兰州大学机构库 >药学院
新白叶藤碱衍生物 Z24对灰葡萄孢菌的作用机制及靶标研究
Alternative TitleStudy on the mechanism of action and target of Neocryptolepine derivatives Z24 against Botrytis cinerea
赵文斌
Subtype博士
Thesis Advisor刘映前
2023-09-02
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name理学博士
Degree Discipline药物化学生物学
Keyword新白叶藤碱衍生物Z24 neocryptolepine derivative Z24 灰葡萄孢菌 B. cinerea 分子靶标 molecular target 硫胺噻唑合酶 thiaminothiazole synthase 可变剪接 variable splicing 硫胺素代谢 thiamine metabolism 虚拟筛选 virtual screening
Abstract

血红白叶藤(Cryptolepis sanguinolenta (Lindl.) Schlechter)是广泛分布在非洲的传统草药,在西非传统医药中占有重要地位,其具有抗炎、降血脂、抗菌、抗疟、抗肿瘤等多种药理作用。近年来,课题组对血红白叶藤的主要有效成分新白叶藤碱、白叶藤碱和异白叶藤碱等进行了系统的结构优化与抗菌活性评价研究,并首次发现了新白叶藤碱对植物病原真菌具有良好的抗菌活性,以此为先导化合物,通过结构衍生与优化后得到了抗菌谱更广、抗菌活性更优和毒性更低的候选化合物 Z24。据此,本论文旨在研究和明晰候选先导化合物 Z24对
灰葡萄孢菌(Botrytis cinerea Pers.)的作用机制及其靶标,为进一步生物合理性设计基于新白叶藤碱类生物碱的抗菌剂奠定基础。 
本研究以 B. cinerea Pers.为供试菌株,通过 DARTS和 LC-MS/MS联用技术对 Z24的直接作用靶标进行鉴定,并借助酶学、抗体制备、分子对接、转录组学、可变剪接、代谢组学和外源施加代谢物等手段对鉴定得到的疑似靶标蛋白进行了系统深入的研究。研究的主要内容和结果如下: 
1、新白叶藤碱衍生物 Z24为高效、低毒的抗真菌剂 
体外活性研究结果发现,相较新白叶藤碱而言,Z24对 4种重大植物病原真菌表现出更加优异的抗真菌活性,其中 Z24对 B. cinerea Pers.的抗菌活性最优(EC50=0.56 μg/mL),优于阳性对照药嘧霉胺(EC50=4.45 μg/mL)。在活体实验中,Z24在 100 μg/mL浓度下能够显著抑制 B. cinerea Pers.对番茄果实的侵染,具有较好的保护作用。盆栽实验中,100 μg/mL Z24显著抑制 B. cinerea Pers.对
黄瓜叶片的侵染,其保护作用可达到与嘧霉胺相当的防治效果。与此同时,Z24能够浓度依赖性抑制 B. cinerea Pers.孢子的萌发,并诱导孢子中活性氧的积累。Z24也显著诱导了 B. cinerea Pers.菌丝细胞的凋亡,并造成菌丝细胞膜的完整性丢失和细胞显微结构的破坏。此外,Z24在 HIEC细胞、HL7702细胞和 GES-1细胞中具有比新白叶藤碱更低的细胞毒性,其中在 HIEC细胞和 GES-1细胞中的毒性低于嘧霉胺。大鼠急性经口毒性 LD50>2000 mg/kg,并且 2000 mg/kg Z24未造成大鼠组织病理学的改变,也未引起大鼠血清中碱性磷酸酶、谷草转氨酶、尿素氮和谷丙转氨酶肝肾损伤指标的异常变化。最后,Z24也表现出相对较低的植物毒性。 
2、基于 DARTS和 LC-MS/MS技术获得了 Z24作用于灰葡萄孢菌的疑似靶标蛋白 Bcthi4 
采用 DARTS、考马斯亮蓝染色、银染和质谱分析等技术手段鉴定出了 Z24的直接作用靶标蛋白-硫胺噻唑合酶(Bcthi4),其中 Bcthi4蛋白在鉴定列表中相对丰度占比为 37.11%。随后利用 docking法验证了 Z24与 Bcthi4同源蛋白有较好的结合能力,对接评分为-4.302 Kcal/mol。KEGG分析发现,Z24富集到的这类蛋白主要参与代谢途径、次级代谢产物的生物合成、核糖体、抗生素的生物合成、不同环境中微生物的代谢、碳代谢、氨基酸的生物合成、糖酵解/糖原异生和丙酮酸代谢等生物过程。 
3、确证了灰葡萄孢菌 Bcthi4蛋白为 Z24的直接作用靶标蛋白

在靶标蛋白的确证中,首先通过参考 GenBank已公布的 B. cinerea B05.10 Bcthi4基因序列(Gene ID:5430120)设计引物,利用 PCR技术扩增灰葡萄孢菌Bcthi4基因后连接至 pET-B2M亚克隆载体,构建了重组质粒 Bcthi4-pET-B2M。将 Bcthi4-pET-B2M质粒转入 E. coli BL21(DE3)感受态细胞,经 IPTG诱导培养后获得了纯度较高的 Bcthi4重组蛋白。动物免疫后,经间接 ELISA和Western blotting检测发现,制备所得的灰葡萄孢菌 Bcthi4基因兔多克隆抗体具有灵敏度高和特异性强的特性,可用于下一步的 Western blotting检测。然后通过 Western blotting法对 Z24和 Bcthi4的结合反应进行了特异性检测,为此,我们设计并应用了 CETSA和 DARTS技术确认了 Z24与 Bcthi4蛋白的结合反应,发现 Z24与Bcthi4蛋白具有较好的结合活性,且具有浓度依赖性。此外,0.05 μg/mL和 0.1 μg/mL Z24能显著抑制 B. cinerea Pers.和 B. cinerea B05.10 Bcthi4活性。综上所述,通过 Western blotting和 ELISA等方法的联合验证,证实硫胺噻唑合酶(Bcthi4)确实为 Z24的直接作用靶点。 
4、Z24通过影响硫胺素焦磷酸与 Bcthi4核糖开关的结合来调控硫胺素代谢 
Bcthi4作为硫胺素生物合成途径中的核糖开关位点,在真菌中是通过可变剪接的方式进行基因的表达调控。差异可变剪接基因的 KEGG Pathway分析发现,富集最显著的通路确实为硫胺素代谢途径,并且有 3个差异可变剪接基因富集到硫胺素代谢通路,包括 Bcthi4、Bcthi6和 Bcrpb8,表明它们在硫胺素代谢中发挥关键作用。值得注意的是,负责硫胺素噻唑环合成的核糖开关 Bcthi4中检测到了显著上调的可变剪接事件(Alternative Splicing, AS),且呈现 A5SS剪
接模式,表明 Z24处理引起灰葡萄孢菌基因 Bcthi4前体 mRNA的选择性剪接,并造成 Bcthi4前体 mRNA剪接的增加。此外,灰葡萄孢菌在 Z24胁迫生长过程。中诱导了靶标基因 Bcthi4的高水平表达,表明菌丝细胞处于硫胺素饥饿的状态,通过提高硫胺素合成相关基因的表达水平才能够部分恢复灰葡萄孢菌的生长,其作用方式与市售真菌剂戊唑醇类似。上述结果表明,Z24通过影响 TPP与Bcthi4核糖开关的结合来进一步调控灰葡萄孢菌的硫胺素代谢过程。 
5、Z24是一种硫胺素生物合成通路的抑制剂 
向含有 Z24的 PDA平板和 PD水培养基中外源施加硫胺素后显著降低了 Z24对 B. cinerea Pers.和 B. cinerea B05.10的抑制活性,而硫胺素本身不能够促进灰葡萄孢菌的生长,从而表明 Z24是一种硫胺素生物合成通路的抑制剂。此外,B. cinerea Pers.和 B. cinerea B05.10在 Z24胁迫生长下均发生密集生长后,菌丝细胞内硫胺素及其活性形式硫胺素焦磷酸随化合物浓度的增加呈现不断积累的变化趋势,表明菌丝细胞是处于硫胺素饥饿的状态,只有通过积累硫胺素等代谢产物才能够恢复灰葡萄孢菌的生长。此外,非靶向代谢组学表明,硫胺素代谢异常进一步影响了灰葡萄孢菌的次生代谢物的生物合成、氨基酸生物合成、丙氨酸、天门冬氨酸和谷氨酸代谢、ABC转运蛋白、氨基酰基-tRNA生物合成等生物过程。 
6、基于虚拟筛选获得了潜在的 Bcthi4小分子抑制剂 
灰葡萄孢菌 Bcthi4蛋白的三维结构目前还未被解析,通过前期的文献调研发现,Bcthi4的 AlphaFold预测三维结构评分为 70分以上的占比很高,表明AlphaFold预测的结构可靠性良好(AlphaFold ID:AF-A0A384JNK6-F1)。因此,通过 Bcthi4的 AlphaFold预测结构(其结合口袋选自 Bcthi4蛋白与底物的结合口袋,其关键氨基酸为 CYS89/GLU110/GLY118/V AL183/HIS234/MET286等)进行计算机虚拟筛选。通过 Schrö dinger软件的 LigPrep Module模块将78.8 K个化合物的 2D格式进行加氢、能量优化等处理,输出 3D结构进行虚拟筛选,最终获得了与靶蛋白 Bcthi4结合力强的 Top 200小分子化合物。将打分前 5名化合物(Z1601701772、Z2506212851、Z1738460435、HY-Q01831、HY-Q03084)与Bcthi4蛋白的结合模式进行 2D、3D作图,进一步分析两者间的相互作用模式和预测小分子配体的生物活性,为进一步探索基于 Bcthi4蛋白的全新化学实体的设计合成与抗真菌药物开发奠定基础。 

Other Abstract

Cryptolepis sanguinolenta (Lindl.) Schlechter is a traditional plant found throughout Africa that is frequently utilized in traditional medicine. It has a wide range of pharmacological activities, including anti-inflammatory, hypolipidemic, 
antibacterial, antimalarial, and anti-tumor effects. In recent years, neocryptolepine, cryptolepine, isocryptolepine, and other key active ingredients of Cryptolepis sanguinolenta (Lindl.) Schlechter have undergone systematic structural optimization and antifungal activity testing. This work revealed for the first time that neocryptolepine has good antifungal activity against plant pathogenic fungi, and with this as the lead compound, structural derivation and optimization were used to create the candidate chemical Z24, which has a larger antifungal spectrum, improved antifungal activity, and decreased toxicity. As a result, the purpose of this research is to examine and clarify the target and mechanism of action of a prospective lead chemical Z24 against B. cinerea Pers., which will serve as a basis for future biorational designs of neocryptolepine-based antifungal medicines. 

B. cinerea Pers. was used as the test strain in this work, and DARTS and LC-MS/MS were used to identify the direct target of Z24. The hypothesized target proteins were thoroughly investigated using enzymology, antibody preparation,molecular docking, transcriptomics, variable splicing, metabolomics, and exogenous metabolites. The primary findings and results of this study are as follows: 
1. Z24, a derivative of neocryptolepine, is a highly effective and low toxic antifungal agent 
According to the results of an in vitro activity study, Z24 outperformed cryptolepine in its superior antifungal efficacy against four major plant pathogenic fungi, outperforming pyrimethanil (EC50=4.45 g/mL), with the highest antifungal 
activity against B. cinerea Pers. (EC50=0.56 g/mL). In an in vivo investigation, Z24 has a high protective effect and can significantly reduce B. cinerea Pers. infection of tomato 
fruits when applied at a concentration of 100 g/mL. In the pot experiment, 100 g/mL Z24 effectively suppressed B. cinerea Pers. infection on cucumber leaves, and its protective efficacy was comparable to pyrimethanil. Simultaneously, Z24 can limit the spore germination of B. cinerea Pers. and induce the accumulation of reactive oxygen species in the spores. Z24 also dramatically results in the loss of mycelium membrane integrity and cell microstructure disintegration, promoting apoptosis in B. cinerea Pers. mycelium cells. In addition, Z24 had lower cytotoxicity in HIEC cells, HL7702 cells, and GES-1 cells than neocryptolepine, which was less toxic in HIEC cells and GES-1 
cells than pyrimethanil. Acute oral toxicity of LD50>2000 mg/kg and 2000 mg/kg. Z24 did not elicit histological alterations in rats, nor aberrant changes in AKP/ALP , GOT, 
Urea Nitrogen, and GPT damage indices in rat serum. Finally, the seed germination test revealed that Z24 had comparatively low plant toxicity. 
2. Based on DARTS and LC-MS/MS techniques, the suspected target protein 
Bcthi4 of Z24 was obtained DARTS, Coomassie bright blue staining, silver staining, and mass spectrometry were used to identify the direct target protein-thiaminothiazole synthase (Bcthi4) of Z24, and the relative abundance of Bcthi4 protein in the identification list was 37.11%. The docking approach was then utilized to confirm that the Z24 and Bcthi4 homologous 
proteins exhibited high bonding ability, with a docking score of -4.302 Kcal/mol. 
According to KEGG analysis, these Z24-enriched proteins were primarily involved in biological processes such as metabolic pathways, secondary metabolite biosynthesis, ribosomes, antibiotic biosynthesis, microbial metabolism in different environments, carbon metabolism, amino acid biosynthesis, glycolysis/glycoconeogenesis, and pyruvate metabolism. 
3. B. cinerea's Bcthi4 protein was identified as a direct target protein of Z24. 
In the confirmation of the target protein, the primer was first designed by referring to the B. cinerea B05.10 Bcthi4 gene sequence (Gene ID:5430120) published by GenBank, the recombinant plasmid Bcthi4-pET-B2M was constructed after the Bcthi4 gene was amplified by PCR and connected to the pET-B2M subclonal vector. Bcthi4-pET-B2M plasmid was transferred into E. coli BL21 (DE3) receptor cells, and high 
purity Bcthi4 recombinant protein was obtained after induction culture by IPTG. After animal immunization, indirect ELISA and Western blotting detection showed that the prepared rabbit polyclonal antibody against Bcthi4 gene of B. cinerea had high sensitivity and strong specificity, and could be used for further Western blotting. Then, the specific binding reaction of Z24 and Bcthi4 was detected by the Western blotting method. For this purpose, we designed and applied CETSA and DARTS technology to confirm the binding reactions of Z24 and Bcthi4 protein, and found that Z24 and Bcthi4 proteins had good binding activity in a concentration dependent manner. Moreover, 0.05 μg/mL and 0.1 μg/mL Z24 significantly inhibited B. cinerea Pers. and B. cinerea B05.10 Bcthi4 activity. In summary, thiaminothiazole synthase (Bcthi4) was indeed the direct target of Z24 through the joint verification of Western blotting and ELISA. 
4. Z24 regulates thiamine metabolism by affecting the binding of TPP to Bcthi4 ribose switches 
Bcthi4, a ribose switching site in the thiamine biosynthesis pathway, controls gene expression in fungi by variable splicing. The thiamine metabolic pathway was significantly enriched in the KEGG pathway analysis of differentially variable splicing genes, and three differentially variable splicing genes, including Bcthi4, Bcthi6, and Bcrpb8, were enriched into this pathway, indicating that they play a crucial role in thiamine metabolism. Notably, significant upregulation of AS events was detected in Bcthi4, the ribose switch responsible for thiamine thiazole ring synthesis, as well as the A5SS splicing pattern, indicating that Z24 treatment induced selective splicing and 
increased splicing of Bcthi4 precursor mRNA of B. cinerea Pers. gene. Furthermore, during Z24 stress development, B. cinerea Pers. produced a high level of expression of the target gene Bcthi4, showing that mycelial cells were thiamine starved, B. cinerea Pers. growth could be partially restored by boosting the expression of genes involved in thiamine synthesis, similar to how the commercially marketed fungal drug tebuconazole works. These findings suggest that Z24 regulates thiamine metabolism in B. cinerea via modulating the binding of TPP and the Bcthi4 ribose switch. 
5. Z24 is a thiamine biosynthesis pathway inhibitor 

Exogenous thiamine application to PDA plates and PD water medium containing Z24 significantly reduced Z24's inhibitory activity against B. cinerea Pers. and B. cinerea B05.10, whereas thiamine alone could not promote B. cinerea growth, indicating that Z24 is an inhibitor of the thiamine biosynthesis pathway. Furthermore, after dense growth of B. cinerea Pers. and B. cinerea B05.10 under Z24 stress, thiamine and its active form thiamine pyrophosphate in mycelial cells showed a trend of 
continuous accumulation with increasing compound concentration, indicating that mycelial cells were in a state of thiamine deficiency, and that only the accumulation of  metabolites such as thiamine can restore B. cinerea growth. In addition, non-targeted metabolomics showed that abnormal thiamine metabolism further affected secondary metabolites biosynthesis, amino acid biosynthesis, alanine, aspartate and glutamate metabolism, ABC transporter, aminoacyl-tRNA biosynthesis and other biological processes in B. cinerea Pers.
6. Based on virtual screening, potential Bcthi4 small molecule inhibitors were identified 
The three-dimensional structure of the B. cinerea Bcthi4 protein has yet to be determined, the prior literature analysis revealed that the fraction of Bcthi4 AlphaFold prediction three-dimensional structure score was more than 70 points, indicating that the structural reliability of AlphaFold prediction was good (AlphaFold ID: AF-A0A384JNK6-F1). Therefore, computer virtual screening is performed by Bcthi4's AlphaFold predictive structure (The binding pocket is selected from the binding pocket of Bcthi4 protein and substrate, the key amino acids are CYS89/GLU110/GL Y118/V AL183/HIS234/MET286, etc). The LigPrep Module of Schrö dinger software was used to process 2D formats of 78.8K compounds for hydrogenation and energy optimization, and 3D structures were output for virtual screening, yielding the Top 200 small molecule compounds with strong binding force to the target protein Bcthi4. The top 5 compounds (Z1601701772, Z2506212851, 
Z1738460435, HY-Q01831, HY-Q03084) and Bcthi4 protein binding patterns were mapped in 2D and 3D to further study the interaction patterns between the two and predict the biological activity of small molecule ligands, it lays the groundwork for future research into the design and synthesis of novel chemical entities based on the Bcthi4 protein, as well as the development of antifungal agents. 

MOST Discipline Catalogue理学 - 化学
URL查看原文
Language中文
Other Code262010_120190907441
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/538098
Collection药学院
Affiliation
兰州大学药学院
Recommended Citation
GB/T 7714
赵文斌. 新白叶藤碱衍生物 Z24对灰葡萄孢菌的作用机制及靶标研究[D]. 兰州. 兰州大学,2023.
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