loops were not defined in the electron density
[Fig. 5C(ii) and fig. S15, D and E]. Moreover,
His49 (the equivalent ofPf YRS His70) adopts
a position (magenta in Fig. 5D) similar to the
position adopted by His70 in chain A of ML901-
Tyr-boundPf YRS (compare figs. S13, G and H,
and S15E), further suggesting that this config-
uration is associated with increased loop mobil-
ity. A comparison of the interaction networks
(figs. S13, B to D, and S15, B and C) reveals no-
tably fewer interactions with the ML901 moiety
in HsYRS compared with Pf YRS and specific
interactions are poorly conserved between the
two enzymes.
We also solved the structure ofPf YRSS234C
in complex with synthetic ML901-Tyr (fig. S16).
Similar toHsYRS the KMSKS loops of both
monomers were not defined in the electron
density (fig. S16, E and F), and His70 adopts a
rotamer that is not consistent with a struc-
tured KMSKS loop [green in Fig. 5, C(iii) and D;
compare figs. S13, G and H, and S16, G and H].
Potency and selectivity of ML901 forPf YRS
thus appears to be associated with a stabilized
loop over the active site. That is, the decreased
susceptibility ofHsYRS andPf YRSS234Cto
reaction hijacking by ML901 is associated
with mobility of the KMSSS/KMSKS loop,
which is in turn associated with rotation of
the His49/70 side chain. These conforma-
tional changes may promote dissociation of
the charged tRNA, thereby preventing the
hijacking reaction.
The pyrazolopyrimidine sulfamate chemo-
type is an attractive starting point for a malaria
drug discovery program, based on our obser-
vation that the specific inhibition ofPfYRS by
ML901 is lethal to disease-causing and trans-
missible stages ofP. falciparum,andthat
ML901 exhibits a long in vivo half-life, under-
pinning its single-dose efficacy in a murine
model of human malaria. Further explora-
tion of substitutions at the 7-position of the
pyrazolopyrimidine sulfamates class is ex-
pected to allow for identification of com-
pounds with reduced activity against human
Atg7 and thus even higher specificity for
plasmodium. We note that the HIAQ and
KMSKS motifs are conserved across api-
complexan and kinetoplastid parasites but
not in metazoan organisms (fig. S17). This
suggests that ML901-like compounds could
exhibit cross-pathogen inhibitory activity. Use
of sulfamates in a drug combination could pre-
vent evolution of resistant mutants.
Our finding that nucleoside sulfamates can
hijack Class I and Class II tRNA aaRSs—as well
as E1s—opens up the possibility of designing
bespoke membrane permeable AFE inhibitors
with small molecular weight and adjustable
specificity. In addition to charging tRNA and
activating ubiquitin, AFEs are involved in
activating fatty acids for degradation, biosyn-
thesis of natural products, and other diverse
pathways ( 21 ). Thus, nucleoside sulfamates may
find applications in a broad range of infectious,
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ACKNOWLEDGMENTS
We thank the following colleagues. Cellular assays: A. Koley,
P. Dhar, P. Mukherjee, TCG Lifesciences, India; R. van der Laak,
A. Sturm, TropIQ Health Sciences. Support with the SCID mouse
and panel assays: U. Lehmann, C. Scheurer, Swiss Tropical and
Public Health Institute, Switzerland. Support with the bioanalytical
determination and PK evaluation in mouse blood samples by
LC/MS-MS: A. Kress, I. Barme, M. Enzler, C. Siethoff, Swiss
BioQuant, Switzerland. Plasmodium screening: B. Nare, SCYNEXIS
Inc, USA. Technical support and lead-in experiments: Y.-F. Mok
(Melbourne Protein Facility), C. Dogovski, M. Gorman, T. Ahmed,R. Manhas, S. Liu, University of Melbourne; D. Baud, Medicines
for Malaria Venture. Useful advice: P. Gleeson, S. Ralph, M. Dixon,
N. Spillman, University of Melbourne; R.J. Griffin, Takeda
Pharmaceuticals; L. Ribas, IRCC Barcelona. We thank C.n Doerig,
RMIT University, for providing eIK1 genetically disrupted parasites.
This research was partly undertaken on the MX2 and SAXS/WAXS
beamlines at the Australian Synchrotron, part of the Australian
Nuclear Science and Technology Organization, and made use of the
ACRF Detector on the MX2 beamline. We thank the beamline staff for
their assistance. Millennium Pharmaceuticals, a wholly owned
subsidiary of Takeda Pharmaceuticals Company Limited, provided
access to ML901 under a Materials Transfer Agreement with the
University of Melbourne.Funding:This work was funded by the
following: the Global Health Innovative Technology Fund, Japan
(H2019-104 to L.T., A.E.G., and S.B.); National Health and Medical
Research Council (NHMRC, 1139884 to L.T.); Medicines for Malaria
Venture (MMV RD/15/0007 to S.B., RD-08-2800 to J.B.); and
Millennium Pharmaceuticals (to A.E.G. and S.P.L.), a wholly owned
subsidiary of Takeda Pharmaceuticals Company Limited. J.B. is
supported by an Investigator Award from Wellcome (100993/
B/13/Z); M.W.P. is an NHMRC Research Fellow (APP1117183) and
Investigator (APP1194263). The project was also supported by the
Malaria Drug Accelerator (MalDA, BMGF OPP1054480 to M.R.L., S.O.,
K.K., M.C.S.L., J.C.N., and E.A.W.); L.T. was supported by an Australian
Research Council Laureate Fellowship (FL150100106); M.R.L. was
supported by a Ruth L. Kirschstein Institutional National Research
Award from the National Institute for General Medical Sciences (T32
GM008666);D.J.C.wassupportedbyaNHMRCSynergyGrant
(APP1185354)Author contributions:Conceptualization: S.C.X.,
R.D.M., E.D., C.J.M., S.W., J.C.N., M.C.S.L., J.B., S.O., E.A.W., D.J.C., N.W.,
S.B., S.P.L., L.R.D., M.D.W.G., A.E.G., L.T.; Investigation: S.C.X., R.D.M.,
E.D., S.-C.H., T.P., Y.D., S.N., M.R.L., L.M., M.-S.K., C.F.A.P.,
K.K., C.G., F.J.H., A.C., M.T.F., D.C.B., S.D., D.L.G., C.C.K., W.N.;
Analysis: S.C.X., R.D.M., E.D., C.J.M., S.-C.H., M.R.L., M.C.S.L.,
S.O., A.C., E.A.W., S.B., L.R.D., M.D.W.G., A.E.G., L.T.; Funding
acquisition: J.C.N., M.C.S.L., J.B., D.J.C., E.A.W., S.B., M.W.P.,
M.D.W.G., S.P.L., A.E.G., L.T.; Writing: S.C.X., R.D.M., E.D., C.J.M.,
M.W.P., L.R.D., M.D.W.G., S.P.L., A.E.G., L.T.Competing interests:
S.-C.H., L.M., M.-S.K., S.P.L., and A.E.G. are (or were) employees
and shareholders of Takeda. ML901 is exemplified (as compound
I-27) in patent application, PCT/US2017/061094 ( 22 ). Data
and materials availability:Coordinate files and structure factors
have been deposited in the PDB:PfYRS/AMP-Tyr: 7ROR;PfYRS/
ML901-Tyr: 7ROS;PfYRSS234C/ML901-Tyr: 7ROT;HsYRS/
ML901-Tyr: 7ROU. All other data are available in the main text
or the supplementary materials. The structure and synthesis of
ML901 are detailed in the paper. For supply of materials developed
as part of this work, please contact the corresponding authors.
A Materials Transfer Agreement may be required for some
materials.License information:Copyright © 2022 the authors,
some rights reserved; exclusive licensee American Association
for the Advancement of Science. No claim to original US
government works. https://www.science.org/about/science-
licenses-journal-article-reuseSUPPLEMENTARY MATERIALS
science.org/doi/10.1126/science.abn0611
Materials and Methods
Figs. S1 to S17
Tables S1 to S8
References ( 23 – 72 )
MDAR Reproducibility ChecklistView/request a protocol for this paper fromBio-protocol.Submitted 3 November 2021; accepted 1 April 2022
10.1126/science.abn0611Xieet al., Science 376 , 1074–1079 (2022) 3 June 2022 6of6
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