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: 6784604
• 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/6784604
• 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（由申请人提供）：革兰氏阴性败血症是全身性细菌感染的常见且严重的后遗症，是死亡的主要原因，仅在美国每年就造成约 200,000 人死亡。革兰氏阴性菌感染性休克的发病机制是由于宿主对革兰氏阴性菌表面存在的内毒素或脂多糖（LPS）的反应所致。迄今为止，还没有 FDA 批准的针对内毒素本身来预防或治疗这种疾病的治疗方案。我们已经证明，相对简单且易于合成的脂多胺类分子可以特异性地与 LPS 结合，并在体外和败血性休克动物模型中中和其毒性。然而，脂多胺对 LPS 的亲和力相对较弱（2-10 (M)）。在本提案中，我们的目标是鉴定具有非脂多胺支架的高亲和力 LPS 结合剂，作为治疗革兰氏阴性脓毒症的新先导药物。将使用以 HTS 格式实施的完善的荧光置换方法来筛选约 6000 种化合物的集中库，每种化合物都具有 LPS 结合的主要药效团。然而，结合并不一定体现在其中。为了中和 LPS 的毒性，HTS 中确定的额外的、适当位置的长链脂肪族基团将被适当烷基化，以在体外产生 LPS 中和化合物（螯合剂）。我们将在上述筛选中确定的一组选定的有希望的先导化合物中表征先导化合物抑制 LPS 介导的促炎细胞因子（例如肿瘤坏死因子）释放的效力。将通过显示相关上游细胞信号转导事件被阻断来验证抑制 LPS 毒性的作用机制是通过其隔离。然后将在两个成熟的革兰氏阴性败血症鼠模型中检查特别有前途的分子的保护作用。我们将在精心挑选的一组体外测定中系统地评估测试化合物的毒性。将应用分子建模技术，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（例如结合几何形状、氢键、静电、疏水性和范德华对结合自由能的贡献）关联起来。基于来自初级筛选、计算机建模和生物测定的数据，我们将结合重点虚拟库筛选和经典药物化学方法，围绕有希望的先导化合物合成一系列类似物。