Gram-Negative Sepsis: Pharmacophore-Based Therapeutics

描述（由申请人提供）：革兰氏阴性脓毒症是全身性细菌感染的常见和严重后遗症，是死亡的主要原因，仅在美国每年就有约200，000例死亡。革兰氏阴性菌感染性休克的发病机制是由于宿主对存在于革兰氏阴性菌表面上的内毒素或脂多糖（LPS）的反应。迄今为止，没有FDA批准的靶向内毒素本身的治疗选择来预防或治疗这种疾病。我们已经表明，相对简单，合成容易获得的脂多胺类分子特异性结合LPS和中和其毒性在体外和动物模型的感染性休克。然而，脂多胺对LPS的亲和力相对较弱（2-10（M））。在这个建议中，我们的目标是确定高亲和力的LPS结合剂与非脂多胺支架作为治疗革兰氏阴性脓毒症的新线索。将使用以HTS形式实施的完善的荧光置换法筛选约6000种化合物的集中文库，每种化合物均具有LPS结合的主要药效团。然而，结合不一定表现为LPS毒性的中和。为了中和，额外的、适当定位的长链脂肪族基团是必要的。在HTS中鉴定的高亲和力结合剂（“命中物”）将被适当烷基化以产生LPS中和化合物（螯合剂）。在体外测定中，将表征先导化合物抑制LPS介导的促炎细胞因子如肿瘤坏死因子释放的效力。在上述筛选中鉴定的有希望的先导物的选择子集中，我们将通过显示相关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螯合。特别有前途的分子的保护作用，然后将在两个良好建立的革兰氏阴性脓毒症小鼠模型进行检查。我们将在精心选择的一组体外试验中系统地评价供试化合物的毒性。将应用分子建模技术，努力将实验观察到的供试化合物结合亲和力与分子相互作用特征（如结合几何结构、H键、静电、疏水和货车范德华力对结合自由能的贡献）相关联。基于来自初步筛选、计算机模拟和生物测定的数据，我们将使用集中的虚拟库筛选和经典药物化学方法的组合，围绕有希望的先导化合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.