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Biochemistry | Chemistry


Lisa Gentile


The discovery of the SARS-CoV-2 virus during the COVID-19 pandemic required scientists to develop medical solutions to reduce infectivity, severity of symptoms, and death. Although vaccines and drugs provided urgent assistance, the need to continue developing better drugs is necessary long term, and understanding the structure of the virus and finding potential inhibitors would prove vital to discovering solutions to this worldwide health problem. This experimental project focuses on targeting the unique accessory protein, Open Reading Frame 8 (ORF8) in SARS-CoV-2 through studying its interactions with a repurposable drug, novobiocin. Importantly, ORF8 specializes in helping evade immune system checks by downregulating major histocompatibility complex I (MHC-I) after infection, and is also involved in inflammatory responses from the cytokine storm, the most prominent cause of fatalities from the virus. Additionally, the ORF8 protein has been proposed to act as a histone mimic at the histone-H3 ARKS motif that causes post-translational changes in chromatin, further worsening these problems through gene transcription. Previous in silico and in vitro work from our lab has shown that novobiocin [Kd = 54.5 ± 3.14 μM] and three other computationally verified ligands (iohexol [Kd = 1185 ± 193 μM], kaempferol-7-O-glucoside [Kd = 135 ± 10.9 μM] & lercanidipine hydrochloride [Kd = 52.7 ± 5.71 μM]) bind to. To probe the role of Arg in the histone-H3 ARKS motif, specific mutation was done in position 52 from Arg to Met, Glu and Leu respectively. The above results in drastic changes in intermolecular forces such as opposing charge, polarity and different sizes of these side chains that negatively affect novobiocin’s ability to bind to the ORF8 pocket , which this research aims to experimentally prove this hypothesis. In silico analyses for the mutagenic ORF8 were initially found to require more energy to bind novobiocin to the ARKS motif than wild-type, but was still possible to dock according to Swissdock. Primers for the ORF8 R52 mutants were then designed and mutagenic plasmids were constructed and sequence verified. Each of the mutant ORF8 proteins was overexpressed, purified, and Kd values for binding to novobiocin were determined via intrinsic fluorescence spectroscopy and compared with wild-type binding. This data will help further understand the role of SARS-CoV-2 ORF8 protein ARKS motif and how its interactions affected novobiocin binding, potentially benefitting future studies attempting to repurpose novobiocin and other ligands for treatment of the spreading virus.