Science - USA (2022-06-03)

INSIGHTS | PERSPECTIVES

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needed to activate arteminisin, resistant
P. falciparum strains have emerged with
mutations that extend their ring life cycle
stage. At ring stage, they do not consume
host hemoglobin and therefore have a low
basal activation of arteminisin ( 6 ).
Another traditional approach to treat ma-
laria relies on covalent electrophilic com-
pounds that target the catalytic cysteine of
P. falciparum proteases that degrade hemo-
globin. Electrophilic compounds are alkyl-
ating agents that form covalent bonds with
the nucleophilic cysteine, lysine, histidine,
tyrosine, and serine amino acid side chains.
For example, the natural product galinomide
A alkylates the catalytic cysteine of falcipain
and shows antimalarial activity in vitro and
in vivo ( 7 ). Galinomide A is specific to fal-
cipain and ensures the selective alkylation
of its catalytic cysteine. Although consider-
able specificity for falcipain can be achieved,
the major challenge of electrophilic drugs
is the rampant proteome-wide reactivity at
concentrations as low as 10 μM, leading to
nonspecific cellular toxicity ( 8 ). This non-
specific reactivity may prevent the drug use
at therapeutically needed doses and stems
from the abundance of many nucleophilic
residues such as cysteine, serine, lysine, his-
tidine, and tyrosine in the cellular environ-
ment. Moreover, nonspecific alkylation of
intracellular glutathione can also deactivate
the drug and cause liver toxicity, as occurs
with the analgesic drug acetaminophen.
To overcome the nonspecific reactivity of
electrophilic drugs with intracellular nu-
cleophiles, one possible solution is to swap
the polarity of the drug and make it nucleo-
philic. Such a drug would not react with the
fellow nucleophiles in the cell but rather
react with cellular electrophiles such as en-
zyme intermediates and cofactors or those
on protein surfaces, such as sulfenic acids
(–S–OH) ( 9 , 10 ). Perhaps the best-known
example is the antibiotic nucleocidin that
contains a sulfamate functional group

(–O–SO 2 –NH 2 ) ( 11 ). Upon deprotonation of

the –NH 2 group, the resulting nucleophile

R–O–SO 2 –NH– can react, for example, with

electrophilic thioesters in the cell. This

property of sulfamates has been success-

fully used to design adenosine 5 ́-sulfa-

mates as activity-based probes and inhibi-

tors for ubiquitin-like proteins ( 12 , 13 ).

Xie et al. tested the cytotoxicity of ad-

enosine 5 ́-sulfamate (ASM) against P. fal-

ciparum and discovered that ASM is very

toxic to P. falciparum [median inhibitory

concentration (IC 50 ) 1.8 nM] and human

cells (IC 50 26 nM). To identify compounds

with more selective toxicity for P. falci-

parum, they screened an additional 2000

sulfamates and discovered an ASM de-

rivative, ML901, that is 800 to 5000 times

more toxic to P. falciparum than to a panel

of human cell lines. ML901 reacts with the

tyrosine-charged tRNA in the presence of

the P. falciparum tyrosine aminoacyl-tRNA

synthetase (Pf tyrosine-AS) to produce co-

valent ML901-Tyr conjugate. ML901-Tyr

acts as a bivalent inhibitor of Pf tyrosine-

AS, preventing the incorporation of tyro-

sine in nascent proteins during translation.

Notably, the equivalent human tyrosine-AS

enzyme did not catalyze the formation of

ML901-Tyr conjugates, suggesting that ML-

901 will not inhibit protein translation in

human cells. Therefore, a large therapeutic

window for ML901 can be achieved. This

distinct mode of action is exciting because

there are 20 AS enzymes (one for each

amino acid), therefore each P. falciparum

AS could be selectively inhibited with sul-

famates by using the mechanism similar to

that of ML901. This should lead to the inhi-

bition of protein synthesis in P. falciparum

and toxicity. Prior work has shown that the

inhibition of the Pf methionine AS is also

toxic to P. falciparum ( 14 ).

The ability to target different Pf AS en-

zymes provides an opportunity to also ad-

dress potential drug resistance to ML901.

Xie et al. show that prolonged treatment

of P. falciparum cell cultures with ML901

leads to a Ser^234 Cys mutation in Pf tyro-

sine-AS in drug-resistant clones. The result-

ing mutant Pf tyrosine-AS enzyme was less

efficient at stimulating the formation of co-

valent ML901-Tyr conjugates and was also

less sensitive to the inhibition by ML901-

Tyr conjugates. Such resistance can be ad-

dressed by switching to the inhibitor for a

different Pf AS enzyme or alternatively treat

P. falciparum with the cocktail containing

different combinations of Pf AS inhibitors,

arteminisin, falcipain, and hemozoin inhib-

itors. Given that ASM displays antibacterial

properties, a similar strategy could perhaps

be used to design antibiotics. j

REFE RENCES AND NOTES

1. S. C. Xie et al., Science 376 , 1074 (2022).


2. S. E. Francis, D. J. Sullivan Jr., D. E. Goldberg, Annu. Rev.
   Microbiol. 51 , 97 (1997).


3. B. Meunier, A. Robert, Acc. Chem. Res. 43 , 1444 (2010).


4. A. Robert, F. Benoit-Vical, C. Claparols, B. Meunier, Proc.
   Natl. Acad. Sci. U.S.A. 102 , 13676 (2005).


5. J. Wang et al., Nat. Commun. 6 , 10111 (2015).


6. S. Mok et al., Science 347 , 431 (2015).


7. A. S t o y e et al., J. Med. Chem. 62 , 5562 (2019).


8. B. R. Lanning et al., Nat. Chem. Biol. 10 , 760 (2014).


9. M. L. Matthews et al., Nat. Chem. 9 , 234 (2017).


10. Y. Shi, K. S. Carroll, Acc. Chem. Res. 53 , 20 (2020).


11. U. Ngivprom et al., RSC Advances 11 , 3510 (2021).


12. H. An, A. V. Statsyuk, J. Am. Chem. Soc. 135 , 16948
    (2013).


13. M. Misra et al., Structure 25 , 1120 (2017).


14. X. Chen et al., Proc. Natl. Acad. Sci. U.S.A. 103 , 14548
    (2006).

ACKNOWLEDGMENTS

I th ank V. E. Mgbemena for helpful comments. A.V.S. is funded

by R01GM115632 and 1R01CA256543-01A1 (co-investigator).

10.1126/science.abq4457

Hemoglobin

Steps of the P. falciparum feeding pathway targeted by antimalarial drugs

Electrophile

Plasmepsins

I and II

Falcipain

Heme release Hemozoin

Small peptides Amino acids

Protein synthesis

ML901

Arteminisin

Galinomide A

Polymerization

Activation

Radical toxicity

Nucleophile

Tyrosine aminoacyl tRNA synthetase

N

N

N N

H 2 N O

OO O

O

OH

S

O NH–

HO OH OH

O

F

F

N

N

N N

H 2 N

H 2 N

H 2 N

O

OOO

S

O N

H

OH

OH

HO

O

F

F

Paracitophorous vacuole

Ty r t R N A

Acylation

reaction

ML901 prodrug

ML901-Tyr conjugate inhibits parasite translation

“ML901-Tyr ... [prevents] the

incorporation of tyrosine

in nascent [Pf] proteins during

translation.”

GRAPHIC: KELLIE HOLOSKI/

SCIENCE

Covalent drugs to treat malaria
The feeding pathway of Plasmodium falciparum can be inhibited at various stages by covalent drugs such as arteminisin, galinomide A, and ML901. ML901 is an adenosine
5′-sulfamate analog that is selectively toxic to P. falciparum but not human cells. This is because Pf tyrosine aminoacyl-tRNA synthetase conjugates ML901 to tyrosine, and
the resulting ML901-tyrosine conjugate prevents the incorporation of tyrosine into nascent proteins.

1050 3 JUNE 2022 • VOL 376 ISSUE 6597