A pan-coronavirus fusion inhibitor targeting the HR1 domain of human coronavirus spike

Xia, Shuai; Yan, Lei; Xu, Wei; Agrawal, Anurodh Shankar; Algaissi, Abdullah; Tseng, Chien-Te K.; Wang, Qian; Du, Lanying; Tan, Wenjie; Wilson, Ian A.; Jiang, Shibo; Yang, Bei; Lu, Lu

It was previously reported that introduction of negatively and positively charged amino acids Glu (E) and Lys (K), at the i to i + 3 or i to i + 4 positions in a helix, into an HIV-1 fusion inhibitory peptide can form intramolecular E-K or K-E salt bridges that result in substantial enhancement of the peptide's stability, solubility, and antiviral activity (30, 31) . Using a similar design, we further optimized the sequence of OC43-HR2P by introducing Glu or Lys at appropriate sites in the peptide to increase the solubility and thereby the antiviral activity of the peptide. Moreover, on the basis of the structure of MERS-CoV S 6-HB, we also introduced mutations at some sites that are not expected to be involved in HR1 binding, such as 4Q, 14Y, 32D, and 36L, to further enhance the fusion inhibitory activity of the peptide (table S2) . Among the series of optimized peptides, peptides EK0-1, EK0-2, EK0-3, and EK1 showed gradually increased solubility and excellent inhibitory activity in cell-cell fusion assays. The final peptide, EK1, exhibited the most potent pan-CoV antiviral fusion activity with IC 50 values in the range of 0.19 to 0.62 M (table S2) . In some HCoV cell-cell fusion assays, EK1 exhibited even more effective inhibitory activity than the autologous peptide, such as MERS-HR2P and SARS-HR2P. Akin to its ancestor OC43-HR2P, EK1, which is derived from the short 6-HB fusion core of OC43, was also able to potently inhibit the cell-cell fusion mediated by 229E and NL63 S proteins, both of which harbor the long fusion core. These results further underscore the broad-spectrum anti-HCoV potential of EK1 (Fig. 2 , A to E, and table S2). Furthermore, EK1 also had superior pharmaceutical properties and solubility in phosphatebuffered saline (PBS) and water, which increased by 478-fold and 3.5-fold, respectively, as compared to the original OC43-HR2P. Together, these results establish an important framework and platform for subsequent development of EK1 as a potential therapeutic (table S2) .