Targeting a Human Acyltransferase for Broad-Spectrum Antivirals

摘要 SARS CoV-2和相关冠状病毒劫持宿主蛋白以促进细胞进入、复制和 免疫抑制靶向被劫持的宿主蛋白是一种有吸引力的发展策略 因为相同的蛋白质通常被多种抗病毒药物靶向， 病毒。最近，一项全球SARS-CoV-2/人类蛋白质/蛋白质相互作用分析显示， 这样的目标：人酰基转移酶DHHC 5，催化蛋白质的棕榈酰化， 调节内吞途径中的运输，显示与SARS-CoV-2 Spike相互作用 蛋白这一发现本身令人兴奋，但鉴于多条证据表明， DHHC 5在质膜定位蛋白质方面的作用被各种病原体劫持， 获得进入细胞，这是令人信服的理由，发展DHHC 5抑制剂测试， 抗病毒药 我们建议开发选择性DHHC 5抑制剂作为先导分子，在动物模型中进行测试。 冠状病毒感染。目前，针对这一目标的小分子药物发现工作的工具 缺乏：关于DHHC家族成员的结构信息非常少，没有可靠的HTS 测定。本R21提案的目标是开发和验证发现所需的工具 DHHC5抑制剂并启动先导发现计划为了解决这个问题，我们将开发和 验证一个强大的，定量的高通量DHHC 5酶测定的基础上耦合 荧光偏振免疫测定法（FPIA），将作为筛选和 进行电极导线碰撞/SAR研究。我们将使用该测定来筛选多样性文库，验证 命中，并根据理化性质、效力、选择性、机制 行动和功能活动。此外，我们将在2015年生产DHHC 5的共晶结构。 与经过验证的命中分子复合。这些目标的完成将确立技术可行性 为了追求结构驱动的设计方法来发现靶向DHHC 5的先导分子， 这可能最终导致开发具有广谱活性的抗病毒药物， SARS-CoV-2和未来出现的冠状病毒病原体。

Abstract SARS CoV-2 and related coronaviruses hijack host proteins to facilitate cell entry, replication and immune suppression. Targeting hijacked host proteins is an attractive strategy for development of broad-spectrum antiviral drugs because the same proteins are often targeted by multiple viruses. A global SARS-CoV-2/human protein/protein interaction analysis recently revealed one such target: the human acyltransferase DHHC5, which catalyzes palmitoylation of proteins to regulate trafficking in the endocytic pathway, was shown to interact with the SARS-CoV-2 Spike protein. This finding was exciting in itself, but in light of multiple lines of evidence indicating that DHHC5’s role in localizing proteins at the plasma membrane is hijacked by diverse pathogens to gain entry into the cell, it is compelling rationale for developing DHHC5 inhibitors to test as antivirals. We propose to develop selective DHHC5 inhibitors as lead molecules to test in animal models of coronavirus infection. Currently, the tools for a small molecule drug discovery effort for this target are lacking: there is very little structural information on DHHC family members and no robust HTS assays. The goals of this R21 proposal are to develop and validate the tools needed for discovery of DHHC5 inhibitors and initiate a lead discovery program. To address this, we will develop and validate a robust, quantitative high throughput DHHC5 enzymatic assay based on a coupled fluorescence polarization immunoassay (FPIA) that will serve as a critical tool for screening and performing hit-to-lead/SAR studies. We will use the assay to screen a diversity library, validate the hits and prioritize them based on physicochemical properties, potency, selectivity, mechanism of action and functional activity. Additionally, we will produce a cocrystal structure of DHHC5 in complex with a validated hit molecule. Completion of these aims will establish technical feasibility for pursuing a structure-driven design approach to discover lead molecules targeting DHHC5, which could ultimately result in the development of antivirals with broad spectrum activity against SARS-CoV-2 and coronavirus pathogens that emerge in the future.