Optimization of pyrazolopyridine 4-carboxamides with potent antimalarial activity for which resistance is associated with the P. falciparum transporter ABCI3.

Emerging resistance to current antimalarials is reducing their effectiveness and therefore there is a need to develop new antimalarial therapies. Toward this goal, high throughput screens against the P. falciparum asexual parasite identified the pyrazolopyridine 4-carboxamide scaffold. Structure-activity relationship analysis of this chemotype defined that the N1-tert-butyl group and aliphatic foliage in the 3- and 6-positions were necessary for activity, while the inclusion of a 7'-aza-benzomorpholine on the 4-carboxamide motif resulted in potent anti-parasitic activity and increased aqueous solubility. A previous report that resistance to the pyrazolopyridine class is associated with the ABCI3 transporter was confirmed, with pyrazolopyridine 4-carboxamides showing an increase in potency against parasites when the ABCI3 transporter was knocked down. The low metabolic stability intrinsic to the pyrazolopyridine scaffold and the slow rate by which the compounds kill asexual parasites resulted in poor performance in a P. berghei asexual blood stage mouse model. Lowering the risk of resistance and mitigating the metabolic stability and cytochrome P450 inhibition will be challenges in the future development of the pyrazolopyrimidine antimalarial class.

Discovery of antiplasmodial pyridine carboxamides and thiocarboxamides.

Malaria continues to be a significant burden, particularly in Africa, which accounts for 95% of malaria deaths worldwide. Despite advances in malaria treatments, malaria eradication is hampered by insecticide and antimalarial drug resistance. Consequently, the need to discover new antimalarial lead compounds remains urgent. To help address this need, we evaluated the antiplasmodial activity of twenty-two amides and thioamides with pyridine cores and their non-pyridine analogues. Twelve of these compounds showed in vitro anti-proliferative activity against the intraerythrocytic stage of Plasmodium falciparum, the most virulent species of Plasmodium infecting humans. Thiopicolinamide 13i was found to possess submicromolar activity (IC50 = 142 nM) and was >88-fold less active against a human cell line. The compound was equally effective against chloroquine-sensitive and -resistant parasites and did not inhibit ß-hematin formation, pH regulation or PfATP4. Compound 13i may therefore possess a novel mechanism of action.

A pH Fingerprint Assay to Identify Inhibitors of Multiple Validated and Potential Antimalarial Drug Targets.

New drugs with novel modes of action are needed to safeguard malaria treatment. In recent years, millions of compounds have been tested for their ability to inhibit the growth of asexual blood-stage Plasmodium falciparum parasites, resulting in the identification of thousands of compounds with antiplasmodial activity. Determining the mechanisms of action of antiplasmodial compounds informs their further development, but remains challenging. A relatively high proportion of compounds identified as killing asexual blood-stage parasites show evidence of targeting the parasite's plasma membrane Na+-extruding, H+-importing pump, PfATP4. Inhibitors of PfATP4 give rise to characteristic changes in the parasite's internal [Na+] and pH. Here, we designed a "pH fingerprint" assay that robustly identifies PfATP4 inhibitors while simultaneously allowing the detection of (and discrimination between) inhibitors of the lactate:H+ transporter PfFNT, which is a validated antimalarial drug target, and the V-type H+ ATPase, which was suggested as a possible target of the clinical candidate ZY19489. In our pH fingerprint assays and subsequent secondary assays, ZY19489 did not show evidence for the inhibition of pH regulation by the V-type H+ ATPase, suggesting that it has a different mode of action in the parasite. The pH fingerprint assay also has the potential to identify protonophores, inhibitors of the acid-loading Cl- transporter(s) (for which the molecular identity(ies) remain elusive), and compounds that act through inhibition of either the glucose transporter PfHT or glycolysis. The pH fingerprint assay therefore provides an efficient starting point to match a proportion of antiplasmodial compounds with their mechanisms of action.

Optimization of 2,3-Dihydroquinazolinone-3-carboxamides as Antimalarials Targeting PfATP4.

There is an urgent need to populate the antimalarial clinical portfolio with new candidates because of resistance against frontline antimalarials. To discover new antimalarial chemotypes, we performed a high-throughput screen of the Janssen Jumpstarter library against the Plasmodium falciparum asexual blood-stage parasite and identified the 2,3-dihydroquinazolinone-3-carboxamide scaffold. We defined the SAR and found that 8-substitution on the tricyclic ring system and 3-substitution of the exocyclic arene produced analogues with potent activity against asexual parasites equivalent to clinically used antimalarials. Resistance selection and profiling against drug-resistant parasite strains revealed that this antimalarial chemotype targets PfATP4. Dihydroquinazolinone analogues were shown to disrupt parasite Na+ homeostasis and affect parasite pH, exhibited a fast-to-moderate rate of asexual kill, and blocked gametogenesis, consistent with the phenotype of clinically used PfATP4 inhibitors. Finally, we observed that optimized frontrunner analogue WJM-921 demonstrates oral efficacy in a mouse model of malaria.

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 PfATP4G358S 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 PfATP4G358S parasites, and that their combination with unrelated antimalarials may mitigate against resistance development.

Discovery of spirooxadiazoline oxindoles with dual-stage antimalarial activity.

Malaria remains a prevalent infectious disease in developing countries. The first-line therapeutic options are based on combinations of fast-acting artemisinin derivatives and longer-acting synthetic drugs. However, the emergence of resistance to these first-line treatments represents a serious risk, and the discovery of new effective drugs is urgently required. For this reason, new antimalarial chemotypes with new mechanisms of action, and ideally with activity against multiple parasite stages, are needed. We report a new scaffold with dual-stage (blood and liver) antiplasmodial activity. Twenty-six spirooxadiazoline oxindoles were synthesized and screened against the erythrocytic stage of the human malaria parasite P. falciparum. The most active compounds were also tested against the liver-stage of the murine parasite P. berghei. Seven compounds emerged as dual-stage antimalarials, with IC50 values in the low micromolar range. Due to structural similarity with cipargamin, which is thought to inhibit blood-stage P. falciparum growth via inhibition of the Na + efflux pump PfATP4, we tested one of the most active compounds for anti-PfATP4 activity. Our results suggest that this target is not the primary target of spirooxadiazoline oxindoles and further studies are ongoing to identify the main mechanism of action of this scaffold.

An Open Drug Discovery Competition: Experimental Validation of Predictive Models in a Series of Novel Antimalarials.

The Open Source Malaria (OSM) consortium is developing compounds that kill the human malaria parasite, Plasmodium falciparum, by targeting PfATP4, an essential ion pump on the parasite surface. The structure of PfATP4 has not been determined. Here, we describe a public competition created to develop a predictive model for the identification of PfATP4 inhibitors, thereby reducing project costs associated with the synthesis of inactive compounds. Competition participants could see all entries as they were submitted. In the final round, featuring private sector entrants specializing in machine learning methods, the best-performing models were used to predict novel inhibitors, of which several were synthesized and evaluated against the parasite. Half possessed biological activity, with one featuring a motif that the human chemists familiar with this series would have dismissed as "ill-advised". Since all data and participant interactions remain in the public domain, this research project "lives" and may be improved by others.

Identifying the major lactate transporter of Toxoplasma gondii tachyzoites.

Toxoplasma gondii and Plasmodium falciparum parasites both extrude L-lactate, a byproduct of glycolysis. The P. falciparum Formate Nitrite Transporter, PfFNT, mediates L-lactate transport across the plasma membrane of P. falciparum parasites and has been validated as a drug target. The T. gondii genome encodes three FNTs that have been shown to transport L-lactate, and which are proposed to be the targets of several inhibitors of T. gondii proliferation. Here, we show that each of the TgFNTs localize to the T. gondii plasma membrane and are capable of transporting L-lactate across it, with TgFNT1 making the primary contribution to L-lactate transport during the disease-causing lytic cycle of the parasite. We use the Xenopus oocyte expression system to provide direct measurements of L-lactate transport via TgFNT1. We undertake a genetic analysis of the importance of the tgfnt genes for parasite proliferation, and demonstrate that all three tgfnt genes can be disrupted individually and together without affecting the lytic cycle under in vitro culture conditions. Together, our experiments identify the major lactate transporter in the disease causing stage of T. gondii, and reveal that this transporter is not required for parasite proliferation, indicating that TgFNTs are unlikely to be targets for anti-Toxoplasma drugs.

A 4-cyano-3-methylisoquinoline inhibitor of Plasmodium falciparum growth targets the sodium efflux pump PfATP4.

We developed a novel series of antimalarial compounds based on a 4-cyano-3-methylisoquinoline. Our lead compound MB14 achieved modest inhibition of the growth in vitro of the human malaria parasite, Plasmodium falciparum. To identify its biological target we selected for parasites resistant to MB14. Genome sequencing revealed that all resistant parasites bore a single point S374R mutation in the sodium (Na+) efflux transporter PfATP4. There are many compounds known to inhibit PfATP4 and some are under preclinical development. MB14 was shown to inhibit Na+ dependent ATPase activity in parasite membranes, consistent with the compound targeting PfATP4 directly. PfATP4 inhibitors cause swelling and lysis of infected erythrocytes, attributed to the accumulation of Na+ inside the intracellular parasites and the resultant parasite swelling. We show here that inhibitor-induced lysis of infected erythrocytes is dependent upon the parasite protein RhopH2, a component of the new permeability pathways that are induced by the parasite in the erythrocyte membrane. These pathways mediate the influx of Na+ into the infected erythrocyte and their suppression via RhopH2 knockdown limits the accumulation of Na+ within the parasite hence protecting the infected erythrocyte from lysis. This study reveals a role for the parasite-induced new permeability pathways in the mechanism of action of PfATP4 inhibitors.

Characterization of the ATP4 ion pump in Toxoplasma gondii.

The Plasmodium falciparum ATPase PfATP4 is the target of a diverse range of antimalarial compounds, including the clinical drug candidate cipargamin. PfATP4 was originally annotated as a Ca2+ transporter, but recent evidence suggests that it is a Na+ efflux pump, extruding Na+ in exchange for H+ Here we demonstrate that ATP4 proteins belong to a clade of P-type ATPases that are restricted to apicomplexans and their closest relatives. We employed a variety of genetic and physiological approaches to investigate the ATP4 protein of the apicomplexan Toxoplasma gondii, TgATP4. We show that TgATP4 is a plasma membrane protein. Knockdown of TgATP4 had no effect on resting pH or Ca2+ but rendered parasites unable to regulate their cytosolic Na+ concentration ([Na+]cyt). PfATP4 inhibitors caused an increase in [Na+]cyt and a cytosolic alkalinization in WT but not TgATP4 knockdown parasites. Parasites in which TgATP4 was knocked down or disrupted exhibited a growth defect, attributable to reduced viability of extracellular parasites. Parasites in which TgATP4 had been disrupted showed reduced virulence in mice. These results provide evidence for ATP4 proteins playing a key conserved role in Na+ regulation in apicomplexan parasites.

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 stages and actively motile extracellular forms that move through tissue. Parasite cytosolic Ca2+ signalling activates motility, but how this is switched off after invasion is complete to allow for replication to begin is not understood. Here, we show that the cyclic adenosine monophosphate (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 PKAc1 is sequestered to the parasite periphery by dual acylation of PKA regulatory subunit 1 (PKAr1). Upon genetic depletion of PKAc1 we show that newly invaded parasites exit host cells shortly thereafter, in a perforin-like protein 1 (PLP-1)-dependent fashion. Furthermore, we demonstrate that loss of PKAc1 prevents rapid down-regulation of cytosolic [Ca2+] levels shortly after invasion. We also provide evidence that loss of PKAc1 sensitises parasites to cyclic GMP (cGMP)-induced Ca2+ signalling, thus demonstrating a functional link between cAMP and these other signalling modalities. Together, this work provides a new paradigm in understanding how Toxoplasma and related apicomplexan parasites regulate infectivity.

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 4 (PF4), has intrinsic antiplasmodial activity but also undesirable chemokine properties. We engineered a peptide containing the isolated PF4 antiplasmodial domain, 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 membranes 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.

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

The antimalarial activity of chemically diverse compounds, including the clinical candidate cipargamin, has been linked to the ATPase PfATP4 in the malaria-causing parasite Plasmodium falciparum 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 P. falciparum 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 P. falciparum 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 [Ca2+]. With an apparent Km for ATP of 0.2 mm and an apparent Km 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 Km for Na+ is slightly elevated. Our study provides new insights into the biochemical properties and chemical sensitivity of an important new antimalarial drug target.

Diverse antimalarials from whole-cell phenotypic screens disrupt malaria parasite ion and volume homeostasis.

Four hundred structurally diverse drug-like compounds comprising the Medicines for Malaria Venture's 'Pathogen Box' were screened for their effect on a range of physiological parameters in asexual blood-stage malaria (Plasmodium falciparum) parasites. Eleven of these compounds were found to perturb parasite Na+, pH and volume in a manner consistent with inhibition of the putative Na+ efflux P-type ATPase PfATP4. All eleven compounds fell within the subset of 125 compounds included in the Pathogen Box on the basis of their having been identified as potent inhibitors of the growth of asexual blood-stage P. falciparum parasites. All eleven compounds inhibited the Na+-dependent ATPase activity of parasite membranes and showed reduced efficacy against parasites carrying mutations in PfATP4. This study increases the number of chemically diverse structures known to show a 'PfATP4-associated' phenotype, and adds to emerging evidence that a high proportion (7-9%) of the structurally diverse antimalarial compounds identified in whole cell phenotypic screens share the same mechanism of action, exerting their antimalarial effect via an interaction with PfATP4.

Cell Swelling Induced by the Antimalarial KAE609 (Cipargamin) and Other PfATP4-Associated Antimalarials.

For an increasing number of antimalarial agents identified in high-throughput phenotypic screens, there is evidence that they target PfATP4, a putative Na+ efflux transporter on the plasma membrane of the human malaria parasite Plasmodium falciparum For several such "PfATP4-associated" compounds, it has been noted that their addition to parasitized erythrocytes results in cell swelling. Here we show that six structurally diverse PfATP4-associated compounds, including the clinical candidate KAE609 (cipargamin), induce swelling of both isolated blood-stage parasites and intact parasitized erythrocytes. The swelling of isolated parasites is dependent on the presence of Na+ in the external environment and may be attributed to the osmotic consequences of Na+ uptake. The swelling of the parasitized erythrocyte results in an increase in its osmotic fragility. Countering cell swelling by increasing the osmolarity of the extracellular medium reduces the antiplasmodial efficacy of PfATP4-associated compounds, consistent with cell swelling playing a role in the antimalarial activity of this class of compounds.

A forward genetic screen identifies a negative regulator of rapid Ca2+-dependent cell egress (MS1) in the intracellular parasite Toxoplasma gondii.

Toxoplasma gondii, like all apicomplexan parasites, uses Ca2+ signaling pathways to activate gliding motility to power tissue dissemination and host cell invasion and egress. A group of "plant-like" Ca2+-dependent protein kinases (CDPKs) transduces cytosolic Ca2+ flux into enzymatic activity, but how they function is poorly understood. To investigate how Ca2+ signaling activates egress through CDPKs, we performed a forward genetic screen to isolate gain-of-function mutants from an egress-deficient cdpk3 knockout strain. We recovered mutants that regained the ability to egress from host cells that harbored mutations in the gene Suppressor of Ca2+-dependent Egress 1 (SCE1). Global phosphoproteomic analysis showed that SCE1 deletion restored many Δcdpk3-dependent phosphorylation events to near wild-type levels. We also show that CDPK3-dependent SCE1 phosphorylation is required to relieve its suppressive activity to potentiate egress. In summary, our work has uncovered a novel component and suppressor of Ca2+-dependent cell egress during Toxoplasma lytic growth.

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

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, Plasmodium falciparum. 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 Xenopus laevis 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.

Biochemical and Structural Characterization of Selective Allosteric Inhibitors of the Plasmodium falciparum Drug Target, Prolyl-tRNA-synthetase.

Plasmodium falciparum (Pf) prolyl-tRNA synthetase (ProRS) is one of the few chemical-genetically validated drug targets for malaria, yet highly selective inhibitors have not been described. In this paper, approximately 40,000 compounds were screened to identify compounds that selectively inhibit PfProRS enzyme activity versus Homo sapiens (Hs) ProRS. X-ray crystallography structures were solved for apo, as well as substrate- and inhibitor-bound forms of PfProRS. We identified two new inhibitors of PfProRS that bind outside the active site. These two allosteric inhibitors showed >100 times specificity for PfProRS compared to HsProRS, demonstrating this class of compounds could overcome the toxicity related to HsProRS inhibition by halofuginone and its analogues. Initial medicinal chemistry was performed on one of the two compounds, guided by the cocrystallography of the compound with PfProRS, and the results can instruct future medicinal chemistry work to optimize these promising new leads for drug development against malaria.

Molecular Mechanisms for Drug Hypersensitivity Induced by the Malaria Parasite's Chloroquine Resistance Transporter.

Mutations in the Plasmodium falciparum 'chloroquine resistance transporter' (PfCRT) confer resistance to chloroquine (CQ) and related antimalarials by enabling the protein to transport these drugs away from their targets within the parasite's digestive vacuole (DV). However, CQ resistance-conferring isoforms of PfCRT (PfCRTCQR) also render the parasite hypersensitive to a subset of structurally-diverse pharmacons. Moreover, mutations in PfCRTCQR that suppress the parasite's hypersensitivity to these molecules simultaneously reinstate its sensitivity to CQ and related drugs. We sought to understand these phenomena by characterizing the functions of PfCRTCQR isoforms that cause the parasite to become hypersensitive to the antimalarial quinine or the antiviral amantadine. We achieved this by measuring the abilities of these proteins to transport CQ, quinine, and amantadine when expressed in Xenopus oocytes and complemented this work with assays that detect the drug transport activity of PfCRT in its native environment within the parasite. Here we describe two mechanistic explanations for PfCRT-induced drug hypersensitivity. First, we show that quinine, which normally accumulates inside the DV and therewithin exerts its antimalarial effect, binds extremely tightly to the substrate-binding site of certain isoforms of PfCRTCQR. By doing so it likely blocks the normal physiological function of the protein, which is essential for the parasite's survival, and the drug thereby gains an additional killing effect. In the second scenario, we show that although amantadine also sequesters within the DV, the parasite's hypersensitivity to this drug arises from the PfCRTCQR-mediated transport of amantadine from the DV into the cytosol, where it can better access its antimalarial target. In both cases, the mutations that suppress hypersensitivity also abrogate the ability of PfCRTCQR to transport CQ, thus explaining why rescue from hypersensitivity restores the parasite's sensitivity to this antimalarial. These insights provide a foundation for understanding clinically-relevant observations of inverse drug susceptibilities in the malaria parasite.

Globally prevalent PfMDR1 mutations modulate Plasmodium falciparum susceptibility to artemisinin-based combination therapies.

Antimalarial chemotherapy, globally reliant on artemisinin-based combination therapies (ACTs), is threatened by the spread of drug resistance in Plasmodium falciparum parasites. Here we use zinc-finger nucleases to genetically modify the multidrug resistance-1 transporter PfMDR1 at amino acids 86 and 184, and demonstrate that the widely prevalent N86Y mutation augments resistance to the ACT partner drug amodiaquine and the former first-line agent chloroquine. In contrast, N86Y increases parasite susceptibility to the partner drugs lumefantrine and mefloquine, and the active artemisinin metabolite dihydroartemisinin. The PfMDR1 N86 plus Y184F isoform moderately reduces piperaquine potency in strains expressing an Asian/African variant of the chloroquine resistance transporter PfCRT. Mutations in both digestive vacuole-resident transporters are thought to differentially regulate ACT drug interactions with host haem, a product of parasite-mediated haemoglobin degradation. Global mapping of these mutations illustrates where the different ACTs could be selectively deployed to optimize treatment based on regional differences in PfMDR1 haplotypes.