Fig. 3: NCP26-resistance selection and whole genome sequence analysis.

a In vitro activity of NCP26 against ABS P. falciparum Dd2-2D4 (parent) or subclones from three independent resistance selection experiments (S1-3). S1 did not yield resistant parasites and S1—clone A8 was included for comparison. b In vitro activity of reference compounds against Dd2-2D4 (parent) or subclones from each selection (S1-3). Reference compounds include PfcProRS inhibitors (halofuginone and halofuginol) and non-PfcProRS inhibitors dihydroartemisinin (DHA) and borrelidin (P. falciparum threonyl-tRNA synthetase inhibitor). c Residues T512S (S2) and F405L (S3) were identified by whole genome sequence analysis to mediate NCP26-resistance and are mapped to the co-crystal structure of NCP26 (green) and proline (white) bound to wild-type PfcProRS (PDB: 6T7K). Both amino acid side chains directly interact with the pyrazinamide core. d Whole genome sequence analysis revealed amplification of the intra-chromosomal region harboring the PfcProRS locus on chromosome 12 that is observed in all S3 clones. No copy number variations were observed in other chromosomes for S3 clones or in any chromosomes for Dd2-2D4 (parent), S1 (no NCP26-resistance observed), or S2 (PfcProRS^T512S). Detailed CNV data are available in Source Data—CNV. ABS growth assay data in a are expressed as mean ± s.d. (n = 3 biological replicates) and are representative of at least two independent experiments. ABS growth assay data in b are expressed as the mean IC₅₀ values from independent experiments ± s.d. (n = 3 biological replicates per independent experiment) and include at least two independent experiments, except for DHA in S2—clone B9 which only was only tested once. For a, b, and d, clonal parasite lines isolated from the same selection flask are shown as different shades of the same color (S1 = pink, S2 = shades of green, and S3 = shades of blue).