RESUMO
Drug discovery for malaria has been transformed in the last 5 years by the discovery of many new lead compounds identified by phenotypic screening. The process of developing these compounds as drug leads and studying the cellular responses they induce is revealing new targets that regulate key processes in the Plasmodium parasites that cause malaria. We disclose herein that the clinical candidate (+)-SJ733 acts upon one of these targets, ATP4. ATP4 is thought to be a cation-transporting ATPase responsible for maintaining low intracellular Na(+) levels in the parasite. Treatment of parasitized erythrocytes with (+)-SJ733 in vitro caused a rapid perturbation of Na(+) homeostasis in the parasite. This perturbation was followed by profound physical changes in the infected cells, including increased membrane rigidity and externalization of phosphatidylserine, consistent with eryptosis (erythrocyte suicide) or senescence. These changes are proposed to underpin the rapid (+)-SJ733-induced clearance of parasites seen in vivo. Plasmodium falciparum ATPase 4 (pfatp4) mutations that confer resistance to (+)-SJ733 carry a high fitness cost. The speed with which (+)-SJ733 kills parasites and the high fitness cost associated with resistance-conferring mutations appear to slow and suppress the selection of highly drug-resistant mutants in vivo. Together, our data suggest that inhibitors of PfATP4 have highly attractive features for fast-acting antimalarials to be used in the global eradication campaign.
Assuntos
Antimaláricos/farmacologia , ATPases Transportadoras de Cálcio/metabolismo , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Isoquinolinas/farmacologia , Malária/tratamento farmacológico , Modelos Moleculares , Plasmodium/efeitos dos fármacos , Antimaláricos/farmacocinética , ATPases Transportadoras de Cálcio/genética , Senescência Celular/efeitos dos fármacos , Descoberta de Drogas , Resistência a Medicamentos/genética , Eritrócitos/efeitos dos fármacos , Citometria de Fluxo , Compostos Heterocíclicos de 4 ou mais Anéis/farmacocinética , Ensaios de Triagem em Larga Escala , Isoquinolinas/farmacocinética , Estrutura MolecularRESUMO
BACKGROUND: Drugs that kill or inhibit Plasmodium gametocytes in the human host could potentially synergize the impact of other chemotherapeutic interventions by blocking transmission. To develop such agents, reliable methods are needed to study the in vitro activity of compounds against gametocytes. This study describes a novel assay for characterizing the activity of anti-malarial drugs against the later stages of Plasmodium falciparum gametocyte development using real-time PCR (qPCR). METHODS: Genes previously reported to be transcribed at the different sexual stages of the gametocytogenesis were selected for study and their mRNA expression was measured in a gametocytogenesis course by qPCR. Genes mainly expressed in the later stages of gametocyte development were used as a surrogate measurement of drug activity. To distinguish between cidal and static drug effects, two different experiments were performed in parallel, one with constant drug pressure throughout the experiment (144 h), and another in which the gametocyte cultures were exposed to the compound for only 48 h. RESULTS: Four P. falciparum genes coding for proteins Pf77, ROM3, Pfs25, and Pfg377 with transcription specific for late-stage gametocyte development were identified. The in vitro anti-malarial activity of compounds against such gametocytes was assessed by measuring mRNA levels of these genes using qPCR. The assay was validated against standard anti-malarial drugs (epoxomicin, dihydroartemisinin, chloroquine, thiostrepton, and methylene blue) and compounds from the GSK compound library with known anti-gametocyte activity. CONCLUSIONS: This study describes a novel assay for characterizing the activity of anti-malarial drugs against the later stages of P. falciparum gametocyte development using qPCR in genetically unmodified parasites. The method described is a reliable and user-friendly technique with a medium throughput that could be easily implemented in any laboratory.
Assuntos
Antimaláricos/isolamento & purificação , Antimaláricos/farmacologia , Descoberta de Drogas/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Plasmodium falciparum/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sobrevivência Celular/efeitos dos fármacos , Perfilação da Expressão Gênica , Plasmodium falciparum/fisiologiaRESUMO
Target-based approaches have traditionally been used in the search for new anti-infective molecules. Target selection process, a critical step in Drug Discovery, identifies targets that are essential to establish or maintain the infection, tractable to be susceptible for inhibition, selective towards their human ortholog and amenable for large scale purification and high throughput screening. The work presented herein validates the Plasmodium falciparum mRNA 5' triphosphatase (PfPRT1), the first enzymatic step to cap parasite nuclear mRNAs, as a candidate target for the development of new antimalarial compounds. mRNA capping is essential to maintain the integrity and stability of the messengers, allowing their translation. PfPRT1 has been identified as a member of the tunnel, metal dependent mRNA 5' triphosphatase family which differs structurally and mechanistically from human metal independent mRNA 5' triphosphatase. In the present study the essentiality of PfPRT1 was confirmed and molecular biology tools and methods for target purification, enzymatic assessment and target engagement were developed, with the goal of running a future high throughput screening to discover PfPRT1 inhibitors.
Assuntos
Antimaláricos , Descoberta de Drogas , Plasmodium falciparum , Antimaláricos/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Plasmodium falciparum/enzimologia , Descoberta de Drogas/métodos , Humanos , Ensaios de Triagem em Larga Escala/métodos , RNA Mensageiro/genética , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , Inibidores Enzimáticos/farmacologia , Hidrolases Anidrido ÁcidoRESUMO
Phenotypic screening has produced most of the new chemical entities currently in clinical development for malaria, plus many lead compounds active against Plasmodium falciparum asexual stages. However, lack of knowledge about the mode of action of these compounds delays and may even hamper their future development. Identifying the mode of action of the inhibitors greatly helps to prioritise compounds for further development as novel antimalarials. Here we describe a whole-cell method to detect inhibitors of the mitochondrial electron transport chain, using oxygen consumption as high throughput readout in 384-well plate format. The usefulness of the method has been confirmed with the Tres Cantos Antimalarial Compound Set (TCAMS). The assay identified 124 respiratory inhibitors in TCAMS, seven of which were novel anti-plasmodial chemical structures never before described as mitochondrial inhibitors.
Assuntos
Antimaláricos/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Mitocôndrias/efeitos dos fármacos , Plasmodium falciparum/efeitos dos fármacos , Descoberta de Drogas/métodos , Avaliação Pré-Clínica de Medicamentos/instrumentação , Complexo de Proteínas da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Humanos , Concentração Inibidora 50 , Malária/tratamento farmacológico , Malária/parasitologia , Malária Falciparum , Oxigênio/metabolismo , Plasmodium falciparum/citologiaRESUMO
In order to maximise compliance, the future antimalarial treatment should ideally require just a single-dose administration. This, in turn, demands new fast-acting effective drugs. Currently, methods to measure the in vitro killing rate of antimalarials are based on parasite growth. We have developed and validated a method to determine and classify antimalarial agents based on their cidal or static activity following quantitative Real Time PCR (RT-PCR) analysis. The method described here is a fast, reliable and user-friendly technique with a medium throughput. Metabolic activity of the parasite is followed by measuring mRNA expression levels of several genes during 5 parasite life cycles. mRNA from the parasite culture is then retrotranscribed to cDNA and quantified by RT-PCR. This new method provides a rapid and reproducible way to accurately measure the antimalarial activity of new compounds in vitro against Plasmodium falciparum.
Assuntos
Antimaláricos/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Plasmodium falciparum/efeitos dos fármacos , RNA Mensageiro/análise , Antimaláricos/classificação , Gametogênese/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Genes de Protozoários/genética , Plasmodium falciparum/genética , RNA Mensageiro/metabolismoRESUMO
The virulence of Candida albicans mutants lacking one or both copies of RAD52, a gene involved in homologous recombination (HR), was evaluated in a murine model of hematogenously disseminated candidiasis. In this study, the virulence of the rad52Delta mutant was dependent upon the inoculum concentration. Mice survived at a cell inoculum of 1 x 10(6), but there was a decrease in survival time at dosages of 1.5 x 10(6) and especially at 3 x 10(6) cells per animal. The heterozygote RAD52/rad52 behaved like wild type, whereas a reintegrant strain was intermediate in its ability to cause death compared to these strains and to the avirulent rad52/rad52 null at inocula of 1 x 10(6) and 1.5 x 10(6) cells. A double mutant, lig4/lig4/rad52/rad52, was avirulent at all inocula used. PCR analysis of the RAD52 and/or LIG4 loci showed that all strains recovered from animals matched the genotype of the inoculated strains. Analysis of the electrophoretical karyotypes indicated that the inoculated, reintegrant strain carried a large deletion in one copy of chromosome 6 (the shortest homologue, or Chr6b). Interestingly, truncated Chr6b was regenerated in all the strains recovered from moribund animals using the homologue as a template. Further, regeneration of Chr6b was paralleled by an increase in virulence that was still lower than that of wild type, likely because of the persistent loss of heterozygosity in the regenerated region. Overall, our results indicate that systemic candidiasis can develop in the absence of HR, but simultaneous elimination of both recombination pathways, HR and nonhomologous end-joining, suppresses virulence even at very high inocula.