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

描述(申请人提供)：NEF是一种HIV-1辅助因子，对HIV感染细胞的病毒致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着HIV治疗的一种新方法。在这项响应PAR-12-058(高通量筛选检测请求发现化学探针)的申请中，我们建议使用基于细胞的方法进行Nef二聚化，以发现Nef功能的化学探针，这些探针也可能代表新的抗逆转录病毒药物线索。在本实验中，Nef与YFP的非荧光互补片段融合，并在同一细胞群中共表达。Nef的二聚化导致YFP片段的并置和荧光团的重组，这一过程被称为双分子荧光互补(BIFC)。为了简化自动化检测，两个Nef-YFP融合蛋白加上一个MRFP报告从单一载体表达，该载体驱动单一转录本中所有三种蛋白质的同等翻译。验证研究表明，表达野生型Nef的细胞与二聚化缺陷Nef突变体的Nef-BIFC/MRFP比率非常清楚地分开，Z因子始终在0.6-0.7范围内。对约3,000种化合物的全自动中试筛选确定了几种活性结构，它们可以在低微摩尔范围内重复阻止Nef二聚反应。二次分析表明，这些化合物直接与Nef结合，并抑制其与HIV感染性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，并评估它们对这种关键的HIV毒力因子的活性，具体目标如下：1.使用基于细胞的BIFC方法筛选大量不同的化学集合以寻找Nef二聚的抑制剂。我们将实施Nef-BIFC检测，从两个互补的文库中全自动筛选60,000个离散化合物。其中包括ChemDiv的10,000种非肽多肽仿制药，旨在阻断不同的蛋白质-蛋白质接口，以及从ChemBridge 410,000化合物核心库中挑选的50,000种结构，以最大限度地提高结构多样性，同时增强预测的物理化学性质。2.进行后续实验，寻找选择性最强、细胞活性最强的Nef二聚抑制剂，并探讨其作用机制。这一目标将确定HIV-1Nef功能最有效和最具选择性的化学探针。第一组检测将确定每个HIT化合物是否直接与Nef蛋白相互作用并影响其在体外和电子计算机中的自结合，而第二组检测将探索基于细胞的系统中的抗逆转录病毒活性和机制。该项目的成功完成将使我们更接近我们的长期目标，即发现选择性和有效地抑制尽可能多的HIV-1 Nef功能的细胞活性化合物。

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.