抑制剂调节M2通道荧光强度的机制研究及靶向流感新药高通量筛选的方法建立

Cross-species infection of influenza viruses, for example, the H7N9 avian flu, has been on the rise globally. Its high mortality rate, even for the young and healthy, combined with its likelihood to acquire mutations to spread between humans, poses serious threat to the public health. Specific vaccines take time to develop, and cannot provide one hundred percent protection. Anti-flu medications, on the other hand, face the increasingly rampant drug-resistant challenge. Development of new-generation flu drugs is a key part of the solution. Amantadine, a FDA proved flu medicine, is an inhibitor of the M2 proton channel which is crucial to the propagation of the virus. The majority of the currently circulating viruses have gained drug resistance to amantadine, 95% of which have the S31N single residue mutation. Development of inhibitors for S31N has been slow due to two reasons: on one hand, the structures were not solved until recently, and the channel is in a highly dynamic equilibrium among multiple conformations, hence not an easy target for the structure-based drug design; on the other hand, reliable high-throughput screening (HTS) methods had been lacking, with the reported methods affected by factors like compound toxicity. In the preliminary experiments, we have demonstrated that the tryptophan fluorescence of S31N was quenched upon compound binding, and the degree of the quenching was correlated to the degree of the compound inhibition of the channel conductance. Based on this phenomenon, we propose to generate an in vitro HTS method. We plan to elucidate the mechanism of the fluorescence quenching in order to prove its generality, then with a conservative mutation to a fluorophore more suitable for the HTS format, establish the proper screening conditions and verify the quality of the assay method. The expected results from this HTS method development will aid in the discovery of new lead compounds targeting simultaneously wild type and S31N channels, as well as the development of new-generation native-born flu medications.

近年来全球屡发高致死率的跨种族流感传染，并伴随日益严重的病毒耐药性问题。例如流感M2质子通道的耐药性突变已经使得金刚烷胺对流行病毒无效。然而耐药性M2抑制剂的研究进展缓慢，原因之一就是缺乏可靠的高通量筛选方法。文献报道的药物筛选方法或者通量和灵敏度低，或者受化合物毒性等因素影响产生假阳性和假阴性。这些问题可以通过荧光检测和体外筛选的方法来避免。申请人在结构研究中发现耐药性M2的色氨酸荧光强度随抑制剂的结合而减弱，荧光减弱的程度与化合物抑制通道功能的程度相关。这种现象的成因尚不明确，因此本项目计划阐明抑制剂调节色氨酸荧光强度的机制，从理论角度验证荧光变化与抑制剂活性的联系；进而选择更适合高通量筛选、且不影响通道结构功能的荧光基团来取代色氨酸；并优化筛选条件，以统计方法确认筛选技术的可靠性。本项目的研究成果将为发现新的先导化合物提供可靠的高通量筛选手段，促进具有自主知识产权的流感新药的研发。

通过测量不同pH条件下抑制剂结合与M2通道色氨酸荧光强度的关系，我们发现组氨酸质子化并不是荧光减弱的唯一影响因素。系列稀释实验证明色氨酸间的自身荧光共振能量转移是更重要的荧光强度决定因素，尤其是在与通道活性相关的酸性条件下。基于此发现，我们采用了更简便的NBD取代方案，以及更可靠的荧光偏振参数进行测量，并在此过程中建立了一种基于荧光偏振的跨膜蛋白自聚合状态的简单快速的通用定量方法。该方法有助于加快跨膜蛋白这一类重要药物靶点的结构活性研究，相关论文发表于2017年，而其物理原理已进一步被一篇J Phys Chem B论文阐述，从一个侧面说明了科学共同体对这个新方法的兴趣。我们从合作实验室得到了一些在蛙卵电生理实验中针对耐药性通道表现出较强抑制活性的化合物，期望可以作为耐药性通道的阳性对照。通过测量NBD标记耐药性通道的荧光偏振随抑制剂结合的变化，优化了pH、表面活性剂组成、温度等实验条件。不过这些化合物在我们的体系中活性都不够高，无法在合理浓度范围内确立耐药性通道的阳性对照，导致随后的统计研究无法进行。有趣的是这些化合物在流感动物模型中亦都未显示活性。而当以金刚烷胺作为野生型通道的阳性对照、无活性的化合物作为阴性对照，在最优实验条件下及化合物浓度25 µM时，各项统计测试结果都完全满足高通量筛选可靠性的要求。因此我们推测这个基于荧光偏振的体外检测体系很可能比电生理实验更准确地反映了化合物对于M2通道的作用强度，并且有极大潜力成为一个高通量筛选耐药性通道抑制剂的可靠方法。

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