The Malaria Parasite's Lactate Transporter PfFNT Is the Target of Antiplasmodial Compounds Identified in Whole Cell Phenotypic Screens

Hapuarachchi, Sanduni V.; Cobbold, Simon A.; Shafik, Sarah H.; Dennis, Adelaide S. M.; McConville, Malcolm J.; Martin, R. L.; Kirk, Kiaran; Lehane, Adele M.

doi:10.1371/journal.ppat.1006180

Cited by 39 publications

(84 citation statements)

References 55 publications

(79 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The situation is quite similar to malaria parasites, which express PfFNT from a single FNT encoding gene (2). Drug-like inhibitors of PfFNT led to accumulation of the metabolic end product L-lactate and to acidification of the cytosol killing the parasites (9,36). By making use of the preferential orientation of PfFNT in the proteoliposome membrane, we could now show pH-dependent transport via PfFNT (and EhFNT) in the physiological direction, i.e.…”

Section: Discussionmentioning

confidence: 79%

Formate–nitrite transporters carrying nonprotonatable amide amino acids instead of a central histidine maintain pH-dependent transport

Helmstetter

Arnold

Höger

et al. 2019

Journal of Biological Chemistry

View full text Add to dashboard Cite

Microbial formate-nitrite transporter-type proteins (FNT) exhibit dual transport functionality. At neutral pH, electrogenic anion currents are detectable, whereas upon acidification transport of the neutral, protonated monoacid predominates. Physiologically, FNT-mediated proton co-transport is vital when monocarboxylic acid products of the energy metabolism, such as L-lactate, are released from the cell. Accordingly, Plasmodium falciparum malaria parasites can be killed by small-molecule inhibitors of PfFNT. Two opposing hypotheses on the site of substrate protonation are plausible. The proton relay mechanism postulates proton transfer from a highly conserved histidine centrally positioned in the transport path. The dielectric slide mechanism assumes decreasing acidity of substrates entering the lipophilic vestibules and protonation via the bulk water. Here, we defined the transport mechanism of the FNT from the amoebiasis parasite Entamoeba histolytica, EhFNT, and also show that BtFdhC from Bacillus thuringiensis is a functional formate transporter. Both FNTs carry a nonprotonatable amide amino acid, asparagine or glutamine, respectively, at the central histidine position. Despite having a nonprotonatable residue, EhFNT displayed the same substrate selectivity for larger monocarboxylates including L-lactate, a low substrate affinity as is typical for FNTs, and, strikingly, proton motive force-dependent transport as observed for PfFNT harboring a central histidine. These results argue against a proton relay mechanism, indicating that substrate protonation must occur outside of the central histidine region, most likely in the vestibules. Furthermore, EhFNT is the sole annotated FNT in the Entamoeba genome suggesting that it could be a putative new drug target with similar utility as that of the malarial PfFNT.

show abstract

Section: Discussionmentioning

confidence: 79%

Formate–nitrite transporters carrying nonprotonatable amide amino acids instead of a central histidine maintain pH-dependent transport

Helmstetter

Arnold

Höger

et al. 2019

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…2). Two Malaria Box compounds (MMV007839 and MMV000972) that do not display the ionic signature of PfATP4 inhibition and that have been found to inhibit the unrelated (non-ATP-hydrolyzing) P. falciparum lactate/H ϩ transporter PfFNT (21) were included as controls. Neither of these compounds (each tested at 4 M) affected Na ϩ -dependent ATPase activity (Fig.…”

Section: Inhibition Of Na ؉ -Dependent Atpase Activity In Parasite Mementioning

confidence: 99%

Biochemical characterization and chemical inhibition of PfATP4-associated Na+-ATPase activity in Plasmodium falciparum membranes

Rosling

Ridgway

Summers

et al. 2018

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

The antimalarial activity of chemically diverse compounds, including the clinical candidate cipargamin, has been linked to the ATPase PfATP4 in the malaria-causing parasite The characterization of PfATP4 has been hampered by the inability thus far to achieve its functional expression in a heterologous system. Here, we optimized a membrane ATPase assay to probe the function of PfATP4 and its chemical sensitivity. We found that cipargamin inhibited the Na-dependent ATPase activity present in membranes from WT parasites and that its potency was reduced in cipargamin-resistant PfATP4-mutant parasites. The cipargamin-sensitive fraction of membrane ATPase activity was inhibited by all 28 of the compounds in the "Malaria Box" shown previously to disrupt ion regulation in in a cipargamin-like manner. This is consistent with PfATP4 being the direct target of these compounds. Characterization of the cipargamin-sensitive ATPase activity yielded data consistent with PfATP4 being a Na transporter that is sensitive to physiologically relevant perturbations of pH, but not of [K] or [Ca]. With an apparent for ATP of 0.2 mm and an apparent for Na of 16-17 mm, the protein is predicted to operate at below its half-maximal rate under normal physiological conditions, allowing the rate of Na efflux to increase in response to an increase in cytosolic [Na]. In membranes from a cipargamin-resistant PfATP4-mutant line, the apparent for Na is slightly elevated. Our study provides new insights into the biochemical properties and chemical sensitivity of an important new antimalarial drug target.

show abstract

“…Many of the microorganisms in which these FNT proteins are found are pathogens ( Waight et al, 2010 ; Czyzewski and Wang, 2012 ; Lü et al, 2012b ; Marchetti et al, 2015 ; Wu et al, 2015 ). Moreover, because FNT channels are not found in higher eukarya, this makes them suitable drug targets and recent studies on the PfFNT lactate channel in the malaria parasite Plasmodium falciparum has validated this prospect ( Golldack et al, 2017 ; Hapuarachchi et al, 2017 ). Notwithstanding their future potential importance in helping combat some infectious diseases, the study of these membrane proteins is of broad general interest for our understanding of the biophysical properties of membrane protein folding, the bioenergetics of substrate translocation through these channels and their role in anaerobic metabolism…”

Section: Introductionmentioning

confidence: 99%

The C-terminal Six Amino Acids of the FNT Channel FocA Are Required for Formate Translocation But Not Homopentamer Integrity

et al. 2017

View full text Add to dashboard Cite

FocA is the archetype of the pentameric formate-nitrite transporter (FNT) superfamily of channels, members of which translocate small organic and inorganic anions across the cytoplasmic membrane of microorganisms. The N- and C-termini of each protomer are cytoplasmically oriented. A Y-L-R motif is found immediately after transmembrane helix 6 at the C-terminus of FNT proteins related to FocA, or those with a role in formate translocation. Previous in vivo studies had revealed that formate translocation through FocA was controlled by interaction with the formate-producing glycyl-radical enzyme pyruvate formate-lyase (PflB) or its structural and functional homolog, TdcE. In this study we analyzed the effect on in vivo formate export and import, as well as on the stability of the homopentamer in the membrane, of successively removing amino acid residues from the C-terminus of FocA. Removal of up to five amino acids was without consequence for either formate translocation or oligomer stability. Removal of a sixth residue (R280) prevented formate uptake by FocA in a strain lacking PflB and significantly reduced, but did not prevent, formate export. Sensitivity to the toxic formate analog hypophosphite, which is also transported into the cell by FocA, was also relieved. Circular dichroism spectroscopy and blue-native PAGE analysis revealed, however, that this variant had near identical secondary and quaternary structural properties to those of native FocA. Interaction with the glycyl radical enzyme, TdcE, was also unaffected by removal of the C-terminal 6 amino acid residues, indicating that impaired interaction with TdcE was not the reason for impaired formate translocation. Removal of a further residue (L279) severely restricted formate export, the stability of the protein and its ability to form homopentamers. Together, these studies revealed that the Y278-L279-R280 motif at the C-terminus is essential for bidirectional formate translocation by FocA, but that L279 is both necessary and sufficient for homopentamer integrity.

show abstract

The Malaria Parasite's Lactate Transporter PfFNT Is the Target of Antiplasmodial Compounds Identified in Whole Cell Phenotypic Screens

Cited by 39 publications

References 55 publications

Formate–nitrite transporters carrying nonprotonatable amide amino acids instead of a central histidine maintain pH-dependent transport

Formate–nitrite transporters carrying nonprotonatable amide amino acids instead of a central histidine maintain pH-dependent transport

Biochemical characterization and chemical inhibition of PfATP4-associated Na+-ATPase activity in Plasmodium falciparum membranes

The C-terminal Six Amino Acids of the FNT Channel FocA Are Required for Formate Translocation But Not Homopentamer Integrity

Contact Info

Product

Resources

About