DEVELOPMENT OF NEW LIPID A BINDING AGENTS

描述：脓毒症影响着数十万人的生命 在美国，革兰氏阴性菌引起的败血症都是由 对内毒素的脂质A（LA）部分的不利宿主免疫应答。 对LA具有高亲和力的化合物，包括多粘菌素B（PMB）和 衍生物，能够解毒LA和保护宿主免受 败血症 通过结合LA，这些化合物还使革兰氏阴性菌 细菌对疏水性抗生素的反应。 然而，PMB的治疗用途 由于其毒性而受到限制。 本研究的目的是开发 能够与LA强和选择性缔合的小分子， 用于治疗脓毒症和作为对抗细菌感染的手段。 本研究以PMB与LA的相互作用为模型， 基于胆酸的简单化合物的制备 支架，能够模拟PMB的LA结合行为，但 没有抗生素的毒性。 基于简单胆酸的LA 结合剂与报道的LA结合分子相比具有许多优点 包括：易于制备和衍生化， 生物稳定性和潜在的口服生物利用度。 胆酸 衍生物被设计为模拟PMB的构象，据信PMB是 在与LA的关系中，初步实验， 胆酸衍生物证明了它们敏化革兰氏阴性菌的能力。 阴性菌对疏水性抗生素，LA结合的行为 剂. LA结合特性的优化将通过以下方式实现： 制备由与胆酸连接的氨基酸组成的化合物文库 酸性支架 将使用以下方法筛选文库的LA结合： 亲和层析 亲和色谱固定相 将由通过疏水相互作用固定的LA组成， C18-二氧化硅颗粒或聚苯乙烯珠粒。 氨基酸的种类 将通过质谱法测定有效LA结合剂中的含量。 新 将测试LA结合剂致敏革兰氏阴性杆菌的能力。 对疏水性抗生素呈阴性的细菌和/或抑制作用 LA对人单核细胞（特别是白细胞介素1b的生产）。 PMB和新的LA结合剂与LA和LA衍生物的缔合 将使用微量滴定量热法进行比较。

DESCRIPTION: Sepsis affects the lives of hundreds of thousands of people each in the U.S. Sepsis caused by Gram-negative bacteria results from adverse host immune response to the Lipid A (LA) portion of endotoxin. Compounds with high affinity for LA, including polymyxin B (PMB) and derivatives, are capable of detoxifying LA nd protection a host against sepsis. By binding LA, these compounds also sensitize Gram-negative bacteria to hydrophobic antibiotics. However, therapeutic use of PMB is limited due to its toxicity. The aim of this research is to develop small molecules capable of strong and selective associating with LA for use in treatment of sepsis and as means of fighting bacterial infection. Taking the interactions of PMB with LA as a model, this research focuses on the preparation of simple compounds, based on cholic acid scaffolding, capable of mimicking the LA-binding behavior of PMB but lacking the toxicity of the antibiotic. Simple cholic-acid based LA binders will present many advantages over reported LA binding molecules including: ease of preparation and derivatization, greater control over biological stability, and potential oral bioavailablity. Cholic acid derivatives were designed to mimic a conformation of PMB believed to be important in its association with LA. Preliminary experiments with cholic acid derivatives demonstrate their ability to sensitize Gram- negative bacteria to hydrophobic antibiotics, a behavior of LA binding agents. Optimization of LA-binding characteristics will be achieved by preparing libraries of compounds made up by amino acids linked to cholic acid scaffold. The libraries will be screened for LA binding using affinity chromatography. The affinity chromatography stationary phase will be made up of LA immobilized through hydrophobic interactions of C18- silica particles or polystyrene beads. The types of amino acids in effective LA binders will be determined via mass spectroscopy. New LA-binding agents will be tested for the ability to sensitize Gram- negative bacteria to hydrophobic antibiotics and/or inhibit the effects of LA on human monocytes (specifically interleukin 1b production). Association of PMB and the new LA-binders with LA and LA derivatives will be compared using microtitration calorimetry.