RESUMO
Plasmodium species, the causative agent of malaria, rely on glucose for energy supply during blood stage. Inhibition of glucose uptake thus represents a potential strategy for the development of antimalarial drugs. Here, we present the crystal structures of PfHT1, the sole hexose transporter in the genome of Plasmodium species, at resolutions of 2.6 Å in complex with D-glucose and 3.7 Å with a moderately selective inhibitor, C3361. Although both structures exhibit occluded conformations, binding of C3361 induces marked rearrangements that result in an additional pocket. This inhibitor-binding-induced pocket presents an opportunity for the rational design of PfHT1-specific inhibitors. Among our designed C3361 derivatives, several exhibited improved inhibition of PfHT1 and cellular potency against P. falciparum, with excellent selectivity to human GLUT1. These findings serve as a proof of concept for the development of the next-generation antimalarial chemotherapeutics by simultaneously targeting the orthosteric and allosteric sites of PfHT1.
Assuntos
Proteínas de Transporte de Monossacarídeos/ultraestrutura , Plasmodium falciparum/metabolismo , Plasmodium falciparum/ultraestrutura , Proteínas de Protozoários/ultraestrutura , Sequência de Aminoácidos , Animais , Antimaláricos , Transporte Biológico , Glucose/metabolismo , Humanos , Malária , Malária Falciparum/parasitologia , Proteínas de Transporte de Monossacarídeos/química , Proteínas de Transporte de Monossacarídeos/metabolismo , Parasitos , Plasmodium falciparum/genética , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Açúcares/metabolismoRESUMO
Artemisinin-resistant malaria parasites have emerged and have been spreading, posing a significant public health challenge. Antimalarial drugs with novel mechanisms of action are therefore urgently needed. In this report, we exploit a "selective starvation" strategy by inhibiting Plasmodium falciparum hexose transporter 1 (PfHT1), the sole hexose transporter in P. falciparum, over human glucose transporter 1 (hGLUT1), providing an alternative approach to fight against multidrug-resistant malaria parasites. The crystal structure of hGLUT3, which shares 80% sequence similarity with hGLUT1, was resolved in complex with C3361, a moderate PfHT1-specific inhibitor, at 2.3-Å resolution. Structural comparison between the present hGLUT3-C3361 and our previously reported PfHT1-C3361 confirmed the unique inhibitor binding-induced pocket in PfHT1. We then designed small molecules to simultaneously block the orthosteric and allosteric pockets of PfHT1. Through extensive structure-activity relationship studies, the TH-PF series was identified to selectively inhibit PfHT1 over hGLUT1 and potent against multiple strains of the blood-stage P. falciparum Our findings shed light on the next-generation chemotherapeutics with a paradigm-shifting structure-based design strategy to simultaneously target the orthosteric and allosteric sites of a transporter.
Assuntos
Antimaláricos/química , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 3/ultraestrutura , Malária Falciparum/tratamento farmacológico , Proteínas de Transporte de Monossacarídeos/ultraestrutura , Proteínas de Protozoários/ultraestrutura , Sítio Alostérico , Sequência de Aminoácidos/genética , Animais , Cristalografia por Raios X , Glucose/metabolismo , Transportador de Glucose Tipo 1/antagonistas & inibidores , Transportador de Glucose Tipo 1/química , Transportador de Glucose Tipo 3/química , Malária Falciparum/genética , Malária Falciparum/parasitologia , Proteínas de Transporte de Monossacarídeos/antagonistas & inibidores , Proteínas de Transporte de Monossacarídeos/genética , Plasmodium falciparum/química , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Plasmodium falciparum/patogenicidade , Conformação Proteica/efeitos dos fármacos , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/genética , Relação Estrutura-AtividadeRESUMO
Antimalarial drugs have thus far been chiefly derived from two sources-natural products and synthetic drug-like compounds. Here we investigate whether antimalarial agents with novel mechanisms of action could be discovered using a diverse collection of synthetic compounds that have three-dimensional features reminiscent of natural products and are underrepresented in typical screening collections. We report the identification of such compounds with both previously reported and undescribed mechanisms of action, including a series of bicyclic azetidines that inhibit a new antimalarial target, phenylalanyl-tRNA synthetase. These molecules are curative in mice at a single, low dose and show activity against all parasite life stages in multiple in vivo efficacy models. Our findings identify bicyclic azetidines with the potential to both cure and prevent transmission of the disease as well as protect at-risk populations with a single oral dose, highlighting the strength of diversity-oriented synthesis in revealing promising therapeutic targets.
Assuntos
Antimaláricos/síntese química , Antimaláricos/farmacologia , Azetidinas/uso terapêutico , Descoberta de Drogas , Estágios do Ciclo de Vida/efeitos dos fármacos , Malária Falciparum/tratamento farmacológico , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/crescimento & desenvolvimento , Animais , Antimaláricos/administração & dosagem , Antimaláricos/uso terapêutico , Compostos Azabicíclicos/administração & dosagem , Compostos Azabicíclicos/síntese química , Compostos Azabicíclicos/farmacologia , Compostos Azabicíclicos/uso terapêutico , Azetidinas/administração & dosagem , Azetidinas/efeitos adversos , Azetidinas/farmacologia , Citosol/enzimologia , Modelos Animais de Doenças , Feminino , Fígado/efeitos dos fármacos , Fígado/parasitologia , Macaca mulatta/parasitologia , Malária Falciparum/prevenção & controle , Malária Falciparum/transmissão , Masculino , Camundongos , Fenilalanina-tRNA Ligase/antagonistas & inibidores , Compostos de Fenilureia/administração & dosagem , Compostos de Fenilureia/síntese química , Compostos de Fenilureia/farmacologia , Compostos de Fenilureia/uso terapêutico , Plasmodium falciparum/citologia , Plasmodium falciparum/enzimologia , SegurançaRESUMO
Inflammation disrupts tissue architecture and function, thereby contributing to the pathogenesis of diverse diseases; the signals that promote or restrict tissue inflammation thus represent potential targets for therapeutic intervention. Here, we report that genetic or pharmacologic Hedgehog pathway inhibition intensifies colon inflammation (colitis) in mice. Conversely, genetic augmentation of Hedgehog response and systemic small-molecule Hedgehog pathway activation potently ameliorate colitis and restrain initiation and progression of colitis-induced adenocarcinoma. Within the colon, the Hedgehog protein signal does not act directly on the epithelium itself, but on underlying stromal cells to induce expression of IL-10, an immune-modulatory cytokine long known to suppress inflammatory intestinal damage. IL-10 function is required for the full protective effect of small-molecule Hedgehog pathway activation in colitis; this pharmacologic augmentation of Hedgehog pathway activity and stromal IL-10 expression are associated with increased presence of CD4+Foxp3+ regulatory T cells. We thus identify stromal cells as cellular coordinators of colon inflammation and suggest their pharmacologic manipulation as a potential means to treat colitis.
Assuntos
Colite/metabolismo , Sulfato de Dextrana/efeitos adversos , Proteínas Hedgehog/metabolismo , Interleucina-10/metabolismo , Transdução de Sinais , Animais , Antígenos CD4/metabolismo , Colite/induzido quimicamente , Colite/tratamento farmacológico , Modelos Animais de Doenças , Progressão da Doença , Fatores de Transcrição Forkhead/metabolismo , Proteínas Hedgehog/efeitos dos fármacos , Humanos , Camundongos , Mutação , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/administração & dosagem , Bibliotecas de Moléculas Pequenas/farmacologia , Linfócitos T Reguladores/metabolismo , Proteína GLI1 em Dedos de Zinco/genéticaRESUMO
The synthesis of a 2-methyl-substituted analogue of the natural product, neopeltolide, is reported in an effort to analyze the importance of molecular conformation and ligand-target interactions in relation to biological activity. The methyl substitution was incorporated via highly diastereoselective ester enolate alkylation of a late-stage intermediate. Coupling of the oxazole sidechain provided 2-methyl-neopeltolide and synthetic neopeltolide via total synthesis. The substitution was shown to maintain the conformational preferences of its biologically active parent compound through computer modeling and NMR studies. Both compounds were shown to be potential antimalarial compounds through the inhibition of mitochondrial respiration in P. falciparum parasites.
Assuntos
Antimaláricos/síntese química , Antimaláricos/farmacologia , Produtos Biológicos/farmacologia , Desenho de Fármacos , Macrolídeos/farmacologia , Mitocôndrias/efeitos dos fármacos , Plasmodium falciparum/efeitos dos fármacos , Antimaláricos/química , Produtos Biológicos/síntese química , Produtos Biológicos/química , Respiração Celular/efeitos dos fármacos , Macrolídeos/síntese química , Macrolídeos/química , Mitocôndrias/metabolismo , Conformação Molecular , Testes de Sensibilidade Parasitária , Plasmodium falciparum/metabolismoRESUMO
Pancreatic ductal adenocarcinoma (PDA) is the most lethal of common human malignancies, with no truly effective therapies for advanced disease. Preclinical studies have suggested a therapeutic benefit of targeting the Hedgehog (Hh) signaling pathway, which is activated throughout the course of PDA progression by expression of Hh ligands in the neoplastic epithelium and paracrine response in the stromal fibroblasts. Clinical trials to test this possibility, however, have yielded disappointing results. To further investigate the role of Hh signaling in the formation of PDA and its precursor lesion, pancreatic intraepithelial neoplasia (PanIN), we examined the effects of genetic or pharmacologic inhibition of Hh pathway activity in three distinct genetically engineered mouse models and found that Hh pathway inhibition accelerates rather than delays progression of oncogenic Kras-driven disease. Notably, pharmacologic inhibition of Hh pathway activity affected the balance between epithelial and stromal elements, suppressing stromal desmoplasia but also causing accelerated growth of the PanIN epithelium. In striking contrast, pathway activation using a small molecule agonist caused stromal hyperplasia and reduced epithelial proliferation. These results indicate that stromal response to Hh signaling is protective against PDA and that pharmacologic activation of pathway response can slow tumorigenesis. Our results provide evidence for a restraining role of stroma in PDA progression, suggesting an explanation for the failure of Hh inhibitors in clinical trials and pointing to the possibility of a novel type of therapeutic intervention.
Assuntos
Carcinoma Ductal Pancreático/metabolismo , Proteínas Hedgehog/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais , Animais , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Proteínas Hedgehog/antagonistas & inibidores , Proteínas Hedgehog/genética , Humanos , Camundongos , Camundongos Knockout , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas p21(ras)/genéticaRESUMO
Antimalarial drug discovery has until recently been driven by high-throughput phenotypic cellular screening, allowing millions of compounds to be assayed and delivering clinical drug candidates. In this review, we will focus on target-based approaches, describing recent advances in our understanding of druggable targets in the malaria parasite. Targeting multiple stages of the Plasmodium lifecycle, rather than just the clinically symptomatic asexual blood stage, has become a requirement for new antimalarial medicines, and we link pharmacological data clearly to the parasite stages to which it applies. Finally, we highlight the IUPHAR/MMV Guide to MALARIA PHARMACOLOGY, a web resource developed for the malaria research community that provides open and optimized access to published data on malaria pharmacology.
Assuntos
Antimaláricos , Malária , Humanos , Malária/tratamento farmacológico , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Descoberta de Drogas , Ensaios de Triagem em Larga EscalaRESUMO
Widespread Plasmodium falciparum resistance to first-line antimalarials underscores the vital need to develop compounds with novel modes of action and identify new druggable targets. Here, we profile five compounds that potently inhibit P. falciparum asexual blood stages. Resistance selection studies with three carboxamide-containing compounds, confirmed by gene editing and conditional knockdowns, identify point mutations in the parasite transporter ABCI3 as the primary mediator of resistance. Selection studies with imidazopyridine or quinoline-carboxamide compounds also yield changes in ABCI3, this time through gene amplification. Imidazopyridine mode of action is attributed to inhibition of heme detoxification, as evidenced by cellular accumulation and heme fractionation assays. For the copy-number variation-selecting imidazopyridine and quinoline-carboxamide compounds, we find that resistance, manifesting as a biphasic concentration-response curve, can independently be mediated by mutations in the chloroquine resistance transporter PfCRT. These studies reveal the interconnectedness of P. falciparum transporters in overcoming drug pressure in different parasite strains.
Assuntos
Antimaláricos , Antagonistas do Ácido Fólico , Malária Falciparum , Parasitos , Quinolinas , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Heme , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Proteínas de Membrana Transportadoras/genética , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Quinolinas/farmacologiaRESUMO
Gli transcription factors within the Hedgehog (Hh) signaling pathway direct key events in mammalian development and promote a number of human cancers. Current therapies for Gli-driven tumors target Smoothened (SMO), a G protein-coupled receptor-like protein that functions upstream in the Hh pathway. Although these drugs can have remarkable clinical efficacy, mutations in SMO and downstream Hh pathway components frequently lead to chemoresistance. In principle, therapies that act at the level of Gli proteins, through direct or indirect mechanisms, would be more efficacious. We therefore screened 325 120 compounds for their ability to block the constitutive Gli activity induced by loss of Suppressor of Fused (SUFU), a scaffolding protein that directly inhibits Gli function. Our studies reveal a family of bicyclic imidazolium derivatives that can inhibit Gli-dependent transcription without affecting the ciliary trafficking or proteolytic processing of these transcription factors. We anticipate that these chemical antagonists will be valuable tools for investigating the mechanisms of Gli regulation and developing new strategies for targeting Gli-driven cancers.
Assuntos
Imidazóis/farmacologia , Proteína GLI1 em Dedos de Zinco/antagonistas & inibidores , Animais , Compostos Heterocíclicos com 2 Anéis/síntese química , Compostos Heterocíclicos com 2 Anéis/farmacologia , Compostos Heterocíclicos com 3 Anéis/síntese química , Compostos Heterocíclicos com 3 Anéis/farmacologia , Imidazóis/síntese química , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Estrutura Molecular , Células NIH 3T3 , Fosforilação Oxidativa/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
Resistance has developed in Plasmodium malaria parasites to every antimalarial drug in clinical use, prompting the need to characterize the pathways mediating resistance. Here, we report a framework for assessing development of resistance of Plasmodium falciparum to new antimalarial therapeutics. We investigated development of resistance by P. falciparum to the dihydroorotate dehydrogenase (DHODH) inhibitors DSM265 and DSM267 in tissue culture and in a mouse model of P. falciparum infection. We found that resistance to these drugs arose rapidly both in vitro and in vivo. We identified 13 point mutations mediating resistance in the parasite DHODH in vitro that overlapped with the DHODH mutations that arose in the mouse infection model. Mutations in DHODH conferred increased resistance (ranging from 2- to ~400-fold) to DHODH inhibitors in P. falciparum in vitro and in vivo. We further demonstrated that the drug-resistant parasites carrying the C276Y mutation had mitochondrial energetics comparable to the wild-type parasite and also retained their fitness in competitive growth experiments. Our data suggest that in vitro selection of drug-resistant P. falciparum can predict development of resistance in a mouse model of malaria infection.
Assuntos
Inibidores Enzimáticos/uso terapêutico , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Parasitos/enzimologia , Animais , Di-Hidro-Orotato Desidrogenase , Modelos Animais de Doenças , Resistência a Medicamentos/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Feminino , Camundongos SCID , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Parasitos/efeitos dos fármacos , Fenótipo , Plasmodium falciparum , Mutação Puntual/genética , Pirimidinas/química , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Triazóis/química , Triazóis/farmacologia , Triazóis/uso terapêuticoRESUMO
Drug resistance has been reported for every antimalarial in use highlighting the need for new strategies to protect the efficacy of therapeutics in development. We have previously shown that resistance can be suppressed with a population biology trap: by identifying situations where resistance to one compound confers hypersensitivity to another (collateral sensitivity), we can design combination therapies that not only kill the parasite but also guide its evolution away from resistance. We applied this concept to the Plasmodium falciparum dihydroorotate dehydrogenase ( PfDHODH) enzyme, a well validated antimalarial target with inhibitors in the development pipeline. Here, we report a high-throughput screen to identify compounds specifically active against PfDHODH resistant mutants. We additionally perform extensive cross-resistance profiling allowing us to identify compound pairs demonstrating the potential for mutually incompatible resistance. These combinations represent promising starting points for exploiting collateral sensitivity to extend the useful lifespan of new antimalarial therapeutics.
Assuntos
Antimaláricos/isolamento & purificação , Antimaláricos/farmacologia , Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Di-Hidro-Orotato Desidrogenase , Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios de Triagem em Larga Escala/métodosRESUMO
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.
Assuntos
Antimaláricos/farmacologia , Quimioprevenção , Descoberta de Drogas , Malária/prevenção & controle , Plasmodium/efeitos dos fármacos , Antimaláricos/química , Antimaláricos/isolamento & purificação , Antimaláricos/uso terapêutico , Avaliação Pré-Clínica de Medicamentos , Humanos , Mitocôndrias/efeitos dos fármacos , Plasmodium/crescimento & desenvolvimentoRESUMO
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.
Assuntos
Antimaláricos/farmacologia , Resistência a Medicamentos/genética , Genoma de Protozoário , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Ativação Metabólica , Alelos , Variações do Número de Cópias de DNA , Evolução Molecular Direcionada , Resistência a Múltiplos Medicamentos/genética , Genes de Protozoários , Metabolômica , Mutação , Plasmodium falciparum/crescimento & desenvolvimento , Seleção Genética , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Given that resistance to all drugs in clinical use has arisen, discovery of new antimalarial drug targets is eagerly anticipated. The Plasmodium mitochondrion has been considered a promising drug target largely based on its significant divergence from the host organelle as well as its involvement in ATP production and pyrimidine biosynthesis. However, the functions of Plasmodium mitochondrial protein complexes and associated metabolic pathways are not fully characterized. Here, we report the development of novel and robust bioenergetic assay protocols for Plasmodium falciparum asexual parasites utilizing a Seahorse Bioscience XFe24 Extracellular Flux Analyzer. These protocols allowed us to simultaneously assess the direct effects of metabolites and inhibitors on mitochondrial respiration and glycolytic activity in real-time with the readout of oxygen consumption rate and extracellular acidification rate. Using saponin-freed parasites at the schizont stage, we found that succinate, malate, glycerol-3-phosphate, and glutamate, but not pyruvate, were able to increase the oxygen consumption rate and that glycerol-3-phosphate dehydrogenase had the largest potential as an electron donor among tested mitochondrial dehydrogenases. Furthermore, we revealed the presence of a glucose-regulated metabolic shift between oxidative phosphorylation and glycolysis. We measured proton leak and reserve capacity and found bioenergetic evidence for oxidative phosphorylation in erythrocytic stage parasites but at a level much lower than that observed in mammalian cells. Lastly, we developed an assay platform for target identification and mode of action studies of mitochondria-targeting antimalarials. This study provides new insights into the bioenergetics and metabolomics of the Plasmodium mitochondria.
Assuntos
Antimaláricos/farmacologia , Descoberta de Drogas/métodos , Glucose/metabolismo , Malária Falciparum/parasitologia , Mitocôndrias/metabolismo , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/metabolismo , Animais , Metabolismo Energético , Glicólise/efeitos dos fármacos , Humanos , Redes e Vias Metabólicas/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Fosforilação Oxidativa/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Plasmodium falciparum/genética , Plasmodium falciparum/crescimento & desenvolvimentoRESUMO
Cytoplasmic dyneins 1 and 2 are related members of the AAA+ superfamily (ATPases associated with diverse cellular activities) that function as the predominant minus-end-directed microtubule motors in eukaryotic cells. Dynein 1 controls mitotic spindle assembly, organelle movement, axonal transport, and other cytosolic, microtubule-guided processes, whereas dynein 2 mediates retrograde trafficking within motile and primary cilia. Small-molecule inhibitors are important tools for investigating motor protein-dependent mechanisms, and ciliobrevins were recently discovered as the first dynein-specific chemical antagonists. Here, we demonstrate that ciliobrevins directly target the heavy chains of both dynein isoforms and explore the structure-activity landscape of these inhibitors in vitro and in cells. In addition to identifying chemical motifs that are essential for dynein blockade, we have discovered analogs with increased potency and dynein 2 selectivity. These antagonists effectively disrupt Hedgehog signaling, intraflagellar transport, and ciliogenesis, making them useful probes of these and other cytoplasmic dynein 2-dependent cellular processes.
Assuntos
Dineínas do Citoplasma/antagonistas & inibidores , Dineínas do Citoplasma/química , Animais , Proteínas Hedgehog/fisiologia , Camundongos , Estrutura Molecular , Células NIH 3T3 , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/química , Quinazolinonas/química , Quinazolinonas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Microbial resistance to chemotherapy has caused countless deaths where malaria is endemic. Chemotherapy may fail either due to pre-existing resistance or evolution of drug-resistant parasites. Here we use a diverse set of antimalarial compounds to investigate the acquisition of drug resistance and the degree of cross-resistance against common resistance alleles. We assess cross-resistance using a set of 15 parasite lines carrying resistance-conferring alleles in pfatp4, cytochrome bc1, pfcarl, pfdhod, pfcrt, pfmdr, pfdhfr, cytoplasmic prolyl t-RNA synthetase or hsp90. Subsequently, we assess whether resistant parasites can be obtained after several rounds of drug selection. Twenty-three of the 48 in vitro selections result in resistant parasites, with time to resistance onset ranging from 15 to 300 days. Our data indicate that pre-existing resistance may not be a major hurdle for novel-target antimalarial candidates, and focusing our attention on fast-killing compounds may result in a slower onset of clinical resistance.
Assuntos
Resistência a Medicamentos , Parasitos/fisiologia , Plasmodium falciparum/fisiologia , Animais , Antimaláricos/farmacologia , Células Clonais , Resistência a Medicamentos/efeitos dos fármacos , Mutação INDEL/genética , Mutação/genética , Parasitos/efeitos dos fármacos , Plasmodium falciparum/efeitos dos fármacos , Polimorfismo de Nucleotídeo Único/genéticaRESUMO
Aminopyrazoles are a new class of antimalarial compounds identified in a cellular antiparasitic screen with potent activity against Plasmodium falciparum asexual and sexual stage parasites. To investigate their unknown mechanism of action and thus identify their target, we cultured parasites in the presence of a representative member of the aminopyrazole series, GNF-Pf4492, to select for resistance. Whole genome sequencing of three resistant lines showed that each had acquired independent mutations in a P-type cation-transporter ATPase, PfATP4 (PF3D7_1211900), a protein implicated as the novel Plasmodium spp. target of another, structurally unrelated, class of antimalarials called the spiroindolones and characterized as an important sodium transporter of the cell. Similarly to the spiroindolones, GNF-Pf4492 blocks parasite transmission to mosquitoes and disrupts intracellular sodium homeostasis. Our data demonstrate that PfATP4 plays a critical role in cellular processes, can be inhibited by two distinct antimalarial pharmacophores, and supports the recent observations that PfATP4 is a critical antimalarial target.