自2019年底COVID-19疫情爆发以来，全球已有超7亿人感染，700多万人死亡。尽管疫苗接种在遏制病毒传播和降低重症率方面展现出显著成效，然而病毒快速变异、疫苗保护效力随时间递减以及特定人群免疫应答不足等挑战的持续存在，凸显了抗病毒药物在疫情防控体系中的不可替代性。开发安全有效的新药不仅能提供更多治疗选择，减少重症和死亡风险，还能为无法接种疫苗或免疫脆弱人群提供额外保护。因此，深入研究抗新冠病毒药物不仅是应对当前疫情的迫切需求，更是为未来可能的公共卫生危机做好科学准备。“老药新用”策略因其已知的药代动力学特性和安全性数据，为突发传染病防控提供了快速响应方案，而基于先导化合物开发的活性衍生物，则可进一步突破原药在药效、选择性或耐药性等方面的局限，实现治疗方案的持续优化。

本研究采用了基于细胞的抗病毒筛选策略，对经典抗菌药物氯福克酚 (Clofoctol) 的衍生物和多活性天然产物石蒜碱 (Lycorine) 的衍生物进行逐一筛选，并利用RT-qPCR、RNA-seq、WB、TEM、DARTS和CETSA等实验，对其中两个优势先导化合物的作用机制进行了研究。

研究从134个氯福克酚衍生物和14个石蒜碱衍生物中发现了22个具有显著抗SARS-CoV-2活性 (即半数效应浓度EC₅₀≤10 μM) 的先导化合物，分别为氯福克酚衍生物 (1-10) 及石蒜碱衍生物 (3, 5-13, 15, 16)。对氯福克酚衍生物10的研究表明，其对SARS-CoV-2原型株及四个变异株 (Alpha、Beta、Delta和Omicron BA.1) 均具有显著的抗病毒活性。化合物10的EC₅₀在0.5至2.2 μM之间，半数细胞毒性浓度 (CC₅₀) 为44.1 μM，选择性指数 (SI) 达到20.0到88.2之间，较原药氯福克酚提升4.4-27.6倍。对石蒜碱衍生物7的研究表明，其对SARS-CoV-2原型株及四个变异株均具有显著的抗病毒活性。其EC₅₀在0.57至1.15 μM之间，CC₅₀为79.81 μM，原始株的SI值达到109.3，较原药石蒜碱提升1.4倍。进一步对氯福克酚衍生物10与石蒜碱衍生物7的体内活性进行评价，发现二者均能有效降低金黄地鼠肺部的病毒载量，并缓解肺部炎症。

进一步的机制研究表明，化合物10显著抑制了多个脂代谢相关基因的表达，尤其是脂肪酸合成酶 (FASN) 、长链脂酰辅酶A合成酶4 (ACSL4) 和硬脂酰辅酶A去饱和酶1 (SCD1)。TEM观察显示，化合物10处理后的细胞中，病毒复制中心 (ROs) 的形态发生显著改变：其中双层膜囊泡 (DMVs) 数量减少且异常增大，并产生了更多的异常复制细胞器 (AROs)，表明化合物10通过干扰脂肪酸代谢并影响了DMV的形成和稳定性，进而抑制了病毒RNA的复制。此外，本研究还通过脂肪酸补充实验进一步验证了化合物10的抗病毒机制。在无脂肪酸的培养基中，化合物10的抗病毒效果显著减弱，但依然存在。而外源性补充棕榈酸 (PA) 、花生四烯酸 (ARA) 和油酸 (OA) 等脂肪酸则部分逆转了化合物10的抗病毒作用，这一结果显示化合物10对脂代谢相关基因的抑制是其发挥抗病毒活性的重要机制。此外，DARTS实验联合CETSA与CHX追踪实验表明，化合物10能够通过靶向并降解核受体共激活因子1 (NCOA1)，实现对过氧化物酶体增殖物激活受体γ (PPARγ) 的抑制及对下游FASN、ACSL4和SCD1的表达调控，从而发挥抗病毒作用，具有作为抗SARS-CoV-2候选药物的潜力。

此外，对石蒜碱衍生物7的机制研究表明，化合物7通过特异性结合宿主抗病毒蛋白ZAP-S (关键位点E111/E115/F549) ，增强其稳定性，进而抑制病毒复制必需的-1 PRF过程。该作用通过阻断ORF1a到ORF1b的移码，减少病毒复制核心酶 (如nsp12) 的生成。这些发现表明，化合物7通过靶向ZAP-S调控的-1 PRF通路发挥广谱抗病毒作用，具备良好开发前景。

本研究通过系统筛选，不仅验证了宿主脂代谢和移码调控机制在SARS-CoV-2复制中的重要作用，还成功鉴定出两种靶向不同宿主通路的抗病毒候选药物，为COVID-19治疗提供了有潜力的先导化合物。

Since the outbreak of the COVID-19 pandemic at the end of 2019, more than 700 million people have been infected globally, with over 7 million deaths. Although vaccination has demonstrated significant efficacy in curbing viral transmission and reducing severe disease rates, persistent challenges—such as rapid viral mutation, waning vaccine protection over time, and inadequate immune responses in specific populations—highlight the indispensable role of antiviral drugs in pandemic control systems. Developing safe and effective new drugs not only provides additional treatment options to mitigate severe outcomes and mortality but also offers supplemental protection for vaccine-ineligible or immunocompromised individuals. Thus, in-depth research on anti-SARS-CoV-2 therapeutics addresses both an urgent need for the current pandemic and a scientific preparedness strategy for future public health crises. The "Drug Repurposing" approach, leveraging known pharmacokinetic profiles and safety data, enables rapid responses to emerging infectious diseases, while active derivatives developed from lead compounds can overcome limitations of parent drugs in efficacy, selectivity, or resistance, facilitating continuous therapeutic optimization.

This study employed a cell-based antiviral screening platform to evaluate derivatives of the classic antibacterial agent clofoctol and the multi-active natural product lycorine. Two promising lead compounds were further characterized through comprehensive mechanistic studies employing RT-qPCR, RNA-seq, western blotting, transmission electron microscopy (TEM), drug affinity responsive target stability (DARTS), and cellular thermal shift assay (CETSA).

From 134 clofoctol derivatives and 14 lycorine derivatives, we identified 22 lead compounds exhibiting potent anti-SARS-CoV-2 activity (EC₅₀ ≤10 μM), including clofoctol derivatives (1-10) and lycorine derivatives (3, 5-13, 15, and 16). Clofoctol derivative 10 demonstrated broad-spectrum activity against SARS-CoV-2 prototype strain and four variants (Alpha, Beta, Delta, and Omicron BA.1), with EC₅₀ values ranging from 0.5 to 2.2 μM. The compound also showed favorable cytotoxicity profiles (CC₅₀ = 44.1 μM), resulting in significantly enhanced selectivity indices (SI = 20.0-88.2) that represented a remarkable 4.4- to 27.6-fold improvement compared to the clofoctol. Parallel studies revealed lycorine derivative 7 showed consistent antiviral potency against all tested strains (EC₅₀ from 0.57 to 1.15 μM, and CC₅₀ = 79.81 μM), achieving an SI of 109.3 against the prototype strain (1.4-fold enhancement versus lycorine). Furthermore, both compounds significantly reduced lung viral loads and attenuated pulmonary inflammation in golden Syrian hamster models.

Mechanistic studies revealed that compound 10 substantially downregulated lipid metabolism-related genes, particularly FASN, ACSL4, and SCD1. TEM analysis demonstrated the morphology of viral replication organelles (ROs) was significantly altered: the number of double-membrane vesicles (DMVs) decreased while their size became abnormally enlarged, accompanied by an increase in aberrant replication organelles (AROs). These findings indicate that compound 10 interferes with fatty acid metabolism, disrupts DMVs formation and stability, and thereby suppresses viral RNA replication. Fatty acid supplementation experiments confirmed this mechanism: the antiviral effect of compound 10 diminished in fatty acid-free medium but persisted, while exogenous palmitic acid (PA), arachidonic acid (ARA), and oleic acid (OA) partially reversed its activity. DARTS/CETSA combined with CHX chase assays identified NCOA1 as the direct target, through which compound 10 degrades NCOA1 to suppress PPARγ-mediated expression of FASN/ACSL4/SCD1, establishing its candidacy as a clinical anti-SARS-CoV-2 agent.

For compound 7, mechanistic studies revealed its specific binding to host antiviral protein ZAP-S (key residues E111/E115/F549), enhancing ZAP-S stability to inhibit viral -1 programmed ribosomal frameshifting (-1 PRF). This blockade prevents ORF1a-to-ORF1b frameshifting, reducing core replicase (e.g., nsp12) production. These findings position compound 7 as a promising broad-spectrum antiviral candidate acting through ZAP-S-mediated -1 PRF inhibition.

This study employed systematic screening to validate the critical roles of host lipid metabolism and the -1 PRF regulatory mechanism in SARS-CoV-2 replication. We successfully identified two promising antiviral candidates targeting distinct host pathways, providing potential lead compounds for COVID-19 drug development.