Bahamontes-Rosa Noemí, Gomez-Lorenzo María G, Lelièvre Joël, Rodriguez Alejandre Ane, Almela María Jesus, Lozano Sonia, Herreros Esperanza, Gamo Francisco-Javier
Tres Cantos Medicines Development Campus, Diseases of the Developing World, GlaxoSmithKline, Tres Cantos, 28760, Madrid, Spain.
Malar J. 2016 Jul 22;15(1):385. doi: 10.1186/s12936-016-1429-9.
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).
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.
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.
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.
能够杀死或抑制人类宿主体内疟原虫配子体的药物，有可能通过阻断传播来增强其他化疗干预措施的效果。为了开发此类药物，需要可靠的方法来研究化合物对配子体的体外活性。本研究描述了一种使用实时定量聚合酶链反应（qPCR）来表征抗疟药物对恶性疟原虫配子体发育后期活性的新检测方法。
选择先前报道在配子体发生的不同性阶段转录的基因进行研究，并通过qPCR在配子体发生过程中测量其mRNA表达。主要在配子体发育后期表达的基因用作药物活性的替代测量指标。为了区分杀菌和抑菌药物作用，并行进行了两个不同的实验，一个在整个实验过程（144小时）中保持恒定的药物压力，另一个是将配子体培养物仅暴露于化合物48小时。
鉴定出四个编码Pf77、ROM3、Pfs25和Pfg377蛋白的恶性疟原虫基因，其转录特异性针对配子体发育后期。通过使用qPCR测量这些基因的mRNA水平来评估化合物对此类配子体的体外抗疟活性。该检测方法针对标准抗疟药物（环氧霉素、双氢青蒿素、氯喹、硫链丝菌素和美蓝）以及来自葛兰素史克化合物库中具有已知抗配子体活性的化合物进行了验证。
本研究描述了一种使用qPCR在未基因修饰的寄生虫中表征抗疟药物对恶性疟原虫配子体发育后期活性的新检测方法。所描述的方法是一种可靠且用户友好的技术，具有中等通量，可在任何实验室轻松实施。
