Biology subjects

Lehane, A. M.

Publications and source records attributed to Lehane, A. M..

6 recordsLinked to original sources

bioRxiv2026

Mutations within and outside the PfFNT transport pathway drive MMV007839 resistance in Plasmodium falciparum

Plasmodium falciparum, the parasite responsible for the majority of malaria cases and deaths worldwide, poses a major global health challenge due to its ability to rapidly develop resistance to antimalarial drugs. To circumvent existing resistance pathways, it is of interest to identify both new inhibitors and new therapeutic targets within the parasite. One such target is the Plasmodium falciparum Formate-Nitrite Transporter (PfFNT), which mediates essential lactate and proton (H+) export from the parasite cytosol during the intra-erythrocytic stage. Several inhibitors of PfFNT have been identified that block lactate/H transport and/or kill parasites in vitro, including MMV007839, which binds within the substrate transport pathway as revealed by cryo- EM structures. However, three resistance-conferring mutations have previously been identified following prolonged in vitro exposure of parasites to PfFNT inhibitors. Here, we performed further in vitro evolution experiments with MMV007839 in P. falciparum and identified two additional mutations in PfFNT, V200L and L198M, the latter of which is located outside the substrate transport pathway. These mutations are associated with 6-fold and 13-fold changes in the IC50 of MMV007839. Using molecular dynamics simulations, and thermodynamic integration, we show that these mutations, together with previously identified resistance mutations, destabilise inhibitor binding within the transport pathway; and we suggest the molecular mechanisms underlying this destabilisation. Finally, using both molecular docking and additional simulations, we suggest new sites where additional mutations that confer resistance to PfFNT inhibitors may arise. Author SummaryMalaria parasites are becoming resistant to existing drugs, creating an urgent need for new treatments. One promising drug target is the Plasmodium falciparum Formate-Nitrite Transporter (PfFNT), which the parasite needs to remove waste products and survive during infection. Although some compounds that block PfFNT have been identified, several mutations in the protein can make these compounds ineffective. To better understand how resistance might develop, we exposed malaria parasites to the PfFNT inhibitor MMV007839 in the laboratory and identified two new resistance-related mutations, one of which lies outside the MMV007839-binding site. We also used computer models to predict other regions where resistance-conferring mutations could occur. These findings help assess PfFNT as a drug target and provide guidance for designing new drugs that are less likely to be undermined by resistance.

biophysics↗

bioRxiv2026

A proton transfer mechanism in the malaria parasite lactate/H+ symporter reveals a channel-like transporter without conformational changes

The malaria parasite Plasmodium falciparum relies on anaerobic glycolysis for energy during the intraerythrocytic stage, producing lactate and H+ that must be extruded via its formate-nitrite transporter (PfFNT) to prevent death by cytosolic acidification and swelling. Unlike bacterial homologues classified as channels, PfFNT mediates saturable lactate transport and was classified as a transporter, raising questions about its transport mechanism and what distinguishes channels from transporters. Here, we combine over 720 s of molecular dynamics simulations, quantum chemical calculations, and transport assays in Xenopus laevis oocytes to characterize the transport cycle of PfFNT. We show a transport mechanism in which protonation of a central histidine (H230) enables lactate binding, and proton transfer from H230 to lactate is required to release the substrate as neutral lactic acid. This mechanism is supported by data indicating that neutral compounds such as lactamide do not enter the cavity, while anions like iodide, which are unlikely to protonate under physiological conditions, bind in the cavity but are not released and thus block lactate transport. As neither lactate nor lactic acid can diffuse passively through the protein, it is the requirement for proton transfer defines PfFNT as a lactate/H+ transporter rather than a channel. Notably, the PfFNT transport process occurs without significant protein conformational changes. Furthermore, our data indicates that under acidic conditions, small neutral molecules such as formic acid can passively diffuse through the protein. This suggests PfFNT challenges standard definitions of channels and transporters and differs from existing examples of channel-like transporters as it i) operates as a transporter without conformational changes and ii) can switch between transporter and channel modes within the same transport pathway.

biophysics↗

bioRxiv2025

Identification of α-azacyclic acetamide-based inhibitors of P. falciparum Na+ pump (PfATP4) with fast-killing asexual blood-stage antimalarial activity by phenotypic screening

Malaria treatments are compromised by drug resistance, creating an urgent need to discover new drugs. We used a phenotypic high-throughput screening (HTS) platform to identify new antimalarials, uncovering three related pyrrole-, indole-, and indoline-based series with a shared -azacyclic acetamide core. These compounds showed fast-killing activity on asexual blood-stage Plasmodium falciparum parasites, were not cytotoxic, and disrupted parasite intracellular pH and Na+ regulation similarly to cipargamin (KAE609), a clinically advanced inhibitor of the P. falciparum Na+ pump (PfATP4). PfATP4 is localized to the parasite plasma membrane and is essential for maintaining a low cytosolic Na+ concentration. Resistance selections on P. falciparum parasites with two -azacyclic acetamide analogs identified mutations in PfATP4, and cross-resistance was observed across the -azacyclic acetamides and KAE609, confirming PfATP4 as the target. PfATP4 is a well-established antimalarial target, and identification of additional PfATP4 inhibitors provides alternative avenues to disrupt its function.

microbiology↗

bioRxiv2024

PfATP2 is an essential flippase on the Plasmodium falciparum surface that influences parasite sensitivity to antiplasmodial compounds

Antimalarials play a crucial role in the fight against malaria. However, resistance of the most virulent malaria parasite, Plasmodium falciparum, to front-line antimalarials is spreading. To identify new antimalarials, millions of compounds have been screened for their ability to inhibit the growth of blood-stage P. falciparum parasites. To gain insight into the mode of action of novel compounds and the ease by which parasites can acquire resistance to them, many have been tested in in vitro evolution experiments, in which parasites are exposed to the compound for a prolonged period of time. In a recent study, parasite resistance to two compounds, MMV007224 and MMV665852, was associated with amplification of the pfatp2 gene, implicating PfATP2, a putative phospholipid flippase, as a parasite drug target or resistance determinant. These two compounds, along with MMV665794 (which is structurally related to MMV007224) had previously been reported to dysregulate pH in parasites. Here, we show that PfATP2 localises to the parasite surface and is essential for parasite growth. We demonstrate that parasites genetically engineered to overexpress PfATP2 display reduced sensitivity to MMV665794, MMV007224 and MMV665852 compared to parasites with a normal expression level of the protein, and that parasites in which PfATP2 is knocked down become hypersensitive to the three compounds. We show that PfATP2 expression level does not affect the cytosolic pH of parasites, or the potency by which MMV665794 or MMV007224 dysregulate parasite pH. We show that PfATP2-overexpressing parasites internalise a fluorescent phosphatidylserine analogue (NBD-PS) at a greater rate than parasites with a normal expression level of PfATP2, and that parasites in which PfATP2 is knocked down have a reduced rate of NBD-PS uptake. Further, we provide evidence that MMV665794 and MMV007224 inhibit both ATP-dependent and ATP-independent NBD-PS internalisation mechanisms, the former at lower concentrations. Together, our data are consistent with PfATP2 serving as a major ATP-dependent phosphatidylserine internalisation mechanism at the parasite plasma membrane, and being a target of MMV665794 and MMV007224.

microbiology↗

bioRxiv2022

Generation and characterisation of P. falciparum parasites with a G358S mutation in the PfATP4 Na+ pump and clinically relevant levels of resistance to some PfATP4 inhibitors

Small-molecule inhibitors of PfATP4, a Plasmodium falciparum protein that is believed to pump Na+ out of the parasite while importing H+, are on track to become much-needed new antimalarial drugs. The spiroindolone cipargamin is poised to become the first PfATP4 inhibitor to reach the field, having performed strongly in Phase 1 and 2 clinical trials. Previous attempts to generate cipargamin-resistant parasites in the laboratory have yielded parasites with reduced susceptibility to the drug; however, the highest 50% inhibitory concentration reported to date is 24 nM. Here, we show that P. falciparum parasites can acquire a clinically-significant level of resistance to cipargamin that enables them to withstand micromolar concentrations of the drug. Independent experiments to generate high-level cipargamin resistance using different protocols and strains led to the same change each time - a G358S mutation in PfATP4. Parasites with this mutation showed high-level resistance not only to cipargamin, but also to the dihydroisoquinolone (+)-SJ733. However, for certain other (less clinically advanced) PfATP4-associated compounds the G358S mutation in PfATP4 conferred only moderate resistance or no resistance. The G358S mutation in PfATP4 did not affect parasite susceptibility to antimalarials that do not target PfATP4. The G358S mutation in PfATP4, and the equivalent mutation in the Toxoplasma gondii ATP4 homologue (G419S), decreased the sensitivity of the Na+-ATPase activity of ATP4 to inhibition by cipargamin and (+)-SJ733, and decreased the sensitivity of parasites expressing these ATP4 mutations to disruption of parasite Na+ regulation by cipargamin- and (+)-SJ733. The G358S mutation in PfATP4 reduced the affinity of the protein for Na+ and was associated with an increase in the parasites resting cytosolic Na+ concentration; however, no significant defect in parasite growth rate was observed. Our findings suggest that codon 358 in pfatp4 should be monitored closely in the field as a molecular marker for cipargamin resistance, and that PfATP4 inhibitors in clinical development should be tested for their activity against PfATP4G358S parasites.

microbiology↗

bioRxiv2018

Protein Kinase A Negatively Regulates Ca2+ signalling in Toxoplasma gondii

The phylum Apicomplexa comprises a group of obligate intracellular parasites that alternate between intracellular replicating forms and actively motile extracellular forms that move through tissue. Parasite cytosolic Ca2+ signalling activates motility, but how this is switched off after invasion is not understood. Here we show that the cAMP-dependent Protein Kinase A catalytic subunit 1 (PKAc1) of Toxoplasma is responsible for suppression of Ca2+ signalling upon host cell invasion. We demonstrate that that PKAc1 is sequestered to the parasite periphery by dual acylation of its regulatory subunit PKAr1. Newly invaded PKAc1-deficient parasites exit host cells shortly thereafter in a perforin-like protein 1 (PLP-1)-dependent fashion. We demonstrate that loss of PKAc1 results in an inability to rapidly downregulate cytosolic Ca2+ levels shortly after invasion. Furthermore, we demonstrate that PKAc1 also specifically negatively regulates resting cytosolic Ca2+ in conditions that mimic intracellularity. We also show that cAMP and cGMP have opposing role in microneme secretion, further supporting evidence that cAMP signalling has a suppressive role during motility. Together, this work provides a new paradigm in understanding how Toxoplasma and related apicomplexan parasites regulate infectivity.

microbiology↗