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

目前的联合抗逆转录病毒疗法（cART）可以抑制血液中的病毒水平，但不能根除 HIV前病毒基因组整合拷贝的细胞库。这些细胞之所以能存活，部分原因是 前病毒保持一种逃避免疫应答和病毒致细胞病变作用的潜伏状态。方法来 通过重新激活潜伏细胞清除贮库提供了潜伏期可以在体内逆转的证据， 然而，仅逆转潜伏期还不足以减少潜伏性水库。目前， 将潜伏期再激活与根除感染细胞的策略结合起来-例如通过设计和激活 更有效的抗HIV细胞毒性T淋巴细胞（CTL）。另一个关键角色是Nef，一种辅助蛋白 由HIV编码，这是我们拟议研究的主要重点。因为Nef抑制了抗- HIV CTL，这种蛋白质的有效抑制剂将有助于实现HIV根除。的主要功能之一 Nef是下调主要组织相容性复合体I类编码蛋白（MHC-I）， 从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。联合治疗 潜伏拮抗剂加Nef抑制剂可以协同作用清除HIV储库。迄今为止，没有Nef抑制剂 已经在Nef存在下实现了MHC-I的有效恢复。我们开发了一种高通量检测方法， 鉴定Nef介导的MHC-I下调的抑制剂，并筛选天然产物提取物（NPE） 产生10个具有Nef抑制活性的命中。我们鉴定了一些相关的化合物， 这些提取物中的几种成分。纯天然产品有效地恢复MHC-1的表面表达， 我在Nef的存在，而不抑制其其他活动。我们测试了一些结构相关的 该天然产物家族中的化合物，并鉴定了两种在人体中具有pM至nM效力的化合物， 主要细胞基于这一强有力的初步数据，我们认为进一步增强Nef抑制活性 通过类似物的开发，这些分子将产生一种安全的抗Nef药物。因此，我们计划（A） 通过进一步分离和表征抗Nef效应与脱靶活性来优化这些抑制剂， 鉴定用于开发的先导候选药物和（B）确定抑制剂 干扰Nef介导的MHC-I下调，从而可以更智能地进行优化。这些 目标将通过以下具体目标来实现：（1）进行先导化合物结构优化 以改善药物性质。(2)对所有有希望的类似物进行详细的功能分析， 确定理想的先导化合物;（3）确定天然产物衍生的抑制剂 破坏Nef介导的MHC-I下调，包括靶标鉴定和生物化学研究。从这个 工作，我们希望产生一类新的化合物，是有效的Nef抑制剂，具有高的药物活性。 潜力将Nef抑制性化合物添加到当前的cART鸡尾酒中预期可增强免疫应答。 清除病毒储存库，导致长期难以治愈的艾滋病毒。

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.