脊髓肌肉萎縮症的治療藥物篩選及藥物作用機制之探討

Drug Screening for Spinal Muscular Atrophy and Investigation of the Mechanism of Drug Effect

陳郁佳(Yu-Chia Chen)

指導教授 ： 張建國

高雄醫學大學/醫學院/醫學研究所/碩士(2009年)

摘要

脊髓肌肉萎縮症(Spinal muscular atrophy, 簡稱SMA)是一種體隱性遺傳疾病，由於脊髓神經的前角細胞退化，造成肌肉無力及萎縮的病症。大約每六千個新生兒中，就會有一個是脊髓肌肉萎縮症患者，為目前第二常見的體隱性遺傳疾病，同時也是造成新生兒死亡最常見的遺傳疾病。SMA的致病原因主要是在第五對染色體q臂上的survival motor neuron 1 (SMN1)基因產生突變或缺失，使得由SMN1基因所產生的SMN蛋白質不足所造成。同樣在第五對染色體長臂上還有一個SMN2基因，然而因為exon 7上的第六個核苷酸由原本的C變成了T，造成在RNA剪接的過程中，exon 7容易被剪掉，產生truncated form的SMN蛋白質，這樣的SMN蛋白質無法穩定存在體內去彌補全長的SMN蛋白質功能。由於SMA病人體內都至少存在SMN2基因，利用藥物促進SMN2 promoter的轉錄或改變SMN2的mRNA剪接，使得全長的SMN蛋白質表現量增加，是目前用來治療SMA的可行方法之一。我們利用在運動神經細胞NSC34中送入表現SMN2 minigene (exon 6-exon 8)- luciferase質體的藥物篩選系統找到了四種潛力藥物：oxa-32、oxa-19、CH-15及#91，進一步再SMA病人的細胞中測試，卻發現oxa-19、CH-15對SMN FL mRNA沒有顯著影響，其中oxa-32雖然可以促進SMN FL mRNA表現，對SMN蛋白質表現卻也沒有顯著效果；只有#91化合物，經實驗已證明#91化合物不僅促進了冷光酵素活性(luciferase activity)，在SMA病人的細胞中也發現#91化合物可以促進包含exon 6到exon 8的全長SMN mRNA表現。進一步探討#91化合物對serine/arginine-rich protein (SR protein)的影響，發現#91化合物會抑制heterogeneous nuclear ribonucleoprotein (hnRNP) A1的表現，由目前已知的SMN2 exon 7剪接的模式，我們推測#91化合物是透過抑制hnRNPA1來增加SMN2全長mRNA表現。除此之外，我們另外在NSC34運動神經細胞中送入表現SMN2 promoter-minigene-luciferase的質體，這樣的系統讓我們可以同時篩選促進SMN2 promoter的轉錄或改變SMN2的mRNA剪接而使得全長的SMN表現量增加的藥物。在測試過數百個化合物之後，我們也發現了三種可以促進luciferase activity的化合物：WTC1467q、CYL840h、Bifido 15476，進一步利用這些化合物刺激SMA病人的細胞並偵測mRNA及蛋白質的表現，發現WTC1467q、CYL840h無法有效影響mRNA及蛋白質的表現，而Bifido 15476在一定濃度範圍內會增加SMN FL mRNA的表現，對SMN蛋白質表現則沒有顯著效果，因此是否有發展成藥物的潛力需要再經過評估。我們會利用NSC34/SMN2 promoter-minigene-luciferase系統繼續篩選藥物，從中挑選有效促進SMN2 promoter的轉錄或使得全長的SMN表現量增加的藥物繼續探討其作用機制。

關鍵字

脊髓肌肉萎縮症 ； 藥物篩選系統 ； 運動神經細胞 ； SMN1基因 ； SMN2基因 ； SMN蛋白質 ； RNA剪接

並列摘要

Spinal muscular atrophy (SMA) is an autosomal recessive disease characterized by the degeneration of motor neurons in the spinal cord, leading to muscular atrophy. SMA is caused by deletions/mutations in the telomeric copy of the survival motor neuron gene (SMN1) on chromosome 5q13. A second centromeric copy of the SMN gene (SMN2) also exists on chromosome 5, and both genes can produce functional protein. However, due to the alternative splicing of the exon 7, the majority of SMN protein produced by SMN2 is unstable and unable to compensate for the loss of SMN1. Increasing full-length SMN protein production by promoting the exon 7 inclusion in SMN2 mRNA or increasing SMN2 gene transcription could be a therapeutic approach for SMA. We screened for hundreds of compounds by using NSC34 motor neuron cell lines with SMN2 minigene (exon 6-exon 8)-luciferase system, and had identified the four compounds: oxa-32, oxa-19, CH-15 and #91 that significantly increased the luciferase activity. We also showed that oxa-32 and #91 promotes SMN2 exon 7 inclusion in SMA patient-derived cell lines, but no significant effect for oxa-19, CH-15. However, oxa-32 has no significant effect on SMN protein expression. To explore the mechanism of drug effect, we analyzed if #91 affected SR and hnRNP protein expression in SMA cells. We found that #91 down-regulates hnRNPA1. According to the models of SMN2 exon 7 splicing, we suggest that the compound #91 enhances SMN2 exon 7 inclusion by suppressing hnRNPA1 expression. In addition, we constructed a new system for drug screening. The new system was built in NSC34 motor neuron cell containing SMN2 promoter and SMN2 minigene-luciferase. By using this system, we can screen drugs that promoting the exon 7 inclusion in SMN2 mRNA and/or increasing SMN2 gene transcription at the same time. We screened for more than four hundred compounds and identified three compounds, WTC1467q, CYL840h, and Bifido 15476, that increase luciferase activity. We detected RNA and protein expression to figure out the effect of these compounds is to promote the exon 7 inclusion in SMN2 mRNA or increase SMN2 gene transcription. The results showed that WTC1467q and CYL840h had no significant effects of promoting the exon 7 inclusion in SMN2 mRNA or increasing SMN2 gene transcription. Bifido 15476 has the ability to increase SMN FL mRNA, but causes no significant change in SMN protein expression. We will confirm the potential of Bifido 15476 as a therapeutic drug. We will also screen more compounds and investigate the mechanism of those compounds that can promote the exon 7 inclusion in SMN2 mRNA and/or increase SMN2 gene transcription.

並列關鍵字

spinal muscular atrophy ； SMN1 gene ； SMN2 gene ； alternative splicing ； system for drug screening ； SMN protein ； NSC34 motor neuron cell

參考文獻

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