流感病毒血凝素共价抑制剂的设计、合成及活性研究

doi:10.5246/jcps.2024.01.001

中国药学(英文版) ›› 2024, Vol. 33 ›› Issue (1): 1-14.DOI: 10.5246/jcps.2024.01.001

• 【研究论文】 • 下一篇

流感病毒血凝素共价抑制剂的设计、合成及活性研究

汪念¹^,², 李晨宁²^,³, 程晨曦²^,⁴, 陈建忠², 王鹏飞², 吕迅²^,³^,^*(), 李学兵²^,³^,^*()

^1. 山东第一医科大学(山东省医学科学院)研究生部, 山东济南 250117
^2. 中国科学院微生物研究所, 北京 100101
^3. 中国科学院大学存济医学院, 北京 100049
^4. 沈阳药科大学中药学院, 辽宁沈阳 110016

收稿日期:2023-10-25 修回日期:2023-11-13 接受日期:2023-12-08 出版日期:2024-01-31 发布日期:2024-01-31
通讯作者: 吕迅, 李学兵

Design, synthesis, and biological evaluation of covalent inhibitors targeting influenza virus hemagglutinin

Nian Wang¹^,², Chenning Li²^,³, Chenxi Cheng²^,⁴, Jianzhong Chen², Pengfei Wang², Xun Lv²^,³^,^*(), Xuebing Li²^,³^,^*()

¹ Graduate School of Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan 250117, Shandong, China
² Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
³ Savaid Medical School, University of Chinese Academy of Sciences, Beijing 100049, China
⁴ Shenyang Pharmaceutical University, College of Traditional Chinese Medicine, Shenyang 110016, Liaoning, China

Received:2023-10-25 Revised:2023-11-13 Accepted:2023-12-08 Online:2024-01-31 Published:2024-01-31
Contact: Xun Lv, Xuebing Li
Supported by:
National Natural Science Foundation of China (Grant No. 22377143 and 82341088) and the National Key R&D Program of China (Grant No. 2021YFC2300700 and 2022YFC2303100).

摘要/Abstract

摘要：

近年来流感频发, 针对传统靶点抗流感药物(如扎那米韦和奥司他韦)的耐药病毒大量出现, 迫切需求开发新靶点、新作用机制的抗流感药物。本研究针对流感病毒血凝素(HA)底物结合位点附近的赖氨酸(Lys133和Lys222), 设计了系列具有共价结合潜力的唾液酸类化合物(1a–1h)。活性研究表明该系列化合物对两种流感病毒HA蛋白的结合力较母体化合物唾液酸(SA)有明显提升, 其中化合物1d与阳性化合物2,6-唾液酸乳糖胺(2,6-SLN)持平(K_D = 38.2和39.6 μM); 该系列化合物安全无毒(对MDCK细胞CC₅₀ > 1 mM), 其中化合物1d可有效抑制流感病毒在MDCK细胞中的复制。本研究提出了基于流感病毒HA的共价抑制新策略, 深入发掘有望获得新型强效HA抑制剂。

关键词: 唾液酸, 流感病毒, 血凝素, 抑制剂, 共价结合

Abstract:

The ongoing evolution of influenza strains and the limited effectiveness of existing anti-influenza drugs underscore the imperative for novel therapeutics. In addressing this, we developed and synthesized several sialic acid (SA) analogs, designed as potential covalent inhibitors by targeting lysine residues (Lys133 and Lys222) proximal to the substrate binding site of hemagglutinin (HA). The affinities of these compounds for HA were meticulously assessed using surface plasmon resonance (SPR). Intriguingly, all target compounds exhibited superior affinities for HA compared to the parent compound SA. Notably, compound 1d demonstrated a binding potency similar to that of the positive control (2,6-SLN), with K_D values of 38.2 μM and 39.6 μM, respectively. Importantly, all compounds demonstrated non-cytotoxicity to MDCK cells, and compound 1d displayed robust inhibitory effects on the influenza virus in vitro. The development of covalent inhibitors targeting HA, as outlined in this study, presented a rational and promising strategy for the creation of potent HA inhibitors.

Key words: Sialic acid, Influenza virus, Hemagglutinin, Inhibitors, Covalent binding

Supporting:

汪念, 李晨宁, 程晨曦, 陈建忠, 王鹏飞, 吕迅, 李学兵. 流感病毒血凝素共价抑制剂的设计、合成及活性研究[J]. 中国药学(英文版), 2024, 33(1): 1-14.

Nian Wang, Chenning Li, Chenxi Cheng, Jianzhong Chen, Pengfei Wang, Xun Lv, Xuebing Li. Design, synthesis, and biological evaluation of covalent inhibitors targeting influenza virus hemagglutinin[J]. Journal of Chinese Pharmaceutical Sciences, 2024, 33(1): 1-14.

图/表 6

Figure 1. The mechanism of action of HA-specific covalent inhibitors.

Figure 2. Design and structural formulas of HA-specific covalent inhibitors. (A) The X-ray crystal structure of SA in complex with the H1-HA (PDB ID: 2WRH); (B) The oxygen atom at SA position 2 has a distance of 10.8 Å from H1-Lys222 side chain amino group and a distance of 12.0 Å from H1-Lys133 side chain amino group; (C) Proposed covalent mechanism of the target compound; (D) Structure of covalent inhibitors 1a–1h.

Scheme 1. Synthesis route of compounds 1a–1h. Reagents and conditions: a. DCC, DMAP, dry DCM, rt; b. K2CO3, KI, dry DMF, 80 °C; c. bis(pinacol)diborane, 1,4-dioxane, PdCl2(dppf), 90 °C; d. phenylboronic acid, THF/MeOH, rt; e. CuCl, BTTPS, DCM/MeOH, rt.

Figure 3. Covalent binding of target compounds to lysine at the molecular level (with compounds 1a and 1h as examples).

Table 1. Affinity of compound 1 to HA and cytotoxicity on MDCK cellsa.

Figure 4. The target compounds inhibit the replication of influenza viruses. Inhibition rates of compounds 1a–1h against virus replication (A/Puerto Rico/8/34 H1N1 and A/Moscow/10/99 H3N2) in MDCK cells at 500 μM. All assays were performed in triplicate, and the results were reported as mean ± SD.

参考文献 30

[1]	Laver, G.; Garman, E. The origin and control of pandemic influenza. Science. 2001, 293, 1776–1777.
[2]	Xu, R.; McBride, R.; Paulson, J.C.; Basler, C.F.; Wilson, I.A. Structure, receptor binding, and antigenicity of influenza virus hemagglutinins from the 1957 H2N2 pandemic. J. Virol. 2010, 84, 1715–1721.
[3]	Gerdil, C. The annual production cycle for influenza vaccine. Vaccine. 2003, 21, 1776–1779.
[4]	Von, I.M. The war against influenza: discovery and development of sialidase inhibitors. Nat. Rev. Drug Discov. 2007, 6, 967–974.
[5]	Meijer, A.; Lackenby, A.; Hungnes, O.; Lina, B.; van der Werf, S.; Schweiger, B.; Opp, M.; Paget, J.; van de Kassteele, J.; Hay, A.; Zambon, M. Oseltamivir-resistant influenza virus A (H1N1), Europe, 2007–08 season. Emerg. Infect. Dis. 2009, 15, 552–560.
[6]	Hai, R.; Schmolke, M.; Leyva-Grado, V.H.; Thangavel, R.R.; Margine, I.; Jaffe, E.L.; Krammer, F.; Solórzano, A.; García-Sastre, A.; Palese, P.; Bouvier, N.M. Influenza a (H7N9) virus gains neuraminidase inhibitor resistance without loss of in vivo virulence or transmissibility. Nat. Commun. 2013, 4, 2854.
[7]	Collins, P.J.; Haire, L.F.; Lin, Y.P.; Liu, J.F.; Russell, R.J.; Walker, P.A.; Skehel, J.J.; Martin, S.R.; Hay, A.J.; Gamblin, S.J. Crystal structures of oseltamivir-resistant influenza virus neuraminidase mutants. Nature. 2008, 453, 1258–1261.
[8]	Wu, Y.; Bi, Y.H.; Vavricka, C.J.; Sun, X.M.; Zhang, Y.F.; Gao, F.; Zhao, M.; Xiao, H.X.; Qin, C.F.; He, J.H.; Liu, W.J.; Yan, J.H.; Qi, J.X.; Gao, G.F. Characterization of two distinct neuraminidases from avian-origin human-infecting H7N9 influenza viruses. Cell Res. 2013, 23, 1347–1355.
[9]	Das, K.; Aramini, J.M.; Ma, L.C.; Krug, R.M.; Arnold, E. Structures of influenza A proteins and insights into antiviral drug targets. Nat. Struct. Mol. Biol. 2010, 17, 530–538.
[10]	Sigal, G.B.; Mammen, M.; Dahmann, G.; Whitesides, G.M. Polyacrylamides bearing pendant α-sialoside groups strongly inhibit agglutination of erythrocytes by influenza virus: the strong inhibition reflects enhanced binding through cooperative polyvalent interactions. J. Am. Chem. Soc. 1996, 118, 3789–3800.
[11]	Lee, Y.C.; Lee, R.T. Carbohydrate-protein interactions: basis of glycobiology. Acc. Chem. Res. 1995, 28, 321–327.
[12]	Marra, A.; Moni, L.S.; Pazzi, D.; Corallini, A.; Bridi, D.; Dondoni, A. Synthesis of sialoclusters appended to calix [4] arene platforms via multiple azide-alkyne cycloaddition. New inhibitors of hemagglutination and cytopathic effect mediated by BK and influenza A viruses. Org. Biomol. Chem. 2008, 6, 1396–1409.
[13]	Collins, B.E.; Paulson, J.C. Cell surface biology mediated by low affinity multivalent protein-glycan interactions. Curr. Opin. Chem. Biol. 2004, 8, 617–625.
[14]	Lundquist, J.J.; Toone, E.J. The cluster glycoside effect. Chem. Rev. 2002, 102, 555–578.
[15]	Oka, H.; Onaga, T.; Koyama, T.; Guo, C.T.; Suzuki, Y.; Esumi, Y.; Hatano, K.; Terunuma, D.; Matsuoka, K. Sialyl α (2 → 3) lactose clusters using carbosilane dendrimer core scaffolds as influenza hemagglutinin blockers. Bioorg. Med. Chem. Lett. 2008, 18, 4405–4408.
[16]	Umemura, M.; Makimura, Y.; Itoh, M.; Yamamoto, T.; Mine, T.; Mitani, S.; Simizu, I.; Ashida, H.; Yamamoto, K. One-step synthesis of efficient binding-inhibitor for influenza virus through multiple addition of sialyloligosaccharides on chitosan. Carbohydr. Polym. 2010, 81, 330–334.
[17]	Bauer, R.A. Covalent inhibitors in drug discovery: from accidental discoveries to avoided liabilities and designed therapies. Drug Discov. Today 2015, 20, 1061–1073.
[18]	Liu, J.F.; Stevens, D.J.; Haire, L.F.; Walker, P.A.; Coombs, P.J.; Russell, R.J.; Gamblin, S.J.; Skehel, J.J. Structures of receptor complexes formed by hemagglutinins from the Asian Influenza pandemic of 1957. Proc. Natl. Acad. Sci. USA. 2009, 106, 17175–17180.
[19]	Tzarum, N.; de Vries, R.P.; Peng, W.J.; Thompson, A.J.; Bouwman, K.M.; McBride, R.; Yu, W.L.; Zhu, X.Y.; Verheije, M.H.; Paulson, J.C.; Wilson, I.A. The 150-loop restricts the host specificity of human H10N8 influenza virus. Cell Rep. 2017, 19, 235–245.
[20]	Tzarum, N.; de Vries, R.P.; Zhu, X.Y.; Yu, W.L.; McBride, R.; Paulson, J.C.; Wilson, I.A. Structure and receptor binding of the hemagglutinin from a human H6N1 influenza virus. Cell Host Microbe. 2015, 17, 369–376.
[21]	Xu, R.; de Vries, R.P.; Zhu, X.Y.; Nycholat, C.M.; McBride, R.; Yu, W.L.; Paulson, J.C.; Wilson, I.A. Preferential recognition of avian-like receptors in human influenza A H7N9 viruses. Science. 2013, 342, 1230–1235.
[22]	Dalton, S.E.; Dittus, L.; Thomas, D.A.; Convery, M.A.; Nunes, J.; Bush, J.T.; Evans, J.P.; Werner, T.; Bantscheff, M.; Murphy, J.A.; Campos, S. Selectively targeting the kinome-conserved lysine of PI3Kδ as a general approach to covalent kinase inhibition. J. Am. Chem. Soc. 2018, 140, 932–939.
[23]	Choi, S.; Connelly, S.; Reixach, N.; Wilson, I.A.; Kelly, J.W. Chemoselective small molecules that covalently modify one lysine in a non-enzyme protein in plasma. Nat. Chem. Biol. 2010, 6, 133–139.
[24]	Akçay, G.; Belmonte, M.A.; Aquila, B.; Chuaqui, C.; Hird, A.W.; Lamb, M.L.; Rawlins, P.B.; Su, N.; Tentarelli, S.; Grimster, N.P.; Su, Q.B. Inhibition of Mcl-1 through covalent modification of a noncatalytic lysine side chain. Nat. Chem. Biol. 2016, 12, 931–936.
[25]	Pineux, F.; Federico, S.; Klotz, K.N.; Kachler, S.; Michiels, C.; Sturlese, M.; Prato, M.; Spalluto, G.; Moro, S.; Bonifazi, D. Targeting G protein-coupled receptors with magnetic carbon nanotubes: the case of the A₃ adenosine receptor. ChemMedChem. 2020, 15, 1909–1920.
[26]	Gavrilyuk, J.I.; Wuellner, U.; Barbas, C.F. β-Lactam-based approach for the chemical programming of aldolase antibody 38C2. Bioorg. Med. Chem. Lett. 2009, 19, 1421–1424.
[27]	Spiegel, D.; Parker, C. U.S. Patent 20120269766A1. 2012.
[28]	Ribeiro, J.; Pau, W.; Pifferi, C.; Renaudet, O.; Varrot, A.; Mahal, L.; Imberty, A. Characterization of a high-affinity sialic acid-specific CBM40 from Clostridium perfringens and engineering of a divalent form. Biochem. J. 2016, 473, 2109–2118.
[29]	Wei, J.H.; Zheng, L.T.; Lv, X.; Bi, Y.H.; Chen, W.W.; Zhang, W.; Shi, Y.; Zhao, L.; Sun, X.M.; Wang, F.; Cheng, S.H.; Yan, J.H.; Liu, W.J.; Jiang, X.Y.; Gao, G.F.; Li, X.B. Analysis of influenza virus receptor specificity using glycan-functionalized gold nanoparticles. ACS Nano. 2014, 8, 4600–4607.
[30]	Lv, X.; Wang, P.F.; Li, C.N.; Cheng, S.H.; Bi, Y.H.; Li, X.B. Zanamivir-cholesterol conjugate: a long-acting neuraminidase inhibitor with potent efficacy against drug-resistant influenza viruses. J. Med. Chem. 2021, 64, 1740.

流感病毒血凝素共价抑制剂的设计、合成及活性研究

Design, synthesis, and biological evaluation of covalent inhibitors targeting influenza virus hemagglutinin

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 6

参考文献 30

相关文章 15

编辑推荐

Metrics

本文评价

[1]	迟骁玮, 黎奇, 钟毅, 龚桐, 衣楚潇, 张亮仁, 刘振明. 基于虚拟筛选发现潜在的黄嘌呤氧化酶抑制剂[J]. 中国药学(英文版), 2023, 32(8): 626-635.
[2]	朱玉霞, 张玲建, 胡懿鸣, 刘卫华, 关丽萍, 林琳. 海洋中药厚藤中柚皮素(naringenin)衍生物合成及胆碱酯酶抑制活性研究[J]. 中国药学(英文版), 2023, 32(8): 636-644.
[3]	吴崇昊, 黄攀杰, 杨春琦, 高川, 曾鸣. 恶性胸膜间皮瘤免疫检查点抑制剂疗效及安全性meta分析[J]. 中国药学(英文版), 2023, 32(4): 291-301.
[4]	李晓辉, 王倩, 刘琛楠, 韩江雪, 刘思含, 刘天俊, 王倩, 关艳, 肖春玲, 王潇, 刘忆霜. 一种新型KPC-2抑制剂: PHT427[J]. 中国药学(英文版), 2023, 32(12): 989-997.
[5]	李楷, 唐冰洁, 柴新龙, 平洋, 王丽红, 苏瑾. 唾液酸功能化靶向给药系统: 与唾液酸结合免疫球蛋白样凝集素或选择素受体结合的肿瘤和炎症治疗进展[J]. 中国药学(英文版), 2023, 32(10): 773-795.
[6]	付颖, 刘思默, 马燕, 吴南楠. 钠-葡萄糖共转运蛋白2抑制剂卡格列净在2型糖尿病治疗中的新进展[J]. 中国药学(英文版), 2022, 31(8): 569-588.
[7]	刘文彬, 刘东, 王雪, 杨照亮, 何筠, 杨杨. 奥司他韦修饰纳米金棒快速可视化检测流感病毒[J]. 中国药学(英文版), 2022, 31(7): 491-498.
[8]	王倩, 刘琛楠, 韩江雪, 刘思含, 肖春玲, 关艳, 李兴华, 王颖, 王潇, 蒙建州, 甘茂罗, 刘忆霜. 一种新型NDM-1抑制剂: (–)-表儿茶素没食子酸酯[J]. 中国药学(英文版), 2021, 30(9): 716-724.
[9]	李春杏, 朱元明, 刘桦, 徐钊. 促动力药联合质子泵抑制剂与单独质子泵抑制剂治疗胃食管反流疾病疗效的荟萃分析[J]. 中国药学(英文版), 2021, 30(4): 347-356.
[10]	骆少红, 董凉凉, 李逸元, 徐丹, 陈敏. 酪氨酸激酶抑制剂 (吉非替尼、厄洛替尼、阿法替尼和奥希替尼)一线治疗表皮生长因子受体突变的晚期非小细胞肺癌的成本-效果分析[J]. 中国药学(英文版), 2021, 30(3): 253-263.
[11]	周景, 张洪, 李晓娇, 宋相仕, 张萌萌, 丁艳华. HCV耐药突变在三个Ib期临床试验中对DAA单药给药后短期疗效影响的分析[J]. 中国药学(英文版), 2021, 30(2): 133-145.
[12]	周艳, 宋恒文, 邵志超, 尹博民, 付西美, 谢典佑, 韦利军. 一种新的细胞周期蛋白依赖性激酶(CDK)9抑制剂QHRD107对急性髓系白血病的临床前疗效[J]. 中国药学(英文版), 2021, 30(2): 146-156.
[13]	北京大学药学院天然药物及仿生药物国家重点实验室. 杨晓达教授团队在基于钒活性基团的蛋白磷酸酶特异性抑制剂及其生物效应方面取得重要进展[J]. 中国药学(英文版), 2021, 30(11): 932-933.
[14]	刘琛楠, 王倩, 韩江雪, 刘思含, 肖春玲, 关艳, 李兴华, 王颖, 王潇, 蒙建州, 甘茂罗, 刘忆霜. 金属-β-内酰胺酶L1抑制剂的虚拟筛选和高通量实验测试[J]. 中国药学(英文版), 2021, 30(10): 806-812.
[15]	北京大学药学院天然药物及仿生药物国家重点实验室. 汤新景团队在环状反义寡聚核苷酸作为microRNA抑制剂的研究方面取得进展[J]. 中国药学(英文版), 2021, 30(10): 855-856.