RESUMO
Concerns about malaria parasite resistance to treatment with artemisinin drugs (ARTs) have grown with findings of prolonged parasite clearance t1/2s (>5 h) and their association with mutations in Plasmodium falciparum Kelch-propeller protein K13. Here, we describe a P. falciparum laboratory cross of K13 C580Y mutant with C580 wild-type parasites to investigate ART response phenotypes in vitro and in vivo. After genotyping >400 isolated progeny, we evaluated 20 recombinants in vitro: IC50 measurements of dihydroartemisinin were at similar low nanomolar levels for C580Y- and C580-type progeny (mean ratio, 1.00; 95% CI, 0.62-1.61), whereas, in a ring-stage survival assay, the C580Y-type progeny had 19.6-fold (95% CI, 9.76-39.2) higher average counts. In splenectomized Aotus monkeys treated with three daily doses of i.v. artesunate, t1/2 calculations by three different methods yielded mean differences of 0.01 h (95% CI, -3.66 to 3.67), 0.80 h (95% CI, -0.92 to 2.53), and 2.07 h (95% CI, 0.77-3.36) between C580Y and C580 infections. Incidences of recrudescence were 57% in C580Y (4 of 7) versus 70% in C580 (7 of 10) infections (-13% difference; 95% CI, -58% to 35%). Allelic substitution of C580 in a C580Y-containing progeny clone (76H10) yielded a transformant (76H10C580Rev) that, in an infected monkey, recrudesced regularly 13 times over 500 d. Frequent recrudescences of ART-treated P. falciparum infections occur with or without K13 mutations and emphasize the need for improved partner drugs to effectively eliminate the parasites that persist through the ART component of combination therapy.
Assuntos
Antimaláricos/farmacologia , Artemisininas/farmacologia , Malária Falciparum/parasitologia , Plasmodium falciparum/efeitos dos fármacos , Animais , Aotidae , Cruzamentos Genéticos , Resistência a Medicamentos , Regulação da Expressão Gênica , Mutação , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismoRESUMO
Genetic screening using random transposon insertions has been a powerful tool for uncovering biology in prokaryotes, where whole-genome saturating screens have been performed in multiple organisms. In eukaryotes, such screens have proven more problematic, in part because of the lack of a sensitive and robust system for identifying transposon insertion sites. We here describe quantitative insertion-site sequencing, or QIseq, which uses custom library preparation and Illumina sequencing technology and is able to identify insertion sites from both the 5' and 3' ends of the transposon, providing an inbuilt level of validation. The approach was developed using piggyBac mutants in the human malaria parasite Plasmodium falciparum but should be applicable to many other eukaryotic genomes. QIseq proved accurate, confirming known sites in >100 mutants, and sensitive, identifying and monitoring sites over a >10,000-fold dynamic range of sequence counts. Applying QIseq to uncloned parasites shortly after transfections revealed multiple insertions in mixed populations and suggests that >4000 independent mutants could be generated from relatively modest scales of transfection, providing a clear pathway to genome-scale screens in P. falciparum QIseq was also used to monitor the growth of pools of previously cloned mutants and reproducibly differentiated between deleterious and neutral mutations in competitive growth. Among the mutants with fitness defects was a mutant with a piggyBac insertion immediately upstream of the kelch protein K13 gene associated with artemisinin resistance, implying mutants in this gene may have competitive fitness costs. QIseq has the potential to enable the scale-up of piggyBac-mediated genetics across multiple eukaryotic systems.
Assuntos
Elementos de DNA Transponíveis , Sequenciamento de Nucleotídeos em Larga Escala , Análise de Sequência de DNA , Sequência de Bases , Biblioteca Gênica , Ontologia Genética , Mutagênese Insercional , Fenótipo , Plasmodium falciparum/genéticaRESUMO
Malaria kills approximately 1 million people a year, mainly in sub-Saharan Africa. Essential steps in the life cycle of the parasite are the development of gametocytes, as well as the formation of oocysts and sporozoites, in the Anopheles mosquito vector. Preventing transmission of malaria through the mosquito is necessary for the control of the disease; nevertheless, the vast majority of drugs in use act primarily against the blood stages. The study described herein focuses on the assessment of the transmission-blocking activities of potent antierythrocytic stage agents derived from the 4(1H)-quinolone scaffold. In particular, three 3-alkyl- or 3-phenyl-4(1H)-quinolones (P4Qs), one 7-(2-phenoxyethoxy)-4(1H)-quinolone (PEQ), and one 1,2,3,4-tetrahydroacridin-9(10H)-one (THA) were assessed for their transmission-blocking activity against the mosquito stages of the human malaria parasite (Plasmodium falciparum) and the rodent parasite (P. berghei). Results showed that all of the experimental compounds reduced or prevented the exflagellation of male gametocytes and, more importantly, prevented parasite transmission to the mosquito vector. Additionally, treatment with ICI 56,780 reduced the number of sporozoites that reached the Anopheles salivary glands. These findings suggest that 4(1H)-quinolones, which have activity against the blood stages, can also prevent the transmission of Plasmodium to the mosquito and, hence, are potentially important drug candidates to eradicate malaria.
Assuntos
Acridinas/farmacologia , Anopheles/efeitos dos fármacos , Antimaláricos/farmacologia , Estágios do Ciclo de Vida/efeitos dos fármacos , Malária Falciparum/prevenção & controle , Malária/prevenção & controle , Quinolonas/farmacologia , Acridinas/síntese química , Animais , Anopheles/parasitologia , Antimaláricos/síntese química , Feminino , Humanos , Insetos Vetores , Estágios do Ciclo de Vida/fisiologia , Malária/parasitologia , Malária/transmissão , Malária Falciparum/parasitologia , Malária Falciparum/transmissão , Masculino , Camundongos , Testes de Sensibilidade Parasitária , Plasmodium berghei/efeitos dos fármacos , Plasmodium berghei/crescimento & desenvolvimento , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/crescimento & desenvolvimento , Quinolonas/síntese química , Glândulas Salivares/efeitos dos fármacos , Glândulas Salivares/parasitologia , Relação Estrutura-AtividadeRESUMO
With the exception of primaquine, tafenoquine, and atovaquone, there are very few antimalarials that target liver stage parasites. In this study, a transgenic Plasmodium berghei parasite (1052Cl1; PbGFP-Luc(con)) that expresses luciferase was used to assess the anti-liver stage parasite activity of ICI 56,780, a 7-(2-phenoxyethoxy)-4(1H)-quinolone (PEQ), as well as two 3-phenyl-4(1H)-quinolones (P4Q), P4Q-146 and P4Q-158, by using bioluminescent imaging (BLI). Results showed that all of the compounds were active against liver stage parasites; however, ICI 56,780 and P4Q-158 were the most active, with low nanomolar activity in vitro and causal prophylactic activity in vivo. This potent activity makes these compounds ideal candidates for advancement as novel antimalarials.
Assuntos
Antimaláricos/síntese química , Antimaláricos/farmacologia , Malária/tratamento farmacológico , Plasmodium berghei/efeitos dos fármacos , Quinolonas/síntese química , Quinolonas/farmacologia , Esporozoítos/efeitos dos fármacos , Animais , Feminino , Genes Reporter , Células Hep G2 , Hepatócitos/efeitos dos fármacos , Hepatócitos/parasitologia , Humanos , Concentração Inibidora 50 , Cinética , Fígado/efeitos dos fármacos , Fígado/parasitologia , Luciferases , Malária/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Organismos Geneticamente Modificados , Plasmodium berghei/genética , Plasmodium berghei/crescimento & desenvolvimento , Esporozoítos/crescimento & desenvolvimentoRESUMO
New drugs to treat malaria must act rapidly and be highly potent against asexual blood stages, well tolerated, and affordable to residents of regions of endemicity. This was the case with chloroquine (CQ), a 4-aminoquinoline drug used for the prevention and treatment of malaria. However, since the 1960s, Plasmodium falciparum resistance to this drug has spread globally, and more recently, emerging resistance to CQ by Plasmodium vivax threatens the health of 70 to 320 million people annually. Despite the emergence of CQ resistance, synthetic quinoline derivatives remain validated leads for new drug discovery, especially if they are effective against CQ-resistant strains of malaria. In this study, we investigated the activities of two novel 4-aminoquinoline derivatives, TDR 58845, N(1)-(7-chloro-quinolin-4-yl)-2-methyl-propane-1,2-diamine, and TDR 58846, N(1)-(7-chloro-quinolin-4-yl)-2,N(2),N(2)-trimethylpropane-1,2-diamine and found them to be active against P. falciparum in vitro and Plasmodium berghei in vivo. The P. falciparum clones and isolates tested were susceptible to TDR 58845 and TDR 58846 (50% inhibitory concentrations [IC(50)s] ranging from 5.52 to 89.8 nM), including the CQ-resistant reference clone W2 and two multidrug-resistant parasites recently isolated from Thailand and Cambodia. Moreover, these 4-aminoquinolines were active against early and late P. falciparum gametocyte stages and cured BALB/c mice infected with P. berghei. TDR 58845 and TDR 58846 at 40 mg/kg were sufficient to cure mice, and total doses of 480 mg/kg of body weight were well tolerated. Our findings suggest these novel 4-aminoquinolines should be considered for development as potent antimalarials that can be used in combination to treat multidrug-resistant P. falciparum and P. vivax.
Assuntos
Aminoquinolinas/farmacologia , Antimaláricos/farmacologia , Cloroquina/análogos & derivados , Estágios do Ciclo de Vida/efeitos dos fármacos , Malária/tratamento farmacológico , Plasmodium berghei/efeitos dos fármacos , Plasmodium falciparum/efeitos dos fármacos , Plasmodium vivax/efeitos dos fármacos , Administração Oral , Aminoquinolinas/síntese química , Animais , Antimaláricos/síntese química , Camboja , Cloroquina/síntese química , Cloroquina/farmacologia , Esquema de Medicação , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Eritrócitos/parasitologia , Humanos , Concentração Inibidora 50 , Malária/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Plasmodium berghei/crescimento & desenvolvimento , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium vivax/crescimento & desenvolvimento , Taxa de Sobrevida , TailândiaRESUMO
Severe malaria is caused by the apicomplexan parasite Plasmodium falciparum. Despite decades of research, the distinct biology of these parasites has made it challenging to establish high-throughput genetic approaches to identify and prioritize therapeutic targets. Using transposon mutagenesis of P. falciparum in an approach that exploited its AT-rich genome, we generated more than 38,000 mutants, saturating the genome and defining mutability and fitness costs for over 87% of genes. Of 5399 genes, our study defined 2680 genes as essential for optimal growth of asexual blood stages in vitro. These essential genes are associated with drug resistance, represent leading vaccine candidates, and include approximately 1000 Plasmodium-conserved genes of unknown function. We validated this approach by testing proteasome pathways for individual mutants associated with artemisinin sensitivity.
Assuntos
Genes de Protozoários , Malária Falciparum/parasitologia , Plasmodium falciparum/genética , Reprodução Assexuada/genética , Animais , Antimaláricos/farmacologia , Artemisininas/farmacologia , Sequência Conservada , Resistência a Medicamentos/genética , Eritrócitos/parasitologia , Genes Essenciais , Aptidão Genética , Humanos , Vacinas Antimaláricas/genética , Mutagênese , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/crescimento & desenvolvimentoRESUMO
Malaria remains one of the most devastating parasitic diseases worldwide, with 90% of the malaria deaths in Africa in 2013 attributable to Plasmodium falciparum. The clinical symptoms of malaria include cycles of fever, corresponding to parasite rupture from red blood cells every 48 h. Parasite pathways involved in the parasite's ability to survive the host fever response, and indeed, the functions of ~40% of P. falciparum genes as a whole, are still largely unknown. Here, we evaluated the potential of scalable forward-genetic screening methods to identify genes involved in the host fever response. We performed a phenotypic screen for genes linked to the parasite response to febrile temperatures by utilizing a selection of single-disruption P. falciparum mutants generated via random piggyBac transposon mutagenesis in a previous study. We identified several mutants presenting significant phenotypes in febrile response screens compared to the wild type, indicating possible roles for the disrupted genes in this process. We present these initial studies as proof that forward genetics can be used to gain insight into critical factors associated with parasite biology. IMPORTANCE Though the P. falciparum genome sequence has been available for many years, ~40% of its genes do not have informative annotations, as they show no detectable homology to those of studied organisms. More still have not been evaluated via genetic methods. Scalable forward-genetic approaches that allow interrogation of gene function without any pre-existing knowledge are needed to hasten understanding of parasite biology, which will expedite the identification of drug targets and the development of future interventions in the face of spreading resistance to existing frontline drugs. In this work, we describe a new approach to pursue forward-genetic phenotypic screens for P. falciparum to identify factors associated with virulence. Future large-scale phenotypic screens developed to probe other such interesting phenomena, when considered in parallel, will prove a powerful tool for functional annotation of the P. falciparum genome, where so much remains undiscovered.
RESUMO
The spread of Plasmodium falciparum multidrug resistance highlights the urgency to discover new targets and chemical scaffolds. Unfortunately, lack of experimentally validated functional information about most P. falciparum genes remains a strategic hurdle. Chemogenomic profiling is an established tool for classification of drugs with similar mechanisms of action by comparing drug fitness profiles in a collection of mutants. Inferences of drug mechanisms of action and targets can be obtained by associations between shifts in drug fitness and specific genetic changes in the mutants. In this screen, P. falciparum, piggyBac single insertion mutants were profiled for altered responses to antimalarial drugs and metabolic inhibitors to create chemogenomic profiles. Drugs targeting the same pathway shared similar response profiles and multiple pairwise correlations of the chemogenomic profiles revealed novel insights into drugs' mechanisms of action. A mutant of the artemisinin resistance candidate gene - "K13-propeller" gene (PF3D7_1343700) exhibited increased susceptibility to artemisinin drugs and identified a cluster of 7 mutants based on similar enhanced responses to the drugs tested. Our approach of chemogenomic profiling reveals artemisinin functional activity, linked by the unexpected drug-gene relationships of these mutants, to signal transduction and cell cycle regulation pathways.