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

0093806 Barron生物工程的一个前沿领域是设计非天然的、序列控制的聚合物，这些聚合物可以有效地模拟天然蛋白质的一些结构和活性，但作为体内治疗剂是非免疫原性的和稳定的。 这种探索性的研究需要从生物化学和生物物理学的方法与生物化学和生物医学工程的原则和范式的整合。 提出了一个集中的，医学相关的研究项目，即使只有部分成功，将有深远的，仿生聚合物工程的新领域的基本影响，一个重要的和易处理的问题被选中：对改进的合成类似物的人类表面活性蛋白SP-B和SP-C的criticalneed。 初步数据显示，并提出了新的战略，对发展新的SP-模拟物的基础上的多肽：非天然聚合物的基础上的肽骨架，但不同的是，侧链键合到骨架氮，而不是a-碳。 具有蛋白质侧链的N-取代类肽先前已显示出极强的蛋白酶抗性，并且进一步仅在体内引起非常低水平的免疫应答。 在研究者的实验室和其他地方，一些类肽序列已被证明在水溶液或有机溶液中采用稳定的螺旋二级结构。 序列特异性类肽可以很容易地合成，成本比肽低得多，容易获得不同的侧链化学。天然肺表面活性剂覆盖哺乳动物肺的内表面，使其能够正常呼吸。 它是由95%的脂质和5%的表面活性剂特异性蛋白（SP）组成的复杂混合物。 蛋白质和磷脂组分在肺表面活性物质的生理特性中起着关键作用，通过调节肺泡气液界面的表面张力（作为其表面积的函数）来减少呼吸功。 两亲性螺旋表面活性剂蛋白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，促进磷脂单层在整个呼吸循环中的再扩散，并调节单层的相行为以产生尽可能低的肺泡表面张力。 肺泡表面的肺表面活性物质的缺乏或功能障碍导致呼吸窘迫综合征（RDS），其中肺不顺应并且容易塌陷。 严重RDS患者不能在不损伤肺组织的情况下进行机械通气，需要立即进行表面活性剂替代治疗。 早产儿（30周）出生时肺部不成熟，缺乏表面活性剂，经常患有严重的RDS。 这些患者通常在出生时接受动物源性表面活性剂制剂的表面活性剂替代疗法。 合成制剂存在，但在实现适当的肺功能方面比天然表面活性剂明显更不有效，主要是因为它们缺乏SP-B和SP-C的有效功能模拟物。 动物来源的药物引起了对跨物种病原体传播可能性和免疫原性的担忧。 因此，开发一种有效的人工合成仿生表面活性剂替代物是当前的迫切需要，为满足这一需求，同时开展重要的基础研究工作，主要包括：（1）设计、合成、纯化和表征螺旋型两亲性肺表面活性蛋白SP-B和SP-C的类肽模拟物的二级结构。 方法包括有机合成、分析和制备HPLC、质谱和圆二色性（CD）;（2）确认并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定法和朗缪尔-威廉米表面测定法。(3)根据这些仔细重复的初步结果，准备另一份建议书给NSF生物工程理事会，以继续此后的研究项目。

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.