(table S4). Among these“on-target”down-
regulated genes were those involved in key par-
asite processes, such as egress and invasion,
cytoadherence, parasite protein export, and in-
volvement in sexual stages, as well as house-
keeping functions including metabolism, RNA
processing, lipid modification, and mitochon-
drial function (Fig. 4E and table S4). Of the 696
“on-target”genes identified as down-regulated
byPfCLK3 inhibition (table S4), 425 matched
those that have recently been determined to be
essential for asexualP. falciparumsurvival ( 12 )
(table S4).
Gene ontology enrichment analysis was used
to determine biological functions that were dis-
proportionally down-regulated byPfCLK3 inhi-
bition. In this analysis, genes associated with
key biological functions, particularly protein
modification, phospholipid biosynthesis, and
lipid modification, were significantly overrep-
resented among those genes that were down-
regulated (fig. S8 and table S5). We found that93% of the“on-target”down-regulated genes
contained introns (Fig. 4F and table S4), versus
52% of genes in theP. falciparumgenome that
are annotated as containing introns ( 32 )(Fig.4G).
Hence,PfCLK3 inhibition significantly affected
the transcription of genes that contained introns
(P< 0.0001, Pearsonc^2 test), further supporting
its role in splicing.
In addition to the nearly 700 genes down-
regulated in response toPfCLK3 inhibition,
therewere154genesthatweresignificantlyAlamet al.,Science 365 , eaau1682 (2019) 30 August 2019 5of8
Fig. 4. Inhibition ofPfCLK3 prevents trophozoite-to-schizont
transition, kills the parasite with rapid kinetics, and disrupts gene
transcription.(A) Smears of synchronized blood-stageP. falciparum
cultures after treatment with TCMDC-135051 (2mM) were taken at
the indicated times after TCMDC-135051 administration. (B) The in vitro
parasite reduction rate in the presence of 10 × EC 50 of TCMDC-135051
was used to determine the onset of action and rate of killing. Data are
means ± SEM. Previous results reported on standard antimalarials tested
at 10 × EC 50 using the same conditions are shown for comparison ( 44 ).
(CandD) Illustration of the genes that are designated as significantly
changing (moderatettest,n= 4) in transcription after treatment with
TCMDC-135051 (1mM, 60 min) of either (C) parent Dd2 parasites or (D)
TM051C mutant parasites. Each line represents the log 2 fold change in the
probes used in the microarray. Numbers of genes represented by the
probes are indicated. (E) Summary of the parasite processes associated
with the genes where transcription is statistically significantly down-regulated
after TCMDC-135051 treatment. (F) Assessment of intron-containing genes
among genes that are up-regulated and down-regulated in Dd2 parasites
after TCMDC-135051 treatment. (G) Assessment of intron-containing genes
in theP. falciparumgenome (data derived from Plasmodb).RESEARCH | RESEARCH ARTICLE