Potent acridone antimalarial against all three life stages of Plasmodium

Domain: biology
Published: 2026-04-14
Editorial outcome: minor_revision

Abstract
Antimalarial therapeutics ideally should target all three major Plasmodium life cycle stages. Here we
present an acridone antimalarial chemotype that is potent against blood, liver, and mosquito stages of
malaria parasites. Attributes of lead candidate T111 include potent in vitro activity against cultured
parasites, ex vivo activity against clinical isolates, oral single-dose cure in an asexual blood-stage rodent
model, inhibition of sexual blood-stage parasites, activity against relapsing parasites in non-human
primate liver cells, prevention of parasite development in mosquitoes, and synergy in combination with
tafenoquine against blood- and liver-stage parasites. Analysis of parasites selected for resistance to T111
suggests inhibition of the mitochondrial electron transport chain, with a mechanism distinct from that of
other antimalarials in use or under development. The safety profile of T111, including toxicology
evaluations in rats, absence of hemolytic toxicity, and low genotoxicity and cardiotoxicity potential,
demonstrates a favorable therapeutic index. Overall, T111 emerges as a promising candidate for treatment
and prevention of malaria, with potential for single-dose cure of bloodstream infections, radical cure of
liver infections, and interruption of transmission to mosquitoes.

Introduction
Malaria has plagued humans for millennia and remains one of the deadliest diseases in modern
times, resulting in approximately a quarter billion clinical cases and over a half million deaths annually1.
The most vulnerable group is children under the age of five, with the estimation that a child dies from
malaria roughly every minute. Plasmodium falciparum is responsible for the majority of malaria mortality
worldwide, with Plasmodium vivax the most widespread human malaria parasite, and other plasmodial
species that infect humans each presenting different challenges2. Despite commendable progress in past
decades, malaria control continues to face many challenges, including alarming resistance to artemisinin
(ART) and some artemisinin-based combination therapy (ARCT) partner drugs3,4, declining effectiveness
of insecticides5, safety concerns associated with the limited radical cure options for relapsing P. vivax and
Plasmodium ovale infections6,7, lack of transmission-inhibiting drugs8, and inability to provide full
immunity with newly implemented vaccines9. Ideally, a chemotherapy approach to combat all stages and
species of Plasmodium parasites would offer broad benefits toward the control and ultimate elimination
of malaria10,11.

Malaria is a mosquito-borne infectious disease caused by Plasmodium parasites, with life cycles
involving both vertebrate hosts and mosquito vectors11-13. Human infection begins when an infected
female Anopheles mosquito bites a person, injecting sporozoites which enter the blood stream and invade
liver cells, multiply asexually, and mature into schizonts. Thousands of merozoites are then released into
the blood, where they invade red blood cells, multiply asexually, and cause clinical illness. Some blood-
stage parasites develop into sexual-stage parasites (called gametocytes) that are taken up by biting
mosquitoes. Inside the mosquito, the parasites undergo sexual reproduction to develop into oocysts and
then sporozoites. When the mosquito bites another human, sporozoites are injected, enabling continued
transmission. Relapsing malaria, seen only with P. vivax and P. ovale, follows the reactivation of liver
hypnozoites after extended periods of dormancy, leading to recurrent asexual bloodstream infection14.

Most of the antimalarial drugs currently in use target blood-stage parasites, which are responsible
for clinical manifestations11,15. The only available causal prophylactics that target liver-stage infection are
Malarone (atovaquone (ATV) + proguanil (PG)), primaquine (PQ), and recently approved tafenoquine
(TQ). Of these, PQ and TQ are the only drugs that offer radical cures, killing hypnozoites to prevent
relapsing P. vivax and P. ovale infections. In addition, low dose PQ is now recommended as a malaria
transmission-inhibiting drug in some settings7,16,17. Unfortunately, PQ and TQ are both 8-aminoquinolines
that cause hemolytic toxicity in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency, a
common enzyme abnormality18,19.

The advantages of antimalarial agents that eliminate malaria parasites in multiple life-cycle stages
are self-evident, however the discovery of such highly desirable drugs remains an arduous challenge11,20.
We have discovered and developed an acridone antimalarial chemotype, with T111 as the lead candidate,
that exhibits potent activity against liver (including hypnozoite), blood (both asexual and sexual), and
mosquito stage parasites, with single-dose cure in a murine model as well as the potential to prevent
relapsing infection and interrupt transmission.

Results
In vitro activity against asexual blood-stage P. falciparum. In our previous report21, T111 exhibited
potent in vitro antiplasmodial activity against a panel of asexual blood-stage P. falciparum parasites that
included pan-sensitive strain D6, multi-drug resistant (MDR) strain Dd2, and ATV-resistant strain Tm90-
C2B, with sub- to low-nanomolar IC values. Considering the rising spread of ART resistance22, here we
50
tested T111, alongside dihydroartemisinin (DHA), against two recently isolated P. falciparum K13 mutant
parasites, using a ring-stage survival assay (RSA), the Growth, Resistance, and Recovery Assay
(GRRA)23 to generate fine-scale dose-response curves (GRRA ). SP045 and SP060 are K13 mutant
50
clones from Rwanda24,25. SP045 (V555A) is relatively susceptible to ART (ART-S), and SP060 (R561H)
is highly ART-resistant (ART-R)25. As demonstrated in Figure 1A and 1B, ART-R SP060 has a DHA
GRRA value of 25 nM and ART-S SP045 has a GRRA of 7.7 nM. T111 exhibits approximately 10-
50 50
to 29-fold greater potency than DHA, with sub-nanomolar effectiveness against both parasites, and
indicates no cross-resistance between T111 and DHA in these clinically relevant isolates (GRRA = 0.78
50
nM for SP045 and 0.87 nM for SP060). T111 was also tested against the ART-R lab-derived P. falciparum
clones DR1 and DR4, which were selected in the Dd2 strain with escalating DHA pressure26. DHA had
decreased activity against these two resistant parasites (IC = 90 nM for DR1 and 173 nM for DR4
50
compared to 8.3 nM for Dd2)26. As shown in Figure 1C, T111 displayed excellent potency, against Dd2
(IC = 0.028 nM), DR1 (IC = 0.11 nM), and DR4 (IC = 0.11 nM).
50 50 50

Ex vivo activity against asexual blood-stage P. falciparum clinical isolates. The activity of T111
against fresh P. falciparum isolates collected from patients diagnosed with falciparum malaria was
evaluated in Burkina Faso and Uganda27,28. T111 demonstrated picomolar activity, with median IC
50
values of 0.062 nM (0.0013–0.49 nM, n=78) in Burkina Faso and 0.049 nM (0.0038–0.66 nM, n=81) in
Uganda, with ex vivo activities similar to in vitro activities against the Dd2 and 3D7 control strains (Figure
1D). Ex vivo testing in the field provides real-time data on drug potency. Importantly, all patient isolates
were highly susceptible to T111, with sub-nanomolar IC values.
50

In vivo oral efficacy against asexual blood-stage P. yoelii in a rodent model. To investigate oral
efficacy against blood-stage infections, we employed a well-established model21,29-31 using 4–5-week-old
CF1 mice inoculated with P. yoelii (3.5 × 104 per inoculum via tail vein injection). As shown in
Supplementary Figure 1, T111 (via oral treatment) exhibited excellent antimalarial efficacy with low ED
50
and ED values in the 4-day model (ED = 0.35 and ED = 0.50 mg/kg/day in female mice; ED = 0.33
90 50 90 50
and ED = 0.46 mg/kg/day in male mice), and was curative at 10 mg/kg/day for four consecutive days.
90
With interest in a single-dose curative regimen, we also tested T111 administered orally once, 24 h post-
inoculation31-33. One of four mice treated with a single oral dose of 40 mg/kg, and all of four mice treated
with 50 mg/kg were cured, with no parasites detected over 28 days (Figure 1E).

In vitro activity against sexual blood-stage P. falciparum gametocytes. The activity of T111 against
sexual blood-stage gametocytes was investigated after asexual stage P. falciparum NF54 parasites were
stressed by starvation to induce gametocytogenesis34. The drug was added daily on Days 5 to 7 post
gametocyte induction when the gametocytes were approximately stage II to III, and gametocytemia (stage
V gametocytes) was determined on Day 14, when control parasites had matured into late-stage
gametocytes. T111 inhibited the formation of stage V gametocytes by 75% and 93% at 1 µM and 10 µM,
respectively (Figure 1F).

Standard membrane feeding assay (SMFA). The transmission-inhibitory activity of T111 was
investigated in a standard direct membrane feeding assay35, in which the drug was included during
mosquito feeding with blood samples from a naturally infected P. falciparum NF54 strain gametocyte
carrier. T111 inhibited oocyst development in a dose-dependent manner and eliminated oocyst formation
at 4.0 ng/mL (9.5 nM) concentration (Figure 1G).

Tarsal contact assay. In a tarsal contact assay36, female Anopheles gambiae G3 strain mosquitoes were
allowed to land on a thin film surface incorporated with a T111 prodrug at 5% by weight (structure shown
in Supplementary Figure 4) for 6 min and one hour later fed a blood meal carrying P. falciparum NF54
gametocytes. Midguts of the infected mosquitoes were dissected on Day 7. There was a significant, nearly
complete reduction in oocyst prevalence (92.5% inhibition) and intensity (97.6% inhibition) in
mosquitoes after brief tarsal contact with the T111 prodrug, whereas nearly all control mosquitoes showed
a high intensity infection (Figure 1H).

In vitro activity against relapsing Plasmodium cynomolgi in rhesus hepatocytes. P. cynomolgi is a
non-human primate (NHP) malaria parasite closely related to P. vivax and is widely used as surrogate
model for relapsing malaria due to its ability to form dormant liver-stage hypnozoites. To evaluate the
activity of T111 against relapsing liver-stage malaria, we employed a high-throughput in vitro assay using
P. cynomolgi sporozoites infecting primary rhesus hepatocytes37, assessing both prophylaxis (prevention
of liver-stage establishment) and radical cure (elimination of existing hypnozoites and developing liver-
stage schizonts) dosing paradigms. Under prophylactic conditions, T111 exhibited potent activity against
the establishment of liver-stage hypnozoites, comparable or superior to that of the reference compounds
TQ38, ATV39, and the Plasmodium PI4 kinase (PI4K) inhibitor KDU69140,41 (Table 1). In addition,
prophylactic activity inhibiting schizont formation with low-nanomolar IC values was observed.
50
Considering radical cure treatment against dormant hypnozoites, the potency of T111 was superior to the
known hypnozoitocidal reference drug TQ for inhibition of both hypnozoites and schizonts. Cytotoxicity
of T111 was assessed in parallel by photometric measurements of host hepatocyte viability, revealing
moderate in vitro profile against rhesus hepatocytes (CC = 0.56 µM; Table 1), however, the cytotoxicity
50
IC value vs human HepG2 hepatocyte cells was > 200 µM (Supplementary Table 1).
50

In vitro synergy of the T111/TQ combination against asexual blood-stage P. falciparum parasites.
Synergy was assessed by isobolar analysis with a well-established fixed ratio platform42,43, using a SYBR
Green based assay44. Drug interactions were quantified by calculating the Fractional Inhibitory
Concentration (FIC) for each drug, defined as the IC of the drug in combination divided by its IC alone.
50 50
The ΣFIC index (the sum of the individual FICs) was used to determine the nature of the interaction,
where a ΣFIC value < 1.0 indicates synergy. As shown in Figure 2A, relative synergy between T111 and
TQ was observed against pan-sensitive D6 (ΣFIC index = 0.69), MDR Dd2 (ΣFIC index = 0.62), and
ATV-resistant Tm90-C2B (ΣFIC index = 0.46).

In vitro synergy of the T111/TQ combination against relapsing P. cynomolgi parasites. Using the in
vitro P. cynomolgi assay described above37, for prophylaxis, synergy was evident in the inhibition of both
hypnozoite and schizont development, with ΣFIC index values for the TQ/T111 combination at 0.39 and
0.41, respectively. For radical cure, synergy was also observed in inhibiting hypnozoite and schizont
development, with ΣFIC index values at 0.58 and 0.72, respectively (Figure 2B).

In vivo potentiation of TQ against blood-stage P. yoelii in the 4-day suppression murine model. We
compared the activity of TQ alone and in the presence of sub-therapeutic oral dosages of T111 in the 4-
day suppression model. T111 potentiated the oral efficacy of TQ, with an ~34-fold reduction in ED
50
values at 0.12 mg/kg/day (Figure 2C). The dose-response curves for oral efficacy of T111 alone are shown
in Supplementary Figure 1.

In vivo single-dose cure synergy of the T111/TQ combination against P. yoelii in mice. We also
investigated the combination of T111 and TQ as a single-dose cure in the P. yoelii model. TQ achieved
single-dose cure in all treated mice at 40 mg/kg (Table 2). T111 achieved single-dose cure in all treated
mice at 50 mg/kg. Various lower dosage combinations of TQ with T111 achieved single-dose cures (Table
2).

In vivo synergy of the T111/TQ combination against liver-stage Plasmodium berghei in mice. We
previously reported in vivo oral efficacy of T111 against liver-stage Plasmodium infection21. A real-time
in vivo imaging system (IVIS) using transgenic bioluminescent parasites to quantify parasite development
was employed to study liver-stage infection in live anesthetized P. berghei infected mice45-47. Potential
synergy between T111 and TQ was investigated using the same model. Luciferase-expressing P. berghei
sporozoites were inoculated in 4- to 6-week-old Albino C57BL/6 mice on Day 0, with oral doses of drugs
administered on Day -1 (1 day prior to inoculation), Day 0 (day of inoculation), and Day 1 (1-day post-
inoculation) and bioluminescence signals were measured at 24 and 48 h after inoculation for liver-stage
development and at 72 h for blood-stage infection in anesthetized mice. Strong bioluminescence signals
were detected in untreated mice at both 24 and 48 h in the liver, followed by intense whole-body signals
at 72 h (Figure 3). Treatment with TQ at 1 mg/kg/day or 2.5 mg/kg/day, and with T111 at 1 mg/kg/day or
4 mg/kg/day did not clear liver-stage infection, and all treated mice developed blood-stage infection.
Treatment with TQ or T111 at 10 mg/kg/day provided full protection from liver-stage infections.
Treatment with the combination of 1 mg/kg/day TQ + 4 mg/kg/day T111 provided full protection, without
visible parasites in the liver at 24 and 48 h or the blood over 31 days after inoculation. The same results
were observed with the combination of 2.5 mg/kg/day TQ + 1 mg/kg/day T111.

In vitro metabolic stability and in vivo pharmacokinetic (PK) profile. T111 was evaluated for in vitro
metabolic stability by measuring the disappearance of the parent compound after incubation with pooled
human, mouse and rhesus liver microsomes (HLM, MLM and RLM)48,49. T111 demonstrated good
metabolic stability in all microsomal systems (t > 60 min in HLM and MLM; and t = 34.0 min in
1/2 1/2
RLM). In vivo PK studies of T111 were conducted following a single oral administration in both male
and female ICR-CD1 mice at 40 mg/kg with blood and liver samples taken at 11 time points, using well-
established methods21,31,50. PK profiles of T111 were similar in male and female mice. Considering the
reported PK data for ATV (t in mice plasma ~ 13 h51 ), T111 demonstrated a relatively long half-life in
1/2
both liver and plasma, with higher concentration in liver than plasma (Table 3), which is consistent with
its excellent in vivo oral efficacy against liver-stage infection21. The development of prodrugs of T111
with enhanced PK profiles is underway.

In vitro safety evaluations. Assessment of cytotoxicity against human hepatic HepG2 cells using the
MTT assay52 showed no apparent toxicity, with IC > 200 µM, indicating a selectivity index > 10,000
50
(Supplementary Table 1). hERG (human-ether-a-go-go-related gene) channel inhibition assessment using
an automatic parallel patch clamp system53 showed low risk of cardiotoxicity, with estimated IC value
50
higher than 100 µM (Supplementary Table 2). The Ames test against Salmonella typhimurium TA98 and
TA100 strains with and without S9 activation54 showed negative results at the tested concentration of 10
µM. No increase above the background reversion rate was observed, indicating a low risk of mutagenicity
(Supplementary Figure 2). Evaluation of eryptosis as a marker for potential drug induced hemolytic
toxicity, characterized by cell shrinkage, oxidative stress, and phosphatidylserine translocation to cell
surface55, did not show the toxicity seen with TQ against G6PD-deficient RBCs (Figure 4).

In vivo toxicology and toxicokinetic (TK) analysis in rats. Murine blood-stage and liver-stage efficacy
studies included observations of animal weight, grooming, posture, and locomotion. No overt clinical
toxicity or behavior change was observed in any T111 treated mice (highest tested dose: 30 mg/kg × 4
days and 80 mg/kg × 1 day). In addition, safety evaluation of T111 was conducted in male and female
rats, with both single dose and repeat dose toxicology studies. In the maximum tolerated dose (MTD)
determination study, a single oral dose of T111 was administered by gavage at 30, 100, 200, and 400
mg/kg to male and female Sprague Dawley rats, and animals were observed for two days. Clinical
observations were performed immediately, 2–4 h and 24 h post dosing. T111 was well tolerated, with no
clinical signs suggesting toxicity. A single dose MTD could not be established due to limited solubility
of T111 in the vehicle (PEG-400), but it is considered to be greater than 400 mg/kg, the highest dose
tested. Based on the observed effects and T111 solubility limitations in the MTD studies, the dose levels
selected for 7-day repeat dose studies were 25, 100 and 400 mg/kg. Male and female Sprague Dawley rats
were given T111 daily for 7 consecutive days via oral gavage then euthanized and evaluated for body
weight changes, clinical signs, plasma drug concentrations, organ weights, gross and microscopic
examinations of selected tissues, and clinical pathology (hematology, clinical chemistry). These repeated
daily oral administrations were well tolerated, albeit with minor clinical signs, specifically, shoveling and
ruffled fur in T111-treated animals. Repetitive motions were observed in T111-treated females at 100
mg/kg and 400 mg/kg and in one control male and one treated male in the 400 mg/kg treatment group.
Pink discharge in the eye and/or nostril areas (chromodacryorrhea) was observed with increased frequency
in T111-treated females relative to controls, as well as two males from the 25 mg/kg treatment group and
one male from the 100 mg/kg treatment group. No significant alterations in body weight, organ weight,
clinical pathology, or histopathology were observed. For TK analysis, T111 plasma levels were
determined at 6 time points on Day 1 and Day 7 in male and female rats. T111 was not completely cleared
within 24 h, indicating potential accumulation upon repeated daily dosing (Supplementary Table 3).
Maximum systemic exposure was obtained in the 100 mg/kg dose group, likely due to the limited
solubility at 400 mg/kg, which was administered as a suspension (the lower doses were administered as
solutions). The TK profile of T111 in these studies was consistent with the PK parameters in mice (Table
3), which indicated a long half-life and rapid absorption21.

Drug resistance selection. To gain insight into the mechanism of action and propensity for drug
resistance selection, we cultured P. falciparum Dd2 strain under continuous and incrementally increasing
concentrations of T111. Over 16–18 months, the parasites acquired resistance to T111 in an ordered and
sequential manner (Figure 5). Because earlier iterations of development of our acridone chemotype had a
presumed target of Plasmodium cytochrome b (cyt b)30, we directly sequenced pfcytb in parasites
emerging from drug selections with decreased susceptibilities to T111. Multiple mutations in the cyt b
gene were identified in these resistant parasites (Figure 5). Initial selection led to modest resistance in
parasites with a single mutation, V259L or G131S. Further selection yielded parasites with the
combination of these two mutations, and subsequent selection with high nanomolar concentrations of
T111 yielded parasites with 3 or 4 pfcytb mutations, including two novel mutations V140I and I119L. All
mutations selected, except L87V, are located in the cyt b Q site56.
o

To confirm that cyt b mutant haplotypes confer T111 resistance, we tested susceptibility of the
mutant lines to T111 and other compounds known to target the mitochondrial electron transport chain
(ETC) (Table 4). T111 retained low nanomolar potency against parasites with two cyt b mutations, but a
third mutation conferred high level resistance. T111-resistant mutant lines did not show significant cross
resistance with ATV (Q site inhibitor), but had various degrees of cross resistance with other Q site
o o
inhibitors (ELQ-400 and myxothiazol (MYX)). Two T111-resistant mutant lines, Dd2-C I119L-G131S-V259L
0
and Dd2-C G131S-V140I-V259L, exhibited significant cross resistance with ELQ-300 (Q site inhibitor) and
1 i
DSM265 (DHODH inhibitor), and sequencing revealed that they also carried the pfdhodh C276F mutation.
There was no cross resistance with the clinically deployed antimalarials chloroquine (CQ), piperaquine
(PIP), lumefantrine (LUM), or DHA (Supplementary Table 4), which do not act against the ETC.

Whole genome sequencing (WGS) and analysis. To determine whether T111 selection induces
mutations in other genomic regions and to assess their relationship with the T111 resistance phenotype,
six clones carrying multiple pfcytb mutations after prolonged selection with T111 and the unselected
parental strain were evaluated with WGS (Supplementary Table 5). All mutations previously identified
by direct sequencing of pfcytb and pfdhodh were confirmed by WGS. The only gene in which mutations
were observed in all independently selected drug resistance lines was pfcytb, offering strong evidence that
PfCYTB is a primary target of T11111,57. We observed other mutations elsewhere in the genome, but not
in each selected line. Additional mutations occurred in two clones in the pfdhodh gene, consistent with
findings from Sanger sequencing. Importantly, PfDHODH was the only protein with selected mutations
other than CYT B that is a known drug target. Mutations in pfdhodh have previously been associated with
resistance to cyt b inhibitors58. Supplementary Table 5 identifies 15 other genes that acquired mutations
in at least one of the drug selected lines. These sporadic mutations were likely attributable to spontaneous
genetic variation that developed over a year of continuous in vitro culture and not to selection by T111.

Cross-resistance profile with Plasmodium mitochondrial electron transport chain (ETC) inhibitors
with different site preference. To study cross-resistance between T111 and other ETC inhibitors, we
used a panel of P. falciparum parasites with different cyt b Q /Q site sequences (Table 5). Clinical isolate
o i
Tm90-C2B carries a point mutation at the Q site of cyt b (Y268S) that confers ATV resistance59. The
o
Dd2-D I22L clone was selected by culturing P. falciparum Dd2 under ELQ-300 drug pressure, leading to
1
an I22L mutation in the Q region of cyt b contributing to loss of sensitivity to ELQ-30060. P. falciparum
i
transgenic D10yDHODH parasites, which bypass the parasite’s ETC fueled pyrimidine synthesis pathway,
are therefore completely resistant to all mitochondrial ETC inhibitors61,62. We also included pan-sensitive
(D6) and MDR (Dd2) strains of P. falciparum as reference parasites. T111 exhibited decreased activity
against ATV-resistant parasites, but retained nanomolar potency (IC vs Tm90-C2B = 4.0 nM).
50
Interestingly, T111 had enhanced activity against parasites resistant to the Q site inhibitor ELQ-300 (IC
i 50
of T111 vs Dd2-D I22L = 0.00073 nM). In contrast, ATV and ELQ-300 had decreased activity against
1
Tm90-C2B (ATV IC = 8,538 nM) and Dd2-D I22L (ELQ-300 IC = 245 nM), respectively. Similar to
50 1 50
the ETC inhibitors ATV, ELQ-300, and DSM161,63, T111 had reduced activity against D10yDHODH,
with a resistance index >1000 (vs D6 or Dd2), confirming that T111 targets the mitochondrial ETC.
To further validate the target, T111 was tested in the 4-day suppression rodent model using an ATV-
resistant P. yoelii clone (ATVr) harboring Y268S mutations corresponding to the Q site of cyt bc
o 1
corresponding to known ATV-resistant mutations in P. falciparum64 and an ELQ-300 resistant P. yoelii
clone (ELQ-300r) containing an I22L mutation at the Q region of cyt bc , corresponding to known ELQ-
i 1
300 resistant mutations in P. falciparum generated in vitro64. As shown in Supplementary Table 6,
although both ATVr and ELQ-300r P. yoelii strains were highly resistant to their respective parent
compounds, ATVr parasites remained fully sensitive to ELQ-300 and vice versa64. T111 exhibited slightly
reduced oral efficacy against ATV-resistant parasites (ED = 0.24 mg/kg/day vs wild type; ED = 0.31
50 50
vs ATVr ), with no cross-resistance with ELQ-300 (ED = 0.25 vs ELQ-300r ), which is consistent
Y268S 50 I22L
with our observations from in vitro evaluation.

Docking analysis of T111-selected cyt b mutants. We used AutoDock 4.0 to model the predicted binding
of T111 to P. falciparum cyt b. Based on the homologous crystal structure of S. cerevisiae cyt b, the Q
o
binding site in P. falciparum cyt b was modeled by substituting amino acids at corresponding positions65.
T111 readily docked into the modeled P. falciparum cyt b Q binding site, where residues G131 and V259
o
surrounded the ligand (Supplementary Figure 3). However, introduction of mutations identified in T111-
selected resistant strains revealed potential steric clashes: the V140I substitution clashed with the head
group of T111, whereas the I119L substitution clashed with the tail end of the compound, destabilizing
its binding (Supplementary Figure 3). Consistent with these predictions, parasite lines harboring either
mutation exhibited the highest T111 IC values (Table 4). Together, the IC measurements and
50 50
molecular docking results indicate that selection of the third mutation confers high-level resistance to
T111.

Methods
The research was conducted under an IACUC-approved animal use protocol in an AAALAC
International-accredited facility with a Public Health Services Animal Welfare Assurance and in
compliance with the Animal Welfare Act and other federal statutes and regulations relating to laboratory
animals. In vivo asexual blood-stage antimalarial studies were carried out at VA Portland Health Care
System (VAPORHCS), and the procedures were conducted under protocols approved by the
VAPORHCS’s IACUC. In vivo liver-stage antimalarial studies and in vivo PK were carried out at Walter
Reed Army Institute of Research (WRAIR), and the procedures were conducted under protocols approved
by the WRAIR/Naval Medical Research Command (NMRC)’s IACUC in accordance with national and
Department of Defense guidelines. In vivo safety and TK studies were carried out at SRI International
(SRI), and the procedures were conducted under protocols approved by the SRI’s IACUC.

Synthesis of T111 and its prodrug. The original synthetic method for preparing T111 was described in
our previous publication21. Details of the revised synthetic approach (Supplementary Figure 4), including
the experimental procedures, and structural characterization data (Supplementary Methods and
Supplementary Figures 5–14) for the scale-up production of T111, as well as the synthetic method for its
prodrug, are provided in the Supplementary Information.

In vitro GRRA against ART-resistant clinical isolates. T111 was evaluated against two P. falciparum
K13 mutants (SP045 and SP060) originally obtained from symptomatic malaria patients in the Huye
District, Southern Provence, Rwanda, during a 2019 molecular surveillance study. These isolates carry
the validated R561H Kelch 13 propeller domain mutation associated with delayed parasite clearance in
East Africa24,25. Using the 6-hour GRRA23, all strains were thawed, frozen, and cultured under standard
conditions at 5% hematocrit. Samples parasitemia was measured by flow cytometry. Parasite cultures that
were predominantly segmented schizonts were synchronized using a single Percoll layer method23.
Cultures that met quality control requirements of ≥70% early ring stage and ≥1% parasitemia were
adjusted to 0.25% parasitemia at 2% hematocrit. Treated samples were exposed starting at 2800 nM to
DHA in 0.02% DMSO and 10 subsequent two-fold dilutions along with a no-drug control, or starting at
400 nM of T111 in 0.02% DMSO followed by 10 two-fold dilutions; untreated (no-drug control) samples
were exposed to the same amount of DMSO (0.02%) alone. The GRRA implements 11 RSA
50
experiments to generate a dose-response curve for each parasite line per drug. Master mixes of samples
were aliquoted into 2 technical replicates of 275 µL each in 96 well plates (2 treated each condition, 2
untreated). At 6 h post-exposure, 200 µL of media was removed from the wells and replaced with 200 µL
of incomplete media and spun for 3 min at 800g to rinse. The rinse was repeated a total of 3 times, after
which 200 µL complete media was added back to the wells. At 120 h post-treatment, thin microscopy
smears were made using 2 µL of culture for each sample and 20 µL of culture was transferred to PCR
strips and stored at -80 ℃ until qPCR quantification. A standard curve of known parasitemia was
generated using the NF54 parasite line for each qPCR quantification for conversion from Cycle threshold
(C) to parasitemia and comparison between qPCR experiments. Replicate viability was calculated
t
according to the GRRA method23. Each individual reaction mixture and cycling conditions were set to
previously used methods. C was calculated using AB1 SDA 2.4.1. software. All graphs and regressions
t
were performed in the software GraphPad Prism 10 (version 10.5.0). We find that commonly reported
thresholds (cutoffs) for resistance (e.g., 1% survival in traditional RSA) may not be broadly applicable,
especially with lower thresholds subject to potential artifacts due to markedly low survival rates of ART-
sensitive and low-level ART-resistant parasites. Regular RSA relies on a single drug concentration, 700
nM. To gain a more fine-scale quantification to compare drug susceptibilities with relevance to therapeutic
success and failure, we developed the GRRA
50
to generate full dose-response curves for precise
quantitative comparisons.

Ethical statement for Rwandan clinical isolates. The P. falciparum clinical isolates used in this study
(including SP045 and SP060) were originally collected during a molecular surveillance study conducted
between September and December 2019 in the Huye District, Southern Province, Rwanda. Ethical
Clearance for the primary collection and subsequent use of these isolates was granted by the Rwanda
National Ethics Committee (Kigali, Rwanda, approval ref no. 416/RNEC/2017 and 686/RNEC/2019).
Prior to enrollment, written informed consent was obtained from all adult participants or from the parents
or legal guardians of child participants. Additionally, written assent was obtained from all minor
participants aged 7–18 years, in accordance with the guidance of the Rwanda National Ethics Committee.
All samples were de-identified prior to use in the current study.

In vitro drug susceptibility assay against ART-resistant parasites. Drug susceptibility assay against
ART-resistant P. falciparum clones was performed using a SYBR Green-based assay44,74. Synchronized
ring stage parasites were cultured for 72 h in the presence of the test drug. The assay was performed in
96-well plates at 2% hematocrit and 0.5% starting parasitemia. Each experiment was conducted in
triplicate and repeated with three biological replicates. Parasite growth at 72 h was quantified via SYBR
Green I staining. The obtained counts were plotted against the logarithm of the compound concentration
and a curve was fitted with non-linear regression (sigmoidal dose-response/variable slope equation) to
determine IC (half-maximal inhibitory concentration) values. The data was analyzed using GraphPad
50
Prism software 8.0.

Ex vivo drug susceptibility assay against clinical isolates. The ex vivo antiplasmodial activity of T111
was tested against fresh P. falciparum isolates using a 72-h growth inhibition assay with SYBR Green
detection27,28. Isolates were collected from patients with uncomplicated falciparum malaria in Bobo-
Dioulasso, Burkina Faso and Tororo, Uganda. For parasitemia culture, parasitemia was identified with
Giemsa-stained thin films using a light microscope. Only samples with at least 0.3% parasitemia were
analyzed. Isolates were stored at 4 ℃ and assayed within 24 h of collection. To measure ex vivo drug
susceptibilities, a 72-h microplate growth inhibition assay was used with SYBR Green detection. Study
compounds were dissolved in DMSO as 10 mM stocks and stored at -20 ℃. Drugs were serially diluted,
including drug-free and parasite-free control wells, with concentrations optimized to capture full dose-
response curves. Cultures were diluted with uninfected O+ erythrocytes to achieve 0.2% parasitemia and
2% hematocrit. After 72 h, wells were resuspended and culture from each well was transferred to 96-well
plates containing SYBR Green lysis buffer and mixed. Plates were incubated for 1 h in the dark at room
temperature and fluorescence was measured with a plate reader (485 nm excitation and 530 nm emission).
IC values were derived by plotting fluorescence intensity against log drug concentration and fit to a non-
50
linear curve using a four-parameter Hill equation in Prism (version 9.0).

Ethical statement for Burkina Faso and Uganda clinical isolates. The P. falciparum clinical isolates
used for ex vivo susceptibility testing in this study were obtained from symptomatic patients with
uncomplicated malaria in Burkina Faso and Uganda. Ethical clearance for the collection and subsequent
use of these isolates was granted by the Institutional Ethics Committee of the Institut de Recherche en
Sciences de la Santé (IRSS) (Bobo-Dioulasso, Burkina Faso), the Makerere University School of
Biomedical Sciences Research Ethics Committee (Kampala, Uganda), and the Uganda National Council
for Science and Technology. In Burkina Faso, isolates were collected during the 2021 and 2022 malaria
transmission seasons from patients seeking treatment at health centers in Bobo-Dioulasso. In Uganda,
isolates were collected longitudinally from government health facilities in the Tororo, Busia, Mbale, and
Agago districts (2010–2024). Prior to enrollment, written informed consent was obtained from all adult
participants or from the parents or legal guardians of child participants. Written assent was also obtained
from all minor participants (aged 8–17 years) in accordance with the guidance of the respective National
Ethics Committees. All samples were de-identified and assigned unique study identifiers prior to
laboratory analysis.

In vivo antimalarial efficacy against blood-stage P. yoelii in rodent model. The efficacy of T111 and
the T111/TQ combination was determined using two well-established murine models against P. yoelii
parasites: 1) a 4-day suppression model21,29-31; and 2) a single-dose cure model31-33. Animals were housed
at the VAPORHCS in Thoren Maxi-Miser microisolator cages (78 sq. in. floor space) with a maximum
density of five females or four males per cage. Cages were maintained on a 12-h light/dark cycle and
provided with autoclaved nesting materials (nestlets) for environmental enrichment. All mice had ad
libitum access to food and water. Four- to five-week-old female and male outbred CF1 mice (Charles
River Laboratories, Raleigh, NC, USA) were randomly assigned to treatment and control groups using a
simple randomization procedure (e.g., computer-generated random numbers) to ensure balanced
distribution of baseline body weights and starting parasitemia levels across all cohorts. Groups of four
mice (n=4, mean body weight of each group was ~30 g) were infected intravenously with 3.5 × 104 P.
yoelii (Kenya strain, MRA-428) from a donor animal. Drug administration commenced the day after the
animals were inoculated (Day 1). The test drug or the drug combination was dissolved in PEG-400 and
administered by oral gavage once daily for 4 successive days in the 4-day suppression model, but only
once on Day 1 in the single-dose cure model. Blood was collected via the tail vein with the aid of a syringe
needle on Day 5 and then at weekly intervals through Day 28. Blood films were Giemsa-stained and
examined microscopically to determine the level of parasitemia. All mice were observed daily to assess
clinical signs. Animals with observable parasitemia following the experiments were euthanized; animals
cleared of parasitemia from their bloodstream were observed daily with assessment of parasitemia
performed weekly until Day 28, at which point, the animal(s) were scored as cured of the infection and
then humanely euthanized. All treated mice with a negative smear on Day 28 were considered cured (100%
protection). ED and ED values (mg/kg/day) were derived graphically from the dose required to reduce
50 90
the parasite burden by 50% and 90%, respectively, relative to drug-free controls.

Animals and ethical statement at VAPORHCS. In vivo asexual blood-stage antimalarial efficacy
studies were conducted at the VAPORHCS under a protocol reviewed and approved by the VA Portland
Institutional Animal Care and Use Committee (IACUC). This work was originally approved under local
animal protocol #6067-22 (IRBNet ID #1673475-5) on March 26, 2022. A triennial renewal was
subsequently granted under protocol #6067-24 (IRBNet ID #1673475-13) effective December 18, 2024,
which remains valid through December 17, 2027. This research also received federal oversight and
approval from the U.S. Army Medical Research and Development Command (USAMRDC) Animal Care
and Use Review Office (ACURO) under protocol #PR210491.e001. The protocol was originally approved
by ACURO on May 10, 2022, with the most recent renewal approval granted on February 26, 2025. All
research was conducted in an AAALAC International-accredited facility in strict accordance with the
Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals (NRC Publication, Eighth
edition)75. All research described herein with all relevant ethical regulations.

In vitro antiplasmodial activity against sexual blood-stage P. falciparum gametocytes. P. falciparum
NF54 was used to generate gametocytes. The erythrocytic stage of the parasite was cultured following
standard laboratory protocols. Gametocyte production was carried out as described by Miura et al34. P.
falciparum NF54 asexual parasites were cultured in RPMI 1640 medium (KD Medical) containing L-
glutamine, 25 mM HEPES, and 50 µg/mL hypoxanthine, and supplemented with 0.21% sodium
bicarbonate (from a 7.5% stock solution: KD Medical), 2 µg/mL gentamicin (from a 10 mg/mL stock
solution: KD Medical), and 0.25% (w/v) Albumax II. Cultures were maintained in washed human O+ red
blood cells at a final hematocrit of 2%76. For gametocyte cultures, parasites were induced using serum-
free human media34. Gametocyte cultures were maintained in washed human O+ red blood cells at a final
hematocrit of 5%. Cultures were initiated at an asexual parasitemia of 1.5% in serum-free gametocyte
media and allowed to crash and form mature gametocytes over 14 days77,78. All cultures were maintained
at 37 ℃ in a Billups chamber under a gas mixture of 90% N , 5% CO , and 5% O , with media changes
2 2 2
daily. On Days 5 through 7 post gametocyte induction, gametocyte cultures were treated with T111 at
concentrations of 1 µM or 10 µM. Fresh T111 was added at each media change. DMSO diluted in
gametocyte media was used as the control. To remove T111, cultures were washed once with RPMI 1640,
centrifuged at 1000 × g for 3 min, and resuspended in warm serum-free gametocyte media without drug.
Gametocytemia was assessed on Day 14 post-induction using blood smears stained with the Hema 3TM
Manual Staining System (Fisher) and analyzed by light microscopy.

Membrane feeding assay (SMFA) in mosquitoes. The SMFA assay was performed using P. falciparum
NF54 gametocytes produced in vitro35. P. falciparum NF54 gametocytes were grown in human blood
cultured in RPMI 1640 GlutaMax medium (Gibco) supplemented with 25 mM HEPES, hypoxanthine,
and human serum35. Deidentified human blood and serum were obtained from the Australian Red Cross
Blood Service. Mature (stage V) gametocytes were quantified in Giemsa-stained blood smears.
Immediately prior to infection, a feeding mixture comprising 45% human serum and 55% human blood
containing 0.15% stage V gametocytes and the desired concentration of drug or vehicle control, was
prepared and maintained at 37 ℃. Adult female Anopheles stephensi (MR4) mosquitoes reared under the
standard insectary conditions79 were placed in mesh-topped cups and allowed to feed on the gametocyte
mixtures in prewarmed glass feeders wrapped with parafilm. Mosquitoes that failed to feed in 20 min
were removed. Mosquito midguts were dissected 7 days after infection, stained in 0.2% mercurochrome35,
and oocysts were counted using a light microscope.

Ethical statement for human blood product used for SMFA assay. The use of de-identified human
blood products for the in vitro SMFA assay was conducted at the University of Melbourne. This work
was performed under the University of Melbourne Human Research Ethics Committee (HREC) project
ID number 22013. All blood products were provided by the Australian Red Cross Lifeblood in accordance
with national ethical guidelines and specific deeds of agreement.

Tarsal contact assay in mosquitoes. The tarsal assay using a thin film was performed36. For preparation
of a thin film, 25 mL xylenes and 1.0 g low density polyethylene polymer were combined on the lid of a
glass petri dish (6 cm) and heated at 150 ℃ until the polymer was dissolved. Xylenes were occasionally
replenished upon evaporation. A solution of xylenes containing T111 prodrug was then added to the above
solution. Excess xylenes were evaporated slowly and the resulting crude polymer films were allowed to
dry overnight. The crude polymer thin film was then put underneath the glass petri dish, and the assembled
apparatus was heated on a hot plate with a weight placed on top. The resulting extruded polymer thin film
containing the 5% (w/w) T111 prodrug in low-density polyethylene (LDPE) was allowed to cool and
removed from the petri dish lid. Next, the T111 prodrug film was placed in a 6 cm glass petri dish fitted
with a transparent lid to contain mosquitoes. 4–7-day old female Anopheles gambiae G3 strain mosquitoes
were aspirated into this container and allowed to land on the T111 prodrug thin film for six min. After
exposure, the mosquitoes were immediately transferred to a cage and one hour later provided with cultured
P. falciparum NF54 gametocyte-infected erythrocytes using a membrane feeder. Infected mosquitoes
were housed in a secure infection glovebox for the remainder of the experiments and provided with 10%
w/v glucose water solution ad libitum. On Day 7 post-infection, mosquitoes were aspirated into 80%
ethanol and incubated at -20 ℃ for 10 min before being transferred to 1X PBS for dissection. Midguts
were dissected into 1X PBS, stained with 0.2% w/v mercurochrome (mercury dibromofluorescein
disodium salt, Sigma-Aldrich) in double-distilled water for 14 min, then mounted in 0.02%
mercurochrome for microscopy. Midguts were imaged and oocysts counted at 100× magnification on an
Olympus Inverted CKX41 microscope to quantify oocyst prevalence and intensity.

In vitro liver-stage P. cynomolgi assay. The liver-stage P. cynomolgi assay was designed to identify
anti-hypnozoite drugs utilizing a 384-well in vitro culture system37,31. Briefly, cryopreserved primary
simian hepatocytes (donor lots XGB, OQB, and QHO: BioIVT, Inc.) were seeded in collagen-coated 384-
well plates and infected with P. cynomolgi bastianellii (B strain) sporozoites dissected from infected
Anopheles dirus mosquitoes. Test compounds were solubilized in 100% DMSO and serially diluted 2-
fold to generate up to a 12-point dose-response curve, maintaining a final DMSO concentration of 0.1%
across all wells. Two distinct treatment modes were evaluated. For the prophylactic assay, to assess the
prevention of infection, compound exposure began 1-hour post-infection with daily media changes on
Days 1 and 2. For the radical cure assay, to assess activity against established liver forms, drugs were
administered daily from Day 4 through Day 7 post-infection. In both treatment modes, the highest
concentration tested was 20 µM for KDU691, T111, and TQ, 10 µM for ATV and MAD. For readout on
Day 8, plates were fixed and stained via immunofluorescence to visualize parasite forms (hypnozoites
and schizonts) and hepatocyte nuclei, and images were acquired using an Operetta CLS high-content
imaging system (PerkinElmer). Host hepatocyte viability was measured in parallel to determine
compound toxicity. Parasite forms were automatically quantified using Harmony 4.9 software, and
percent inhibition and toxicity were calculated relative to in-plate DMSO vehicle controls. IC and CC
50 50
(half-maximal cytotoxic concentrations) were determined by fitting dose-response data to a four-
parameter logistic (variable slope) model using a custom Python script incorporating a grid search
algorithm.

Ethical statement for P. cynomolgi sporozoites production at Armed Forces Research Institute of
Medical Sciences (AFRIMS). Anopheles dirus mosquitoes infected with the B strain of P. cynomolgi
were produced by the Department of Entomology at the AFRIMS (Bangkok, Thailand). All procedures
involving non-human primates (NHP) and mosquitoes were conducted under an animal use protocol
reviewed and approved by the USAMD-AFRIMS Institutional Animal Care and Use Committee (IACUC)
and received formal oversight and approval from the U.S. Army Medical Research and Development
Command (USAMRDC) Animal CARE and Use Review Office (ACURO) on September 19, 2022.
Animals were maintained in strict accordance with the Guide for the Care and Use of Laboratory Animals
(NRC Publication, Eighth edition)75 and the Animal for Scientific Purposes Act (Thailand). The USAMD-
AFRIMS animal care and use program is fully accredited by AAALAC International. In compliance with
U.S. Department of Defense and institutional security policies, specific internal protocol numbers are
maintained by the USAMD-AFRIMS IACUC Office and are available for independent verification by
the Editorial Office upon request.

In vivo antimalarial efficacy against liver-stage P. berghei in rodent model. The liver-stage efficacy
of T111 or the T111/TQ combinations was evaluated as prophylaxis in a murine model using the IVIS
system21,30,31,45,46. Four- to six-week-old female inbred Albino C57BL/6 mice (18–22 g, Jackson
Laboratories, Bar Harbor, ME, USA) were utilized. Animals were assigned unique study numbers via ear
tags and cage cards and were randomly assigned to treatment or control groups (n=5 per group) using a
weigh-stratified randomization procedure. All animals were quarantined and acclimatized for 7 days prior
to study initiation and were housed in a 12:12 light/dark cycle with food and water provided ad libitum.
Mice were infected intravenously with 104 luciferase-expressing P. berghei sporozoites (ANKA strain)
via tail vein injection on Day 0. Drugs were dissolved in PEG-400 and administered orally once per day
on Day -1, Day 0, and Day 1. Liver-stage efficacy was determined by assessing the percent reduction of
bioluminescence signals emitted in the liver area at 24 and 48 h post-infection, while the early blood-
stage efficacy was assessed at 72 h by examining signals emitted from the whole body of the animal.
Infection reduction was assessed by comparing the bioluminescence signals emitted by treated groups
with signals emitted by the vehicle control-treated group at that time point. Later blood-stage parasitemia
was determined via flow cytometry. In this model, an animal is considered cured if the liver-stage
bioluminescence signal is below the IVIS limit of detection at 48 h post-infection and blood-stage
parasitemia is below the flow cytometer limit of detection at 30 days post-infection. Long-term blood-
stage efficacy was evaluated starting on Day 6 post-infection and continued for up to 30 days using flow
cytometry.

Animals and ethical statements at Walter Reed Army Institute of Research (WRAIR). The animal
protocols for research conducted for liver-stage efficacy and PK studies at WRAIR were reviewed and
approved by the WRAIR/Naval Medical Research Command (NMRC) Institutional Animal Care and Use
Committee (IACUC) and received formal oversight and approval from the U.S. Army Medical Research
and Development Command (USAMRDC) (approved March 27, 2024). The experiments reported herein
were conducted in compliance with the Animal Welfare Act and per the principles set forth in the “Guide
for Care and Use of Laboratory Animals,” Institute of Laboratory Animals Resources, National Research
Council, National Academy Press, 1996. Research was conducted in an AAALAC International-
accredited facility in strict accordance with the Guide for the Care and Use of Laboratory Animals (NRC
Publications, 8th edition, 2011)75. Department of Defense (DoD) regulations, and the Animal Welfare Act.
In compliance with DoD and institutional security policies, specific internal protocol numbers are
maintained by the WRAIR Office of Animal Care and Compliance and are available for independent
verification by the Editorial Office upon request.

In vitro drug combination studies against blood-stage P. falciparum and liver-stage P. cynomolgi.
For in vitro T111/TQ drug combination studies, blood-stage and liver-stage drug susceptibility tests were
performed as described above, using a fixed-ratio platform for drug combinations42,43,80. For the asexual
blood-stage assays, parasites were maintained in de-identified human A+ red blood cells purchased from
Lampire Biological Laboratories (Pipersville, PA, USA). Briefly, drugs were diluted in fixed ratios (4:1,
3:2, 2:3, and 1:4) of starting concentrations predetermined to generate well-defined concentration
response curves. Intrinsic dose-response curve for each drug alone and four different fixed-ratio
combination dose-response curves yielded corresponding IC values. The fractional inhibitory
50
concentrations (FICs) were then calculated by the following formulas: FIC (A) = IC of drug A in
50
combination/IC of drug A alone; FIC (B) = IC of drug B in combination/IC of drug B alone; ΣFIC
50 50 50
index = FIC (A) + FIC (B). The isobolograms were constructed by plotting a pair of FICs for each
combination. An interpretation of a straight diagonal line (ΣFIC index = 1) on the isobologram indicates
an additive effect between two drugs. A concave curve below the line (ΣFIC index < 1.0) indicates synergy
of the combination, while a convex curve above the line (ΣFIC >1.0) indicates antagonism. All
isobolograms were generated using Microsoft Excel (version 16.66.1).

Ethical Statement for human red blood cells used in blood-stage susceptibility test. The use of
commercially available, de-identified human red blood cells for these in vitro assays was determined to
be exempt from IRB oversight by the VA Portland Health Care System IRB, as the research did not
involve “human subject” as defined by 45 CFR 46.102(f).

In vivo drug combination studies against blood-stage P. yoelii and liver-stage P. berghei in mice. In
vivo antimalarial efficacy studies were performed as described above. In the blood-stage 4-day
suppression model, the effect of T111 on TQ oral efficacy was conducted by comparing dose-response
curves of TQ alone and in the presence of sub-therapeutic oral doses of T111, and the potentiation effect
was expressed as the ratio of ED values for TQ and in the presence of T111. In the blood-stage single-
50
dose cure model, various oral dose combinations of T111 with TQ were administered in mice 24 h post
infection, and corresponding curative effects (no parasites detected) were determined on Day 28. For
liver-stage in vivo oral efficacy studies, T111 alone, TQ alone, and sub-therapeutic combinations were
administered to mice on Days -1, 0, and +1 after parasite inoculation. Parasite loads in the liver at 24 and
48 h were determined for liver-stage protection, and blood-stage cure was determined on Day 31.

In vitro metabolic stability. The half-lives (t ) of T111 in liver microsomes (HLM, MLM and RLM)
1/2
were determined using our well-established methods48,49. Sample stocks at 20 µM in DMSO were diluted
to a final concentration of 1.0 µM with a mixture containing 0.5 mg/mL prewarmed pooled human or
mouse or monkey liver microsomes (BD Gentest), 1.3 mM NADP (Sigma), 3.3 mM MgCl (Sigma), and
2
100 mM potassium phosphate buffer (pH 7.4) using a TECAN Genesis robotic liquid handler. The
reaction was started with the addition of 1 U/mL glucose-6-phosphate dehydrogenase (G6PD). The
mixture was incubated on a shaking platform at 37 ℃, and aliquots were taken and quenched with the
addition of an equal volume of cold acetonitrile at 0, 10 -, 20-, 30-, and 60-min. Samples were centrifuged
at 37,000 rpm for 10 min at 20 ℃ to remove debris. Sample quantification was carried out by LC-MS,
and the metabolic half-life (t ) was calculated by log plots of the total ion chromatography area remaining.
1/2

In vivo pharmacokinetic (PK) studies. PK studies were conducted using well-established methods21,31,50.
Four to five weeks old male and female outbred ICR-CD1 mice (Charles River Laboratories. Inc. Raleigh,
NC) weighing 23–35 g were used for PK studies. Animals were assigned unique study numbers via ear
tags and cage cards and were randomly assigned to treatment or control groups (n=3 per group) using a
weigh-stratified randomization procedure. All animals were quarantined and acclimatized for 7 days prior
to study initiation and were housed in a 12:12 light/dark cycle with food and water provided ad libitum.
T111 was formulated in sterile water containing 5% ethanol, 5% CRM, and 0.5% HECT, and given orally
using intragastric administration. A single 40 mg/kg dose of T111 was administered to three mice at each
experimental endpoint. At each time point, a terminal whole blood sample was collected by cardiac
puncture using heparinized syringes (Heparin, Hospira, Lake Forest, IL), while mice were under
anesthesia in accordance with the approved IACUC protocol. Following the separation of appropriate
aliquots, plasma was obtained from the whole blood via centrifugation at 16,200 rpm for 10 min at 4 ℃.
Mice were humanely euthanized as described in the protocol, after which the liver samples were also
collected and weighed at each experimental time point. All tissue samples were immediately preserved
on dry ice and later stored at -80 ℃ until analytical work was performed. Drug concentrations were
determined for each sample taken from animals dosed with the test compound. PK parameters such as
time to maximum plasma concentration (T ), maximum plasma concentration (C ), the apparent
max max
elimination half-life (t ), area under the curve (AUC), etc., were obtained using noncompartmental
1/2
analysis via the Phoenix WinNonlin software package (version 6.4; Pharsight Corp., Mountain View, CA).

In vitro cytotoxicity assay. Cytotoxicity was evaluated in human HepG2 liver cells, using an MTT
assay52. Briefly, HepG2 cells were cultured in complete Dulbecco’s modified Eagle’s medium (DMEM;
Gibco-Invitrogen, No. 11995-065) prepared by supplementing DMEM with 10% heat inactivated FBS
(Gibco-Invitrogen, No. 16000-036), 100 units/mL penicillin, and 100 µg/mL streptomycin. Cell viability
was assessed using trypan blue. Ninety-six-well plates were seeded with 2 × 104 cells per well and
incubated at 37 ℃ overnight in a humidified 5% CO atmosphere. Test compound plates were prepared
2
with an INTEGRA assist plus station and 96-well plates containing 11 duplicate 2-fold serial dilutions of
test compound suspended in DMSO. The diluted test compound was then added to the corresponding well
preseeded with HepG2 cells. Cells were exposed to test compound concentrations ranging from 200 µM
to 0.2 µM for 24 h. Next, the media and test compounds in each well were removed and plates were then
added with fresh drug-free medium and continued to incubate for an additional 24 h. After incubation, 20
µL of a 0.1 mg/mL solution of resazurin diluted in PBS was added to each well, and all plates were
subsequently incubated for 3 to 4 h in an incubator. Absorbance was determined with the Molecular
Devices SpectraMax plate reader. Three independent experiments, each in duplicate, were performed, and
the IC values were determined using GraphPad Prism software (Version 10) using the non-linear
50
regression equation (sigmoidal dose-response, variable slope).

hERG channel inhibition assay. The hERG assay was performed at Eurofins Panlabs, Inc., St. Charles,
MO, United States. T111 was tested using the standard hERG potassium channel assay53 at six
concentrations from a top concentration of 100 µM. Verapamil was used as a positive control.

Ames assay. Mutagenicity was assessed using the Ames assay54 (EBPI, Ontario, Canada) against S.
typhimurium TA100 and TA98, with and without S9 activation. The tester strain S. typhimurium (TA100
and TA98) cultures were inoculated from the lyophilized pellets and grown in nutrient broth provided by
EBPI. Each inoculated vial (5 mL volume with antibiotics) was placed overnight in the incubator at 37 ℃.
The positive controls for the assay were 1.0 µg/µL 2-aminoanthracene (2-AA) for incubations with the
S9 mix and 5 µg/mL sodium azide (NaN , used for TA100) and 300 µg/mL 2-nitrofluorene (2NF, used
3
for TA98) for incubations without the S9 mix. The tester strains (5 µL) were mixed with 2.5 mL reaction
mixture and, where appropriate, 2.0 mL of S9 mixture, T111 (200 µL/reaction), was then added to each
reaction tube to give a final volume of 20 mL. An aliquot (0.2 mL) of the mixture was then added to each
well of a 96-well plate. The 96-well plates were covered, sealed in zip-lock bags, and incubated for 5 days
at 37 ℃. Plates were scored visually, and the positive wells against background mutations were
statistically analyzed.

Eryptosis assay. In vitro evaluation of eryptosis as a marker for induced hemolytic toxicity was
performed using deidentified human blood samples obtained from the Clinical Trial Center at WRAIR,
using established methods55,81,82. Fresh Li-heparin-anticoagulated blood samples was centrifuged and the
platelet and leukocyte-containing supernatant was removed. Erythrocytes were incubated with TQ or
T111 in Ringer solution at 37 ℃ for 48 h. Flow cytometry was employed to estimate stimulators of
eryptosis: phosphatidylserine exposure at the cell surface from annexin-V-binding, reactive oxygen
species (ROS) formation from 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) dependent
fluorescence, and cell volume from forward scatter. Annexin-V is a small protein that has a high affinity
for phosphatidylserine and annexin-V binding is used to measure the presence of phosphatidylserine on
the cell membrane surface and subsequently the eryptotic activity of the cell.

Annexin-V-binding assay for detection of phosphatidylserine translocation. After incubation under
the respective experimental condition, cell suspension was washed in Ringer solution containing CaCl
2
and then stained with Annexin-V-FITC at 37 ℃ for 15 min under protection from light. The annexin-V-
abundance at the erythrocyte surface was subsequently determined on a FACS Calibur. Annexin-V-
binding was measured at an excitation wavelength of 488 nm and an emission wavelength of 530 nm. A
marker (M1) was placed to set an arbitrary threshold between annexin-V-binding cells and control cells.
The same threshold was used for untreated and drug treated erythrocytes.

Reactive oxygen species (ROS) detection assay. Oxidative stress was determined utilizing DCFDA.
After incubation, a 150 µL suspension of erythrocytes was washed in Ringer solution and stained with
DCFDA in Ringer solution containing DCFDA at a final concentration of 10 µM. Erythrocytes were
incubated at 37 ℃ for 30 min in the dark and washed two times in Ringer solution. The DCFDA-loaded
erythrocytes were resuspended in Ringer solution and ROS-dependent fluorescence intensity was
measured at an excitation wavelength of 488 nm and an emission wavelength of 530 nm on a FACS
Calibur. Subsequently, the geomean of the DCFDA dependent fluorescence was determined.

Quantification of cell size. Erythrocyte volume was estimated utilizing forward scatter (FSC) as a
measure of light diffraction. After incubation in the presence or absence of the test compounds, the
erythrocyte suspension was washed and resuspended in Ringer solution. Cells were identified and gated
based on their light-scatter characteristics (FSC-linear vs. SSC-linear) on a FACS Calibur to exclude
debris and cellular aggregates. For the analysis of cell volume, an FSC-linear histogram was generated
from the gated population. The FSC gain was adjusted to fix the geometric mean (Geo Mean) of the
untreated control erythrocytes at approximately 500 arbitrary units (AU). The same gain and threshold
settings were maintained for both untreated and drug-treated normal and G6PD-deficeinet erythrocytes.
Shifts in the Geo Mean of the FSC signal were subsequently determined to quantify changes in cell
volume relative to the baseline control.

Ethical Statement for use of human erythrocytes in the eryptosis assay. The study protocol was
approved by the WRAIR Institutional Review Board in compliance with all applicable Federal regulations
governing the protection of human subjects. Specifically, the study protocol for the use of volunteer-
derived human erythrocytes (normal and G6PD-deficient RBCs) was reviewed and approved by the Ethics
Committee of the WRAIR under specimen collection protocol #2567.04, titled ′Blood Collection for
G6PD Antimalarial Assay′ (version 1.0, approved 21 December 2018). Written informed consent was
obtained from all participants prior to collection in accordance with institutional and federal regulations
(45 CFR 46). All blood products were provided as de-identified specimens and assigned unique study
identifiers prior to laboratory analysis.

In vivo toxicology and toxicokinetic (TK) studies in rats. In vivo toxicology and TK studies in rats
were performed at SRI International, using the well-established methods83-85. Rats were housed in
microisolator cages with hardwood chip bedding (Sani-Chips; Envigo Teklad) and maintained on a 12-h
light/dark cycle. Environmental conditions were controlled at 20–26 ℃ (68–79°F) with 30–70% relative
humidity. Animals were group-housed, except where individual housing access to a certified global 18%
protein rodent diet (Envigo Teklad) and purified water. T111 was dissolved in PEG-400 and continuously
stirred in a heated water bath between 85–98 ℃ for 1–4 h, until a clear solution or a homogeneous
formulation was obtained. Male and female Sprague Dawley outbred rats were randomly assigned to
treatment and control groups using a simple randomization procedure to ensure a balanced distribution of
baseline weights across all cohorts. For the MTD study, each group of two male and two female Sprague
Dawley rats were administered a single dose of 30, 100, 200 or 400 mg/kg T111 by oral gavage and the
animals were monitored immediately post dose, 2–4 h and 24 h post dose for clinical signs, with body
weights recorded pre-dose and on Day 1 for each dose level group. For the 7-day repeat dose study, each
group of eight male and eight female Sprague Dawley rats were administered a single oral dose of T111
(25, 100 or 400 mg/kg) or vehicle control daily for 7 consecutive days and evaluated for body weight
changes (pre-dose, on Day 8 and 14, and prior to euthanasia), clinical observations (immediately, 2–4 h
post dose, and daily on Day 1 to 7), plasma drug levels (on Day 1 and 7), clinical pathology (hematology,
clinical chemistry on Day 8 and 14), organ weights (brain, heart, kidney, liver, spleen; on Day 8 and 14),
and gross and microscopic examination of the selected tissues (brain, cecum, colon, duodenum, heart,
ileum, jejunum, kidneys, liver, lungs, lymph nodes, rectum, spleen, forestomach, glandular stomach; on
Day 8 and 14). For TK studies, blood was collected from animals at 6 time points post dose on Day 1 (0.5,
1, 2, 4, 8, and 24 h) and on Day 7 (0.5, 2, 8, 24, 48, and 96 h). Drug levels were determined in collected
plasma samples using bioanalytical method developed at SRI. The plasma drug level data were analyzed
using Phoenix WinNonlin (version 8.3) software to perform noncompartmental data analysis.

Animals and ethical statements at SRI International (SRI). All rat safety and TK studies were
conducted at SRI International (Menlo Park, CA, USA) in an AAALAC-accredited facility. All
procedures were reviewed and approved by the SRI International Institutional Animal Care and Use
Committee (IACUC Protocol #02006) and received formal oversight and approval from the U.S. Army
Medical Research and Development Command (USAMRDC) Animal Care and Use Review Office
(ACURO Protocol #PR210491.e002) on September 21, 2023. Studies were performed in strict accordance
with the Guide for the Care and Use of Laboratory Animals (NRC Publication, Eighth edition)1 and the
U.S. Department of Agriculture (USDA) Animal Welfare Act.

Drug resistance selection studies. Drug resistance selection studies were performed by culturing P.
falciparum Dd2 parasite under incremental and continuous drug pressure of T111, using a well-
established method30,86. Four independent cultures of P. falciparum Dd2 were maintained in the presence
of T111 (0.1, 0.5, or 2 nM), with a non-drug exposed control grown in parallel. The concentration of T111
was incrementally increased when parasites replicated at the same rate as the untreated parent line for
three life cycles. Selection experiments were completed when T111 pressure reached 1 µM, at which
point the culture was cryopreserved. Cryopreserved parasites from specific T111 concentrations were
thawed and cloned by limiting dilution. DNA extractions were performed on clonal parasites using the
QIAamp DNA extraction kit (QIAGEN Inc, Valencia, CA) or ZYMO Quick gDNA Miniprep kit (Zymo
Research Corp, Irvine, CA). For sequencing of the open reading frame of pfcytb, PCR was performed
with genomic DNA using forward and reverse flanking primers for cyt b primer 1: 5’-
TTCCTGATTATCCAGACGCT-3’ and primer 2: 5’-TGTTCCGCTCAATACTCAGA-3’. PCR products
were subjected to enzymatic cleanup with ExoSAP-IT (Thermo Fisher Scientific) and sent for Sanger
sequencing (Eurofins Genomics, Louisville, KY) with PCR primer 2 paired with an internal forward
primer: 5’-GAGTTATTGGGGTGCAACTG-3’ and an internal reverse primer: 5’-
CACTCACAGTATATCCTCCACA-3’. Sequences were assembled using 4Peaks software (Nucleobytes,
Amsterdam, Netherlands) and aligned with Clustal Omega (EMBL-EBI). For sequencing of the open
reading frame of pfdhodh, PCR was performed with genomic DNA using forward primer: 5’-
GTGATAGATAGCTCCAGTCGATTTC-3’ and reverse primer: 5’-
TTTGCGCACTTATGTGTCGCCC-3’. PCR products were prepared for Sanger sequencing and
analyzed as described above, except using two forward primers: 5’-GCTATTAATGTAAGCTCCC-3’
and 5’-CCATTCGGTGTTGCTGC-3’ and with two reverse primers: 5’-
GCATTACCCGTTTGGCCCCTTGGGG-3’ and 5’-GGAGCTTACATTAATAG-3’. Newly generated
T111-resistant P. falciparum clones and parental strains described in this study are available from the
corresponding author upon reasonable request for non-commercial research purposes, subject to standard
Material Transfer Agreements (MTA).

Genomic DNA isolation, sequencing, and whole genome sequencing (WGS) analysis. T111 mutants
and the parental strain were evaluated with Illumina whole genome sequencing. Genomic DNA was
isolated from six clones resulting from drug selection experiments (Qiagen DNeasy) and concentrated per
the manufacturer’s instructions (Zymo Research Corporation Genomic DNA Clean & ConcentratorTM
Kit-25). Libraries for Illumina short-read sequencing were prepared and sequenced (Illumina NovaSeq X
Plus, Novogene). Genomic sequence data were aligned to the Dd2 genome (version 51 from
PlasmoDB87,88), sorted with samtools (v1.11)89, duplicates marked with picrad (v2.25.0)90, variants called
with freebayes (v1.3.5), filtered for quality, and annotated with SnpEff (v5.0). Single-nucleotide variants
(SNVs) in protein-coding regions identified in the parental Dd2 strain were subtracted from those
identified in each T111-resistant strain to identify those unique to each resistant strain. To identify SNVs
implicated in T111 resistance, SNVs present in less than 90% of the reads of a particular strain were
excluded.

P. falciparum cyt b modeling and autodocking calculations. Automated docking simulations were
conducted using the AutoDock 4.0 program suite91. The sequence of P. falciparum cyt b is highly
homologous to yeast cyt b and thus the crystal structure of yeast cyt b (PDB ID 4PD4) was used for ligand
docking studies with corresponding residues replaced with those of P. falciparum cyt b around the ATV
binding pocket. The 3D structures of compounds T63, T111, ELQ 300 and ELQ400 were modeled based
on molecular formulas in PubChem database. The crystal structures of ATV, MYX, or other model
structures were roughly placed in the binding pocket as described in the yeast cyt b-ATV complex, which
was used as the initial starting point to minimize the complexes using the Lamarckian genetic algorithm.
The crystal structure of bovine cyt b-MYX complex (PDB ID 1SQP) was also used in docking study as a
reference. In all cases, a grid box size of 60 × 60 × 60 points with a grid-spacing of 0.375Å was
implemented, which was centered on the ligand to cover the whole pocket. Thirty docked structures, i.e.,
30 runs, were generated by using genetic algorithm searches. A default protocol was applied, with an
initial population of 50 randomly placed individuals, a maximum number of 2.5 × 105 energy evaluations,
and a maximum number of 2.7 × 104 generations. A mutation rate of 0.02 and a crossover of 0.8 were
used. Results differing by less than 1.0 Å in positional root-mean-square deviation (RMSD) were
clustered together. A ligand conformation with the lowest docking energy and catalytically binding to the
protein was considered to be docked.

Drug susceptibility testing for cross-resistance profile. Drug susceptibility assays against P. falciparum
strains were performed using a SYBR Green based assay44. D6 and Dd2 were obtained from MR4. Tm90-
C2B was obtained from WRAIR. Dd2-D I22L and ELQ-300 were obtained from VAPORHCS.
1
D10yDHODH was obtained as a courtesy from Dr. Akhil Vaidya at Drexel University. P. yoelii ATVr
and ELQ-300r clones were obtained from VAPORHCS.

Statistical analysis and sample size determination. Sample sizes for in vivo efficacy and PK studies
were determined by a priori power analysis using G*power software (version 3.1.9.7). Based on the
difference between two independent means observed in previous experiments (Cohen’s d = 2.34), a one-
tailed t-test (α = 0.05, power = 0.80) was used to justify a group size of n=4 for blood-stage efficacy and
n=5 for liver-stage efficacy. All statistical comparisons were performed in GraphPad Prism (versions 8.0–
10) or Microsoft Excel (version 16.66.1). For eryptosis assay (Figure 4), significance was determined
using two-way ANOVA. For gametocyte inhibition assays, the non-parametric Kruskal-Wallis with
Dunn’s post-hoc test was used. Mosquito infection intensity (SMFA) was analyzed using the Friedman
test. Tarsal contact assay was analyzed with two-tailed Mann-Whitney test, while infection prevalence
was compared using two-tailed Fisher’s exact tests. Data are presented as mean ± SEM or mean ± SD as
indicated in the figure legends. p values < 0.05 were considered statistically significant.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript describes the evaluation of an antimalarial drug candidate. The main selling points are: i) a picomolar activity against disease-causing asexual blood stages; ii) activity against other major life-cycle stages, including gametocytes, liver stages and insect stage, albeit with highly variable efficacies; iii) transmission blocking activity; iv) efficacy in a mouse malaria model system; and v) favorable safety profiles in acute and repeat toxicology studies in rats. The study features multiple assay systems to support the broad-spectrum efficacy and safety of the compound. The results of several different assay systems are shown to support the broad potency of the compound and its safety in relevant systems. However, some important data are either missing or contradictory. Therefore, further investigations are necessary to fully assess the therapeutic potential of the compound.\n1. History of malaria. The opening sentence of the introduction should be corrected. Malaria is an ancient disease of humanity, with evidence suggesting it may have coexisted with early humans for over 2.4 million years, as indicated by findings such as Plasmodium-infected mosquitoes preserved in amber. Please revise the sentence to accurately reflect this.\n2. Determination of DHA responsiveness. The standard growth inhibition assay is insufficient for evaluating artemisinin responsiveness, as it fails to capture the delayed clearance phenotype. The ring-stage survival assay (RSA) is a more appropriate and widely accepted method. It is recommended that RSA be used to benchmark the compound's activity against dihydroartemisinin (DHA).\n3. Responsiveness of field isolates. The more than 150-fold variation in IC50 values among filed isolates is surprising and requires further explanation. This range could indicate early signs of resistance. It is recommended to perform genetic analysis on low-responder isolates, focusing at minimum on the cyt b and pfdhodh loci. Whole genome sequencing (WGS) of representative isolates would be even more informative.\n4. Discrepancy between in vitro and in vivo activity. The marked discrepancy between the in vitro activity (IC50 values in the picomolar range) and the required in vivo curative dose in a mouse malaria model system (50 mg/kg) raises several questions that require further investigation. Possible explanation include: poor oral bioavailability of the compound, high metabolic turn-over in mice, poor responsiveness of P. yoelii due to altered uptake or metabolic stability of the compound. To address these critical issues the following experiments should be performed: i) PK in mice including determination of whether parasite killing is Cmax or AUC driven; ii) metabolism of the compound using liver microsomes from mice and humans; iii) uptake and efflux kinetics of the compound by P. falciparum infected human erythrocytes and P. yoelii infected mice erythrocytes (Is uptake mediated by NPP?); iii) target validation in P. yoelii with regard to cyt b and pfdhodh; and iv stability of the compound in short term P. falciparum and P. yoelii cultures\n5. Molecular targets of the compound. It is stated that cyt b and possibly pfdhodh are targeted by the compound. It is unclear whether this statement is based on whole genome sequencing of resistant lines or on targeted sequencing of just the two loci. If the latter is the case, than other important resistance conferring mutations may have been missed. It is therefore recommended to determine the whole genome of some resistant line, possible those that display different levels of responsiveness to evaluate revolution of resistance. It is further recommended to evaluate the contribution of the two resistance associated genes independently using e.g., CRISPR Cas9 genome editing technology.\n6. Inhibition of cyt b by the compound. Molecular dynamics simulations should be performed to better understand the interaction of the compound with cyt b at the molecular level and how mutations in the protein cause resistance. Additionally, in vitro binding studies using purified cyt b protein would help clarify the mechanism of inhibition and the impact of resistance-associated mutations on binding kinetics.\n7. PK/PD model in humans. The development of a human PK/PD model is critical to predict clinical efficacy and inform dose selection. The authors should construct such a model to define the relationship between dosage, efficacy, and safety in humans.", "reviewer_2": "1. A consistent number of significant figures should be used for the IC50 and EC50 data.\n2. The authors should generate in vitro ADME/PK data or discuss existing in vitro ADME/PK data for their lead compound T111 and its prodrug.\n3. Why did the authors not assess if T111 also had activity against hypnozoites?", "reviewer_3": "Review of ‘Potent Acridone Antimalarial against All Three Life Stages of Plasmodium” by Kancharla et al.\nBrief summary of the most noteworthy results:\nThe authors of this manuscript have performed extensive experimental testing of the potential new antimalarial compound T111 using different malaria species and different stages of the life cycle (blood stage, gametocytes, mosquito stages, liver stages), using a combination of in vitro and in vivo experiments. All the malaria species in all the stages of the life cycle were eliminated by this compound, even hypnozoites in radical cure mode (the most difficult stage to treat) were susceptible. Several drug resistant P. falciparum strains were tested and almost all of those were still susceptible to treatment with T111. Induction of resistance was difficult and could be pinpointed to a set of mutations in CytB Q0 site.\nToxicity testing showed hardly any toxicity issues for this compound, making the compound very promising.\nThe experimental design is good and execution of the experiments is sound. New antimalarials are desperately needed and this compound could be beneficial for a lot of people. Hopefully this product will proceed to clinical testing and can make a difference in the combat against malaria in all its shapes and forms.\nValidity\nRelevant numbers of replicates have been performed for all of the experiments, the data look consistent and standard deviations are within the expected range.\nSignificance\nThe combat against malaria could benefit from the use of this compound in a clinical setting. This set of experiments is showing the potential of T111 as new antimalarial in several in vitro/vivo models. Especially interesting is the observation of activity of T111 against hypnozoites in a radical cure setting, as this would be a new scaffold replacing the much debated 8 aminoquinolines. Or it could be given as a combination drug thereby lowering the tafenoquine dose drastically.\nData and methodology\nThe data are presented clearly and the number of biological and technical replicates are sufficient.\nIt surprised me that, although many P. falciparum strains carrying various drug resistance mutations were tested in blood stage assays, no experiments were performed testing the efficacy on P. vivax blood stages (or P. cynomolgi as a proxy for vivax).\nI also have some remarks on the methodology of the in vivo testing of the synergy between tafenoquine/T111. The model system used (P. yoelii in mice for blood stage testing and P. berghei in mice for liver stage testing). To my knowledge tafenoquine alone is not used to treat blood stages because of the high dose needed to eliminate all gametocytes. Rather than looking at clearance of the blood stage alone, it would have been desirable to test the effect of the drug combinations on transmission. Moreover, the liver stage testing of tafenoquine and T111 in P. berghei infected mice does not include testing of T111 against hypnozoites, as this stage of the life cycle is not observed in this model. Although the in vitro activity against hypnozoites is promising, it is not supported by any in vivo evidence.\nAnalytical approach\nIn general, the analyses are clear and straightforward.\nI only wonder about the Kruskal-Wallis test used for significance calculation in fig 1E, to compare oocysts of untreated vs oocysts of treated. Does not seem to me that a multiple-comparison test is needed and significance also for the lower dose would probably be shown if the statistical analysis would include a one-way ANOVA test (BUT I’m not a statistician!).\nSuggested improvements\nThis manuscript is well written, contains a huge amount of data, which are presented very clearly and without any doubt T111 is a promising compound for progression into clinical testing.\nThe inclusion of in vivo testing of T111 as a radical cure would test the hypothesis of the activity of this compound against established hypnozoites. For this I would propose to include a set of experiments in which both prophylactic and radical cure testing of T111 (either by itself or in combination with tafenoquine) is performed on either humanized mice/P. vivax or rhesus monkeys/P.cynomolgi.\nI have categorized other minor improvements or questions according to the sequence in the manuscript\nRegarding blood stage testing\nIn Fig 1B the authors show that there is quite a lot of variation in the IC50 measured in the clinical isolates, although the IC50’s are still low. Would that be an indication of the presence of parasites already carrying one or two mutations in the CytBQ0 site?\nRegarding the in vivo testing against P. yoelii blood stages (page 6 and fig 1C), I wondered how it was decided upon the treatment doses (40 and 50 mg/kg) tested in the mouse experiments. Linked to that, were there any Pk/PD tests performed beforehand using these doses? I’m asking because it looks like the difference in treatment results between the 40 and 50 mg/kg are quite striking, so I wondered if this can be explained somehow.\nAlso, the authors state that there is no male-female difference in Pk/PD parameters, but it looks to me that the T1/2 is shorter in females (table 3), can you check if there is a statistically relevant difference in T1/2?\nIn the text it is written ‘long half-life’ (p17). Do the authors think 15-18hr half-life is long? If you compare this to the T1/2 of other antimalarials (T1/2 of chloroquine is 20-40 days, T1/2 of tafenoquine is 14 days T1/2 of atovaquone is 5-6 days), the T1/2 is not very long.\nGametocyte stages\nSuggestion for the layout of the paper: It would be more logical to put the mosquito stage testing after the gametocytes if the manuscript follows the mosquito life cycle, which it did up to this point.\nLiver stages\nThe activity of T111 is compared to the reference drugs Atovaquone and tafenoquine. So why is maduromycin in the table? The authors also state in the discussion that atovaquone is not active against hypnozoites, but the IC50 of atovaquone against hypnozoites is quite low. That is contradictory.\nDoes T111 eliminate the hypnozoites from the cultures completely or do the authors still see tiny parasites after prophylactic or radical cure treatment?\nMosquito stages\nIn the tarsal contact tests, reduced oocyst numbers were seen. But do these still result in sporozoites? And if so, are the sporozoites infective?\nWas the transmission blocking effect tested only by adding the compound to infected blood? I think these kinds of tests are suitable as a first screening, but feeding on infected/treated animals would be additionally informative with regards to transmission testing.\nFig 1E what is the oocyst load of the untreated control?\nSynergy experiments\nThe synergy between T111 and tafenoquine is obvious. Inhibition of the Q0 by T111 increases the parasite’s sensitivity for tafenoquine. Does this also help to identify the mode of action of tafenoquine?\nFig 2C Are the T111 alone curves not included?\nI found a typo in page 29\nHeader: Drug subspeciality testing for cross-resistance profile.\nI assume the authors mean susceptibility here\nWith kind regards\nAnne-Marie Zeeman"}, "author_response": "Potent Acridone Antimalarial against All Three Life Stages of Plasmodium\nNCOMMS-25-44081-T\nPoint-by-Point Response to Reviewers Comments\nReviewer #1 (Remarks to the Author):\nThe manuscript describes the evaluation of an antimalarial drug candidate. The main\nselling points are: i) a picomolar activity against disease-causing asexual blood stages; ii) activity\nagainst other major life-cycle stages, including gametocytes, liver stages and insect stage, albeit\nwith highly variable efficacies; iii) transmission blocking activity; iv) efficacy in a mouse\nmalaria model system; and v) favorable safety profiles in acute and repeat toxicology studies in\nrats. The study features multiple assay systems to support the broad-spectrum efficacy and safety\nof the compound. The results of several different assay systems are shown to support the broad\npotency of the compound and its safety in relevant systems. However, some important data are\neither missing or contradictory. Therefore, further investigations are necessary to fully assess the\ntherapeutic potential of the compound.\n1. History of malaria. The opening sentence of the introduction should be corrected. Malaria is\nan ancient disease of humanity, with evidence suggesting it may have coexisted with early\nhumans for over 2.4 million years, as indicated by findings such as Plasmodium-infected\nmosquitoes preserved in amber. Please revise the sentence to accurately reflect this.\nResponse: We are appreciative of this note and the opening sentence has been revised\naccordingly.\n2. Determination of DHA responsiveness. The standard growth inhibition assay is insufficient for\nevaluating artemisinin responsiveness, as it fails to capture the delayed clearance phenotype. The\nring-stage survival assay (RSA) is a more appropriate and widely accepted method. It is\nrecommended that RSA be used to benchmark the compound's activity against\ndihydroartemisinin (DHA).\nResponse: The RSA has proved problematic for ex vivo assays of fresh African parasites,\nand in fact standard dose response curves, as included for our ex vivo assays of parasites\nfrom Uganda and Burkina Faso, are better indicators of ART-resistance (see PMID\n40783405). However, based on the reviewer’s appropriate suggestion, T111 was tested in\nvitro against two P. falciparum clinical isolates, ART-sensitive SP045 and ART-R SP060,\nusing the recently described Growth, Resistance, and Recovery Assay (GRRA), an\noptimized higher-throughput version of the RSA that allows us to generate a dose response\ncurve (11 replicated RSAs per drug/parasite combinations). As expected, DHA showed\nreduced susceptibility in the ART-R mutant, but T111 was far more potent than DHA and\nmaintained comparable activity against both ART-sensitive and ART-resistant parasites.\n(Figure 1A and 1B of the revised manuscript).\n3. Responsiveness of field isolates. The more than 150-fold variation in IC values among filed\n50\nisolates is surprising and requires further explanation. This range could indicate early signs of\nresistance. It is recommended to perform genetic analysis on low-responder isolates, focusing at\nminimum on the cyt b and pfdhodh loci. Whole genome sequencing (WGS) of representative\nisolates would be even more informative.\nResponse: We appreciate the reviewers reasonable concerns, but in our experience, ex vivo\nassays routinely include uncommon outliers with unusually high or low IC s, likely due to\n50\ncompound solubility limitations, relatively poor parasite growth, human error, or other\nfactors that cannot be corrected, as assays necessarily cannot be repeated. We present all\ndata, but appreciate that, as has been the case with a few dozen other compounds studied\nby our group (see PMID 34339273, 36094216, 37158739), uncommon outliers may be\nmisleading. With this understanding, because resources are not available for the requested\ngenomic studies, and most importantly because all “outliers” had sub-nanomolar IC s (i.e.,\n50\nall isolates were extremely susceptible to T111) we request that these studies be deferred.\nAdditional clarification has been added in the revised manuscript.\n4. Discrepancy between in vitro and in vivo activity. The marked discrepancy between the in\nvitro activity (IC values in the picomolar range) and the required in vivo curative dose in a\n50\nmouse malaria model system (50 mg/kg) raises several questions that require further\ninvestigation. Possible explanation include: poor oral bioavailability of the compound, high\nmetabolic turn-over in mice, poor responsiveness of P. yoelii due to altered uptake or metabolic\nstability of the compound. To address these critical issues the following experiments should be\nperformed: i) PK in mice including determination of whether parasite killing is Cmax or AUC\ndriven; ii) metabolism of the compound using liver microsomes from mice and humans; iii)\nuptake and efflux kinetics of the compound by P. falciparum infected human erythrocytes and P.\nyoelii infected mice erythrocytes (Is uptake mediated by NPP?); iii) target validation in P. yoelii\nwith regard to cyt b and pfdhodh; and iv stability of the compound in short term P. falciparum\nand P. yoelii cultures.\nResponse: Per suggestion, we studied in vitro metabolic stability of T111 using liver\nmicrosomes from human, mouse, and rhesus as well as in vivo PK studies in both male and\nfemale mice. The results are included in the revised manuscript. We agree that the\ndiscrepancy between the in vitro activity and in vivo curative dose may be the result of\npoor oral bioavailability and we have started the development of prodrugs of T111 to\nenhance oral efficacy and PK profiles. This approach was recently validated by early\ninvestigation of prodrugs using a T111 analog (T226), with marked enhancement in both in\nvivo oral antimalarial efficacy and PK profiles (e.g., C and AUC) in both plasma and\nmax\nliver (the findings were reported in our recent publication PMID: 40666482). In addition,\nwe tested T111 in mice infected with P. yoelii parasites carrying cyt b mutations at either\nthe Q or Q site, and the result is consistent with our in vitro observations. The findings are\no i\nincluded in the revised manuscript. It is important to emphasize that the significance of our\nwork is the novelty and overall profile of the acridone antimalarial chemotype. Given the\npaucity of anti-relapsing compounds and narrow safety profiles with tafenoquine, our\nacridone antimalarial offers potential for a novel radical cure (killing relapsing liver stage\nparasites) drug. T111 is presented as a prototype. Recent prodrug development and\noptimization, beyond the scope of this manuscript, should result in substantive\nimprovements in PK profiles and oral efficacy. Other in-depth studies suggested by the\nreviewer (e.g., uptake and efflux kinetics of the compound by P. falciparum infected human\nerythrocytes and P. yoelii infected murine erythrocytes) will be fully explored upon the\nselection of a late-stage preclinical candidate. In response to the reviewer’s concerns, we\nhave added to the Discussion in depth consideration of the limitations of T111 and the\nlikelihood that these limitations can be surmounted by a prodrug strategy that is now\nunderway.\n5. Molecular targets of the compound. It is stated that cyt b and possibly pfdhodh are targeted by\nthe compound. It is unclear whether this statement is based on whole genome sequencing of\nresistant lines or on targeted sequencing of just the two loci. If the latter is the case, than other\nimportant resistance conferring mutations may have been missed. It is therefore recommended to\ndetermine the whole genome of some resistant line, possible those that display different levels of\nresponsiveness to evaluate revolution of resistance. It is further recommended to evaluate the\ncontribution of the two resistance associated genes independently using e.g., CRISPR Cas9\ngenome editing technology.\nResponse: Per the Reviewer’s recommendation, we conducted whole genome sequence on 6\ndrug pressured clones and now report genome-wide mutations in Supplemental Table S3.\nThe T111 mutants were generated by Dr. Roland Cooper’s team, and sadly he passed away\nin 2024. Recently, we were able to locate the T111-selected mutant lines that were\ngenerated by his team and to submit them for whole genome sequencing. We focused WGS\non six of the T111 mutant lines with 3 or 4 pfcytb mutations. All mutations previously\nidentified by targeted Sanger sequencing of pfcytb and pfdhodh were confirmed by WGS.\nImportantly, no additional recurrent mutations outside of pfcytb or pfdhodh were identified\nthat could be clearly linked to drug resistance. The remaining variants detected appear to\nbe sporadic, randomly distributed across the genome, and likely reflect background\nmutations accumulated during prolonged continuous in vitro culture (over a year) rather\nthan drug-selected resistance mechanisms. We agree that using CRISPR Cas9 genome\nediting technology to further investigate the T111 mutant lines would be exciting, but\ngenetic modification of the cytb is not possible because it is located on the mitochondrial\ngenome in P. falciparum and, to date, modification of this genome has not been reported\n(clarification on this has been provided in the revised manuscript). Indeed, we already have\na good understanding of the mechanism of action of T111; the data presented (and new\nstructural data; see response #6 below) suggest that the cytochrome b Q site is the primary\no\ntarget, as Q site mutations are found in all of the resistant clones. Further mechanistic\no\nstudies are planned, but we feel that these will best be done after candidate selection, rather\nthan with our current lead compound.\n6. Inhibition of cyt b by the compound. Molecular dynamics simulations should be performed to\nbetter understand the interaction of the compound with cyt b at the molecular level and how\nmutations in the protein cause resistance. Additionally, in vitro binding studies using purified cyt\nb protein would help clarify the mechanism of inhibition and the impact of resistance-associated\nmutations on binding kinetics.\nResponse: We have developed molecular docking models of T111 and studied the selected\ncyt b mutations to predict binding of T111 to the crystal structure of S. cerevisiae cyt b,\nsince the sequence of P. falciparum cyt b is highly homologous to that of yeast cyt b. The\nresults, showing relevant Q residues that were associated with T111-resistance, are now\no\nincluded in the revised manuscript. Furthermore, a team led by an expert structural\nbiologist, Dr. Alexander Mühleip at University of Helsinki, recently studied binding\nstructures of atovaquone, ELQ-300, and T111 using the cryo-EM structure of pfbc1. The\nresults indicated that the binding pockets for T111 primarily reside in the Q region. Given\no\nthis major breakthrough in the determination of the pfbc1 cryo-EM structure to study\nETC antimalarial inhibitors, a comprehensive analysis with comparison of atovaquone,\nELQs, and T111 analogs will best be reported by Dr. Mühleip’s group in a future\nmanuscript focusing on high-resolution visualization of inhibitor-bound binding sites.\n7. PK/PD model in humans. The development of a human PK/PD model is critical to predict\nclinical efficacy and inform dose selection. The authors should construct such a model to define\nthe relationship between dosage, efficacy, and safety in humans.\nResponse: Our manuscript is intended to present the breakthrough discovery of a novel\nantimalarial chemotype. As mentioned above, it is the overall drug profile (in particular\naction against all parasite stages) rather than the single compound that is our focus. We\noffer T111 as a proof-of-concept, not as a therapeutic candidate. In response to the\nreviewer’s concerns, we have added to the Discussion in depth consideration of the\nlimitations of T111 and the likelihood that these limitations can be surmounted by a\nprodrug strategy that is now underway.\nReviewer #2:\n1. A consistent number of significant figures should be used for the IC and EC data.\n50 50\nResponse: The numbers in all the tables have been input with consistent number of\nsignificant figures in the revised manuscript.\n2. The authors should generate in vitro ADME/PK data or discuss existing in vitro ADME/PK\ndata for their lead compound T111 and its prodrug.\nResponse: The ADME/PK data for T111 has been added in the revised manuscript. The\ndevelopment of T111 prodrugs is underway and the findings will be reported in our future\npublications.\n3. Why did the authors not assess if T111 also had activity against hypnozoites?\nResponse: We believe that these extensive studies are most appropriate after selection of a\ncandidate compound, either the T111 prodrug or another analog, and thus that these\nstudies are beyond the scope of the current manuscript. A prodrug of T111 is being tested\nfor anti-relapse efficacy and synergy with tafenoquine using non-human primates infected\nwith P. cynomolgi. The results, as well as extensive optimization of the acridone prodrugs,\nwill be reported in future publications.\nReviewer #3:\nReview of ‘Potent Acridone Antimalarial against All Three Life Stages of Plasmodium” by\nKancharla et al.\nBrief summary of the most noteworthy results:\nThe authors of this manuscript have performed extensive experimental testing of the potential\nnew antimalarial compound T111 using different malaria species and different stages of the life\ncycle (blood stage, gametocytes, mosquito stages, liver stages), using a combination of in vitro\nand in vivo experiments. All the malaria species in all the stages of the life cycle were eliminated\nby this compound, even hypnozoites in radical cure mode (the most difficult stage to treat) were\nsusceptible. Several drug resistant P. falciparum strains were tested and almost all of those were\nstill susceptible to treatment with T111. Induction of resistance was difficult and could be\npinpointed to a set of mutations in CytB Q0 site.\nToxicity testing showed hardly any toxicity issues for this compound, making the compound\nvery promising.\nThe experimental design is good and execution of the experiments is sound. New antimalarials\nare desperately needed and this compound could be beneficial for a lot of people. Hopefully this\nproduct will proceed to clinical testing and can make a difference in the combat against malaria\nin all its shapes and forms.\nValidity\nRelevant numbers of replicates have been performed for all of the experiments, the data look\nconsistent and standard deviations are within the expected range.\nSignificance\nThe combat against malaria could benefit from the use of this compound in a clinical setting.\nThis set of experiments is showing the potential of T111 as new antimalarial in several in\nvitro/vivo models. Especially interesting is the observation of activity of T111 against\nhypnozoites in a radical cure setting, as this would be a new scaffold replacing the much debated\n8 aminoquinolines. Or it could be given as a combination drug thereby lowering the tafenoquine\ndose drastically.\nData and methodology\nThe data are presented clearly and the number of biological and technical replicates are\nsufficient.\nIt surprised me that, although many P. falciparum strains carrying various drug resistance\nmutations were tested in blood stage assays, no experiments were performed testing the efficacy\non P. vivax blood stages (or P. cynomolgi as a proxy for vivax).\nResponse: We agree that assessment of blood-stage activity against P. vivax would be\nvaluable. However, routine continuous in vitro culture of P. vivax blood stages is not\npossible, and ex vivo assays are limited by low throughput and availability. We therefore\nfocused our blood-stage resistance profiling on P. falciparum, where well-validated panels\nof drug-resistant strains exist, and complemented this with studies of the liver stage of P.\ncynomolgi, a close relative of P. vivax that is easier to study and the accepted surrogate for\nP. vivax relapse biology. Together, these approaches allowed us to interrogate both\nresistance liability and radical cure potential within practical experimental constraints. For\nin vivo assessment in the animals, a prodrug of T111 is being tested currently for anti-\nrelapse efficacy and synergy with tafenoquine using non-human primates infected with P.\ncynomolgi. The results, as well as extensive optimization of the acridone prodrugs,\nwill be reported in our future publications. Further clarification has been added in the Discussion.\nI also have some remarks on the methodology of the in vivo testing of the synergy between\ntafenoquine/T111. The model system used (P. yoelii in mice for blood stage testing and P.\nberghei in mice for liver stage testing). To my knowledge tafenoquine alone is not used to treat\nblood stages because of the high dose needed to eliminate all gametocytes. Rather than looking\nat clearance of the blood stage alone, it would have been desirable to test the effect of the drug\ncombinations on transmission. Moreover, the liver stage testing of tafenoquine and T111 in P.\nberghei infected mice does not include testing of T111 against hypnozoites, as this stage of the\nlife cycle is not observed in this model. Although the in vitro activity against hypnozoites is\npromising, it is not supported by any in vivo evidence.\nResponse: Our prodrug approach has produced compounds with substantially enhanced\nPK profile and oral efficacy. A pilot safety study of T111 prodrug in rhesus monkeys\nsuggested that the drug was well-tolerated by the animals. Validation of anti-relapse\nefficacy of the acridone prodrug and synergy with tafenoquine in non-human primates\ninfected with P. cynomolgi is underway in collaboration with WRAIR and AFRIMS.\nHowever, these studies are not complete, and are beyond the scope of this manuscript. The\nresults will be reported in our future publications focusing on the optimization of the\nprodrugs. Further clarification has been added in the Discussion.\nAnalytical approach\nIn general, the analyses are clear and straightforward. I only wonder about the Kruskal-Wallis\ntest used for significance calculation in fig 1E, to compare oocysts of untreated vs oocysts of\ntreated. Does not seem to me that a multiple-comparison test is needed and significance also for\nthe lower dose would probably be shown if the statistical analysis would include a one-way\nANOVA test (BUT I’m not a statistician!).\nResponse: A one-way ANOVA with Kruskal-Wallis multiple comparison test was used in\nFigure 1D (now 1F in the revised manuscript), not Figure 1E. There was no comparison\nbetween untreaded oocysts vs treated oocysts in this study, as the experiment was for\nsexual blood stage gametocytes. In the sexual blood stage gametocytes assay, we used a\nKruskal–Wallis test followed by post hoc comparisons versus the DMSO control because\nmultiple treatment groups were analyzed simultaneously and data did not meet\nassumptions required for parametric testing given the small sample size. While a one-way\nANOVA is less conservative, post hoc multiple-comparison correction is still required when\nmaking pairwise comparisons, and we therefore kept the nonparametric approach. In\nFigure 1E (now Figure 1G in the revised manuscript), the Friedman test – a test similar to\nANOVA, but for non-parametric data, was used to study the inhibition of P. falciparum\noocyst development in mosquitoes after direct membrane feeding.\nSuggested improvements\nThis manuscript is well written, contains a huge amount of data, which are presented very clearly\nand without any doubt T111 is a promising compound for progression into clinical testing.\nThe inclusion of in vivo testing of T111 as a radical cure would test the hypothesis of the activity\nof this compound against established hypnozoites. For this I would propose to include a set of\nexperiments in which both prophylactic and radical cure testing of T111 (either by itself or in\ncombination with tafenoquine) is performed on either humanized mice/P. vivax or rhesus\nmonkeys/P.cynomolgi.\nResponse: As mentioned above, validation of anti-relapse efficacy of a T111 prodrug and\nsynergy with tafenoquine using non-human primates infected with P. cynomolgi is\nunderway in collaboration with WRAIR and AFRIMS. However, these studies are not\ncomplete, and are beyond the scope of this manuscript. The results will be reported in our\nfuture publications focusing on the optimization of the prodrugs. Further clarification has\nbeen added in the Discussion.\nI have categorized other minor improvements or questions according to the sequence in the\nmanuscript\nRegarding blood stage testing\nIn Fig 1B the authors show that there is quite a lot of variation in the IC50 measured in the\nclinical isolates, although the IC50’s are still low. Would that be an indication of the presence of\nparasites already carrying one or two mutations in the CytBQ0 site?\nResponse: Please see comment #3 to Reviewer 1 above.\nRegarding the in vivo testing against P. yoelii blood stages (page 6 and fig 1C), I wondered how\nit was decided upon the treatment doses (40 and 50 mg/kg) tested in the mouse experiments.\nLinked to that, were there any Pk/PD tests performed beforehand using these doses? I’m asking\nbecause it looks like the difference in treatment results between the 40 and 50 mg/kg are quite\nstriking, so I wondered if this can be explained somehow.\nResponse: The difference in oral efficacy in mice was only observed in blood stage testing\nin the single dose cure model, and we suspect that the poor aqueous solubility of T111\ncontributed to variability. When the compound was dosed via gavage it was not completely\ndissolved in the vehicle, and so provided as a suspension. To address this concern, we have\nadded this limitation to our Discussion. Of note, T111 prodrugs provided much improved\nsolubility and consequently enhanced PK profile and oral efficacy. These data will be\ndisclosed in a future publication focusing on the development of T111 prodrugs.\nAlso, the authors state that there is no male-female difference in Pk/PD parameters, but it looks\nto me that the T1/2 is shorter in females (table 3), can you check if there is a statistically relevant\ndifference in T1/2?\nResponse: We have now included the PK study with data for male and female mice. PK\nprofiles of T111 were similar in male and female mice (Table 3). We also conducted a\ncomparison of oral efficacy of T111 in the 4-day suppression rodent model with male and\nfemale mice infected with P. yoelii. The ED and ED values in male and female mice were\n50 90\nalmost identical (Figure S1). These results are included in the revised manuscript.\nIn the text it is written ‘long half-life’ (p17). Do the authors think 15-18hr half-life is long? If\nyou compare this to the T1/2 of other antimalarials (T1/2 of chloroquine is 20-40 days, T1/2 of\ntafenoquine is 14 days T1/2 of atovaquone is 5-6 days), the T1/2 is not very long.\nResponse: The listed T values of other antimalarials by the reviewer are in humans.\n1/2\nTafenoquine has a T of 50 h in mouse plasma and 75 h in mouse liver (internal data from\n1/2\nWRAIR). The T for chloroquine in healthy mice is about 46 h (PMID: 21646487;\n1/2\nPMCID: PMC3147625). The T for atovaquone in healthy mice is about 13 h (PMID:\n1/2\n23292347; PMCID: PMC4344550). T111 T in mouse plasma ranges from 17 h to 26 h\n1/2\n(Table 3). To clarify this point, we added to the revised manuscript that this is a relatively\nlong half-life for an antimalarial compound in mice.\nGametocyte stages\nSuggestion for the layout of the paper: It would be more logical to put the mosquito stage testing\nafter the gametocytes if the manuscript follows the mosquito life cycle, which it did up to this\npoint.\nResponse: This is a great suggestion and we have moved the mosquito life cycle section\nimmediately following the gametocytes studies.\nLiver stages\nThe activity of T111 is compared to the reference drugs Atovaquone and tafenoquine. So why is\nmaduromycin in the table? The authors also state in the discussion that atovaquone is not active\nagainst hypnozoites, but the IC of atovaquone against hypnozoites is quite low. That is\n50\ncontradictory.\nResponse: The in vitro anti-relapse activity in the radical cure mode for tafenoquine\nheavily depends on P450. Maduromycin was included as a positive control because it has\npotent activities against hypnozoites and schizonts in both prophylactic mode and radical\ncure mode. However, the anti-relapse efficacy of tafenoquine was validated using P.\ncynomolgi infected rhesus monkeys. Although atovaquone shows in vitro activity against\nhypnozoites, it does not provide a durable radical cure and is ineffective at preventing\nrelapse in vivo. In our P. cynomolgi rhesus hepatocyte system, however, this weak activity\n(IC values > 2 µM) occurred at concentrations that overlap with hepatocyte cytotoxicity\n50\n(Table 1, Source data file), resulting in a narrow or absent therapeutic window.\nFurthermore, independent literature provides in vitro liver-stage atovaquone IC₅₀ values\nconsistent with those observed by us (≈2 µM) in primary hepatocyte P. cynomolgi assays,\nPMID: 21483865). Importantly, those studies did not report a therapeutic index, and as\nnoted in our work, apparent atovaquone hypnozoite inhibition occurs at concentrations\noverlapping with cytotoxicity. Since the submission of our original manuscript, additional\nexperiments with independent biological replicates have been performed and Table 1 has\nbeen updated. We also added an additional reference drug KDU691 (PI4K) inhibitor) with\nprophylactic activity against liver stage parasites, but ineffective as a radical cure agent\nagainst relapsing hypnozoites (PMID: 26926645).\nDoes T111 eliminate the hypnozoites from the cultures completely or do the authors still see tiny\nparasites after prophylactic or radical cure treatment?\nResponse: The high throughput in vitro hypnozoitocidal assay developed by WRAIR using\nP. cynomolgi sporozoites and non-human primate rhesus hepatocytes was conducted with\nimmunofluorescence staining and imaging by an Operetta CLS high-content imager with\nhigh sensitivity. Following T111 treatment in prophylactic mode, no morphologically\ndetectable hypnozoites were observed above background levels at the assay endpoint (raw\ncounts available in the Source data file), although we cannot exclude the presence of non-\nviable residual structures below detection limits. Furthermore, following T111 treatment in\nradical cure mode, a small residual population (~10-20%) of hypnozoites remained with the\nhighest concentrations tested, as shown in the source data file. Future studies will be\nconducted to determine whether the residual hypnozoite population observed under radical\ncure conditions remains viable using extended-duration liver stage assays.\nMosquito stages\nIn the tarsal contact tests, reduced oocyst numbers were seen. But do these still result in\nsporozoites? And if so, are the sporozoites infective?\nResponse: We did not assess sporozoite numbers in these experiments, so we cannot be\ncertain whether these few oocysts produced infectious sporozoites. However, we can be\ncertain that the vast majority of tarsally exposed mosquitoes will produce no sporozoites,\ngiven the 92.5% reduction in oocyst prevalence, and that those mosquitoes with\nbreakthrough infections will produce few sporozoites, given the 97.6% reduction in oocyst\nintensity. Oocyst counts following the standard membrane feeding assay (SMFA) are\nconsidered the gold standard for transmission blocking studies (PMID: 24042109, PMID:\n25890243, PMID: 29247222) and are positively correlated with subsequent sporozoite\nnumbers (PMID: 31060594, PMID: 38272943, PMID:38517746). Moreover, it has been\nshown that sporozoite transmission is rare at inoculums below ~10,000 sporozoites (PMID:\n32453765). Given the extremely low prevalence and intensity of oocysts in these\nexperiments, we expected that these three infected mosquitoes would likely not reach this\nthreshold of sporozoites, and we therefore chose not to measure sporozoites in this\nexperiment. We do not expect that pre-infection T111 tarsal contact would have activity\nagainst sporozoites directly because it has an estimated half-life of ~18 hours. However, it is\npossible that development of the few breakthrough oocysts we observed and their\nsubsequent sporozoite infectivity may be impaired in some way by the lingering impact of\nT111 exposure during initial development. Due to the low infection prevalence and\nintensity, we would be unable to isolate sufficient sporozoites to perform robust liver stage\ninfections to assess this possibility, and we therefore chose not to discuss this in the text.\nFurther, we think that extensive additional experiments to precisely characterize\nquantitative impacts on transmission will be most appropriate with a T111 prodrug after\ncandidate selection, as discussed above, rather than at this early stage in drug development.\nWas the transmission blocking effect tested only by adding the compound to infected blood?\nI think these kinds of tests are suitable as a first screening, but feeding on infected/treated animals\nwould be additionally informative with regards to transmission testing.\nResponse: The transmission blocking effects of T111 were assessed using three different\nassays: 1) sugar bait feeding assay where drugs were mixed into a sucrose-water solution\nand fed to infected mosquitos (reported by our collaborator Dr. Dean Goodman in previous\npublication PMID: 40449870); 2) direct membrane feeding assay (SMFA) that simulates a\nblood meal taken by the mosquitos from a treated human (presented in this manuscript),\nand 3) the tarsal contact assay that mimics uptake of the drugs via mosquito’s legs after\nlanding on antimalarial infused bed nets, even with short exposure time (presented in this\nmanuscript). SMFA is the gold standard for testing transmission blocking in a preclinical\nstudy. This assay specifically tests the impact of a drug on the ability of functional\ngametocytes to infect mosquitoes and avoids confounding effects of the drug on asexual\ngrowth and gametocyte development prior to infection. Specific impacts on asexual and\ngametocyte development are demonstrated in other experiments (Figure 1). Treating\nanimals prior to transmission is possible, but the impacts of the drug on asexual growth\nand gametocyte development prior to infection confound the analysis of stage specific\neffects, so these trials are not usually done at this stage.\nFig 1E what is the oocyst load of the untreated control?\nResponse: The oocyst loads of the untreated control vary between biological repeats, so the\ndata have been normalized to better compare the impact of the drug across multiple trials.\nRaw counts of oocyst load can be found in the source data file for Figure 1G in the revised\nmanuscript.\nSynergy experiments\nThe synergy between T111 and tafenoquine is obvious. Inhibition of the Q by T111 increases\n0\nthe parasite’s sensitivity for tafenoquine. Does this also help to identify the mode of action of\ntafenoquine?\nResponse: This is a great question. The mechanism of action of tafenoquine is not fully\nunderstood, but its radical curative activity is likely due to its metabolites, which generate\nreactive oxygen species that may damage the parasite's cellular components. Studies\nsuggest that tafenoquine may also cause mitochondrial dysfunction and an increase in\nintracellular calcium levels within the parasite. One possible explanation of synergy is that\nthe mitochondrial disruption caused by tafenoquine leads to vulnerability to other drugs\ntargeting mitochondrial ETC. But, to our knowledge, other ETC inhibitors such as\natovaquone and ELQs are not synergetic with tafenoquine. Our studies differentiating\nbetween T111 and tafenoquine in their effects on G6PD deficient erythrocytes suggest that\nT111 does not produce redox cycling. Dr. Dennis Shanks, who conducted the first field trial\nof tafenoquine, is investigating the effect of T111 and its prodrugs on the metabolism and\nredox cycling of tafenoquine. The results will best be reported by Dr. Shanks’ group in a\nfuture manuscript including a large panel of standard antimalarials.\nFig 2C Are the T111 alone curves not included?\nResponse: This figure is intended to focus on the leftward shift (potency increase) achieved\nby the addition of T111, compared to tafenoquine alone. The inclusion of T111 alone curve\nwould interfere visually. However, the dose-response curves for T111 alone are shown in\nFigure S1, which was added in the revised manuscript. Importantly, the doses of T111\nused in this combination study were below therapeutic level (ED /ED of T111 = 0.27/0.39\n50 90\nmg/kg/d).\nI found a typo in page 29\nHeader: Drug subspeciality testing for cross-resistance profile.\nI assume the authors mean susceptibility here\nResponse: Thanks! The typo has been fixed in the revised manuscript."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have substantially strengthened the revised manuscript, significantly improving both the depth and breadth of the data supporting their central claim. In particular, the additional experimental evidence convincingly supports the conclusion that T111 represents a previously unexploited chemotype with stage-transcending antimalarial activity, a distinct resistance mechanism, and a favorable safety profile.\nMy initial recommendations have been comprehensively addressed, and the revisions have enhanced the impact of the study.", "reviewer_3": "I have read the rebuttal and the revised manuscript. All the points have been addressed. I have no further comments"}, "author_response": ""}]

Decision letter