Broad-Spectrum Inhibition of SARS-CoV-2 Variants by Dibutyl Phthalate Through Allosteric Disruption of Spike-ACE2 Interface

Jiafan Chen^1,2Dekuan Guo³Xiaoxuan Guo^1,2Lijun Zhao^1,2Geng Li^3,4Helu Liu^1,2Shaobo Wang^5,6*Zizhao Lao^1,2,4*Meiling Zhu^1,2*

• ¹Shenzhen Clinical College of Integrated Chinese and Western Medicine, Guangzhou University of Chinese Medicine, Shenzhen, China
• ²Shenzhen Traditional Chinese Medicine Hospital, Shenzhen ，Guangdong, China
• ³State Key Laboratory of Traditional Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou, China
• ⁴Chinese Medicine Guangdong Laboratory, Guangdong Hengqin, China
• ⁵Guangzhou National Laboratory, Department of Basic Research, Guangzhou International Bio-Island, Guangzhou, China
• ⁶The First Affiliated Hospital of Guangzhou Medical University, State Key Laboratory of Respiratory Diease, Guangzhou Laboratory Clinical Base, Guangzhou Medical University, Guangzhou, China

SARS-CoV-2 continues to exhibit persistent low-level circulation punctuated by localized clusters of outbreaks, driven by its high mutation rate. These mutations confer immune evasion and enhanced transmissibility, escalating the risk of reinfection even among vaccinated individuals. Emerging evidence highlights a heightened susceptibility to Long COVID (LC)—characterized by prolonged fatigue, cognitive dysfunction, and reduced exercise tolerance—following reinfection. Traditional Chinese Medicine (TCM) volatile oils, rich in bioactive compounds, have long been utilized for antiviral therapies. Their volatility and biocompatibility enable efficient nasal delivery, positioning them as promising agents for targeting upper respiratory tract infections. This study investigates the inhibitory effects of TCM volatile oil-derived compounds on the interaction between the SARS-CoV-2 S protein (Spike) and its host receptor ACE2. Through virtual screening of 47 structurally diverse TCM volatile compounds, we identified ten potential inhibitors. Among them, dibutyl phthalate (DBP) exhibited the strongest activity, suppressing Spike-mediated membrane fusion and pseudovirus infection. Mechanistically, DBP disrupted the binding of the Spike S1 receptor-binding domain (RBD) to ACE2 without altering ACE2 enzymatic activity. Molecular docking predicted that DBP binds to the RBD/ACE2 interface, and this mechanism was definitively established by site-directed mutagenesis, which validated Tyr453 and Tyr495 as essential residues for DBP's function. Surface plasmon resonance (SPR) confirmed DBP competitively inhibits Spike trimer-ACE2 binding (from KD = 8.28 nM without DBP to KD = 86.7 nM with DBP). Notably, DBP demonstrated broad-spectrum activity against SARS-CoV-2 variants, including SARS-CoV-2 Delta (IC50 = 49.22 μM) and SARS-CoV-2 Omicron XBB.1.5 (IC50 = 53.70 μM). Our findings elucidate a previously uncharacterized mechanism of DBP-mediated inhibition of viral entry, distinct from existing therapeutics targeting viral replication. This study advances our understanding of SARS-CoV-2 pathogenesis and identifies DBP as a promising lead compound for antiviral therapies.