Table 2 Mutations identified in MMV688533-selected resistant P. falciparum clones and validated using CRISPR-Cas9 gene editing.

Four parasite clones (sel. 533-CL1 from flask 1, sel. 533-CL2 and 533-CL3 from flask 2, and sel. 533-CL4 from flask 3) were generated from selections (sel.) and named after the last three digits of the selecting compound (MMV688533) followed by the clone number. These clones were then chosen for whole-genome sequencing. Fold IC50 increases compared to the parent 3D7-A10 are indicated below the clone names. P. falciparum ACG1W286R, ACG1G98V, and EHDD218Y strains were gene-edited (ed.) using CRISPR-Cas9 to introduce the designated mutation into 3D7-A10 parasites. The sel. ed. 533-CL1EHD-D218Y clone was generated by CRISPR-Cas9 editing the EHD D218Y mutation into the selected 533-CL1 clone. wt, wild-type; ATP, adenosine 5′-triphosphate.

Gene productGene IDAmino acid substitution
sel.
533-CL2
ed. 3D7
ACG1W286R
sel.
533- CL3
sel.
533- CL4
ed. 3D7
ACG1G98V
ed. 3D7
EHDD218Y
sel. 533-
CL1
sel. ed.
533-CL1EHD-D218Y
3.1 × IC501.7 × IC502.5 × IC504.6 × IC501.8 × IC501.2 × IC502.2 × IC506.2 × IC50
Conserved Plasmodium
protein (PfACG1)
PF3D7_0910300W286RW286RT92*G98VG98VwtG98VG98V
EHD-containing protein
(PfEHD)
PF3D7_0304200wtwtwtD218YwtD218YwtD218Y
Conserved Plasmodium
protein
PF3D7_0510100wtwtwtwtwtwtN1042HN1042H
RNA pseudouridylate
synthase, putative
PF3D7_0511500wtwtK2762Ewtwtwtwtwt
ATP synthase (C/AC39)
subunit, putative
PF3D7_1464700L260Iwtwtwtwtwtwtwt

*Stop mutation resulting from a deletion-induced frameshift.