Prevention of Candida biofilms by localized delivery of aurein analogues

摘要 念珠菌是从人类分离的最常见的真菌病原体， 医院获得性感染，主要是由于包括导管在内的留置医疗器械的定植。 当前预防念珠菌属的策略。与医疗器械相关的感染包括全身性感染， 抗真菌药物，但这些药物遭受严重的毒性和副作用，耐药菌株 涌现我们项目的目标是开发一种新的策略来预防C。白色念珠菌生物膜形成 导管通过设计一种新的抗真菌药物结构模板上的天然广谱 抗菌肽（AMP）aurein 1.2.与许多AMP一样，aurein 1.2采用螺旋结构，并选择性地将其转化为AMP。 通过阳离子和疏水相互作用使微生物膜透化。然而，努力发展 AMP转化为药物在很大程度上是不成功的，因为这些化合物在生理上具有低稳定性。 环境.在这里，我们建议合成具有折叠模式的α/β-肽金霉素1.2模拟物 其以与天然AMP几乎相同的方式呈递侧链，因此允许使用α/β-肽 以天然抗微生物肽序列为模板的类似物作为先导化合物。α/β-肽 比天然抗微生物肽结构更稳定，并且对蛋白水解降解具有抗性， 药物开发的优势。此外，我们建议制定一项战略， 药物从导管表面，局部治疗，以抑制生物膜的形成。在之前的工作中，PI确定 β-氨基酸的阳离子两亲性低聚物（称为β-肽）可以表现出高水平的特异性， 活性C.与哺乳动物细胞相比，尽管努力达到了效力的极限， 的特异性与共同研究者在该项目上的合作证明了从导管持续释放 表面并抑制体外和体内生物膜形成。在这里，我们将扩展我们的发现和方法， 由α-和β-氨基酸组成的α/β-肽。在本项目的头两年（R21 阶段），我们将产生α/β-aurein类似物，并确定如何改变疏水性，净电荷， 螺旋稳定性影响抗抗药性C.对白色念珠菌的特异性较强;白色念珠菌与哺乳动物 细胞然后，我们将评估这些化合物是否从可渗透多层（PEM）聚合物中释放 导管表面的薄膜抑制C.体外和体内白念珠菌生物膜形成。的其余部分 项目（R33期）将进一步改变α-和β-氨基酸序列，联合收割机结合α/β-金霉素的特性 类似物鉴定影响活性和特异性在C.优化广谱抗真菌药物 活性和特异性，在其他致病性念珠菌属。最后，我们将开发聚合物膜介导的 释放α/β-金霉素类似物以持续抑制念珠菌属。体外模型中的生物膜， 然后评价生物膜预防在中心静脉导管感染的大鼠模型中的有效性。在一起， 这些结果将开发出一种预防装置相关念珠菌血症的新策略。

ABSTRACT Candida spp. are the most common fungal pathogens isolated from humans and are leading causes of hospital-acquired infections, largely due to colonization of indwelling medical devices including catheters. Current strategies to prevent Candida spp. infections associated with medical devices include systemic antifungals, but these drugs suffer from severe toxicity and side effects, and drug-resistant strains have emerged. The goal of our project is to develop a novel strategy to prevent C. albicans biofilm formation on catheters by designing a new antifungal drug structurally templated on the natural broad-spectrum antimicrobial peptide (AMP) aurein 1.2. Like many AMPs, aurein 1.2 adopts a helical structure and selectively permeabilizes microbial membranes via cationic and hydrophobic interactions. However, efforts to develop AMPs into drugs have been largely unsuccessful since these compounds possess low stability in physiologic environments. Here, we propose to synthesize α/β-peptide aurein 1.2 mimetics which exhibit folding patterns that present side chains in virtually identical manner to native AMPs, and thus allow the use of α/β-peptide analogues templated on native antimicrobial peptide sequences as lead compounds. α/β-peptides are much more structurally stable than native antimicrobial peptides and are resistant to proteolytic degradation, offering significant advantages for drug development. Furthermore, we propose to develop a strategy to release this drug from catheter surfaces, localizing the treatment to inhibit biofilm formation. In prior work the PI identified that cationic, amphiphilic oligomers of β-amino acids (called β-peptides) can exhibit high levels of specific activity against C. albicans as compared to mammalian cells, although efforts reached a limit of potency and specificity. Collaborations with co-investigators on this project demonstrated sustained release from catheter surfaces and inhibition of biofilm formation in vitro and in vivo. Here, we will extend our findings and approach to α/β-peptides which are composed of both α− and β-amino acids. In the first two years of this project (R21 phase) we will generate α/β-aurein analogues and determine how varying hydrophobicity, net charge, and helical stability affect activity against drug-resistant C. albicans and specificity for C. albicans vs. mammalian cells. Then we will assess whether release of these compounds from polyelectrolyte multilayer (PEM) polymer films on a catheter surface inhibits C. albicans biofilm formation in vitro and in vivo. The remainder of the project (R33 phase) will further vary α- and β-amino acid sequence and combine features of α/β-aurein analogues identified to affect activity and specificity in C. albicans to optimize broad-spectrum antifungal activity and specificity in additional pathogenic Candida spp. Finally, we will develop polymer film-mediated release of the α/β-aurein analogues for sustained inhibition of Candida spp. biofilms in an in vitro model and then evaluate effectiveness of biofilm prevention in a rat model of central venous catheter infections. Together, these results will develop a novel strategy for prevention of device-associated candidemia.