pfap2tf-10 [pfap2-i (37)]. Although not all compounds resulted in metabolic responses, the
general concordance between metabolic changes
and resistance mutations supports the combined
use of these approaches for compound target
Discussion and conclusions
Our study represents a controlled examination
of antimalarial drug resistance acquisition by
P. falciparum. Prior studies examining the parasite’s genetic response during drug resistance
development have evaluated the response only
to known antimalarials (31, 38, 55, 56) or have
focused on coding mutations in one target
gene in response to a single compound class
(8, 13, 32, 40, 57–60). It is likely that the genes
that are identified here will prove important in
both clinical studies and the process of drug
One noteworthy finding from this data set is
the high enrichment of mutations under positive
selection. We focused here on genes for which
there were multiple lines of supporting evidence
across our study; however, it is likely that other
important genes were identified. Excluding genes
contained within CNVs, the 57 singleton genes
with nonsynonymous mutations encode potential
druggable targets, such as guanosine triphosphatases, mRNA decapping enzymes, components of
V-type ATPase complexes, kinases, and ubiquitin
ligases (table S5). Although some of these nonsynonymous changes could have been maintained
within the population by chance, some are plausible targets of selection. For example, although
resistance to ACT-451840 is conferred by mutations in pfmdr1 (61), a mutation was observed in
the gene encoding a putative replication factor C
subunit 4 (PF3D7_1241700) that results in a S42T
amino acid change near the ATP-binding pocket
in two independent ACT-451840–resistant clones
(61). Replication factor C subunit 4 is the subunit
ATPase in the clamp-loading DNA polymerase
complex, is likely essential, and is a good drug
target. Modeling shows that ACT-451840 could
bind between subunits 1 and 4 of replication
factor C, potentially disrupting ATPg ingress (fig.
S10). Our findings also highlight the large number
of uncharacterized genes in the P. falciparum
genome and the need for further functional
Not all mutations discovered in this study will
confer resistance—they may compensate, spe-
cifically or nonspecifically, for fitness losses that
result from a resistance-conferring mutation.
Alternatively, they may be associated with ad-
aptation to long-term in vitro culture. Genome
editing or creating recombinant lines may be
used to confirm that alleles provide resistance
(8, 15, 27, 40, 58, 62, 63). On the other hand,
given the multigenic nature of resistance, it may
be difficult to recreate a resistance phenotype (7 ).
For example, the nonsynonymous changes that
emerged in phenylalanine tRNA ligase during
development of resistance to BRD1095 were
accompanied by copy number changes at un-
related sites, which potentially explains why at-
tempts at validation through genome editing
Beyond changes in coding sequences, the low
frequency at which noncoding variants were
found in our data set suggests a possible role. We
identified only 21 silent mutations within genes
that are plausible drug targets, including those
encoding protein kinase 4, Ark3 kinase, cytochrome
c oxidase 3, a histone-lysine N-methyltransferase, a
putative serine threonine protein kinase, a DNA
helicase, and an 18S ribosomal RNA. Several of the
genes with silent mutations were highly expressed,
including the cytochrome c oxidase subunit 3 gene,
in which the mutation substitutes a rare codon
for a frequently used one [acA (23.8% usage for
threonine) to acG (3.6%)]. Silent mutations in hu-
man mdr1 can confer resistance to cancer drugs
owing to altered protein folding (64). Further work
will be needed to establish whether these silent
substitutions play a role in drug resistance in
P. falciparum. Similarly, intergenic and intronic
mutations were also common. Although in most
cases, resistance was explained by the presence
of nonsynonymous coding mutations in the tar-
get or resistance genes, intergenic mutations
were also common. Likewise, intron variants were
more likely to be found in the core genome than
in subtelomeric regions (64:5), suggesting a pos-
sible functional role.
Last, it is likely that among the genes that we
identified, several may contribute to clinical resist-
ance at some level. Although pharmacokinetics are
different in vivo than in vitro, we repeatedly re-
discovered mutations in genes important for
clinical resistance. It is therefore likely that pre-
viously unidentified genes are candidates to be
under selection in clinical isolates. Mutations in
3247 clinical P. falciparum isolates reveal that
four mediators of resistance (the ABC trans-
porter gene pfabcI3, the putative amino acid
transporter gene pfaat2, the AP2 transcription
factor gene pfap2tf-6b, and the farnesyltransferase
gene pfftb) have nonsynonymous-to-synonymous
ratios greater than 2:1, suggesting that they are
under positive selection (65) (table S15). However,
the role of these genes in clinical drug resistance
is unknown. In addition, our data highlight the
importance of CNVs in conferring multidrug re-
sistance and suggest that high-coverage genome
sequencing of clinical isolates will provide infor-
mation on selective pressures in the field (66).
It is notable that we were able to identify a likely
target or resistance gene for every compound for
which resistant parasites were generated. The
haploid genome and the lack of transcriptional
feedback loops suggest that P. falciparum is a
particularly good model for both target identifica-
tion and resistome studies. Our characterization
of the chemogenetic landscape of P. falciparum
will guide the design of small-molecule inhib-
itors against this deadly eukaryotic pathogen.
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This work was supported by grants from the Bill and Melinda Gates
Foundation (OPP1040406 and OPP1119049), the National
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