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 含有HIV前病毒基因组完整拷贝的细胞的储存库。这些细胞之所以持续存在，部分原因是 前病毒保持一种潜伏状态，逃避免疫反应和病毒的细胞病变效应。实现以下目标的方法 通过重新激活潜伏细胞来清除蓄水池的证据表明，潜伏期在体内是可以逆转的， 然而，仅靠潜伏期的逆转并不足以减少潜在的储集层。现在，努力已经到位。 将潜伏期重新激活与根除受感染细胞的策略相结合-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)。另一个关键因素是Nef，一种辅助蛋白质 由HIV编码，这是我们提议的研究的主要重点。因为Nef抑制抗心磷脂的活性 艾滋病毒CTL是这种蛋白的有效抑制物，将有助于实现艾滋病毒的根除。的主要功能之一 NEF是主要组织相容性复合体I类编码蛋白(MHC-I)的下调，掩蔽 来自宿主免疫系统的感染，并使感染艾滋病毒的细胞持续存在。联合治疗与 潜伏期拮抗剂和Nef抑制剂可以协同作用清除艾滋病毒宿主。到目前为止，还没有Nef抑制剂 已经在Nef存在的情况下实现了MHC-I的有效恢复。我们开发了一种高通量的检测方法来 确定Nef介导的MHC-I下调的抑制剂，并筛选天然产物提取物(NPE) 获得了10次具有Nef抑制活性的Hit。我们鉴定了一些相关的化合物，作为活性物质 其中几种提取物中的成分。纯天然产物能有效恢复MHC的表面表达。 I在Nef存在的情况下，不抑制其其他活性。我们测试了一些结构上相关的 这一天然产物家族中的化合物，并鉴定出两种在人类中具有PM到NM活性的化合物 原生细胞。基于这一强劲的初步数据，我们认为进一步增强Nef的抑制活性 这些分子通过类比开发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非靶标活性的抗Nef效应来优化这些抑制剂 确定可供开发的候选先导药物，以及(B)确定抑制剂的作用机制 中断Nef介导的MHC-I降调，以便更智能地进行优化。这些 将通过以下具体目标实现目标：(1)进行牵头化合物结构优化 以提高药用性能。(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和(3)确定天然产物衍生的抑制剂的机理 干扰Nef介导的MHC-I下调，包括靶标识别和生化研究。由此 工作中，我们希望产生一类新的化合物，这些化合物是高效的Nef抑制剂，具有很高的药效 潜力。在目前的CART鸡尾酒中添加Nef抑制化合物有望增强免疫力 清除病毒库，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.