Design of pH-responsive Peptide Assembly for Acid-activatable Antimicrobial Therapy

细菌可以感染所有身体部位，包括pH值低于中性的部位，如尿路、口腔、呼吸道上皮、皮肤和其他软组织。虽然许多抗微生物剂在中性pH下具有最佳杀菌活性，但不寻常的酸性pH会损害药物的抑菌和杀菌活性并促进细菌耐药性。抗菌肽（Antimicrobial Peptides，AMP）具有广谱抗菌活性，不易产生耐药性，是目前解决全球性传染病问题的有效药物。尽管AMP被认为是传统抗生素的有前途的替代品，但它们的广泛使用和转化为临床应用受到几种内在限制的阻碍，包括它们对蛋白酶的敏感性和对宿主细胞的中度至重度毒性。人们普遍认为，需要新的设计和工程策略来克服传统抗菌肽的内在局限性，用于靶向抗菌治疗。PI的团队最近开发了一种自组装抗菌纳米纤维（SAAN）的技术平台，以规避传统抗生素和AMP的缺点。SAAN是多结构域肽（MDP）的超分子组装体，其可以经历程序化自组装成纳米结构的细丝。SAAN在杀死广谱耐药细菌的同时，在改善的稳定性、精确控制的AMP负载能力、最小的细胞毒性和大大改善的血液相容性方面提供了优于传统单链AMP的关键优势。最近，PI的小组提出了基本的设计原则，以开发一种新的酸活化的SAAN使用pH响应的MDP作为分子构建块。长期目标是将SAAN作为稳定的全身性纳米载体用于靶向AMP递送。本提案的研究目标是扩展含有pH响应性非天然氨基酸的SAAN家族，以进一步调节材料的稳定性和活性，并开发具有协同抗菌作用的更有效的pH依赖性多组分SAAN，以在其优选的酸性环境中根除细菌而不影响健康组织和细胞。在德克萨斯大学阿灵顿分校，这是一个西班牙裔服务机构，PI有助于增加STEM领域的多样性教育，特别是鼓励代表性不足的学生参与研究，学习成绩差沿着她的其他推广和教育工作。技术概述NSF项目的重点是合成和自组装pH响应肽与非天然氨基酸，显示触发微生物活性在弱酸性pH范围。 该项目是由我们最近发现的酸活化自组装抗菌纳米纤维（AA-SAAN）驱动的，AA-SAAN是通过pH响应性多结构域肽（MDP）的自组装产生的。MDP在生理条件下组装成纳米纤维并且是无活性的。在局部pH降低的酸性感染部位，AA-SAAN被触发以分解并释放活化的MDP，从而与细菌细胞膜相互作用并杀死细菌。在本提案中，我们将通过使用具有扩展化学的MDP来开发更有效的AA-SAAN来验证和推进基本设计原则。本课题的研究目标是：1）利用含非天然离子氨基酸的新型pH响应型MDP构建AA-SAAN; 2）制备和评价具有协同抗菌活性的多组分AA-SAAN; 3）评价这些材料的pH依赖性体外生物活性。这些努力将提供关键的洞察到基本的分子和超分子化学，管理AA-SAAN的结构和抗菌活性。此外，这一战略对于合理设计和合成具有内置pH响应特性的功能材料，以探测pH梯度发挥重要作用的各种复杂生物过程具有高度变革性。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Non-technical SummaryBacteria can infect all body parts, including those with pH values below neutral, such as the urinary tract, oral cavity, respiratory epithelia, skin, and other soft tissues. While many antimicrobial agents have optimal bactericidal activity at a neutral pH, the unusual acidic pH can impair the bacteriostatic and bactericidal activities of pharmaceutical drugs and promote bacterial resistance. Antimicrobial peptides (AMPs) have emerged as an alternative solution to conventional antibiotics to address the global problem of infectious diseases due to their broad-spectrum antimicrobial activity and less tendency for bacteria to develop resistance. Although AMPs are considered promising alternatives to conventional antibiotics, their widespread use and translation into clinical application is hampered by several intrinsic limitations, including their susceptibility to proteases and moderate to severe toxicity towards host cells. It is generally believed that new design and engineering strategy is needed to overcome the intrinsic limitation of traditional AMPs for targeted antimicrobial therapy. The PI’s group recently developed a technology platform of self-assembling antimicrobial nanofibers (SAANs) to circumvent the shortcomings of both traditional antibiotics and AMPs. SAANs are supramolecular assemblies of multidomain peptides (MDPs) that can undergo programmed self-assembly into nanostructured filaments. While killing broad-spectrum antibiotic-resistant bacteria, SAANs offer key advantages over traditional single chain AMPs in terms of improved stability, precisely controlled AMP loading capacity, minimal cytotoxicity and greatly improved hemocompatibility. More recently, the PI’s group advance the fundamental design principles to develop a novel acid-activatable SAANs using pH-responsive MDPs as the molecular building block. The long-term goal is to apply SAANs as stable systemic nanocarriers for targeted AMP delivery. The research objectives of the present proposal are to expand the family of SAANs containing pH-responsive non-natural amino acids to further modulate materials stability and activity, and to develop more potent pH-dependent multicomponent SAANs with synergistic antimicrobial effect to eradicate bacteria at their preferred acidic environment without affecting healthy tissues and cells. At the University of Texas at Arlington which is a Hispanic-serving institution, the PI contributes to education of increased diversity in STEM fields, particularly encouraging research participation from underrepresented students with weakly performing academic record along with her other outreach and education efforts. Technical SummaryThe NSF project is focused on the synthesis and self-assembly of pH-responsive peptides with non-natural amino acids that show triggered antimicrobial activity in the weakly acidic pH range. The project is driven by our recent discovery of Acid-Activatable Self-Assembled Antimicrobial Nanofibers (AA-SAANs), which is generated through the self-assembly of pH-responsive multidomain peptides (MDPs). The MDPs are assembled into nanofibers at the physiological condition and being inactive. At the acidic infection site where the local pH decreases, AA-SAANs are triggered to dissemble and release the activated MDPs to interact with the bacterial cell membrane and kill bacteria. In this proposal we will validate and advance the fundamental design principle by using MDPs with expanded chemistry to develop more potent AA-SAANs. The research objectives of this proposal are: 1) Establish AA-SAANs using new pH-responsive MDPs containing non-natural ionic amino acids; 2) Fabrication and evaluation of multicomponent AA-SAANs with synergistic antimicrobial activity; 3) Evaluate the pH-dependent biological activity of these materials in vitro. These efforts will provide critical insight into the fundamental molecular and supramolecular chemistry that govern the structure and antimicrobial activity of AA-SAANs. Further, this strategy is highly transformative for the rational design and synthesis of functional materials with built-in pH-responsive properties to probe various complex biological processes in which pH gradient plays an important role.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.