Structure function analysis of cyclotide scaffolds as novel antimicrobial agents

环状肽如多粘菌素作为抗菌剂具有相当大的潜力：然而，它们具有具有挑战性的药效学特性，并且在目标细菌中迅速产生耐药性。植物衍生的环核苷酸，如环堇菜酸O，可再生胱氨酸结（ICK）支架，由头尾环化和三个二硫桥形成。这种支架已被用于其他治疗应用，但仅在非常有限的程度上用于抗菌剂。该项目将致力于i)了解驱动这些肽折叠的潜在特征，ii)修改支架以最大限度地提高抗菌和药效学性能，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月开发环violacin O及其新衍生物的合成路线，包括确保肽[1]正确折叠的方法。该学生将探索环长度和序列的变化如何影响肽折叠成活性ICK构象[2]的能力，并将开发一个集中的肽库，其中包含将增强抗菌活性和最小化毒性的氨基酸（Tabor）。18 - 24月该肽库将通过筛选级联进行抗菌活性评估，该筛选级联是由申请人（Sutton）[3]开发和使用的，用于评估抗菌肽的功能、耐药性出现的可能性和作用/耐药性机制。Patchclampstudies (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。基于生物学和生物物理研究的信息，第二多肽库将被合成和筛选，以增强抗菌和药效学特性。此外，合成的多肽将包含(i)酰基链以增强膜相互作用（ii）共聚焦显微镜的荧光团（iii）光反应性氨基酸以调节抑制剂活性[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].