High-throughput assays and small-molecule discovery of antiviral candidates targeting influenza hemagglutinin

针对流感血凝素的抗病毒候选药物的高通量测定和小分子发现

• 批准号: 10612773
• 负责人: IAN A WILSON
• 金额: $ 67.85万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10612773
• 关键词: Affinity; Animal Model; Animals; Antibodies; Antiviral Agents; Appearance; Binding; Binding Sites; Biochemical; Biological Assay; Biophysics; Biotechnology; Birds; Cells; Collection; Communities; Complementarity Determining Regions; Complex; Cyclic Peptides; Development; Disease; Drug Targeting; Economic Burden; Epidemic; Exhibits; Fluorescence Polarization; Fluorescence Resonance Energy Transfer; Foundations; Future; Goals; Half-Life; Head; Health; Hemagglutinin; Housing; Human; Hydrogen Bonding; Hydrophobicity; Infection; Influenza; Influenza A Virus, H1N1 Subtype; Influenza A Virus, H2N2 Subtype; Influenza A Virus, H3N2 Subtype; Influenza A Virus, H5N1 Subtype; Influenza A Virus, H7N7 Subtype; Influenza A Virus, H7N9 Subtype; Influenza A Virus, H9N2 Subtype; Influenza A virus; Influenza B Virus; Influenza Hemagglutinin; Journals; Label; Laboratories; Lead; Libraries; Membrane; Membrane Glycoproteins; Mission; Mutation; Nature; Neuraminidase; Peptides; Pharmaceutical Chemistry; Pharmaceutical Preparations; Point Mutation; Predisposition; Property; Proteins; Publishing; Recurrence; Reporter; Resistance; Roentgen Rays; Science; Seasons; Serotyping; Signal Transduction; Site; Specificity; Structure; Surface; Surface Antigens; United States National Institutes of Health; Viral; Viral Physiology; Virus; X-Ray Crystallography; Zoonoses; active method; anti-influenza; combat; combinatorial; cost effective; design; drug discovery; feature detection; fitness; flu; fluorophore; high throughput screening; improved; influenza infection; influenzavirus; inhibitor; innovation; interest; iterative design; mutant; nanomolar; neutralizing antibody; new pandemic; novel; novel therapeutics; pandemic disease; pandemic influenza; pandemic potential; pathogenic virus; prevent; rational design; receptor binding; scaffold; seasonal influenza; small molecule; small molecule libraries; stem; stoichiometry; therapeutic candidate; tool

PROJECT SUMMARY / ABSTRACT Influenza A viruses exhibit extreme diversity as exemplified by the multiple serotypes of the hemagglutinin (HA, H1-H18) and neuraminidase (NA, N1-N11) surface antigens. To date, only 3 of 198 possible combinations of HA and NA in avian and other animal reservoirs have been associated with human pandemics (H1N1, H2N2, H3N2). Recent appearances of H5N1, H6N1, H7N7, H7N9, H9N2, and H10N8 in humans are constant reminders of the potential for devastating new pandemics. Influenza B viruses with its two lineages further increase the health and economic burdens of seasonal influenza. No effective antiviral drugs are currently available for preventing entry of influenza A or B viruses into host cells (scientific premise). However, relatively recent discoveries of broadly neutralizing antibodies to human influenza viruses and concomitant structural studies have identified sites-of-vulnerability on the HA in pandemic, seasonal, and emerging influenza viruses. These HA surface sites include the receptor binding site and membrane-proximal stem housing the fusion machinery, both of which are essential for cellular infection. Common features for recognition of these sites can now be exploited in design of small molecules to ultimately develop broadly applicable influenza antivirals. Here, we will employ this structural information into the optimization and execution of high-throughput assays to identify new small-molecule scaffolds that target the highly conserved and vulnerable stem-binding site. High- throughput screening will be performed in parallel on representative HAs from influenza A group 1 against 600K structurally diverse molecules (SA1). We will also subject group 2 and influenza B HAs to a 300K compound screen (SA2). Validated hit compounds will be prioritized based on affinity and breadth across HAs and top candidates will be rigorously optimized into lead molecules by x-ray structure-based design cycled with medicinal chemistry. Biophysical binding, cellular infectivity and resistance assays (e.g., combinatorial viral libraries of HA mutants) will aid in iterative design, selection, and characterization of potential novel therapeutic candidates with favorable drug-like properties. All of these methods are actively employed in the Wolan and Wilson laboratories. As proof-of-concept for this approach, we identified a molecule with modest affinity to the stem of group 1 HAs with an HT assay of our own design. Its co-crystal structure with HA provided critical information towards design and synthesis of a focused compound library, which we used to produce a stereoselective molecule with nanomolar affinity and antiviral activity. Our overall goal is to identify and improve molecules with broad potency against the stem of groups 1 and 2 as well as flu B HAs. To our knowledge, we are the first to design an assay against group 2 and flu B HAs amenable to HTS (innovation). We anticipate that several classes of stem-targeted compound scaffolds will be identified with nanomolar affinity to HAs with cellular antiviral activity and suitable PK-ADME properties. Future efforts will include animal models of influenza infections to further validate our antivirals with the ultimate goal of combatting future influenza pandemics and seasonal epidemics.

项目摘要/摘要 甲型流感病毒表现出极大的多样性，例如血凝素的多种血清型 (HA，H1-H18）和神经氨酸酶（NA，N1-N11）表面抗原。到目前为止，198种可能的组合中只有3种 禽和其他动物宿主中的HA和NA与人类大流行（H1N1，H2 N2， H3N2）。最近在人类中出现的H5 N1、H6 N1、H7 N7、H7N9、H9 N2和H10 N8不断提醒人们 新的毁灭性流行病的可能性。流感B病毒及其两个谱系进一步增加了 季节性流感的健康和经济负担。目前没有有效的抗病毒药物可用于 防止甲型或B型流感病毒进入宿主细胞（科学前提）。然而，相对最近， 人类流感病毒的广泛中和抗体的发现和伴随的结构研究 已经确定了大流行性、季节性和新出现的流感病毒在HA上的脆弱部位。这些 HA表面位点包括受体结合位点和容纳融合机器的膜近端茎， 这两者都是细胞感染所必需的。识别这些网站的共同特征现在可以 在小分子设计中利用，以最终开发广泛适用的流感抗病毒药物。 在这里，我们将采用这种结构信息的优化和高通量检测的执行 以鉴定新的小分子支架，靶向高度保守和脆弱的茎结合位点。高- 将对来自甲型流感病毒1组的代表性HA平行进行通量筛选，以对抗600 K 结构多样的分子（SA 1）。我们还将对第2组和B型流感HA进行300 K化合物治疗 屏幕（SA 2）。将根据HA之间的亲和力和广度对经验证的热门化合物进行优先排序， 候选物将通过基于X射线结构的设计与药物循环， 化学.生物物理结合、细胞感染性和抗性测定（例如，HA组合病毒文库 突变体）将有助于迭代设计，选择和表征潜在的新的治疗候选物， 有利的药物样性质。所有这些方法都被Wolan和Wilson实验室积极采用。 作为这种方法的概念验证，我们鉴定了对第1组HA的茎具有适度亲和力的分子。 用我们自己设计的HT检测它与HA的共晶结构为设计提供了关键信息 并合成了一个聚焦的化合物库，我们用它来生产一种立体选择性分子， 纳摩尔亲和力和抗病毒活性。我们的总体目标是鉴定和改进具有广泛效力的分子 针对组1和2的茎以及流感B HA。据我们所知，我们是第一个设计出 抗2型流感和流感B A的药物对HTS（创新）有效。我们预计，几类茎靶向 化合物支架将被鉴定为对HA具有纳摩尔亲和力，具有细胞抗病毒活性， PK-ADME属性。未来的工作将包括流感感染的动物模型，以进一步验证我们的研究。 抗病毒药物的最终目标是对抗未来的流感大流行和季节性流行病。