Development of a Biomimetic Lung Surfactant Replacement

描述(由申请人提供)：几种呼吸系统疾病和疾病可以通过一种有效和低成本的仿生肺表面活性物质(LS)替代品来更好和更广泛地治疗。在目前的医疗实践中，LS替代物(主要用于治疗新生儿呼吸窘迫综合征)源自动物肺，价格昂贵，并带有病毒传播和免疫反应的风险。我们建议继续开发一类新的基于聚N-取代甘氨酸(肽)的肺表面活性蛋白的功能模拟物，这是一种在固相合成的类似于肽的序列特异性杂聚体。类肽是抗蛋白酶的，通过适当的序列设计可以形成稳定的螺旋二级结构，抵抗非特异性聚集。在过去的3.5年里，我们创造和研究了两类不同的新型螺旋两亲性类肽低聚物，它们的序列和结构与(1)SP-B(残基1-25)和(2)SP-C(残基5-32)相似。对这些基于类肽的SP模拟物在LS类脂膜中的表面活性的体外生物物理研究表明，每类模拟物中的最佳设计与它们要取代的天然多肽的表现相似。虽然初步结果是非常有希望的，但仍有许多工作要做，以开发基于类肽的SP模拟物，用于临床上有用的仿生LS替代品。我们现在必须更深入地了解正在研究的两类两亲性类肽SP模拟物的结构-活性关系。我们建议通过改变关键的结构特征和对表面活性的影响进行详细的研究来实现这一点，同时进行分子动力学模拟。对于SP-C模拟物，我们将研究疏水螺旋长度、侧链化学和总螺旋度的影响，也将模拟天然SP-C的棕榈酰化反应。所提出的SP-B模拟物将具有越来越多的类蛋白质序列和侧链化学成分，我们将检验二聚化将提高其活性的假设。我们将使用CD、脉动气泡表面测定法、Langmuir槽/Wilhelmy表面天平、荧光显微镜、单层囊泡和X射线反射/衍射对含有类肽的表面活性物质进行体外研究，以绘制构效关系图并研究类肽-脂相互作用。我们将研究含有这两种表面活性物质模拟物的表面活性物质配方，我们将研究类肽表面活性物质与肺上皮细胞和RDS动物模型的双相容和体内疗效。

DESCRIPTION (provided by applicant): Several respiratory diseases and disorders could be better and more widely treated with an effective and low-cost biomimetic lung surfactant (LS) replacement. In present medical practice, LS replacements (used predominantly to treat neonatal respiratory distress syndrome) derive from animal lungs, are expensive, and carry risks of viral transmission and immune response. We propose to continue the development of a novel class of functional mimics of the lung surfactant proteins based on poly-N-substituted glycines (peptoids), which are sequence-specific heteropolymers synthesized on solid phase, similarly to peptides. Peptoids are protease resistant, and with proper sequence design can form stable, helical secondary structures that resist non-specific aggregation. Over the past 3.5 years, we have created and studied two different, novel classes of helical, amphipathic peptoid oligomers with sequence and structural similarity to (1) SP-B (residues 1-25) and (2) SP-C (residues 5-32). In vitro biophysical studies of the surface activities of these peptoid-based SP mimics in an LS-like lipid film demonstrate that the best designs within each class of mimics perform similarly to the natural peptides they are designed to replace. While initial results are very promising, much work remains to be done to develop peptoid-based SP mimics for a clinically useful, biomimetic LS replacement. We must now gain a deeper understanding of structure-activity relationships for the two classes of amphiphilic peptoid SP mimics under study. We propose to do this by varying key structural features and performing detailed studies of the effects on surface activity, in parallel with Molecular Dynamics simulations. For SP-C mimics, we will study the effects of hydrophobic helix length, side chain chemistry and overall helicity, and will also mimic the palmitoylation of natural SP-C. Proposed SP-B mimics will have increasingly protein-like sequences and side chain chemistries, and we will test the hypothesis that dimerization will improve their activity. We will carry out in vitro studies of peptoid-containing surfactant using CD, pulsating bubble surfactometry, a Langmuir trough/Wilhelmy surface balance, fluorescence microscopy, unilamellar vesicles, and X-ray reflectivity/diffraction to map out structure-activity relationships and study peptoid-lipid interactions. We will investigate surfactant formulations containing mimics of both SPs, and we will study the bicompatibility and in vivo efficacy of peptoid surfactants with lung epithelial cells and animal models of RDS.