Gram-Negative Sepsis: Pharmacophore-Based Therapeutics

革兰氏阴性脓毒症：基于药效团的治疗

• 批准号: 6848719
• 负责人: Sunil A David
• 金额: $ 36万
• 依托单位: UNIVERSITY OF KANSAS LAWRENCE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2008-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6848719
• 关键词: alkylation; biological signal transduction; cations; clinical research; combinatorial chemistry; computer simulation; cooperative study; disease /disorder model; drug discovery /isolation; endotoxins; high throughput technology; human tissue; hydrogen bond; interleukin 1; intermolecular interaction; laboratory mouse; lipopolysaccharides; microorganism disease chemotherapy; molecular site; nitric oxide; polyamines; septic shock; septicemia; tumor necrosis factor alpha

DESCRIPTION (provided by applicant): Gram-negative sepsis, a common and serious sequel of systemic bacterial infections is the leading cause of mortality, accounting for some 200,000 fatalities annually in the US alone. The pathogenesis of Gram-negative septic shock is due to the host response to endotoxins, or lipopolysaccharides (LPS), present on the surface of gram-negative bacteria. There are, to date, no FDA-approved therapeutic options targeting the endotoxin itself to prevent or treat this disease. We have shown that relatively simple, and synthetically easily accessible molecules of the lipopolyamine class specifically bind to LPS and neutralize its toxicity both in vitro and in animal models of septic shock. The affinity of the lipopolyamines toward LPS, however, is relatively weak (2-10 (M). In this proposal, our goal is to identify high-affinity LPS binders with nonlipopolyamine scaffolds as novel leads for the therapy of Gram-negative sepsis. A focused library of ~6000 compounds, each possessing the primary pharmacophore for LPS binding will be screened using a well-established fluorescence displacement method implemented in HTS formats. Binding, however, does not necessarily manifest in neutralization of LPS toxicity. For neutralization, an additional, appropriately positioned long-chain aliphatic group is essential. High-affinity binders ("hits") identified in HTS will be alkylated appropriately to generate LPS-neutralizing compounds (sequestrants). In in vitro assays, the potency of lead compounds in inhibiting the release of LPS-mediated proinflammatory cytokines such as tumor necrosis factor will be characterized. In a select subset of promising leads identified in the screens described above, we will verify that the mechanism of action of inhibition of LPS toxicity is via its sequestration by showing that relevant upstream cell-signaling events are blocked. The protective effects of particularly promising molecules will then be examined in two well-established murine models of gram-negative sepsis. We will systematically evaluate the toxicity of the test-compounds in a carefully chosen panel of in vitro assays. Molecular modeling techniques will be applied in an effort to correlate experimentally observed binding affinities of the test compounds with features of molecular interaction such as binding geometry, H-bonds, electrostatic, hydrophobic, and van der Waals contributions to the free energy of binding. Based on the data from the primary screen, in silico modeling, and biological assays, we will synthesize a series of analogues around promising leads using a combination of focused virtual library screening and classical medicinal chemistry approaches.

描述(申请人提供)：革兰氏阴性败血症是一种常见和严重的系统性细菌感染的后遗症，是导致死亡的主要原因，仅在美国每年就有大约20万人死亡。革兰氏阴性感染性休克的发病机制是由于宿主对革兰氏阴性细菌表面存在的内毒素或脂多糖(LPS)的反应。到目前为止，还没有FDA批准的针对内毒素本身的治疗方案来预防或治疗这种疾病。我们已经证明，在体外和败血症休克动物模型中，相对简单且易于合成的脂多胺类分子可以与内毒素特异结合，并中和其毒性。然而，脂多胺对脂多糖的亲和力相对较弱(2-10(M)。在这项建议中，我们的目标是确定高亲和力的脂多糖结合非脂多胺支架作为治疗革兰氏阴性脓毒症的新线索。将使用在HTS格式中实施的成熟的荧光置换方法来筛选具有~6000个化合物的焦点文库，每个化合物都具有与内毒素结合的主要药效团。然而，结合并不一定表现为中和内毒素毒性。对于中和，额外的、适当定位的长链脂肪族基团是必不可少的。HTS中确定的高亲和力粘结剂(“HITS”)将被适当烷基化，以产生内毒素中和化合物(隔离剂)。在体外试验中，将表征先导化合物在抑制脂多糖介导的促炎细胞因子(如肿瘤坏死因子)释放方面的效力。在上述筛选中确定的一组有希望的导联中，我们将通过显示相关的上游细胞信号事件被阻断来验证抑制内毒素毒性的作用机制是通过其封存。然后，将在两个公认的革兰氏阴性脓毒症小鼠模型中检验特别有希望的分子的保护作用。我们将在精心挑选的体外测试小组中系统地评估测试化合物的毒性。分子模拟技术将被应用于努力将实验观察到的测试化合物的结合亲和力与分子相互作用的特征相关联，如结合几何形状、H键、静电、疏水性和范德华对结合自由能的贡献。基于来自初级筛选、电子模型和生物分析的数据，我们将使用聚焦虚拟文库筛选和经典药物化学方法相结合的方法，围绕有希望的线索合成一系列类似物。