Science - USA (2022-06-03)

0.4 nM) both validatesPf YRS as the target
and points to an extremely potent inhibitory
interaction.
ML901 inhibits protein translation in
P. falciparumschizonts (as monitored by
O-propargyl-puromycin incorporation) ( 14 )
(Fig. 3C), consistent withPf YRS as the target.
The IC50_3hvalue (50 nM) correlates well with
that for parasite-killing potency (Fig. 3C). An-

other protein translation inhibitor, cyclohexi-

mide, has a similar profile (fig. S7A), whereas

the folate pathway inhibitor WR99210 kills

parasites with no immediate effect on protein

translation (fig. S7B). ML901 triggers eIF2aphos-

phorylation in wild-type (WT)P. falciparum

(Cam3.II_rev; Fig. 3D), consistent with the

presence of uncharged tRNA ( 10 , 15 ). In eIK1

(GCN2 equivalent) ( 16 ) knockout parasites,

the amino acid starvation pathway is disrupted

and ML901 treatment does not result in eIF2a

phosphorylation (fig. S7C), consistent with an

aaRS target (fig. S7D).

YRSs from WT (Pf YRS), mutant (Pf YRSS234C),

and human (matureHsYRS) ( 17 ) were pro-

duced inE. coli. Biophysical characterization

revealed well-folded dimers (figs. S8 and S9

and table S6).Pf tRNATyrandHstRNATyr( 18 )

Xieet al., Science 376 , 1074–1079 (2022) 3 June 2022 3of6

Fig. 2. ML901 exhibits potent activity against

P. falciparumin vivo.(A) Structure of the

pyrazolopyrimidine ribose sulfamate, ML901.

(B) Pharmacokinetics profile (in blood) over the first

day for SCID mice engrafted with human RBCs

infected withP. falciparumfollowing treatment

with ML901 at 50 mg/kg i.p. (C) Therapeutic

efficacy of ML901 in the SCID mouseP. falciparum

model, dosed with ML901 at 50 mg/kg i.p. in

comparison with the gold standard antimalarial

chloroquine, dosed at 50 mg/kg p.o.

A B

024624

0

2000

4000

6000

8000

Time after first administration (h)

ML901 (ng/ml)

50 mg/kg i.p.

ML901

C

01234567

0.01

0.1

1

10

100

Post-infection (Days)

0

Parasitaemia (%)

Untreated control

ML901 (1 x 50 mg/kg i.p.)

CQ (1 x 50 mg/kg p.o.)

Detection limit

Fig. 3. ML901 targetsPfYRS and inhibits protein

translation.(A andB) Sensitivity to ML901 exposure

(72 hours) for a cloned wildtype line (Dd2) and

three CRISPR-edited clones harboringPfYRSS234C(A)

or an aptamer-regulatablePfYRS line upon addition

of aTc, with data normalized to a no-drug control (B);

see table S5 for data values. (C) RBCs infected with

schizont stage (43 to 46 hours p.i.)P. falciparum

(Cam3.II-rev) were exposed to ML901 for 3 hours.

Protein translation was assessed in the second two

hours of the incubation through the incorporation of

OPP. Aliquots of inhibitor-exposed cultures were

washed and returned to cultures, and viability was

estimated at the trophozoite stage of the next cycle.

IC 50 (translation) = 65 nM, IC 50 (viability) = 56 nM.

Data are representative of three independent

experiments. Error bars correspond to the range of

technical duplicates. (D) Schizont stage Cam3.II_rev

parasites were incubated with DMSO (mock), 1mM

DHA, 200 nM borrelidin (BOR) or 200 nM ML901 for

3 hours and Western blots of lysates were probed

for phosphorylated-eIF2awithPfBiP as a loading

control. The blot is typical of data from three

independent experiments.

A

C

D

kDa

38

62

Anti-phospho-eIF2α

Anti-PfBip

MockDHA BORML901

B

5

2

10

-0.5 0.0 0.5 1.0 1.5 2.0

0.0

0.2

0.4

0.6

0.8

1.0

1.2

log [ML901] (nM)

Viability

Dd2 parent

Mutant clone E12

Mutant clone F

Mutant clone G1

-1.5 -1.0 -0.5 0.0 0.5 1.0 1.5

0

20

40

60

80

100

120

log 10 [ML901] (nM)

Parasite proliferation (%)

50 nM aTc

1 nM aTc

no aTc

-101234

0.0

0.2

0.4

0.6

0.8

1.0

1.2

log 10 [ML901] (nM)

Translation activity / Viability

Translation

Viability

RESEARCH | RESEARCH ARTICLE