Theta-defensins Novel HIV-1 Uptake Inhibitors

描述（由申请人提供）：我们拟研究非人类灵长类动物宿主对人类HIV-1及相关逆转录病毒的免疫机制。我们的长期目标是：1)确定参与灵长类HTV-1感染发病机制的先天免疫系统分子。2)表征α -防御素和α -防御素的碳水化合物结合特异性，并将这一特性与它们抗HIV-1的活性联系起来。3)为未来的体内研究确定有效的β -防御素，以评估其治疗效果。本基金不建议进行体内研究。-防御素是由旧大陆猴和其他一些非人类灵长类动物的白细胞表达的环状八肽。它们识别某些碳水化合物结构（聚糖）的能力允许一些9-防御素以高亲和力结合HIV-1的gp120和CD4，并阻止R5和X4株HIV-1进入其他易感的人类细胞。人类基因组中至少存在60个0-防御素基因，而β -防御素mRNA存在于人的骨髓、胸腺和脾脏中。然而，人类-防御素基因及其mRNA含有一个过早停止密码子，人类细胞不产生-防御素肽。逆转录细胞周期蛋白1-3是人类在沉默突变（过早停止密码子）缺失的情况下可以产生的代表防御素的合成肽。逆转录周期蛋白-2对HIV-1原代分离株有效，这些分离株代表A、B、C和CRF01_AE亚型，这些亚型在世界范围内引起大多数HIV-1感染。由于反转录细胞周期素对阴道乳酸菌无细胞毒性和无损伤，它们为未来的药物开发提供了有趣的先导化合物。本提案中包含的研究旨在提供一个知识平台，以促进未来治疗性β -防御素的发展。具体目标是：1)。合成新的-防御素，包括基于非人类灵长类动物的-防御素基因序列和人类反转录细胞周期蛋白类似物的肽，并测试它们对HIV-1的活性和细胞毒性。2）. 鉴定灵长类动物防御素结合的糖和寡糖，并将其与抗逆转录病毒特性相关联。目的1将通过固相肽合成和使用外周血单个核细胞和指示细胞系进行体外保护实验来完成。特异性目标2将通过微化学技术的结合来实现，包括表面等离子体共振，串联质谱，以及最先进的电组学和肽组学。

DESCRIPTION (provided by applicant): We propose to study the immune mechanism of defensins in host resistance to human HIV-1 and related retroviruses of non-human primates. Our long-term objectives are: 1) to identify innate immune system molecules involved in the pathogenesis of HTV-1 infections in primates. 2) to characterize the carbohydrate-binding specificity of theta- and alpha-defensins, and to correlate this property with their activity against HIV-1. 3) to identify potent theta-defensins for future in vivo studies designed to assess their therapeutic usefulness. No in vivo studies are proposed hi this grant. Theta-defensins are cyclic octadecapeptides expressed by leukocytes of Old World monkeys and some other non human primates. Their ability to recognize certain carbohydrate structures (glycans) allows some 9-defensins to bind both gp120 of HIV-1 and CD4 with high affinity, and to prevent the entry of R5 and X4 strains of HIV-1 into otherwise susceptible human cells. At least 6 0-defensin genes exist in the human genome, and theta-defensin mRNA exists in human bone marrow, thymus, and spleen. However, human theta-defensin genes and their mRNA contain a premature stop codon, and human cells do not produce theta-defensin peptides. Retrocyclins 1-3 are synthetic peptides that represent the theta-defensins that humans could produce if the silencing mutation (the premature stop codon) were absent. Retrocyclin-2 is effective against HIV-1 primary isolates representing subtypes A,B,C and CRF01_AE, which cause most HIV-1 infections worldwide. Because retrocyclins are noncytotoxic and noninjurious to vaginal lactobacilli, they provide intriguing lead compounds for future pharmaceutical development. The studies contained in this proposal are intended to provide a knowledge-platform that will facilitate the development of therapeutic theta-defensins in the future. The specific aims are: 1). To synthesize novel theta-defensins, including peptides based on theta-defensin gene sequences in non-human primates and analogues of human retrocyclins, and to test them for activity against HIV-1 and cytotoxicity. 2). To identify the sugars and oligosaccharides that primate theta-defensins bind, and correlate binding with their antiretroviral properties. Aim 1 will be accomplished by solid phase peptide synthesis and in vitro protection assays using peripheral blood mononuclear cells and indicator cell lines. Specific Aim 2 will be realized by a combination of microchemical techniques, including surface plasmon resonance, tandem mass spectrometry, and state-of-the-art lectinomic and peptidomic chemistry.