Research Center for Proteinaceous Materials (RCPM), Chosun University, Kwangju 501-759, Republic of Korea.
Biomaterials. 2014 Jan;35(3):1025-39. doi: 10.1016/j.biomaterials.2013.10.035. Epub 2013 Oct 28.
In response to the growing problem of multidrug-resistant pathogenic microbes, much attention is being paid to naturally occurring and synthetic antimicrobial peptides (AMPs) and the effects of their structural modification. Among these modifications, amino acid substitution is a simple approach to enhancing biological activity and reducing cytotoxicity. An earlier study indicated that HPA3, an analog of HP (2-20) derived from the N-terminus of Helicobacter pylori ribosomal protein L1, forms large pores and shows considerable cytotoxicity. However, HPA3P, in which a proline (Pro) is substituted for glutamic acid (Glu) at position 9 of HPA3, shows markedly less cytotoxicity. This may be attributable to the presence of a Pro-kink into middle of the HPA3P structure within the membrane environment. Unfortunately, HPA3P is not an effective antibacterial agent in vivo. We therefore designed a helix-PXXP-helix structure (HPA3P2), in which Pro was substituted for the Glu and phenylalanine (Phe) at positions 9 and 12 of HPA3, yielding a molecule with a flexible central hinge. As compared to HPA3P, HPA3P3 exhibited dramatically increased antibacterial activity in vivo. ICR mice infected with clinically isolated multidrug-resistant Pseudomonas aeruginosa showed 100% survival when administered one 0.5-mg/kg dose of HPA3P2 or three 0.1-mg/kg doses of HPA3P2. Moreover, in a mouse model of septic shock induced by P. aeruginosa LPS, HPA3P2 reduced production of pro-inflammatory mediators and correspondingly reduced lung (alveolar) and liver tissue damage. The changes in HPA3 behavior with the introduction of Pro likely reflects alterations of the mechanism of action: i) HPA3 forms pores in the bacterial cell membranes, ii) HPA3P permeates the cell membranes and binds to intracellular RNA and DNA, and iii) HPA3P2 acts on the outer cellular membrane component LPS. Collectively, these results suggest HPA3P2 has the potential to be an effective antibiotic for use against multidrug-resistant bacterial strains.
针对日益严重的多药耐药性病原微生物问题，人们越来越关注天然存在和合成的抗菌肽 (AMPs) 及其结构修饰的效果。在这些修饰中，氨基酸取代是提高生物活性和降低细胞毒性的一种简单方法。早期的一项研究表明，HP (2-20) 的 N 端衍生的 Helicobacter pylori 核糖体蛋白 L1 的类似物 HPA3 形成大孔并表现出相当大的细胞毒性。然而，HPA3P 中，HPA3 的 9 位谷氨酸 (Glu) 被脯氨酸 (Pro) 取代，显示出明显较低的细胞毒性。这可能是由于在膜环境中 HPA3P 结构的中间存在 Pro 扭曲。不幸的是，HPA3P 不是体内有效的抗菌剂。因此，我们设计了一个螺旋-PXXP-螺旋结构 (HPA3P2)，其中 Pro 取代了 HPA3 的 9 位和 12 位的 Glu 和苯丙氨酸 (Phe)，产生了一个具有灵活中心铰链的分子。与 HPA3P 相比，HPA3P3 在体内表现出显著增强的抗菌活性。用临床分离的多药耐药性铜绿假单胞菌感染的 ICR 小鼠，给予 0.5mg/kg 剂量的 HPA3P2 或 0.1mg/kg 剂量的 HPA3P2 三次，100%存活。此外，在铜绿假单胞菌 LPS 诱导的脓毒症休克小鼠模型中，HPA3P2 降低了促炎介质的产生，相应地减少了肺 (肺泡) 和肝组织损伤。引入 Pro 后 HPA3 行为的变化可能反映了作用机制的改变：i) HPA3 在细菌细胞膜上形成孔，ii) HPA3P 渗透细胞膜并与细胞内的 RNA 和 DNA 结合，iii) HPA3P2 作用于细胞膜外的 LPS 成分。总之，这些结果表明 HPA3P2 有可能成为一种有效的抗生素，用于治疗多药耐药性细菌菌株。
