Verdaguer Ignasi, Chen Cathy, Filho Maurício Mazzine, Santos Matheus, Castro Gabriela, Yamaguchi Lydia, de Oliveira Sandra, Peres Manoel, Hernández Agustín, Kronenberger Thales, Bargieri Daniel, Angeli Claudia, Hodge Dana, Palmisano Giuseppe, Izquierdo Luis, Azevedo Luciana, John Audrey Odom, Katzin Alejandro, Crispim Marcell
University of São Paulo.
Children's Hospital of Philadelphia.
Res Sq. 2025 Jun 5:rs.3.rs-6574048. doi: 10.21203/rs.3.rs-6574048/v1.
Fosmidomycin was proposed as an antimalarial drug but failed in clinical trials due to recrudescence, a phenomenon whose causes remain poorly understood. The mechanism of action of fosmidomycin is the inhibition of the methylerythritol 4-phosphate (MEP) pathway, essential for producing isoprenoids in parasites. The key isoprenoids produced by the MEP pathway are farnesyl and geranylgeranyl pyrophosphates (FPP and GGPP), vital for protein isoprenylation, ubiquinone, and dolichol biosynthesis. studies have demonstrated that prenols, like farnesol (FOH) and geranylgeraniol (GGOH), can temporarily circumvent the MEP pathway, rescuing parasites from fosmidomycin effects. Our group identified a parasitic prenol kinase (PolK), responsible for converting FOH and GGOH into their active pyrophosphate forms. Additionally, GGOH's human plasma concentration is sufficient to affect MEP inhibitors. This suggests that the parasite's uptake of host prenols could diminish fosmidomycin effectiveness against malaria. To test this hypothesis, we generated PolK knockout parasites (ΔPolK). These transgenic parasites were viable but could not utilize exogenous prenols for protein prenylation and caused a form of murine malaria that responded more effectively to fosmidomycin therapy compared to parasites preserving PbPolK. Consequently, we explored compounds that could inhibit the parasite utilization of exogenous prenols, using biochemical and bioinformatics approaches, as well as in vitro assays in wild type and ΔPfPolK P. falciparum strains. Geraniol inhibits PolK activity and the incorporation of GGOH into P. falciparum. Moreover, geraniol enhanced fosmidomycin antimalarial effect in , even in the presence of GGOH. ΔPolK strains exhibited profound metabolic dysregulation in carbon metabolism, as assessed by proteomics. Taking all together, findings here presented demonstrate that the prenol salvage pathway is a modulatory mechanism of metabolic homeostasis, facilitates prenol utilization from the host, and contributes to the limited efficacy of fosmidomycin in malaria treatment.
磷霉素曾被提议作为一种抗疟药物，但由于复发现象，在临床试验中失败了，而复发这一现象的原因仍知之甚少。磷霉素的作用机制是抑制磷酸甲基赤藓糖醇（MEP）途径，该途径对寄生虫中类异戊二烯的产生至关重要。MEP途径产生的关键类异戊二烯是法呢基焦磷酸和香叶基香叶基焦磷酸（FPP和GGPP），它们对蛋白质异戊烯化、泛醌和多萜醇生物合成至关重要。研究表明，异戊烯醇，如法呢醇（FOH）和香叶基香叶醇（GGOH），可以暂时绕过MEP途径，使寄生虫免受磷霉素的影响。我们的研究小组鉴定出一种寄生性异戊烯醇激酶（PolK），负责将FOH和GGOH转化为它们的活性焦磷酸形式。此外，GGOH在人体血浆中的浓度足以影响MEP抑制剂。这表明寄生虫对宿主异戊烯醇的摄取可能会降低磷霉素对疟疾的疗效。为了验证这一假设，我们构建了PolK基因敲除寄生虫（ΔPolK）。这些转基因寄生虫是有活力的，但不能利用外源性异戊烯醇进行蛋白质异戊烯化，并且引发了一种鼠疟形式，与保留PbPolK的寄生虫相比，这种鼠疟对磷霉素治疗的反应更有效。因此，我们使用生化和生物信息学方法以及在野生型和ΔPfPolK恶性疟原虫菌株中的体外试验，探索了能够抑制寄生虫对外源性异戊烯醇利用的化合物。香叶醇抑制PolK活性以及GGOH掺入恶性疟原虫。此外，即使在存在GGOH的情况下，香叶醇也增强了磷霉素在 中的抗疟效果。通过蛋白质组学评估，ΔPolK菌株在碳代谢中表现出严重的代谢失调。综上所述，此处呈现的研究结果表明，异戊烯醇补救途径是代谢稳态的一种调节机制，促进了从宿主摄取异戊烯醇，并导致磷霉素在疟疾治疗中的疗效有限。
