时间： 2022年5月20日（星期五）8:30-11:45

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主办：华中农业大学、洪山实验室

承办：生命科学技术学院

报告人1：姚霞 8:30-9:15    

题目：基于结构的靶向BTK激酶和电压门控钙离子通道的药物发现

Structure-based drug discovery targeting Bruton’s tyrosine kinase and voltage-gated Ca²⁺ channels

摘要：蛋白激酶和离子通道是两类重要的药物靶点，相关药物广泛用于治疗肿瘤、自身免疫性疾病、以及精神类疾病等。根据靶点蛋白与药物复合物的三维结构不仅可以获知药物结合的细节信息，还可以预测药物空间或极性改变后对药效的潜在影响，因此为药物的理性设计与优化提供了更加经济、有效的途径。应用基于结构的药物发现策略，我合成了一系列新型的4-氨基喹啉类BTK激酶抑制剂并验证了其在治疗自身免疫疾病-类风湿性关节炎中的应用前景。BTK激酶属于非受体酪氨酸激酶家族，是多种细胞通路特别是B细胞受体通路中的关键信号分子,对B淋巴细胞的分化、活化、增殖与生存均有至关重要的作用。抑制BTK活性可以有效治疗自身免疫性疾病以及B细胞恶性肿瘤。我合成的4-氨基喹啉化合物不仅具有良好的成药性与安全性，且在小鼠关节炎模型中表现出与临床药物地塞米松相当的体内活性，未来有潜力应用于类风湿性关节炎等多种自身免疫疾病的治疗。不同于激酶靶点，获取离子通道的三维结构信息十分困难。2013年后冷冻电镜的革命性发展使其成为高分辨膜蛋白结构解析的主流手段并加速了基于结构的膜蛋白靶向药物的发展。电压门控钙离子通道是一类广泛分布的细胞膜离子通道蛋白，通过将电信号转化成钙离子流，控制着肌肉收缩、神经递质释放以及激素分泌等多种重要的生命过程。钙离子通道功能异常会引发心血管疾病以及精神紊乱等。靶向药物被FDA批准治疗高血压、癫痫、难治性疼痛等。通过异源表达体系，我们首例获得了多个高分辨的人源钙离子通道三元复合物的冷冻电镜结构，并且解析了其被多肽类以及小分子药物调控的精细分子机制。未来我将应用仿真细胞体系-脂质体捕获离子通道的多种工作构象，为电压-机械力耦合机制的研究以及新型离子通道药物的开发开辟新路径。

Protein kinases and ion channels are two major families of drug targets. Active compounds, by modulating the functions of protein kinases and ion channels, have been proved as effective therapeutics for a broad spectrum of diseases, such as cancers, autoimmune disorders, and neurological problems. Structure-based drug discovery provides an efficient and economical method for rational drug design and optimization. It is based on the three-dimension structural information that reveals how the drugs interact with the targets and how their shape and charge changes can cause the improvement or impairment of the medical effects. Using the strategy of structure-based drug discovery, I synthesized a series of 4-aminoquinoline compounds as novel Bruton’s tyrosine kinase (BTK) inhibitors and validated their therapeutic potential for rheumatoid arthritis, the most common autoimmune disease. BTK belongs to the non-receptor tyrosine kinase family and plays important roles in various signal pathways, especially for the B cell receptor pathway, which regulates the differentiation, activation, proliferation, and survival of B lymphocytes. Inhibiting BTK has been well validated as effective therapeutic for autoimmune diseases and B cell lymphoma. Our 4-aminoquinoline compounds display favorable drug-like properties, good safety profiles, and excellent in vivo efficacy that is comparable to dexamethasone in mouse collagen-induced arthritis model. Taken together, our BTK inhibitors are promising for the treatment of rheumatoid arthritis and other autoimmune diseases in the future. Compared to proteins kinases, structural analysis for ion channels is much more difficult. Since 2013, cryogenic electron microscopy (cryo-EM) has become a mainstream means for high-resolution structural elucidation of membrane proteins and speeded up the structure-based drug discovery. The membrane widely distributed voltage-gated Ca²⁺ (Ca_v) channels can transduce electrical activity into calcium influx, thus playing a critical role in diverse physiological processes, such as muscle contraction, neurotransmitter release, and hormone secretion. Consistent with their physiological importance, aberrant regulation or malfunction of Ca_v channels is associated with cardiac and neurological disorders. Ca_v drugs have been approved by FDA for the treatment of hypertension, seizures, and intractable pains. Using recombinant expression system, we for the first time solved high-resolution cryo-EM structures of different types of human ternary Ca_v complex and revealed the molecular basis for the modes of actions of peptide and small-molecule drugs. In the future, I will attempt to capture different conformations of Ca_v channels by reconstituting proteins in cell-like proteoliposomes. The structure analysis of Ca_v conformations in different working states not only facilitates the study of electromechanical mechanism, but also pave a new avenue for the drug discovery targeting ion channels.

报告人2：张睿彬 9:15-10:00

题目：农业害虫生物防治技术研究及其应用前景

Biological control technology of agricultural pests and its application prospect

摘要：近年来，农业害虫为害是制约农业生产持续稳定发展的重要因素之一。农业农村部提出了“两增两减”的防治策略，即增加统防统治、绿色防控覆盖率，减少病虫危害损失、化学农药使用量。生物防治是利用有益生物及其产物控制有害生物种群数量的一种防治技术，包括昆虫行为调控技术、以菌治虫技术、转基因及生物育种抗虫技术等，这些防治技术均符合绿色防控的要求，在农业可持续发展中具有重要作用。因此，亟需加大生物防治技术的研究力度，为生物防治技术的更新与升级奠定基础。昆虫嗅觉识别机制研究是昆虫行为调控技术的理论基石，我们以蚜虫保守的报警现象为切入点，首次鉴定了蚜虫感受报警信息素的嗅觉受体及神经，从分子和细胞水平揭示了蚜虫识别报警信息素的机制，并证实了以气味受体为靶标筛选昆虫行为调控剂的可行性，为发展绿色环保的害虫防治策略提供了新思路。在以菌治虫方面，苏云金芽胞杆菌（Bacillus thuringiensis, Bt）制剂是目前应用最成功的微生物杀虫剂。典型的Bt菌株芽胞与晶体产生于同一细胞，晶体的产生依赖于芽胞的形成。那么，是否存在不依赖于芽胞的形成，新型的杀虫蛋白高效表达体系呢？我们以一株具有独特分化表型的Bt LM1212菌株为切入点，鉴定了负责LM1212菌株细胞分化过程以及cry基因表达的转录因子CpcR，并利用转录因子CpcR及其指导的cry基因启动子构建了全新的非芽胞杀虫蛋白表达体系，为新一代Bt制剂的遗传改良开辟新途径。

In recent years, the harm of agricultural pests is one of the important factors that restrict the sustainable and stable development of agricultural production. The Ministry of Agriculture and Rural Affairs put forward the prevention and control strategy of "two increases and two decreases", that is, to increase the coverage of unified control and green prevention and control, reduce the loss of plant diseases and agricultural pests, and reduce the use of chemical pesticides. Biological control is a kind of technology using beneficial organisms and their products to control the population of harmful organisms, including insect behavior regulation technology, pests control with microorganisms, transgenic and biological breeding pest control technology, etc. These control technologies all meet the requirements of green control, which play an important role in the sustainable development of agriculture. Therefore, it is urgent to increase the research intensity of biological control technology, and lay a foundation for the updating and upgrading of biological control technology. The olfactory recognition mechanism of insects is the theoretical foundation of insect behavior control technology. Based on the conserved alarm behavior in aphids, we identified the odorant receptors and neurons sensing alarm pheromone for the first time, revealing the mechanism of alarm pheromone recognition from molecular and cellular levels in aphid. Our results confirmed the feasibility for screening insect behavior regulation agents by using insect odorant receptor as target, providing a new idea for the development of pest green control strategies. As for using microorganisms to control pests, the preparation of Bacillus thuringiensis (Bt) is the most successful microbial insecticide. The spores and crystals of typical Bt strains were produced in the same cell, and the production of crystals depended on the formation of spores. So, is there a new efficient expression system of insecticidal proteins independent of sporulation? Taking a Bt LM1212 strain with a unique differentiated phenotype as the entry point, we identified the transcription factor CpcR responsible for the cell differentiation and cry gene expression in LM1212 strain, and constructed a novel non-sporulating insecticidal protein expression system using the transcription factor CpcR and cry gene promoter, which opened up a new approach for genetic improvement of new generation Bt agents.

报告人3：刘衍军 10:00-10:45    

题目：生物正交剪切反应介导的蛋白质在体激活技术

In situ protein activation in living systems via bioorthogonal cleavage reactions

摘要：作为生命活动的执行者，蛋白质几乎参与了所有细胞事件的调控，并与疾病的发生发展息息相关。如何实现活细胞内目标蛋白质功能的原位调控和扰动，对于揭示其作用机制具有重要意义，也极具挑战性。近年来，“活性位点”脱笼策略在蛋白质原位激活领域大放异彩，其利用遗传密码子拓展技术与生物正交剪切反应的有机结合，实现了活细胞内特定蛋白质活性的时空调控。在此基础上，我们开发了基于计算机辅助设计的新一代蛋白质通用激活平台——“邻近位点”脱笼策略。不同于传统“活性位点”脱笼策略，我们将非天然氨基酸引入到蛋白质活性残基的邻近位置，利用位阻效应，实现了蛋白质功能的高时空分辨率调控。这一技术不再局限于蛋白质活性位点氨基酸种类，只需一种非天然氨基酸，即可实现对荧光素酶、激酶、去甲基化酶、水解酶、金属蛋白酶等多种蛋白质的原位激活，具有很强的通用性。利用这一技术，我们构建了一套生物正交、时空可控的邻近标记酶，解决了Turbo内源标记背景和生物素饥饿导致的毒性问题。并在此基础上，通过邻近标记技术与磷酸化富集技术的偶联，开发了首个基于生物正交邻近标记的亚细胞磷酸化蛋白质组捕获技术，成功实现了活细胞中亚细胞水平的磷酸化蛋白质组动态捕获，并将其拓展至神经元及活体动物等复杂体系。

Proteins, as molecule machines for life, underpin most biological processes and are highly related to the occurrence and development of diseases. Therefore, it is of great significance and challenging to manipulate protein functions in situ for mechanism researches. In recent years, the ‘active-site’ decaging strategy has made great strides in protein activation area owing to its generality, easy to design and less influence to proteins. Inspired by ‘activate-site’ decaging, we developed ‘proximal decaging’ strategy, the new generation of protein activation platform based on computer-aided design. Different from ‘active site’ decaging strategy, the activity of protein was blocked by incorporated a caged amino acid in close proximity to active site as a temporal ‘bump’ and rescued promptly by biorthogonal cleavage reactions. We demonstrated the wide applicability of our method on diverse protein families, such as luciferase, kinase, demethylase, hydrolase, metalloprotease and so on. At last, a bioorthogonally controlled proximity-labeled enzymes was constructed using this strategy. By further adopting an integrated orthogonal pull-down strategy with quantitative mass spectrometry, we developed a subcellular-specific uncaging-assisted biotinylation and mapping of phosphoproteome (SubMAPP) strategy to monitor the phosphorylation dynamics of subcellular proteome in living cells and animals.