1Department of Cell Biology and Physiology, University of North Carolina School of Medicine, Neuroscience Research Building, Room 5119 115 Mason Farm Rd., Campus Box 7545, Chapel Hill, NC 27599-7545 USA.
2Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University School of Pharmacy and the Purdue Center for Cancer Research, West Lafayette, IN USA.
Mol Autism. 2018 Aug 17;9:45. doi: 10.1186/s13229-018-0228-2. eCollection 2018.
Angelman syndrome (AS) is a severe neurodevelopmental disorder lacking effective therapies. AS is caused by mutations in ubiquitin protein ligase E3A (), which is genomically imprinted such that only the maternally inherited copy is expressed in neurons. We previously demonstrated that topoisomerase I (Top1) inhibitors could successfully reactivate the dormant paternal allele of in neurons of a mouse model of AS. We also previously showed that one such Top1 inhibitor, topotecan, could unsilence paternal in induced pluripotent stem cell-derived neurons from individuals with AS. Although topotecan has been well-studied and is FDA-approved for cancer therapy, its limited CNS bioavailability will likely restrict the therapeutic use of topotecan in AS. The goal of this study was to identify additional Top1 inhibitors with similar efficacy as topotecan, with the expectation that these could be tested in the future for safety and CNS bioavailability to assess their potential as AS therapeutics.
We tested 13 indenoisoquinoline-derived Top1 inhibitors to identify compounds that unsilence the paternal allele of in mouse neurons. Primary cortical neurons were isolated from embryonic day 14.5 (E14.5) mice with a fluorescent tag on the paternal allele ( mice) or mice that lack the maternal allele and hence model AS ( mice). Neurons were cultured for 7 days, treated with drug for 72 h, and examined for paternal UBE3A protein expression by Western blot or fluorescence immunostaining. Dose responses of the compounds were determined across a log range of drug treatments, and cytotoxicity was tested using a luciferase-based assay.
All 13 indenoisoquinoline-derived Top1 inhibitors unsilenced paternal . Several compounds exhibited favorable paternal unsilencing properties, similar to topotecan, and of these, indotecan (LMP400) was the most effective based on estimated E (maximum response of unsilencing paternal ) and EC (half maximal effective concentration).
We provide pharmacological profiles of indenoisoquinoline-derived Top1 inhibitors as paternal unsilencers. All 13 tested compounds were effective at unsilencing paternal , although with variable efficacy and potency. Indotecan (LMP400) demonstrated a better pharmacological profile of unsilencing compared to our previous lead compound, topotecan. Taken together, indotecan and its structural analogues are potential AS therapeutics whose translational potential in AS treatment should be further assessed.
天使综合征（AS）是一种严重的神经发育障碍，缺乏有效的治疗方法。AS 是由泛素蛋白连接酶 E3A () 的基因突变引起的，该基因在基因组中被印记，使得只有母系遗传的拷贝在神经元中表达。我们之前的研究表明，拓扑异构酶 I (Top1) 抑制剂可以成功地重新激活 AS 小鼠模型中休眠的父系 等位基因。我们还之前表明，拓扑异构酶 I 抑制剂之一拓扑替康可以使 AS 患者诱导多能干细胞衍生的神经元中父系沉默。尽管拓扑替康已被广泛研究并获得 FDA 批准用于癌症治疗，但它在中枢神经系统中的有限生物利用度可能会限制拓扑替康在 AS 中的治疗用途。本研究的目的是鉴定具有与拓扑替康相似疗效的其他 Top1 抑制剂，期望未来可以对这些药物进行安全性和中枢神经系统生物利用度的测试，以评估它们作为 AS 治疗药物的潜力。
我们测试了 13 种吲哚异喹啉衍生的 Top1 抑制剂，以鉴定能够使小鼠神经元中父系沉默的化合物。从胚胎第 14.5 天（E14.5）的小鼠中分离出初级皮质神经元，这些神经元在父系等位基因上带有荧光标记（ 小鼠）或缺乏母系 等位基因，因此模拟 AS（ 小鼠）。神经元培养 7 天，用药物处理 72 小时，然后通过 Western blot 或荧光免疫染色检测父系 UBE3A 蛋白的表达。在对数范围内测试化合物的剂量反应，并使用基于荧光素酶的测定法测试细胞毒性。
所有 13 种吲哚异喹啉衍生的 Top1 抑制剂均使父系沉默。几种化合物表现出与拓扑替康相似的有利的父系沉默特性，其中吲哚替康（LMP400）是最有效的，根据最大反应（沉默父系 ）和半最大有效浓度（EC）进行估计。
我们提供了吲哚异喹啉衍生的 Top1 抑制剂作为父系沉默剂的药理学特征。所有 13 种测试化合物都能有效地沉默父系 ，尽管效果和效力不同。吲哚替康（LMP400）在沉默 方面表现出比我们之前的先导化合物拓扑替康更好的药理学特征。总的来说，吲哚替康及其结构类似物是潜在的 AS 治疗药物，其在 AS 治疗中的转化潜力应进一步评估。
