Balanced impacts of fitness and drug pressure on the evolution of PfMDR1 polymorphisms in Plasmodium falciparum.

BACKGROUND: Anti-malarial drug resistance may be limited by decreased fitness in resistant parasites. Important contributors to resistance are mutations in the Plasmodium falciparum putative drug transporter PfMDR1. METHODS: Impacts on in vitro fitness of two common PfMDR1 polymorphisms, N86Y, which is associated with sensitivity to multiple drugs, and Y184F, which has no clear impact on drug sensitivity, were evaluated to study associations between resistance mediators and parasite fitness, measured as relative growth in competitive culture experiments. NF10 P. falciparum lines engineered to represent all PfMDR1 N86Y and Y184F haplotypes were co-cultured for 40 days, and the genetic make-up of the cultures was characterized every 4 days by pyrosequencing. The impacts of culture with anti-malarials on the growth of different haplotypes were also assessed. Lastly, the engineering of P. falciparum containing another common polymorphism, PfMDR1 D1246Y, was attempted. RESULTS: Co-culture results were as follows. With wild type (WT) Y184 fixed (N86/Y184 vs. 86Y/Y184), parasites WT and mutant at 86 were at equilibrium. With mutant 184 F fixed (N86/184F vs. 86Y/184F), mutants at 86 overgrew WT. With WT N86 fixed (N86/Y184 vs. N86/184F), WT at 184 overgrew mutants. With mutant 86Y fixed (86Y/Y184 vs. 86Y/184F), WT and mutant at 86 were at equilibrium. Parasites with the double WT were in equilibrium with the double mutant, but 86Y/Y184 overgrew N86/184F. Overall, WT N86/mutant 184F parasites were less fit than parasites with all other haplotypes. Parasites engineered for another mutation, PfMDR1 1246Y, were unstable in culture, with reversion to WT over time. Thus, the N86 WT is stable when accompanied by the Y184 WT, but incurs a fitness cost when accompanied by mutant 184F. Culturing in the presence of chloroquine favored 86Y mutant parasites and in the presence of lumefantrine favored N86 WT parasites; piperaquine had minimal impact. CONCLUSIONS: These results are consistent with those for Ugandan field isolates, suggest reasons for varied haplotypes, and highlight the interplay between drug pressure and fitness that is guiding the evolution of resistance-mediating haplotypes in P. falciparum

Drug susceptibility of Plasmodium falciparum in eastern Uganda: a longitudinal phenotypic and genotypic study

Background: Treatment and control of malaria depends on artemisinin-based combination therapies (ACTs) and is challenged by drug resistance, but thus far resistance to artemisinins and partner drugs has primarily occurred in southeast Asia. The aim of this study was to characterise antimalarial drug susceptibility of Plasmodium falciparum isolates from Tororo and Busia districts in Uganda. Methods: In this prospective longitudinal study, P falciparum isolates were collected from patients aged 6 months or older presenting at the Tororo District Hospital (Tororo district, a site with relatively low malaria incidence) or Masafu General Hospital (Busia district, a high-incidence site) in eastern Uganda with clinical symptoms of malaria, a positive Giemsa-stained blood film for P falciparum, and no signs of severe disease. Ex-vivo susceptibilities to ten antimalarial drugs were measured using a 72-h microplate growth inhibition assay with SYBR Green detection. Relevant P falciparum genetic polymorphisms were characterised by molecular methods. We compared results with those from earlier studies in this region and searched for associations between drug susceptibility and parasite genotypes. Findings: From June 10, 2016, to July 29, 2019, 361 P falciparum isolates were collected in the Busia district and 79 in the Tororo district from 440 participants. Of 440 total isolates, 392 (89%) successfully grew in culture and showed excellent drug susceptibility for chloroquine (median half-maximal inhibitory concentration [IC50] 20·0 nM [IQR 12·0-26·0]), monodesethylamodiaquine (7·1 nM [4·3-8·9]), pyronaridine (1·1 nM [0·7-2·3]), piperaquine (5·6 nM [3·3-8·6]), ferroquine (1·8 nM [1·5-3·3]), AQ-13 (24·0 nM [17·0-32·0]), lumefantrine (5·1 nM [3·2-7·7]), mefloquine (9·5 nM [6·6-13·0]), dihydroartemisinin (1·5 nM [1·0-2·0]), and atovaquone (0·3 nM [0·2-0·4]). Compared with results from our study in 2010-13, significant improvements in susceptibility were seen for chloroquine (median IC50 288·0 nM [IQR 122·0-607·0]; p\u3c0·0001), monodesethylamodiaquine (76·0 nM [44·0-137]; p\u3c0·0001), and piperaquine (21·0 nM [7·6-43·0]; p\u3c0·0001), a small but significant decrease in susceptibility was seen for lumefantrine (3·0 nM [1·1-7·6]; p\u3c0·0001), and no change in susceptibility was seen with dihydroartemisinin (1·3 nM [0·8-2·5]; p=0·64). Chloroquine resistance (IC50\u3e100 nM) was more common in isolates from the Tororo district (11 [15%] of 71), compared with those from the Busia district (12 [4%] of 320; p=0·0017). We showed significant increases between 2010-12 and 2016-19 in the prevalences of wild-type P falciparum multidrug resistance protein 1 (PfMDR1) Asn86Tyr from 60% (391 of 653) to 99% (418 of 422; p\u3c0·0001), PfMDR1 Asp1246Tyr from 60% (390 of 650) to 90% (371 of 419; p\u3c0·0001), and P falciparum chloroquine resistance transporter (PfCRT) Lys76Thr from 7% (44 of 675) to 87% (364 of 417; p\u3c0·0001). Interpretation: Our results show marked changes in P falciparum drug susceptibility phenotypes and genotypes in Uganda during the past decade. These results suggest that additional changes will be seen over time and continued surveillance of susceptibility to key ACT components is warranted. Funding: National Institutes of Health and Medicines for Malaria Venture

Associations between Varied Susceptibilities to PfATP4 Inhibitors and Genotypes in Ugandan Plasmodium falciparum Isolates.

Among novel compounds under recent investigation as potential new antimalarial drugs are three independently developed inhibitors of the Plasmodium falciparum P-type ATPase (PfATP4): KAE609 (cipargamin), PA92, and SJ733. We assessed ex vivo susceptibilities to these compounds of 374 fresh P. falciparum isolates collected in Tororo and Busia districts, Uganda, from 2016 to 2019. Median IC50s were 65 nM for SJ733, 9.1 nM for PA92, and 0.5 nM for KAE609. Sequencing of pfatp4 for 218 of these isolates demonstrated many nonsynonymous single nucleotide polymorphisms; the most frequent mutations were G1128R (69% of isolates mixed or mutant), Q1081K/R (68%), G223S (25%), N1045K (16%), and D1116G/N/Y (16%). The G223S mutation was associated with decreased susceptibility to SJ733, PA92, and KAE609. The D1116G/N/Y mutations were associated with decreased susceptibility to SJ733, and the presence of mutations at both codons 223 and 1116 was associated with decreased susceptibility to PA92 and SJ733. In all of these cases, absolute differences in susceptibilities of wild-type (WT) and mutant parasites were modest. Analysis of clones separated from mixed field isolates consistently identified mutant clones as less susceptible than WT. Analysis of isolates from other sites demonstrated the presence of the G223S and D1116G/N/Y mutations across Uganda. Our results indicate that malaria parasites circulating in Uganda have a number of polymorphisms in PfATP4 and that modestly decreased susceptibility to PfATP4 inhibitors is associated with some mutations now present in Ugandan parasites

Antimalarial proteasome inhibitor reveals collateral sensitivity from intersubunit interactions and fitness cost of resistance.

We describe noncovalent, reversible asparagine ethylenediamine (AsnEDA) inhibitors of the Plasmodium falciparum proteasome (Pf20S) β5 subunit that spare all active subunits of human constitutive and immuno-proteasomes. The compounds are active against erythrocytic, sexual, and liver-stage parasites, against parasites resistant to current antimalarials, and against P. falciparum strains from patients in Africa. The β5 inhibitors synergize with a β2 inhibitor in vitro and in mice and with artemisinin. P. falciparum selected for resistance to an AsnEDA β5 inhibitor surprisingly harbored a point mutation in the noncatalytic β6 subunit. The β6 mutant was resistant to the species-selective Pf20S β5 inhibitor but remained sensitive to the species-nonselective β5 inhibitors bortezomib and carfilzomib. Moreover, resistance to the Pf20S β5 inhibitor was accompanied by increased sensitivity to a Pf20S β2 inhibitor. Finally, the β5 inhibitor-resistant mutant had a fitness cost that was exacerbated by irradiation. Thus, used in combination, multistage-active inhibitors of the Pf20S β5 and β2 subunits afford synergistic antimalarial activity with a potential to delay the emergence of resistance to artemisinins and each other

Development of a Highly Selective Plasmodium falciparum Proteasome Inhibitor with Anti-malaria Activity in Humanized Mice.

Plasmodium falciparum proteasome (Pf20S) inhibitors are active against Plasmodium at multiple stages-erythrocytic, gametocyte, liver, and gamete activation stages-indicating that selective Pf20S inhibitors possess the potential to be therapeutic, prophylactic, and transmission-blocking antimalarials. Starting from a reported compound, we developed a noncovalent, macrocyclic peptide inhibitor of the malarial proteasome with high species selectivity and improved pharmacokinetic properties. The compound demonstrates specific, time-dependent inhibition of the β5 subunit of the Pf20S, kills artemisinin-sensitive and artemisinin-resistant P. falciparum isolates in vitro and reduces parasitemia in humanized, P. falciparum-infected mice

Preclinical characterization and target validation of the antimalarial pantothenamide MMV693183.

Drug resistance and a dire lack of transmission-blocking antimalarials hamper malaria elimination. Here, we present the pantothenamide MMV693183 as a first-in-class acetyl-CoA synthetase (AcAS) inhibitor to enter preclinical development. Our studies demonstrate attractive drug-like properties and in vivo efficacy in a humanized mouse model of Plasmodium falciparum infection. The compound shows single digit nanomolar in vitro activity against P. falciparum and P. vivax clinical isolates, and potently blocks P. falciparum transmission to Anopheles mosquitoes. Genetic and biochemical studies identify AcAS as the target of the MMV693183-derived antimetabolite, CoA-MMV693183. Pharmacokinetic-pharmacodynamic modelling predict that a single 30 mg oral dose is sufficient to cure a malaria infection in humans. Toxicology studies in rats indicate a \u3e 30-fold safety margin in relation to the predicted human efficacious exposure. In conclusion, MMV693183 represents a promising candidate for further (pre)clinical development with a novel mode of action for treatment of malaria and blocking transmission

Fitness Consequences of Plasmodium falciparum pfmdr1 Polymorphisms Inferred from Ex Vivo Culture of Ugandan Parasites

Polymorphisms in the Plasmodium falciparum multidrug resistance 1 (pfmdr1) gene impact sensitivity to multiple antimalarials. In Africa, polymorphisms at N86Y and D1246Y are common and have various impacts on sensitivity to different drugs. To gain insight into the fitness consequences of these polymorphisms, we cultured parasites isolated from children with malaria in Tororo, Uganda, where the multiplicity of infection is high, and used pyrosequencing to follow polymorphism prevalences in culture over time. Of 71 cultures, parasites in 69 were successfully analyzed at N86Y and parasites in 68 were successfully analyzed at D1246Y over 3 to 36 days of culture. For position 86, the sequences of 39/69 (56.5%) parasites remained stable (>90% prevalence over 2 to 17 time points), with 82.1% of these being stable for the 86Y mutation. For position 1246, the sequences of 31/68 (45.6%) parasites remained stable, with 64.5% of these being stable for the wild-type D1246 sequence (P = 0.0002 for comparison of stable mutant genotypes for the two alleles). Defining allele selection as a ≥15% change in prevalence between the first and last samples assessed, for position 86, 11 samples showed selection, with selection toward 86Y occurring in 72.7% of alleles; for position 1246, 14 samples showed selection, with selection toward D1246 occurring in 64.3% of alleles (P = 0.11 for comparison of selection of mutations at the two alleles). Among the 7 samples with selection at both alleles, 5 showed selection for both 86Y and D1246. Overall, consistent trends in the direction of selection were seen, although differences were not statistically significant. Our results suggest fitness advantages for parasites with the pfmdr1 86Y mutation and wild-type D1246, highlighting the complex interplay between drug resistance and fitness in malaria parasites. (This study has been registered at ClinicalTrials.gov under registration no. NCT00948896 and NCT00993031.)

Balanced impacts of fitness and drug pressure on the evolution of PfMDR1 polymorphisms in Plasmodium falciparum.

BackgroundAnti-malarial drug resistance may be limited by decreased fitness in resistant parasites. Important contributors to resistance are mutations in the Plasmodium falciparum putative drug transporter PfMDR1.MethodsImpacts on in vitro fitness of two common PfMDR1 polymorphisms, N86Y, which is associated with sensitivity to multiple drugs, and Y184F, which has no clear impact on drug sensitivity, were evaluated to study associations between resistance mediators and parasite fitness, measured as relative growth in competitive culture experiments. NF10 P. falciparum lines engineered to represent all PfMDR1 N86Y and Y184F haplotypes were co-cultured for 40 days, and the genetic make-up of the cultures was characterized every 4 days by pyrosequencing. The impacts of culture with anti-malarials on the growth of different haplotypes were also assessed. Lastly, the engineering of P. falciparum containing another common polymorphism, PfMDR1 D1246Y, was attempted.ResultsCo-culture results were as follows. With wild type (WT) Y184 fixed (N86/Y184 vs. 86Y/Y184), parasites WT and mutant at 86 were at equilibrium. With mutant 184 F fixed (N86/184F vs. 86Y/184F), mutants at 86 overgrew WT. With WT N86 fixed (N86/Y184 vs. N86/184F), WT at 184 overgrew mutants. With mutant 86Y fixed (86Y/Y184 vs. 86Y/184F), WT and mutant at 86 were at equilibrium. Parasites with the double WT were in equilibrium with the double mutant, but 86Y/Y184 overgrew N86/184F. Overall, WT N86/mutant 184F parasites were less fit than parasites with all other haplotypes. Parasites engineered for another mutation, PfMDR1 1246Y, were unstable in culture, with reversion to WT over time. Thus, the N86 WT is stable when accompanied by the Y184 WT, but incurs a fitness cost when accompanied by mutant 184F. Culturing in the presence of chloroquine favored 86Y mutant parasites and in the presence of lumefantrine favored N86 WT parasites; piperaquine had minimal impact.ConclusionsThese results are consistent with those for Ugandan field isolates, suggest reasons for varied haplotypes, and highlight the interplay between drug pressure and fitness that is guiding the evolution of resistance-mediating haplotypes in P. falciparum