SGER: Novel Protein Mimics for a Biomimetic Lung Surfactant Replacement: Design, Synthesis, and Biophysical Characterization

目前生物工程的一个前沿是设计非天然的、序列可控的聚合物，这种聚合物可以有效地模拟天然蛋白质的一些结构和活性，但与体内疗法一样，是非免疫原性的和稳定的。这项探索性研究需要将生物化学和生物物理学中的原理和范例与生物化学和生物医学工程的方法相结合。一个有重点的、与医学相关的研究项目被提出，即使只是部分成功，也将对仿生聚合物工程的新领域产生深远的、根本性的影响，选择了一个重要而容易处理的问题：对人类表面活性蛋白SP-B和SP-C的改进合成类似物的迫切需要。给出了初步数据，并提出了开发基于多肽的新型SP-模拟物的新策略：基于多肽骨架的非天然聚合物，但不同之处在于侧链与主链氮而不是a-碳键合。N-取代的类肽与蛋白质来源的侧链以前已被证明是极具抗蛋白酶的，并进一步在体内只引起非常低水平的免疫反应。在研究人员的实验室和其他地方，一些类肽序列已经被证明在水溶液或有机溶液中采用稳定的螺旋二级结构。序列特异的类肽类化合物可以很容易地合成，成本比肽低得多，而且很容易获得各种侧链化学物质。天然的肺表面活性物质覆盖在哺乳动物肺的内表面，使正常的呼吸得以实现。它是由95%的脂类和5%的表面活性物质特异性蛋白(SP)组成的复杂混合物。蛋白质组分和磷脂组分在肺表面活性物质S的生理特性中都起着关键作用，它们通过调节肺泡气-液界面的表面张力来减少呼吸功，作为其表面积的函数。两亲性螺旋表面活性蛋白SP-B和SP-C(分别为79和35个氨基酸)促进磷脂快速吸附到气-液界面，促进磷脂单分子层在整个呼吸周期中的重新铺展，并调节单分子层的相行为，以获得尽可能低的肺泡表面张力。肺泡表面活性物质的缺失或功能障碍会导致呼吸窘迫综合征(RDS)，在这种综合征中，肺的顺应性很差，容易崩溃。患有严重RDS的患者无法在不损害肺组织的情况下进行机械呼吸，因此需要立即进行表面活性物质替代治疗。早产儿(30周)出生时肺表面活性物质缺乏，通常患有严重的RDS。这些患者通常在出生时接受表面活性物质替代治疗，并使用动物来源的表面活性物质配方。合成制剂存在，但在使肺功能正常方面明显不如天然表面活性物质有效，主要是因为它们缺乏有效的SP-B和SP-C的功能模拟物。来自动物的药物引起了人们对跨物种病原体传播的可能性以及免疫原性的担忧。因此，开发一种有效的仿生表面活性物质替代品是当前的需要。满足这一需求以及开展重要基础研究的目标是：(1)设计、合成、提纯和表征螺旋两亲性肺表面活性物质蛋白SP-B和SP-C的肽类模拟物的二级结构。方法包括有机合成、分析和制备高效液相、质谱学和圆二色谱(CD)；(2)确认和进一步表征基于类肽的SP-B和SP-C模拟物作为仿生磷脂混合物的铺展剂的体外生物物理功能。实验方法包括脉动气泡表面测量法和Langmuir-Wilhelmy表面测量法。(3)根据这些重复的初步结果，准备另一份提案，提交给NSF生物工程委员会，以继续进行该研究项目。

0093806BarronA current frontier in bioengineering is the design of non-natural, sequence-controlled polymers that can effectively mimic some of the structures and activities of natural proteins, but that are non-immunogenic and stable as in vivo therapeutics. This exploratory research requires an integration of principles and paradigms taken from biochemistry and biophysics with methods of biochemical and biomedical engineering. A focused, medically-relevant research project is proposed that, even if only partially successful, will have far-reaching, fundamental implications for the new field of biomimetic polymer engineering, An important and tractable problem is chosen: The criticalneed for improved synthetic analogs of the human surfactant proteins SP-B and SP-C. Preliminary data is shown and new strategies are proposed towards the development of novel SP-mimics based on polypeptoids: non-natural polymers based on a peptide backbone, yet differing in that sidechains are bonded to backbone nitrogens rather than to a-carbons. N-substituted peptoids with proteinogenic sidechains previously have been shown to be extremely protease-resistant, and further to raise only very low-level immune response in vivo. In the investigator's lab and elsewhere, some peptoid sequences have been shown to adopt stable, helical secondary structure in aqueous or organic solution. Sequence-specific peptoids can be synthesized easily, at substantially lower cost than peptides, with facile access to diverse sidechain chemistries.Natural lung surfactant coats the internal surfaces of mammalian lungs and enables normal breathing. It is a complex mixture composed of 95% lipids and 5% surfactant-specific proteins (SP). Both protein and phospholipid fractions play critical roles in lung surfactant s physiological properties, providing a decrease in the work of breathing by regulating surface tension at the air-liquid interface of alveoli as a function of their surface area. The amphipathic, helical surfactant proteins SP-B and SP-C (79 and 35 amino acids, respectively) promote rapid phospholipid adsorption to the air-liquid interface, facilitate respreading of the phospholipid monolayer throughout the respiration cycle, and regulate the phase behavior of the monolayer to yield the lowest possible alveolar surface tension. The absence or dysfunction of lung surfactant on alveolar surfaces leads to respiratory distress syndrome (RDS) in which lungs are incompliant and vulnerable to collapse. Patients with severe RDS cannot be mechanically ventilated without damage to lung tissue, and require immediate surfactant replacement therapy. Premature infants ( 30 weeks) are born with immature lungs lacking surfactant, and often suffer from severe RDS. These patients typically receive surfactant replacement therapy at birth with an animal-derived surfactant formulation. Synthetic formulations exist, but are significantly less efficacious than natural surfactant in enabling proper lung functioning, primarily because they lack effective functional mimics of SP-B and SP-C. Medicines sourced from animals raise concerns about a possibility for cross-species pathogen transmission and also for immunogenicity. Therefore, the development of an effective synthetic biomimetic surfactant replacement is a present need.Aims to address this need, as well as to carry out important fundamental research are:(1) To design, synthesize, purify, and characterize the secondary structure of peptoid-based mimics of the helical, amphipathic lung surfactant proteins SP-B and SP-C. Methods include organic synthesis, analytical and preparative HPLC, mass spectroscopy, and circular dichroism (CD);(2) To confirm and further characterize the in vitro biophysical functioning of peptoid-based SP-B and SP-C mimics as spreading agents for biomimetic phospholipid admixtures. Experimental approaches include both pulsating bubble surfactometry and Langmuir-Wilhelmy surfactometry.(3) Based on these carefully-repeated preliminary results, to prepare another proposal to the NSF Directorate of Bioengineering to continue the research project thereafter.