As shown in Fig.1, we generated a combined database with the 41,384 chemical compounds from the Target Mol, Selleck and MCE companies. After standardization operation for the chemical compounds, we obtained the 3D version of each single- molecule compound. We initially screened the compounds by the Lipinski’s rule14 (rule of five) and PAINS filtration15 (to remove the compounds that might have non-specific interactions with proteins), which generated a subset of 5,762 molecules for subsequent structural docking.
We selected the conserved nsp8-binding cavity on nsp12 subunit as the target site for molecular docking using the GOLD16 software (Fig. 1a). Among the 50 top-ranking compounds in docking scores, we selected the 10 water-soluble hints as the prioritized candidates for activity assays (Extended Data Table 1).
We produced the full-length SARS-CoV–2 nsp12 protein using the baculovirus expression system, and expressed the nsp7 and nsp8 subunits in Escherichia coli (Chinese SARS-CoV–2 isolate BetaCov/Wuhan/WH01/2019 (EPI_ISL_406798). All these proteins were purified to high purity and homogeneity as shown by size-exclusion chromatography (SEC) and SDS-PAGE assays (Extended Data Fig. 3). Subsequently, surface plasmon resonance (SPR) experiments were performed to test the binding capacities of the 10 candidate compounds to the immobilized nsp12 protein. Among the 10 compounds, six of them showed obvious binding response, including polymyxin B sulfate (CAS number, 1405–20–5), LTX–315 (Chemical Abstracts Service (CAS) number, 1345407–05–7), Caspofungin Acetate (CAS number, 179463–17–3), Endomorphin 1 (CAS number, 189388–22–5), Fast Green FCF (CAS number, 2353–45- 9), Erioglaucine disodium salt (CAS number, 3844–45–9). Further experiments with serial-diluted concentration gradients revealed the binding affinities of the 6 candidates are 15.1 uM, 9.8 uM, 19.2 uM, 2.01 uM, 18.2 uM and 11.2 uM, respectively (Fig. 2). In addition, the binding affinities of nsp8 to nsp12 is 1.19 uM (Extended Data Fig. 4).
The in vitro processive RNA synthesis activity assay was performed as previously reported17, to evaluate the inhibition efficacy of the 6 nsp12-bound chemical compounds. Two of the candidate drugs, Caspofungin Acetate and LTX–315 showed good inhibition activities for RNA synthesis in vitro (Fig. 3). When the molar ratio of nsp12: nsp7L8: LTX–315 or Caspofungin Acetate is 1:1:50, LTX–315 or Caspofungin Acetate can inhibit polymerase activities completely; while the molar ratio is 1:1:25, LTX–315 or Caspofungin Acetate can inhibit about half of the activities.
We further carried out standard assays to evaluate the cytotoxicity of the two enzymatically inhibitory compounds and to test the inhibition efficacy for SARS-CoV-2 replication at cell level. The cytotoxicity of the compounds in vero cells (ATCC, CCL-81) was determined by the CCK–8 assay. Then, vero cells were infected with BetaCov/Wuhan/WH01/2019 in the presence of drugs with varying concentrations. Efficacies were evaluated by quantifying the virus copy numbers in the cell supernatant via quantitative real-time RT-PCR (qRT-PCR). LTX–315 (half-maximal effective concentration (EC50) = 32.17 μM, the half cytotoxic concentration (CC50) > 50 μM) can inhibit the virus replication in cells but has strong cytotoxicity under the 100 μM concentration that show nearly 100% inhibition activity (Fig. 4). Notably, Caspofungin Acetate (EC50 = 19.41 μM, CC50 > 100 μM) can effectively inhibit virus replication in cells (Fig. 4) without obvious cytotoxicity.
LTX–315 is currently tested in phase I/II clinical trials, as a potential first-in-class oncolytic 9-mer peptide18. Therefore, LTX–315 can result in cytotoxicity in vero cells under high concentrations, as vero cells are transformed cells that have some characteristics of cancer cells. LTX–315 is a derivative of the host defense peptide (HDP) bovine lactoferricin19. HDPs have been found in a wide variety of species as part of the host defense system against pathogens and are rich in basic and hydrophobic amino acid residues20. It has been revealed that the biological activity of the HDPs often relies on the peptide-membrane interactions in addition to possible intracellular targets21. The instability of LTX–315 in human plasma results from the sequential exopeptidase- mediated cleavage at the N-terminus, and the half-life of LTX–315 was determined to be 160 minutes20. The animal protection experiment should be conducted in the future to test the in vivo efficacy of LTX–315. Molecular docking of LTX–315 on nsp12 structure shows that the peptide fills the nsp8 binding cavity and the C-terminal portion forms intensive interactions with the open edge of the cavity (Extended Data Fig. 5). The information of LTX–315 provides a promising starting point for designing anti- coronavirus drugs, and also gives a drug choice of compassionate use for the infected cancer patients.
Caspofungin has been approved to treat the candidal esophagitis and deep-seated candidal infections, and is also an alternative therapy for Aspergillus infections22. The inhibition mechanism of Caspofungin is to non-competitively inhibit the synthesis of 1,3-β-glucans which are key components of fungal cell walls23. The enzymatic system for 1,3-β-glucan synthesis is absent in mammalian cells22. Caspofungin is a cyclic- hexapeptide compound with a fatty acid side chain and is available in an intravenous formulation but lacks an oral formulation24. Molecular docking of Caspofungin in the nsp8 binding cavity on nsp12 structure shows that the cyclic-hexapeptide moiety is accommodated within the cavity and the fatty acid side chain reaches out to interact with the edge region (Extended Data Fig. 5).
Recent clinical studies have revealed that severe SARS-CoV–2 infected patients probably have fungal coinfection during the disease progression25. Thus, the discovery of Caspofungin against SARS-CoV–2 infection can provide a nice choice for the dual- function treatment of the patients, inhibiting both SARS-CoV–2 and fungi infections. Since Caspofungin has been used in human patients with a safety track record, we suggest that it should be evaluated in patients suffering from the COVID–19.