Science - USA (2022-06-03)

were generated through in vitro transcription
(fig. S10). When ML901 was incubated with
Pf YRS in the presence of all other substrates
[i.e., Tyr, adenosine triphosphate (ATP), and
tRNATyr], the apparent protein melting point
(Tm)—measured by differential scanning flu-
orimetry (DSF)—increased by 15°C (Fig. 4, A
and B, and fig. S11A). The increase in thermal
stability is even greater than that induced by
the tightly bound adenylate intermediate
AMP-Tyr (table S7). Although formation of
the AMP adenylate requires only Tyr and ATP,
the thermal stabilization induced by ML901
requires all three substrates (Tyr, ATP, and
tRNATyr). This result is consistent with a hijack-
ing mechanism that requires charged tRNATyr
(Tyr-tRNATyr; see Fig. 1A). Notably, recombi-
nantHsYRS was not stabilized in the presence

of ML901 plus substrates, suggesting that the

inhibitory species is not produced by, or does not

bind to, the human enzyme (Fig. 4B, red curves).

The mutantPf YRSS234Cwas less well stabilized

(fig. S11A and table S7), suggesting weaker

binding of the inhibitory species, consistent with

the mutant parasite’s resistance to ML901.

ML901 exerts its activity by hijacking the

active site–bound reaction product

We examined the abilities of the recombinant

YRSs to consume ATP—i.e., to form and

release AMP-Tyr in the initial reaction phase.

HsYRS andPf YRSS234Cshow higher activity (in

the absence of tRNA) thanPf YRS (fig. S11B).

This difference suggests that AMP-Tyr is bound

less tightly to theHs and mutantPf YRS active

sites. Upon addition of the cognate tRNATyr,

ATP consumption is enhanced, consistent with

productive aminoacylation. Acylation of the

cognate tRNATyrto radiolabeled tyrosine ( 19 ),

occurs at a similar level in all three enzymes

(fig. S11C). ML901 inhibits ATP consumption

by Pf YRSwhenaddedinthepresenceof

PftRNATyrbut not in its absence (fig. S11D).

Similarly, ML901 inhibits tRNATyracylation

to tyrosine in vitro byPf YRS but not byHsYRS

(Fig.4C).SyntheticallygeneratedML901-Tyr

conjugate is able to inhibit the activity of both

Pf YRS andHsYRS (fig. S11, E and F), suggesting

that althoughHsYRS is unable to generate the

ML901-Tyr inhibitor, it can bind the pre-

formed conjugate.

To confirm that recombinantPf YRS can

generate the ML901-Tyr conjugate, we incubated

Pf YRS with substrates and ML901 and then

Xieet al., Science 376 , 1074–1079 (2022) 3 June 2022 4of6

Fig. 4. ML901 inhibits
PfYRS by a reaction-
hijacking mechanism.
(A) The apparent
melting temperature
(Tm)ofPfYRS after
incubation at 37°C for
3 hours with the indi-
cated reactants:
ML901 (50mM),
ATP (50mM), tyrosine
(100mM), and
PftRNATyr(4 mM). Data
represent the average
of three independent
assays and error bars
correspond to SD.
(B) First derivatives of
melting curves for
PfYRS andHsYRS with
or without pre-
incubation with ML901
(50mM), ATP (50mM),
tyrosine (100mM),
andPftRNATyr(4 mM)
or HstRNATyr(20mM).
Data are representative
of three independent
assays. (C) Effects
of increasing concen-
trations of ML901
on tyrosine acylation of
the cognate tRNATyr
by PfYRS andHsYRS
with YRS (0.25mM),
ATP (10mM), tyrosine
(100 to 200mM),
cognate tRNATyr
(24mM), and pyrophos-
phatase (1 unit/mL),
at 37°C for 1 hour. IC 50 (PfYRS) = 53mM; IC 50 (HsYRS) > 500mM. Data represent the average of eight independent assays and error bars correspond to SEM.
(D) Structure of ML901-Tyr. (E) PfYRS was incubated with ML901 (50mM), ATP (10mM), tyrosine (20mM), andPftRNATyr(8 mM). Following urea denaturation and
TFA precipitation, the supernatant was subjected to LCMS analysis, revealing the expected protonated ML901-Tyr ion. The profile is typical of data from three
independent experiments. (F) Schematic of reaction-hijacking mechanism.

AB

C D

50

55

60

65

T

m

(°C)

ATP –

Tyrosine

+

PftRNATyr

ML901

• ++
  –+– ++
  ––––+
  ––+++

30 40 50 60 70 80

-1 104

0

1104

2104

-1 104

0

1104

2104

3104

4104

Temperature (°C)

d(RFU)/dT

Pf

YRS

d(RFU)/dT

Hs

YRS

no ML901 + ML901

E

ML901-Tyr

Mass error = 2.4 ppm

Relative Abundance

575576577578579

m/z

0

10

20

30

40

50

60

70

80

90

100

576.1331

z=1

577.1358

z=1

578.1292

z=1

F

PPi

tRNATyr

AMP

PfYRS (dimer)

ATP

Tyr

tRNATyr

ML901-Tyr inhibited PfYRS

Tyr

ML901

Tyr Tyr Tyr

0123

0

20

40

60

80

100

120

log 10 [ML901] (μM)

Tyr-tRNA production (%)HsYRS

PfYRS

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