RESUMEN
African trypanosomiasis is a disease caused by Trypanosoma brucei parasites with limited treatment options. Trypanosoma is unable to synthesize purines de novo and relies solely on their uptake and interconversion from the host, constituting purine nucleoside analogues a potential source of antitrypanosomal agents. Here we combine structural elements from known trypanocidal nucleoside analogues to develop a series of 3'-deoxy-7-deazaadenosine nucleosides, and investigate their effects against African trypanosomes. 3'-Deoxytubercidin is a highly potent trypanocide in vitro and displays curative activity in animal models of acute and CNS-stage disease, even at low doses and oral administration. Whole-genome RNAi screening reveals that the P2 nucleoside transporter and adenosine kinase are involved in the uptake and activation, respectively, of this analogue. This is confirmed by P1 and P2 transporter assays and nucleotide pool analysis. 3'-Deoxytubercidin is a promising lead to treat late-stage sleeping sickness.
Asunto(s)
Desoxiadenosinas/farmacología , Proteínas de Transporte de Nucleósidos/metabolismo , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/efectos de los fármacos , Tripanosomiasis Africana/tratamiento farmacológico , Tubercidina/farmacología , Animales , Antimetabolitos Antineoplásicos/química , Antimetabolitos Antineoplásicos/farmacología , Línea Celular , Supervivencia Celular , Desoxiadenosinas/química , Quimioterapia Combinada , Femenino , Humanos , Ratones , Estructura Molecular , Proteínas de Transporte de Nucleósidos/genética , Proteínas Protozoarias/genética , Tripanocidas/química , Tripanocidas/farmacología , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/fisiología , Tripanosomiasis Africana/parasitología , Tubercidina/químicaRESUMEN
In Trypanosoma brucei, the generation of knockout mutants is relatively easy compared to other organisms as transfection methods are well established. These methods have their limitations, however, when it comes to the generation of genome-wide libraries that require a minimum of several hundred thousand transformants. Double-strand breaks with the meganuclease ISce-I dramatically increase transformation efficiency, but are not widely in use as cell lines need to be generated de novo before each transfection. Here we show that zinc finger nucleases are a robust and stable tool that can enhance transformation in bloodstream forms by more than an order of magnitude.
Asunto(s)
Desoxirribonucleasas/metabolismo , Marcación de Gen/métodos , Transformación Genética , Trypanosoma brucei brucei/enzimología , Dedos de ZincRESUMEN
African trypanosomes, which divide their life cycle between mammals and tsetse flies, are confronted with environments that differ widely in temperature, nutrient availability and host responses to infection. In particular, since trypanosomes cannot predict when they will be transmitted between hosts, it is vital for them to be able to sense and adapt to their milieu. Thanks to technical advances, significant progress has been made in understanding how the parasites perceive external stimuli and react to them. There is also a growing awareness that trypanosomes use a variety of mechanisms to exchange information with each other, thereby enhancing their chances of survival.
