Histidine-rich Modification of a Scorpion-derived Peptide Improves Bioavailability and Inhibitory Activity against HSV-1.
Histidine-rich Modification of a Scorpion-derived Peptide Improves Bioavailability and Inhibitory Activity against HSV-1.
复制标题
蝎子衍生肽的富含组氨酸修饰可提高 HSV-1 的生物利用度和抑制活性
作者：
Zeng Z;Zhang R;Hong W;Cheng Y;Wang H;Lang Y;Ji Z;Wu Y;Li W;Xie Y;Cao Z
基本原理：单纯疱疹病毒是传播最广泛的人类病毒病原体之一。HSV-1感染了很大一部分人类人口，并导致严重的疾病。目前HSV-1的临床治疗以核苷类似物为基础，由于耐药性、副作用和生物利用度低等原因，其使用受到限制。AMPS已被确定为可能克服这些限制的潜在抗病毒药物。因此，我们从蝎子来源的AMP文库中筛选出抗HSV-1多肽，并将其中一个候选基因改造成富含组氨酸的多肽，显著提高了抗病毒活性和开发潜力。方法：以中国所在的云南产全蝎为材料，构建毒腺基因文库。从该文库中鉴定了6个可能的AMPs，并通过空斑形成实验对合成的多肽进行了筛选，以确定其杀灭病毒的能力。根据HSV-1感染过程的加入时间实验，确定感兴趣的多肽的作用模式。利用螺旋轮模型和圆二色谱对多肽进行了结构分析，设计了富含组氨酸的修饰。用流式细胞仪和共聚焦显微镜分别检测细胞摄取和分布情况。结果：在HSV-1空斑减少试验中，Eval418肽具有较高的清除活性。Eval418对HSV-1的灭活呈剂量依赖性和时间依赖性，并呈剂量依赖性抑制HSV-1与宿主细胞的附着。然而，由于细胞摄取能力差，Eval418几乎不能抑制已建立的HSV-1感染。我们进一步设计并修饰Eval418为四个富含组氨酸的衍生肽，具有更强的抗病毒活性和更低的细胞毒性。所有的衍生肽都能抑制已建立的HSV-1感染。其中一种多肽Eval418-FH5不仅具有较强的病毒灭活活性和增强的附着抑制活性，而且对细胞内HSV-1也具有较高的抑制活性，这与共聚焦显微镜和流式细胞仪证实其改善的细胞内摄取和分布是一致的。结论：我们成功地从蝎毒多肽文库中鉴定了抗HSV-1多肽Eval418，并设计了富含组氨酸的Eval418衍生物，为进一步开发抗HSV-1药物奠定了基础。这一成功的修饰可以为提高多肽制剂的生物利用度、细胞分布和抗病毒活性提供一种设计策略。
Rationale: HSV is one of the most widespread human viral pathogens. HSV-1 infects a large portion of the human population and causes severe diseases. The current clinical treatment for HSV-1 is based on nucleoside analogues, the use of which is limited due to drug resistance, side effects and poor bioavailability. AMPs have been identified as potential antiviral agents that may overcome these limitations. Therefore, we screened anti-HSV-1 peptides from a scorpion-derived AMP library and engineered one candidate into a histidine-rich peptide with significantly improved antiviral activity and development potential. Methods: A venomous gland cDNA library was constructed from the scorpion Euscorpiops validus in the Yunnan Province of China. Six putative AMPs were characterized from this cDNA library, and the synthesized peptides were screened via plaque-forming assays to determine their virucidal potential. Time of addition experiments according to the infection progress of HSV-1 were used to identify the modes of action for peptides of interest. The histidine-rich modification was designed based on structural analysis of peptides by a helical wheel model and CD spectroscopy. Peptide cellular uptake and distribution were measured by flow cytometry and confocal microscopy, respectively. Results: The peptide Eval418 was found to have high clearance activity in an HSV-1 plaque reduction assay. Eval418 exhibited dose-dependent and time-dependent inactivation of HSV-1 and dose-dependent inhibition of HSV-1 attachment to host cells. However, Eval418 scarcely suppressed an established HSV-1 infection due to poor cellular uptake. We further designed and modified Eval418 into four histidine-rich derivative peptides with enhanced antiviral activities and lower cytotoxicities. All of the derivative peptides suppressed established HSV-1 infections. One of these peptides, Eval418-FH5, not only had strong viral inactivation activity and enhanced attachment inhibitory activity but also had high inhibitory activity against intracellular HSV-1, which was consistent with its improved intracellular uptake and distribution as confirmed by confocal microscopy and flow cytometry. Conclusion: We successfully identified an anti-HSV-1 peptide, Eval418, from a scorpion venom peptide library and designed a histidine-rich Eval418 derivative with significantly improved potential for further development as an anti-HSV-1 drug. This successful modification can provide a design strategy to improve the bioavailability, cellular distribution and antiviral activity of peptide agents.