!"#$!%&&
'()$*&

+(!"&
-./*0$1&

!"#$!%&

'(22/3&
45676895&

$,-./*0$1&

@%&"4&

!5=795&%&

f
Troph

4000

Schizont

0.1

(+)-SJ733
Pyrimethamine
Halofantrine
Artesunate

0.01

EC50 (M)

(+)-(3H)SJ733 in pellet (nM)

Ring

0.001

0.0001

10

20

30

Time of Treatment (Hours)

log(Forward Scatter)

300 +M ouabain
4d

2
4.8

4.9

1000

10

100

(+)-(3H)SJ733 in supernatent (nM)

untreated
4d

4.7

2000

3000

40

5.0

4.7

log(Side Scatter)

4.8

4.9

5.0

!"#$!%&&
+()$*&
+3@&"4&

!5=795&A&

(+)-SJ733 Dose (mg/kg)

96

0
100
200
0
100
200
0
100
200
0
100
200
0
100
200
0
100
200
0
100
200
0
100
200

Assay Value

80

(+)-SJ733, P. berghei (pbatp4->pfatp4)

(+)-SJ733, P. berghei (pbatp4->pfatp4)
Clodronoate depleted
Chloroquine, P. berghei

100

(+)-SJ733, P. berghei (pbatp4->pfatp4)

Chloroquine, P. berghei

Normalized % Parasitemia

Normalized % Parasitemia

(+)-SJ733, P. berghei

100

1000