Theta-defensins Novel HIV-1 Uptake Inhibitors

描述(申请人提供)：我们建议研究防御素在宿主抵抗人类HIV-1和非人类灵长类动物相关逆转录病毒中的免疫机制。我们的长期目标是：1)确定与灵长类动物HTV-1感染发病相关的先天免疫系统分子。2)表征theta和α-防御素的碳水化合物结合专一性，并将这一特性与其抗HIV-1活性相关联。3)为将来的体内研究确定有效的theta防御素，以评估它们的治疗有效性。在这项拨款中，没有建议进行体内研究。Theta-Defensins是由东半球猕猴和其他一些非人类灵长类动物的白细胞表达的环状十八肽。它们识别某些碳水化合物结构(多糖)的能力允许一些9-防御素以高亲和力结合HIV-1和CD4的gp120，并阻止HIV-1的R5和X4毒株进入原本敏感的人类细胞。人类基因组中至少存在60-防御素基因，人的骨髓、胸腺和脾中存在theta-防御素基因。然而，人类theta防御素基因及其mRNA包含一个过早终止密码子，人类细胞不产生theta防御素多肽。逆转录周期蛋白1-3是一种合成肽，代表人类在沉默突变(过早终止密码子)缺失的情况下可能产生的theta防御素。Retroclin-2对代表A、B、C和CRF01_AE亚型的HIV-1初级分离株有效，这些亚型导致全球大多数HIV-1感染。由于逆转录周期素对阴道乳杆菌无细胞毒性和无害，它们为未来的药物开发提供了有趣的先导化合物。这项提案中包含的研究旨在提供一个知识平台，促进未来治疗性theta防御素的发展。具体目标是：1)。合成新的theta防御素，包括基于非人类灵长类动物theta防御素基因序列的多肽和人类逆转录周期蛋白的类似物，并测试它们对HIV-1的活性和细胞毒性。2)。鉴定与Theta-Defensins结合的灵长类糖和寡糖，并将结合与其抗逆转录病毒特性相关联。目标1将通过固相多肽合成和使用外周血单核细胞和指示细胞系的体外保护分析来实现。具体目标2将通过微化学技术的组合来实现，包括表面等离子激元共振、串联质谱学以及最先进的凝集素和多肽化学。

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.