A G358S mutation in the Plasmodium falciparum Na⁺ pump PfATP4 confers clinically-relevant resistance to cipargamin

Diverse compounds target the Plasmodium falciparum Na(+) pump PfATP4, with cipargamin and (+)-SJ733 the most clinically-advanced. In a recent clinical trial for cipargamin, recrudescent parasites emerged, with most having a G358S mutation in PfATP4. Here, we show that PfATP4(G358S) parasites can withstand micromolar concentrations of cipargamin and (+)-SJ733, while remaining susceptible to antimalarials that do not target PfATP4. The G358S mutation in PfATP4, and the equivalent mutation in Toxoplasma gondii ATP4, decrease the sensitivity of ATP4 to inhibition by cipargamin and (+)-SJ733, thereby protecting parasites from disruption of Na(+) regulation. The G358S mutation reduces the affinity of PfATP4 for Na(+) and is associated with an increase in the parasite’s resting cytosolic [Na(+)]. However, no defect in parasite growth or transmissibility is observed. Our findings suggest that PfATP4 inhibitors in clinical development should be tested against PfATP4(G358S) parasites, and that their combination with unrelated antimalarials may mitigate against resistance development

Impact of opioid-free analgesia on pain severity and patient satisfaction after discharge from surgery: multispecialty, prospective cohort study in 25 countries

Background: Balancing opioid stewardship and the need for adequate analgesia following discharge after surgery is challenging. This study aimed to compare the outcomes for patients discharged with opioid versus opioid-free analgesia after common surgical procedures.Methods: This international, multicentre, prospective cohort study collected data from patients undergoing common acute and elective general surgical, urological, gynaecological, and orthopaedic procedures. The primary outcomes were patient-reported time in severe pain measured on a numerical analogue scale from 0 to 100% and patient-reported satisfaction with pain relief during the first week following discharge. Data were collected by in-hospital chart review and patient telephone interview 1 week after discharge.Results: The study recruited 4273 patients from 144 centres in 25 countries; 1311 patients (30.7%) were prescribed opioid analgesia at discharge. Patients reported being in severe pain for 10 (i.q.r. 1-30)% of the first week after discharge and rated satisfaction with analgesia as 90 (i.q.r. 80-100) of 100. After adjustment for confounders, opioid analgesia on discharge was independently associated with increased pain severity (risk ratio 1.52, 95% c.i. 1.31 to 1.76; P < 0.001) and re-presentation to healthcare providers owing to side-effects of medication (OR 2.38, 95% c.i. 1.36 to 4.17; P = 0.004), but not with satisfaction with analgesia (beta coefficient 0.92, 95% c.i. -1.52 to 3.36; P = 0.468) compared with opioid-free analgesia. Although opioid prescribing varied greatly between high-income and low- and middle-income countries, patient-reported outcomes did not.Conclusion: Opioid analgesia prescription on surgical discharge is associated with a higher risk of re-presentation owing to side-effects of medication and increased patient-reported pain, but not with changes in patient-reported satisfaction. Opioid-free discharge analgesia should be adopted routinely

Defense peptides engineered from human platelet factor 4 kill Plasmodium by selective membrane disruption

Malaria is a serious threat to human health and additional classes of antimalarial drugs are greatly needed. The human defense protein, platelet factor\ua04 (PF4), has intrinsic antiplasmodial activity but also undesirable chemokine properties. We engineered a peptide containing the isolated PF4 antiplasmodial\ua0domain, which through cyclization, retained the critical structure of the parent protein. The peptide, cPF4PD, killed cultured blood-stage Plasmodium falciparum with low micromolar potency by specific disruption of the parasite digestive vacuole. Its mechanism of action involved selective penetration and accumulation inside the intraerythrocytic parasite without damaging the host cell or parasite membranes; it did not accumulate in uninfected cells. This selective activity was accounted for by observations of the peptide's specific binding and penetration of\ua0membranes with exposed negatively charged phospholipid headgroups. Our findings highlight the tremendous potential of the cPF4PD scaffold for developing antimalarial peptide drugs with a distinct and selective mechanism of action

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

<div><p>In this study the ‘Malaria Box’ chemical library comprising 400 compounds with antiplasmodial activity was screened for compounds that perturb the internal pH of the malaria parasite, <i>Plasmodium falciparum</i>. Fifteen compounds induced an acidification of the parasite cytosol. Two of these did so by inhibiting the parasite’s formate nitrite transporter (PfFNT), which mediates the H⁺-coupled efflux from the parasite of lactate generated by glycolysis. Both compounds were shown to inhibit lactate transport across the parasite plasma membrane, and the transport of lactate by PfFNT expressed in <i>Xenopus laevis</i> oocytes. PfFNT inhibition caused accumulation of lactate in parasitised erythrocytes, and swelling of both the parasite and parasitised erythrocyte. Long-term exposure of parasites to one of the inhibitors gave rise to resistant parasites with a mutant form of PfFNT that showed reduced inhibitor sensitivity. This study provides the first evidence that PfFNT is a druggable antimalarial target.</p></div

Fluorescence traces showing the effects of DMSO (solvent control), MMV007839 and MMV000972 on the cytosolic pH (pH_cyt) of 3D7 <i>P</i>. <i>falciparum</i> trophozoites under varying conditions.

<p>Each trace is representative of those obtained in at least three independent experiments. (A-C) The effects of DMSO (0.1% v/v; solvent control; A), MMV007839 (1 μM; B) and MMV000972 (1 μM; C) on the cytosolic pH of parasites suspended in (glucose-containing) Experimental Saline Solution (pH 7.10) at 37°C. (D-F) The effects of DMSO (0.25% v/v; solvent control; D), MMV007839 (2.5 μM; E), and MMV000972 (2.5 μM; F) on the cytosolic pH of parasites suspended (for 10 min prior to commencing recording, and throughout the initial recording period) in glucose-free Experimental Saline Solution (pH 7.10, 37°C), and the effects of the subsequent addition of glucose (22 mM) to the parasite suspension. (G-I) The effects of adding DMSO (0.25% v/v; solvent control; G), MMV007839 (2.5 μM; H), and MMV000972 (2.5 μM; I), followed by sodium L-lactate (10 mM), on the cytosolic pH of parasites suspended at 4°C in Experimental Saline Solution (pH 7.10).</p

L-[¹⁴C]lactate influx into PfFNT^Gly107Ser mutant parasites and Dd2 parental parasites.

<p>(A) The uptake of L-[¹⁴C]lactate by isolated trophozoite-stage PfFNT^Gly107Ser mutant parasites (white bars) and Dd2 parental parasites (black bars) in the absence of compound (0.4% v/v DMSO; solvent control) and in the presence of the non-specific anion transport inhibitor NPPB (100 μM). The mean distribution ratio (i.e., [intracellular L-[¹⁴C]lactate]/[extracellular L-[¹⁴C]lactate]) and SEM from nine (parent) or seven (mutant) independent experiments for the control, and from three independent experiments for NPPB, are shown. The results of unpaired t-tests are shown; ***<i>P</i> < 0.001. (B-C) The effects of MMV007839 (B) and MMV000972 (C) on L-[¹⁴C]lactate uptake into isolated trophozoite-stage PfFNT^Gly107Ser mutant parasites (white symbols) and Dd2 parental parasites (black symbols). L-[¹⁴C]lactate uptake in the presence of each concentration of MMV compound is expressed as a percentage of that observed when there was no inhibition of transport. The data shown are the average ± SEM from <i>n</i> independent experiments performed on different days, where <i>n</i> was seven for the MMV007839 data with parental parasites, three for the MMV007839 data with PfFNT^Gly107Ser mutant parasites, three for the MMV000972 data with parental parasites, and four for the MMV000972 data with PfFNT^Gly107Ser mutant parasites (with the exception of the lowest concentration, for which there was one less experiment in all cases). In all cases (panels A-C), the experiments were performed at 4°C in pH 6.1 Experimental Saline Solution, and parasites were preincubated with the compound (at the concentration indicated) or DMSO (0.4% v/v; solvent control) for 1 min prior to the addition of L-[¹⁴C]lactate (1.3 μM). The concentrations of compound and DMSO after the addition of L-[¹⁴C]lactate were the same as those present during the preincubation. The uptake of L-[¹⁴C]lactate was measured over 20 s.</p

Representative traces illustrating the strategy employed to screen the Malaria Box for compounds that perturb the cytosolic pH (pH_cyt) of the parasite.

<p>Malaria Box compounds (1 μM; from the May 2012 Malaria Box batch; identified here by their positions on the plates) were added successively to BCECF-loaded isolated 3D7 <i>P</i>. <i>falciparum</i> trophozoites (suspended at 37°C in pH 7.10 Experimental Saline Solution). A maximum of nine inactive compounds were tested on a single batch of parasites before the V-type H⁺-ATPase inhibitor concanamycin A (conA; 100 nM) was added as a positive control to ensure that a pH change was still detectable (A). A batch of parasites was also replaced with a new batch after a hit was detected (B) or a compound caused an optical effect (seen as an instantaneous perturbation of the fluorescence signal; C). In the latter case concanamycin A (100 nM) was added before changing to another batch of parasites. The purpose of adding the concanamycin A was to confirm (or otherwise) that any such optical effect did not obscure the pH-responsiveness of the fluorescence signal.</p

The effects of MMV007839 and MMV000972 on PfFNT-mediated L-[¹⁴C]lactate uptake by <i>Xenopus</i> oocytes expressing native PfFNT or PfFNT^Gly107Ser.

<p>The PfFNT-mediated uptake of L-[¹⁴C]lactate by oocytes expressing native PfFNT (black symbols) or oocytes expressing PfFNT^Gly107Ser (white symbols) was measured in the presence of a range of different concentrations of MMV007839 (A) and MMV000972 (B). The component of transport attributable to PfFNT (or to PfFNT^Gly107Ser) was determined by subtracting the uptake of L-[¹⁴C]lactate into non-injected oocytes (averaged from the values obtained across the full range of inhibitor concentrations tested, noting that the uptake of L-[¹⁴C]lactate into non-injected oocytes was unaffected by either inhibitor; <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006180#ppat.1006180.s006" target="_blank">S4 Fig</a>) from the uptake of L-[¹⁴C]lactate measured (at each inhibitor concentration) into oocytes expressing either native PfFNT or PfFNT^Gly107Ser. In all panels, the PfFNT-mediated uptake of L-[¹⁴C]lactate is expressed as a percentage of that determined for native PfFNT in the absence of MMV compound (0.4% v/v DMSO; solvent control). To enable a visual comparison between the native PfFNT and PfFNT^Gly107Ser datasets, only part of the concentration range used for the latter is shown in the main panels. The insets show the data obtained for PfFNT^Gly107Ser over the full concentration range tested. In all cases, the data are averaged from four independent experiments (using oocytes from different frogs) and are shown ± SEM. The uptake was measured at 27.5°C over 10 min at pH 6.4 using 2.7 μM L-[¹⁴C]lactate and 600 μM unlabelled L-lactate.</p

The effect of MMV007839 on the metabolite profile of parasitised erythrocytes.

<p>(A) Relative amounts of intracellular metabolites in MMV007839-treated parasitised erythrocytes (infected with the 3D7 strain) and untreated parasitised erythrocytes. The data shown are the mean log₂ ratios of the level of each metabolite in the MMV007839-treated samples relative to its level in the untreated samples (+ MMV007839/- MMV007839), determined 1 h, 3 h and 6 h after the addition of MMV007839, obtained from three independent experiments. The concentration of MMV007839 used (6 μM) corresponds to approximately 20× the IC₅₀ value for growth inhibition of 3D7 parasites. (B) The log₂ ratios of metabolite levels in MMV007839-treated samples relative to those in untreated samples for all metabolites of known identity for which a statistically significant difference in abundance was observed between MMV007839-treated and untreated infected erythrocytes (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006180#ppat.1006180.s009" target="_blank">S1 Data</a> for statistical analysis). The data shown are the mean + SEM from three independent experiments. GL3P, sn-glycerol-3-phosphate; Pyr, pyruvate; Lac, lactate; R5P, ribose-5-phosphate; AcCoA, acetyl coenzyme A; Gln, glutamine; VD, Val-Asp; PE, Pro-Glu; PVQ, Pro-Val-Gln; PDAV, Pro-Asp-Ala-Val; TNVK, Thr-Asn-Val-Lys; Oro, orotate; O5P, orotidine-5-phosphate; DC, deoxycytidine; GGPP, geranylgeranyl-pyrophosphate. (C) Relative amounts of metabolites in the extracellular medium in suspensions containing MMV007839-treated and untreated parasitised erythrocytes. The data shown are the mean log₂ ratios (+ MMV007839/- MMV007839) + SEM from three independent experiments. There was a statistically significant difference in the concentrations of 3-phosphoglycerate and phosphoenolpyruvate in the extracellular medium between MMV007839-treated and untreated cell suspensions (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006180#ppat.1006180.s009" target="_blank">S1 Data</a>). DHAP, glycerone phosphate; PEP, phosphoenolpyruvate; 3PG, 3-phosphoglycerate; Cit, citrate; αKG, 2-ketoglutarate; Mal, malate; Pip, pipecolate.</p

PfFNT mutation and growth inhibition by various compounds in parasites generated by exposure to increasing concentrations of MMV007839.

<p>PfFNT mutation and growth inhibition by various compounds in parasites generated by exposure to increasing concentrations of MMV007839.</p