An RNAi-mediated screen identifies novel targets for next-generation antiepileptic drugs based on increased expression of the homeostatic regulator pumilio.
An RNAi-mediated screen identifies novel targets for next-generation antiepileptic drugs based on increased expression of the homeostatic regulator pumilio.
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DOI：
10.1080/01677063.2018.1465570
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发表时间：
2018-03
影响因子：
1.9
通讯作者：
Baines RA
中科院分区：
文献类型：
--
作者：
Lin WH;He M;Fan YN;Baines RA
尽管抗癫痫药物（AED）种类繁多，但只有约三分之二的癫痫患者对药物治疗反应良好。因此，需要新的目标来催化下一代AED的设计。在果蝇中，通过增强Pumilio（Pum）活性来操纵神经元放电率稳态已被证明是有效的抗惊厥剂。在这项研究中，我们在S2 R+细胞中进行了全基因组RNAi筛选，使用基于dPum活性的报告基因，并确定了1166个参与dPum调控的基因。在这些基因中，我们集中在699个基因，敲低，增强dPum活性/表达。在该亚组中，基于与先前通过RNA测序鉴定为活性依赖性的基因的比较，101个基因是活性依赖性的。功能聚类分析显示这些基因在DNA损伤、细胞周期调控和蛋白酶体蛋白催化等途径中富集。为了测试抗惊厥活性，我们在体内使用了RNA干扰方法。RNAi介导的敲除显示，57/101个基因（61%）足以显著减少特征性癫痫发作突变体parabss的癫痫发作持续时间。我们进一步表明，化学抑制剂的蛋白质产品的一些基因的目标是类似的抗惊厥。最后，为了确定所鉴定的化合物的抗惊厥活性是否由增加的dpum转录引起，我们进行了基于内切酶的测定以监测dpum启动子活性。暴露于氟化钠、吉西他滨、二甲双胍、bestatin、WP 1066或丙戊酸的三龄幼虫都显示出增加的dpum启动子活性。因此，本研究验证了Pum作为AED设计的有利目标，并且鉴定了能够增加这种稳态调节剂的表达的许多先导化合物。
Despite availability of a diverse range of anti-epileptic drugs (AEDs), only about two-thirds of epilepsy patients respond well to drug treatment. Thus, novel targets are required to catalyse the design of next-generation AEDs. Manipulation of neuron firing-rate homoeostasis, through enhancing Pumilio (Pum) activity, has been shown to be potently anticonvulsant in Drosophila. In this study, we performed a genome-wide RNAi screen in S2R + cells, using a luciferase-based dPum activity reporter and identified 1166 genes involved in dPum regulation. Of these genes, we focused on 699 genes that, on knock-down, potentiate dPum activity/expression. Of this subgroup, 101 genes are activity-dependent based on comparison with genes previously identified as activity-dependent by RNA-sequencing. Functional cluster analysis shows these genes are enriched in pathways involved in DNA damage, regulation of cell cycle and proteasomal protein catabolism. To test for anticonvulsant activity, we utilised an RNA-interference approach in vivo. RNAi-mediated knockdown showed that 57/101 genes (61%) are sufficient to significantly reduce seizure duration in the characterized seizure mutant, parabss. We further show that chemical inhibitors of protein products of some of the genes targeted are similarly anticonvulsant. Finally, to establish whether the anticonvulsant activity of identified compounds results from increased dpum transcription, we performed a luciferase-based assay to monitor dpum promoter activity. Third instar larvae exposed to sodium fluoride, gemcitabine, metformin, bestatin, WP1066 or valproic acid all showed increased dpum promoter activity. Thus, this study validates Pum as a favourable target for AED design and, moreover, identifies a number of lead compounds capable of increasing the expression of this homeostatic regulator.
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影响因子：
4.3
作者：
Follwaczny P;Schieweck R;Riedemann T;Demleitner A;Straub T;Klemm AH;Bilban M;Sutor B;Popper B;Kiebler MA
通讯作者：
Kiebler MA
影响因子：
15.1
作者：
Engel, Tobias;Martinez-Villarreal, Jaime;Henshall, David C.
通讯作者：
Henshall, David C.
DOI：
10.1016/b978-0-12-804066-9.00024-9
发表时间：
2017-01-01
期刊：
MODELS OF SEIZURES AND EPILEPSY, 2ND EDITION
影响因子：
--
作者：
Baines, Richard A.;Giachello, Carlo N. G.;Lin, Wei-Hsiang
通讯作者：
Lin, Wei-Hsiang
DOI：
10.1093/bioinformatics/btp101
发表时间：
2009-04-15
期刊：
Bioinformatics (Oxford, England)
影响因子：
--
作者：
Bindea G;Mlecnik B;Hackl H;Charoentong P;Tosolini M;Kirilovsky A;Fridman WH;Pagès F;Trajanoski Z;Galon J
通讯作者：
Galon J
影响因子：
6.1
作者：
Grabenstatter HL;Del Angel YC;Carlsen J;Wempe MF;White AM;Cogswell M;Russek SJ;Brooks-Kayal AR
通讯作者：
Brooks-Kayal AR