Eltrombopag is a potential target for drug intervention in SARS-CoV-2 spike protein

Abstract

The COVID-19 pandemic, caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), is a current global threat for which there is an urgent need to search for an effective therapy. The transmembrane spike (S) glycoprotein of SARS-CoV-2 directly binds to the host angiotensin-converting enzyme 2 (ACE2) and mediates viral entrance, which is therefore considered as a promising drug target. Considering that new drug development is a time-consuming process, drug repositioning may facilitate rapid drug discovery dealing with sudden infectious diseases. Here, we compared the differences between the virtual structural proteins of SARS-CoV-2 and SARS-CoV, and selected a pocket mainly localizing in the fusion cores of S2 domain for drug screening. A virtual drug design algorithm screened the Food and Drug Administration-approved drug library of 1234 compounds, and 13 top scored compounds were obtained through manual screening. Through in vitro molecular interaction experiments, eltrombopag was further verified to possess a high binding affinity to S protein plus human ACE2 and could potentially affect the stability of the ACE2-S protein complex. Hence, it is worth further exploring eltrombopag as a potential drug for the treatment of SARS-CoV-2 infection.

Keywords: Homology Modeling; SARS-CoV-2; Surface plasmon resonance; Virtual drug design.

Graphical abstract

Fig. 1

The tertiary structure of SARS-CoV-2 Spike (S) protein and the pocket for drug screening. The tertiary structure of SARS-CoV-2 S protein was acquired via homology modeling (A) or ab initio model (B, cyan). (C) Comparison of SARS-CoV-2 S protein (cyan) and SARS-CoV S protein 5 × 58 (mauve). (D) Identification of SARS-CoV-2 S protein RBD (cyan) by comparing to SARS-CoV S protein 6CRV (mauve). (E) Comparison of SARS-CoV-2 S protein RBD (cyan) with SARS-CoV 5WRG RBD (mauve). (F) Comparison of RBD amino acid sequences between different strains of SARS-CoV and SARS-CoV-2. (G) The RBD of S protein (green area) show insufficient stability for future drug screening. (H) The selected binding pocket (yellow area) for drug screening,

Fig. 2

Interaction sites between drugs and SARS-CoV-2 S protein. The interaction between different drugs and SARS-CoV-2 S protein were virtualized to calculate binding affinity. The structure of each drug and the corresponding amino acids interacting with each drug are shown: (A) Dactinomycin (DB00970, PubChem CID: 457193); (B) Glycyrrhizic acid (DB13751, PubChem CID: 14982); (C) Eltrombopag (DB06210, PubChem CID: 135449332); (D) Azilsartan medoxomil (DB08822, PubChem CID: 25210270).

Fig. 3

SPR analysis of interactions of different drugs and SARS-CoV-2 S protein. (A, B) Representative SPR data for various concentrations of eltrombopag binding to S1 + S2 domain or S2 domain at pH 5.5. (C) Representative SPR data for various concentrations of glycyrrhizic acid binding to S2 domain at pH 5 0.5. RU, resonance units.

Fig. 4

SPR analysis of interactions of different drugs and human ACE2. (A) Representative SPR data for various concentrations of eltrombopag binding to human ACE2 at pH 4.5. (B, C) Representative SPR data for various concentrations of glycyrrhizate, glycyrrhizin binding to human ACE2 at pH 4.5.

Fig. 5

Eltrombopag affects the stability of RBD-ACE2 complex. (A, B) The Tm of RBD-ACE2 complex with different metal ions (Black: Control; Red: 5 mM ZnCl₂; Green: 5 mM MgCl₂; 5 mM CaCl₂) as control were tested by TSA. (C, D) The Tm of RBD-ACE2 complex with different compounds treatment (Red: 100 μM eltrombopag; Green: 100 μM EGCG (epigallocatechin gallate); Black: 1% DMSO) were tested by TSA. A decreased Tm value can be observed after eltrombopag treatment.

Fig. S2

Amino acid sequence differences between SARS-CoV-2 and SARS-CoV. Amino acid sequences of SARS-CoV-2 or SARS-CoV S protein (A), M protein (B), E protein, (C) and N protein (N) were downloaded and compared.

Fig. S3

TM score of modeling and reported S protein structure of SARS-CoV-2. The structure of spike model (QHD43416.pdb) was compared to the reported S protein (6LZG.pdb) via TM-align. Two different chains, chain 1 (A316309) and chain 2 (B316309), were used, respectively.

Fig. S4

Tertiary structures of SARS-CoV-2 and SARS-COV M, E, N protein. M, E, N protein structure of SARS-CoV-2 were acquired through ab initio modeling. (A) M protein. (B) E protein (Green: SARS-CoV-2, Blue: SARS-CoV 2MM4). (C) N protein.

Fig. S5

Ramachandran plot of SARS-CoV-2 proteins. The protein structure M protein (A), N protein (B), and E protein (C) were assessed by Procheck and depicted using a Ramachandran plot.

Fig. S6

Ramachandran plot statistics. The Ramachandran plot statistics of M protein (A), N protein (B), and E protein (C) were listed. The percentage or residues in the most favored regions indicated the quality of model (>90% is thought to possess great quality), while the value of G factors provides information on unusual property (<−0.5 represents unusual, and <−1.0 represents high unusual).

Fig. S7

Interaction models of different drugs and SARS-CoV-2 S protein. The interaction between different drugs and SARS-CoV-2 S protein were virtualized to calculate binding affinity. The structure and interacting spots of each drug are shown. (A) Bictegravir (DB11799, PubChem CID: 129626368); (B) Temsirolimus (DB06287, PubChem CID: 134812825); (C) Dolutegravi (DB08930, PubChem CID: 54726191); (D) Elbasvir (DB11574, PubChem CID: 71661251); (E) Irbesartan (DB01029, PubChem CID: 3749); (F) Gliquidone (DB01251, PubChem CID: 91610); (G) Lanreotide (DB06791, PubChem CID: 71349); (H) Tasosartan (DB01349, PubChem CID: 60919); (I) Velpatasvir (DB11613, PubChem CID: 67683363).

Fig. S8

SPR analysis of interactions of other drugs and SARS-CoV-2 S protein. SPR data for various concentrations of different drugs: (A) Bictegravir, (B) Dolutegravir, (C) Temsirolimus, (D) Elbasvir, (E) Irbesartan, (F) Gliquidone, (G) Velpatasvir, (H) Azilsartan Medoxomil binding to SARS-CoV-2 S2 domain at pH 5.5.

Fig. S9

Experiment details of thermo shift assay (TSA). (A) Prepared S-RBD-mFB (sino) and ACE-2-His protein sample. (B) Concentrate S-RBD-ACE2 complex. S-RBD (mFc tag): 51.5 kD, ACE2: 85.1 kD. Collect lane 5– 7 as concentrated protein complex, based on SDS-PAGE result.