Caspofungin and LTX-315 inhibit SARS-CoV-2 replication by targeting the nsp12 polymerase

The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, previously designated as 2019-nCoV) outbreak has caused global concern1. Currently, there are no clinically approved speci�c drugs or vaccines available for this virus. The viral polymerase is a promising target for developing broad-spectrum antiviral drugs. Here, based on the highly similar structure of SARS-CoV non-structural protein 12 (nsp12) polymerase subunit2, we applied virtual screen for the available compounds, including both the FDA-approved and under-clinic drugs, to identify potential antiviral molecules against SARS-CoV-2. We found two drugs, the clinically approved anti-fungi drug Caspofungin Acetate (Cancidas) and the oncolytic peptide LTX-315, can bind SARS-CoV-2 nsp12 protein to block the polymerase activity in vitro. Further live virus assay revealed that both Caspofungin Acetate and LTX-315 can effectively inhibit SARS-CoV-2 replication in vero cells. These �ndings present promising drug candidates for treatment of related diseases and would also stimulate the development of pan-coronavirus antiviral agents. Authors Min Wang, Fei Ye, Jiaqi Su, Jingru Zhao, and Bin Yuan contributed equally to this work.


Introduction
In early December 2019, a novel coronavirus SARS-CoV-2 emerged in Wuhan city in China3, and quickly spread to other regions of China and also other more than 100 countries including USA, Canada, Germany, France, Japan and South Korea1,4,5.The origins of the infection are yet to be determined.The infected patients have typical clinical symptoms such as fever, dry cough, dyspnea, headache and pneumonia6, which was designated as Corona Virus Disease 2019 (COVID-19)1.As of 27th March 2020, it has reported more than 500,000 human infections with more than 20,000 deaths.To date, no clinically approved vaccines or drugs are available for prevention and treatment of the novel coronavirus infection.
In the past years, six coronavirus species have been reported to infect humans.Among them, infections with human coronavirus-229E (HCoV-229E), HCoV-OC43, HCoV-NL63 and HCoV-HKU1 usually cause mild upper respiratory diseases similar to the common cold, while the other two highly pathogenic coronaviruses, which are responsible for severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), have caused two large-scale epidemics in the last two decades7,8.SARS-CoV-2 is the seventh human coronavirus which shows high sequence identity with the SARS-related coronaviruses found in bats9, and it uses the same cell entry receptor, angiotensin-converting Enzyme 2 (ACE2), as SARS-CoV1,10.However, the SARS-CoV-2 exhibited different pathogenesis properties compared with SARS-CoV, including higher transmission ability, and lower fatality.Therefore, it is urgent to understand the virus infection process which is important for the control and prevention of the outbreak.In addition, the emerging SARS-CoV-2 outbreak further highlights the necessity of developing broad-spectrum antiviral strategies targeting the conserved elements of the virus replication cycle.
The multi-subunit CoV RNA synthesis machinery is relatively conserved among different coronaviruses, which is responsible for the replication and transcription of the positive-sense RNA genome.The core components of this machinery include the nsp12 RNA-dependent RNA polymerase and its cofactors nsp7 and nsp8, which is competent for nucleotide polymerization.Other additional nsps, such as nsp10, nsp13, nsp14 and nsp16, are likely necessary to carry out the full replication and transcription activities and guarantee the delity of RNA synthesis11-13.
The amino acid sequences of SARS-CoV-2 nsp12, nsp7 and nsp8 proteins are highly similar to their counterparts in SARS-CoV, with 96%, 97% and 99% sequence identities, respectively (Extended Data Fig. 1-2).Therefore, we can use the available structure of SARS-CoV nsp12 bound to nsp7 and nsp8 cofactors2 for virtual screening of potential drugs against the emerging SARS-CoV-2, especially within those clinically approved drugs for the emergent treatment of patients.

Results And Discussion
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 ve) and PAINS ltration15 (to remove the compounds that might have nonspeci c 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).
The in vitro processive RNA synthesis activity assay was performed as previously reported17, to evaluate the inhibition e cacy 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).
We further carried out standard assays to evaluate the cytotoxicity of the two enzymatically inhibitory compounds and to test the inhibition e cacy 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.E cacies 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) = 32.17μM, 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 = 19.41μM, 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 rst-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 peptidemembrane 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 e cacy of LTX-315.Molecular docking of LTX-315 on nsp12 structure shows that the peptide lls 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 Assay Interference Compounds (PAINS) structure 15.The remaining compounds were docked into the nsp8-binding site of nsp12 using GOLD16 program.The parameter les for docking was prepared using Hermes (ref. ).The box dimension for docking calculation was set to 10 Å (centered at residue P116) to allow enough space for orientation sampling.

Protein expression and puri cation
The genes for full-length nsp7 and nsp8 of SARS-CoV-2 isolate BetaCov/Wuhan/WH01/2019 (EPI_ISL_406798) were synthesized by Synbio Technologies, which were codon-optimized for Escherichia coli E. coli) and cloned into pET-21a plasmid for protein expression.The nsp8 subunit was fused with an N-terminal 6 × His-tag to facilitate puri cation.The nsp7L8 fusion construct was generated by introducing a 6×His-linker between nsp7 and nsp8 coding sequences.Both the nsp8 subunit and nsp7L8 fusion protein were expressed in E. coli BL21 (DE3).
The transformed bacteria were cultivated at 37 °C to an OD600 nm of 0.6 and then supplemented with 1 mM isopropyl β-D-1-thiogalactopyranoside to induce protein expression at 16 °C for an additional 14-18 h.Cells were harvested by centrifugation and resuspended in a buffer containing 20 mM HEPES pH 7.5, 500 mM NaCl, 2 mM Tris(2-carboxyethyl)phosphine (TCEP), which were homogenized at 4 °C with a ultrahigh-pressure cell disrupter (JNBIO, China).The lysate was cleared by centrifugation at 12,000× rpm for 60 min and passed through 0.22-μm lter lms (Millipore).Recombinant proteins were captured by Ni-NTA a nity chromatography using a HisTrapHP column (GE Life Sciences), and further puri ed by sizeexclusion chromatography (SEC) using a Superdex 200 increase 10/300 column (GE Life Sciences).The eluted fractions were analyzed by SDS-PAGE and concentrated with an Amicon Ultra concentrator (Millipore).
The gene for SARS-CoV-2 nsp12 was chemically synthesized with codon optimization for insect cells (Spodoptera frugiperda) by Synbio Technologies.The sequence was fused with a C-terminal thrombin cleavage site, a 6×His-tag and a 2×Strep-tag (LVPRGSHHHHHHGWSHPQFEKGGGSGGGSGGSAWSHPQFEKGS), and incorporated into pFastbac-1 plasmid.Recombinant protein was expressed with High Five cells at 27 °C for 48 h post infection.Cells were harvested by centrifugation at 2,000× rpm for 30 min, and resuspended in a buffer consisting of 25 mM HEPES pH 7.4, 1 M NaCl, 1 mM MgCl2 and 2mM TCEP.An equal volume of the same buffer supplementaed with 0.2% (v/v) Igepal CA-630 (Anatrace) was added and incubated at 4 ° C for 10 min.Cells were lysed by sonication and the lysate was clari ed by ultracentrifugation at 30,000× rpm for 2 h.Cleared lysates were passed through a 0.22-μm lter lm before further puri cation.The protein was puri ed by tandem a nity chromatography using a StrepTrapHP (GE Life Sciences) and SEC using a Superdex 200 increase 10/300 column (GE Life Sciences).The SEC buffer consists of 25 mM HEPES pH 7.5, 300 mM NaCl, 0.1 mM MgCl2 and 2 mM TCEP.The eluted fractions were analyzed by SDS-PAGE and concentrated with an Amicon Ultra concentrator (Millipore).

Figures Figure 1
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