25-Hydroxycholesterol-Conjugated EK1 Peptide with Potent and Broad-Spectrum Inhibitory Activity against SARS-CoV-2, Its Variants of Concern, and Other Human Coronaviruses

Abstract

The COVID-19 pandemic caused by SARS-CoV-2 infection poses a serious threat to global public health and the economy. The enzymatic product of cholesterol 25-hydroxylase (CH25H), 25-Hydroxycholesterol (25-HC), was reported to have potent anti-SARS-CoV-2 activity. Here, we found that the combination of 25-HC with EK1 peptide, a pan-coronavirus (CoV) fusion inhibitor, showed a synergistic antiviral activity. We then used the method of 25-HC modification to design and synthesize a series of 25-HC-modified peptides and found that a 25-HC-modified EK1 peptide (EK1P4HC) was highly effective against infections caused by SARS-CoV-2, its variants of concern (VOCs), and other human CoVs, such as HCoV-OC43 and HCoV-229E. EK1P4HC could protect newborn mice from lethal HCoV-OC43 infection, suggesting that conjugation of 25-HC with a peptide-based viral inhibitor was a feasible and universal strategy to improve its antiviral activity.

Oxysterols in the Immune Response to Bacterial and Viral Infections

“…Several mechanisms of action for 25-OHC regarding inhibiting the viral entry of many enveloped viruses—for instance, Porcine reproductive and respiratory syndrome virus [ 79 ]; hepatitis B virus [ 49 ]; ZIKV [ 69 ]; and, more recently, SARS-CoV-2 [ 30 , 68 ]—have been suggested. The majority of these studies demonstrate that the alteration of cholesterol on the plasma membrane is the key to restriction for viral entry, although various studies have demonstrated that 25-OHC is able to be localized within the plasma membrane [ 27 ], directly affecting membrane properties [ 31 , 80 ].…”

“…Therefore, this mode of action appears to be conserved across viral and bacterial infections [ 31 , 68 ]. In addition, the benefits of utilizing 25-OHC in combination with other viral inhibitors have been studied recently on human coronaviruses [ 30 ]. The authors conjugated 25-OHC with a peptide-based viral inhibitor with a different mode of action and tested its inhibitory efficacy against a broad range of coronaviruses.…”

“…The authors conjugated 25-OHC with a peptide-based viral inhibitor with a different mode of action and tested its inhibitory efficacy against a broad range of coronaviruses. The resulting 25-OHC-conjugated lipopeptide (EK1P4HC) demonstrated a synergistic antiviral effect on inhibiting viral entry against SARS-CoV-2 and its variants as well as other human coronaviruses [ 30 ]. Apart from inducing cholesterol remodeling in plasma membranes, 25-OHC also localizes within late endosomes, where it prevents SARS-CoV-2-mediated entry to the cytosol through inhibiting cholesterol export [ 67 ].…”

Targeting an evolutionarily conserved “E-L-L” motif in the spike protein to develop a small molecule fusion inhibitor against SARS-CoV-2

“…Huang et al, 2020). This is why peptides that competitively inhibit HR1-HR2 interactions can effectively block 6-HB formation and hence, viral fusion (Lan et al, 2021; Outlaw et al, 2020; S Xia et al, 2019; Shuai Xia, Liu, et al, 2020; Shuai Xia, Zhu, et al, 2020; Y Zhu et al, 2020; Yuanmei Zhu et al, 2021). Although these studies highlight the importance of the non-covalent interactions in the fusion process, the use of peptides as an effective therapeutic drug has several limitations compared to small molecule inhibitors, which are often more potent, stable, have better pharmacokinetics, and are easier to produce (Fosgerau & Hoffmann, 2015; Otvos & Wade, 2014).…”

“…Huang et al, 2020). This is why peptides that competitively inhibit HR1-HR2 interactions can effectively block 6-HB formation and hence, viral fusion (Lan et al, 2021;Outlaw et al, 2020;S Xia et al, 2019;Yuanmei Zhu et al, 2021).…”