RESUMO
Plasmodium falciparum calcium-dependent protein kinase 4 (PfCDPK4) is essential for the exflagellation of male gametocytes. Inhibition of PfCDPK4 is an effective way of blocking the transmission of malaria by mosquitoes. A series of 5-aminopyrazole-4-carboxamide analogues are demonstrated to be potent inhibitors of PfCDPK4. The compounds are also able to block exflagellation of Plasmodium falciparum male gametocytes without observable toxicity to mammalian cells.
Assuntos
Antimaláricos/química , Antimaláricos/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Proteínas Quinases/metabolismo , Pirazóis/química , Pirazóis/farmacologia , Animais , Linhagem Celular , Culicidae/parasitologia , Humanos , Malária Falciparum/prevenção & controle , Malária Falciparum/transmissão , Masculino , Plasmodium falciparum/fisiologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologiaRESUMO
Gametocytes of the malaria parasite Plasmodium are taken up by the mosquito vector with an infectious blood meal, representing a critical stage for parasite transmission. Calcium-independent protein kinases (CDPKs) play key roles in calcium-mediated signaling across the complex life cycle of the parasite. We sought to understand their role in human parasite transmission from the host to the mosquito vector and thus investigated the role of the human-infective parasite Plasmodium falciparum CDPK4 in the parasite life cycle. P. falciparum cdpk4- parasites created by targeted gene deletion showed no effect in blood stage development or gametocyte development. However, cdpk4- parasites showed a severe defect in male gametogenesis and the emergence of flagellated male gametes. To understand the molecular underpinnings of this defect, we performed mass spectrometry-based phosphoproteomic analyses of wild-type and Plasmodium falciparum cdpk4- late gametocyte stages to identify key CDPK4-mediated phosphorylation events that may be important for the regulation of male gametogenesis. We further employed in vitro assays to identify these putative substrates of Plasmodium falciparum CDPK4. This indicated that CDPK4 regulates male gametogenesis by directly or indirectly controlling key essential events, such as DNA replication, mRNA translation, and cell motility. Taken together, our work demonstrates that PfCDPK4 is a central kinase that regulates exflagellation and thereby is critical for parasite transmission to the mosquito vector. IMPORTANCE Transmission of the malaria parasite to the mosquito vector is critical for the completion of the sexual stage of the parasite life cycle and is dependent on the release of male gametes from the gametocyte body inside the mosquito midgut. In the present study, we demonstrate that PfCDPK4 is critical for male gametogenesis and is involved in phosphorylation of proteins essential for male gamete emergence. Targeting PfCDPK4 and its substrates may provide insights into achieving effective malaria transmission-blocking strategies.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Gametogênese/fisiologia , Mosquitos Vetores , Plasmodium falciparum/enzimologia , Plasmodium falciparum/metabolismo , Animais , Sinalização do Cálcio , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Culicidae , Gametogênese/genética , Células Germinativas/metabolismo , Estágios do Ciclo de Vida , Malária Falciparum/parasitologia , Malária Falciparum/transmissão , Masculino , Fosforilação , Plasmodium falciparum/genética , Proteínas de Protozoários/genéticaRESUMO
Malaria remains a major health concern for a large percentage of the world's population. While great strides have been made in reducing mortality due to malaria, new strategies and therapies are still needed. Therapies that are capable of blocking the transmission of Plasmodium parasites are particularly attractive, but only primaquine accomplishes this, and toxicity issues hamper its widespread use. In this study, we describe a series of pyrazolopyrimidine- and imidazopyrazine-based compounds that are potent inhibitors of PfCDPK4, which is a calcium-activated Plasmodium protein kinase that is essential for exflagellation of male gametocytes. Thus, PfCDPK4 is essential for the sexual development of Plasmodium parasites and their ability to infect mosquitoes. We demonstrate that two structural features in the ATP-binding site of PfCDPK4 can be exploited in order to obtain potent and selective inhibitors of this enzyme. Furthermore, we demonstrate that pyrazolopyrimidine-based inhibitors that are potent inhibitors of the in vitro activity of PfCDPK4 are also able to block Plasmodium falciparum exflagellation with no observable toxicity to human cells. This medicinal chemistry effort serves as a valuable starting point in the development of safe, transmission-blocking agents for the control of malaria.