Table 2.
Phenotypic and genotypic profiles of recrudescent P. falciparum 3D70087/N9 parasites from humanized NSG mice treated with ZY19489, MMV693183, M5717, or ZY19489-based combination therapies
| Parasite Line IDa | NSG mouse treatment group and dosing regimena | DoRb | IC50 ± SEM (nM)c | Genetic changesd | |
|---|---|---|---|---|---|
| ZY19489 + MMV693183 | ZY19489 | MMV693183 | |||
| TAD_022 | None (control) | NA | 11.1 ± 0.6 | 2.6 ± 0.3 | Parental reference |
| TAD_746 | 1 × 40 mg/kg ZY19489 + 1 × 1.5 mg/kg MMV693183 | 15.6 | 11.1 ± 0.5 | 2.3 ± 0.2 | None |
| TAD_752 | 1 × 20 mg/kg ZY19489 + 1 × 5.0 mg/kg MMV693183 | 17.0 | 11.2 ± 0.7 | 2.6 ± 0.2 | None |
| TAD_755 | 1 × 20 mg/kg ZY19489 + 1 × 5.0 mg/kg MMV693183 | 16.6 | 12.3 ± 0.8 | 2.9 ± 0.1 | None |
| TAD_758 | 1 × 40 mg/kg ZY19489 + 1 × 1.5 mg/kg MMV693183 | 17.5 | 11.2 ± 0.2 | 2.8 ± 0.2 | None |
| TAD_761 | 1 × 40 mg/kg ZY19489 + 1 × 5.0 mg/kg MMV693183 | 18.0 | 11.8 ± 0.8 | 2.8 ± 0.3 | None |
| TAD_764 | 2 × 40 mg/kg ZY19489 + 1 × 1.5 mg/kg MMV693183 | 22.3 | 11.7 ± 0.3 | 2.6 ± 0.3 | None |
| TAD_767 | 1 × 40 mg/kg ZY19489 + 1 × 5.0 mg/kg MMV693183 | 20.9 | 11.9 ± 0.9 | 2.6 ± 0.2 | None |
| TAD_770 | 3 × 2.5 mg/kg MMV693183 | 26.2 | 11.2 ± 0.5 | 2.6 ± 0.2 | None |
| TAD_773 | 1 × 20 mg/kg ZY19489 + 3 × 2.5 mg/kg MMV693183 | 26.5 | 11.6 ± 0.8 | 2.9 ± 0.4 | None |
| TAD_779 | 2 × 40 mg/kg ZY19489 + 3 × 2.5 mg/kg MMV693183 | 30.7 | 11.0 ± 0.3 | 2.5 ± 0.3 | Y1020H in PF3D7_0825000, N636Y in PF3D7_1416100 |
| TAD_782 | 2 × 40 mg/kg ZY19489 + 3 × 2.5 mg/kg MMV693183 | 30.7 | 11.7 ± 1.2 | 2.9 ± 0.2 | None |
| ZY19489 + Ferroquine | ZY19489 | Ferroquine | |||
| TAD_022 | None (control) | NA | 6.7 ± 0.6 | 10.3 ± 1.6 | Parental reference |
| TAD_494 | 4 × 40 mg/kg ZY19489 | 25.5 | 7.2 ± 0.5 | 8.3 ± 0.2 | None |
| TAD_496 | 4 × 40 mg/kg ZY19489 + 1 × 15 mg/kg Ferroquine | 28.3 | 6.9 ± 1.2 | 9.5 ± 1.1 | D1698N in PF3D7_0210200, PF3D7_1224000 (loss of 3.5 copies) |
| TAD_497 | 4 × 40 mg/kg ZY19489 + 1 × 15 mg/kg Ferroquine | 27.5 | 7.1 ± 0.9 | 8.9 ± 0.6 | N1580S in PF3D7_1329100, G48A in PF3D7_1408600 |
| ZY19489 + M5717 | |||||
| TAD_030 | None (control) | NA | - | - | Parental reference |
| TAD_377 | 1 × 3 mg/kg M5717 | 14.1 | - | - | No mutations in PF3D7_1451100 |
| TAD_382 | 1 × 20 mg/kg M5717 | 20.2 | - | - | G373S in PF3D7_0320500, P490L in PF3D7_1032000, R149T in PF3D7_1312600, Y186N in PF3D7_1451100* |
| TAD_384 | 1 × 40 mg/kg M5717 | 21.6 | - | - | Y186N in PF3D7_1451100* |
| TAD_378 | 1 × 20 mg/kg ZY19489 + 1 × 3 mg/kg M5717 | 14.2 | - | - | G373S in PF3D7_0320500, P490L in PF3D7_1032000 |
| TAD_379 | 1 × 20 mg/kg ZY19489 + 1 × 3 mg/kg M5717 | 14.5 | - | - | P490L in PF3D7_1032000 |
| TAD_386 | 1 × 20 mg/kg ZY19489 + 1 × 20 mg/kg M5717 | 23.7 | - | - | P490L in PF3D7_1032000 |
| TAD_400 | 1 × 20 mg/kg ZY19489 + 1 × 20 mg/kg M5717 | 28.0 | - | - | P490L in PF3D7_1032000, R149T in PF3D7_1312600, Y186N in PF3D7_1451100* |
aRecrudescent parasite lines were recovered from humanized NSG mice infected with the P. falciparum 3D70087/N9 strain and treated with ZY19489, MMV693183, M5717, or ZY19489-based combinations (ZY19489 + MMV693183, ZY19489+Ferroquine, or ZY19489 + M5717). Parental control lines (TAD_022 and TAD_030) were sampled from untreated mice.
bDoR, Day of Recrudescence: Day of experiment when parasites reappear in circulating blood after initial complete clearance. Day 1 corresponds to the first day of treatment administration. NA, not applicable.
cIC50 values (nM) were determined for the parental controls and drug-exposed recrudescent parasites using standard 72-h in vitro dose-response assays following culture adaptation. Values represent means ± SEM from 3 to 5 independent experiments, each performed in duplicate. Recrudescent parasites from the ZY19489 + M5717 treatment group were not available for phenotypic analysis. Statistical significance was assessed relative to parental controls (TAD-022 or TAD_030) using two-tailed unpaired t-tests. No significant differences in susceptibility to ZY19489, MMV693183, or ferroquine were observed in any tested recrudescent line.
dSingle nucleotide polymorphisms (SNPs) and copy number variations (CNVs) were identified by whole-genome sequencing of all recrudescent lines relative to the respective 3D70087/N9 parental genomes (TAD-022 or TAD-030), except for TAD_377 and TAD_384 that were not whole-genome sequenced. All samples from the ZY19489 + M5717 group, including TAD_377 and TAD_384, were analyzed by targeted amplicon sequencing of the PfeEF2 (PF3D7_1451100) locus. The following genes were affected: PF3D7_0825000 (conserved Plasmodium protein of unknown function); PF3D7_1416100 (SEY1, a putative Plasmodium endoplasmic reticulum-shaping protein); PF3D7_0210200 (conserved Plasmodium protein of unknown function); PF3D7_1329100 (Myosin F, putative); PF3D7_1408600 (40S ribosomal protein S8e, putative); PF3D7_0320500 (nicotinamidase, putative); PF3D7_1032000 (ribosome maturation factor RimM, putative); PF3D7_1312600 (2-oxoisovalerate dehydrogenase subunit alpha, mitochondrial, putative); PF3D7_1451100 (P. falciparum elongation factor 2, PfeEF2) – the known target of M5717. Additionally, a 3.5-fold reduction in copy number was observed in a ~ 1.8 kb segment of chromosome 12 harboring the gch1 gene (PF3D7_1224000) in the TAD-496 genome. None of the observed genomic differences were associated with phenotypic resistance to ZY19489, MMV693183, or ferroquine.
*The PfeEF2 Y186N mutation identified in M5717-exposed recrudescent lines TAD_382, TAD_384, and TAD_400 is known to confer high-grade resistance to M5717, with an ~16,000-fold increase in IC50.