COVID-19 is a disease caused by the highly transmissible and pathogenic SARS-CoV-2 virus. Since its first case was documented in 2019, it has rapidly widespread and has caused millions of deaths worldwide. Many intervention strategies targeting these proteins have been developed. However, frequently mutation of SARS-CoV-2 poses a challenge to the effectiveness of current treatments. Therefore, it is critical to develop new therapeutic drugs against this disease. In this present study, in silico approach was used to study the interaction between RetroMAD1™and SARS-CoV-2 proteins including Spike proteins (S), 3-chymotrypsin-like protease (3CLpro) and papain-like protease (PLpro). The interaction of these viral proteins and RetroMAD1™ was performed through HDOCK server and visualised using PyMOL. Docking results revealed that all the complexes of SARS-CoV-2 proteins binding with RetroMAD1™ have relatively high docking scores. The binding energy of RetroMAD1™ complexes with SARS-CoV-2 S, 3CLpro, PLpro were − 15, -12.3 and − 15.4, respectively. RetroMAD1™antiviral efficiency and cytotoxicity was also evaluated using EpiAirway™ Model. In vitro validation of viral inhibitory effect of RetroMAD1™was performed with 3CLpro Inhibition Assay. The outcome showed that RetroMAD1™ represents a potential drug candidate against SARS-CoV-2 for its promising viral inhibitory effect.
Article
Anti-viral chimeric protein RetroMAD1™ potently block SARS-CoV-2 viral entry and propagation
https://doi.org/10.21203/rs.3.rs-2712307/v1
This work is licensed under a CC BY 4.0 License
Journal Publication
published 17 Nov, 2023
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RetroMAD1™
SARS-CoV-2
COVID-19
anti-viral protein
Spike
3CLPro
PLPro
MAP-30
Dermaseptin S-1
Retrocyclin-101
In late 2019, an outbreak of pneumonia of unknown aetiology had occurred in Wuhan, China and then spread rapidly and globally, causing millions of deaths. The cause of the outbreak was later determined to result from a coronavirus, which then named as SARS-CoV-2 and the disease was called as coronavirus disease (COVID-19). Whilst the introduction of COVID-19 vaccinations in early 2021 was a watershed moment in the worldwide pandemic's struggle, two oral antiviral treatments — molnupiravir and Ritonavir-boosted nirmatrelvir (Paxlovid) — have been approved at the end of 2021 for therapeutic management of non-hospitalized adults with mild to moderate COVID-19 who are at high risk of disease progression [1, 2]. As these antiviral pills navigate their way into pharmacies throughout the world, experts are already planning for the other antiviral drugs that will eventually replace them [1, 3]. Joining the race for antiviral drugs to beat COVID-19, here we presented our chimeric protein RetroMAD1™ (Patent Number: US 2013/0336955A1) which delivered orally proven to be effective against Feline Leukemia Virus (FeLV) naturally infecting the cats [4], has also proven to be effective in inhibiting SARS-CoV-2 replication.
RetroMAD1™ is a 41.3 kDa protein expressed by pET-26b (+) and pET-22b (+) using the Escherichia coli expression system [5]. This chimeric protein was invented on the basis of three different anti-microbial proteins. It consists of Retrocyclin-101 linked to the N-terminal of momordica anti-human immunodeficiency virus (HIV) protein of 30 kDa (MAP-30) via a linker peptide, and Dermaseptin-S1 directly join to MAP-30 at the C-terminal (Fig. 1) [5]. Retrocyclins are stable, 18-residue defensin peptide, cationic with a ß-sheet structure are robust and resistant to boiling, acidic conditions, and other severe situations [6]. Synthetically synthesized by Cole et al. (2002) [7] based on the pseudogene mRNA sequence expressed by human bone marrow homologous to rhesus monkey circular minidefensins (Ɵ-defensins), retrocyclin specifically block HIV-1 replication in human cell at an entry step [8]. Differed by a single arginine to lysine substitution, Retrocyclin-101, a non-haemolytic and minimally cytotoxic shows superior potency antiviral activity against HIV-1 by binding to gp120 at 25-fold greater affinity compared to Retrocyclin [9, 10]. Retrocyclin-101 has also been reported to be effectively inhibit; flaviviruses, at either the entry or both entry and replication steps [11], influenza viruses through Toll-like receptors (TLR)-dependent and TLR‐independent mechanisms [12], bacterial vaginosis by toxin inhibition and biofilm retardation [13], adherence, survival, and proliferation of Staphylococcus aureus on human nasal epithelia [14], Herpes Simplex viruses by inhibiting viral adhesion to gB2 at virus entry step [15], and SARS-CoV-2 through inhibition of Spike to its receptor, angiotensin-converting enzyme 2 (ACE2) at entrance step [16].
Extracted from Momordica charantia seeds, MAP-30 is reported to possess anti-HIV infection and replication [17, 18], and anti-tumor activity (Lee-Huang et al., 1995, Lee-Huang et al., 2000, Jiang et al., 2018) [18, 19, 20] inhibiting the infection and replication of Herpes Simplex Virus HSV-1, HSV-2 as well as acyclovir-resistant HSV strains [21]. MAP-30 also significantly reduced the dosage of chloramphenicol and erythromycin required to inhibit the growth of Staphylococcus aureus, Enterococcus faecalis, Salmonella typhimurium, Salmonella enteritidis, and Pseudomonas aeruginosa [22], inhibits the growth of Kaposi's sarcoma-associated herpes virus (KSHV)-infected tumor cells from AIDS patients by inhibiting the expression of viral and cellular genes essential for proliferation and expansion of the virus infected tumor cells [23], and effectively suppressing Hepatitis B virus (HBV) gene expression and genome replication [24]. Belonging to the family of type-1-ribosomal inactivating proteins (RIPs), MAP-30 has N-glycosidase activity that depurinates adenine base–ribose glycosidic bond at position A-4324 in universally conserved α-sarcin /ricin loop of 28S ribosomal (r)RNA [25, 26]. This depurination irreversibly inactivates the ribosome, hence inhibiting ribosomal protein synthesis [25, 26, 27, 28]. Nevertheless, MAP-30 RIP RNA N-glycosidase (RNG) reaction mechanism is unrelated to its anti-HIV/tumor activity [17, 18, 29]. Instead, MAP-30 inhibit the replication of viral DNA by downregulating replicative intermediate and reduce viral covalently closed circular DNA (cccDNA). The capacity of MAP30 to disrupt critical topological interconversions of viral DNA and ribosomal function of rRNA in virally infected cells may give novel pathways for its antiviral activities [24]. Because MAP30 toxicity is limited to virally infected or tumor-transformed cells and has no impact on healthy cells, making it a promising option for clinical trials [21, 29, 30].
A 34 residue anti-microbial defensin peptide of Dermaseptin-S1 (ALWKTMLKKLGTMALHAGKAALGAAADTISQGTQ), first purified by Mor et al. (1991) [31] from the skin extract of the South American arboreal frog Phyllomedusa Sauuagii. Dermaseptin-S1 have shown a to be non-hemolytic but potent activity against pathogenic fungi (Aspergillus fumigatus and Arthroderma simii) [31], Gram positive bacterium (Staphylococcus aureus), Gram negative bacterium (Escherichia coli) [32], the protozoon (Leishmania major) [32, 33], and genital pathogens (Trichomonas vaginalis, Herpes simplex virus, Papillomavirus) [34]. The anti-microbial activity of dermaseptin has been shown to be mediated by the selective interaction of basic and amphipathic α-helix moieties with plasma membrane phospholipids, resulting in permeabilization and lysis [32]. The antiviral activity of Dermaseptin-S1 is promoted by its amphipathic α-helix that could alter the virus capsidic structures or cell surface receptor, hence inhibit virus-cell surface interactions during the initial infectious entry phase [34].
In this study, RetroMAD1™ was subjected to molecular docking against the SARS-CoV-2 Spike (S), non-structural proteins namely 3-chymotrypsin-like protease (3CLpro) and papain-like protease (PLpro) to explore its anti-viral effect against SARS-CoV-2 virus. As these viral proteins play dominant roles in different stages of viral replication, they are also important targets for COVID-19 treatment. Several drugs that proved to have effective effect for SARS-CoV-2 proteins (Andrographolide, Ivermectin, Neobavaisoflavone) were used as reference drugs to compare the interaction. Our study here provided evidence that RetroMAD1™strongly blocked the SARS-CoV-2 (Wuhan-Hu-1) infection using an in vitro model of healthy, human-derived tracheal/bronchial epithelial (TBE) cells. Further study proof that inhibition of SARS-CoV-2 (Wuhan-Hu-1) replication is through direct blocking of the 3CLpro enzyme activity by RetroMAD1™. Our findings suggest that RetroMAD1™may prove to be a candidate drug for treatment of SARS-CoV-2.
RetroMAD1™ Structure Modelling
The structure o RetroMAD1™ was predicted using default parameters of ColabFold coupled with Google Colab [35, 36]. The ColabFold replaced AlphaFold2 homology search with MMseqs2 search against both UniRef and Environment sequence database using both paired and unpaired sequences [37, 38]. The models were visualized using YASARA Structure Version (20.10.4) [39] and Pymolv2.5 (PyMOL, 2020; https://pymol.org/2/) [40]. The predicted structure consists of complete domain of Retrocyclin 101, MAP30 and Dermaseptin S-1 model was selected for molecular docking (Fig. 1).
Preparation of SARS-CoV-2 Proteins
The crystal structure of SARS-CoV-2 Spike Protein (S) (PDB ID = 7DF4), 3-chymotrypsin-like protease (3CLpro) (PDB ID: 6LU7), Papain-like protease (PLpro) (PDB ID: 6WUU) of Wuhan-Hu-1 variant are readily in the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB) (https://www.rcsb.org/) and were retrieved in the .pdb format. Swiss Model web server was used for repairing the missing residues in the structures [41]. All the crystal structures were prepared by removing existing co-crystallised ligands, water molecules and hetero atoms by PyMOLv2.5 (PyMOL, 2020; https://pymol.org/2/) [40].
Preparation of Reference Drugs
The 3D structure of reference drugs in this study (Andrographolide, Ivermectin, Neobavaisoflavone) were retrieved from the PubChem database (www.puchem.ncbi.nlm.nih.gov) [42]. These ligands were then further converted into the .pdb format using Open Babel tool [43].
Protein-protein docking
HDOCK server (http://hdock.phys.hust.edu.cn/) was used to carry out protein-protein docking between RetroMAD1™ as a ligand and proteins of SARS-CoV2 Wuhan-Hu-1 as a receptor. Then, these docking results were compared with positive control docking. Based on HDOCK the top 5 docked protein-protein complexes with higher docking scores were assessed for the docking poses and visualized using PyMOLv2.5 (PyMOL, 2020; https://pymol.org/2/) [40]. Prodigy website (https://wenmr.science.uu.nl/prodigy/) was used to predict the values of binding affinity (ΔG) kcal/mol) at 25.0°C.
In vitro Evaluation of Antiviral efficiency and cytotoxicity in EpiAirway™ Model
Compound: The RetroMAD1™ was diluted to the test dilutions of 400, 300, 200, 150, 100, 75, 50, and 37.5 µg/mL in the MatTek culture medium (AIR-100-MM). Remdesivir (MedChemExpress, cat# HY-104077) was tested in singlet wells at 1, 0.1, 0.01, and 0.001 µg/mL as the positive control. Virus: The USA-WA1/2020 strain of SARS-CoV-2 was passaged three times in Vero 76 cells to create the virus stock. Virus was diluted in AIR-100-MM medium before infection, yielding a multiplicity of infection (MOI) of approximately 0.03 CCID50 per cell. Determination of virus titers from each treated cell culture: Vero 76 cells (African green monkey (Chlorocebus aethiops) kidney; ATCC CRL-1587) were seeded in 96-well plates and grown overnight (37°C, 5% CO2) to 90% confluence. Samples containing virus were diluted in 10-fold increments in infection medium and 200 µL of each dilution transferred into respective wells of a 96-well microtiter plate. Four microwells were used for each dilution to determine 50% viral endpoints. After 7 days of incubation, each well was scored positive for virus if any cytopathic effect (CPE) was observed as compared with the uninfected control, and counts were confirmed for endpoint on day 10. The virus dose that was able to infect 50% of the cell cultures (CCID50 per 0.1 mL) was calculated by the Reed- Muench method [44]. The day 7 values are reported. Untreated, uninfected cells were used as the cell controls.
Cell Culture
The EpiAirway™ Model consists of normal, human-derived tracheal/bronchial epithelial (TBE) cells which have been cultured to form a multi layered, highly differentiated model which closely resembles the epithelial tissue of the respiratory tract. The cell cultures were made to order by MatTek Life Sciences (https://www.mattek.com) (Ashland, MA) and arrived in kits with either 12- or 24-well inserts each. The TBE cells were grown on 6mm mesh disks in transwell inserts. During transportation the tissues were stabilized on a sheet of agarose, which was removed upon receipt. One insert was estimated to consist of approximately 1.2 x 106 cells. Kits of cell inserts (EpiAirwayTM AIR-100, AIR-112) originated from a single donor, # 9831, a 23-year-old, healthy, non-smoking, Caucasian male. The cells have unique properties in forming layers, the apical side of which is exposed only to air and that creates a mucin layer. Upon arrival, the cell transwell inserts were immediately transferred to individual wells of a 6-well plate according to manufacturer’s instructions, and 1 mL of MatTek’s proprietary culture medium (AIR-100- MM) was added to the basolateral side, whereas the apical side was exposed to a humidified 5% CO2 environment. The TBE cells were cultured at 37°C for one day before the start of the experiment. After the 24 hours equilibration period, the mucin layer, secreted from the apical side of the cells, was removed by washing with 400 µL pre-warmed 30 mM HEPES buffered saline solution 3X. Culture medium was replenished to the basal side following the wash steps.
Experimental design
Each compound treatment (120 µL) and virus (120 µL) were applied to the apical side, and compound treatment only was applied to the basal side (1 mL), for a 2 h incubation. As a virus control, 3 of the cell wells were treated with placebo (cell culture medium only). Following the 2 h infection, the apical medium was removed, and the basal side was replaced with fresh compound or medium. The cells were maintained at the air- liquid interface. On day 6, the medium was removed and discarded from the basal side. Virus released into the apical compartment of the tissues was harvested by the addition of 400 µL of culture medium that was pre-warmed at 37°C. The contents were incubated for 30 minutes, mixed well, collected, thoroughly vortexed and plated on Vero 76 cells for virus yield reduction (VYR) titration assay. Triplicate and singlet wells were used for virus control and cell controls, respectively.
Assay of cytotoxicity
Uninfected cells treated with each concentration of test compound were ran in parallel with the infected, treated wells. The cytotoxicity was determined microscopically, the toxicity control cells also be examined microscopically for any changes in cell appearance compared to normal control cells run in the same plate. These changes may be enlargement, granularity, cells with ragged edges, a filmy appearance, rounding, detachment from the surface of the well, or other changes. These changes are given a designation of T (100% toxic), PVH (partially toxic–very heavy–80%), PH (partially toxic–heavy–60%), P (partially toxic–40%), Ps (partially toxic–slight–20%), or 0 (no toxicity–0%), conforming to the degree of cytotoxicity observed.
3CLpro Inhibition Assay
The RetroMAD1™ was diluted to the test dilutions of 400, 300, 200, 160, 128, 102.4, 81.92, 65.54, 52.43, 41.94, 33.55 µg/mL in the Dulbecco′s Modified Eagle′s Medium/Nutrient Mixture F-12 Ham (DMEM/F12) non-phenol red cell culture media (Gibco™, Thermo Fisher Scientific, USA). The 3CLpro inhibitor, feline antiviral drug (GC376) provided by BPS Bioscience was used as an inhibitor control. The Untagged 3CL Protease Assay Kit (BPS Bioscience #78042-1) was used to assess the effect of RetroMAD1™ in inhibiting the activity of the 3CLpro. The assay was performed according to the manufacturer’s instructions. Briefly, 3CLpro was diluted in assay buffer (with 1 mM DTT) at 0.5 ng/µl (15 ng per reaction). 30 µl of the diluted 3CL Protease enzyme solution was added to wells designated as “Positive Control”, “Inhibitor Control” and “Test Sample”. 30 µl of the Assay Buffer (with 1 mM DTT) was added to the “Blank” well. 10 µl GC376 (500µM) was added to the wells labelled “Inhibitor Control”. 10 µl of RetroMAD1™ at specific diluted concentrations was added to each well designated “Test Sample”. 10 µl of Assay Buffer with DTT was added to “Blank” and “Positive Control” wells. The enzyme and inhibitors were preincubated for 30 minutes at room temperature with slow shaking. The reaction was started by adding 10 µl of the 3CL Protease substrate solution (200 µM) to each well. The sealed plate was incubated for 4 hours at room temperature with slow shaking. The fluorescence intensity was measured by M3 multi-mode microplate reader (SpectraMax® Molecular Devices) at an excitation wavelength of 360 nm and detection of emission at a wavelength 460 nm.
Data and Statistical analysis
The GraphPad Prism 9 software (GraphPad Software Inc., San Diego, CA, USA) was used for data and statistical analysis. The non-linear regression of inhibitor’s concentration- response curve fit model was used to determine the half maximal effective concentration (EC50) and half maximal inhibitory concentration (IC50) of RetroMAD1™. The EC90 was calculated using GraphPad Prism 9’s calculator based on EC50 value and hill slope obtained by non-linear regression. An unpaired t-test with Welch’s correction was performed for statistical analysis and represented as the mean ± SD. P values < 0.05 considered statistically significant.
Binding complex of RetroMAD1™ with SARS-CoV-2 Spike (Wuhan-Hu-1)
By using HDOCK server (http://hdock.phys.hust.edu.cn/), we analysed the potential of RetroMAD1™ to inhibit spike-ACE2 interaction by performing molecular docking of Spike RBD-RetroMAD1™ complex using the SARS-CoV-2 Spike-ACE2 complex from PDB (PDB ID: 7DF4). The docking of original structure of SARS-CoV-2 Spike RBD-ACE2 (PDB: 7DF4) was used for comparison. The docking results show that docking score of Spike RBD-RetroMAD1™ complex (-331.22) is slightly lower than the Spike RBD-ACE2 complex (-298.58) (Table 1). As more negative scores mean more a possible binding model, this suggests that RetroMAD1™ would serve as a well-matched competitor of ACE2 to bind with RBD region. We then performed similar docking study of ACE2-RetroMAD1™ complex, and compared with non-peptide human ACE2 (hACE2) inhibitor, N-(2-aminoethyl)-1-aziridine-ethanamine (NAAE). Results showed that docking scores of RetroMAD1™ and were − 257.47 and − 87.84, respectively, suggesting that RetroMAD1™ formed a relatively more stable binding to hACE2 comparing to NAAE.
|
Target |
Ligand/peptide |
HDOCK Docking Score |
Binding affinity, ΔG (kcal mol− 1) |
|---|---|---|---|
|
SARS-CoV-2 Spike RBD (7DF4) |
RetroMAD1™ ACE2 |
-331.22 -298.58 |
-15.0 -14.3 |
|
hACE2 in Spike-ACE2 complex (7DF4) |
RetroMAD1™ NAAE (C6H15N3) |
-257.47 -87.84 |
-14.5 -5.2 |
The docking complexes above were then evaluated their binding affinities and dissociation constant using PRODIGY server. Table 1 shows the binding affinity for SARS-CoV-2 spike RBD-RetroMAD1™ complex is -15.0 kcal mol− 1 compared with Spike RBD-ACE2, -14.3 kcal mol− 1. The interacting residues of both SARS-CoV-2 spike RBD-RetroMAD1™ complex and SARS-CoV-2 spike RBD-ACE2 complex are listed in Fig. 2. There are 30 interacting residues detected in SARS-CoV-2 spike RBD-ACE2 complex while there are 41 interacting residues detected in SARS-CoV-2 spike-RetroMAD1™ complex. Seventeen identical interacting residues were shared by both complexes including Arg403, Gly446, Tyr449, Tyr453, Leu455, Phe456, Tyr489, Phe490, Gln493, Ser494, Gly496, Gln498, Thr500, Asn501, Gly502, Val503, Tyr505. It is worth to notice that among these residues, Gln493, Tyr505, Gln498, Asn501, Thr500, Tyr449, Tyr489, Phe456, Leu455 that have been identified as key interacting residues of RBD in complex with hACE2 [45].
Binding complexes of RetroMAD1™ with SARS-CoV-2 3CLpro
3CLpro enzyme is highly conserved enzyme in coronaviruses as the sequence of 3CLpro of SARS-CoV-2 and SARS-CoV share 96% similarity [46]. It is one of the main drug targets for its essential role in cleavage process of 11 distinct sites to generate various non-structural proteins. 3CLpro is a homodimer which each of the monomer has 306 residues and is composed of three domains. Domains I (residues 8-101) and II (resides 102–184) contain six stranded antiparallel β-sheet structure [46, 47]. On the other hand, domain III (residue 201–303) contains five α-helices and functions for enzyme dimerization [46, 47]. The His41-Cys145 catalytic dyad, which serves as binding site of the substrate is located in a pocket between domain I and II [48].
The complex of SARS-CoV-2 3CLpro with RetroMAD1™ was subjected to HDOCK server. Among the top 10 highest scores, the one with its best binding post to interact with the catalytic site (His41-Cys145) or other 25 important catalytic pocket residues (Thr24, Thr25, Thr26, Leu27, His41, Met49, Tyr54, Phe140, Leu141, Asn142, Gln143, Ser144, Cys145, His163, His164, Met165, Glu166, Leu167, Pro168, His172, Asp187, Arg188, Gln189, Thr190, Ala191, Gln192) [49]. Andrographolide and Ivermectin which had been proven to have effectivity to 3CLpro were served as reference drugs. The docking scores of SARS-CoV-2 3CLpro complexes with RetroMAD1™, Andrographolide and Ivermectin were − 219.74, -117.10 and − 42.85 respectively(Fig. 3). While the binding affinity for each complex was − 12.3, -5.43, -5.42 kcal mol− 1, respectively(Table 2). RetroMAD1™’s binding affinity was relatively higher, and it binds to the His41-Cys145 catalytic dyad and other catalytic pocket residues.
Table 2: Docking scores of RetroMAD1™ in complex with SARS-CoV-2 3CLpro
Notes: Interacting residues of 3CLpro active sites are bolded. Interacting residues of allosteric site are underlined.
Binding complexes of RetroMAD1™ with SARS-CoV-2 PLpro
SARS-CoV-2 PLpro is an essential protease for viral replication. The catalytic sites of SARS-CoV-2 PLpro that comprises of a canonical catalytic triad (Cys111-His272-Asp 286) could be served as the potential target for therapy agents [50]. SARS-CoV-2 PLpro (PDB ID 6WUU) in complex with RetroMAD1™ was used for docking and the catalytic triad was set as the specific receptor binding site residues. From the top 10 models generated, the model with highest score and the best gesture that bind near to the targeted sites was selected. Neobavaisoflavone was used as a control drugs. HDOCK scores obtained for SARS-CoV-2 PLpro complexes with RetroMAD1™ and Neobavaisoflavone were − 216.85 and − 126.83, respectively (Table 3). PRODIGY-predicted binding energy of RetroMAD1™ and Neobavaisoflavone were − 15.4 and 5.4 kcal mol− 1. Figure 4 shows the binding gesture of RetroMAD1™ and Neobavaisoflavone on catalytic site of PLpro. We found that RetroMAD1™’s MAP30 compound interacted with two (His272, Asp286) of three PLpro catalytic binding site residues while Neobavaisoflavone was found to interact with two (Cys-111, His272) of the three key residues of active site. Considering this, RetroMAD1™ might be a potential inhibitor to PLpro that prevent the function of PLpro.
RetroMAD1™ Inhibit SARS-CoV-2 Infection
To demonstrate RetroMAD1™’s antiviral properties in a more physiologically relevant model for infection of human cells, we used the EpiAirway™ Model, which consists of normal, human derived TBE cells cultured to form a multi-layered, highly differentiated model that closely resembles respiratory epithelial tissue. On human-derived TBE cells, no microscopic cytotoxicity was seen at any of the tested RetroMAD1™doses (microscopy images not shown). RetroMAD1™’s antiviral efficacy in preventing SARS-CoV-2 infection in human-derived TBE cells was evaluated by assessing its inhibitory impact on SARS-CoV-2 at different concentrations and comparing it to Remdesivir's efficacy (Table 4).
Table 3: Docking scores of RetroMAD1™ in complex with SARS-CoV-2 PLpro
Notes: Residues of active sites are bolded.
Table 4. Antiviral efficiency of RetroMAD1™ against SARS-CoV-2 .
|
Test Compounds |
Concentration (µg/mL) |
aLog10 CCID50 virus per 0.2 mL |
bEC90 (µg/mL) |
|
RetroMAD1™ |
400 |
2.5 |
157 |
|
300 |
2.67 |
||
|
200 |
3.5 |
||
|
150 |
3.67 |
||
|
100 |
3.67 |
||
|
75 |
4 |
||
|
50 |
4.3 |
||
|
37.5 |
4 |
||
|
Remdesivir |
1 |
1.67 |
0.006 |
|
0.1 |
2.67 |
||
|
0.01 |
3.5 |
||
|
0.001 |
3.67 |
||
|
Virus Control |
NA |
4.4 |
NA |
Notes: The data representing the mean of three biological replicates. Each well was scored positive for virus if any CPE was observed as compared with the uninfected control. Vero-76 cells were scored on day 7 and confirmed on day 10.
aTiter results from the virus yield reduction assay.
bEC90 = 90% effective concentration (concentration to reduce virus yield by 1-log10) determined by regression analysis.
NA=not applicableWhile the primary cytotoxicity screen on TBE cells revealed that RetroMAD1™was safe on human cells at high concentrations tested, 300 to 400µg/ml, the viral yield reduction assay in Vero-76 cells using virus-containing supernatant revealed that RetroMAD1™at those concentrations efficiently inhibited SARS-CoV-2 replication, with log10 CCID50 of 2.67 and 2.5, respectively (Table 4: highlighted in light grey). In the absence of any anti-viral compound, cells exposed to infection media yielded log10 CCID50, 4.4. Overall, these data in physiologically relevant human bronchial cell model show that RetroMAD1™effectively reduced viral reproduction by 41.2 percent, which is equal to Remdesivir's capability to suppress SARS-CoV-2 replication at 0.1 µg/ml (Table 4: highlighted in dark grey). An analysis of multiple concentration-response yielded an EC90 of 157 µg/ml for RetroMAD1™ to reduce SARS-CoV-2 infectivity by 1-log10.
RetroMAD1™ inhibit 3CLpro enzyme activity
To further evaluate the mechanism of action of RetroMAD1™ in inhibiting SARS-CoV-2 replication, we examined the inhibitory activity of our compound and compared to GC376, a protease inhibitor of the 3CLpro enzyme (Table 5). An unpaired t-test with Welch’s correction statistical analysis on the 3CLpro enzyme inhibition by RetroMAD1™ at tested concentration of 128, 160, 200, 300 and 400 µg/ml (Table 5: highlighted dark grey) independently compared to GC376 at 500µM (Table 5: highlighted light grey) found no significant differences in inhibiting the 3CLpro enzyme activity. A concentration-response of 3CLpro activity observed with RetroMAD1™showing an IC50 of 86.89µg/ml (Fig. 5).
Table 5: 3CLpro enzyme Inhibition by RetroMAD1™
|
Test Compounds |
Concentration |
3CLpro Inhibition (%) |
SD |
Unpaired t test with Welch's correction, p-value |
|
GC376 |
500μM |
101.35 |
1.08 |
|
|
RetroMAD1™ |
33.55μg/ml |
28.35 |
1.60 |
<0.0001* |
|
41.94μg/ml |
22.29 |
19.65 |
0.0197 * |
|
|
52.43μg/ml |
24.48 |
6.75 |
0.0021 * |
|
|
65.54μg/ml |
28.79 |
4.14 |
0.0006 * |
|
|
81.92μg/ml |
51.29 |
5.72 |
0.0033 * |
|
|
102.4μg/ml |
91.03 |
1.59 |
0.0013 * |
|
|
128μg/ml |
99.63 |
1.47 |
0.1842 |
|
|
160μg/ml |
98.19 |
1.86 |
0.0787 |
|
|
200μg/ml |
101.13 |
1.12 |
0.8173 |
|
|
300μg/ml |
103.44 |
0.60 |
0.0581 |
|
|
400μg/ml |
104.30 |
1.47 |
0.0531 |
Notes: The data representing the mean of three biological replicates. Unpaired t test with Welch's correction statistically was analysed between GC376 and RetroMAD1™ at each tested concentration. *= P<0.05 indicates statistically significant.
RetroMAD1™ complexes with Omicron XBB 1.5
To investigate whether if RetroMAD1™ is a relevant anti-viral drug to combat recently evolved “kraken” Omicron XBB 1.5, we retrieved nucleotide sequence of EPI_ISL_16993645 classified as Omicron XBB 1.5 from publicly open database GISAID (https://gisaid.org/) (Shu and McCauley, 2017) on Feb 23,2023. Its Spike 3D structure modelled using SWISS-MODEL server, applying the most fitted protein template, SARS-CoV-2 Omicron BA.2 variant (7XIW.1. A). We then performed similar docking study described above. Result shows the HDOCK docking score was − 333.26, hence slightly better complexion form than the Wuhan-Hu-1 Spike RBD- RetroMAD1™ shown in Table 1.
The present COVID-19 remains as a global challenge and thus highlights the crucial need for the development of both effective vaccinations and treatments against evolved variant. Among SARS-CoV-2 viral proteins, spike S protein, 3CL protein and PL protein play essential role in viral infection and replication. The S protein is a structural protein. Interaction of SARS-CoV-2 spike protein with its host cellular receptor, the angiotensin-converting enzyme 2 (ACE2) mediates viral uptake and fusion into the cells [51]. During virus infection, cellular furin cleaves S protein at subunit 1 (S1) and subunit 2 (S2) cleavage site. S1 contains receptor-binding domain (RBD) which binds directly to the ACE2, while S2 responsible for membrane fusion [52]. Therefore, interfering the binding of RBD with ACE2 receptor is most straightforward strategy to inhibit virus infection. On the other hand, 3CLpro and PLpro are non-structural proteins. 3CLpro cleaves at least 11 sites on large viral polyproteins that are required for replication and transcription [53, 54]. PLpro can affect the innate immune response by cleaving ubiquitin and interferon-stimulated gene [55]. Based on their importance, S protein, 3CLpro and PLpro have been targeted by many research groups that seeking effective treatment to neutralize SARS-CoV-2 infection [48, 56].
Many plants and animals-derived proteins have been identified to exhibit antiviral activity. Here, we investigated the effect of a chimeric protein in which its compounds Retrocyclin-101, MAP-30 and Dermaseptin-S1 derived from rhesus monkeys, bitter gourd Momordica charantia and Hylid frog’s skin secretion, respectively. RetroMAD1™was invented to express three anti-viral peptide fusion protein that enable synergistical effect in anti-viral activity. Since 2020, RetroMAD1™ has been commercialized worldwide for therapeutic used against FeLV and FCoV infection in cats. Our docking study showed that RetroMAD1™ has a stronger affinity for Spike, 3CLpro and PLpro compared to their natural receptor or drug control. Previous study has reported that inhibition of Spike-mediated membrane fusion through Retrocyclin-101 that bind to viral Spike protein hence inhibit its interaction with ACE2 [16]. While Dermaseptin-S1 is not only able to bind to Spike protein, but also able to alter its conformation, promoted its unfolding and precipitation, thus negatively affected Spike/ACE2 interaction at early stage of viral entrance [16, 57]. Although currently there is lacking of study of MAP30’s effectivity in inhibiting SARS-CoV-2, our docking result suggest that MAP30 effectively binds to the SARS-CoV-2 proteins.
Recognizing that the Spike protein is required for vaccine design, the mutation frequency of the Spike protein in each emergent SARS-CoV-2 variant such as E484K, N501Y, G446S, G339D, N440K, S371L, and K417N are associated with immune escape, posing a challenge to COVID-19 vaccines in combating viral infection [58, 59, 60]. While specific vaccine effectiveness estimates against Omicron XBB 1.5 are unknown, a warning from WHO indicate, this variant may have the highest immune escape to date [61]. As a result, RetroMAD1™ benefited as a therapeutic therapy for COVID-19 patients. Despite being subjected to these evolutionary processes, it is envisaged that the Spike protein will be inactivated and made non-infectious as RetroMAD1™ directly attach to the RBD of viral Spike protein, hence preventing the entry of all SARS-CoV-2 variants.
In addition, our in vitro data from human derived TBE cells also suggested that high potency of RetroMAD1™ at 300 to 400µg/ml against SARS-CoV-2 (Wuhan-Hu-1) infection. The effectiveness of RetroMAD1™ shown to be at the same rate as Remdesivir at 0.1 µg/ml, the only drug that is approved by the Food and Drug Administration (FDA) for the treatment of COVID-19. No cytotoxicity was seen at the highest RetroMAD1™ concentration given of 400ug/ml on human derived TBE cells. However, higher concentration would be necessary to specify an actual CC50, if any cytotoxicity can be identified.
Like other anti-viral therapies, a major challenge that may militate against the widespread use of antiviral drug such as Remdisivir is the possible development of resistance against evolved variant of SARS-CoV-2 [62, 63]. The feasibility of this has been shown by Agostini et al. (2018) [64] which reported the EC50 of remdesivir need to be increased from 0.01 to 0.06 µM in evolved variant of SARS-CoV cultures carrying mutations within nonstructural proteins 12 (nsp12) of viral RdRp. Thus, a combination of repurposed drugs that could reduce the risk of drug-resistance and increase therapeutic efficacy to facilitate progression into clinical trials would be a better option [65]. This brought advantage to RetroMAD1™ as it is unlikely allow the development of viral resistance due to its distinct modes of actions against broad spectrum of viruses.
In this article, docking study of RetroMAD1™ in complex with SARS-CoV-2 viral proteins including spike, 3CLpro and PLpro was conducted. Each docking model suggested that RetroMAD1™ has a high affinity to SARS-CoV-2 proteins. Additional, in vitro study on the normal, human derived TBE cells strongly ensure the candidacy of RetroMAD1™as a promising antiviral therapy option for COVID-19. Overall, the results suggested that RetroMAD1™ could be a potential inhibitor bind to SARS-CoV-2, and highly relevant despite of high evolutionary rate of Omicron variant. Further extensive clinical research and clinical trials are suggested.
Acknowledgements
We would like to extend our gratitude to Dr. Kie-Hoon Jung, the Study Director at the Institute for Antiviral Research, Utah State University, Logan, Utah, United States of America, for providing the study report on the antiviral efficacy of RetroMAD1™ against SARS-CoV-2 in human-derived tracheal/bronchial epithelial cells. Additionally, we would like to acknowledge Professor Dr. Ivy Chung, Director of Cytus SDN. BHD., Malaysia, for providing the report on RetroMAD1™ inhibition of SARS-CoV-2 using the 3CL Protease Untagged (SARS-CoV-2) Assay Kit.
Author contributions
CLC and ASMY performed the docking analysis, visualized the figures, conducted formal analysis, summarized all findings, and composed the main manuscript text. ASMY and AMSAB conceptualized the study design. SS and RSRY confirmed the DNA sequence of the plasmid containing RetroMAD1™. TCL modelled the 3D structure of RetroMAD1™. All authors reviewed the manuscript.
Funding
This research was funded by Biovalence Technologies Pte. Ltd.
Competing interests
CLC and ASMY are employees of Biovalence SDN. BHD. AMSAB is an inventor of RetroMAD1™. SS, RSRY, and TCL have declared no conflicts of interest with regard to this publication involving RetroMAD1™.
Data availability
The datasets generated and/or analysed during the current study are available in the GenBank repository, BankIt2688861 RetroMAD1 OQ714810.
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Competing interest reported. CLC and ASMY are employees of Biovalence SDN. BHD. AMSAB is an inventor of RetroMAD1™. SS, RSRY, and TCL have declared no conflicts of interest with regard to this publication involving RetroMAD1™.