RESUMEN
To contribute to the global effort to develop new antimalarial therapies, we previously disclosed initial findings on the optimization of the dihydroquinazolinone-3-carboxamide class that targets PfATP4. Here we report on refining the aqueous solubility and metabolic stability to improve the pharmacokinetic profile and consequently in vivo efficacy. We show that the incorporation of heterocycle systems in the 8-position of the scaffold was found to provide the greatest attainable balance between parasite activity, aqueous solubility, and metabolic stability. Optimized analogs, including the frontrunner compound S-WJM992, were shown to inhibit PfATP4-associated Na+-ATPase activity, gave rise to a metabolic signature consistent with PfATP4 inhibition, and displayed altered activities against parasites with mutations in PfATP4. Finally, S-WJM992 showed appreciable efficacy in a malaria mouse model and blocked gamete development preventing transmission to mosquitoes. Importantly, further optimization of the dihydroquinazolinone class is required to deliver a candidate with improved pharmacokinetic and risk of resistance profiles.
RESUMEN
With resistance to most antimalarials increasing, it is imperative that new drugs are developed. We previously identified an aryl acetamide compound, MMV006833 (M-833), that inhibited the ring-stage development of newly invaded merozoites. Here, we select parasites resistant to M-833 and identify mutations in the START lipid transfer protein (PF3D7_0104200, PfSTART1). Introducing PfSTART1 mutations into wildtype parasites reproduces resistance to M-833 as well as to more potent analogues. PfSTART1 binding to the analogues is validated using organic solvent-based Proteome Integral Solubility Alteration (Solvent PISA) assays. Imaging of invading merozoites shows the inhibitors prevent the development of ring-stage parasites potentially by inhibiting the expansion of the encasing parasitophorous vacuole membrane. The PfSTART1-targeting compounds also block transmission to mosquitoes and with multiple stages of the parasite's lifecycle being affected, PfSTART1 represents a drug target with a new mechanism of action.
Asunto(s)
Acetamidas , Antimaláricos , Plasmodium falciparum , Proteínas Protozoarias , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Plasmodium falciparum/crecimiento & desarrollo , Acetamidas/farmacología , Acetamidas/química , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Antimaláricos/farmacología , Antimaláricos/química , Animales , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genética , Mutación , Malaria Falciparum/parasitología , Malaria Falciparum/prevención & control , Malaria Falciparum/tratamiento farmacológico , Humanos , Resistencia a Medicamentos/genética , Resistencia a Medicamentos/efectos de los fármacos , Estadios del Ciclo de Vida/efectos de los fármacosRESUMEN
To combat the global burden of malaria, development of new drugs to replace or complement current therapies is urgently required. Here, we show that the compound MMV1557817 is a selective, nanomolar inhibitor of both Plasmodium falciparum and Plasmodium vivax aminopeptidases M1 and M17, leading to inhibition of end-stage hemoglobin digestion in asexual parasites. MMV1557817 can kill sexual-stage P. falciparum, is active against murine malaria, and does not show any shift in activity against a panel of parasites resistant to other antimalarials. MMV1557817-resistant P. falciparum exhibited a slow growth rate that was quickly outcompeted by wild-type parasites and were sensitized to the current clinical drug, artemisinin. Overall, these results confirm MMV1557817 as a lead compound for further drug development and highlights the potential of dual inhibition of M1 and M17 as an effective multi-species drug-targeting strategy.IMPORTANCEEach year, malaria infects approximately 240 million people and causes over 600,000 deaths, mostly in children under 5 years of age. For the past decade, artemisinin-based combination therapies have been recommended by the World Health Organization as the standard malaria treatment worldwide. Their widespread use has led to the development of artemisinin resistance in the form of delayed parasite clearance, alongside the rise of partner drug resistance. There is an urgent need to develop and deploy new antimalarial agents with novel targets and mechanisms of action. Here, we report a new and potent antimalarial compound, known as MMV1557817, and show that it targets multiple stages of the malaria parasite lifecycle, is active in a preliminary mouse malaria model, and has a novel mechanism of action. Excitingly, resistance to MMV15578117 appears to be self-limiting, suggesting that development of the compound may provide a new class of antimalarial.
Asunto(s)
Aminopeptidasas , Antimaláricos , Plasmodium falciparum , Plasmodium vivax , Antimaláricos/farmacología , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/enzimología , Animales , Ratones , Plasmodium vivax/efectos de los fármacos , Plasmodium vivax/enzimología , Aminopeptidasas/antagonistas & inhibidores , Aminopeptidasas/metabolismo , Resistencia a Medicamentos , Humanos , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/parasitología , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , FemeninoRESUMEN
Malaria is a devastating disease that causes significant morbidity worldwide. The development of new antimalarial chemotypes is urgently needed because of the emergence of resistance to frontline therapies. Independent phenotypic screening campaigns against the Plasmodium asexual parasite, including our own, identified the aryl amino acetamide hit scaffold. In a prior study, we identified the STAR-related lipid transfer protein (PfSTART1) as the molecular target of this antimalarial chemotype. In this study, we combined structural elements from the different aryl acetamide hit subtypes and explored the structure-activity relationship. It was shown that the inclusion of an endocyclic nitrogen, to generate the tool compound WJM-715, improved aqueous solubility and modestly improved metabolic stability in rat hepatocytes. Metabolic stability in human liver microsomes remains a challenge for future development of the aryl acetamide class, which was underscored by modest systemic exposure and a short half-life in mice. The optimized aryl acetamide analogs were cross resistant to parasites with mutations in PfSTART1, but not to other drug-resistant mutations, and showed potent binding to recombinant PfSTART1 by biophysical analysis, further supporting PfSTART1 as the likely molecular target. The optimized aryl acetamide analogue, WJM-715 will be a useful tool for further investigating the druggability of PfSTART1 across the lifecycle of the malaria parasite.
Asunto(s)
Antimaláricos , Proteínas Portadoras , Malaria Falciparum , Malaria , Ratas , Ratones , Humanos , Animales , Antimaláricos/química , Plasmodium falciparum , Malaria Falciparum/tratamiento farmacológico , Malaria/tratamiento farmacológico , Acetamidas/farmacología , LípidosRESUMEN
The Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) is the current leading blood-stage malaria vaccine candidate. PfRH5 functions as part of the pentameric PCRCR complex containing PTRAMP, CSS, PfCyRPA and PfRIPR, all of which are essential for infection of human red blood cells (RBCs). To trigger RBC invasion, PfRH5 engages with RBC protein basigin in a step termed the RH5-basigin binding stage. Although we know increasingly more about how antibodies specific for PfRH5 can block invasion, much less is known about how antibodies recognizing other members of the PCRCR complex can inhibit invasion. To address this, we performed live cell imaging using monoclonal antibodies (mAbs) which bind PfRH5 and PfCyRPA. We measured the degree and timing of the invasion inhibition, the stage at which it occurred, as well as subsequent events. We show that parasite invasion is blocked by individual mAbs, and the degree of inhibition is enhanced when combining a mAb specific for PfRH5 with one binding PfCyRPA. In addition to directly establishing the invasion-blocking capacity of the mAbs, we identified a secondary action of certain mAbs on extracellular parasites that had not yet invaded where the mAbs appeared to inactivate the parasites by triggering a developmental pathway normally only seen after successful invasion. These findings suggest that epitopes within the PfCyRPA-PfRH5 sub-complex that elicit these dual responses may be more effective immunogens than neighboring epitopes by both blocking parasites from invading and rapidly inactivating extracellular parasites. These two protective mechanisms, prevention of invasion and inactivation of uninvaded parasites, resulting from antibody to a single epitope indicate a possible route to the development of more effective vaccines.
Asunto(s)
Basigina , Merozoítos , Humanos , Animales , Plasmodium falciparum , Anticuerpos Monoclonales , EpítoposRESUMEN
The malaria parasite uses actin-based mechanisms throughout its lifecycle to control a range of biological processes including intracellular trafficking, gene regulation, parasite motility and invasion. In this work we assign functions to the Plasmodium falciparum formins 1 and 2 (FRM1 and FRM2) proteins in asexual and sexual blood stage development. We show that FRM1 is essential for merozoite invasion and FRM2 is required for efficient cell division. We also observed divergent functions for FRM1 and FRM2 in gametocyte development. Conditional deletion of FRM1 leads to a delay in gametocyte stage progression. We show that FRM2 controls the actin and microtubule cytoskeletons in developing gametocytes, with premature removal of the protein resulting in a loss of transmissible stage V gametocytes. Lastly, we show that targeting formin proteins with the small molecule inhibitor of formin homology domain 2 (SMIFH2) leads to a multistage block in asexual and sexual stage parasite development.
Asunto(s)
Actinas , Plasmodium falciparum , Actinas/genética , Forminas , Plasmodium falciparum/genética , División Celular , CitoesqueletoRESUMEN
A key element of Plasmodium biology and pathogenesis is the trafficking of ~10% of the parasite proteome into the host red blood cell (RBC) it infects. To cross the parasite-encasing parasitophorous vacuole membrane, exported proteins utilise a channel-forming protein complex termed the Plasmodium translocon of exported proteins (PTEX). PTEX is obligatory for parasite survival, both in vitro and in vivo, suggesting that at least some exported proteins have essential metabolic functions. However, to date only one essential PTEX-dependent process, the new permeability pathways, has been described. To identify other essential PTEX-dependant proteins/processes, we conditionally knocked down the expression of one of its core components, PTEX150, and examined which pathways were affected. Surprisingly, the food vacuole mediated process of haemoglobin (Hb) digestion was substantially perturbed by PTEX150 knockdown. Using a range of transgenic parasite lines and approaches, we show that two major Hb proteases; falcipain 2a and plasmepsin II, interact with PTEX core components, implicating the translocon in the trafficking of Hb proteases. We propose a model where these proteases are translocated into the PV via PTEX in order to reach the cytostome, located at the parasite periphery, prior to food vacuole entry. This work offers a second mechanistic explanation for why PTEX function is essential for growth of the parasite within its host RBC.
Asunto(s)
Parásitos , Plasmodium falciparum , Animales , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Vacuolas/metabolismo , Transporte de Proteínas , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Eritrocitos/parasitología , Parásitos/metabolismo , Péptido Hidrolasas/metabolismoRESUMEN
With emerging resistance to frontline treatments, it is vital that new antimalarial drugs are identified to target Plasmodium falciparum. We have recently described a compound, MMV020291, as a specific inhibitor of red blood cell (RBC) invasion, and have generated analogues with improved potency. Here, we generated resistance to MMV020291 and performed whole genome sequencing of 3 MMV020291-resistant populations. This revealed 3 nonsynonymous single nucleotide polymorphisms in 2 genes; 2 in profilin (N154Y, K124N) and a third one in actin-1 (M356L). Using CRISPR-Cas9, we engineered these mutations into wild-type parasites, which rendered them resistant to MMV020291. We demonstrate that MMV020291 reduces actin polymerisation that is required by the merozoite stage parasites to invade RBCs. Additionally, the series inhibits the actin-1-dependent process of apicoplast segregation, leading to a delayed death phenotype. In vitro cosedimentation experiments using recombinant P. falciparum proteins indicate that potent MMV020291 analogues disrupt the formation of filamentous actin in the presence of profilin. Altogether, this study identifies the first compound series interfering with the actin-1/profilin interaction in P. falciparum and paves the way for future antimalarial development against the highly dynamic process of actin polymerisation.
Asunto(s)
Antimaláricos , Malaria Falciparum , Humanos , Plasmodium falciparum/metabolismo , Actinas/genética , Actinas/metabolismo , Profilinas/genética , Profilinas/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/prevención & control , Malaria Falciparum/genética , Eritrocitos/parasitología , Antimaláricos/farmacologíaRESUMEN
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.
Asunto(s)
Antimaláricos , Malaria Falciparum , Malaria , Animales , Ratones , Antimaláricos/farmacología , Antimaláricos/uso terapéutico , Plasmodium falciparum , Homeostasis , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/parasitologíaRESUMEN
The development of new antimalarials is required because of the threat of resistance to current antimalarial therapies. To discover new antimalarial chemotypes, we screened the Janssen Jumpstarter library against the P. falciparum asexual parasite and identified the 7-N-substituted-3-oxadiazole quinolone hit class. We established the structure-activity relationship and optimized the antimalarial potency. The optimized analog WJM228 (17) showed robust metabolic stability in vitro, although the aqueous solubility was limited. Forward genetic resistance studies uncovered that WJM228 targets the Qo site of cytochrome b (cyt b), an important component of the mitochondrial electron transport chain (ETC) that is essential for pyrimidine biosynthesis and an established antimalarial target. Profiling against drug-resistant parasites confirmed that WJM228 confers resistance to the Qo site but not Qi site mutations, and in a biosensor assay, it was shown to impact the ETC via inhibition of cyt b. Consistent with other cyt b targeted antimalarials, WJM228 prevented pre-erythrocytic parasite and male gamete development and reduced asexual parasitemia in a P. berghei mouse model of malaria. Correcting the limited aqueous solubility and the high susceptibility to cyt b Qo site resistant parasites found in the clinic will be major obstacles in the future development of the 3-oxadiazole quinolone antimalarial class.
Asunto(s)
Antimaláricos , Antagonistas del Ácido Fólico , Malaria Falciparum , Quinolonas , Animales , Ratones , Antimaláricos/farmacología , Citocromos b , Antagonistas del Ácido Fólico/metabolismo , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/parasitología , Plasmodium falciparum , Quinolonas/farmacologíaRESUMEN
The cation efflux pump Plasmodium falciparum ATPase 4 (PfATP4) maintains Na+ homeostasis in malaria parasites and has been implicated in the mechanism of action of many structurally diverse antimalarial agents, including >7% of the antimalarial compounds in the Medicines for Malaria Venture's 'Malaria Box' and 'Pathogen Box'. Recent screens of the 'Malaria Box' and 'Pathogen Box' revealed that many PfATP4 inhibitors prevent parasites from exiting their host red blood cell (egress) or entering new host cells (invasion), suggesting that these compounds may have additional molecular targets involved in egress or invasion. Here, we demonstrate that five PfATP4 inhibitors reduce egress but not invasion. These compounds appear to inhibit egress by blocking the activation of protein kinase G, an enzyme that, once stimulated, rapidly activates parasite egress. We establish a direct link between egress and PfATP4 function by showing that the inhibition of egress is attenuated in a Na+-depleted environment and in parasites with a mutation in pfatp4. Finally, we show that PfATP4 inhibitors induce host cell lysis when administered prior to the completion of parasite replication. Since host cell lysis mimics egress but is not followed by invasion, this phenomenon likely explains why several PfATP4 inhibitors were previously classified as invasion inhibitors. Collectively, our results confirm that PfATP4-mediated Na+ efflux is critical to the regulation of parasite egress.
Asunto(s)
Antimaláricos , Proteínas de Transporte de Catión , Malaria Falciparum , Malaria , Animales , Humanos , Plasmodium falciparum/genética , Esquizontes/metabolismo , Adenosina Trifosfatasas/genética , Antimaláricos/farmacología , Malaria Falciparum/parasitología , Eritrocitos/parasitología , Malaria/metabolismo , Proteínas de Transporte de Catión/genética , Sodio/metabolismo , Sodio/uso terapéutico , Iones/metabolismoRESUMEN
Plasmodium falciparum parasites which cause malaria, traffic hundreds of proteins into the red blood cells (RBCs) they infect. These exported proteins remodel their RBCs enabling host immune evasion through processes such as cytoadherence that greatly assist parasite survival. As resistance to all current antimalarial compounds is rising new compounds need to be identified and those that could inhibit parasite protein secretion and export would both rapidly reduce parasite virulence and ultimately lead to parasite death. To identify compounds that inhibit protein export we used transgenic parasites expressing an exported nanoluciferase reporter to screen the Medicines for Malaria Venture Malaria Box of 400 antimalarial compounds with mostly unknown targets. The most potent inhibitor identified in this screen was MMV396797 whose application led to export inhibition of both the reporter and endogenous exported proteins. MMV396797 mediated blockage of protein export and slowed the rigidification and cytoadherence of infected RBCs-modifications which are both mediated by parasite-derived exported proteins. Overall, we have identified a new protein export inhibitor in P. falciparum whose target though unknown, could be developed into a future antimalarial that rapidly inhibits parasite virulence before eliminating parasites from the host.
Asunto(s)
Antimaláricos , Malaria Falciparum , Malaria , Parásitos , Animales , Antimaláricos/metabolismo , Antimaláricos/farmacología , Antimaláricos/uso terapéutico , Eritrocitos/parasitología , Humanos , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/parasitología , Parásitos/metabolismo , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismoRESUMEN
Plasmepsin X (PMX) is an aspartyl protease that processes proteins essential for Plasmodium parasites to invade and egress from host erythrocytes during the symptomatic asexual stage of malaria. PMX substrates possess a conserved cleavage region denoted by the consensus motif, SFhE (h=hydrophobic amino acid). Peptidomimetics reflecting the P3 -P1 positions of the consensus motif were designed and showed potent and selective inhibition of PMX. It was established that PMX prefers Phe in the P1 position, di-substitution at the ß-carbon of the P2 moiety and a hydrophobic P3 group which was supported by modelling of the peptidomimetics in complex with PMX. The peptidomimetics were shown to arrest asexual P. falciparum parasites at the schizont stage by impairing PMX substrate processing. Overall, the peptidomimetics described will assist in further understanding PMX substrate specificity and have the potential to act as a template for future antimalarial design.
Asunto(s)
Antimaláricos , Antagonistas del Ácido Fólico , Malaria Falciparum , Peptidomiméticos , Aminoácidos , Antimaláricos/química , Antimaláricos/farmacología , Ácido Aspártico Endopeptidasas , Carbono , Humanos , Malaria Falciparum/tratamiento farmacológico , Peptidomiméticos/química , Peptidomiméticos/farmacología , Plasmodium falciparum/metabolismo , Inhibidores de Proteasas/química , Proteínas ProtozoariasRESUMEN
Infection with Plasmodium falciparum parasites results in approximately 627,000 deaths from malaria annually. Key to the parasite's success is their ability to invade and subsequently grow within human erythrocytes. Parasite proteins involved in parasite invasion and proliferation are therefore intrinsically of great interest, as targeting these proteins could provide novel means of therapeutic intervention. One such protein is P113 which has been reported to be both an invasion protein and an intracellular protein located within the parasitophorous vacuole (PV). The PV is delimited by a membrane (PVM) across which a plethora of parasite-specific proteins are exported via the Plasmodium Translocon of Exported proteins (PTEX) into the erythrocyte to enact various immune evasion functions. To better understand the role of P113 we isolated its binding partners from in vitro cultures of P. falciparum. We detected interactions with the protein export machinery (PTEX and exported protein-interacting complex) and a variety of proteins that either transit through the PV or reside on the parasite plasma membrane. Genetic knockdown or partial deletion of P113 did not significantly reduce parasite growth or protein export but did disrupt the morphology of the PVM, suggesting that P113 may play a role in maintaining normal PVM architecture.
Asunto(s)
Malaria Falciparum , Parásitos , Animales , Eritrocitos/parasitología , Humanos , Malaria Falciparum/parasitología , Parásitos/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Transporte de Proteínas/genética , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Vacuolas/metabolismoRESUMEN
The emerging resistance to combination therapies comprised of artemisinin derivatives has driven a need to identify new antimalarials with novel mechanisms of action. Central to the survival and proliferation of the malaria parasite is the invasion of red blood cells by Plasmodium merozoites, providing an attractive target for novel therapeutics. A screen of the Medicines for Malaria Venture Pathogen Box employing transgenic P. falciparum parasites expressing the nanoluciferase bioluminescent reporter identified the phenylsulfonyl piperazine class as a specific inhibitor of erythrocyte invasion. Here, we describe the optimization and further characterization of the phenylsulfonyl piperazine class. During the optimization process we defined the functionality required for P. falciparum asexual stage activity and determined the alpha-carbonyl S-methyl isomer was important for antimalarial potency. The optimized compounds also possessed comparable activity against multidrug resistant strains of P. falciparum and displayed weak activity against sexual stage gametocytes. We determined that the optimized compounds blocked erythrocyte invasion consistent with the asexual activity observed and therefore the phenylsulfonyl piperazine analogues described could serve as useful tools for studying Plasmodium erythrocyte invasion.
Asunto(s)
Antimaláricos/farmacología , Eritrocitos/efectos de los fármacos , Malaria Falciparum/tratamiento farmacológico , Piperazinas/farmacología , Plasmodium falciparum/efectos de los fármacos , Plasmodium knowlesi/efectos de los fármacos , Animales , Antimaláricos/síntesis química , Antimaláricos/química , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Eritrocitos/parasitología , Células Hep G2 , Humanos , Ratones , Microsomas Hepáticos/química , Microsomas Hepáticos/metabolismo , Estructura Molecular , Pruebas de Sensibilidad Parasitaria , Piperazinas/síntesis química , Piperazinas/química , Solubilidad , Relación Estructura-ActividadRESUMEN
With emerging resistance to frontline treatments, it is vital that new drugs are identified to target Plasmodium falciparum. One of the most critical processes during parasites asexual lifecycle is the invasion and subsequent egress of red blood cells (RBCs). Many unique parasite ligands, receptors and enzymes are employed during egress and invasion that are essential for parasite proliferation and survival, therefore making these processes druggable targets. To identify potential inhibitors of egress and invasion, we screened the Medicines for Malaria Venture Pathogen Box, a 400 compound library against neglected tropical diseases, including 125 with antimalarial activity. For this screen, we utilised transgenic parasites expressing a bioluminescent reporter, nanoluciferase (Nluc), to measure inhibition of parasite egress and invasion in the presence of the Pathogen Box compounds. At a concentration of 2 µM, we found 15 compounds that inhibited parasite egress by >40% and 24 invasion-specific compounds that inhibited invasion by >90%. We further characterised 11 of these inhibitors through cell-based assays and live cell microscopy, and found two compounds that inhibited merozoite maturation in schizonts, one compound that inhibited merozoite egress, one compound that directly inhibited parasite invasion and one compound that slowed down invasion and arrested ring formation. The remaining compounds were general growth inhibitors that acted during the egress and invasion phase of the cell cycle. We found the sulfonylpiperazine, MMV020291, to be the most invasion-specific inhibitor, blocking successful merozoite internalisation within human RBCs and having no substantial effect on other stages of the cell cycle. This has significant implications for the possible development of an invasion-specific inhibitor as an antimalarial in a combination based therapy, in addition to being a useful tool for studying the biology of the invading parasite.
Asunto(s)
Antimaláricos/farmacología , Evaluación Preclínica de Medicamentos , Plasmodium falciparum/efectos de los fármacos , Animales , Eritrocitos/parasitología , Humanos , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/parasitología , Merozoítos/efectos de los fármacos , Piperazina , Piperazinas/farmacología , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/metabolismo , Esquizontes/efectos de los fármacosRESUMEN
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.
Asunto(s)
Eritrocitos/efectos de los fármacos , Isoquinolinas/síntesis química , Plasmodium falciparum/efectos de los fármacos , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Membrana Celular/efectos de los fármacos , Resistencia a Medicamentos/efectos de los fármacos , Eritrocitos/parasitología , Isoquinolinas/química , Isoquinolinas/farmacología , Modelos Moleculares , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Mutación Puntual , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Sodio , ATPasa Intercambiadora de Sodio-Potasio/química , ATPasa Intercambiadora de Sodio-Potasio/genética , Secuenciación Completa del GenomaRESUMEN
Plasmodium spp. parasites that cause malaria disease remain a significant global-health burden. With the spread of parasites resistant to artemisinin combination therapies in Southeast Asia, there is a growing need to develop new antimalarials with novel targets. Invasion of the red blood cell by Plasmodium merozoites is essential for parasite survival and proliferation, thus representing an attractive target for therapeutic development. Red blood cell invasion requires a co-ordinated series of protein/protein interactions, protease cleavage events, intracellular signals, organelle release and engagement of an actin-myosin motor, which provide many potential targets for drug development. As these steps occur in the bloodstream, they are directly susceptible and exposed to drugs. A number of invasion inhibitors against a diverse range of parasite proteins involved in these different processes of invasion have been identified, with several showing potential to be optimised for improved drug-like properties. In this review, we discuss red blood cell invasion as a drug target and highlight a number of approaches for developing antimalarials with invasion inhibitory activity to use in future combination therapies.
Asunto(s)
Sistemas de Liberación de Medicamentos , Eritrocitos/parasitología , Plasmodium/fisiología , Antimaláricos/farmacología , Interacciones Huésped-Parásitos/efectos de los fármacos , Humanos , Malaria/parasitología , Malaria/prevención & controlRESUMEN
The ability of Plasmodium parasites to egress from their host red blood cell is critical for the amplification of these parasites in the blood. Previous forward chemical genetic approaches have implicated the subtilisin-like protease (SUB1) and the cysteine protease dipeptidyl aminopeptidase 3 (DPAP3) as key players in egress, with the final step of SUB1 maturation thought to be due to the activity of DPAP3. In this study, we have utilized a reverse genetics approach to engineer transgenic Plasmodium falciparum parasites in which dpap3 expression can be conditionally regulated using the glmS ribozyme based RNA-degrading system. We show that DPAP3, which is expressed in schizont stages and merozoites and localizes to organelles distinct from the micronemes, rhoptries and dense granules, is not required for the trafficking of apical proteins or processing of SUB1 substrates, nor for parasite maturation and egress from red blood cells. Thus, our findings argue against a role for DPAP3 in parasite egress and indicate that the phenotypes observed with DPAP3 inhibitors are due to off-target effects.