Heterologous Expression of a Novel Drug Transporter from the Malaria Parasite Alters Resistance to Quinoline Antimalarials

Abstract

Antimalarial drug resistance hampers effective malaria treatment. Critical SNPs in a particular, putative amino acid transporter were recently linked to chloroquine (CQ) resistance in malaria parasites. Here, we show that this conserved protein (PF3D7_0629500 in Plasmodium falciparum; AAT1 in P. chabaudi) is a structural homologue of the yeast amino acid transporter Tat2p, which is known to mediate quinine uptake and toxicity. Heterologous expression of PF3D7_0629500 in yeast produced CQ hypersensitivity, coincident with increased CQ uptake. PF3D7_0629500-expressing cultures were also sensitized to related antimalarials; amodiaquine, mefloquine and particularly quinine. Drug sensitivity was reversed by introducing a SNP linked to CQ resistance in the parasite. Like Tat2p, PF3D7_0629500-dependent quinine hypersensitivity was suppressible with tryptophan, consistent with a common transport mechanism. A four-fold increase in quinine uptake by PF3D7_0629500 expressing cells was abolished by the resistance SNP. The parasite protein localised primarily to the yeast plasma membrane. Its expression varied between cells and this heterogeneity was used to show that high-expressing cell subpopulations were the most drug sensitive. The results reveal that the PF3D7_0629500 protein can determine the level of sensitivity to several major quinine-related antimalarials through an amino acid-inhibitable drug transport function. The potential clinical relevance is discussed.

Novel Combinations of Agents Targeting Translation That Synergistically Inhibit Fungal Pathogens

“…We reasoned that this strain should be hyper-sensitive to a synergistic action which involves tryptophan starvation. As observed previously ( Khozoie et al, 2009 ; Tindall et al, 2018 ), the auxotrophic trp1 Δ mutant was sensitive to quinine alone (only a small sensitization was observable at the quinine concentration used here) ( Figures 7A,B ). However, trp1 Δ cells were no more sensitive to the quinine + bicarbonate combination, relative to quinine alone, than the wild type ( Figures 7A,C ).…”

“…Therefore, we focused on inhibitors targeting aromatic and sulfur-containing amino acids. Besides sulfate transport inhibitors (chromate, molybdate, orthovanadate, oxalate, malonate, probenecid, bicarbonate, and selenate), which provoke cysteine and methionine starvation ( Markovich, 2001 ; Holland et al, 2010 ; Moreno-Martinez et al, 2015 ), we examined amino acid transport inhibitors [quinine and eugenol inhibit aromatic amino acid uptake ( Khozoie et al, 2009 ; Darvishi et al, 2013 ; Tindall et al, 2018 )], amino acid biosynthesis inhibitors [cyprodinil inhibits methionine synthesis ( Masner et al, 1994 ; FRAC, 2018 )], non-proteinogenic amino acids [ L -DOPA, DL-ethionine, norvaline compete with proteinogenic amino acids ( Hartman et al, 2007 )], and agents that modify thiol containing amino acids [thiram, ziram, mancozeb ( Santos et al, 2009 ; Dias et al, 2010 ; Roede and Jones, 2014 ) and copper; copper additionally targets FeS biosynthesis required for synthesis of certain amino acids ( Alhebshi et al, 2012 ; Vallieres et al, 2017 )]. These agents were tested in combination with either: (i) another agent that impairs availability of functional amino acids, or (ii) an aminoglycoside antibiotic.…”

“…The hypothesized mode of action of the combinations was perturbation of mRNA translation fidelity, via amino acid depletion. The principal mode(s) of quinine drug action is unresolved, but one effect detected first in the yeast model is that quinine competes with tryptophan to cause tryptophan starvation ( Khozoie et al, 2009 ; Tindall et al, 2018 ). To test a role for tryptophan here, we used a S. cerevisiae trp1 Δ mutant defective for tryptophan biosynthesis, so reliant on uptake of the amino acid from the growth medium.…”

“…Quinine uptake was assayed by measuring quinine absorbance at 350 nm as described previously ( Tindall et al, 2018 ). Briefly, overnight cultures were diluted to OD 600 ∼0.5 in fresh YPD medium and cultured for a further 4 h with shaking.…”

Hybrid molecules with potential in vitro antiplasmodial and in vivo antimalarial activity against drug‐resistant Plasmodium falciparum

“…Quinoline antimalarials such as quinine (QN), ferroquine (FQ), chloroquine (CQ), amodiaquine (AQ), mefloquine (MQ) and primaquine (PQ) could kill P falciparum by blocking hemozoin formation or by docking into grooves on the hemozoin crystal and preventing further crystal growth . Quinoline antimalarials are used widely in clinics for the treatment of malaria, but like other antimalarials, P falciparum strains have already generated severe resistance against quinoline group . Among the drug‐resistant P falciparum strains, chloroquine‐resistant (CQR) parasites are very common in hospital.…”