Tubeimosides are pan-coronavirus and filovirus inhibitors that can block their fusion protein binding to Niemann-Pick C1

SARS-CoV-2 and filovirus enter cells via the cell surface angiotensin-converting enzyme 2 (ACE2) or the late-endosome Niemann-Pick C1 (NPC1) as a receptor. Here, we screened 974 natural compounds and identified Tubeimosides I, II, and III as pan-coronavirus and filovirus entry inhibitors that target NPC1. Using in-silico, biochemical, and genomic approaches, we provide evidence that NPC1 also binds SARS-CoV-2 spike (S) protein on the receptor-binding domain (RBD), which is blocked by Tubeimosides. Importantly, NPC1 strongly promotes productive SARS-CoV-2 entry, which we propose is due to its influence on fusion in late endosomes. The Tubeimosides’ antiviral activity and NPC1 function are further confirmed by infection with SARS-CoV-2 variants of concern (VOC), SARS-CoV, and MERS-CoV. Thus, NPC1 is a critical entry co-factor for highly pathogenic human coronaviruses (HCoVs) in the late endosomes, and Tubeimosides hold promise as a new countermeasure for these HCoVs and filoviruses.


Supplemental Figures
Fig. S1.HIV-1 psedovirions were produced from HEK293T cells by transfection with indicated vectors and virions were purified by ultracentrifugation.Viral protein expression was determined by WB.Experiments were repeated 3 times independently, and representative results are shown.

Fig. S2 .
Fig. S2.A library containing 974 plant-sourced small compounds was screened at 10 µM for their antiviral activity to HIV-1 luciferase (Luc) reporter pseudovirions expressing Ebola virus glycoprotein (EBOV-GP) or HIV-1 envelope glycoprotein (HIV-1 Env).The screening for anti-EBOV (red) was conducted in Vero-E6 cells, and the screening for anti-HIV-1 was done in TZM-bI cells.Results shown are from one representative screening from these experiments.

Fig. S3 .
Fig. S3.Mixed Cathepsin B and L substrates were incubated with cell lysate from A549 cells treated with Tubs I, II, and III, and their activity was determined.E-64-d ethyl ester (EST) was used at 10 µM as a positive control.

Fig. S5 .
Fig. S5.CHO cells were transduced with a lentiviral vector expressing human ACE2 and stable clones were obtained after selection with blasticidin.CHO (WT) cells and CHO expressing ectopic ACE2 (Wt-A1) were then transduced with another lentiviral vector expressing CRISPR/Cas9 and NPC1 sgRNAs and stable knockout (KO) clones were obtained after selection with puromycin.Results shown are western blots for selection of these cell clones.Experiments were repeated 3 times independently, and representative results are shown.

Fig. S6 .
Fig. S6.A549 cells were transduced with a lentiviral vector expressing human ACE2 and stable clones were obtained after selection with blasticidin.A549 (WT) cells and A549 expressing ectopic ACE2 (Wt-A10) were then transduced with another lentiviral vector expressing CRISPR/Cas9 and NPC1 sgRNAs and stable knockout (KO) clones were obtained after selection with puromycin.Results shown are western blots for the selection of these cell clones.Experiments were repeated 3 times independently, and representative results are shown.
Fig. S8.Vero-E6 cells were transduced with lentiviral vectors expressing human ACE2 (A) or TMPRSS2 (T), and stable clones were obtained after selection with blasticidin.Vero-WT cells and Vero-WT-A+T cells were then transduced with another lentiviral vector expressing CRISPR/Cas9 and NPC1 sgRNAs and stable knockout (KO) clones were obtained after selection with puromycin.Results shown are western blots for selection of these cell clones.Experiments were repeated 3 times independently, and representative results are shown.

RLUFig. S10 .
Fig. S10.Local view of the binding interface of SARS2-S (left) and Omicron-S (right) are presented..The SARS2-S and Omicron-S structure are shown in cyan, and their binding interfaces, predicted to directly interact with NPC1-C, are highlighted in red as a surface.
Fig.S12.NPC1 and its deletion mutants that only express its N-terminal 1-620 or 1-377 amino acids were expressed with S proteins from indicated SARS2 variants in HEK293T cells.These NPC1 proteins were immunoprecipitated and proteins were detected by western blots.Experiments were repeated 3 times independently, and representative results are shown.

Fig. S13 .
Fig. S13.Omicron/NPC1-C complex sequence coverage and pLDDT.(a) The Omicron variant spans residues 1 to 1270, and the NPC1-C is represented by residues 1271 to 1514, totaling 244 residues.Both sequences exhibit a reliable multiple sequence alignment depth, surpassing 100.(b) The interacting region of Omicron (residue 340-510) and the entire structure of NPC1-C are predominantly in this range.
Fig. S14.Detection of the exogenous cholesterol activity in SARS2 infection.Indicated Caco2 cells were infected with SARS2-S pseudoviruses and treated with cholesterol at indicated concentrations.After 48 hours, viral infection was determined by measuring the intracellular luciferase activity.RLU, relative light unit.

Table . S1
. Binding interface of WT-S and Omicron