The HIV-1 Nef accessory factor is critical to the viral life cycle in vivo and promotes immune escape of infected cells via downregulation of cell-surface MHC-I. Previously, we discovered small molecules that bind directly to Nef and block many of its functions, including enhancement of viral infectivity and replication in T cell lines. These compounds also restore cell-surface MHC-I expression in HIV-infected CD4 T cells from AIDS patients, enabling recognition and killing by autologous cytotoxic T lymphocytes (CTLs). In this study, we describe the synthesis and evaluation of a diverse set of analogs based on the original hydroxypyrazole Nef inhibitor core. All analogs were screened for the interaction with recombinant HIV-1 Nef by surface plasmon resonance (SPR) and for antiretroviral activity in TZM-bl reporter cells infected with HIV-1. Active analogs were ranked on the basis of an activity score that integrates three aspects of the SPR data (affinity, residence time, and extent of binding) with antiretroviral activity. The top scoring compounds bound tightly to Nef by SPR, with KD values in the low nM to pM range, and displayed very slow dissociation from their Nef target. These analogs also suppressed HIV-1 replication in donor peripheral blood mononuclear cells (PBMCs) with IC50 values in the 1-10 nM range without cytotoxicity, inhibited Nef-mediated IL-2-inducible tyrosine kinase (Itk) and hematopoietic cell kinase (Hck) activation, and rescued MHC-I downregulation in a Nef-transfected T cell line. The development of Nef inhibitors based on the structure-activity relationships defined here has promise as a new approach to antiretroviral therapy that includes a path to eradication of HIV-infected cells via the adaptive immune response.

中文翻译：

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紧密结合的羟基吡唑HIV-1 Nef抑制剂抑制供体单核细胞中的病毒复制，并逆转Nef介导的MHC-1下调。

HIV-1 Nef辅助因子对于体内病毒生命周期至关重要，并通过下调细胞表面MHC-1来促进被感染细胞的免疫逃逸。以前，我们发现了与Nef直接结合并阻断其许多功能的小分子，包括增强病毒感染性和在T细胞系中复制。这些化合物还可以恢复来自AIDS患者的HIV感染的CD4 T细胞中细胞表面MHC-1的表达，从而能够被自体细胞毒性T淋巴细胞（CTL）识别和杀死。在这项研究中，我们描述了基于原始羟基吡唑Nef抑制剂核心的多种类似物的合成和评估。通过表面等离振子共振（SPR）筛选所有类似物与重组HIV-1 Nef的相互作用，并筛选感染HIV-1的TZM-b1报告基因细胞中的抗逆转录病毒活性。根据将SPR数据的三个方面（亲和力，停留时间和结合程度）与抗逆转录病毒活性相结合的活性评分对活性类似物进行排名。得分最高的化合物通过SPR与Nef紧密结合，KD值在nM至pM的较低范围内，并且显示出与Nef靶标非常缓慢的解离。这些类似物还抑制了供体外周血单核细胞（PBMC）中的HIV-1复制，IC50值在1-10 nM范围内，而没有细胞毒性，抑制了Nef介导的IL-2诱导的酪氨酸激酶（Itk）和造血细胞激酶（Hck ）激活，并挽救了Nef转染的T细胞系中MHC-1的下调。