高通量筛选和优化新型无义突变通读诱导剂用于治疗遗传病和肿瘤

This research aims to use high throughput screening (HTS) technology to screen and develop compounds with therapeutic potential that can read through nonsense mutations caused by primary premature termination codons and restore affected protein expression, which, in principle, can be used to treat all the diseases caused by nonsense mutations. Nonsense mutation accounts for about 30% of human disease-causing alleles and is one of most frequent cause of genetic disorders. Nonsense mutations are also found in >480 critical cancer genes that either cause cancer (e.g. familial adenomatous polyposis, FAP) or occur during cancer progression (e.g., p53). Unfortunately, there is no effective treatment for most of genetic disorders and cancer now. Interestingly, certain compounds, including aminoglycosides (e.g., gentamicin) and nonaminoglycosides (PTC124, RTC13 and RTC14), showed the ability to read through nonsense mutations, which allows translation of full-length functional protein. Extensive studies on these compounds have successfully demonstrated that chemical-induced nonsense mutation readthrough has great potential as a treatment strategy for genetic diseases and cancer caused by nonsense mutations, especially for diseases like ataxia telangiectasia (A-T), where restoring even a small amount of protein can significantly reduce disease severity. However, despite the promise of this approach, only a few readthrough compounds (RTCs) are identified, and their therapeutic benefits still remain unclear. Completed clinical trials of aminoglycosides and PTC124 failed to show significant clinical benefit. RTC13 and RTC14 identified by our previous study demonstrated activities in multiple genes, but need to be further evaluated. Therefore, given the great potential of readthrough strategy in genetic disorders and cancer and a very few RTCs available to study, there is urgent unmet need to identify novel RTCs with better therapeutic properties. Identifying novel RTCs is critical for moving readthrough strategy forward to clinic. Therefore, as an effort to identify novel RTCs, in this study we propose to 1) employ an optimized HTS strategy to screen new RTCs from chemical libraries with better chemistry design and drug-like properties, including NIH-approved drug library and natural product libraries; 2) we will then evaluate all the positive hits for their activities using A-T cell lines containing ATM nonsense mutations as a genetic disease model, and FAP cell lines with APC nonsense mutations as a cancer model, to select the best hit for structure optimization and toxicity studies; 3) lastly, we will perform structure-activity studies for the selected RTC to generate more active analogs; early absorption, distribution, metabolism, excretion and toxcity (ADMET) studies will also be conducted to help select the most promising drug candidates for future in vivo studies.

30%人类致病基因突变属无义突变。无义突变不但是遗传病主要病因，也与肿瘤发病密切相关。研究显示少数化合物可诱导无义突变通读，使突变基因继续翻译而恢复功能。此类化合物多属氨基糖苷类抗生素，此外还有若干小分子化合物。对此类化合物的研究已证明化学诱导的无义突变通读对众多无义突变所致遗传病和肿瘤是一种前景广阔的治疗策略。然而，当前相关研究主要受阻于可测试的活性通读剂数量极少，且所有已知通读剂的临床试验或失败，或待开展。因此研发新型通读剂对发展本治疗策略至关重要。本研究旨在基于过去工作基础上使用更优化的高通量筛选方案，对更具药物特性的多个化学文库进行筛查，从而发现新型通读剂；并在共济失调毛细血管扩张病(A-T,ATM突变引起)和家族性腺瘤性息肉病(FAP，APC突变引起)两种疾病病人细胞内进行功能测试；最后对最具活性的通读剂进行构效关系研究以优化结构、活性和毒性，从而发展具有自主知识产权的候选药物。

30%人类致病基因突变属无义突变。基因编码区发生的无义突变可致蛋白翻译在无义突变位点提前终止，导致蛋白功能缺失，引起包括遗传病、肿瘤在内的众多疾病。某些化合物如氨基糖苷类抗生素、非氨基糖苷类抗生素及其他小分子化合物能诱导无义突变通读，从而翻译出有功能的全长蛋白。化合物诱导的无义突变通读对于遗传病及肿瘤治疗是一个非常有潜力的治疗策略,但目前没有几种化合物显示出确切的临床疗效。当前相关研究主要受阻于可测试的活性通读剂数量极少，且所有已知通读剂的临床试验或失败，或待开展。因此，急需筛选新型、更具药物特性的化合物，推动通读治疗走向临床。本研究基于过去工作基础上，建立了稳定表达含无义突变位点的虫荧光素酶细胞株，对购自上海中科院生化细胞所的更具药物特性的多个化学文库进行筛查，同时使用PTT-ELISA方法进一步鉴定，目前已筛选出了7种新型通读剂，包括灭瘟素、3,3'-二吲哚甲烷、考迈斯托醇、H01、H02、H03、Y01。这些活性通读剂在含有APC基因853位密码子无义突变的结直肠癌细胞株HT-29中，可以恢复APC蛋白表达。我们重点研究了灭瘟素的通读活性，发现随着灭瘟素浓度升高，无义突变虫荧光素酶mRNA及蛋白表达水平不同程度上升，虫荧光素酶活性得到部分恢复，且通读效果显著高于G418。在HT29肿瘤细胞中，灭瘟素同样可以显著恢复无义突变APC基因mRNA及蛋白表达，同时下调了下游信号通路β-catenin与c-myc基因蛋白水平。细胞划痕实验、Transwell方法发现，灭瘟素处理后，肿瘤细胞迁移能力下降，MTT方法表明肿瘤细胞生长速度减慢。接着用流式细胞术、TUNEL方法分析灭瘟素细胞毒性，提示不高于G418。最后我们根据已发现的活性通读剂结构特点，根据药物开发潜力，选取了7种黄酮类化合物、5种吲哚类同系化合物，以优化其活性。从这些化合物中，初步发现了H4、Y1活性接近PTC124。总之，应用无义突变虫荧光素酶稳定表达细胞株、PTT-ELISA方法，筛选出了9种活性通读剂，可作为发展具有自主知识产权的候选通读剂药物，以治疗无义突变所致疾病。