Structure function analysis of cyclotide scaffolds as novel antimicrobial agents

环肽，如多粘菌素，具有相当大的抗微生物潜力：然而，它们具有挑战性的药效学特性，并在目标细菌中迅速产生耐药性。植物来源的环肽，如环维菌素O，头尾环化形成的可获得的胱氨酸结(ICK)支架和三个二硫键。这种支架已经被用于其他治疗应用，但仅在非常有限的程度上用于抗微生物。这个项目的目标是：i)了解驱动这些多肽折叠的潜在特征，ii)修饰支架以最大化抗菌和药效学特性，iii)通过详细的微生物学和膜相互作用研究来表征作用机制。10-24月10-24开发合成环紫素O和新衍生物的路线，包括确保多肽正确折叠的方法[1]。学生将探索环长和序列的变化如何影响多肽折叠成其活性ICK构象的能力[2]，并将开发一个集中的多肽库，其中包含将增强抗菌活性和将毒性降至最低的氨基酸(TABOR)。18-24个月将在申请者(Sutton)[3]以前开发和使用的筛选级联中评估多肽库的抗菌活性，以评估抗菌肽的功能、耐药出现的可能性和作用/耐药机制。24-36个月的膜片钳研究(Mason)[3]将用于了解环肽类似物如何与细菌膜相互作用或穿透细菌膜，以及这与它们的生物活性有何关系。30-48个月根据生物学和生物物理研究的信息，将合成和筛选第二个多肽文库，以增强抗菌和药效学特性。此外，还将合成含有(I)酰基链以增强膜相互作用的多肽(Ii)用于共聚焦显微镜的荧光团(Iii)调节抑制剂活性的光反应性氨基酸[4，5]。

Cyclic peptides such as the polymyxins have considerable potential as antimicrobials:however, they have challenging pharmacodynamic properties and rapidly generateresistance in target bacteria. Plant-derived cyclotides such as cycloviolacin O, havestable cystine knot (ICK) scaffolds formed by head to tail cyclisation and three disulfidebridges. Such scaffolds have been exploited for other therapeutic applications, butonly to a very limited extent for antimicrobials. This project will aim to i) understandthe underlying features that drive the folding of these peptides, ii) modify the scaffoldto maximise antimicrobial and pharmacodynamic properties, iii) characterise themechanism of action through detailed microbiology and membrane interaction studies.Months 10 - 24 Development of synthetic routes to cycloviolacin O and novelderivatives, including methods that ensure the correct folding of the peptide [1]. Thestudent will explore how changes in the loop length and sequence affect the ability ofthe peptide to fold into its active ICK conformation [2], and will develop a focussedpeptide library, incorporating amino acids which will enhance antibacterial activity andminimise toxicity (Tabor). Months 18 - 24 The peptide library will be assessed forantimicrobial activity in a screening cascade developed and used previously by theapplicants (Sutton) [3] to evaluate antimicrobial peptide function, the potential forresistance emergence and mechanisms of action/resistance. Months 24 - 36 Patchclampstudies (Mason) [3] will be used to understand how the cyclotide analoguesinteract with or penetrate through bacterial membranes and how this relates to theirbiological activity. Months 30 - 48 Based on the information from the biological andbiophysical studies, a second library of peptides will be synthesised and screened toenhance antimicrobial and pharmacodynamic properties. In addition, peptides will besynthesised incorporating (i) acyl chains to enhance membrane interactions (ii)fluorophores for confocal microscopy (iii) photoreactive amino acids to modulate theinhibitor activity [4,5].