High-throughput Discovery of Chemical Probes for HIV-1 Nef Function

描述（由申请人提供）：Nef是HIV-1辅助因子，对HIV感染细胞的病毒发病和免疫逃逸至关重要。许多Nef功能需要自结合（二聚化），干扰Nef二聚化的小分子可能代表HIV治疗的新途径。在这个响应PAR-12-058（高通量筛选发现化学探针的检测方法征集）的应用程序中，我们建议使用基于细胞的Nef二聚化检测来发现Nef功能的化学探针，这也可能代表新的抗逆转录病毒药物线索。在这个实验中，Nef与非荧光的、互补的YFP片段融合，并在相同的细胞群中共表达。Nef的二聚化导致YFP片段并置和荧光团的重建，这一过程被称为双分子荧光互补（BiFC）。为了简化自动化分析，两个Nef-YFP融合蛋白加上一个mRFP报告蛋白在单个载体上表达，从而驱动来自单个转录物的所有三种蛋白的等效翻译。验证研究显示，表达野生型Nef与二聚化缺陷Nef突变体的细胞产生的Nef- bifc /mRFP比率非常明显地分开，z因子一致在0.6-0.7范围内。约3000种化合物的全自动先导筛选鉴定出几种活性结构，可在低微摩尔范围内重复阻断Nef二聚化。二级分析表明，这些化合物直接结合Nef并抑制其与HIV感染和受体下调相关的功能。在这里，我们建议筛选大量不同的化合物，并评估它们对这一关键的HIV毒力因子的活性，具体目的如下：使用基于细胞的bbc方法筛选大量不同的Nef二聚化抑制剂的化学集合。我们将实施Nef-BiFC检测，全自动筛选来自两个互补文库的60,000个独立化合物。其中包括来自ChemDiv的10,000种非肽类肽模拟物，旨在阻断多种蛋白质界面，以及从ChemBridge的410,000种化合物核心文库中选择的50,000种结构，以最大限度地提高结构多样性，同时提高预测的物理化学性质。2. 进行后续分析以确定最具选择性和最有效的Nef二聚化细胞活性抑制剂，并探索其作用机制。这一目标将鉴定出最有效和选择性的HIV-1 Nef功能的化学探针。第一组试验将确定每种命中化合物是否直接与Nef蛋白相互作用，并在体外和计算机上影响其自我结合，而第二组试验将探索基于细胞的系统中的抗逆转录病毒活性和机制。这个项目的成功完成将使我们更接近我们的长期目标，即发现细胞活性化合物，选择性地和有效地抑制尽可能多的HIV-1 Nef功能。

DESCRIPTION (provided by applicant): Nef is an HIV-1 accessory factor essential for viral pathogenesis and immune escape of HIV- infected cells. Many Nef functions require self-association (dimerization), and small molecules that interfere with Nef dimerization may represent a new approach to HIV therapeutics. In this application, which is responsive to PAR-12-058 (Solicitation of Assays for High Throughput Screening to Discover Chemical Probes), we propose to use a cell-based assay for Nef dimerization to discover chemical probes for Nef function that may also represent new antiretroviral drug leads. In this assay, Nef is fused to non-fluorescent, complementary fragments of YFP and co-expressed in the same cell population. Dimerization of Nef results in juxtaposition of the YFP fragments and reconstitution of the fluorophore, a process known as bimolecular fluorescence complementation (BiFC). To simplify the assay for automation, the two Nef-YFP fusion proteins plus an mRFP reporter are expressed from a single vector that drives equivalent translation of all three proteins from a single transcript. Validation studies revealed that Nef-BiFC/mRFP ratios resulting from cells expressing wild-type Nef vs. a dimerization-defective Nef mutant were very clearly separated, with Z-factors consistently in the 0.6-0.7 range. Fully automated pilot screens of ~3,000 compounds identified several active structures that reproducibly blocked Nef dimerization in the low micromolar range. Secondary assays showed that these compounds bind directly to Nef and inhibit its functions related to HIV infectivity and receptor down- regulation. Here we propose to screen a large collection of diverse chemical compounds and evaluate their activity against this critical HIV virulence factor with the following Specific Aims: 1. Screen a large, diverse chemical collection for inhibitors of Nef dimerization using a cell-based BiFC approach. We will implement the Nef-BiFC assay for fully automated screening of 60,000 discrete compounds from two complementary libraries. These include 10,000 non-peptide peptidomimetics from ChemDiv designed to block diverse protein-protein interfaces and 50,000 structures selected from the ChemBridge 410,000 compound core library to maximize structural diversity while enhancing predicted physiochemical properties. 2. Perform follow-up assays to identify the most selective and potent cell-active inhibitors of Nef dimerization, and explore thei mechanism of action. This Aim will identify the most potent and selective chemical probes of HIV-1 Nef function. The first group of assays will determine whether or not each hit compound interacts directly with the Nef protein and impacts its self-association in vitro and in silico, whle the second assay set will explore antiretroviral activity and mechanism in cell-based systems. Successful completion of this project will bring us closer to our long-term goal of discovering cell-active compounds that selectively and potently inhibit as many HIV-1 Nef functions as possible.