Functional genetic evaluation of DNA house-cleaning enzymes in the malaria parasite: dUTPase and Ap4AH are essential in Plasmodium berghei but ITPase and NDH are dispensable

Background: Cellular metabolism generates reactive oxygen species. The oxidation and deamination of the deoxynucleoside triphosphate (dNTP) pool results in the formation of non-canonical, toxic dNTPs that can cause mutations, genome instability, and cell death. House-cleaning or sanitation enzymes that break down and detoxify non-canonical nucleotides play major protective roles in nucleotide metabolism and constitute key drug targets for cancer and various pathogens. We hypothesized that owing to their protective roles in nucleotide metabolism, these house-cleaning enzymes are key drug targets in the malaria parasite.

Methods: Using the rodent malaria parasite Plasmodium berghei we evaluate here, by gene targeting, a group of conserved proteins with a putative function in the detoxification of non-canonical nucleotides as potential antimalarial drug targets: they are inosine triphosphate pyrophosphatase (ITPase), deoxyuridine triphosphate pyrophosphatase (dUTPase) and two NuDiX hydroxylases, the diadenosine tetraphosphate (Ap4A) hydrolase and the nucleoside triphosphate hydrolase (NDH).

Results: While all four proteins are expressed constitutively across the intraerythrocytic developmental cycle, neither ITPase nor NDH are required for parasite viability. dutpase and ap4ah null mutants, on the other hand, are not viable suggesting an essential function for these proteins for the malaria parasite.

Conclusions: Plasmodium dUTPase and Ap4A could be drug targets in the malaria parasite.     

核苷类药物在乙型肝炎后肝硬化治疗中的疗效

目的分析在乙型肝炎后肝硬化治疗中采用核苷类药物的治疗价值。方法将本院在2018年1月—2019年2月期间收治的乙型肝炎后肝硬化患者68例纳入研究,以奇偶法均分为两组,对照组(34例)行常规治疗方案,观察组(34例)行核苷类药物治疗,对比两组乙型肝炎后肝硬化患者的治疗效果。结果对照组HBeAg转阴率14.71%低于观察组44.12%,P<0.05差异有统计学意义。观察组临床指标(ALT、AST、TBIL、ALB、PTA、Child-Pugh)优于对照组患者,P<0.05差异有统计学意义。观察组并发症发生率8.82%低于对照组并发症发生率为41.18%,P<0.05差异有统计学意义。结论在乙型肝炎后肝硬化治疗中采用核苷类药物可以取得显著的治疗效果,可以有效改善患者的临床症状,提升患者的转阴率,安全性较高。     

Contribution of equilibrative nucleoside transporters 1 and 2 to gemcitabine uptake in pancreatic cancer cells

Hepatic arterial infusion (HAI) chemotherapy is expected to be a more effective and safer method to treat the hepatic metastasis of pancreatic cancer than intravenous (iv) administration because of higher tumor exposure and lower systemic exposure. To clarify the uptake mechanism of nucleoside anticancer drugs, including gemcitabine (GEM), in pancreatic cancer, we investigated the uptakes of radiolabeled uridine (a general substrate of nucleoside transporters) and GEM in pancreatic cancer cell lines MIA‐PaCa2 and As‐PC1. Uridine uptake was inhibited by non‐labeled GEM and also by S‐(4‐nitrobenzyl)‐6‐thioinosine (NBMPR; an inhibitor of equilibrative nucleoside transporters, ENTs) in a concentration‐dependent manner, suggesting that ENTs contribute to uridine uptake in pancreatic cancer cells. As for GEM, saturable uptake was mediated by high‐ and low‐affinity components with K_m values of micromolar and millimolar orders, respectively. Uptake was inhibited in a concentration‐dependent manner by NBMPR and was sodium ion‐independent. Moreover, the concentration dependence of uptake in the presence of 0.1 μM NBMPR showed a single low‐affinity site. These results indicated that the high‐ and low‐affinity sites correspond to hENT1 and hENT2, respectively. The results indicated that at clinically relevant hepatic concentrations of GEM in GEM‐HAI therapy, the metastatic tumor exposure of GEM is predominantly determined by hENT2 under unsaturated conditions, suggesting that hENT2 expression in metastatic tumor would be a candidate biomarker for indicating anticancer therapy with GEM‐HAI.     

Sofosbuvir as treatment against dengue?

Dengvaxia^® (CTD‐TDV), the only licensed tetravalent dengue vaccine by Sanofi Pasteur, was made available since 2015. However, administration of CTD‐TDV, in general, has not received the prequalification recommendation from the World Health Organization. Having a universal antidengue agent for treatment will therefore beneficial. Accordingly, the development of nucleoside inhibitors specific to dengue viral polymerase that perturb dengue infection has been studied by many. Alternatively, we have used a marketed anti‐HCV prodrug sofosbuvir to study its in silico and in vitro effects against dengue. As a result, the active metabolite of sofosbuvir (GS‐461203) was predicted to bind to the catalytic motif (Gly‐Asp‐Asp) of dengue viral polymerase with binding affinity of −6.9 kcal/mol. Furthermore, sofosbuvir demonstrated excellent in vitro viral inhibition with an EC₉₀ of 0.4 μm . In addition, this study demonstrated the requirement of specific liver enzymes to activate the prodrug into GS‐461203 to exert its antidengue potential. All in all, sofosbuvir should be subjected to in‐depth studies to provide information of its efficacy toward dengue and its lead potential as DENV polymerase inhibitor in human subjects. In conclusion, we have expended the potential of the clinically available drug sofosbuvir as treatment for dengue.     

Lyophilization provides long-term stability for a lipid nanoparticle-formulated,nucleoside-modified mRNA vaccine

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Identification of Nucleoside Analogs as Inducers of Neuronal Differentiation in a Human Reporter Cell Line and Adult Stem Cells

Nucleoside analogs (NSAs) were among the first chemotherapeutic agents and could also be useful for the manipulation of cell fate. To investigate the potential of NSAs for the induction of neuronal differentiation, we developed a novel phenotypic assay based on a human neuron‐committed teratocarcinoma cell line (NT2) as a model for neuronal progenitors and constructed a NT2‐based reporter cell line that expressed eGFP under the control of a neuron‐specific promoter. We tested 38 structurally related NSAs and determined their activity to induce neuronal differentiation by immunocytochemistry of neuronal marker proteins, live cell imaging, fluorometric detection and immunoblot analysis. We identified twelve NSAs, which induced neuronal differentiation to different extents. NSAs with highest activity carried a halogen substituent at their pyrimidine nucleobase and an unmodified or 2′‐O‐methyl substituted 2‐deoxy‐β‐D‐ribofuranosyl residue as glyconic moiety. Cladribine, a purine nucleoside with similar structural features and in use to treat leukemia and multiple sclerosis, induced also differentiation of adult human neural crest‐derived stem cells. Our results suggest that NSAs could be useful for the manipulation of neuronal cell fate in cell replacement therapy or treatment of neurodegenerative disorders. The data on the structure and function relationship will help to design compounds with increased activity and low toxicity.     

慢性乙型肝炎的抗病毒研究进展

慢性乙型肝炎是由乙型肝炎病毒(hepatitis B virus,HBV)引起的全球性传染病,HBV感染是我国肝硬化、原发性肝癌的重要原因。目前,干扰素类与核苷(酸)类似物抗病毒药物已广泛应用于临床,在一定程度上抑制了病毒的复制并控制了疾病的发展,但仍未从根本上清除病毒;各种治疗性疫苗在抗HBV方面也取得了一定疗效,但临床效果不佳。目前不少研究结果表明,生物免疫治疗可以成功清除体内的HBV,从而为乙肝的治疗带来新的希望。     

核苷酸转运体表达与吉西他滨治疗胰腺癌患者临床疗效相关性的研究进展

目的 探讨核苷酸转运体表达与吉西他滨抗胰腺癌临床疗效相关性的研究进展。方法 通过查阅国内外文献,对相关研究归纳、总结进行综述。结果 吉西他滨是胰腺癌化疗的一线药物,较大个体化差异和有效率偏低是目前临床应用的挑战,核苷酸转运体是吉西他滨摄取入细胞的内吞转运体,它的蛋白或mRNA表达可能与接受吉西他滨治疗胰腺癌患者的临床疗效和不良反应相关,但尚未达成共识。结论 核苷酸转运体表达与吉西他滨抗胰腺癌临床疗效的相关性还需要进一步研究,为吉西他滨在胰腺癌中实现临床个体化治疗提供参考依据。