RESUMEN
We identify the Plasmodium falciparum acetyl-coenzyme A synthetase (PfAcAS) as a druggable target, using genetic and chemical validation. In vitro evolution of resistance with two antiplasmodial drug-like compounds (MMV019721 and MMV084978) selects for mutations in PfAcAS. Metabolic profiling of compound-treated parasites reveals changes in acetyl-CoA levels for both compounds. Genome editing confirms that mutations in PfAcAS are sufficient to confer resistance. Knockdown studies demonstrate that PfAcAS is essential for asexual growth, and partial knockdown induces hypersensitivity to both compounds. In vitro biochemical assays using recombinantly expressed PfAcAS validates that MMV019721 and MMV084978 directly inhibit the enzyme by preventing CoA and acetate binding, respectively. Immunolocalization studies reveal that PfAcAS is primarily localized to the nucleus. Functional studies demonstrate inhibition of histone acetylation in compound-treated wild-type, but not in resistant parasites. Our findings identify and validate PfAcAS as an essential, druggable target involved in the epigenetic regulation of gene expression.
Asunto(s)
Acetato CoA Ligasa/antagonistas & inhibidores , Antimaláricos/farmacología , Inhibidores Enzimáticos/farmacología , Malaria/tratamiento farmacológico , Plasmodium falciparum/efectos de los fármacos , Acetato CoA Ligasa/metabolismo , Antimaláricos/química , Inhibidores Enzimáticos/química , Humanos , Malaria/metabolismo , Modelos Moleculares , Estructura Molecular , Pruebas de Sensibilidad Parasitaria , Plasmodium falciparum/enzimologíaRESUMEN
In malaria, chemical genetics is a powerful method for assigning function to uncharacterized genes. MMV085203 and GNF-Pf-3600 are two structurally related napthoquinone phenotypic screening hits that kill both blood- and sexual-stage P. falciparum parasites in the low nanomolar to low micromolar range. In order to understand their mechanism of action, parasites from two different genetic backgrounds were exposed to sublethal concentrations of MMV085203 and GNF-Pf-3600 until resistance emerged. Whole genome sequencing revealed all 17 resistant clones acquired nonsynonymous mutations in the gene encoding the orphan apicomplexan transporter PF3D7_0312500 (pfmfr3) predicted to encode a member of the major facilitator superfamily (MFS). Disruption of pfmfr3 and testing against a panel of antimalarial compounds showed decreased sensitivity to MMV085203 and GNF-Pf-3600 as well as other compounds that have a mitochondrial mechanism of action. In contrast, mutations in pfmfr3 provided no protection against compounds that act in the food vacuole or the cytosol. A dihydroorotate dehydrogenase rescue assay using transgenic parasite lines, however, indicated a different mechanism of action for both MMV085203 and GNF-Pf-3600 than the direct inhibition of cytochrome bc1. Green fluorescent protein (GFP) tagging of PfMFR3 revealed that it localizes to the parasite mitochondrion. Our data are consistent with PfMFR3 playing roles in mitochondrial transport as well as drug resistance for clinically relevant antimalarials that target the mitochondria. Furthermore, given that pfmfr3 is naturally polymorphic, naturally occurring mutations may lead to differential sensitivity to clinically relevant compounds such as atovaquone.
Asunto(s)
Antimaláricos , Malaria , Antimaláricos/farmacología , Resistencia a Medicamentos , Humanos , Malaria/tratamiento farmacológico , Mutación , Plasmodium falciparum/genéticaRESUMEN
The search for antimalarial chemotypes with modes of action unrelated to existing drugs has intensified with the recent failure of first-line therapies across Southeast Asia. Here, we show that the trisubstituted imidazole MMV030084 potently inhibits hepatocyte invasion by Plasmodium sporozoites, merozoite egress from asexual blood stage schizonts, and male gamete exflagellation. Metabolomic, phosphoproteomic, and chemoproteomic studies, validated with conditional knockdown parasites, molecular docking, and recombinant kinase assays, identified cGMP-dependent protein kinase (PKG) as the primary target of MMV030084. PKG is known to play essential roles in Plasmodium invasion of and egress from host cells, matching MMV030084's activity profile. Resistance selections and gene editing identified tyrosine kinase-like protein 3 as a low-level resistance mediator for PKG inhibitors, while PKG itself never mutated under pressure. These studies highlight PKG as a resistance-refractory antimalarial target throughout the Plasmodium life cycle and promote MMV030084 as a promising Plasmodium PKG-targeting chemotype.
Asunto(s)
Antimaláricos/farmacología , Proteínas Quinasas Dependientes de GMP Cíclico/antagonistas & inhibidores , Resistencia a Medicamentos/efectos de los fármacos , Plasmodium falciparum/efectos de los fármacos , Proteínas Protozoarias/antagonistas & inhibidores , Animales , Antimaláricos/química , Antimaláricos/metabolismo , Sitios de Unión , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Femenino , Hepatocitos/citología , Hepatocitos/metabolismo , Hepatocitos/parasitología , Humanos , Imidazoles/química , Estadios del Ciclo de Vida/efectos de los fármacos , Metabolómica , Ratones , Ratones Endogámicos BALB C , Simulación del Acoplamiento Molecular , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/metabolismo , Proteómica , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismoRESUMEN
We report detailed susceptibility profiling of asexual blood stages of the malaria parasite Plasmodium falciparum to clinical and experimental antimalarials, combined with metabolomic fingerprinting. Results revealed a variety of stage-specific and metabolic profiles that differentiated the modes of action of clinical antimalarials including chloroquine, piperaquine, lumefantrine, and mefloquine, and identified late trophozoite-specific peak activity and stage-specific biphasic dose-responses for the mitochondrial inhibitors DSM265 and atovaquone. We also identified experimental antimalarials hitting previously unexplored druggable pathways as reflected by their unique stage specificity and/or metabolic profiles. These included several ring-active compounds, ones affecting hemoglobin catabolism through distinct pathways, and mitochondrial inhibitors with lower propensities for resistance than either DSM265 or atovaquone. This approach, also applicable to other microbes that undergo multiple differentiation steps, provides an effective tool to prioritize compounds for further development within the context of combination therapies.
Asunto(s)
Antimaláricos/farmacología , Metaboloma/efectos de los fármacos , Metabolómica , Plasmodium falciparum/efectos de los fármacos , Antimaláricos/química , Antimaláricos/metabolismo , Atovacuona/química , Atovacuona/metabolismo , Atovacuona/farmacología , Diseño de Fármacos , Proteínas del Complejo de Cadena de Transporte de Electrón/antagonistas & inhibidores , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Humanos , Estadios del Ciclo de Vida/efectos de los fármacos , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/parasitología , Malaria Falciparum/patología , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/metabolismo , Quinolinas/química , Quinolinas/metabolismo , Quinolinas/farmacologíaRESUMEN
Plasmodium parasites possess a protein with homology to Niemann-Pick Type C1 proteins (Niemann-Pick Type C1-Related protein, NCR1). We isolated parasites with resistance-conferring mutations in Plasmodium falciparum NCR1 (PfNCR1) during selections with three diverse small-molecule antimalarial compounds and show that the mutations are causative for compound resistance. PfNCR1 protein knockdown results in severely attenuated growth and confers hypersensitivity to the compounds. Compound treatment or protein knockdown leads to increased sensitivity of the parasite plasma membrane (PPM) to the amphipathic glycoside saponin and engenders digestive vacuoles (DVs) that are small and malformed. Immuno-electron microscopy and split-GFP experiments localize PfNCR1 to the PPM. Our experiments show that PfNCR1 activity is critically important for the composition of the PPM and is required for DV biogenesis, suggesting PfNCR1 as a novel antimalarial drug target. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).
Asunto(s)
Membrana Celular/metabolismo , Proteína Niemann-Pick C1/metabolismo , Plasmodium falciparum/enzimología , Plasmodium falciparum/crecimiento & desarrollo , Proteínas Protozoarias/metabolismo , Técnicas de Silenciamiento del Gen , Homeostasis , Proteína Niemann-Pick C1/genética , Proteínas Protozoarias/genéticaRESUMEN
To discover leads for next-generation chemoprotective antimalarial drugs, we tested more than 500,000 compounds for their ability to inhibit liver-stage development of luciferase-expressing Plasmodium spp. parasites (681 compounds showed a half-maximal inhibitory concentration of less than 1 micromolar). Cluster analysis identified potent and previously unreported scaffold families as well as other series previously associated with chemoprophylaxis. Further testing through multiple phenotypic assays that predict stage-specific and multispecies antimalarial activity distinguished compound classes that are likely to provide symptomatic relief by reducing asexual blood-stage parasitemia from those which are likely to only prevent malaria. Target identification by using functional assays, in vitro evolution, or metabolic profiling revealed 58 mitochondrial inhibitors but also many chemotypes possibly with previously unidentified mechanisms of action.
Asunto(s)
Antimaláricos/farmacología , Quimioprevención , Descubrimiento de Drogas , Malaria/prevención & control , Plasmodium/efectos de los fármacos , Antimaláricos/química , Antimaláricos/aislamiento & purificación , Antimaláricos/uso terapéutico , Evaluación Preclínica de Medicamentos , Humanos , Mitocondrias/efectos de los fármacos , Plasmodium/crecimiento & desarrolloRESUMEN
Chemogenetic characterization through in vitro evolution combined with whole-genome analysis can identify antimalarial drug targets and drug-resistance genes. We performed a genome analysis of 262 Plasmodium falciparum parasites resistant to 37 diverse compounds. We found 159 gene amplifications and 148 nonsynonymous changes in 83 genes associated with drug-resistance acquisition, where gene amplifications contributed to one-third of resistance acquisition events. Beyond confirming previously identified multidrug-resistance mechanisms, we discovered hitherto unrecognized drug target-inhibitor pairs, including thymidylate synthase and a benzoquinazolinone, farnesyltransferase and a pyrimidinedione, and a dipeptidylpeptidase and an arylurea. This exploration of the P. falciparum resistome and druggable genome will likely guide drug discovery and structural biology efforts, while also advancing our understanding of resistance mechanisms available to the malaria parasite.