Novel Peptides Against Modified Heparan Sulfate

描述(申请人提供)：细胞表面硫酸乙酰肝素(HS)与多种蛋白质相互作用，包括参与附着和/或进入宿主的微生物表面蛋白质。HS作为单纯疱疹病毒(HSV)的附着物和膜融合受体的能力与其结构和功能的多样性有关，这些多样性源于其生物合成过程中的广泛修饰。HS的最终修饰是由3-O-磺基转移酶(3-OSTS)介导的3-O硫代反应。3-OST家族中的7个成员已被鉴定，并被认为每个3-OST都能够识别修饰位点周围的独特糖序列。每种异构体的这种特定部位的功能使它们能够产生自己独特的3-O-硫酸乙酰肝素(3-OS HS)基序。为了鉴定HS和3-OS HS功能的小肽抑制剂，特别是在HSV-1进入HSV-1过程中，我们利用12个噬菌体展示文库筛选了3-O-磺基转移酶-3(3-OST-3)产生的未修饰HS和3-OS HS，并分离出一组具有独特序列多样性的多肽。其中，我们分别对针对未修饰HS和修饰HS的G1和G2多肽进行了鉴定。在眼部HSV-1感染和生殖器疱疹感染的体外和体内模型中，G1和G2肽的表征显示了抑制HSV-1感染的良好结果。然而，在初步筛选中鉴定的几个抗HS和抗30HS多肽尚未被测试是否能对抗HSV-1感染。我们的假设是，对整个抗HS和抗3-OS HS多肽的表征不仅将增加我们分别分离出对HS和修饰HS最有效的抑制剂的机会，而且还将导致开发新的试剂来绘制HS的结构复杂链。该提案的目的1将重点放在抗3-O-硫酸乙酰肝素(3-OS HS)结合多肽的合成和功能鉴定上，以防止HSV-1进入和细胞间传播。我们将生成关于单个多肽干扰HSV-1附着、进入和细胞间传播的能力的详细信息。目的研究3-OS HS多肽对多种3-OST亚型的抗HSV活性。它还将确定这些多肽与3种多肽的相对交叉特异性 其他异构体产生的OS HS。我们的研究将产生一些有价值的试剂来对抗各种3-OS HS的修饰，并对理解HS在人类生物学和传染病中的功能有很大的帮助。

DESCRIPTION (provided by applicant): Cell surface heparan sulfate (HS) interacts with diverse proteins including the microbial surface proteins involved in attachment and/or entry into the host. The ability of HS to act as an attachment as well as membrane fusion receptor for herpes simplex virus (HSV) relates to its structural and functional diversity originating from extensive modifications during its biosynthesis. The final modification of HS is the 3-O sulfation which is mediated by 3-O-sulfotransferases (3-OSTs). Seven members of the 3-OST family have been identified and it has been suggested that each of the 3-OSTs are capable of recognizing unique saccharide sequences around the modification sites. This site-specific function of each isoform allows them to generate their own distinct 3-O-sulfated heparan sulfate (3-OS HS) motifs. In order to identify small peptide inhibitors of HS and 3-OS HS functions especially during HSV-1 entry, we screened both unmodified HS and 3-OS HS generated by 3-O-sulfotransferase-3 (3-OST-3) using 12 mer-phage display library and isolated a panel of peptides with unique sequence diversity. Of which, we characterized the G1 and G2 peptides isolated against unmodified HS and modified HS respectively. The characterization of G1 and G2 peptides demonstrated promising results by inhibiting HSV-1 infections both in vitro and in vivo models of ocular HSV-1 infection and genital herpes infection. However, several of the anti-HS and anti-3O HS peptides identified during initial screening have not been tested against HSV-1 infection. Our hypothesis is that characterization of the entire panel of anti-HS and anti-3-OS HS peptide will not only increase our chance to isolate the most potent inhibitor against HS and modified HS respectively but it will also lead towards the development of novel reagents to map out structurally complex chains of HS. Aim 1 of the proposal will focus on synthesis and functional characterization of anti-3-O-sulfated heparan sulfate (3-OS HS) binding peptides against HSV-1 entry and cell-to-cell spread. We will generate detailed information on the abilities of the individual peptides to interfere with HSV-1 attachment, entry and cell-to-cell spread. Aim 2 will determine the anti-HSV potential of 3- OS HS peptides against multiple 3-OST isoforms. It will also determine the relative cross specificities of the peptides against the 3 OS HS generated by other isoforms. Our study will produce some valuable reagents against various 3-OS HS modifications and help greatly with the understanding of HS functions in human biology and infectious diseases.