A new global pandemic disease named COVID-19 has emerged and is still spreading at alarming rates at the time of this report. COVID-19 can cause severe symptoms such as damaging inflammatory response, fever, or severe respiratory illness and lead to death. The causative agent of COVID-19 was found to be a novel coronavirus closely related to the severe acute respiratory syndrome coronavirus (SARS-CoV) based on the latest phylogenetic analysis [1
]. There are some major essential differences in their genetic makeup that led to their different behaviors. SARS-CoV-2, as it is called now, appears to have high transmissibility from person to person, and antibodies that could inhibit SARS-CoV are not functional on SARS-CoV-2 [2
]. Despite global efforts, we still lack an effective antiviral strategy, drug, or vaccine to fight this virus, with the growing fear that SARS-COV-2 may become another endemic virus in our communities.
To lower the costs and speed up the drug discovery phase, numerous researchers have used in silico tools such as protein–ligand docking software to screen for traditional compounds that could bind to and inhibit the key proteins present in SARS-CoV-2, highlighting their potential antiviral activity [6
]. The major targets for these compounds include SARS-CoV-2 key proteins 3-chymotrypsin-like protease (Mpro), papain-like protease (PLpro), RNA-dependent RNA polymerase (RdRp), small envelope protein (E), membrane protein (M), and spike (S) proteins. The S proteins directly interact with human angiotensin-converting enzyme (ACE2), allowing the virus to enter the cells. The S protein is a class I fusion protein consisting of S1 and S2 domains with the receptor-binding domain (RBD) located on the S1 domain [4
]. The RBD is the main target of antibodies and fusion inhibitors in development such as the human convalescent COVID-19 patient-origin B38 antibody (B38) and plant lectin griffithsin (GRFT). Here, we report the in silico potent binding mechanisms against SARS-CoV-2 key proteins of a previously discussed novel broad-spectrum anti-infective fusion protein between a mutant of the ricin A chain and pokeweed antiviral protein isoform 1 (RTAM-PAP1) from seeds of Ricinus communis
and leaves of Phytolacca americana
, respectively [7
]. RTAM-PAP1 activity was compared with that of the B38, ricin A chain (RTA), pokeweed antiviral protein isolated from leaves (PAP1), and GRFT. Their binding capacities were evaluated against the major key proteins of SARS-CoV-2 using the latest peptide-ligand docking software [8
The three-dimensional (3D) structure of RTAM-PAP1 prediction was obtained as previously described [7
], and those of RTA, PAP1, B38, and GRFT were retrieved in protein data bank (PDB) format from the Research Collaboratory for Structural Bioinformatics (RCSB) website (https://www.rcsb.org/
). A knowledge-based scoring docking prediction was performed for all the compounds against S, S1 RBD, and M using CoDockPP global docking. An additional run was conducted for ACE2 and human SARS-CoV antibody CR3022 against S1 RBD as a reference [5
]. The 3D structures of all the key proteins and ACE2 were already available from the software site in this “COVID-19 targets docking only” version. The peptide/antibody–ligand version was used, as small molecules docking software is not suited for these types of compounds. The generated 3D models of B38, ACE2, and CR3022 bound to S1 RBD were comparable to available crystallography of the same complexes in RCSB (access: 7BZ5, 6M0J, and 6W41, respectively) with respective root mean square deviation (RMSD) varying from 0.7 to 4.311 (A), 0.121 to 2.196 (A), and 0.058 to 3.206 (A). However, the greater binding affinity and fusion inhibiting activity of B38 compared to CR3022 for S1 RBD was observed in accordance with published in vitro results [1
]. B38 was found to have a dissociation constant of 70.1 nM with complete inhibition of ACE2 binding to S1 RBD compared to CR3022′s dissociation constant of 115 nM with no inhibition of ACE2 binding. The difference in inhibition of ACE2 binding to S1 RBD is due to their binding conformation to S1 RBD. However, ACE2 binding to S1 RBD was found to have the smallest dissociation constant in literature, with a value ranging from 4 to 15 nM. The results for the first and last models (out of the top 10 generated) of each compound in complex with S, S1 RBD, and M are presented in Table 1
. B38 has the highest overall binding affinity of the lot with a binding energy ranging from −449 to −300 kcal/mol, as expected. ACE2′s binding energy was between −314 to −246 kcal/mol for S1 RBD. RTAM-PAP1 is comparable to B38, with an overall higher binding affinity (lower binding energy) than all of the other compounds tested against the S, S1 RBD, and M key proteins, sometimes higher than B38 with −469 kcal/mol for M, for example. The high binding affinity of RTAM-PAP1 and B38 to S, S1, and M may be explained by the M epitope being very similar in structure to S1 RBD (Figure 1
]. RTA binding affinity is similar to RTAM-PAP1 to a certain extent and GRFT and PAP1 are very comparable.
The same higher binding affinity behavior for RTAM-PAP1 was observed with Mpro, PLpro, E, and RdRp when compared to PAP1, GRFT, and RTA (Table 2
). All of the tested compounds showed potentially inhibiting binding conformations to the various key proteins based on the 3D structures of the complexes formed (results not shown). These results indicated that the fusion between RTAM and PAP1 allowed RTAM-PAP1 to be more stable across the different possible binding conformations with a higher binding affinity than either of its moieties alone when in complex with SARS-CoV-2 key proteins.
B38 was found to have a 50% inhibition of the cytopathic effect (EC50) against SARS-CoV-2 simultaneous infection in Vero cells in vitro at the concentration of 0.177 µg/mL. It was further demonstrated that B38 was effective in mice post-infection [1
]. GRFT was found to have low pre-infection EC50 on different strains of SARS-CoV in cytoprotection (CPE) assays in vitro (0.6–1.2 µg/mL) and effective in mice pre-infection [14
]. RTA was shown in the literature to have a high binding affinity to many viral proteins [15
]. PAP1 has a broad range of antiviral activity against numerous infections both in vitro and in clinical trials [17
]. An earlier different version of RTAM-PAP1 was shown to have potent broad range antiviral activity at low post-infection EC50 (0.002–12.3 µg/mL) against human immunodeficiency virus-I (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), Zika virus (Zika), and human coronavirus 229E (HCoV229E) in CPE assays in vitro [7
]. RTA and PAP1 produce a drastic increase in viral inhibition activity if administered pre-infection both in vitro and in vivo at sub-toxic dosages [20
], with potent antiviral mechanisms, from viral DNA/RNA depurination, viral proteins synthesis inhibition, viral cell entry inhibition, to apoptosis induction of infected cells via a preferential virus-infected cell entry mechanism [7
This high affinity of RTAM-PAP1 to many key proteins of SARS-CoV-2 is uncommon. Yet, the most surprising part of the generated models was the discovery of unique binding mechanisms of RTAM-PAP1 with potential inhibiting activity by hindering viral entry and cellular machinery. This discovery might explain the previously observed gain of function of RTAM-PAP1 [7
] via the acquired ability to simultaneously bind the target with both moieties with high affinity, i.e., increasing the docking sites from 86 to 102 for single moiety binding and simultaneous binding to S1 RBD, for example. To confirm these findings, RTAM-PAP1 was run against SARS-CoV-2 S1 and M using different docking programs (ZDOCK and HADDOCK2.2) with the known active residues in RCSB. The synergetic binding of RTAM-PAP1 was confirmed, and the generated models for M are shown in Figure 1
B–D. Although the model generated by HADDOCK2.2 returned a more important role for PAP1 than RTAM, the simultaneous binding of both moieties can clearly be seen when in complex with M, with an increase in docking sites from 62 for single moiety binding to 96 for simultaneous binding of both moieties (ZDOCK model). This might significantly increase RTAM-PAP1′s potential anti-SARS-CoV-2 activity. We concluded from these results and those previously acquired in vitro that the fusion of RTAM and PAP1 via the flexible linker conferred greater structure stability, enhanced activities, new binding sites and mechanisms, and, potentially, novel functions to RTAM-PAP1.
For those reasons, the decision to produce highly purified RTAM-PAP1 protein was taken to conduct a short toxicity study in BALB/c mice to determine the potential maximum tolerated dose.
The protein production went well and followed a scheme previously used [7
] with the addition of an endotoxins removal step after purification, as shown in Figure 2
A. Highly purified 6-His tag RTAM-PAP1 was obtained (>95% purity), as shown in Figure 2
B. The bioactivity of the proteins was confirmed using a cell-free protein synthesis inhibition assay at three different concentrations in duplicate and yielded a half maximal inhibitory concentration (IC50) of 0.06 nM at 60 min incubation time, in line with previous results [7
] confirming the time- and concentration-dependent inhibitory activity of RTAM-PAP1 on protein synthesis (data available upon request).
The mice were administered the highly purified RTAM-PAP1 with the 6-His tag and tolerated up to 1 mg/kg with no observable adverse effects. Adverse clinical signs were observed (i.e., weight loss, piloerection, etc.) at a single bolus intravenous administration of 3 mg/kg of RTAM-PAP1 with up-regulation of IP-10, KC, and MCP-1 chemokines from 14 cytokines/chemokines assessed (Figure 3
). These results are in line with previously described homopolymers of ribosome-inactivating proteins and confirm an in vivo behavior intermediate between native ribosome-inactivating proteins and immunotoxins [24