RESUMO
Antimalarial peroxides such as the phytochemical artemisinin or the synthetic ozonides arterolane and artefenomel undergo reductive cleavage of the pharmacophoric peroxide bond by ferrous heme, released by parasite hemoglobin digestion. The generated carbon-centered radicals alkylate heme in an intramolecular reaction and proteins in an intermolecular reaction. Here, we determine the proteinaceous alkylation signatures of artemisinin and synthetic ozonides in Plasmodium falciparum using alkyne click chemistry probes to identify target proteins by affinity purification and mass spectrometry-based proteomics. Using stringent controls and purification procedures, we identified 25 P. falciparum proteins that were alkylated by the antimalarial peroxides in a peroxide-dependent manner, but the alkylation patterns were more random than we had anticipated. Moreover, there was little overlap in the alkylation signatures identified in this work and those disclosed in previous studies. Our findings suggest that alkylation of parasite proteins by antimalarial peroxides is likely to be a nonspecific, stochastic process.
Assuntos
Antimaláricos/farmacologia , Peróxidos/farmacologia , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/análise , Alquilação , Antimaláricos/química , Artemisininas/farmacologia , Química Click , Compostos Heterocíclicos/farmacologia , Espectrometria de Massas , Estrutura Molecular , Plasmodium falciparum/efeitos dos fármacos , Proteômica , Proteínas de Protozoários/química , Processos EstocásticosRESUMO
BACKGROUND: Recently published data suggest that artemisinin derivatives and synthetic peroxides, such as the ozonides OZ277 and OZ439, have a similar mode of action. Here the cross-resistance of OZ277 and OZ439 and four additional next-generation ozonides was probed against the artemisinin-resistant clinical isolate Plasmodium falciparum Cam3.I, which carries the K13-propeller mutation R539T (Cam3.IR539T). METHODS: The previously described in vitro ring-stage survival assay (RSA0-3h) was employed and a simplified variation of the original protocol was developed. RESULTS: At the pharmacologically relevant concentration of 700 nM, all six ozonides were highly effective against the dihydroartemisinin-resistant P. falciparum Cam3.IR539T parasites, showing a per cent survival ranging from <0.01 to 1.83%. A simplified version of the original RSA0-3h method was developed and gave similar results, thus providing a practical drug discovery tool for further optimization of next-generation anti-malarial peroxides. CONCLUSION: The absence of in vitro cross-resistance against the artemisinin-resistant clinical isolate Cam3.IR539T suggests that ozonides could be effective against artemisinin-resistant P. falciparum. How this will translate to the human situation in clinical settings remains to be investigated.
Assuntos
Antimaláricos/farmacologia , Artemisininas/farmacologia , Resistência a Medicamentos , Plasmodium falciparum/efeitos dos fármacos , Adamantano/análogos & derivados , Adamantano/farmacologia , Compostos Heterocíclicos com 1 Anel/farmacologia , Peróxidos/farmacologia , Compostos de Espiro/farmacologiaRESUMO
The singular structure of artemisinin, with its embedded 1,2,4-trioxane heterocycle, has inspired the discovery of numerous semisynthetic artemisinin and structurally diverse synthetic peroxide antimalarials, including ozonides OZ277 (arterolane) and OZ439 (artefenomel). Despite the critical importance of artemisinin combination therapies (ACTs), the precise mode of action of peroxidic antimalarials is not fully understood. However, it has long been proposed that the peroxide bond in artemisinin and other antimalarial peroxides undergoes reductive activation by ferrous heme released during hemoglobin digestion to produce carbon-centered radicals that alkylate heme and parasite proteins. To probe the mode of action of OZ277 and OZ439, this paper now describes initial studies with monoclonal antibodies that recognize the alkylation signature (sum of heme and protein alkylation) of these synthetic peroxides. Immunofluorescence experiments conducted with ozonide-treated parasite cultures showed that ozonide alkylation is restricted to the parasite, as no signal was found in the erythrocyte or its membrane. In Western blot experiments with ozonide-treated Plasmodium falciparum malaria parasites, distinct protein bands were observed. Significantly, no protein bands were detected in parallel Western blot experiments performed with lysates from ozonide-treated Babesia divergens, parasites that also proliferate inside erythrocytes but, in contrast to P. falciparum, do not catabolize hemoglobin. However, subsequent immunoprecipitation experiments with these antibodies failed to identify the P. falciparum proteins alkylated by OZ277 and OZ439. To the best of the authors' knowledge, this shows for the first time that antimalarial ozonides, such as the artemisinins, alkylate proteins in P. falciparum.