BACKGROUND Human papillomavirus (HPV) is a small, non-enveloped double-stranded circular DNA virus. The high-risk types of HPV are claimed to be responsible for over 99% of cervical cancers. One of the essential HPV oncoproteins, E7, is responsible for escaping from G1/S cell cycle arrest in HPV-infected cells by binding to the retinoblastoma protein (pRb) through its LXCXE binding site. OBJECTIVE To design a peptide inhibitor targeting HPV E7 through an in silico approach. METHODS In this study, the LXCXE binding domain of pRb is used as a target to design peptide inhibitors using a reverse structure-based approach. The designed amino acid sequence from the B pocket of pRb, named peptide Y, was further investigated in vitro analysis. The cytotoxicity of the peptide was analysed in two cell lines, namely, CaSki, containing an integrated HPV16 genome, and HaCaT, an immortalized keratinocyte cell. Cell cycle analysis was also carried out in both cell lines treated with peptides. RESULTS In the in silico approach, a 9-amino acids peptide sequence formed 4 conventional hydrogen bonds with LXCXE motif was selected for in vitro assay. Based on the cytotoxicity analysis, the peptide showed low toxicity in both cell lines, where the cell viability remained over 74% when treated with peptide Y. The peptide also caused an accumulation of cells in G0/G1 (+5.4%) and S phase (+10.2%) and a reduction of cells in the G2/M phase (-14.9%) in the CaSki cells with no significant effect on normal cells, indicating it is a potential HPV inhibitor. CONCLUSION A peptide inhibitor, peptide Y, that was designed from the LXCXE binding motif in pRb can inhibit HPV E7 by causing a cell accumulation effect in G0/G1, and S phases of the cell cycle in the HPV transformed cell lines. These findings could contribute to HPV E7 peptide inhibitor in the future.

中文翻译：

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一种基于逆向结构的 HPV E7 抑制剂设计。

背景技术人乳头瘤病毒(HPV)是一种小的、无包膜的双链环状DNA病毒。据称，高危型 HPV 与 99% 以上的宫颈癌有关。一种重要的 HPV 癌蛋白 E7 负责通过其 LXCXE 结合位点与视网膜母细胞瘤蛋白 (pRb) 结合，从而摆脱 HPV 感染细胞中的 G1/S 细胞周期停滞。目的通过计算机方法设计靶向HPV E7的肽抑制剂。方法 在这项研究中，pRb 的 LXCXE 结合结构域被用作目标，使用基于反向结构的方法设计肽抑制剂。在体外分析中进一步研究了来自 pRb B 口袋的设计氨基酸序列，命名为肽 Y。在两种细胞系中分析了肽的细胞毒性，即 CaSki、包含一个整合的 HPV16 基因组和 HaCaT，一种永生化的角质形成细胞。还在用肽处理的两种细胞系中进行细胞周期分析。结果 在计算机方法中，选择了与 LXCXE 基序形成 4 个常规氢键的 9 个氨基酸肽序列进行体外测定。根据细胞毒性分析，该肽在两种细胞系中均表现出低毒性，当用肽 Y 处理时，细胞存活率保持在 74% 以上。该肽还导致细胞在 G0/G1 (+5.4%) 和 S 期积累(+10.2%) 和 CaSki 细胞中 G2/M 期细胞的减少 (-14.9%) 对正常细胞没有显着影响，表明它是一种潜在的 HPV 抑制剂。结论 肽抑制剂，肽 Y，从 pRb 中的 LXCXE 结合基序设计的 HPV 可以通过在 HPV 转化细胞系中引起 G0/G1 和 S 期细胞周期的细胞积累效应来抑制 HPV E7。这些发现可能有助于未来的 HPV E7 肽抑制剂。