Science - USA (2022-06-03)

E1 that activates UBLs—including the Atg8s
( 11 ). We identified several pyrazolopyrimidine
sulfamates with a 7-position substituent (ex-
emplar ML901; Fig. 2A) that possess potent
activity againstP. falciparum.TheML901
50% inhibitory concentration (IC50_72h=
2.0±0.1nM)issimilartothatofDHA(tableS1).
ML901wastestedforcytotoxicityagainst
different mammalian cell lines (table S1) and
showed 800- to 5000-fold selectivity toward
P. falciparum(>1000 times higher selectivity
than AMS). ML901 retained activity against
all strains ofP. falciparumtested, regardless
of their resistance profile and geographical
origin (table S2); it also potently inhibited
transmissible male gametes (table S2) and
prevented development ofP. falciparumin
primary human hepatocytes (table S2). We
confirmed that ML901 exerts activity against
human Atg7 (IC 50 =33nM)buthasmuch
weaker activity against other E1 enzymes
(table S3), as previously reported for nucle-
oside sulfamates with a substitution at the
7position( 11 ), and consistent with the low
mammalian cell cytotoxicity. By contrast, AMS
is a potent inhibitor of each of the E1s tested
(table S3). The rat pharmacokinetic profile of
ML901 (fig. S4 and table S3) is encouraging as
it exhibits low blood clearance and has a long

terminal half-life in blood (T1/2 ∞=41hours)

following intravenous or oral dosing.

We determined the in vivo antimalarial ef-

ficacy of ML901 in severe combined immune

deficient (SCID) mice engrafted withP. falciparum–

infected human RBCs ( 12 , 13 )—the gold stan-

dard for testing in vivo efficacy of malaria drug

candidates. A single dose [50 mg/kg intra-

peritoneal injection (ip)] results in excellent

exposure (area under the curve = 580mMper

hour; Fig. 2B) and achieves reduction of

parasitemia to baseline (Fig. 2C) with no evi-

dence of toxicity. The clearance rate is similar

to that of chloroquine [50 mg/kg oral admin-

istration (po)].

ML901 selectively targets plasmodium

tyrosine tRNA synthetase

To identify the target of ML901 inP. falciparum,

extracts of infected RBCs that had been treated

with 3mM ML901 (3 hours) were subjected to

LCMS to search for amino acid-ML901 con-

jugates. An LCMS peak corresponding to pro-

tonated ML901-Tyr (m/z = 576.1324) was

detected. Synthetic ML901-Tyr was generated

and spiked into the untreatedP. falciparum

lysate to confirm the peak assignment (fig. S5).

None of the other 19 possible amino acid con-

jugates were detected.

To determine whetherP. falciparumtyrosine

tRNA synthetase (Pf YRS) is the critical target in

P. falciparum, we subjected parasite cultures

(3D7 line) to increasing concentrations of the

compound over a period of 4 months, after

which we retrieved parasites with 10-fold re-

duced sensitivity (ML901 IC50_72h=28nM)

(fig. S6A). Whole-genome sequencing of one

parental and three resistant clones revealed

nine newly acquired mutations in the resistant

clones, three of which were in PF3D7_0807900

(position 403,556), corresponding to a Ser234Cys

mutation in cytoplasmicPf YRS (table S4). In-

sertion of an additional Asn in an Asn repeat

in kinesin-5 is considered unlikely to be func-

tionally important (see table S4). No mutations

were observed in any other AFE, including

P. falciparumAtg7. Transfectants were gen-

erated (Dd2 parent) harboring thePf YRSS234C

mutation (fig. S6, B to D), which recapitulated

theresistancephenotype(i.e.,10-folddecreased

sensitivity) (Fig. 3A and table S5). Aptamer-

induced down-modulation ofPf YRS decreased

the growth rate compared with the control

(fig. S6E) and increased sensitivity to ML901

(Fig. 3B and table S5); but not to DHA or the

Thr-RS inhibitor borrelidin (BOR) (fig. S6F and

table S5). The considerable potency of ML901

against the knockdown parasites (IC 50 =

Xieet al., Science 376 , 1074–1079 (2022) 3 June 2022 2of6

Fig. 1. AMS-treated infected RBCs reveal

aaRSs as potential targets.(A) E1 enzymes can

catalyze attack of the sulfamate nitrogen on the

carbonyl carbon of the thioester bond between the

UBL and the E1 to form a UBL conjugate. aaRSs

could catalyze nucleoside sulfamate attacks on

activated amino acids to form an amino acid

adduct. (B) Structure of adenosine 5′-sulfamate.

(C) Trophozoite stage parasites were incubated

with DMSO (mock), different concentrations of

AMS, or BOR. Western blots of lysates were

probed for phosphorylated-eIF2awithPfBiP as a

loading control. The blot is typical of data from

three independent experiments. (D) P. falciparum–

infected RBCs were treated with 10mM AMS

for 3 hours. Extracts were subjected to LCMS

analysis identifying the Tyr-AMS conjugate.

The profile is typical of data from three

independent experiments.

AMS-Tyr

AMS

B C

A

D

509 511 512

0

10

20

30

40

50

60

70

80

90

100

Relative Abundance

510.1394

z=1

511.1427

z=1

512.1352

z=1

510 513

m/z

Mass error = 2.6 ppm

kDa

38

62

Anti-phospho-eIF2α

Anti-PfBip

Mock0.1^110 BOR

AMS (μM)

UBL S

O

E1 + H

2 N

S

O

O O

nucleoside

N

H

S

O

O O

nucleoside

UBL

O

:E1

aa O

O

tRNA+H

2 N

S

O

O O

nucleoside

N

H

S

O

O O

nucleoside

aa

O

aaRS+ :aaRS

tRNA

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