RESUMO
Genotoxicity testing is critical for predicting adverse effects of pharmaceutical, industrial, and environmental chemicals. The alkaline comet assay is an established method for detecting DNA strand breaks, however, the assay does not detect potentially carcinogenic bulky adducts that can arise when metabolic enzymes convert pro-carcinogens into a highly DNA reactive products. To overcome this, we use DNA synthesis inhibitors (hydroxyurea and 1-ß-d-arabinofuranosyl cytosine) to trap single strand breaks that are formed during nucleotide excision repair, which primarily removes bulky lesions. In this way, comet-undetectable bulky lesions are converted into comet-detectable single strand breaks. Moreover, we use HepaRG™ cells to recapitulate in vivo metabolic capacity, and leverage the CometChip platform (a higher throughput more sensitive comet assay) to create the 'HepaCometChip', enabling the detection of bulky genotoxic lesions that are missed by current genotoxicity screens. The HepaCometChip thus provides a broadly effective approach for detection of bulky DNA adducts.
Assuntos
Ensaio Cometa/métodos , Adutos de DNA/análise , Carcinogênese , Linhagem Celular , Quebras de DNA de Cadeia Simples , Reparo do DNA , Humanos , Análise em Microsséries/métodos , Sensibilidade e EspecificidadeRESUMO
We report a systematic, cellular phenotype-based antimalarial screening of the Medicines for Malaria Venture Pathogen Box collection, which facilitated the identification of specific blockers of late-stage intraerythrocytic development of Plasmodium falciparum First, from standard growth inhibition assays, we identified 173 molecules with antimalarial activity (50% effective concentration [EC50] ≤ 10 µM), which included 62 additional molecules over previously known antimalarial candidates from the Pathogen Box. We identified 90 molecules with EC50 of ≤1 µM, which had significant effect on the ring-trophozoite transition, while 9 molecules inhibited the trophozoite-schizont transition and 21 molecules inhibited the schizont-ring transition (with ≥50% parasites failing to proceed to the next stage) at 1 µM. We therefore rescreened all 173 molecules and validated hits in microscopy to prioritize 12 hits as selective blockers of the schizont-ring transition. Seven of these molecules inhibited the calcium ionophore-induced egress of Toxoplasma gondii, a related apicomplexan parasite, suggesting that the inhibitors may be acting via a conserved mechanism which could be further exploited for target identification studies. We demonstrate that two molecules, MMV020670 and MMV026356, identified as schizont inhibitors in our screens, induce the fragmentation of DNA in merozoites, thereby impairing their ability to egress and invade. Further mechanistic studies would facilitate the therapeutic exploitation of these molecules as broadly active inhibitors targeting late-stage development and egress of apicomplexan parasites relevant to human health.
Assuntos
Antimaláricos/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Malária Falciparum/tratamento farmacológico , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/crescimento & desenvolvimento , Fragmentação do DNA/efeitos dos fármacos , Humanos , Merozoítos/efeitos dos fármacos , Testes de Sensibilidade Parasitária , Esquizontes/efeitos dos fármacos , Trofozoítos/efeitos dos fármacosRESUMO
Comet assay is a standard approach for studying DNA damage in malaria, but high-throughput options are not available. The CometChip was previously developed using mammalian cells as a high-throughput version of the comet assay. It is based on the same principle as the comet assay but provides greater efficacy, automated data processing, and improved consistency between experiments. In this protocol, we present MalariaCometChip to quantitatively assess drug-induced DNA damage in Plasmodium falciparum. For complete details on the use and execution of this protocol, please refer to Xiong et al. (2020).
Assuntos
Ensaio Cometa/métodos , Dano ao DNA/genética , Ensaios de Triagem em Larga Escala/métodos , Plasmodium falciparum/genética , Células Cultivadas , Dano ao DNA/efeitos dos fármacos , DNA de Protozoário/análise , DNA de Protozoário/efeitos dos fármacos , DNA de Protozoário/genética , Eletroforese , Desenho de Equipamento , Ensaios de Triagem em Larga Escala/instrumentação , Plasmodium falciparum/citologia , Plasmodium falciparum/efeitos dos fármacosRESUMO
Southeast Asia has been the hotbed for the development of drug-resistant malaria parasites, including those with resistance to artemisinin combination therapy. While mutations in the kelch propeller domain (K13 mutations) are associated with artemisinin resistance, a range of evidence suggests that other factors are critical for the establishment and subsequent transmission of resistance in the field. Here, we perform a quantitative analysis of DNA damage and repair in the malaria parasite Plasmodium falciparum and find a strong link between enhanced DNA damage repair and artemisinin resistance. This experimental observation is further supported when variations in seven known DNA repair genes are found in resistant parasites, with six of these mutations being associated with K13 mutations. Our data provide important insights on confounding factors that are important for the establishment and spread of artemisinin resistance and may explain why resistance has not yet arisen in Africa.
Assuntos
Artemisininas/farmacologia , Dano ao DNA , Reparo do DNA/efeitos dos fármacos , Plasmodium falciparum/efeitos dos fármacos , Proteínas de Protozoários/química , África , Sudeste Asiático , Resistência a Medicamentos/efeitos dos fármacos , Genótipo , Geografia , Humanos , Estágios do Ciclo de Vida/efeitos dos fármacos , Fenótipo , Plasmodium falciparum/genética , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/isolamento & purificação , Domínios Proteicos , Proteínas de Protozoários/genéticaRESUMO
Upon Plasmodium falciparum infection of the red blood cells (RBCs), the parasite replicates and consumes haemoglobin resulting in the release of free heme which is rapidly converted to hemozoin crystallites. The bulk magnetic susceptibility of infected RBCs (iRBCs) is changed due to ferric (Fe3+) paramagnetic state in hemozoin crystallites which induce a measurable change in spin-spin relaxation (transverse relaxation) rate in proton nuclear magnetic resonance (NMR) of iRBCs. Earlier, our group reported that this transverse relaxation rate (R2) can be measured by an inexpensive, portable 0.5 Tesla bench top magnetic resonance relaxometry (MRR) system with minimum sample preparation and is able to detect very low levels of parasitemia in both blood cultures as well as animal models. However, it was challenging to diagnose malaria in human blood using MRR, mainly due to the inherent variation of R2 values of clinical blood samples, caused by many physiological and genotypic differences not related to the parasite infection. To resolve the problem of baseline R2 rates, we have developed an improved lysis protocol for removing confounding molecular and cellular background for MRR detection. With this new protocol and by processing larger volume of blood (>1 ml), we are able to reliably detect very low level of parasitemia (representing early stage of infection, ~0.0001%) with a stable baseline and improved sensitivity using the current MRR system.