Verma Neeraj Kumar, Dewangan Rikeshwer Prasad, Harioudh Munesh Kumar, Ghosh Jimut Kanti
Biochemistry and Structural Biology Division, CSIR-Central Drug Research Institute, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow 226031, India.
Biochemistry and Structural Biology Division, CSIR-Central Drug Research Institute, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow 226031, India.
Bioorg Chem. 2023 May;134:106440. doi: 10.1016/j.bioorg.2023.106440. Epub 2023 Feb 24.
Leucine and glycine residues, at the 9th and 10th positions of helical domain of naturally occurring antimicrobial peptide (AMP), Temporin L were substituted with an unnatural amino acid, β-leucine (homovaline) to improve its serum protease stability, haemolytic/cytotoxic properties and reduce the size to some extent. The designed analogue, L9βl-TL showed either equal or improved antimicrobial activity to TL against different microorganisms including the resistant strains. Interestingly, L9βl-TL also exhibited lower haemolytic and cytotoxic activities against human red blood cells and 3T3 cells, respectively. Moreover, L9βl-TL showed antibacterial activity in presence of 25% (v/v) human serum and showed resistance against proteolytic cleavage in presence of it that suggested the serum protease stability of the TL-analogue. L9βl-TL exhibited un-ordered secondary structures in both bacterial and mammalian membrane mimetic lipid vesicles as compared to the helical structures of TL in these environments. However, tryptophan fluorescence studies demonstrated more selective interaction of L9βl-TL with bacterial membrane mimetic lipid vesicles in comparison to non-selective interactions of TL with both kinds of lipid vesicles. Membrane depolarization studies with live MRSA and bacterial membrane-mimetic lipid vesicles suggested a membrane-disrupting mode of action of L9βl-TL. L9βl-TL showed faster bactericidal mechanism compared to TL against MRSA. Interestingly, L9βl-TL was found as more potent than TL either in inhibiting biofilm formation or in eradicating the mature biofilm formed by MRSA. Overall, the present work demonstrates a simple and useful strategy to design of an analogue of TL, with minimal modifications while maintaining its antimicrobial activity with lesser toxicity and higher stability which could be attempted for other AMPs as well.
在天然存在的抗菌肽（AMP）天蚕素L的螺旋结构域的第9和第10位，亮氨酸和甘氨酸残基被非天然氨基酸β-亮氨酸（高缬氨酸）取代，以提高其血清蛋白酶稳定性、溶血/细胞毒性，并在一定程度上减小其尺寸。设计的类似物L9βl-TL对包括耐药菌株在内的不同微生物显示出与天蚕素L相当或更高的抗菌活性。有趣的是，L9βl-TL对人红细胞和3T3细胞分别表现出较低的溶血和细胞毒性活性。此外，L9βl-TL在25%（v/v）人血清存在下显示出抗菌活性，并在其存在下对蛋白水解切割具有抗性，这表明了TL类似物的血清蛋白酶稳定性。与天蚕素L在这些环境中的螺旋结构相比，L9βl-TL在细菌和哺乳动物膜模拟脂质囊泡中均表现出无序的二级结构。然而，色氨酸荧光研究表明，与天蚕素L与两种脂质囊泡的非选择性相互作用相比，L9βl-TL与细菌膜模拟脂质囊泡的相互作用更具选择性。对活的耐甲氧西林金黄色葡萄球菌（MRSA）和细菌膜模拟脂质囊泡的膜去极化研究表明L9βl-TL具有膜破坏作用模式。与天蚕素L相比，L9βl-TL对MRSA显示出更快的杀菌机制。有趣的是，发现L9βl-TL在抑制生物膜形成或根除由MRSA形成的成熟生物膜方面比天蚕素L更有效。总体而言，目前的工作展示了一种简单而有用的策略，用于设计天蚕素L的类似物，只需进行最小程度的修饰，同时保持其抗菌活性，降低毒性并提高稳定性，这也可用于其他抗菌肽。
