Development of natural product inhibitors of Nef for clearance of HIV reservoirs

开发 Nef 天然产物抑制剂以清除 HIV 病毒库

• 批准号: 10204722
• 负责人: Kathleen L. Collins
• 金额: $ 69.86万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10204722
• 关键词: Affect; Back; Binding; Biochemical; Biological Assay; Blood; Cells; Combined Modality Therapy; Complex; Cytotoxic T-Lymphocytes; Data; Development; Dose; Down-Regulation; Family; Genome; Goals; HIV; HIV-1; Human; Immune response; Immune system; Infection; Intelligence; Lead; Lysosomes; Macrocyclic Compounds; Major Histocompatibility Complex; Masks; Mediating; Modification; Molecular; Natural Products; Outcome; Pathway interactions; Pharmaceutical Preparations; Pharmacologic Substance; Primary Cell Cultures; Property; Proteins; Proviruses; Public Health; Research; Route; Solubility; Structure; Structure-Activity Relationship; Surface; T-Lymphocyte; Testing; Toxic effect; United States; Variant; Viral; Viral Cytopathogenic Effect; Viral reservoir; Viremia; Virus; Work; affinity labeling; analog; antiretroviral therapy; aqueous; base; chemical function; chemical stability; design; drug candidate; experimental study; high throughput screening; immune clearance; improved; in vivo; inhibitor/antagonist; latent HIV reservoir; lipophilicity; novel; pandemic disease; protein transport; reactivation from latency; restoration; scaffold; trafficking; uptake; vacuolar H+-ATPase

Current combined antiretroviral therapies (cART) suppress viral levels in the blood but do not eradicate reservoirs of cells harboring integrated copies of HIV proviral genomes. These cells persist in part because the provirus maintains a latent state that evades the immune response and viral cytopathic effect. Approaches to clear reservoirs by reactivating latent cells have provided evidence that latency can be reversed in vivo, however reversal of latency alone has not been sufficient to reduce latent reservoirs. Efforts are now in place to couple latency reactivation with strategies to eradicate the infected cells – such as by design and activation of more efficacious anti-HIV cytotoxic T lymphocytes (CTLs). Another key player is Nef, an accessory protein encoded by HIV, which is a primary focus of our proposed research. Because Nef inhibits the activity of anti- HIV CTLs, a potent inhibitor of this protein would help achieve HIV eradication. One of the main functions of Nef is the down-modulation of major histocompatibility complex class I encoded proteins (MHC-I), masking infection from the host immune system and allowing HIV infected cells to persist. Combination therapy with latency antagonists plus Nef inhibitors could act synergistically to clear HIV reservoirs. To date, no Nef inhibitor has achieved potent restoration of MHC-I in the presence of Nef. We developed a high-throughput assay to identify inhibitors of Nef-mediated MHC-I downregulation, and a screen of natural product extracts (NPEs) yielded 10 hits with Nef inhibitory activity. We identified a number of related compounds, as the active component in several of these extracts. The pure natural products potently restore surface expression of MHC- I in the presence of Nef without inhibiting its other activities. We tested a number of structurally related compounds within this natural product family and identified two that possess pM to nM potencies in human primary cells. Based on this strong preliminary data, we believe that further enhancing the Nef inhibitory activity of these molecules through analog development will yield a safe anti-Nef drug. Therefore, we plan to (A) optimize these inhibitors by further separating and characterizing the anti-Nef effect from off-target activities to identify a lead drug candidate for development and (B) determine the mechanism by which the inhibitor disrupts Nef-mediated MHC-I downmodulation so that optimization can be conducted more intelligently. These goals will be achieved through the following specific aims: (1) Conduct lead compound structural optimization to improve pharmaceutical properties. (2) Perform a detailed functional analysis of all promising analogs to identify ideal lead compounds and (3) Determine the mechanism by which the natural product-derived inhibitor disrupts Nef-mediated MHC-I downmodulation including target identification and biochemical studies. From this work, we expect to generate a new class of compounds that are potent Nef inhibitors with high pharmaceutical potential. The addition of Nef inhibitory compounds to current cART cocktails is expected to enhance immune clearance of viral reservoirs, leading to the long-elusive HIV cure.

当前的抗逆转录病毒疗法（CART）抑制血液中的病毒水平，但不根除 具有HIV病毒基因组综合副本的细胞储层。这些细胞持续存在，部分原因是 病毒保持了潜在的状态，可逃避免疫反应和病毒细胞病变作用。方法 通过重新激活潜在细胞，清晰的储层提供了证据，表明可以在体内逆转潜伏期， 但是，仅延迟的逆转还不足以减少潜在的储层。现在已经做出了努力 将潜伏期重新激活与消除感染细胞的策略（例如设计和激活） 更有效的抗HIV细胞毒性T淋巴细胞（CTL）的含量。另一个关键参与者是NEF，一种配件蛋白 由艾滋病毒编码，这是我们提出的研究的主要重点。因为NEF抑制了抗抗活性 HIV CTLS，该蛋白质的潜在抑制剂将有助于消除HIV。主要功能之一 NEF是主要的组织相容性复合物I类编码蛋白（MHC-I）的下调 宿主免疫系统的感染并允许艾滋病毒感染的细胞持续存在。结合疗法与 潜伏的拮抗剂和NEF抑制剂可以协同起作用以清除HIV储藏。迄今为止，没有NEF抑制剂 在NEF存在下，已实现了MHC-I的潜在恢复。我们开发了一个高通量测定法 鉴定NEF介导的MHC-1下调的抑制剂，以及天然产物提取物（NPE）的筛选 具有NEF抑制活性的10次命中。我们确定了许多相关化合物，为活动 这些提取物中的几个组成部分。纯天然产物可能会恢复MHC-的表面表达 我在NEF面前没有抑制其其他活动。我们测试了许多与结构相关的 该天然产品家族中的化合物，并确定了两个在人类中具有NM电位的PM的化合物 原代细胞。基于这些强大的初步数据，我们认为进一步增强了NEF抑制活性 通过模拟发展这些分子中的这些分子将产生安全的抗Nef药物。因此，我们计划（a） 通过进一步分离和表征抗靶向活动的抗Nef效应来优化这些抑制剂 识别出发育的主要药物候选药物，（b）确定抑制剂的机制 破坏NEF介导的MHC-I下调，以便更聪明地进行优化。这些 将通过以下特定目的实现目标：（1）进行铅复合结构优化 改善药物特性。 （2）对所有有希望的类似物进行详细的功能分析 确定理想的铅化合物，（3）确定自然产物衍生抑制剂的机制 破坏NEF介导的MHC-I下调，包括靶标识别和生化研究。由此 工作，我们希望产生一类新的化合物，这些化合物是具有高药物的潜在NEF抑制剂 潜在的。在当前购物车鸡尾酒中添加NEF抑制性化合物有望增强免疫力 病毒储量的清除，导致长期难以抗衡的HIV治疗。