Collateral hypersensitivity between ZY19489 and piperaquine neutralizes PfCRT-mediated drug efflux and Plasmodium falciparum resistance

Domain: biology
Published: 2026-04-01
Editorial outcome: minor_revision

Abstract
New antimalarial drugs are needed to combat the current emergence and spread of Plasmodium falciparum parasite resistance to artemisinin-based combination therapies. Here, we characterize ZY19489, a triaminopyrimidine currently in a Phase Ib clinical trial. Asexual blood-stage parasites pressured with ZY19489 acquire low-grade resistance, mediated by a novel mutation in the P. falciparum chloroquine resistance transporter PfCRT that cause slow growth rates and a substantial fitness cost. ZY19489-resistant parasites lose their chloroquine resistance status and become hypersusceptible to piperaquine, an artemisinin-based combination partner drug. Uptake studies in proteoliposomes loaded with drug-resistant PfCRT isoforms demonstrate that ZY19489 can block mutant PfCRT-mediated piperaquine and chloroquine transport. In parasites, PfCRT mutant variants can mediate piperaquine and chloroquine resistance via their efflux out of the digestive vacuole. Our findings evoke a scenario of an evolutionary trap whereby resistance to ZY19489 can block piperaquine and chloroquine efflux and thereby restore their activity. Metabolomic studies show that ZY19489 leads to significantly reduced intracellular levels of short hemoglobin-derived peptides (a natural substrate of PfCRT) and accumulation of pyrimidine deoxynucleotides. Our results present a possible marker for tracking the evolution of clinical resistance to ZY19489 and a rationale for pairing this molecule with piperaquine to generate a resistance-refractory combination.

Introduction
The World Health Organization recently reported an estimated 282 million cases and 610,000 malaria-related deaths globally in 20241, highlighting the continuing neutral to upward trend in malaria infections since 2016. This upsurge in disease burden has been, in part, due to the spread of Plasmodium falciparum alleles associated with loss of parasite sensitivity to artemisinin-based combination therapies (ACTs) and, more recently, the emergence of parasites with deletions in histidine-rich proteins 2 and 3 (pfhrp2/3) that confound HRP2-based case detection1-4. Currently approved malaria vaccines, which offer moderate protection, are not yet widely available5,6. There is an urgent need for new, safe and efficacious antimalarial drugs endowed with distinct modes of action (MoA) to complement current treatment options.

Recent antimalarial drug discovery efforts aim to develop new medicines that can circumvent emerging drug resistance, whilst ensuring longer efficacy and improved dosing convenience. These efforts have yielded several new molecules with novel mechanistic profiles that are currently in various phases of clinical development (https://www.mmv.org/mmv-pipeline-antimalarial-drugs). One such candidate is ZY19489 (Fig. 1a), a fast-killing and long-acting triaminopyrimidine identified from a high-throughput screen of an AstraZeneca library comprising 500,000 compounds tested against P. falciparum asexual blood-stage (ABS) parasites using high-content imaging7. This compound (also known as Zintrodiazine, MMV674253, AZ412, or TAP12) showed good safety, pharmacokinetic, and efficacy results in a first-in-human clinical trial8. ZY19489 also retains potency against P. falciparum field isolates expressing mutant kelch13 alleles that confer artemisinin partial resistance9. Currently, ZY19489 is in a Phase Ib trial in combination with the 4-aminoquinoline, ferroquine, for treatment of uncomplicated P. falciparum malaria (https://classic.clinicaltrials.gov/ct2/show/NCT05911828). If approved, this combination could constitute an important non-ACT treatment option in areas where artemisinin partial resistance has emerged10.

The putative MoA and molecular mechanisms that potentially underlie resistance to ZY19489 remain unknown. In prior in vitro selection experiments using several triaminopyrimidines structurally analogous to ZY19489, one derivative (compound 6; Fig. 1a) yielded resistant parasites that exhibited a 3-fold increase in the half-maximal growth inhibition concentration (IC50) compared to parental Dd27. This low-level resistance was initially associated with a G29V mutation in subunit D of the P. falciparum vacuolar proton-transporting V-type ATPase complex (PfV1-D; PF3D7_1341900), suggesting possible involvement of this protein complex in conferring ZY19489 resistance. However, in a recent study using a “pH fingerprint” assay to elucidate the MoA of candidate antimalarials, ZY19489 did not affect the ability of the V-type H+ ATPase to regulate digestive vacuole (DV) or cytoplasmic pH. In addition, ZY19489 was unable to significantly inhibit ATPase activity attributable to this ATPase pump11. These results suggested that the compound has a different MoA in the parasite.

In this report, we explored ZY19489 by leveraging in vitro resistance selection and gene editing, genetic cross trait mapping, metabolomics, drug transport experiments, parasite growth fitness assays, and molecular dynamics simulations. Our study identified a novel point mutation in the P. falciparum chloroquine resistance transporter (PfCRT; PF3D7_0709000) that confers low-grade resistance to this inhibitor. We provide evidence of a high resistance barrier for ZY19489 based on combination treatments in mice and highlight its efficacy against resistance-conferring mutant pfcrt alleles currently circulating in the field. Metabolomic results offer a window into the mechanistic basis of ZY19489 anti-ABS potency. These findings yield a locus for surveilling possible evolution of clinical resistance to this antimalarial drug and a rationale for potential pairing of this compound with the 4-aminoquinoline piperaquine (PPQ; Fig. 1a) to generate a resistance-refractory combination.

Results
In vitro selection and genome sequencing identify mutant parasites with low-grade resistance to ZY19489
To test whether the prior association between ZY19489 resistance and a mutated form of the P. falciparum V-type ATPase complex7 was causal (see Introduction), we generated a conditional knockdown (cKD) line, NF54V1-D cKD (Supplementary Table 1; Supplementary Fig. 1a-c), with PfV1-D expression regulated by anhydrotetracycline (aTc). Assays with the control V-type ATPase inhibitor bafilomycin A showed the expected reduction in IC50 in parasites with reduced expression of PfV1-D. In contrast, we observed no difference between ZY19489 activity against PfV1-D cKD parasites cultured in high or low concentrations of aTc and control lines under no aTc, further discounting a causal role for the V-type ATPase complex (Supplementary Table 2).

To identify a genetic basis of in vitro resistance to ZY19489, we used a high parasite inoculum strategy and the hypermutable Dd2-Polδ line that has a higher basal mutation rate12 (Supplementary Fig. 2). Parasites were intermittently pressured with 100 nM ZY19489, corresponding to ~10× its IC50. Recrudescent parasites obtained from one of the three flasks after 136 days of selection pressure exhibited a 12-fold IC50 increase compared to Dd2-Polδ. Resistance was confirmed in four parasites cloned by limiting dilution (Fig. 1b; Supplementary Table 3).

Whole-genome sequence (WGS) analysis of the four ZY19489-pressured clones identified single nucleotide polymorphisms (SNPs) in four different genes (Table 1). Of these, the N246H mutation in exon 5 of pfcrt and D233N in the anaphase-promoting complex subunit 10 (pfapc10; PF3D7_1217600) were present in all the clones. Mutations in two other genes were observed in a single clone (C8; Table 1), which also showed amplification of an ~80 kb chromosome 5 segment spanning 15 genes including the P. falciparum multidrug resistance protein 1 (PfMDR1; PF3D7_0523000; Supplementary Table 4).

Genetic validation confirms PfCRT N246H as the primary determinant of ZY19489 resistance
To test the role of pfcrt N246H and pfapc10 D233N, we genetically engineered these mutations into ZY19489-sensitive lines (Supplementary Table 1). N246H was edited into pfcrt in recombinant Dd2 lines expressing either the dominant Southeast Asian (Dd2Dd2crt) or two African (Dd2GB4crt and Dd2FCBcrt) alleles, each of which confer chloroquine (CQ) resistance. The pfcrt Dd2 allele has eight point mutations that differ from the CQ-sensitive wildtype (3D7) allele, whereas GB4 and FCB have six and seven mutations, respectively (lacking N326S in GB4 and I356T in both GB4 and FCB)13. The D233N mutation was edited into pfapc10 in the Dd2Dd2crt line. Gene editing used zinc-finger nucleases for pfcrt14 and a CRISPR/Cas9 system for pfapc12 (Supplementary Fig. 3a, b). Successful editing yielded Dd2Dd2crt+N246H, Dd2GB4crt+N246H, Dd2FCBcrt+N246H, and Dd2Dd2crt+apc10 D233N, as confirmed by Sanger sequencing. We observed an 11-, 8- and 5-fold increase in ZY19489 IC50 values in the gene-edited Dd2Dd2crt+N246H, Dd2GB4crt+N246H and Dd2FCBcrt+N246H lines, respectively, compared to their isogenic parental lines (Fig. 1c; Supplementary Table 5). These shifts reproducibly phenocopied the results from the drug-selected Dd2-Polδ clones and indicated a marginally lower fold change in Dd2GB4crt+N246H and Dd2FCBcrt+N246H. On the other hand, two PfAPC10 mutant clones (with mean ± SEM ZY19489 IC50 values of 7.7 ± 0.7 nM and 8.8 ± 1.0 nM) showed comparable susceptibilities to Dd2Dd2crt (mean ± SEM IC50; 8.5 ± 0.4 nM) (Fig. 1c; Supplementary Table 5). Commercially sourced APC10 and ubiquitin ligase inhibitors tested against Dd2Dd2crt+apc10 D233N and its isogenic parent showed no difference in activity (Supplementary Table 6), further arguing against a modulatory role for the pfapc10 D233N mutation. The K2S42T and T1776I mutations observed in PF3D7_1320800 and PF3D7_1321300, respectively, were not studied further since they were present in only one clone.

Because the WGS analysis had also flagged copy number amplification on chromosome 5 that includes pfmdr1, we also examined the influence of differential copies of this pleotropic efflux transporter on ZY19489 activity by testing the susceptibility of two isogenic FCCB lines that differ in their pfmdr1 copy number and expression levels15. These lines showed no significant difference in susceptibility to ZY19489. The aryl amino alcohols, mefloquine and lumefantrine (Supplementary Fig. 4), both showed a ~2-fold IC50 decrease against the single-copy FCB-KDmdr1 relative to the parental line that expresses two copies, as expected15 (Fig. 1d; Supplementary Table 7). These findings establish the PfCRT N246H mutation as the primary driver of in vitro resistance to ZY19489.

Quantitative trait loci analysis map in vitro ZY19489 resistance to a chromosome 7 segment containing pfcrt
We tested the activity of ZY19489 against two contemporary Cambodian multidrug-resistant clinical isolates and observed 2.3- and 4.7-fold higher mean IC50 ± SEM values against Cam3.II (14.9 ± 3.3 nM) and RF7 (27.3 ± 0.7 nM), respectively, compared to the drug-sensitive African reference line NF54 (5.8 ± 0.2 nM). This suggests the presence of naturally occurring field mutations in Cam3.II and RF7 that might modulate parasite response to this compound.

We therefore analyzed a recent genetic cross16 conducted between the PPQ- and dihydroartemisinin (DHA)-resistant RF7 parental line and the drug-sensitive NF54 parental line and performed quantitative trait locus (QTL) mapping to localize candidate determinants of ZY19489 susceptibility. Phenotype profiling of the 34 recombinant progeny clones recovered from that cross revealed ZY19489 mean IC50 values ranging from 8 to 30 nM (Fig. 1e), which allowed for a phenotype-genotype linkage analysis to uncover QTLs associated with partial in vitro resistance to ZY19489. QTL analysis revealed a significant peak on chromosome 7 that spanned 300 kb and contained 34 genes with non-synonymous mutations between NF54 and RF7 (Supplementary Table 8). The maximum LOD (Logarithm of the Odds) score of 5.3 within this segment mapped to pfcrt, providing evidence of an association between ZY19489 in vitro activity and the pfcrt Dd2+M343L mutant allele present in RF7 (Fig. 1f). These observations, along with the WGS data from the in vitro selected clones and genetic validation experiments, confirm that PfCRT mutations can modulate ZY19489 in vitro activity.

Structural evidence suggests that the N246H mutation alters ZY19489 interactions with the PfCRT central cavity
PfCRT localizes to the ABS parasite’s digestive vacuole (DV) membrane and comprises 10 transmembrane (TM) helices, including four antiparallel pairs that surround a large negatively charged central cavity17. Distinct amino acid substitutions, including those that confer CQ or PPQ resistance, line this cavity and mediate the conversion of drug binding events into transport across the DV membrane17. To explore the effect of the N246H mutation on the PfCRT structure and elucidate the protein-ligand interactions that govern ZY19489 resistance, we performed molecular dynamics simulations with the cryo-EM elucidated 7G8 structure and a separate version into which we introduced the N246H mutation (Supplementary Table 9). This residue maps to TM7 and lines the central cavity close to the parasite cytosol (Fig. 2a). In the open-to-DV conformation, ZY19489 appeared to preferentially reside near the CQ resistance-conferring 76T residue in both the parental (7G8) and N246H-mutated proteins, with no direct interaction with residue 246 (Fig. 2a).With the 7G8 isoform, ZY19489 adopted orientations that facilitated hydrogen bonding with residues such as S94 and the 326D variant, unlike in the 7G8+N246H mutant where no specific ligand-protein hydrogen bonds persisted for more than 15% of the simulation time. This suggests a weaker ZY19489 interaction with the mutated protein than with the 7G8 isoform. Additionally, ZY19489 appeared to disrupt the residue interaction network around 76T (Y68, S72, N75, 76T, H97, 326D, D329, Q352) more substantially in 7G8 than in the 7G8+N246H isoform. Interestingly, the 7G8+N246H protein was predicted to form a salt bridge between K80 and E207, or other nearby residues, particularly in the regions between TM1-TM6, TM5-TM9, and TM6-TM9 (Supplementary Fig. 5). This potential salt bridge formation suggests that the mutant protein might transiently adopt an occluded (partially closed-to-DV) conformation, which might contribute to ZY19489 resistance. Conversely, the 7G8 protein did not appear to adopt this occluded conformation (Supplementary Fig. 5; Supplementary Fig. 6).

In the open-to-cytosol model, our simulations predicted significant differences in the hydrogen bonding patterns, favoring stronger interactions and stabilization of ZY19489 with 7G8 than with the mutated protein. Specifically, hydrogen bonds with the side chain of E232 and the backbone carbonyl group of L245 appeared to be important for stabilizing the ligand in the 7G8 isoform (Fig. 2b; Supplementary Fig. 6). In the 7G8+N246H variant, we observed only one hydrogen bond interaction with Q161 that persisted for more than 30% of the simulation period. This hydrogen bond was not consistently detected in all trajectories; appearing exclusively in only one of the three independent molecular dynamics replicate simulations. This absence of consistent hydrogen-bonding in the 7G8+N246H mutant evokes possibly weaker ligand interactions compared to the 7G8 isoform. These findings suggest that the N246H mutation may significantly alter the binding dynamics of ZY19489 in the open-to-cytosol conformation, potentially reducing the interaction stability of the ligand and subsequently facilitating its transport via the mutated protein, which could result in resistance.

PfCRT N246H confers sensitivity to DV-acting aminoquinoline antimalarials
Certain PfCRT mutant isoforms can mediate reduced parasite sensitivity to CQ and PPQ via their transport-mediated efflux out of the DV17,18. To relate the molecular dynamics simulation results to the experimentally derived influence of PfCRT N246H on various antimalarials, we tested the susceptibility of Dd2Dd2crt+N246H, Dd2FCBcrt+N246H and Dd2GB4crt+N246H to clinically important drugs including CQ, mono-desethylamodiaquine (the primary active metabolite of the ACT partner drug amodiaquine), ferroquine, PPQ, atovaquone (ATQ), lumefantrine, and DHA, (Supplementary Fig. 4). This antimalarial set comprises 4-aminoquinoline ring-containing drugs that are believed to inhibit heme detoxification in the DV (the first four listed) as well as other drugs whose primary targets lie elsewhere. Dd2Dd2crt+N246H and Dd2GB4crt+N246H were 9- and 11-times significantly more susceptible to CQ, respectively, than their parental lines, consistent with ablation of CQ resistance (Fig. 2c; Supplementary Table 10). These lines also exhibited a 4- to 6-fold increased sensitivity to mono-desethylamodiaquine as well as a 2- to 3-fold sensitization to ferroquine and PPQ. Although the same trend was also observed for lumefantrine, the drop in IC50 was modest (20%) and not statistically significant (Supplementary Table 10). There was no significant change in susceptibility towards DHA or ATQ (Fig. 2c; Supplementary Table 10), alluding to a collateral sensitization of the N246H lines that was specific to DV-acting 4-aminoquinolines.

ZY19489 competes with CQ and PPQ in PfCRT-mediated drug efflux
To interrogate whether ZY19489 might influence PfCRT-mediated drug transport across the DV membrane, we leveraged an experimental physiological model that mimics an “inside-out” parasite DV whereby [3H]CQ or [3H]PPQ uptake can be measured in PfCRT-containing proteoliposomes17. We observed a significant 72% reduction in [3H]CQ transport by the CQ-resistant 7G8 isoform upon addition of 1 M ZY19489 (Mann-Whitney U tests; p < 0.0001), suggesting that this compound can directly impede CQ transport (Fig. 2d). Similar interference was also observed against [3H]PPQ transport by the PPQ-resistant isoform 7G8+F145I, with a significant 79% inhibition of PPQ transport achieved with 1 M ZY19489 (Mann-Whitney U tests; p < 0.0001, Fig. 2e). Transport of [3H]CQ and [3H]PPQ by the PfCRT isoforms 7G8 and 7G8+F145I, respectively, was significantly inhibited by the addition of the reciprocal agents PPQ and CQ, to levels comparable to those observed with ZY19489 (Mann-Whitney U tests; p < 0.0001, Fig. 2d and 2e). No competitive inhibition of transport was observed with pyrimethamine or ATQ whose resistance mechanisms (mutations in dihydrofolate reductase and cytochrome bc1, respectively) are unrelated to PfCRT-mediated transport. These results show that ZY19489 can antagonize CQ and PPQ transport and offer evidence of competition for the protein’s binding cavity. They also provide compelling evidence favoring ZY19489 as an agent that can restore CQ or PPQ potency by inhibiting their mutant PfCRT-mediated efflux away from their heme target in the DV.

PfCRT N246H confers a slow growth rate and high fitness cost on ASBS parasites
During routine culturing of the ZY19489-pressured, resistant Dd2-Polδ mutants, we noticed a unique parasite morphology characterized by translucent distended DVs during the development from mid-trophozoites to mid-schizonts (Fig. 3a). This aberrant phenotype was retained in all N246H pfcrt-edited lines but absent in the Dd2-Polδ, Dd2Dd2crt and N233D pfapc10-edited clones, suggesting an interplay between this variant PfCRT isoform and parasite DV physiology.

To assess the impact of the PfCRT N246H mutation on growth rate, we monitored the replication rates of synchronized ring stage parasites over several replication cycles and used it as a proxy for ABS fitness. Cultures of Dd2Dd2crt+N246H, Dd2GB4crt+N246H, Dd2FCBcrt+N246H and their respective parental lines were each seeded at an inoculum equivalent to 8×105 parasites per well and tracked over 8 generations (16 days). Parasitemia measurements and media change were performed on days 3 and 4 and every two days thereafter. Parasite cultures with >2% parasitemia were diluted to 0.2% to reinitiate growth. Dd2Dd2crt, Dd2FCBcrt and Dd2GB4crt exhibited a 6- to 7-fold mean growth rate per generation compared to 2- to 3-fold rates observed in the mutants over the same period. From these data, we quantified the fitness cost of each mutant as a percent reduction in growth rate per generation relative to the isogenic parent (Fig. 3b). Dd2Dd2crt+N246H, Dd2GB4crt+N246H and Dd2FCBcrt+N246H exhibited a mean fitness cost of 75%, 73% and 61%, respectively, consistent with the slow expansion rates in routine cultures. These results suggest that PfCRT N246H confers a severe fitness cost to ABS parasites. We hypothesize that parasites with this mutation would be likely to have a major competitive disadvantage were they to emerge in the field.

ZY19489 retains potency against contemporary mutant pfcrt and pfmdr1 alleles
We next investigated whether parasites with contemporary pfcrt and pfmdr1 alleles known to confer resistance to PPQ or other antiplasmodial agents could also be cross-resistant to ZY19489. Assays included CQ-resistant lines harboring different PfCRT mutations at residues 72-76 as well as PPQ-resistant lines expressing causal PfCRT mutations including C350R, H97Y, F145I, M343L, or G353V18. We extended this screening to naturally occurring or drug-selected pfmdr1 mutants on NF10, NF54, Cam3.II and Dd2 backgrounds (Supplementary Table 1). A750T and S784L are naturally occurring PfMDR1 mutations previously identified in field isolates from Western Cambodia and the Thai-Myanmar border19,20 while the M841I+M924I double mutant was obtained from in vitro drug selection using the piperazine-containing ACT-45184021. The F1072L and S1075I PfMDR1 mutants were obtained from selection with hexahydroquinoline derivatives22.

ZY19489 was consistently potent against all CQ- or PPQ-resistant pfcrt mutant alleles (of diverse geographic origins) expressed in the SE Asian Dd2 parasite background, with mean IC50 values ranging from 7.1 nM (Dd23D7crt) to 13.7 nM (Dd2G353Vcrt; Fig. 3c; Supplementary Table 10). Against South American 7G8 parasites expressing either the CQ-resistant 7G8 or PPQ-resistant 7G8+C350R pfcrt alleles (the 7G87G8crt and PPQ-resistant 7G8C350Rcrt lines, respectively), the ZY19489 mean IC50 was 13.6 nM and 14 nM, respectively, highlighting the equipotency of this compound against mutant pfcrt alleles presently circulating in the field. The sensitivity of mutant PfMDR1 parasites, on the other hand, was more varied. There was no difference in potency against NF54 or NF54M841I+M924Imdr1 parasites (mean IC50s values of 8.3 nM and 9.9 nM, respectively). Similarly, ZY19489 was equipotent against NF10A750Tmdr1 and Cam3.II C580YS784Lmdr1 parasites compared with their NF10 and Cam3.II C580Y parental lines. In contrast, we observed a 3-fold decrease in ZY19489 activity against Dd2F1072Lmdr1 (mean IC50 of 31.3 nM) and Dd2S1075Imdr1 (mean IC50 of 29.6 nM) compared to the parental Dd2-B2 line (mean IC50 of 10.2 nM) (Fig. 3c; Supplementary Table 11).

ZY19489 exhibits a high in vivo barrier to resistance in mice treated in combination with other candidate antimalarials
To assess whether ZY19489-resistant parasites might emerge in vivo following combination treatment, we used a validated mouse model of P. falciparum ABS infection of NOD-scid IL-2Rγnull (NSG) mouse engrafted with human erythrocytes (TADhuMouse®)23. In three independent experiments, humanized NSG mice infected with 3D70087/N9 (a standard P. falciparum strain adapted to grow reproducibly in humanized NSG mice24) were treated for 1 to 4 days with varying doses of ZY19489, the quinoline-carboxamide M571725, the pantothenamide MMV69318326, or ZY19489-based combinations including MMV693183, ferroquine, or M5717 (Table 2; Supplementary Fig. 7).

In the ZY19489 + MMV693183 treatment group, 11 recrudescent parasite lines (10 from various dosing regimens of the combination and 1 from a 3-day MMV693183 monotherapy at 2.5 mg/kg) were culture-adapted for phenotypic screening and WGS. No significant differences in parasite susceptibility to ZY19489 or MMV693183 were observed between the parental 3D70087/N9 strain and any of the recrudescent lines (Table 2). WGS identified genetic changes in only one line, TAD_779, which carried a Y1020H mutation in PF3D7_0825000, encoding a conserved Plasmodium protein of unknown function, and a N636Y mutation in PF3D7_1416100, encoding SEY1, a putative endoplasmic reticulum-shaping protein. Neither mutation was associated with altered compound susceptibility. Notably, no mutations were observed in PF3D7_0627800 encoding the CoA-binding enzyme acetyl-CoA synthetase, a known resistance marker for MMV69318326,27. These findings suggest that recrudescence was likely due to sub-optimal drug exposure rather than acquired resistance to ZY19489 or MMV693183.

Similarly, no significant shifts in IC50 or IC90 values for ZY19489 or ferroquine were observed in lines recrudescing after exposure to 4 × 40 mg/kg ZY19489 alone or combined with one dose of 15 mg/kg ferroquine (Table 2). WGS revealed no consistent mutations across ZY19489- or ZY19489+ferroquine-treated lines, indicating no evidence of acquired resistance to either compound.

In the group treated with either M5717 alone or in combination with ZY19489, seven recrudescent samples were obtained (three from M5717 monotherapy and four from combination regimens). These samples, along with the untreated parental strain, were not available for in vitro phenotypic testing but were genotyped at the PfeEF2 (PF3D7_1451100) locus that encodes P. falciparum translation eukaryotic elongation factor, a known target of M5717. Three lines, TAD_382, TAD_384, and TAD_400, harbored the PfeEF2 Y186N mutation, which confers high-grade M5717 resistance (Table 2)25,28. Two of these (TAD_382 and TAD_384) were from M5717 monotherapies; the third (TAD_400) emerged following 20 mg/kg ZY19489 + 20 mg/kg M5717 combination treatment. WGS was performed on five (TAD_382 and all four from the combination treatment) to identify additional mutations. The PfeEF2 Y186N mutation was confirmed in TAD_382 and TAD_400, consistent with genotyping results from amplicon sequencing. Three additional missense mutations were identified in TAD_382, including a P490L substitution in PF3D7_1032000 (ribosome maturation factor RimM), which was present in all five M5717-exposed lines (Table 2). However, no novel SNPs or CNVs were observed in samples exclusively exposed to the ZY19489+M5717 combination, confirming that PfeEF2 Y186N likely arose from M5717 pressure.

To assess potential cross-resistance between M5717 and ZY19489, we tested three M5717-resistant lines (TAD_470, TAD_462, and TAD_464), which harbored distinct PfeEF2 mutations (I183M, P754S, and I182T, respectively), for susceptibility to ZY19489. No significant differences in IC50 or IC90 values were observed compared with the drug-sensitive parental line (TAD_022), indicating that PfeEF2-mediated M5717 resistance does not affect parasite sensitivity to ZY19489 (Supplementary Table 12). M5717 susceptibility testing confirmed the previously reported resistance phenotypes of these lines, with IC50 shifts of 4.5-fold (TAD_470), 151-fold (TAD_462), and ~260-fold (TAD_464) relative to TAD_022 (Supplementary Table 12)28. Together, these data underscore the high in vivo barrier to resistance against ZY19489.

ZY19489 in vitro ABS potency is maximal against rings and schizonts
We assessed the timing of ZY19489’s anti-ABS action by measuring its potency against synchronized rings, trophozoites, and schizonts that had been treated separately for 12 hours29. For unbiased interpretation of optimal drug activity, we used the drug-sensitive 3D7 strain that expresses wildtype isoforms of most of the proteins associated with antimalarial drug resistance. We included DHA, CQ and the 2-aminopyridine MMV390048 (a phosphatidylinositol 4-kinase inhibitor) as control compounds with established peak activity against early rings, trophozoites and schizonts, respectively29. These compounds yielded mean IC50 values of 1.7 nM, 12.3 nM and 24.4 nM, respectively (Supplementary Table 13). ZY19489 exerted optimal activity against rings and schizonts (mean IC50 values of 9 nM and 12.8 nM, respectively, compared to 28.3 nM against trophozoites; Fig. 4a; Supplementary Table 13), a profile distinct from our control compounds.

ZY19489 inhibition of parasite growth is associated with hemoglobin catabolism inhibition
Our observation of swollen DVs in ZY19489-resistant parasites (Fig. 3a) was reminiscent of a similar morphological feature in PPQ-resistant parasites that was previously associated with defective hemoglobin (Hb) breakdown30. To test whether the ZY19489 MoA might also be linked to Hb catabolism, we incubated synchronized 3D7 and Dd2 trophozoites with 10× IC50 of ZY19489 for 2.5 hours and compared the ensuing metabolic responses with no-drug controls31. Parasites treated with the reference mitochondrial inhibitor ATQ exhibited significant accumulation of N-carbamoyl-aspartate and dihydroorotate, two pyrimidine biosynthetic intermediates upstream of its target, P. falciparum dihydroorotate dehydrogenase (PfDHODH; PF3D7_0603300; Supplementary Table 14; Supplementary Fig. 8). These changes were accompanied by reduced levels of intracellular uridine monophosphate (UMP), diphosphate (UDP) and triphosphate (UTP), consistent with ATQ-mediated inhibition of the mitochondrial electron transport chain complex and concomitant loss of PfDHODH activity32. ZY19489-treated 3D7 and Dd2 parasites, on the other hand, showed significant reductions in short Hb-derived peptides, commonly linked with inhibition of Hb endocytosis and/or catabolism (Fig. 4b; Supplementary Table 14). These short peptides ranged from 2 to 6 amino acids in length, had isoelectric points of four to six, and were negatively charged at pH 5.5 or pH 7.4 (reflecting conditions in the DV lumen or cytosol, respectively) (Supplementary Table 15). We also observed significantly elevated levels of deoxyuridine monophosphate (dUMP), a crucial precursor in the production of thymidine required for DNA replication (Supplementary Table 14; Supplementary Fig. 8). Separately, we explored whether ZY19489 might target cGMP dependent protein kinase (PfPKG; PF3D7_1436600), given this compound’s structural similarity with the aminopyrimidine PfPKG inhibitor ML-1033. ZY19489 was found to be inactive against recombinant purified PfPKG (Supplementary Fig. 9).

Evidence that ZY19489 inhibits hemoglobin breakdown but not heme detoxification
Hb degradation is initiated by hemoglobinases and leads to generation of amino acids for growth, with ‘free’ heme released as a toxic byproduct. In its labile form, ‘free’ heme can cause oxidative damage to the parasite and is promptly sequestered into inert hemozoin (Hz) in a process that may be accelerated by P. falciparum heme detoxification protein (PfHDP; PF3D7_1446800) in complex with hemoglobinases including falcipains34. To understand whether ZY19489 inhibits this phase of Hb catabolism, we tested for possible interactions with PfHDP and/or falcipain 2. We generated an aTc-regulatable PfHDP cKD line (designated herein as Dd2HDP cKD) and observed no difference in ZY19489 susceptibility at various aTc concentrations compared to control lines (Supplementary Fig. 10). These data argue against any link between ZY19489 anti-ABS activity and inhibition of PfHDP. On the other hand, quinine displayed a significant ∼2-fold IC50 shift in PfHDP cKD parasites under no aTc relative to parasites cultured at high aTc concentration. We found no significant difference in ZY19489 activity against 3D7 falcipain 2A or 2B knockout lines35 compared to the parental 3D7 line (Supplementary Fig. 11). As a control, we included E64d that is a membrane-permeable derivative of the epoxysuccinate inhibitor, E64, whose broad-spectrum antiprotease activity includes falcipain inhibition35. E64d exhibited high IC50 values against parasites lacking falcipain 2A or 2B (Supplementary Fig. 11).

We then tested for direct interference of Hz formation by first assessing inhibition of the conversion of hematin to β-hematin in a cell-free set-up36. ZY19489 failed to block β-hematin formation (IC50 >500 μM) in contrast to CQ and amodiaquine that were potent inhibitors in this cell-free assay with mean IC50 values of 19 μM and 6 μM, respectively (Fig. 4c). To examine whether the effect of ZY19489 on the pathway was only discernible in live parasites, we then tested for a concentration-dependent effect of the drug on three heme species, namely Hb (undigested Hb species), ‘free’ heme (potentially oxidative labile species liberated from Hb proteolysis), and Hz (inert crystalline species) fractioned from mature trophozoites37. Inhibitors that complex with heme customarily show a concentration-dependent decrease in Hz levels accompanied by a corresponding increase in ‘free’ heme, which correlates with parasite killing37.

In CQ-treated NF54 parasites, the mean ± SEM of ‘free’ heme iron (Fe) present in femtogram per cell (fg/cell) was significantly higher in the highest treatment concentration compared to the untreated control (8.3 ± 0.3 fg/cell vs 5.5 ± 0.2 fg/cell; p < 0.05; Mann-Whitney U tests) (Fig. 4d; Supplementary Table 16). This increase in ‘free’ heme was concentration-dependent and corresponded with a significant decrease in Hz (Fig. 4d; Supplementary Table 16). To interpret the physiological relevance of these observations we superimposed 72-hour parasite growth inhibition curves on the ‘free’ heme levels and observed a correlation between heme concentrations and parasite growth inhibition (Fig. 4e). These data suggest that CQ-mediated parasite death was attributable to the buildup of toxic heme. We also observed modest but significant elevation in Hb levels that plateaued between CQ concentrations corresponding to 1.5× and 2.5× IC50 (Fig. 4f; Supplementary Table 16).

In contrast, the levels of ‘free’ heme in ZY19489-treated parasites showed no concentration-dependent pattern (5.6 ± 0.2 fg/cell in untreated control vs 6.0 ± 0.1 fg/cell in parasites treated with 2.5× IC50) (Fig. 4g). Decreasing amounts of Hz in ZY19489-treated parasites were only observed at higher concentrations and showed no association with ‘free’ heme levels (Fig. 4g; Supplementary Table 16). Additionally, levels of ‘free’ heme did not correlate with 72-hour parasite survival (Fig. 4h, 4i). Similar lack of inhibition was observed in parasites treated with the antifolate, pyrimethamine (Supplementary Table 16). Nonetheless, treatment with ZY19489 caused significant increases in Hb levels at 1.5× and 2.0× IC50 concentrations but not at the more inhibitory concentration of 2.5× IC50 (Fig. 4i), suggesting some activity of this compound during earlier steps of Hb catabolism in a concentration- and stage-specific manner.

Methods
Inclusion and Ethics Statements
The studies were approved by The Art of Discovery Institutional Animal Care and Use Committee (TAD-IACUC), certified by the Biscay County Government (Bizkaiko Foru Aldundia, Basque Country, Spain) to evaluate animal research projects from Spanish institutions according to point 43.3 from Royal Decree 53/2013, from the 1st of February (BOE-A-2013−1337). All experiments were carried out in accordance with European Directive 2010/63/EU. Animal experiment results were reported following ARRIVE guidelines (https://www.nc3rs.org.uk/arrive-guidelines), except for disclosure of business trade confidential information. The human biological samples were sourced ethically, and their research use was in accord with the terms of the informed consents. For routine P. falciparum culturing, RBCs were purchased from the Interstate Blood Bank (Memphis, TN) as pooled, de-identified, anonymized blood that was washed to remove any residual leukocytes. Approval for this protocol (AAAU3761) was provided on 28 October 2022 by the Columbia University Institutional Review Board, which deemed this work to be Not Human Subjects Research under 45 CFR 46.

Generation of P. falciparum V-type ATPase subunit D conditional knockdown parasite lines
This cKD line was generated by fusing the coding sequence and non-coding RNA aptamer sequences in the 5’ and 3’ untranslated regions (UTRs), permitting translational regulation using the TetR-DOZI system59,60. Gene editing was achieved by CRISPR/SpCas9 using the linear pSN054 vector that contains cloning sites for the left homology region (LHR) and the right homology region (RHR) as well a gene-specific guide RNA under control of the T7 promoter. Cloning into the pSN054 donor vector was carried out as described previously59,60. The vector includes preinstalled V5-2HA epitope tags, a 10 tandem array of TetR aptamers upstream of an Hsp86 3’UTR, and a multicistronic cassette for expression of TetR-DOZI (regulation), blasticidin S-deaminase (the selection marker) and a Renilla luciferase reporter. The LHR and recoded region were installed in-frame with tandem V5-2-hemagglutinin (HA) tags to generate a C-terminal epitope-tagged V1-A coding sequence, upstream of the regulatory aptamer array. The final construct was sequence-verified and further confirmed by restriction digests. Transfection into Cas9- and T7 RNA polymerase-expressing NF54 parasites was carried out by pre-loading erythrocytes with the donor vector61. Parasite culture was maintained continuously in 500 nM anhydrotetracycline (aTc, Sigma-Aldrich 37919). Drug selection with 2.5 g/mL of Blasticidin hydrochloride (RPI Corp B12150-0.1) was initiated four days after transfection. Cultures were monitored by Giemsa smears and Renilla luciferase measurements.

Verification of conditional knockdown cKD strategy
To assess regulation of the PfV1-D protein expression, cKD parasites were cultured in the presence (500 nM) or absence of aTc. Protein samples were extracted after 72 hours via saponin lysis and resuspended in parasite lysis buffer that consists of 4% SDS and 0.5% Triton X-114 in PBS. Proteins were separated on a Mini-PROTEAN® TGX™ Precast Gels (4-15% gradient) in Tris-glycine buffer, transferred to a polyvinylidene fluoride (PVDF) membrane using the Mini Trans-Blot Electrophoretic Transfer Cell system according to the manufacturer’s instructions, and blocked with 100 mg/mL skim milk in TBS/PTween. Membrane-bound proteins were probed with mouse anti-HA (1:3000; Sigma H3663) and rabbit anti-GAPDH (1:5000; Abcam AB9485) primary antibodies, and anti-mouse (1:5000; Thermo Fisher Scientific 62-6520) and anti-rabbit (1:5000; Cell signaling 7074S) horseradish peroxidase (HRP)-conjugated secondary antibodies. Following incubation in SuperSignal® West Pico Chemiluminescent substrate (Thermo Fisher Scientific PI34080), protein blots were imaged and analyzed using the ChemiDoc™ MP System and Image Lab 5.2.0 (Bio-Rad).

Assessment of parasite growth and compound susceptibility assays using cKD lines
Assessment of parasite proliferation rate upon protein perturbations was carried out using luminescence as a readout of growth. Synchronous ring-stage parasites cultured in the presence (50 nM) or absence of aTc were set up in triplicate wells in 96-well U-bottom plates (Corning 62406-121). Luminescence signals were quantified at 0 and 72 hours after incubation using the Renilla-Glo(R) Luciferase Assay System (Promega E2750) and the GloMax® Discover Multimode Microplate Reader (Promega). The luminescence values in the knockdown condition (6 nM aTc) were normalized to 50 nM aTc-treated (100% growth) and 200 nM DHA-treated (no growth) samples. Results were visualized on bar graphs using GraphPad Prism (version 10).

To assess compound activity, ZY19489 and bafilomycin A (a V-type ATPase inhibitor) were serially diluted in complete tissue culture medium. Synchronous ring-stage V1-D cKD and control parasites were maintained in high (50 nM) and low (6 nM) aTc or no aTc (for the control line) and were distributed into the drug plate. DMSO and 200 nM DHA treatments served as reference controls. Luminescence was measured after 72 hours as described above on four different occasions and IC50 values were obtained from dose-response curves using GraphPad Prism.

P. falciparum resistance selections with ZY19489
In vitro selection was conducted using a pulsing (intermittent drug pressure) method on Dd2-Polδ (Supplementary Fig. 2). This is a Dd2 parasite line with defective proof-reading activity following CRISPR/Cas9-based replacement of D308 and E310 - the two conserved catalytic residues of P. falciparum DNA polymerase δ, with alanine. This impaired proof-reading activity endows Dd2-Polδ with a ~5 to 10-fold higher mutability rate in the presence of a drug compared to Dd212. Briefly, 109 parasites in triplicate flasks were exposed to 10× ZY19489 (100 nM) for 11 days until all parasites cleared. The cultures were thereafter passaged in drug-free media with weekly introduction of fresh red blood cells (RBCs) until day 34 when parasites reappeared in two flasks. In vitro drug sensitivity testing confirmed these recrudescent parasites were still susceptible to ZY19489 and subsequently pressured again with 100 nM ZY19489 for another 16 days. During this period, parasites in the third flask also recrudesced but were also exposed to drug since there was no shift in their drug response. Upon clearance on day 50, cultures were again passaged in drug-free media until day 71 when they reappeared. All flasks still had drug-sensitive Dd2-Polδ parasites and were exposed to another round of 100 nM ZY19489 for 19 days until all parasites cleared. Drug pressure was then removed until day 112 when only one flask recrudesced. We further pressured the parasites from this flask for another 12 days before removing drug. On day 136, recrudescent parasites appeared in this flask, which upon testing showed a decrease in drug sensitivity compared to untreated Dd2-Polδ parasites. No parasites were recovered from the other two flasks after day 160 and those selections were halted. Four clones were obtained from the resistant flask by limiting dilution. IC50 values were determined in 72-hour drug assays, with parasitemia measured by flow cytometry of cultures stained with SYBR Green and MitoTracker Deep Red (Life Technologies), with ~10,000 cells analyzed per well using an iQue Plus (Sartorius).

72-hour in vitro drug susceptibility assay
Susceptibility to all drugs tested in this study was measured by incubating asynchronous, ABS parasites plated at 0.2% to 0.3% parasitemia (or 0.8% for the poorly growing ZY19489-resistant lines harboring the pfcrt N246H allele) and 1% hematocrit at 37° C in 96-well plates. The assays were set up across a range of drug concentrations with two-fold dilutions. For the experiments testing for susceptibility of recrudescent parasite lines that emerged in humanized NSG mice to ZY19489, M5717, MMV693183, or ferroquine, parasites were cultured at 2% hematocrit in human O+ or A+ RBCs in RPMI-1640 media, supplemented with 25 mM HEPES (Fisher), 50 mg/L hypoxanthine (Sigma Aldrich), 2 mM L-glutamine (Cambridge Isotope Laboratories, Inc.), 0.21% sodium bicarbonate (Sigma Aldrich), 0.5% (w/v) Albumax (Invitrogen), 8% filtered, heat-inactivated, pooled off-the-clot AB+ human serum (Interstate Blood Bank or New York Blood Centre), and 10 μg/mL gentamicin (Fisher). The parental P. falciparum Pf3D70087/N9 strain that has been adapted to proliferate in this humanized NSG mouse model was cultured in parallel as the drug-sensitive parental control strain. All compound susceptibility assays for the recrudescent lines and parental control strain were performed using 8% serum-containing medium. Parasite growth in each well was assessed after 72 hours using flow cytometry of cultures stained with SYBR Green I and MitoTracker Deep Red (Supplementary Fig. S12). IC50 and IC90 values were determined by nonlinear regression analysis (GraphPad Prism 7). Unless otherwise stated, assays were repeated on four independent occasions with technical duplicates. Statistical comparisons were made using Mann–Whitney U tests.

Whole genome sequence analysis
DNA from infected RBCs was extracted using the QiAmp DNA Blood Mini kit (Qiagen). The samples were then pooled and sequenced on an Illumina MiSeq flow cell to obtain 300 bp paired end reads. The sequence data were aligned to the Pf3D7 reference genome (PlasmoDB 48_Pfalciparum3D7; https://plasmodb.org/plasmo/app/downloads/release-48/Pfalciparum3D7/fasta/) using the Burrows-Wheeler Alignment (BWA version 0.7.17). PCR duplicates and unmapped reads were filtered using Samtools (version 1.13) and Picard MarkDuplicates (GATK version 4.2.2). The reads were realigned around indels using GATK RealignerTargetCreator and base quality scores were recalibrated using GATK Table-Recalibration. GATK HaplotypeCaller (version 3.8) was used to identify all possible variants in clones which were filtered based on quality scores (variant quality as function of depth QD > 1.5, mapping quality > 40) and read depth (depth of read > 5) to obtain high-quality single nucleotide polymorphisms that were annotated using snpEFF. The list of variants from resistant clones were compared against the Dd2-Polδ parental clone to obtain homozygous single nucleotide polymorphisms present exclusively in the resistant clones. Integrated Genome Viewer was used to confirm polymorphisms present in resistant clones. BIC-Seq version 1.1.2 was used to discover copy number variants against the parental strain using the Bayesian statistical model.

Targeted sequencing of P. falciparum eukaryotic elongation factor 2 and analysis of mutations
The gene encoding PfeEF2 (PF3D7_1451100), which mediates resistance to M5717, was PCR-amplified and sequenced as previously described28. Sequences were aligned to wildtype PfeEF2 from the 3D7 genome reference strain and analyzed using Geneious 9.1.8. Electropherograms were visually inspected to identify mixed sequences indicating multiple subpopulations.

Gene editing using zinc-finger nucleases and CRISPR/Cas9 strategies
pfcrt was edited using a customized two-plasmid zinc-finger nuclease approach that replaces the endogenous allele with a recombinant allele containing the mutations of interest14. Mutations were introduced into pfcrtDd2, pfcrtGB4 and pfcrtFCB donor plasmids by site-directed mutagenesis. The D233N mutation was inserted through CRISPR/Cas9 editing, using the previously described “all-in-one” method62, with a 610 bp repair template. Two gRNAs were selected based on their selectivity and their proximity to D233, and gRNA primers were annealed and cloned into the pDC2 vector at the BbsI restriction site as previously described62. Repair templates containing the D233N mutation and silent shield mutations were synthesized and cloned into the pDC2 vector by In-Fusion cloning at the EcoRI/AatII sites. Dd2 P. falciparum ABS parasites were cultured in RBCs in RPMI 1640-based culture media containing 0.5% w/v Albumax. Parasites were cultured at 37° C in an airtight chamber with 5% O2 / 5% CO2 / 90% N2. Ring-stage parasites were electroporated with 50-100 µg of purified plasmid DNA in Cytomix. The donor plasmids carrying the human dhfr marker were selected with 2.5 nM WR99210 (Jacobus Pharmaceuticals), and the zinc-finger nuclease plasmid harboring blasticidin S-deaminase was selected with a 6-day pulse of 2 µg/ml blastidicin hydrochloride. Editing was confirmed using PCR and Sanger sequencing, and clones were obtained by limiting dilution. The primer codes and sequences are provided in Supplementary Table 17).

Quantitative trait locus (QTL) analysis and mapping
The generation of the genetic cross between NF54 and RF7, cloning and WGS analysis of the progeny were conducted as described previously16. Unique recombinants were identified using WGS. The R package (version 2) was used to map QTL peaks. To identify significant QTLs for each drug response phenotype, 1,000 permutations of phenotypic data (IC50) were performed to obtain a distribution of maximum log of the odds (LOD) scores. These scores were then used to calculate the LOD threshold at 95% probability. Fine mapping of the QTL segments was performed using Bayesian interval mapping at a 95% confidence level.

Modeling PfCRT in the open-to-DV and open-to-cytosol conformations
The cryo-EM structure of PfCRT strain 7G8 (PDB: 6UKJ)17 was used as the reference protein template. To include the missing residues (114-122), we employed AlphaFold2, with the original PDB structure serving as the template for modeling. The same approach was used for the N246H mutant, substituting asparagine at position 246 for histidine in the primary sequence. The resulting 7G8 and 7G8+N246H structures were highly similar, with a root-mean-square deviation (RMSD) of less than 1 Å across all residues. The open-to-cytosol conformation of PfCRT was obtained from previous work17. The missing segments and residues (81-88, 110-125, 148-154, 169-180, 198-214, 235-251, 269-322, 336-344, 364-381) were modeled using SwissModel63, except for the 277-315 region, which was truncated due to the presence of two disulfide bridges. The final model exhibited an RMSD of 0.9 Å, calculated over 143 pruned atom pairs, when compared to the open-to-cytosol structure from Berger and colleagues64, indicating a high degree of structural similarity between the two models. The same procedure was employed to model the mutated 7G8+N246H isoform.

Molecular dynamics simulations
The protonation states of the amino acids in all models were assigned using PDB2PQR65, with pH values of 5.5 for the open-to-DV conformation and 7.0 for the open-to-cytosol conformation. The ionization state of ZY19489 was checked using MolGpKa66. To obtain an initial pose of the ligand in each system, molecular docking was performed using GOLD, defining a 12 Å radius around the alpha carbon of residue 246 as the binding site. The initial 3D structure of the ligand was generated with Omega67. The 7G8 and 7G8+N246H docking poses obtained for the open-to-DV and open-to-cytosol systems were then embedded into an explicit lipid bilayer using CHARMM-GUI Membrane Builder68. The membrane composition included 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE) and cholesterol (CHL) in a 4:3:3 ratio, based on the approach from Berger and colleagues64. A 150 mM NaCl concentration was added to neutralize the system. The systems were solvated with TIP3P (transferable intermolecular potential with 3 points) water molecules, resulting in approximately 131k atoms per system.

For the force fields, ff14SB was used for the protein, Lipid21 for the lipids, and GAFF2 for the ligand, with atomic charges derived using AM1-BCC. All-atom molecular dynamics simulations were performed in Amber24 using pmemd.cuda. The systems were initially energy-minimized using the steepest descent method for 10,000 steps. For equilibration, we followed the six-step protocol from CharmmGUI, which includes two NVT simulations with the Langevin thermostat and four NPT simulations at 303.15 K. Finally, a 300 ns production run was performed in triplicate for each system, totaling 3.6 µs of simulation time. To analyze the results from the molecular dynamics simulations, we used cpptraj and MDAnalysis69. Representative conformations of each system were obtained through k-means clustering, generating 10 clusters over 500 iterations, based on the ligand-protein RMSD for heavy atoms only.

Drug transport assays
PfCRT 7G8 and 7G8+F145I variants were purified and inhibition of CQ and PPQ uptake was measured using PfCRT-containing proteoliposomes as described previously17 with the following modifications. Purified PfCRT variants were reconstituted in preformed liposomes made of E. coli total lipids:cholesteryl hemisuccinate in a ratio of 97:3 (w/w) and the lumen of the proteoliposomes was composed of 100 mM KPi, pH 7.5 and 2 mM β-mercaptoethanol. Uptake of 100 nM [3H]CQ (1 Ci/mmol) or 100 nM [3H]PPQ (1 Ci/mmol) was performed by diluting PfCRT-containing proteoliposomes (30 ng of PfCRT per reaction) in an uptake buffer containing 100 mM Tris/MES, pH 5.5, in the presence or absence of 1 μM of the test compounds. In addition, 1 μM valinomycin was added to the reaction to generate a K+ diffusion potential-driven membrane potential (ΔΨ). Reactions were stopped after 30 seconds by the addition of ice-cold 100 mM KPi, pH 6.0, and 100 mM LiCl, and filtered through 0.45 μm nitrocellulose filters (Millipore). Filters were dried and incubated in a scintillation cocktail, and the radioactivity captured on the filters was counted in a Hidex SL300 scintillation counter. The efficiency of detection was calculated with a standard curve of known concentrations of each radiolabeled compound, and this was used to transform decays per minute (dpm) into pmol. The nonspecific interaction of each compound with nitrocellulose filters was determined by measuring mock uptake in the absence of liposomes or proteoliposomes. These values (determined for each experiment) were used to calculate background uptake in liposomes or proteoliposomes. Drug-specific uptake was determined by subtracting the time-dependent accumulation of the tested compounds in control liposomes (lacking PfCRT) from that measured in PfCRT-containing proteoliposomes17.

In vitro growth rate and fitness assay
We measured relative growth rates of the pfcrt mutant parasite lines by tracking the growth of the parasites and their isogenic parents over eight generations. Briefly, all parasites were seeded at 0.2% starting parasitemia and 2% hematocrit on day 0 with fresh RBCs. The parasites were then allowed to grow undisturbed for the first 3 days, after which parasitemia was assessed by flow cytometry. Media was changed without disturbing the settled culture and culturing was continued until day 4 when parasitemia was measured again. The parasite cultures were cut back to 0.2% parasitemia on day 4, with the dilution ratio recorded. Parasitemia measurements were subsequently done on day 6, 8, 10, 12, 14 and 16, with dilutions made back to 0.2% parasitemia for any cultures that had expanded to >2% parasitemia. These experiments were conducted on three different occasions with technical duplicates. Parasite growth rate was computed as the square root of parasitemia on the day of dilution divided by 0.2% (starting parasitemia) for days 4, 8, 12 and 16. Mean growth rate was obtained from the average of this analysis over three biological repeats. The mean growth rate for the mutant lines was then computed as a proportion of the isogenic parent. The mean fitness cost associated with PfCRT N246H mutation was calculated relative to its isogenic parent using the following formula:
% Mean FC pfcrtN246H = 100(1- (MGR mutant ÷ MGR parent))
where FC pfcrtN246H is the fitness cost associated with the N246H mutant relative to the parental line and MGR is the mean growth rate per two-day cycle.

Stage specific profiling of antimalarial activity
Determination of the stage-specific activity of ZY19489 and control compounds was conducted as reported elsewhere with minor modifications29. Briefly, highly synchronized early ring-stage parasites were obtained via 70% Percoll gradient purification of mature schizonts followed 5 hours later by sorbitol treatment of young rings upon reinvasion of RBCs. The 40 - 42 hours ABS cycle duration of 3D7 was exploited to allow for three different parasite incubation phases corresponding to rings (0 - 12 hours), trophozoites (18 - 30 hours) and schizonts (30 - 42 hours). Culture synchronicity was confirmed by microscopic observation. After each incubation period (12 hours), drugs were removed from the culture through three rounds of washing using pre-warmed media followed by plate change. All pipetting and washing steps were performed using a TECAN Freedom Evo 100 to increase throughput and accuracy and avoid cross-contamination of wells. Each set of plates per timepoint was placed in a separate humidified chamber to avoid any delay in growth rate due to temperature variations. For the three different stages, growth inhibition was assessed at the 60-hour time point, when parasites had expanded, reinvaded new RBCs, and developed into trophozoites to allow for accurate quantification by flow cytometry. Parasite survival for both the 72 hours and stage-specific 12-hour exposures was assessed by SYBR Green and MitoTracker Deep Red FM staining (Life Technologies) and subsequent flow-cytometric analysis (IntelliCyt iQue3, Sartorius). IC50 values were derived from growth inhibition data using nonlinear regression (Prism 7, GraphPad) following six independent biological repeats with two technical replicates.

In vivo efficacy studies in the TADhuMouse-Pfalc model
The in vivo efficacy studies were performed in a standardized commercial model of P. falciparum infection in NOD-SCID IL-2Rγnull (NSG) mice engrafted with human erythrocytes (TADhuMouse®-Pfalc), as described previously23. Briefly, 22 - 28 g female NSG mice (Charles River, France) were engrafted with human erythrocytes obtained from Centro Vasco de Transfusiones y Tejidos Humanos (Galdakao, Basque Country, Spain), Centro de Transfusiones de la Comunidad de Castilla y León, (Valladolid, Spain), Centro de Transfusión de la Comunindad de Madrid, (Madrid, Spain) and the Banc de Sang I Teixits (Barcelona, Spain). The erythrocytes were injected by intraperitoneal daily inoculation of 1 mL of 50 - 75% hematocrit cell suspensions in RPMI 1640 medium, 25% (v/v) decomplemented human serum, and 3.1 mM hypoxanthine. When the chimerism of human RBCs in peripheral blood was > 40%, TADhuMouse® were infected by intravenous inoculation of via the lateral vein with 0.3 mL of a suspension containing 1.17 × 108 RBCs parasitized with the competent drug-sensitive strain P. falciparum Pf3D70087/N9 prepared from peripheral blood of CO2-euthanized donor mice harboring 5 - 10% parasitemia. After random assignment of mice to its corresponding group, drug treatments started when TADhuMouse® had ∼1.3% patent parasitemia (Day 1) in peripheral blood. Treatments were administered by oral gavage with 20G straight reusable feeding needles (Fine N Science Tools GmbH) at 10 mL·kg −1 unless otherwise stated. The drugs administered in combination were co-formulated as fine homogeneous suspensions.

Parasitemia was measured by flow cytometry in serial 2 μL samples of peripheral blood collected from the tail lateral vein and expressed as the % of parasitized RBCs with respect to the total RBCs in circulation. A qualitative analysis of the effect of treatment on P. falciparum Pf3D70087/N9 was assessed by microscopy with Giemsa-stained blood smears and flow cytometry by staining with TER-119-Phycoerythrine (a marker of murine erythrocytes) and SYTO-16 (nucleic acid dye) and acquisition in an Attune NxT Acoustic Focusing Flow Cytometer (Invitrogen)70.

The concentrations of drugs were measured in samples of peripheral blood (25 μL) taken at different times during dosing, mixed with 25 μL of Milli-Q H2O, and immediately frozen on a thermal block at −80 °C. The frozen samples were stored at −80 °C until analysis. Blood from control humanized mice was used for the preparation of standard curves, calibration, and quality control purposes. The drugs were extracted from 10 μL of lysates obtained by protein precipitation of blood samples using standard liquid-liquid extraction methods. The samples were analyzed by LC-MS/MS for quantification in a Waters Micromass UPLC-TQD (Waters, Man- chester, U.K.). Blood concentration versus time was analyzed by non-compartmental analysis using Phoenix WinNonlin version 8.2 (Certara) or R or Excel (Microsoft), from which exposure-related values (tmax, Cmax, and AUC0−t) were estimated. The clearance of parasitized RBCs from the peripheral blood of mice was assessed by measuring the parasite reduction ratio calculated as the ratio between parasitemia at Day n + 2 divided by parasitemia at Day n for each individual of the study. The parasiticidal effect of drugs in vivo was assessed by measuring the day of recrudescence as the day at which parasitemia after drug treatment reached the % parasitemia at treatment inception71. Drug-treated mice were deemed cured if there was no detectable parasitemia 60 days after infection.

Sample preparation for untargeted LC-MS metabolomics
3D7 and Dd2 P. falciparum parasite strains were cultured under standard conditions at 2% hematocrit and tested for Mycoplasma using a MycoAlert PLUS Mycoplasma Detection Kit (Lonza). Excluding the cycle preceding sample collection, the Mycoplasma-free parasites were sorbitol-synchronized in each generation for at least two generations and subsequently cultivated to the trophozoite stage with a parasitemia of ~10%. Infected RBCs were then magnetically purified using MACS CS columns and VarioMacs magnets. Metabolite extracts were obtained from the purified infected RBCs treated for 2.5 hours with concentrations corresponding to 10× IC50 of the atovaquone or ZY19489, resulting in a 1 mL extraction composed of 90% MeOH. For each strain, one biological repeat extraction was run in technical triplicate consistent with how previous metabolomics dataset have been obtained for 3D7 and Dd2 strains of P. falciparum in our laboratory31. Extracts were spiked with a 0.5 µM [13C4,15N1]-labeled aspartate standard31, vortexed, and centrifuged for 10 minutes at 4°C at 21, 130× g. The supernatant was then transferred into 1.5 mL tubes, dried under a stream of nitrogen, and stored at -80°C.

Metabolomic profiling of drug-treated parasites via mass spectrometry
Upon thawing on ice, extracts were resuspended in ice-cold HPLC-grade water containing 1µM chlorpropamide to reach a concentration of 106 cells/µL. After vortexing and centrifugation for 10 minutes at 4°C at 21, 130× g , supernatants were transferred into 800 µL CRIMP vials in preparation for mass spectrometry. Samples were fractionated using an XSelect HSS T3 2.5 µm C18 Waters column with a 25-minute gradient of 3% aqueous methanol, 15 mM acetic acid, and 10 mM tributylamine and were processed in negative ionization mode on a Thermo Exactive Plus orbitrap.

Data acquired from the mass spectrometry were imported into the El-MSAVEN software package for peak picking72 and were standardized using the [13C4,15N1] aspartate standard. The metabolites were confirmed by comparing their retention times with those of pure molecular standards run on the same mass spectrometer. Fold changes were computed and uploaded onto Rstudio (http://www.rstudio.com/) employing the Hyperspec and Suprahex packages73. Metabolic profiles, as represented by log2 fold changes, were transferred onto a hexagonal map featuring 113 metabolites.

Inhibition of recombinant P. falciparum cGMP-dependent protein kinase (PfPKG)
Full length PfPKG (PF3D7_1436600) was expressed in OverExpress™ C41(DE3) pLysS Chemically Competent Cells (Sigma Aldrich, CMC0018) using established methodology33,74. Briefly, the N-terminal His-tagged recombinant PfPKG protein was purified using a TALON column (GE Healthcare). The final buffer composition of purified protein was 25 mM HEPES pH 7.5, 20 mM NaCl, 120 mM KCl, and 5% glycerol. Purified protein was concentrated using a 15-mL Amicon Ultra 50 KDa MWCO concentrator (Merck Millipore, UFC905024). PfPKG IC50 assays were performed based on previously described methods using the ADP-Glo Kinase Assay (Promega) to measure ADP formation75. Briefly, 3-fold serial dilutions of each inhibitor were prepared in 100% DMSO, and inhibitors were subsequently diluted into assay buffer (25 mM HEPES pH 7.4, 0.1 mg/mL BSA, 0.01% (v/v) Triton-X 100, 20 mM MgCl2, 2 mM DTT, 10 μM cGMP) to 10× the final required concentration. 0.5 μL of each inhibitor dilution was transferred into a white 384-well plate (Greiner 781075). 4.5 µL of a mix containing ATP, GRTGRRNSI-NH2 and PfPKG in assay buffer was then added to each well. The final 5 μL kinase reaction contained 3 nM PfPKG protein, 10 μM ATP, 20 μM GRTGRRNSI-NH2, 1% (v/v) DMSO and inhibitor in assay buffer. Reactions were incubated for 30 minutes at 22°C.

ADP formation was measured using the ADP-Glo Kinase Kit (Promega). Briefly, 5 μL ADP-Glo reagent was added to each well and incubated for 30 minutes at 22°C to deplete the remaining ATP. 10 μL of Kinase Detection Reagent was then added and the reaction incubated for a further 30 minutes at 22°C. The plate was sealed with an adhesive foil seal for all incubation steps. Luminescent signal was measured using a Spectromax Plate Reader. Data were normalized based on the 100% activity controls (1% DMSO only) and the 100% inhibition controls (10 μM kinase inhibitor Staurosporine). Mean IC50 values were calculated from N, n = 3,4 independent experiments.

Generation and drug susceptibility testing of P. falciparum heme detoxification protein cKD lines
PfHDP cKD parasites were generated using a CRISPR/Cas9-based TetR-DOZI aptamer system60. Briefly, the left homology region fused with recodonized 3’ end of PfHDP and right homology region as well as guide sequence were cloned into a pSN054 donor plasmid containing a 3HA epitope tag at the 3’ end of the coding sequence. The final construct together with the pDC2-cam-coSpCas9-U6-gRNA-hDHFR plasmid carrying the guide sequence was transfected into Dd2 parasites and maintained in 500 nM aTc and 2.5 g/mL blasticidin hydrochloride. Edited parasites were confirmed by site-specific integration PCR.

In vitro drug sensitivities of ABS parasites were determined as described above with minor modifications. Asynchronous Dd2 and PfHDP cKD parasite lines were cultured in the presence of 500 nM aTc and then washed to remove aTc. Asynchronous ABS parasites were plated at 0.3% parasitemia and 1% hematocrit in 96-well plates and were incubated with a ten-point, three-fold range of drug concentrations in duplicates with either 30 nM, 3 nM and 0 nM aTc. Plates were incubated at 37°C for 72 hours and parasite survival was assessed by flow cytometry using SYBR Green (Invitrogen) and MitoTracker Deep Red FM (Life Technologies) as nuclear stain and vital dyes, respectively. IC50 and IC90 values were derived from growth inhibition data using nonlinear regression or linear interpolation (Prism 10, GraphPad) as means ± SEM from four independent biological repeats with two technical replicates.

Inhibition of P. falciparum falcipain 2A and 2B knockout parasites
Falcipain knockout lines were generated as described previously35. Susceptibility to ZY19489 and E64d was measured against the 3D7-A10 and 3D7 FP2A/B KO lines by incubating asynchronous, ABS parasites plated at 0.3% (or 0.5% for 3D7 FP2A/B KO) parasitemia and 1% hematocrit at 37°C in 96-well plates. The assays were set up across a range of drug concentrations with two-fold dilutions. Parasite growth in each well was assessed after 72 hours using flow cytometric analysis of cultures stained with SYBR Green I and MitoTracker Deep Red. IC50 and IC90 values were determined by nonlinear regression analysis. Unless otherwise stated, assays were repeated on four independent occasions in technical duplicates and statistical comparisons were made using Mann-Whitney U tests.

In vitro cell-free beta-hematin inhibition assay
Inhibition of lipid-mediated hemozoin formation was quantified by measuring the formation of β-hematin dimers from hemin chloride, in a detergent-mediated assay that substitutes neutral lipids for the commercially available lipophilic detergent Nonidet P-4036. Unreacted hematin was detected through the formation of bis-pyridyl-Fe(III)PPIX complex which absorbs at a wavelength of 405 nm.

Compound stocks (20 mM) of ZY19489, amodiaquine and pyrimethamine were prepared in DMSO, while CQ was prepared in water. These stocks were then diluted to 2 mM with a water/NP40 detergent solution, resulting in a final solution of compound in 61.1 mM NP40/10% DMSO. Serial dilutions into this same buffer were then made for each compound. A 25 mM hemin stock solution was prepared by sonicating hemin in DMSO for one minute and then suspending 179 μL of this stock in a 1 M acetate buffer (20 mL, pH 4.8). The homogenous suspension was then added to the wells to give final buffer and hemin concentrations of 0.5 M and 100 mM, respectively. Plates were covered and incubated at 37°C for 5 hours to enable -hematin formation. A solution of 50% (v/v) pyridine, 30% (v/v) H2O, 20% (v/v) acetone and 2 M HEPES buffer (pH 7.4) was prepared and 32 μL added to each well to give a final pyridine concentration of 5% (v/v). Acetone (60 μL) was then added to each well to assist with -hematin dispersion. The UV-vis absorbance of the plate wells was read on a SpectraMax P340 plate reader. Sigmoidal concentration-response curves were fitted to the absorbance data using GraphPad Prism to obtain an IC50 value for each compound.

Cellular heme fractionation assay
The IC50 values used here for ZY19489, CQ and pyrimethamine (PYR) were obtained from drug susceptibility experiments using the 72-hour SYBR Green assay described above. The heme profiles of NF54 parasites treated separately with the three antimalarials were determined as described previously37. Briefly, NF54 cultures were synchronized at 48-hour intervals with 5% (w/v) sorbitol, and ring-stage parasites at 5% parasitemia and 2% hematocrit were incubated with drug concentrations corresponding to 0.5×, 1×, 1.5×, 2× and 2.5× their IC50 values. Untreated infected RBCs were included as control. Parasites were harvested 30 hours post incubation, and the mature trophozoites were isolated with 0.05% (w/v) saponin and washed with 1× PBS (pH 7.5) to eliminate traces of residual RBC-derived Hb. The number of trophozoites in these samples were quantified using flow cytometry. The contents of the trophozoite pellet were then released by hypotonic lysis and sonication. Following centrifugation, the supernatants (first fraction) corresponding to membrane-soluble Hb were treated with 4% (w/v) SDS and 2.5% (v/v) pyridine. Pellets were again treated with 4% SDS, 2.5% pyridine, sonicated, and centrifuged. Supernatants corresponding to the ‘free’ heme fraction (second fraction) were then recovered. The remaining hemozoin pellets (third fraction) were then solubilized in 4% SDS and 0.3 M NaOH, neutralized with 0.3 M HCl, sonicated, and treated with 25% pyridine. The UV-visible spectrum of each heme fraction as a Fe(III) heme-pyridine complex was measured using a multiwell plate reader (Spectramax 340PC; Molecular Devices).

The total amount of each heme species was quantified using a heme standard curve37. The mass of each heme Fe species per trophozoite was calculated by dividing the total amount of each heme species by the corresponding number of parasites in that fraction37. Statistical comparisons were made using two-tailed Mann-Whitney U tests using GraphPad Prism.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The authors have investigated the mechanism of resistance and the mode of action of the antimalarial clinical development candidate ZY19489. A comprehensive set of experiments was conducted, including in vivo selection of resistant parasites, whole-genome sequencing, QTL analysis of a genetic cross, and in vivo mutagenesis. These independent approaches all converge on PfCRT as the principal determinant of resistance.\nWhile the identification of PfCRT as a resistance determinant is not inherently surprising—given its known structural flexibility and polyspecific substrate recognition—what makes this study noteworthy is the discovery that the PfCRT variant conferring resistance to ZY19489 loses the ability to transport chloroquine (CQ) and piperaquine (PPQ). This finding of inverse susceptibility is of considerable interest, as it raises the possibility of an evolutionary trap, in which the use of two drugs with opposing PfCRT susceptibilities could limit the evolution of cross-resistance.\nPoints to Be Addressed\n1. Quality of the structural models (Figure 2)\nAdditional information is required to assess the robustness of the molecular models presented in Figure 2. Specifically, please include:\ni) graphical representations of RMSD for both the protein backbone and the ligand across all simulated conformations;\nii) the docking scores for each pose;\niii) solvent-accessible surface area (SASA) values; and\niv) plots showing the distances between the ligand and key interacting residues as a function of simulation time.\n2. Clarification regarding hydrogen bond persistence (Figure 2)\nThe figure legend states that “a significant hydrogen bond persisted for more than 30% of the simulation time.” This implies that the interaction was absent for approximately two-thirds of the trajectory. Please clarify the interpretation: does this mean the hydrogen bond is transient and thus not functionally significant?\n3. Molecular dynamics simulations for ZY19489 versus CQ and PPQ\nTo better understand the mechanistic basis of the observed inverse susceptibility, additional MD simulations should be performed comparing ZY19489, CQ, and PPQ. Do these ligands occupy distinct regions of the substrate-binding cavity, or are their binding poses overlapping? Is there evidence of steric hindrance or mutual exclusion between them?\n4. Verification of metabolites in P. falciparum extracts\nPlease clarify whether the proposed metabolites were experimentally verified in the context of P. falciparum extracts. This is an important control, as many metabolites display altered migration or detection profiles due to matrix effects from parasite extracts.\n5. Experimental testing of the evolutionary trap hypothesis\nHave the authors attempted to test the evolutionary trap hypothesis experimentally—for example, by exposing parasites to combinations of ZY19489 with CQ or PPQ? Such data would significantly strengthen the manuscript’s central claim.\n6. Discussion of literature on subverted inverse susceptibility\nThere are documented cases where inverse susceptibility was eventually overcome by cross-resistance, especially in proteins with high mutational plasticity such as PfCRT. The authors should acknowledge and discuss this possibility, citing relevant work (e.g., Mandt et al., eLife 2023, DOI: 10.7554/eLife.85023; Nichol et al., Nat Commun 2019, DOI: 10.1038/s41467-018-08098-6).\nOverall Assessment\nThis is a well-designed and timely study that contributes valuable insights into the mechanisms of antimalarial resistance and the structural dynamics of PfCRT. The concept of an evolutionary trap mediated by inverse susceptibility is particularly intriguing and potentially impactful. Addressing the points outlined above—especially those concerning the molecular modeling data and experimental verification of key hypotheses—would substantially strengthen the manuscript and broaden its relevance.", "reviewer_2": "Okombo et al describe the characterisation of the mode of resistance and the mode of action of ZY19489, a new antimalarial compound currently in phase II clinical trials. By generating resistant parasites in vitro they discover a novel point mutation N246H in the well-studied PfCRT protein. They convincingly show by multiple approaches including the generation of genome edited parasite lines in different genomic backgrounds that this mutation confers low-level resistance shifting the IC50 by about 12-fold. QTL analysis confirms that PfCRT mutations can modulate ZY19489 activity. No resistance was obtained upon infection and treatment of humanised mice. Based on the available cryo-EM structure they model the consequence of the N246H mutation on PfCRT and the binding of ZY19489. Most interestingly, they observe that the N246H mutation confers sensitivity to aminoquinoline antimalarials and show that ZY19489 competes with chloroquine and piperaquine in CRT drug efflux. The N246H mutation results in a severe growth phenotype and ZY19489 retains potency against many of the crt and mdr1 alleles present in the field, features that are very much in favour for a new potential antimalarial compound and opens interesting perspectives of potential drug combinations with counterbalancing effects of resistance selection.\nThe authors address the mode of action of ZY19489 through metabolomic analysis of treated parasites, identify several pathways and focus on Hb catabolism, given the resistance mechanism conferred by CRT. The results are complex. No direct action on candidate proteins like PfHDP or falcipain 2A or 2B could be detected. Hb peptides are increased but ZY19489 seems not to block beta-hematin formation nor increase free heme levels while there is some augmentation in Hb levels. The authors suggest a possible additional mode of action.\nThis is an excellently performed, controlled, described and presented study. Important new insights into PfCRT activity in resistance to different antimalarial compounds are identified and validated in a large number of genetic backgrounds confirming the complexity of genetic resistance to certain antimalarials and to ZY19489.\nMinor comments:\n- P4 line 91: please give a value of the previously observed low-level resistance\n- P5 line 108: indicate how many clones were generated and sequenced\n- P8 line 180ff: The conclusions here might need some more explanation. How can an occluded form facilitate drug transport? Is there a possibility that ZY19489 could enter the DV through CRT? Is the open-to-cytosol conformation just another term for closed-to-DV? If the mutation reduces ZY19489 interactions close to the cytosol side, do the authors conclude that ligand release into the cytosol is facilitated and would be the reason for resistance through faster transport?\n- P10 Fig 3A: is this a bloated FV phenotype indicative of reduced Hb degradation?\n- P10 line 245: has the N246H mutation been found in the 10,000 Pf field genomes?\n- P11 in vivo experiments: There is no method section for these experiments. How many mice were treated? How many showed recrudescence? Which combinations at which concentrations were done? Perhaps a supp figure similar to S2 for the in vitro selection could help. What is the 3D7-0087/N9 strain? Why was 3D7 used here?\n- P13 stage-dependent activity. I acknowledge the courage to present the observed relatively lower susceptibility of trophozoites to the drug. This observation is somewhat in contradiction to the predicted mode of action of ZY19489 through an effect on Hb degradation or possibly thymidine production that should be predominant in trophozoites. At the current state of knowledge this might be difficult to explain but these conflicting observations should as such briefly been mentioned in the discussion, P18 line 460.\n- P21 resistance selection: please refer to Fig S2.\n- P21 line 544: how many clones?\n- P27 line 694: how many hours between Percoll and sorbitol treatment?\n- P41 figure legend 4D: it would help understanding to indicate in the legend that rings were treated for 30h with drugs before analysis.\n- Fig 4: I suggest to use the same scale (5-9 fg/cell) for free heme in panel 4H than in 4E to facilitate comparison. In 4H green data points could be connected by a dotted line for visibility.\n- Supp Fig S1: panel B: how many hours after aTc? The decrease in GAPDH and HA might actually be similar. The loading control actually shows that the experiment didn’t work, no parasite material in the -aTc sample.\n- Supp Fig S3: for curiosity, N246H is located in which crt exon?", "reviewer_3": "The manuscript investigates the mechanism of action and resistance profile of the novel, long-acting antimalarial ZY19489.\nUsing a combination of growth inhibition assays, metabolomics, heme-fractionation, microscopy, and genetic approaches, the authors show that ZY19489 rapidly kills Plasmodium falciparum asexual stages and affects hemoglobin catabolism in the digestive vacuole rather than directly inhibiting heme detoxification or folate/pyrimidine pathways.\nIn vitro resistance selection (particularly in a hypermutable Dd2-Polδ background) identifies a key mutation in the chloroquine resistance transporter PfCRT (N246H) that confers reduced susceptibility to ZY19489. This mutation, however, comes with a substantial fitness cost and creates strong collateral hypersensitivity to piperaquine and related aminoquinolines. Proteoliposome transport assays support a model in which ZY19489 is a PfCRT substrate and N246H alters its transport properties. In vivo experiments in humanized NSG mouse model show robust efficacy of ZY19489 and suggest a relatively high barrier to resistance under the dosing regimens tested. Based on the collateral sensitivity profile, the authors propose that pairing ZY19489 with piperaquine could act as an “evolutionary trap” and form a non-ACT, resistance-refractory combination therapy.\nThe link from PfCRT-mediated resistance to ZY19489, through to collateral hypersensitivity to piperaquine and other aminoquinolines, is logically developed and supported by both genetic and transport data. This provides a coherent mechanistic explanation and a plausible rationale for combination design.\nThis work directly addresses two critical issues for antimalarial development: (i) mechanism of action of a promising clinical candidate, and (ii) how resistance is likely to emerge and how it might be constrained by rational partner drug choice. The in vivo efficacy and resistance-selection data in humanized mouse models enhance the translational impact.\nWhile the data indicate perturbation of hemoglobin uptake/catabolism and implicate PfCRT in resistance, the direct molecular target(s) of ZY19489 remain undefined. Some key mechanistic conclusions rely on correlative metabolomic and heme-fractionation patterns rather than direct measurements of endocytic or proteolytic steps.\nThe principal resistance mutation (N246H) arises in a hypermutator line under strong selection and carries a large fitness cost and pronounced collateral sensitivity. It is not yet clear how likely such a pathway would be to emerge and spread in natural parasite populations, and no epidemiological data are available.\nThe proposal to use ZY19489 with piperaquine as a resistance-refractory combination is mostly based on in vitro susceptibility and transport data. Dedicated in vivo combination and resistance-selection experiments for this specific pair are limited or absent, so the “trap” remains somewhat theoretical at this stage.\nHowever, given its rigorous multidisciplinary experimentation, clear mechanistic and evolutionary insights into a clinically relevant antimalarial candidate, and strong translational implications for rational, resistance-limiting combination therapy design, I endorse this manuscript for publication in Nature Communications pending satisfactory revision.\nMajor point\nThe metabolomics data and heme-fractionation experiments are interpreted as showing that ZY19489 interferes with Hb endocytosis/catabolism rather than heme detoxification. However, the evidence is still somewhat indirect:\na) The decrease in short Hb-derived peptides after 2.5 h of treatment could reflect reduced peptide production, altered peptide export, or broader DV dysfunction, not necessarily a specific upstream step in Hb uptake or digestion.\nb) The heme fractionation experiments show increased Hb and relatively unchanged “free” heme and hemozoin at relevant concentrations, but this pattern could also arise from changes in growth rate or DV morphology rather than a primary MoA.\nc) Knockdown/KO of PfHDP and falcipains 2A/2B rules out those components, but the identity of the primary molecular target remains undefined.\nCan the authors provide more direct experimental evidence that ZY19489’s primary antiparasitic activity is mediated through disruption of hemoglobin uptake/catabolism (e.g., live imaging of Hb endocytosis, measurement of DV endocytic flux, or genetic/chemical epistasis with known Hb-pathway mutants), or else temper the mechanistic claim accordingly?\nThe manuscript proposes PfCRT N246H as a key resistance determinant but also notes a 60–75% asexual blood-stage fitness cost and strong collateral sensitivity to multiple aminoquinolines:\na) N246H was selected in a hypermutable Dd2-Polδ line under prolonged high-dose pressure, which may not reflect natural selection conditions.\nb) There is no current field surveillance evidence for N246H or closely related mutations in clinical isolates.\nc) The claim that such mutants would be “rapidly outcompeted” in the field is plausible but not quantitatively supported.\nCan the authors provide additional data or analysis addressing the in vivo relevance of PfCRT N246H (e.g., competition assays at lower starting frequencies, modeling of within-host or between-host dynamics, or a deeper scan of existing genomic datasets for N246H or compensatory mutations)? If not, the discussion could better distinguish between experimentally observed resistance pathways and their likely epidemiological probability.\nThe manuscript suggests that ZY19489 + PPQ could constitute an “evolutionary trap” and a resistance-refractory non-ACT combination, based mainly on:\na) In vitro collateral hypersensitivity of PfCRT N246H mutants to CQ/PPQ, and\nb) Proteoliposome transport data showing ZY19489 inhibition of mutant PfCRT-mediated CQ/PPQ efflux.\nHowever:\na) There are no in vivo combination efficacy or resistance-selection experiments specifically with ZY19489 + PPQ (only ferroquine, MMV693183, and M5717).\nb) Potential safety concerns and known PPQ-associated resistance and cardiotoxicity issues are not discussed in detail.\nc) It is not shown that long-term co-exposure cannot select other pfcrt or pfmdr1 variants that might erode this “trap”.\nGiven that no in vivo data are presented for ZY19489 + PPQ, can the authors either (i) provide preliminary combination data (e.g., mouse efficacy or resistance-selection experiments, PK/PD compatibility) or (ii) clearly frame the ZY19489–PPQ combination as a theoretical proposal based on in vitro transport and susceptibility data, rather than a fully supported resistance-refractory strategy?\nMinor points\na) In Table 1 the compound name appears as “ZY91489” rather than “ZY19489”, which looks like a typographical error.\nb) For several key datasets (metabolomics, heme fractionation, stage-specific activity, MD analyses), the exact number of biological replicates and the multiple-testing strategy are not always fully explicit in the main text.\nc) Consider softening causal language (e.g., “suggests” rather than “demonstrates”) where the link between metabolite pattern and mechanism is primarily correlative.\nd) The claim of a “high in vivo barrier to resistance” is largely based on the absence of ZY19489-specific mutations in a limited number of recrudescent lines and combinations. It might be more precise to phrase this as “no overt resistance observed under the regimens tested” and acknowledge that longer selection or different dosing might still identify additional resistance pathways.\ne) Ensure that all axes are clearly labeled with units (e.g., nM vs µM; fg/cell) and that symbols/colors in the legends match those in the panels (for example, some multi-compound curves in Fig. 4c and the supra-hex plots benefit from more explicit color labels in the legend).\nf) Metabolomics data are stated to be deposited in Metabolomics Workbench; it would be helpful to also explicitly state where WGS data for selected lines and the NSG recrudescent lines are deposited (e.g., SRA/BioProject accession), or to commit to deposition.\ng) A careful copy-editing pass would help with small inconsistencies (e.g., spacing around units, occasional missing hyphens such as “drug-selected”, and minor grammatical issues). These do not affect scientific content but will improve readability.\nh) In the Materials and Methods section, the notation for concentration units is inconsistent (e.g., use of both “w/w” and “wt/wt”, and both “v/v” and “vol/vol”). Please standardize to a single format throughout (e.g., “% w/w” and “% v/v”) in accordance with the journal’s style.\ni) Mitochondrial bc1 complex should be written as “mitochondrial bc1 complex”.\nj) Line 410, “in vivo” should be written in italic.\nk) At 2.5× IC₅₀, ZY19489 treatment is shoen to cause a significant decrease in hemoglobin (Hb) levels (Fig 4i). Could the authors clarify why Hb levels are reduced specifically at 2.5× IC₅₀ (e.g., stage-specific effects, timing of sampling, or assay sensitivity), and reconcile this with the broader model of DV Hb handling under ZY19489 exposure?\nl) Can the authors be consistent in writing E64D, as in the figures, and not E64d in the text?\nm) The metabolomics data show alterations in pyrimidine intermediates alongside DV-related changes. Since ZY19489 is proposed primarily to affect DV hemoglobin handling, it would be helpful if the authors could clarify whether the pyrimidine perturbations are interpreted as a secondary consequence of DV dysfuncton(e.g., impaired amino acid supply/energy status) rather than a direct effect on pyrimidine biosynthesis, and briefly outline the mechanistic link they envisage between DV damage and pyrimidine metabolism."}, "author_response": "Okombo et al. Collateral hypersensitivity between ZY19489 and piperaquine neutralizes\nPfCRT-mediated drug efflux and Plasmodium falciparum resistance.\nManuscript# NCOMMS-25-76372, under consideration at Nature Communications.\nRESPONSE TO REVIEWER COMMENTS\nNote – page and line numbering relates to the PDF version of the resubmission with changes\nhighlighted. New text is indicated below in blue, with in some instances the prior text indicated in red.\nNew text is highlighted in yellow in the revised manuscript. A clean copy is also included as a Word\ndocument.\nPart I: Summary\nReviewer #1: The authors have investigated the mechanism of resistance and the mode of action\nof the antimalarial clinical development candidate ZY19489. A comprehensive set of experiments\nwas conducted, including in vivo selection of resistant parasites, whole-genome sequencing, QTL\nanalysis of a genetic cross, and in vivo mutagenesis. These independent approaches all\nconverge on PfCRT as the principal determinant of resistance. While the identification of PfCRT as a resistance\ndeterminant is not inherently surprising—given its known structural flexibility and poly-specific\nsubstrate recognition—what makes this study noteworthy is the discovery that the PfCRT variant\nconferring resistance to ZY19489 loses the ability to transport chloroquine (CQ) and piperaquine\n(PPQ). This finding of inverse susceptibility is of considerable interest, as it raises the possibility of\nan evolutionary trap, in which the use of two drugs with opposing PfCRT susceptibilities could limit\nthe evolution of cross-resistance.\nReviewer #2: Okombo et al describe the characterisation of the mode of resistance and the mode\nof action of ZY19489, a new antimalarial compound currently in phase II clinical trials. By generating\nresistant parasites in vitro they discover a novel point mutation N246H in the well-studied PfCRT\nprotein. They convincingly show by multiple approaches including the generation of genome edited\nparasite lines in different genomic backgrounds that this mutation confers low-level resistance\nshifting the IC50 by about 12-fold. QTL analysis confirms that PfCRT mutations can modulate\nZY19489 activity. No resistance was obtained upon infection and treatment of humanised mice.\nBased on the available cryo-EM structure they model the consequence of the N246H mutation on\nPfCRT and the binding of ZY19489. Most interestingly, they observe that the N246H mutation confers\nsensitivity to aminoquinoline antimalarials and show that ZY19489 competes with chloroquine and\npiperaquine in CRT drug efflux. The N246H mutation results in a severe growth phenotype and\nZY19489 retains potency against many of the crt and mdr1 alleles present in the field, features that\nare very much in favour for a new potential antimalarial compound and opens interesting\nperspectives of potential drug combinations with counterbalancing effects of resistance selection.\nThe authors address the mode of action of ZY19489 through metabolomic analysis of treated\nparasites, identify several pathways and focus on Hb catabolism, given the resistance mechanism\nconferred by CRT. The results are complex. No direct action on candidate proteins like PfHDP or\nfalcipain 2A or 2B could be detected. Hb peptides are increased but ZY19489 seems not to block\nbeta-hematin formation nor increase free heme levels while there is some augmentation in Hb levels.\nThe authors suggest a possible additional mode of action. This is an excellently performed,\ncontrolled, described and presented study. Important new insights into PfCRT activity in resistance\nto different antimalarial compounds are identified and validated in a large number of genetic\nbackgrounds confirming the complexity of genetic resistance to certain antimalarials and to\nZY19489.\nReviewer #3: The manuscript investigates the mechanism of action and resistance profile of the\nnovel, long-acting antimalarial ZY19489. Using a combination of growth inhibition assays,\nmetabolomics, heme-fractionation, microscopy, and genetic approaches, the authors show that\nZY19489 rapidly kills Plasmodium falciparum asexual stages and affects hemoglobin catabolism in\nthe digestive vacuole rather than directly inhibiting heme detoxification or folate/pyrimidine pathways.\nmutation in the chloroquine resistance transporter PfCRT (N246H) that confers reduced\nsusceptibility to ZY19489. This mutation, however, comes with a substantial fitness cost and creates\nstrong collateral hypersensitivity to piperaquine and related aminoquinolines. Proteoliposome\ntransport assays support a model in which ZY19489 is a PfCRT substrate and N246H alters its\ntransport properties. In vivo experiments in humanized NSG mouse model show robust efficacy of\nZY19489 and suggest a relatively high barrier to resistance under the dosing regimens tested. Based\non the collateral sensitivity profile, the authors propose that pairing ZY19489 with piperaquine could\nact as an “evolutionary trap” and form a non-ACT, resistance-refractory combination therapy.\nThe link from PfCRT-mediated resistance to ZY19489, through to collateral hypersensitivity to\npiperaquine and other aminoquinolines, is logically developed and supported by both genetic and\ntransport data. This provides a coherent mechanistic explanation and a plausible rationale for\ncombination design. This work directly addresses two critical issues for antimalarial development: (i)\nmechanism of action of a promising clinical candidate, and (ii) how resistance is likely to emerge and\nhow it might be constrained by rational partner drug choice. The in vivo efficacy and resistance-\nselection data in humanized mouse models enhance the translational impact.\nWhile the data indicate perturbation of hemoglobin uptake/catabolism and implicate PfCRT in\nresistance, the direct molecular target(s) of ZY19489 remain undefined. Some key mechanistic\nconclusions rely on correlative metabolomic and heme-fractionation patterns rather than direct\nmeasurements of endocytic or proteolytic steps.\nThe principal resistance mutation (N246H) arises in a hypermutator line under strong selection and\ncarries a large fitness cost and pronounced collateral sensitivity. It is not yet clear how likely such a\npathway would be to emerge and spread in natural parasite populations, and no epidemiological\ndata are available.\nThe proposal to use ZY19489 with piperaquine as a resistance-refractory combination is mostly\nbased on in vitro susceptibility and transport data. Dedicated in vivo combination and resistance-\nselection experiments for this specific pair are limited or absent, so the “trap” remains somewhat\ntheoretical at this stage. However, given its rigorous multidisciplinary experimentation, clear\nmechanistic and evolutionary insights into a clinically relevant antimalarial candidate, and strong\ntranslational implications for rational, resistance-limiting combination therapy design, I endorse this\nmanuscript for publication in Nature Communications pending satisfactory revision.\nReviewer #1\nComment #1. Quality of the structural models (Figure 2). Additional information is required to assess\nthe robustness of the molecular models presented in Figure 2. Specifically, please include: i)\ngraphical representations of RMSD for both the protein backbone and the ligand across all simulated\nconformations; ii) the docking scores for each pose; iii) solvent-accessible surface area (SASA)\nvalues; and iv) plots showing the distances between the ligand and key interacting residues as a\nfunction of simulation time.\nReply: We thank the reviewer for these suggestions. All analyses used to evaluate model robustness\nwere performed during our molecular dynamics (MD) simulations. These data have now been added\nto the revised version of the Supplementary Information file (please refer to Supplementary Table\n9 and Supplementary Fig. 6). These include a table with ChemPLP docking scores and solvent-\naccessible surface area values. Additionally, we have included figures showing the time evolution of\nligand stability within the binding cavity, residue-level flexibility profiles of the protein, and the\ndistances between ZY19489 and the key interacting residues. These results confirm that the\nmodeled conformations remained stable throughout the trajectories and that the ligand maintains a\nconsistent binding mode across replicates.\nComment #2. Clarification regarding hydrogen bond persistence (Figure 2). The figure legend\nstates that “a significant hydrogen bond persisted for more than 30% of the simulation time.” This\n2\nimplies that the interaction was absent for approximately two-thirds of the trajectory. Please clarify\nthe interpretation: does this mean the hydrogen bond is transient and thus not functionally\nsignificant?\nReply: We thank the reviewer for highlighting this point. The interaction with the Q161 residue in the\nN246H mutant demonstrated approximately 30% hydrogen-bond occupancy in only one of the three\nindependent MD replicates. This interaction was not detected in the remaining two replicates.\nHydrogen-bond persistence values in the range of 20 to 50% are widely considered indicative of\nmeaningful but dynamic stabilization in MD simulations, particularly in protein–ligand systems where\nconformational fluctuations and solvent competition naturally weaken average occupancy. These\nmedium-occupancy hydrogen bonds contribute intermittently but repeatedly to ligand residence\nenergetics and might play a role in the overall binding thermodynamics and recognition process.\nHowever, while a ~30% occupancy supports the notion that this interaction is possible and\nenergetically favorable under certain conformational states, it was only observed in one of the three\nindependent trajectories of the 7G8+N246H isoform. The absence of the hydrogen bond in the other\ntwo replicates suggests that this contact is not consistently sustained in the mutated protein, which\nis in line with our mechanistic interpretation. Our modeling suggests that the N246H mutation\nchanges the environment of the open-to-cytosol cavity, increasing solvent exposure and disrupting\nthe stabilizing interactions observed in the parental 7G8 transporter. As a result, ZY19489 forms\nfewer and less persistent molecular contacts in the 7G8+N246H mutant, leading to weaker binding\nstabilization and facilitating efflux, which aligns with both our putative structural observations and the\nfunctional data indicating resistance. To prevent misunderstanding, we have revised the figure\ncaption and main text. The revised sentence now reads as follows:\nResults (page 8, lines 187 -190): “In the 7G8+N246H variant, we observed only one hydrogen bond\ninteraction with Q161 that persisted for more than 30% of the simulation period. This hydrogen bond\nwas not consistently detected in all trajectories; appearing exclusively in one of the three\nindependent molecular dynamics replicate simulations.”\nThe revised Figure legend now reads as follow:\n“Figure 2 (starting page 42 line 1139): B Hydrogen bonding patterns between ZY19489 and 7G8\nPfCRT in the open-to-cytosol conformation implicate the side chain of E232 and the backbone\ncarbonyl group of L245 as important for stabilizing drug-transporter interactions. Bonding pattern\nwith 7G8+N246H PfCRT, illustrating a single significant hydrogen bond between the carbonyl oxygen\nof the Q161 side chain and the ligand that persisted more than 30% of the simulation in one of the\nthree molecular dynamics simulations replicates. Hydrogen bonds are represented as dashed blue\nlines.”\nComment #3. Molecular dynamics simulations for ZY19489 versus CQ and PPQ. To better\nunderstand the mechanistic basis of the observed inverse susceptibility, additional MD simulations\nshould be performed comparing ZY19489, CQ, and PPQ. Do these ligands occupy distinct regions\nof the substrate-binding cavity, or are their binding poses overlapping? Is there evidence of steric\nhindrance or mutual exclusion between them?\nReply: The available structural and computational data allow us to explore the relationship between\nZY19489, CQ, and PPQ within the PfCRT transport cavity. Kim et al., 2019 (reference #16) used\ncryo-EM and MD studies of chloroquine (CQ) bound to wildtype (WT) PfCRT to propose that this\ndrug occupies the central substrate-binding cavity, interacting with residues surrounding the\ncanonical resistance locus, including residues 72, 75, 76, and 97. In our simulations, ZY19489\nconsistently sampled the same T76-adjacent region of the cavity in the 7G8 protein, indicating that\nZY19489 and CQ engage largely overlapping binding volumes rather than distinct or spatially\nseparated pockets.\nGiven the structural similarities between CQ and piperaquine (PPQ), and prior models of PPQ\ninteraction with PfCRT, PPQ is also expected to occupy the same central cavity. In line with this, the\nresults collected in the proteoliposome assays support this interpretation. ZY19489 was observed to\n3\nstrongly inhibit the transport of radiolabeled CQ and PPQ, and reciprocally CQ and PPQ inhibited\nZY19489 transport. This pattern indicates mutual exclusion, which in the context of PfCRT does not\nimply steric hindrance but rather the intrinsic single-substrate nature of the transporter, in which only\none ligand can occupy the central cavity at a time. Any ligand already bound to that region would\nprevent binding or transport of another. The competition pattern observed experimentally is therefore\nconsistent with all three compounds accessing the same substrate-binding cavity. The published\nstructural data for CQ, the expected PPQ cavity engagement based on structural similarity, and our\nsimulations of ZY19489 in WT PfCRT combine to indicate that the three ligands interact with a shared\ncentral binding region. We have included parts of this response into the Discussion to help readers\nto better understand the mechanistic basis of basis of the observed inverse susceptibility and the\noccupancy of ZY19489 in the cavity.\nDiscussion (starting page 17, line 437): “Curiously, parasites expressing the N246H mutation were\nsensitized to DV-acting antimalarials including PPQ and CQ. In our simulations, ZY19489\nconsistently samples the same T76-adjacent region of the cavity, indicating that this molecule, CQ\nand PPQ engage largely overlapping binding volumes rather than distinct or spatially separated\npockets. This interpretation aligns with our proteoliposome data where transport of CQ and PPQ by\nPfCRT variants that mediate resistance to these drugs was potently inhibited in the presence of\nZY19489. This pattern suggests mutual exclusion which, in the context of PfCRT, does not imply\nsteric hindrance but rather the intrinsic single-substrate nature of the transporter in which only one\nligand can occupy the central cavity at a time and preclude binding or transport of another. The\ncompetition pattern observed experimentally is therefore consistent with all three compounds\naccessing the same substrate-binding cavity.”\nComment #4. Verification of metabolites in P. falciparum extracts. Please clarify whether the\nproposed metabolites were experimentally verified in the context of P. falciparum extracts. This is\nan important control, as many metabolites display altered migration or detection profiles due to matrix\neffects from parasite extracts.\nReply: We agree with the reviewer that this is an important query. The metabolites were confirmed\nby comparing their retention times with those of pure molecular standards run on the same mass\nspectrometer. Matrix effects are challenging to control for, but with replicative and reproducible\nexperiments compared to our molecular standards and control compound effects on the parasite,\nwe report reproducible metabolite measurements for comparative fingerprint profiling. We have\nadded this confirmation in the Materials and Methods section which now reads as follows:\nMaterials and Methods (page 32, lines 816 - 817): “The metabolites were confirmed by comparing\ntheir retention times with those of pure molecular standards run on the same mass spectrometer.”\nComment #5. Experimental testing of the evolutionary trap hypothesis. Have the authors attempted\nto test the evolutionary trap hypothesis experimentally—for example, by exposing parasites to\ncombinations of ZY19489 with CQ or PPQ? Such data would significantly strengthen the\nmanuscript’s central claim.\nReply: The reviewer raises an important point, and we would be very keen to experimentally test\nthis specific hypothesis. Theoretically, one could select for resistance to one compound tested alone\nand then run parallel selections with that compound plus a range of sublethal concentrations of the\nsecond compound to show that parasites could not evolve dual resistance. However, the rarity of\nselecting for resistance to any of these compounds makes this infeasible. For ZY19489, resistance\nselection required pressuring hypermutable Dd2-Polδ parasites with ZY19489 for five months of\nconsecutive culture to obtain a mutant line from one of three flasks with 109 parasites each\n(corresponding to 100 to 200 mL of culture per flask). This line is about 10-fold more mutable then\nstandard Dd2 as shown by Kumpornsin et al., Nature Commun 2023 (reference #12). Repeating this\nexperiment would require liters of culture per day over that time period, combining ZY19489 with\nvarying concentrations of PPQ or CQ. This would be required in order to show that instead of\nobtaining resistance in one (or perhaps two) flasks, we obtained no resistance in any (i.e. reducing\nthe frequency from 1 or 2 to 0). This vast culturing effort would therefore not yield any statistically\n4\nrobust effort. We also note that in the malaria literature there are only two reports where CQ or PPQ\nresistance have ever been generated, both with tailored situations where only a single mutation was\nrequired (Fidock et al., Molec Cell 2000, PMID 11090624; Eastman et al., Antimicrob Agents\nChemother, 2011, PMID 21576453), adding to the complexity of this experiment. These combination\nexperiments have only been possible with compounds (such as PfDHODH inhibitors) for which\nresistance is readily acquired (for example with numbers in the range of 106 to 107 parasites; Lukens\net al., 2014, PNAS, PMID: 24381157).\nIn response to the reviewer, we have toned down our discussion of this evolutionary trap as follows:\nAbstract (page 2, lines 35 - 39):\nFrom “Uptake studies in PfCRT-containing proteoliposomes provide evidence that ZY19489 can\nblock mutant PfCRT-mediated efflux of piperaquine and chloroquine, creating a scenario of an\nevolutionary trap whereby resistance to ZY19489 blocks PfCRT-mediated resistance and restores\nsusceptibility to piperaquine and chloroquine”.\nTo “Uptake studies in proteoliposomes loaded with recombinant purified PfCRT provided evidence\nthat ZY19489 can block transport of piperaquine and chloroquine mediated by drug-specific PfCRT\nvariants. In parasites, these PfCRT variants are thought to mediate resistance by effluxing these\ndrugs out of the digestive vacuole. Our findings evoke a scenario of an evolutionary trap whereby\nresistance to ZY19489 can block piperaquine and chloroquine efflux and thereby restore their\nactivity.”.\nDiscussion (page 18, lines 449 - 454):\nFrom “In such a scenario, our data suggest that two drugs could theoretically constitute an effective\ncombination that might force pfcrt into an “evolutionary trap” and prevent the emergence of high-\ngrade resistance to either. This concept has been demonstrated for other antimalarials, including\nPfDHODH, Pf20S proteasome, and mitochondrial cytochrome bc complex inhibitors11…”\ncombination that would force pfcrt into an “evolutionary trap” and prevent the emergence of high-\ngrade resistance to either. This concept remains theoretical, as the rarity of selecting for resistance\nto these compounds alone or in combination precludes experimental validation. Nevertheless, this\nhypothesis has been validated for other antimalarials, including PfDHODH, Pf20S proteasome and\nmitochondrial cytochrome bc complex inhibitors10…”\ncases where inverse susceptibility was eventually overcome by cross-resistance, especially in\nproteins with high mutational plasticity such as PfCRT. The authors should acknowledge and discuss\nthis possibility, citing relevant work (e.g., Mandt et al., eLife 2023, DOI: 10.7554/eLife.85023; Nichol\net al., Nat Commun 2019, DOI: 10.1038/s41467-018-08098-6).\nReply: We agree with the reviewer on this point. We have subsequently modified the Discussion\nto acknowledge this and included the suggested references. The additional sentences we have\nincluded in the Discussion are as follows:\nDiscussion (starting page 18, line 460):\nFrom “This is instructive to the ongoing Phase II trials in Gabon where mutant pfcrt, which could\nprovide a potential background for N246H to emerge, constituted an estimated 53% of all samples\nin a recent report45. Nonetheless, the high resistance barrier of ZY19489 and steep fitness cost\nassociated with PfCRT N246H suggest that clinical resistance to this antimalarial is unlikely to arise\nas mutant parasites would likely be rapidly outcompeted in the field.”\nTo “This is instructive to the ongoing Phase Ib trials in Gabon where mutant pfcrt, which could provide\na potential background for N246H to emerge, constituted an estimated 53% of all samples in a recent\nreport44. It is important to point out that this inverse susceptibility phenomenon could still be\novercome by cross-resistance, especially in proteins with high mutational plasticity like PfCRT and\nPfDHODH. For instance, in vitro selection experiments with DSM265 in combination with TCMDC-\n125334 (active against all PfDHODH mutant lines) yielded parasites with a PfDHODH V532A\nmutation that were cross-resistant to both compounds and as fit as the wildtype parent45. Additionally,\na mathematical quantification and experimental evolution study in Escherichia coli highlighted that a\n5\nsecond drug can stochastically select for increased susceptibility or increased resistance when\nfollowing a first46. Nonetheless, the absence of overt resistance under the ZY19489-containing\nregimens tested and the steep fitness cost associated with PfCRT N246H suggest that clinical\nresistance to this antimalarial is unlikely to arise as mutant parasites would likely be rapidly\noutcompeted in the field. We, however, urge caution in this interpretation since we acknowledge that\nlonger selection or different dosing might still identify additional resistance pathways. We note that\nthis mutation has not been observed in any of the ~20,000 genomes present in the\nMalariaGEN Pf7 data.”\nOverall Assessment\nThis is a well-designed and timely study that contributes valuable insights into the mechanisms of\nantimalarial resistance and the structural dynamics of PfCRT. The concept of an evolutionary trap\nmediated by inverse susceptibility is particularly intriguing and potentially impactful. Addressing the\npoints outlined above—especially those concerning the molecular modeling data and experimental\nverification of key hypotheses—would substantially strengthen the manuscript and broaden its\nrelevance.\nReply: We thank the reviewer for this positive assessment\nReviewer #2:\nComment #1. - P4 line 91: please give a value of the previously observed low-level resistance.\nReply: We have deleted this line. In its place, we have now provided more context in the Introduction\nsection and quantified the level of resistance referred to by this phrase. The revised lines now read\nas follows:\nIntroduction (starting page 3, line 69):\nFrom “In a previous report, low-level resistance to this molecule was associated with a G29V\nmutation in subunit D of the P. falciparum vacuolar proton-transporting V-type ATPase complex…”\nTo “In prior in vitro selection experiments using several triaminopyrimidines structurally analogous\nto ZY19489, one derivative (compound 6; Fig. 1a) yielded resistant parasites that exhibited a 3-fold\nincrease in the half-maximal growth inhibition concentration (IC ) compared to parental Dd27. This\n50\nlow-level resistance was associated with a G29V mutation in...”\nWe have also included the chemical structure of compound 6 in Figure 1A.\nResults (page 4, lines 91 - 92):\nFrom “Applying a ramping in vitro selection method to P. falciparum Dd2 parasites, a previous study\nobserved low-grade resistance after ~200 days of drug pressure7. Whole-genome sequence (WGS)\nanalysis associated resistance with a G29V mutation in PfV1-D (subunit D of the P. falciparum\nvacuolar proton-transporting V-type ATPase complex). To test whether this association was causal,\nwe generated…”\nTo “To test whether the prior association between ZY19489 resistance and a mutated form of the P.\nfalciparum V-type ATPase complex7 was causal (see Introduction), we generated a...”\nComment #2. - P5 line 108: indicate how many clones were generated and sequenced.\nReply: We have now indicated the number of clones that were generated, phenotype and sent out\nfor whole genome sequencing. This information is in the revised sentences that now read as follow:\nResults (page 5, lines 104 -105):\nFrom “Resistance was confirmed in parasites cloned by limiting dilution (Fig. 1b; Supplementary\nTable 3)”\nTo “Resistance was confirmed in four parasites cloned by limiting dilution (Fig. 1b; Supplementary\nTable 3).\nFrom “WGS analysis of the ZY19489-pressured clones identified single nucleotide…”\nTo “Whole-genome sequence (WGS) analysis of all the four ZY19489-pressured clones identified\nsingle nucleotide...”\nComment #3. P8 line 180ff: The conclusions here might need some more explanation. How can an\noccluded form facilitate drug transport? Is there a possibility that ZY19489 could enter the DV\nthrough CRT? Is the open-to-cytosol conformation just another term for closed-to-DV? If the mutation\nreduces ZY19489 interactions close to the cytosol side, do the authors conclude that ligand release\ninto the cytosol is facilitated and would be the reason for resistance through faster transport?\nReply: We have now expanded on our interpretation as follows:\nResults (page 8, lines 179 -182):\nFrom: “This salt bridge formation alludes to a tendency for the mutant protein to adopt a closed-to-\nDV (occluded) conformation and suggests that the mutation induces resistance to ZY19489 by\nfavoring this closed state that may facilitate drug transport. Conversely, the 7G8 protein does not\nadopt this DV-closed conformation, potentially resulting in reduced transport activity (Supplementary\nFig. 5).”\nTo: “This salt bridge formation suggests that the mutant protein might transiently adopt a occluded\n(partially closed-to-DV) conformation and that the N246H mutation might confer resistance to\nZY19489 by favoring this occluded state. Conversely, the 7G8 protein did not appear to adopt this\noccluded conformation (Supplementary Fig. 5; Supplementary Fig. 6).”\nDiscussion (starting page 17, lines 438): “In our simulations, ZY19489 consistently samples the\nsame T76-adjacent region of the cavity, indicating that this molecule, CQ and PPQ engage largely\noverlapping binding volumes rather than distinct or spatially separated pockets. This interpretation\naligns with our proteoliposome data where transport of CQ and PPQ by PfCRT variants that mediate\nresistance to these drugs was potently inhibited in the presence of ZY19489. This pattern suggests\nmutual exclusion which, in the context of PfCRT, does not imply steric hindrance but rather the\nintrinsic single-substrate nature of the transporter in which only one ligand can occupy the central\ncavity at a time and preclude binding or transport of another. The competition pattern observed\nexperimentally is therefore consistent with all three compounds accessing the same substrate-\nbinding cavity”\nComment #4. P10 Fig 3A: is this a bloated FV phenotype indicative of reduced Hb degradation?\nReply: From previous evidence of PfCRT mutations and the bloated digestive vacuole phenotype,\nwe are confident that this morphological feature is directly linked to impaired ability of the parasite to\noptimally degrade host hemoglobin. We have now added a few lines in the Discussion section to\nhighlight the possible link between this phenotype, PfCRT mutations and poor multiplication rate in\nthe mutant parasites.\nDiscussion (page 19, lines 478 - 490):\nFrom “Further, aberrant accumulation of peptides has been observed in variant PfCRT isoforms\nwhere it is associated with reduced parasite fitness, presumably from deprivation of essential globin-\nderived amino acids28,43,44. Our analysis identified HVDD (a truncated version of HVDDM) and\nVDPVNF, both of which were demonstrated to be putative PfCRT substrates in [3H]-CQ cis-inhibition\nexperiments in Xenopus laevis oocytes45\nTo “Further, our results are consistent with an important role of PfCRT mutations in dictating levels\nof short oligopeptides in parasites. Aberrant accumulation of peptides has been observed in variant\nPfCRT isoforms and is predicted to impact the pool of essential amino acids available for protein\nsynthesis, osmotic integrity, and numbers of merozoites that can develop within the confines of the\ninfected RBCs29,47,48. This suboptimal physiological context agrees with the lower parasite\nmultiplication rate and reduced parasite fitness observed in the PfCRT N246H mutant in our study\nand the PPQ resistance-conferring F145I variant earlier studied49,50. The anomalous peptide\naccumulation in the N246H mutant also suggests that one of the native roles of PfCRT involves\n7\nshuttling of peptides from the DV lumen to the cytosol for subsequent liberation of amino acids and\nalleviation of osmotic stress from the DV, with this function being impaired in drug-resistant PfCRT\nmutants. This interpretation is supported by a study of PfCRT isoforms expressed in Xenopus laevis\noocytes that demonstrated PfCRT-mediated transport of peptides ranging from 4 to 11 amino acids,\nwith the 3D7 isoform (CQ-sensitive) permitting a greater quantity and broader range of peptides than\nthe Dd2 (CQ-resistant) isoform51.”\nComment #5. P10 line 245: has the N246H mutation been found in the 10,000 Pf field genomes?\nReply: No, it has not. We searched for this mutation, without success, in the Pf7 repository which\ncontains P. falciparum genome variation in 20,000 worldwide samples collated from the MalariaGEN\nPf3k database, Plasmodium falciparum Community Project and GenRe Mekong Project. This is now\nmentioned in the Discussion as follows:\nDiscussion (page 19, lines 472 - 473): “We note that this mutation has not been observed in any of\nthe ~20,000 genomes present in the MalariaGEN Pf7 data.”\nComment #6. P11 in vivo experiments: There is no method section for these experiments. How\nmany mice were treated? How many showed recrudescence? Which combinations at which\nconcentrations were done? Perhaps a supp figure similar to S2 for the in vitro selection could help.\nWhat is the 3D7-0087/N9 strain? Why was 3D7 used here?\nReply: We have now provided the method used to generate the in vivo data. This information is now\navailable in the Materials and Methods section (starting page 29, line 754) under the title “In vivo\nefficacy studies in the TADhuMouse®-Pfalc model”. We agree with the reviewer that a figure\nwould help assist readers follow this part of the manuscript. We have therefore included a\nsupplementary file (Supplementary Fig. 7) to show the number of mice that were treated in each\ncategory and the number that showed recrudescence. Additionally, information on the doses used in\neach combination is also included in Table 2 in the main text as well as the new Supplementary\nFig. 7. We further describe in detail the 3D70087/N9 strain as a standard P. falciparum strain adapted\nto grow reproducibly in humanized NOD-scid IL-2Rγnull (NSG) mice. This description is reflected in\nthe Results section, which reads as follows:\nResults (page 11, lines 275 - 278): “To assess whether ZY19489-resistant parasites might emerge\nin vivo following combination treatment, we used a validated mouse model of P. falciparum ABS\ninfection of NOD-scid IL-2Rγnull (NSG) mouse engrafted with human erythrocytes\n(TADhuMouse®)22.”\nComment #7. P13 stage-dependent activity. I acknowledge the courage to present the observed\nrelatively lower susceptibility of trophozoites to the drug. This observation is somewhat in\ncontradiction to the predicted mode of action of ZY19489 through an effect on Hb degradation or\npossibly thymidine production that should be predominant in trophozoites. At the current state of\nknowledge this might be difficult to explain but these conflicting observations should as such briefly\nbeen mentioned in the discussion, P18 line 460.\nReply: We thank the reviewer for this comment and appreciate the possible contradiction in\ninterpreting the mode of action of the molecule. While we agree that Hb degradation peaks in the\ntrophozoite phase, we nonetheless argue that the mode of action of this compound is likely to be in\nthe very early phase of hemoglobin degradation before this process is optimal in trophozoites.\nIndeed, supported by identification of active hemoglobinases (falcipains 2 and 3) at this stage,\nXie et al., 2016 (reference #53) demonstrated that hemoglobin uptake and digestion begins and is active\nin early rings. Additionally, fluorescence and electron tomography studies have shown that, by the\nmid-ring stage, parasites have detectable acidified compartments that contain small hemozoin\ncrystals. It is this presence of a hemoglobin degradation machinery in the rings that we presume\nrenders this stage of the parasite susceptible to ZY19489. Furthermore, our cell fractionation data\nshows a slight concentration-dependent increase in hemoglobin heme but not ‘free’ heme or\nhemozoin, consistent with hemoglobin heme species being a product of early degradation of host\n8\nhemoglobin. We have included parts of this reasoning in the Discussion section, which now reads\nas follows:\nDiscussion (page 20, lines 495 - 508):\nFrom “The absence of direct interaction between ZY19489 and ‘free’ heme, or the putative Hz\nnucleation factor PfHDP, or the hemoglobinases falcipains 2A and 2B, suggests inhibition of other\ntargets within the Hb catabolism pathway. Our metabolomic analysis also revealed an accumulation\nof deoxyuridine monophosphate, which could imply a potential impact on pyrimidine biosynthesis.\nThis likelihood of an additional MoA could help explain the compound’s high barrier for resistance.\nMoreover, Hb uptake and degradation begins in early rings46 and pyrimidine biosynthesis peaks\nduring the trophozoite to schizont transition47, which would align with our observations of stage-\nspecific potency against rings and schizonts.”\nTo “The ZY19489 potency observed against rings and schizonts in the stage-specificity experiments\nis informative. Fluorescence and electron tomography studies have shown that, by the mid-ring\nstage, parasites have detectable acidified compartments that contain small Hz crystals52.\nAdditionally, Hb uptake and degradation has been shown to begin in early rings53. The presence of\nthis active Hb degradation machinery in rings may render this stage highly susceptible to ZY19489.\nOn the other hand, pyrimidine biosynthesis peaks during the trophozoite to schizont transition54. Our\nmetabolomic analysis also revealed an accumulation of deoxyuridine monophosphate, implying a\npotential impact on pyrimidine biosynthesis and corroborating the observed stage-specific potency\nagainst schizonts. This likelihood of an additional MoA could help explain the compound’s high\nbarrier for resistance in the conditions tested. Our combined molecular dynamics simulations, heme\nfractionation and metabolomics studies lead us to propose that ZY19489 might act primarily in the\nparasite cytosol, and that the N246H mutation might enable mutant PfCRT to sequester this\ncompound in the DV, away from its cytosolic site of action. In contrast, evidence suggests that CQ\nand PPQ act primarily in the DV by inhibiting Hz formation, with mutant PfCRT mediating resistance\nby effluxing these drugs out of the DV away from their heme target17.”\nComment #8. P21 resistance selection: please refer to Fig S2.\nReply: To guide readers to where more information on how resistance selection with ZY19489 was\nperformed, we have added the words “Supplementary Fig. 2” to the first line of the Materials and\nMethods section under which resistance selection methodology falls. This section now reads as\nfollows:\nMaterials and Methods (page 23, lines 574 - 575): “In vitro selection was conducted using a pulsing\n(intermittent drug pressure) method on Dd2-Polδ (Supplementary Fig. 2).”\nComment #9. P21 line 544: how many clones?\nReply: We have now indicated the number of clones that were obtained from limiting dilution for IC\n50\ndetermination and subsequent whole genome sequencing. The new sentence now reads as follows:\nResults (page 5, lines 104 -105):\nFrom “Resistance was confirmed in parasites cloned by limiting dilution (Fig. 1b; Supplementary\nTable 3)”\nTo “Resistance was confirmed in four parasites cloned by limiting dilution (Fig. 1b; Supplementary\nTable 3).\nResults (page 5, line 107):\nFrom “WGS analysis of the ZY19489-pressured clones identified single nucleotide…”\nTo “Whole-genome sequence (WGS) analysis of the four ZY19489-pressured clones identified single\nnucleotide...”\nMaterials and Methods (page 23, lines 590 - 591):\nFrom “Clones were obtained from the resistant bulk cultures by limiting dilution.”\nTo “Four clones were obtained from the resistant bulk cultures by limiting dilution.”\nReply: We have now indicated the length of time between the two synchronization treatments. The\nnew sentence now reads as follows:\nMaterials and Methods (page 29, lines 738 - 740):\nFrom “Briefly, mature schizonts were obtained via Percoll gradient purification. Upon reinvasion of\nRBCs, cultures were synchronized by sorbitol treatment to obtain pure early rings.”\nTo “Briefly, highly synchronized early ring-stage parasites were obtained via 70% Percoll gradient\npurification of mature schizonts followed 5 hours later by sorbitol treatment of young rings upon\nreinvasion of RBCs.”\nComment #11. P41 figure legend 4D: it would help understanding to indicate in the legend that rings\nwere treated for 30h with drugs before analysis.\nReply: We have now added this information to the Figure legend. This revised section now reads\nas follows:\nFigure 4 (page 44, lines 1179 - 1180):\nFrom “Heme fractionation of CQ- and ZY19489-treated NF54 parasites with different drug\nconcentrations.”\nTo “Heme fractionation of synchronized NF54 parasites treated for 30 hours with different drug\nconcentrations of ZY19489 or CQ.”\nComment #12. Fig 4: I suggest to use the same scale (5-9 fg/cell) for free heme in panel 4H than in\n4E to facilitate comparison. In 4H green data points could be connected by a dotted line for visibility.\nReply: We thank the reviewer for this suggestion. We have now ensured that the chloroquine (CQ)\nand ZY19489 plots in this figure are draw to comparative scales in all axes. To be specific, Figure\n4D and 4G, 4E and 4H as well as 4F and 4I now use the same scales, respectively. We have also\nconnected the green data points on Figure 4H for better visibility and to assist readers see that the\n‘free’ heme levels do not track with parasite death/survival in ZY19489-treated parasites.\nComment #13. Supp Fig S1: panel B: how many hours after aTc? The decrease in GAPDH and HA\nmight actually be similar. The loading control actually shows that the experiment didn’t work, no\nparasite material in the -aTc sample.\nReply: We thank the reviewer for this comment. Knockdown of V1-D exerts a strong inhibitory effect\non the parasite growth, as evidenced by clearance following removal of aTc from the culture medium.\nTo ensure consistency with the drug assay knockdown conditions, we repeated the Western blot\nusing a low aTc concentration (6 nM). These blots were prepared from cell lysates collected 72 hours\nafter aTc washout. We have now included in Supplementary Fig. S1 the repeated image of the\nWestern blot.\nComment #14. Supplementary Fig. S3: for curiosity, N246H is located in which crt exon?\nReply: Codon 246 is located in exon 5 of pfcrt. We have now added this information on the sentence\nwhere the N246H mutation is first-ever mentioned in the manuscript. The new sentence now reads\nas indicated below:\nResults (page 5, lines 108 - 110):\nFrom “Of these, the N246H mutation in pfcrt and D233N in the anaphase-promoting complex subunit\n10 (pfapc10; PF3D7_1217600) were present in all clones.”\nTo “Of these, the N246H mutation in exon 5 of pfcrt and D233N in the anaphase-promoting complex\nsubunit 10 (pfapc10; PF3D7_1217600) were present in all the clones.”\nComment #1. The metabolomics data and heme-fractionation experiments are interpreted as\nshowing that ZY19489 interferes with Hb endocytosis/catabolism rather than heme detoxification.\nHowever, the evidence is still somewhat indirect:\na) The decrease in short Hb-derived peptides after 2.5 h of treatment could reflect reduced peptide\nproduction, altered peptide export, or broader DV dysfunction, not necessarily a specific upstream\nstep in Hb uptake or digestion.\nb) The heme fractionation experiments show increased Hb and relatively unchanged “free” heme\nand hemozoin at relevant concentrations, but this pattern could also arise from changes in growth\nrate or DV morphology rather than a primary MoA.\nc) Knockdown/KO of PfHDP and falcipains 2A/2B rules out those components, but the identity of\nthe primary molecular target remains undefined.\nCan the authors provide more direct experimental evidence that ZY19489’s primary antiparasitic\nactivity is mediated through disruption of hemoglobin uptake/catabolism (e.g., live imaging of Hb\nendocytosis, measurement of DV endocytic flux, or genetic/chemical epistasis with known Hb-\npathway mutants), or else temper the mechanistic claim accordingly?\nReply: The reviewer raises very valid points. Indeed, it has proven experimentally extremely difficult\nto identify the precise MoA of a number of antimalarials that show an effect on Hb catabolism as\nexemplified most notably by lumefantrine (Combrinck et al., 2013, ACS Chem Biol; PMID: 23043646).\nThe metaprint pattern obtained here with ZY19489 shows a clear effect on peptides, consistent with\nthe earlier report from the Llinas lab showing perturbation of Hb catabolism to be a signature of\nhemozoin inhibitors (Allman et al., 2016, reference #30). We have been unable to generate more\ndirect evidence that ZY19489’s primary MoA is via disruption of Hb catabolism. Instead, as\nsuggested by the Reviewer, we have tempered our mechanistic claims as follows:\nAbstract (page 2 lines 39 - 40):\nFrom “Metabolomic studies revealed that ZY19489 significantly reduces intracellular levels of short\nhemoglobin-derived peptides”\nTo “Metabolomic studies provide evidence that ZY19489 significantly reduces intracellular levels of\nshort hemoglobin-derived peptides…”\nResults (page 14 line 335):\nFrom “ZY19489 inhibits parasite growth by interfering with hemoglobin catabolism”\nTo “ZY19489 inhibition of parasite growth is associated with hemoglobin catabolism inhibition”\nResults (page 14 line 356):\nFrom “ZY19489 inhibits hemoglobin breakdown but not heme detoxification”\nTo “Evidence that ZY19489 inhibits hemoglobin breakdown but not heme detoxification”\nComment #2. The manuscript proposes PfCRT N246H as a key resistance determinant but also\nnotes a 60–75% asexual blood-stage fitness cost and strong collateral sensitivity to multiple\naminoquinolines:\na) N246H was selected in a hypermutable Dd2-Polδ line under prolonged high-dose pressure,\nwhich may not reflect natural selection conditions.\nb) There is no current field surveillance evidence for N246H or closely related mutations in clinical\nisolates.\nc) The claim that such mutants would be “rapidly outcompeted” in the field is plausible but not\nquantitatively supported.\nCan the authors provide additional data or analysis addressing the in vivo relevance of PfCRT\nN246H (e.g., competition assays at lower starting frequencies, modeling of within-host or between-\nhost dynamics, or a deeper scan of existing genomic datasets for N246H or compensatory\nmutations)? If not, the discussion could better distinguish between experimentally observed\nresistance pathways and their likely epidemiological probability.\nconfers, parasites bearing this allele are unlikely to occur in the field and certainly not at high\nfrequency. We however take note of the fact that ZY19489+ferroquine trials are still ongoing and our\nrecommendation would be that in the event of any recrudescence from treatment failure then pfcrt\nshould be among the candidate genes to be sequenced and codon 246 would be of particular\ninterest. We nonetheless acknowledge that this point can be better argued in the Discussion section\nin a manner that distinguishes between experimentally observed resistance pathways and their likely\nepidemiological probability. We have therefore revised part of our Discussion as follows:\nDiscussion (page 19, line 468 – 473): “Nonetheless, the absence of overt resistance under the\nZY19489-containing regimens tested and the steep fitness cost associated with PfCRT N246H\nsuggest that clinical resistance to this antimalarial is unlikely to arise as mutant parasites would likely\nbe rapidly outcompeted in the field. We, however, urge caution in this interpretation since we\nacknowledge that longer selection or different dosing might still identify additional resistance\npathways. We note that this mutation has not been observed in any of the ~20,000 genomes present\nin the MalariaGEN Pf7 data.”\nComment #3. The manuscript suggests that ZY19489 + PPQ could constitute an “evolutionary\ntrap” and a resistance-refractory non-ACT combination, based mainly on:\na) In vitro collateral hypersensitivity of PfCRT N246H mutants to CQ/PPQ, and\nb) Proteoliposome transport data showing ZY19489 inhibition of mutant PfCRT-mediated CQ/PPQ\nefflux.\nHowever:\na) There are no in vivo combination efficacy or resistance-selection experiments specifically with\nZY19489 + PPQ (only ferroquine, MMV693183, and M5717).\nb) Potential safety concerns and known PPQ-associated resistance and cardiotoxicity issues are\nnot discussed in detail.\nc) It is not shown that long-term co-exposure cannot select other pfcrt or pfmdr1 variants that might\nerode this “trap”.\nGiven that no in vivo data are presented for ZY19489 + PPQ, can the authors either (i) provide\npreliminary combination data (e.g., mouse efficacy or resistance-selection experiments, PK/PD\ncompatibility) or (ii) clearly frame the ZY19489–PPQ combination as a theoretical proposal based\non in vitro transport and susceptibility data, rather than a fully supported resistance-refractory\nstrategy?\nReply: Our manuscript already provides data on ZY19489 combined with three other antimalarials\nalready undergoing evaluation in human clinical trials, notably MMV693183 (an acetyl CoA\nsynthethase inhibitor), ferroquine (used in the partner drug Phase Ib combination with ZY19489, and\nM5717 (Table 2). These studies showed no gain of resistance to ZY19489. These studies were\nconducted in the humanized RBC mouse model of P. falciparum infection by The Art of Discovery in\nBilbao, Spain, financed by MMV. These are extremely expensive studies that we cannot directly\nfund. Instead, as proposed by the Reviewer, we have now toned down our text to frame this\n“evolutionary trap” as a theoretical proposal. Our changes are described above in our response to\nReviewer #1 Comment #3.\nMinor points\nComment #4. In Table 1 the compound name appears as “ZY91489” rather than “ZY19489”, which\nlooks like a typographical error.\nReply: We have now corrected this. The table title now reads as follows (page 45, line 1189):\n“Table 1. Mutations identified from whole-genome sequencing of ZY19489-selected Dd2-Polδ\nclones.”\nComment #5. For several key datasets (metabolomics, heme fractionation, stage-specific activity,\nMD analyses), the exact number of biological replicates and the multiple-testing strategy are not\nalways fully explicit in the main text.\nIC testing, heme fractionation, stage specificity assay and metabolomic analysis in the Materials\n50\nand Methods sections. We have also ensured this information appears in the Figure legends where\nplots relating to these data are depicted. In addition, we have included the statistical tests used in\nthe analyses for these data. In some instances in the main text, this information is provided in\nparentheses. For each molecular dynamics simulation, we have now also indicated the number of\nreplicates done in the figure legend of the new Supplementary Fig. 6.\nComment #6. Consider softening causal language (e.g., “suggests” rather than “demonstrates”)\nwhere the link between metabolite pattern and mechanism is primarily correlative.\nReply: We have toned down on this causal language in relation to metabolite/peptide pattern. The\none sentence where this strong deduction was made has been tempered down and now reads as\nfollows:\nDiscussion (page 19, lines 490 - 491):\nFrom “Our analysis identified HVDD (a truncated version of HVDDM) and VDPVNF, both of which\nwere demonstrated to be putative PfCRT substrates in [3H]-CQ cis-inhibition...”\nTo “Our analysis identified HVDD (a truncated version of HVDDM) and VDPVNF, both of which were\nsuggested to be putative PfCRT substrates in [3H]-CQ cis-inhibition...”\nComment #7. The claim of a “high in vivo barrier to resistance” is largely based on the absence of\nZY19489-specific mutations in a limited number of recrudescent lines and combinations. It might be\nmore precise to phrase this as “no overt resistance observed under the regimens tested” and\nacknowledge that longer selection or different dosing might still identify additional resistance\npathways.\nReply: We agree with the reviewer on this point. We have therefore rephrased part of the Discussion\nto reflect this cautious interpretation. The modified segment of the Discussion now reads as follows:\nDiscussion (page 19, lines 468 - 472):\nFrom “Nonetheless, the high resistance barrier of ZY19489 and the steep fitness cost associated\nwith PfCRT N246H suggest that clinical resistance to this antimalarial is unlikely to arise as mutant\nparasites would likely be rapidly outcompeted in the field.”\nTo “Nonetheless, the absence of overt resistance under the ZY19489-containing regimens tested\nand the steep fitness cost associated with PfCRT N246H suggest that clinical resistance to this\nantimalarial is unlikely to arise as mutant parasites would likely be rapidly outcompeted in the field.\nWe, however, urge caution in this interpretation since we acknowledge that longer selection or\ndifferent dosing might still identify additional resistance pathways.”\nComment #8. Ensure that all axes are clearly labelled with units (e.g., nM vs µM; fg/cell) and that\nsymbols/colors in the legends match those in the panels (for example, some multi-compound curves\nin Fig. 4c and the supra-hex plots benefit from more explicit color labels in the legend).\nReply: We thank the reviewer for this comment and have thoroughly revised our Figures and Tables\nto remove these errors.\nComment #9. Metabolomics data are stated to be deposited in Metabolomics Workbench; it would\nbe helpful to also explicitly state where WGS data for selected lines and the NSG recrudescent lines\nare deposited (e.g., SRA/BioProject accession), or to commit to deposition.\nReply: This has now been done. We have submitted the whole-genome sequencing data for the\nresistant clones obtained from in vitro selection pressure using ZY19489 as well as recrudescent\nparasites from the NSG mice experiments. The BioProject ID is PRJNA1376518 and the files are\naccessible via the link https://www.ncbi.nlm.nih.gov/bioproject/1376518. This information has now\nbeen added to the Data availability section of the manuscript which now reads as follows:\nData availability (page 35, lines 914 - 917):\n(Project ID: PR002514). Other datasets analyzed during the current study are provided with this\npaper as Supplementary Material.”\nTo “Metabolomic data are publicly accessible through the NCBI Metabolomics Workbench (Project\nID: PR002514). Raw reads of WGS data used in this study have been deposited in NCBI Sequenced\nRead Archive (SRA) under BioProject accession number PRJNA1376518 and are accessible via the\nlink https://www.ncbi.nlm.nih.gov/bioproject/1376518.\nOther datasets analyzed during the current study are provided with this paper as Supplementary\nMaterial.\nComment #10. A careful copy-editing pass would help with small inconsistencies (e.g., spacing\naround units, occasional missing hyphens such as “drug-selected”, and minor grammatical issues).\nThese do not affect scientific content but will improve readability.\nReply: We thank the Reviewer for this comment. We have now carefully gone through the\nmanuscript and corrected some of the typographical and grammatic errors in the original draft. We\nare convinced that the current version is much-improved, more coherent and reads better.\nComment #11. In the Materials and Methods section, the notation for concentration units is\ninconsistent (e.g., use of both “w/w” and “wt/wt”, and both “v/v” and “vol/vol”). Please standardize to\na single format throughout (e.g., “% w/w” and “% v/v”) in accordance with the journal’s style.\nReply: Nature Communications uses the “% v/v and % w/w” style of concentration and volume\nabbreviation. We have therefore made this correction in the three parts of the manuscript where this\nwas not standard with the rest. This is now written as follows:\nMaterial and Methods (page 35, line 896):\nFrom “...hour intervals with 5% (wt/vol) sorbitol…”\nTo “…hour intervals with 5% (w/v) sorbitol…”\nMaterial and Methods (page 35, line 899):\nFrom “...trophozoites were isolated with 0.05% (wt/vol) saponin and washed …”\nTo “…trophozoites were isolated with 0.05% (w/v) saponin and washed …”\nMaterial and Methods (page 35, line 903):\nFrom “...treated with 4% (wt/vol) SDS and 2.5% (vol/vol) pyridine”\nTo “…treated with 4% (w/v) SDS and 2.5% (v/v) pyridine”\nComment #12. Mitochondrial bc1 complex should be written as “mitochondrial bc1 complex”.\nReply: The reference to “mitochondrial bc1 complex” only appears once in our manuscript. However,\nit is unclear from the reviewer’s comment what revision is requested. Nonetheless, we realize that\nthe “mitochondrial bc1 complex” is more accurately referred to and annotated as “mitochondrial\ncytochrome bc complex”. We can only hope that this was the suggestion the Reviewer was making\n1\nand have subsequently made this change to reflect the more correct annotation. The new sentence\nnow reads as follows:\nDiscussion (page 18, lines 453 - 455):\nFrom “This concept has been demonstrated for other antimalarials, including PfDHODH, Pf20S\nproteasome and mitochondrial cytochrome bc complex inhibitors11, and is one of the rationales\n1\nbehind current triple ACT therapies”\nTo “Nevertheless, this hypothesis has been validated for other antimalarials, including PfDHODH,\nPf20S proteasome and mitochondrial cytochrome bc complex inhibitors10, and is one of the\n1\nrationales behind current triple ACT therapies.”\nReply: This has now been corrected. We have also ensured that this phrase is italicized the other\nfive times it appears in the manuscript. The new line now reflects this correction as follows:\nDiscussion (page 17, line 413): “…compound also did not select for in vivo resistance in\ncombination studies…”\nComment #14. At 2.5× IC , ZY19489 treatment is shown to cause a significant decrease in\n50\nhemoglobin (Hb) levels (Fig 4i). Could the authors clarify why Hb levels are reduced specifically at\n2.5× IC (e.g., stage-specific effects, timing of sampling, or assay sensitivity), and reconcile this with\n50\nthe broader model of DV Hb handling under ZY19489 exposure?\nReply: The reviewer raises and important question. We argue that the effect of ZY19489 on the\nlevels of parasite hemoglobin heme is best observed incrementally between 1.0× to 2.0× IC\n50\nconcentrations (Fig. 4i), suggesting some activity of this compound during earlier steps of\nhemoglobin catabolism. However, we think it is likely that the significant drop in hemoglobin level\nobserved 2.5× IC reflects injurious drug concentrations that inhibit the parasite’s ability to import\n50\nsufficient hemoglobin levels for breakdown inside the digestive vacuole. It is important to note, as\nthe reviewer has alluded, that ZY19489 is highly potent against the ring stage of the parasite which\nis the same parasite stage used to set up these fractionation experiments. This model fits with our\nproposed effect of hemoglobin catabolism, albeit in a concentration-sensitive and stage-specific\nmanner. We have added this clarification in the Results section which now reads as follows.\nResults (page 16, lines 397 - 400): “Nonetheless, treatment with ZY19489 caused significant\nincreases in Hb levels at 1.5× and 2.0× IC concentrations but not at the more inhibitory\n50\nconcentration of 2.5× IC (Fig. 4i), suggesting some activity of this compound during earlier steps\n50\nof Hb catabolism in a concentration- and stage-specific manner.”\nComment #15. Can the authors be consistent in writing E64D, as in the figures, and not E64d in the\ntext?\nReply: This has now been done. We have written this as E64d all through the manuscript and in the\nSupplementary Figure and its Legend.\nSupplementary Figure 11:\nFrom “As a comparator, we tested E64D\nTo “As a comparator, we tested E64d”\nComment #16. The metabolomics data show alterations in pyrimidine intermediates alongside DV-\nrelated changes. Since ZY19489 is proposed primarily to affect DV hemoglobin handling, it would\nbe helpful if the authors could clarify whether the pyrimidine perturbations are interpreted as a\nsecondary consequence of DV dysfunction (e.g., impaired amino acid supply/energy status) rather\nthan a direct effect on pyrimidine biosynthesis, and briefly outline the mechanistic link they envisage\nbetween DV damage and pyrimidine metabolism.\nReply: Our studies with the Llinas lab on a variety of different antimalarials have previously not found\nthis signature of impaired Hb-derived peptide levels and perturbation of pyrimidine metabolism that\nwas observed herein with ZY19489. We feel it is too preliminary to speculate on how these two\nsignals could be related. Nonetheless, we note that ZY19489 and pyrimethamine (an inhibitor of\npyrimidine biosynthesis) share a triaminopyrimidine substructure. We now comment on this in our\nrevised Discussion as follows:\nDiscussion (page 17 lines 415 - 420): “An effect on pyrimidine biosynthesis might relate to the\ntriaminopyrimidine substructure present in ZY19489 that is also observed in the antifolate inhibitor\npyrimethamine (Supplementary Fig. 4), which earlier showed partial inhibition of pyrimidine\nbiosynthesis in addition to its dominant effect on antifolate levels36. However, we do not know\n15\nwhether the evidence of an effect of ZY19489 on Hb catabolism and levels of pyrimidine precursors\nsuggests direct inhibition of both pathways or is indirectly related.”\nReviewer #1: Reviewer #1 (Remarks to the Author):\nThe authors have addressed my previous queries in a professional and objective manner, leading to\nimprovements in clarity, content, and overall scope of the manuscript. However, an oversimplified—and\nin parts potentially incorrect—statement has been introduced in the revised version in response to my\nComment 3 concerning ligand occupation of the substrate-binding cavity of PfCRT. To address\nComment 3, the authors added the following passage to the Discussion:\n“In our simulations, ZY19489 consistently samples the same T76-adjacent region of the cavity, indicating\nthat this molecule, CQ and PPQ engage largely overlapping binding volumes rather than distinct or\nspatially separated pockets. This interpretation aligns with our proteoliposome data where transport of\nCQ and PPQ by PfCRT variants that mediate resistance to these drugs was potently inhibited in the\npresence of ZY19489. This pattern suggests mutual exclusion which, in the context of PfCRT, does not\nimply steric hindrance but rather the intrinsic single-substrate nature of the transporter in which only one\nligand can occupy the central cavity at a time and preclude binding or transport of another. The\ncompetition pattern observed experimentally is therefore consistent with all three compounds accessing\nthe same substrate-binding cavity.”\nThis paragraph raises several significant concerns:\n1. Overgeneralization across PfCRT variants. Conclusions drawn primarily from simulations and\nuptake studies performed with PfCRTGB4 are extended broadly to other geographically distinct PfCRT\nvariants without sufficient justification.\n2. Failure to acknowledge structural and functional differences between PfCRT variants. The\nparagraph does not account for evidence indicating that PfCRTGB4 and PfCRTDd2 possess distinct\nsubstrate-binding cavity architectures. While PfCRTGB4 appears to accommodate only a single ligand\nat a time (either CQ or PPQ), as suggested by the authors’ molecular dynamics simulations, PfCRTDd2\nhas been shown to bind both substrates simultaneously under a partial noncompetitive inhibition model.\nFor PfCRTDd2, this interpretation is supported by extensive transport kinetics and complementary\ncomputational studies, which collectively suggest the presence of at least two distinct interaction sites\nwithin the binding cavity (PMID: 37285379; PMID: 25378409; PMID:\n40140375; https://hdl.handle.net/1885/733780662).\n3. Overinterpretation of transport data. The CQ and PPQ uptake data presented in Figures 2D and\n2E are insufficient to support the conclusion that CQ, PPQ, and potentially ZY19489 act as mutually\ncompetitive inhibitors on PfCRTGB4. Demonstrating true competitive inhibition—and, by extension, a\nsingle binding site—would require more detailed transport kinetic analyses, such as those described in\nthe studies cited above.\n4. Conflation of findings from different PfCRT variants. The manuscript uses PfCRTDd2 for resistance\nselection experiments and PfCRTGB4 for molecular dynamics simulations and uptake assays yet treats\n3\nthese variants as functionally equivalent. This is problematic, given their known differences in\npolymorphisms, transport kinetics, and inferred binding-site organization.\nThe intent of these comments is not to create undue difficulty for the authors, but rather to help prevent\noverstatements or inaccuracies that may undermine the manuscript at a later stage. I therefore\nrecommend that the authors rework the cited paragraph in the Discussion to adopt a more nuanced\ninterpretation, explicitly acknowledge the limitations arising from the use of PfCRT variants with distinct\npolymorphisms and transport properties, and avoid generalizing beyond what the data directly support.\nI am not requesting additional experimental work.\nReply: We thank the reviewer for these insightful comments. As a result, we have now amended the\nmanuscript as follows:\n1. In the Results section, we have made the following changes.\n(page 8; line 179): from “Additionally, ZY19489 disrupts the residue interaction network around 76T…”\nto “Additionally, ZY19489 appeared to disrupt the residue interaction network around 76T…”.\n(page 8; lines 180 - 181): from “Interestingly, the 7G8+N246H protein forms a salt bridge between K80\nand E207…” to “Interestingly, the 7G8+N246H protein was predicted to form a salt bridge between K80\nand E207…”\n(page 8; lines 182 – 184): from “This salt bridge formation suggests that the mutant protein might\ntransiently adopt an occluded (partially closed-to-DV) conformation and that the N246H mutation might\nconfer resistance to ZY19489 by favoring this occluded state” to “This potential salt bridge formation\nsuggests that the mutant protein might transiently adopt an occluded (partially closed-to-DV)\nconformation, which might contribute to ZY19489 resistance.”\n2. To further explain our interpretation of the data, our revised Discussion now states (page 17; lines\n423 - 426): “Using metabolomics and Hb degradation assays, we also observed ZY19489-mediated\nperturbations of Hb catabolism, without a direct effect on downstream inhibition of heme detoxification\n(the primary MoA of CQ, PPQ and amodiaquine). These data suggest that a major MoA of ZY194899 is\nrelated to inhibition of Hb catabolism, which we propose is indicative of a primary action outside of the\nDV.”\n3. In the Discussion section, we have revised the paragraph cited above and included the references\nkindly provided by the reviewer. This paragraph now reads as follows:\n(starting page 18; lines 449 - 470): from “In our simulations, ZY19489 consistently samples the same\nT76-adjacent region of the cavity, indicating that this molecule, CQ and PPQ engage largely\noverlapping binding volumes rather than distinct or spatially separated pockets. This interpretation\naligns with our proteoliposome data where transport of CQ and PPQ by PfCRT variants that mediate\nresistance to these drugs was potently inhibited in the presence of ZY19489. This pattern suggests\nmutual exclusion which, in the context of PfCRT, does not imply steric hindrance but rather the\nintrinsic single-substrate nature of the transporter in which only one ligand can occupy the central\ncavity at a time and preclude binding or transport of another. The competition pattern observed\nexperimentally is therefore consistent with all three compounds accessing the same substrate-\nbinding cavity” to “In our simulations with the solved 7G8 isoform structure and its 7G8+N246H variant, ZY19489 consistently sampled the same T76-adjacent region of the cavity, suggesting that this molecule, CQ, and PPQ might partially\noverlap in their binding to these two PfCRT isoforms. This interpretation aligns with our proteoliposome\ndata showing that ZY19489 inhibited transport of CQ and PPQ by their respective drug-resistant 7G8\nand 7G8+F145I PfCRT variants. One explanation could be mutual exclusion which, in the context of\nthese PfCRT isoforms, may suggest a single-substrate feature in which only one ligand could occupy\n4\nthe central cavity at a time and preclude binding or transport of another. Our proteoliposome competition\ndata suggest that all three compounds have at least partially overlapping cavity-binding properties.\nHowever, demonstrating true competitive inhibition and, by extension, confirmation of a single binding-\nsite model would require more detailed transport kinetic analyses40,43,44. It is important to recall that our\nsimulations and transport studies were conducted on 7G8 isoforms and as such this specific\ninterpretation cannot be extended as a generalization to other geographically distinct PfCRT variants.\nIn fact, variations in CQ and PPQ transport phenotypes have been observed between PfCRT isoforms\nexpressing mutant alleles from different geographic regions41,42, suggesting disparate interactions of\nthese drugs with residues within the central cavity. Additionally, various PfCRT isoforms appear to\npossess distinct substrate-binding cavity architectures43,44. Whereas evidence to data suggest that 7G8\nPfCRT may accommodate only a single ligand (either CQ or PPQ) at a time, transport kinetics and\ncomplementary computational studies have shown Dd2 PfCRT to bind both substrates simultaneously\nunder a partial noncompetitive inhibition model40,43,44, suggesting the presence of at least two distinct\ninteraction sites within the cavity of this isoform. We also note that isogenic parasites expressing Dd2,\nGB4 and FCB pfcrt alleles are known to differ in their CQ resistance profiles13, which also suggest\ndifferences in how these variants interact with antimalarial drugs.”\n5"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed my previous queries in a professional and objective manner, leading to improvements in clarity, content, and overall scope of the manuscript. However, an oversimplified—and in parts potentially incorrect— statement has been introduced in the revised version in response to my Comment 3 concerning ligand occupation of the substrate-binding cavity of PfCRT.\nTo address Comment 3, the authors added the following passage to the Discussion:\n“In our simulations, ZY19489 consistently samples the same T76-adjacent region of the cavity, indicating that this molecule, CQ and PPQ engage largely overlapping binding volumes rather than distinct or spatially separated pockets. This interpretation aligns with our proteoliposome data where transport of CQ and PPQ by PfCRT variants that mediate resistance to these drugs was potently inhibited in the presence of ZY19489. This pattern suggests mutual exclusion which, in the context of PfCRT, does not imply steric hindrance but rather the intrinsic single-substrate nature of the transporter in which only one ligand can occupy the central cavity at a time and preclude binding or transport of another. The competition pattern observed experimentally is therefore consistent with all three compounds accessing the same substrate-binding cavity.”\nThis paragraph raises several significant concerns:\n1. Overgeneralization across PfCRT variants. Conclusions drawn primarily from simulations and uptake studies performed with PfCRTGB4 are extended broadly to other geographically distinct PfCRT variants without sufficient justification.\n2. Failure to acknowledge structural and functional differences between PfCRT variants. The paragraph does not account for evidence indicating that PfCRTGB4 and PfCRTDd2 possess distinct substrate-binding cavity architectures. While PfCRTGB4 appears to accommodate only a single ligand at a time (either CQ or PPQ), as suggested by the authors’ molecular dynamics simulations, PfCRTDd2 has been shown to bind both substrates simultaneously under a partial noncompetitive inhibition model. For PfCRTDd2, this interpretation is supported by extensive transport kinetics and complementary computational studies, which collectively suggest the presence of at least two distinct interaction sites within the binding cavity (PMID: 37285379; PMID: 25378409; PMID: 40140375; https://hdl.handle.net/1885/733780662).\n3. Overinterpretation of transport data. The CQ and PPQ uptake data presented in Figures 2D and 2E are insufficient to support the conclusion that CQ, PPQ, and potentially ZY19489 act as mutually competitive inhibitors on PfCRTGB4. Demonstrating true competitive inhibition—and, by extension, a single binding site—would require more detailed transport kinetic analyses, such as those described in the studies cited above.\n4. Conflation of findings from different PfCRT variants. The manuscript uses PfCRTDd2 for resistance selection experiments and PfCRTGB4 for molecular dynamics simulations and uptake assays, yet treats these variants as functionally equivalent. This is problematic, given their known differences in polymorphisms, transport kinetics, and inferred binding-site organization.\nThe intent of these comments is not to create undue difficulty for the authors, but rather to help prevent overstatements or inaccuracies that may undermine the manuscript at a later stage. I therefore recommend that the authors rework the cited paragraph in the Discussion to adopt a more nuanced interpretation, explicitly acknowledge the limitations arising from the use of PfCRT variants with distinct polymorphisms and transport properties, and avoid generalizing beyond what the data directly support. I am not requesting additional experimental work.", "reviewer_3": "The authors have thoroughly addressed all previous comments and suggestions.\nI have no further remarks and would like to congratulate the authors on an excellent piece of work."}, "author_response": "RESPONSE TO REVIEWER COMMENTS\nReviewer #1: Reviewer #1 (Remarks to the Author):\nThe authors have addressed my previous queries in a professional and objective manner, leading to\nimprovements in clarity, content, and overall scope of the manuscript. However, an oversimplified—and\nin parts potentially incorrect—statement has been introduced in the revised version in response to my\nComment 3 concerning ligand occupation of the substrate-binding cavity of PfCRT. To address\nComment 3, the authors added the following passage to the Discussion:\n“In our simulations, ZY19489 consistently samples the same T76-adjacent region of the cavity, indicating\nthat this molecule, CQ and PPQ engage largely overlapping binding volumes rather than distinct or\nspatially separated pockets. This interpretation aligns with our proteoliposome data where transport of\nCQ and PPQ by PfCRT variants that mediate resistance to these drugs was potently inhibited in the\npresence of ZY19489. This pattern suggests mutual exclusion which, in the context of PfCRT, does not\nimply steric hindrance but rather the intrinsic single-substrate nature of the transporter in which only one\nligand can occupy the central cavity at a time and preclude binding or transport of another. The\ncompetition pattern observed experimentally is therefore consistent with all three compounds accessing\nthe same substrate-binding cavity.”\nThis paragraph raises several significant concerns:\n1. Overgeneralization across PfCRT variants. Conclusions drawn primarily from simulations and\nuptake studies performed with PfCRTGB4 are extended broadly to other geographically distinct PfCRT\nvariants without sufficient justification.\n2. Failure to acknowledge structural and functional differences between PfCRT variants. The\nparagraph does not account for evidence indicating that PfCRTGB4 and PfCRTDd2 possess distinct\nsubstrate-binding cavity architectures. While PfCRTGB4 appears to accommodate only a single ligand\nat a time (either CQ or PPQ), as suggested by the authors’ molecular dynamics simulations, PfCRTDd2\nhas been shown to bind both substrates simultaneously under a partial noncompetitive inhibition model.\nFor PfCRTDd2, this interpretation is supported by extensive transport kinetics and complementary\ncomputational studies, which collectively suggest the presence of at least two distinct interaction sites\nwithin the binding cavity (PMID: 37285379; PMID: 25378409; PMID:\n40140375; https://hdl.handle.net/1885/733780662).\n3. Overinterpretation of transport data. The CQ and PPQ uptake data presented in Figures 2D and\n2E are insufficient to support the conclusion that CQ, PPQ, and potentially ZY19489 act as mutually\ncompetitive inhibitors on PfCRTGB4. Demonstrating true competitive inhibition—and, by extension, a\nsingle binding site—would require more detailed transport kinetic analyses, such as those described in\nthe studies cited above.\n4. Conflation of findings from different PfCRT variants. The manuscript uses PfCRTDd2 for resistance\nselection experiments and PfCRTGB4 for molecular dynamics simulations and uptake assays yet treats\n3\nthese variants as functionally equivalent. This is problematic, given their known differences in\npolymorphisms, transport kinetics, and inferred binding-site organization.\nThe intent of these comments is not to create undue difficulty for the authors, but rather to help prevent\noverstatements or inaccuracies that may undermine the manuscript at a later stage. I therefore\nrecommend that the authors rework the cited paragraph in the Discussion to adopt a more nuanced\ninterpretation, explicitly acknowledge the limitations arising from the use of PfCRT variants with distinct\npolymorphisms and transport properties, and avoid generalizing beyond what the data directly support.\nI am not requesting additional experimental work.\nReply: We thank the reviewer for these insightful comments. As a result, we have now amended the\nmanuscript as follows:\n1. In the Results section, we have made the following changes.\n(page 8; line 179): from “Additionally, ZY19489 disrupts the residue interaction network around 76T…”\nto “Additionally, ZY19489 appeared to disrupt the residue interaction network around 76T…”.\n(page 8; lines 180 - 181): from “Interestingly, the 7G8+N246H protein forms a salt bridge between K80\nand E207…” to “Interestingly, the 7G8+N246H protein was predicted to form a salt bridge between K80\nand E207…”\n(page 8; lines 182 – 184): from “This salt bridge formation suggests that the mutant protein might\ntransiently adopt an occluded (partially closed-to-DV) conformation and that the N246H mutation might\nconfer resistance to ZY19489 by favoring this occluded state” to “This potential salt bridge formation\nsuggests that the mutant protein might transiently adopt an occluded (partially closed-to-DV)\nconformation, which might contribute to ZY19489 resistance.”\n2. To further explain our interpretation of the data, our revised Discussion now states (page 17; lines\n423 - 426): “Using metabolomics and Hb degradation assays, we also observed ZY19489-mediated\nperturbations of Hb catabolism, without a direct effect on downstream inhibition of heme detoxification\n(the primary MoA of CQ, PPQ and amodiaquine). These data suggest that a major MoA of ZY194899 is\nrelated to inhibition of Hb catabolism, which we propose is indicative of a primary action outside of the\nDV.”\n3. In the Discussion section, we have revised the paragraph cited above and included the references\nkindly provided by the reviewer. This paragraph now reads as follows:\n(starting page 18; lines 449 - 470): from “In our simulations, ZY19489 consistently samples the same\nT76-adjacent region of the cavity, indicating that this molecule, CQ and PPQ engage largely\noverlapping binding volumes rather than distinct or spatially separated pockets. This interpretation\naligns with our proteoliposome data where transport of CQ and PPQ by PfCRT variants that mediate\nresistance to these drugs was potently inhibited in the presence of ZY19489. This pattern suggests\nmutual exclusion which, in the context of PfCRT, does not imply steric hindrance but rather the\nintrinsic single-substrate nature of the transporter in which only one ligand can occupy the central\ncavity at a time and preclude binding or transport of another. The competition pattern observed\nexperimentally is therefore consistent with all three compounds accessing the same substrate-\nbinding cavity” to “In our simulations with the solved 7G8 isoform structure and its 7G8+N246H variant, ZY19489 consistently sampled the same T76-adjacent region of the cavity, suggesting that this molecule, CQ, and PPQ might partially\noverlap in their binding to these two PfCRT isoforms. This interpretation aligns with our proteoliposome\ndata showing that ZY19489 inhibited transport of CQ and PPQ by their respective drug-resistant 7G8\nand 7G8+F145I PfCRT variants. One explanation could be mutual exclusion which, in the context of\nthese PfCRT isoforms, may suggest a single-substrate feature in which only one ligand could occupy\n4\nthe central cavity at a time and preclude binding or transport of another. Our proteoliposome competition\ndata suggest that all three compounds have at least partially overlapping cavity-binding properties.\nHowever, demonstrating true competitive inhibition and, by extension, confirmation of a single binding-\nsite model would require more detailed transport kinetic analyses40,43,44. It is important to recall that our\nsimulations and transport studies were conducted on 7G8 isoforms and as such this specific\ninterpretation cannot be extended as a generalization to other geographically distinct PfCRT variants.\nIn fact, variations in CQ and PPQ transport phenotypes have been observed between PfCRT isoforms\nexpressing mutant alleles from different geographic regions41,42, suggesting disparate interactions of\nthese drugs with residues within the central cavity. Additionally, various PfCRT isoforms appear to\npossess distinct substrate-binding cavity architectures43,44. Whereas evidence to data suggest that 7G8\nPfCRT may accommodate only a single ligand (either CQ or PPQ) at a time, transport kinetics and\ncomplementary computational studies have shown Dd2 PfCRT to bind both substrates simultaneously\nunder a partial noncompetitive inhibition model40,43,44, suggesting the presence of at least two distinct\ninteraction sites within the cavity of this isoform. We also note that isogenic parasites expressing Dd2,\nGB4 and FCB pfcrt alleles are known to differ in their CQ resistance profiles13, which also suggest\ndifferences in how these variants interact with antimalarial drugs.”\n5"}]

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