Laboratorio de Estructura-Funcion e Ingenieria de Proteinas, Centro de Investigacion en Biotecnologia Universidad Autonoma del Estado de Morelos, Av. Universidad 2001, 62609, Cuernavaca, Morelos, Mexico.
Protein J. 2013 Aug;32(6):456-66. doi: 10.1007/s10930-013-9505-2.
Cationic antimicrobial peptides (AMPs) have attracted a great interest as novel class of antibiotics that might help in the treatment of infectious diseases caused by pathogenic bacteria. However, some AMPs with high antimicrobial activities are also highly hemolytic and subject to proteolytic degradation from human and bacterial proteases that limit their pharmaceutical uses. In this work a D-diastereomer of Pandinin 2, D-Pin2, was constructed to observe if it maintained antimicrobial activity in the same range as the parental one, but with the purpose of reducing its hemolytic activity to human erythrocytes and improving its ability to resist proteolytic cleavage. Although, the hydrophobic and secondary structure characteristics of L- and D-Pin2 were to some extent similar, an important reduction in D-Pin2 hemolytic activity (30-40 %) was achieved compared to that of L-Pin2 over human erythrocytes. Furthermore, D-Pin2 had an antimicrobial activity with a MIC value of 12.5 μM towards Staphylococcus aureus, Escherichia coli, Streptococcus agalactiae and two strains of Pseudomonas aeruginosa in agar diffusion assays, but it was half less potent than that of L-Pin2. Nevertheless, the antimicrobial activity of D-Pin2 was equally effective as that of L-Pin2 in microdilution assays. Yet, when D- and L-Pin2 were incubated with trypsin, elastase and whole human serum, only D-Pin2 kept its antimicrobial activity towards all bacteria, but in diluted human serum, L- and D-Pin2 maintained similar peptide stability. Finally, when L- and D-Pin2 were incubated with proteases from P. aeruginosa DFU3 culture, a clinical isolated strain, D-Pin2 kept its antibiotic activity while L-Pin2 was not effective.
阳离子抗菌肽 (AMPs) 作为一类新型抗生素引起了极大的关注，它们可能有助于治疗由病原菌引起的传染病。然而，一些具有高抗菌活性的 AMPs 也具有很高的溶血活性，并容易受到来自人和细菌蛋白酶的蛋白水解降解，这限制了它们在药物方面的应用。在这项工作中，构建了 Pandinin 2 的 D-对映异构体，D-Pin2，以观察它是否保持与亲本相同的抗菌活性范围，但目的是降低其对人红细胞的溶血活性并提高其抵抗蛋白水解切割的能力。尽管 L- 和 D-Pin2 的疏水性和二级结构特征在某种程度上相似，但与 L-Pin2 相比，D-Pin2 对人红细胞的溶血活性降低了 30-40%。此外，D-Pin2 在琼脂扩散测定中对金黄色葡萄球菌、大肠杆菌、无乳链球菌和两种铜绿假单胞菌具有抗菌活性，MIC 值为 12.5 μM，但比 L-Pin2 弱一半。然而，D-Pin2 的抗菌活性在微量稀释测定中与 L-Pin2 同样有效。尽管如此，当 D-Pin2 和 L-Pin2 与胰蛋白酶、弹性蛋白酶和全人血清孵育时，只有 D-Pin2 对所有细菌保持抗菌活性，但在稀释的人血清中，L-Pin2 和 D-Pin2 保持相似的肽稳定性。最后，当 L-Pin2 和 D-Pin2 与铜绿假单胞菌 DFU3 培养物（一种临床分离株）的蛋白酶孵育时，D-Pin2 保持了其抗生素活性，而 L-Pin2 则无效。
