Gram-Negative Sepsis: Pharmacophore-Based Therapeutics

描述（由申请人提供）：革兰氏阴性脓毒症是一种常见且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。革兰氏阴性脓毒性休克的发病机制是由于宿主对存在于革兰氏阴性细菌表面的内毒素或脂多糖（LPS）的反应。到目前为止，fda还没有批准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，在体外和感染性休克的动物模型中，相对简单且易于合成的脂多胺类分子特异性地与LPS结合并中和其毒性。然而，脂多胺对LPS的亲和力相对较弱（2-10 (M)）。在这项提议中，我们的目标是鉴定高亲和力LPS结合物与非脂多胺支架作为治疗革兰氏阴性脓毒症的新线索。一个大约6000个化合物的集中文库，每个化合物都具有LPS结合的主要药效团，将使用在HTS格式中实现的成熟的荧光位移方法进行筛选。然而，结合并不一定表现为LPS毒性的中和。为了中和，一个额外的，适当定位的长链脂肪基团是必不可少的。在HTS中确定的高亲和结合物（“命中”）将被适当地烷基化以生成脂多糖中和化合物（隔离物）。在体外实验中，铅化合物抑制lps介导的促炎细胞因子（如肿瘤坏死因子）释放的效力将被表征。在上述筛选中确定的有希望的线索的选择子集中，我们将通过显示相关的上游细胞信号传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来实现的。特别有希望的分子的保护作用将在两种已建立的革兰氏阴性脓毒症小鼠模型中进行检查。我们将系统地评估测试化合物的毒性，在一个精心挑选的小组体外分析。分子建模技术将应用于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（如结合几何、氢键、静电、疏水和范德华对结合自由能的贡献）联系起来。基于主筛选、计算机建模和生物分析的数据，我们将结合虚拟文库筛选和经典药物化学方法，围绕有希望的先导物合成一系列类似物。

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.