The ribosome-inactivating proteins MAP30 and Momordin inhibit SARS-CoV-2

Norman R Watts; Elif Eren; Ira Palmer; Paul L Huang; Philip Lin Huang; Robert H Shoemaker; Sylvia Lee-Huang; Paul T Wingfield

doi:10.1371/journal.pone.0286370

. 2023 Jun 29;18(6):e0286370. doi: 10.1371/journal.pone.0286370

The ribosome-inactivating proteins MAP30 and Momordin inhibit SARS-CoV-2

Norman R Watts ^1,^*,^#, Elif Eren ^1,^#, Ira Palmer ¹, Paul L Huang ², Philip Lin Huang ², Robert H Shoemaker ³, Sylvia Lee-Huang ^4,^*, Paul T Wingfield ¹

Editor: Israel Silman⁵

PMCID: PMC10310010 PMID: 37384752

Abstract

The continuing emergence of SARS-CoV-2 variants has highlighted the need to identify additional points for viral inhibition. Ribosome inactivating proteins (RIPs), such as MAP30 and Momordin which are derived from bitter melon (Momordica charantia), have been found to inhibit a broad range of viruses. MAP30 has been shown to potently inhibit HIV-1 with minimal cytotoxicity. Here we show that MAP30 and Momordin potently inhibit SARS-CoV-2 replication in A549 human lung cells (IC₅₀ ~ 0.2 μM) with little concomitant cytotoxicity (CC₅₀ ~ 2 μM). Both viral inhibition and cytotoxicity remain unaltered by appending a C-terminal Tat cell-penetration peptide to either protein. Mutation of tyrosine 70, a key residue in the active site of MAP30, to alanine completely abrogates both viral inhibition and cytotoxicity, indicating the involvement of its RNA N-glycosylase activity. Mutation of lysine 171 and lysine 215, residues corresponding to those in Ricin which when mutated prevented ribosome binding and inactivation, to alanine in MAP30 decreased cytotoxicity (CC₅₀ ~ 10 μM) but also the viral inhibition (IC₅₀ ~ 1 μM). Unlike with HIV-1, neither Dexamethasone nor Indomethacin exhibited synergy with MAP30 in the inhibition of SARS-CoV-2. From a structural comparison of the two proteins, one can explain their similar activities despite differences in both their active-sites and ribosome-binding regions. We also note points on the viral genome for potential inhibition by these proteins.

Introduction

The continuing emergence of SARS-CoV-2 variants capable of eluding monoclonal antibody therapies, conventional and mRNA vaccines, and the human immune response to infection, has highlighted the importance of identifying additional means of intervention.

Ribosome-inactivating proteins (RIPs) have repeatedly been shown to inhibit a broad range of plant and animal viruses including double-stranded DNA viruses, positive-sense single-stranded RNA viruses, negative-sense RNA viruses, and retroviruses, and to therefore have therapeutic potential (for reviews see [1–6]; for a critique see [7]). Saporin, an RIP derived from soapwort (Saponaria officinalis) has recently been proposed as a therapeutic for SARS-CoV-2, a positive-sense single-stranded RNA virus [8], as has RTAM-PAP1, a fusion protein of the Ricin A-chain and Pokeweed Antiviral Protein isoform 1, obtained from Ricinus communis and Phytolacca americana, respectively [9].

RIPs are RNA N-glycosylases [EC 3.2.2.22] that depurinate eukaryotic and prokaryotic rRNAs, thereby arresting protein synthesis. Some also possess DNA glycosylase activity. RIPs are structurally related but broadly divided into two classes; Type I RIPs composed of a single polypeptide (A-chain) with glycosylase activity, and Type II RIPs composed of a catalytic A-chain and a carbohydrate-binding (lectin) B-chain joined by a disulfide bond. Type I RIPs are present in many plants used for human consumption including rice, maize, and barley and they are generally considered non-toxic when consumed. Type II RIPs are much more toxic due to presence of the B-chain which facilitates cell entry [6, 10, 11].

The Type I RIPs MAP30 and Momordin (54.5% sequence identity over residues 1–244) (Fig 1), both derived from bitter melon (Momordica charantia), a common food and traditional medicine in Asia and Africa, have both antiviral and anticancer properties [10, 12–19]. MAP30 has been shown effective against HIV-1 with 50% inhibitory concentration (IC₅₀) values in the sub-nanomolar range and a therapeutic index of over 1000 [12, 20]. At least 20 other RIPs also inhibit HIV-1 [6]. MAP30 has further been reported to synergize with Dexamethasone and Indomethacin in the inhibition of HIV-1, lowering their IC₅₀ by more than a 1000-fold to 10⁻⁷ and 10⁻⁸ M, respectively [20, 21]. Dexamethasone, a corticosteroid, interacts with eukaryotic initiation factor 4E (eIF4E) [22] and reduces inflammation and mortality in Covid-19 patients [23]. Indomethacin, a non-steroidal anti-inflammatory, has also proven beneficial for Covid-19 patients [24]. MAP30, like other RIPs, is an RNA N-glycosylase that specifically depurinates the first adenine (A-4324) in the α-sarcin-ricin recognition loop (SRL; CGAGAG) of the 28S rRNA of mammalian ribosomes, preventing binding of the elongation factor EF2, mRNA-tRNA translocation, and protein synthesis [25–27]. Precisely how MAP30 engages ribosomes is not yet known. In the case of Ricin, ribosome inactivation first involves the active-site-distal residues R193 and R235 (corresponding to K171 and K215 in MAP30) in engaging the ribosomal stalk P-proteins [28], and then the active-site residue Y80 (Y70 in MAP30) in cleavage of the A-4324 glycosidic bond [10]. An 11-residue peptide (SDDDMGFGLFD) in the C-termini of P-proteins (termed c11-P), normally involved in the binding of elongation factors, has been identified in the binding of Trichosanthin (TCS), an RIP obtained from Chinese cucumber (Trichosanthes kirilowii) [29].

Viral inhibition by RIPs may also be due to depurination of other polynucleotides including DNA, poly-A, mRNA, tRNA, and the viral genome [30, 31]. In the case of SARS-CoV-2, this would be a positive-sense single-stranded RNA [32]. As abasic RNAs are resistant to cleavage, the modified 3D structure of the genome may well affect its translation and other interactions in the cell [8]. MAP30 and α-Momocharin, another RIP present in bitter melon, can cause topological inactivation of HIV-1 DNA [33, 34]. Saporin has been proposed to induce apoptosis by various means including DNA damage; PRP1 activation, NAD+ depletion, and mitochondrial damage; rRNA depurination, ribotoxic stress, and MAPK activation; and without rRNA depurination by binding to the cell surface chemokine receptors CCR1, CCR2B, CCR3, CCR4, CCR5, and CXCR4 [8]. More recently, in silico docking studies have also predicted direct interactions between RIPs and the SARS-CoV-2 Spike, Mpro, PLpro, RdRp, and E proteins [9]. In addition to the various cellular and viral targets mentioned above, MAP30 may also inhibit SARS-CoV-2 in another way. Specific peptides from both MAP30 and Momordin, that have been shown to reduce hypertension in a rat model and to competitively inhibit angiotensin-converting enzyme (ACE), a homolog of ACE2 [35] and the key receptor for SARS-CoV-2, have the potential to block viral entry. During our earlier structure determination of MAP30 by means of nuclear magnetic resonance spectroscopy [10] it also became evident that MAP30 is an exceptionally robust protein. As MAP30 is present in a widely consumed food, and as we had also previously found MAP30 to be rather non-toxic, due to an apparent inability to enter uninfected cells [36], these observations all initially suggested that it might be developed as an aerosolized SARS-CoV-2 inhibitor.

In this study, we have assessed MAP30 and Momordin inhibition of SARS-CoV-2 in a nanoluciferase reporter virus (NLRV) assay using A549 human lung cells expressing ACE2. We have assessed both proteins, appending a Tat cell-penetration peptide to each, synergy with Dexamethasone and Indomethacin, and mutating residues in both the active-site and the presumptive ribosome-binding-site of MAP30. We observed similar potent viral inhibition by both proteins with minimal toxicity at the active concentration. Structural analysis of the two proteins can explain their consistently similar activities despite substantial differences in sequence. We also identify sites on the viral genome as potential points for inhibition by these proteins.

Materials and methods

Recombinant protein production

MAP30 and variants were all well expressed in E. coli. Wild type protein and the mutants MAP30.D43A, MAP30.Y70A, and MAP30.K171A, K215A were purified as previously described using ion-exchange chromatography and gel filtration (S1 Fig) [10]. For the double mutant, some adjustments in pH of ion-exchange columns were required due to a shift in protein isoelectric point. MAP30 with a C-terminal his-tag (MAP30-hist) was purified using Ni-Sepharose chromatography and gel filtration. MAP30 with a C-terminal Tat cell-penetration peptide (GRKKRRQRRRPQ) required urea extraction from bacterial cell extracts and 4 M urea was included in chromatography steps. The urea was removed by step wise dialysis (3, 2, 1 and 0 M urea) in PBS. Momordin and Momordin-Tat (expressed mostly soluble) were purified using the same procedure as MAP30. All proteins were single peaks on gel filtration columns and were concentrated using Amicon Ultra-4, 10,000 NMWL centrifugal filtration units and sterile filtered using Whatman Anotop 10, 0.1 μm filters prior to use. Soluble recombinant human ACE2 (APN01) was provided by Apeiron Biologics.

Analytical centrifugation

A Beckman Optima XL-I analytical ultracentrifuge, absorption optics, an An-60 Ti rotor and standard double-sector centerpiece cells were used. Sedimentation equilibrium measurements at 20°C were made at 17,500 rpm for MAP30 and 11,000 rpm for soluble ACE2 receptor with data collection every 4 h to 16 h with overspeed at 40,000 for 4 h to establish a baseline. Partial specific volumes (ν-bar), calculated from the amino acid compositions, and solvent densities estimated using the program SEDNTERP (http://www.rasmb.bbri.org/).

Circular dichroism

Circular dichroism and thermal denaturation measurements were made using a Jasco-715 spectropolarimeter equipped with a PTC-343W1 Peltier-type thermostatic cell holder. Far-UV spectra were recorded at ~ 1 mg/ml protein concentration using 0.02 cm pathlength cells. Temperature response was monitored at 222 nm using a 1-cm path-length cell with a Teflon stopper (Hellma). Cooling circulating water was supplied using a Neslab RTE-100 thermostatic circulator. Proteins (~ 0.1 OD 280 nm) were heated at 1–2°C per minute with a temperature slope of 20–90°C. The step resolution was 1°C, the response time 1 sec, the bandwidth 2 nm, and the sensitivity 100 mdeg. Temperatures at the transition midpoints, i.e., the melting temperature (T_m), were estimated from first derivative plots of the melting curves.

MAP30 interaction with soluble ACE2

MAP30-hist was immobilized on a HiTrap IMAC Sepharose FF column (GE Healthcare) in PBS buffer. Soluble recombinant human ACE2 receptor (APN01), obtained from Apeiron Biologics, was loaded, and the column was washed with buffer then eluted with PBS plus 0.3 M imidazole. Alternatively, the MAP30-hist and ACE2 were mixed, loaded on the column, washed with buffer then eluted with imidazole. Under both conditions, the ACE receptor was in the wash fractions, indicating no interaction with the immobilized MAP30 protein. In another approach, the MAP30 and ACE2 proteins were mixed and applied to a Superdex 200 gel filtration column (GE Healthcare). The proteins eluted separately and according to their molecular weights 184,000 (dimeric ACE2) and 30,000 (monomeric MAP30) with no interaction complexes detected.

Molecular dynamics simulations

MD simulations were carried out using the Desmond-Maestro simulation package (Schrödinger Release 2021–4). Protein and ligand were prepared by Protein Preparation Wizard, and the optimized potentials for liquid simulation force field (OPLS_2005) parameters were used in restraint minimization and system building [37]. The system was set up for simulation using a predefined water model (TIP3P) as a solvent. The electrically neutral system for simulation was built with 0.15 M NaCl in 20 Å buffer. The NPT ensemble with 300°K, and a pressure of 1 bar was applied in the run. The simulation was performed for 250 ns, and the trajectory sampling was done at an interval of 5 ps. The short-range coulombic interactions were analyzed using a cutoff value of 9.0 Å using the short-range method. The smooth particle mesh Ewald method was used for handling long-range coulombic interactions. The stability of MD simulation was monitored by RMSD of the ligand and protein atom positions in time. Hydrogen-bond and salt-bridge formation was analyzed using Visual Molecular Dynamics (VMD) [38].

Model assessment

Modeled protein-peptide interfaces were examined with PDBePISA [39]. Protein stabilities were calculated with FoldX/Yasara [40]. RNA 2D structure predictions were made with RNAfold [41]. RNA 3D structure predictions were made with 3dRNA [42]. Molecular illustrations were prepared with UCSF Chimera [43].

Cell culture

A549 cells expressing ACE2 (obtained from Ralph Baric at UNC) [44] were grown in DMEM high glucose supplemented with 20% (HI) FBS, 1% NEAA, 100 μg/ml Blasticidin and split 1:6 every three days. Blasticidin was removed from the media one passage before using the cells in the assay. On the day of assay, cells were harvested in assay medium (DMEM supplemented with 2% HI FBS, 1% HEPES, 1% Pen/Strep). Total cell number and percent viability determinations were performed using a Luna cell viability analyzer (Logos Biosystems) and trypan blue dye exclusion. Cell viability greater than 95% was required for the cells to be utilized in the assays.

Sample preparation

The stock solution was serially diluted 2-fold resulting in 10 concentrations in a 384-well plate, each at 6X of the final assay concentration. Dilutions were performed in assay medium. A 5 μL aliquot was taken from each well containing compound and transferred to corresponding wells of the assay plate (384 well Corning #3764). Two plates were prepared for the anti-viral assay and one plate for the cytotoxicity assay. The plates containing samples for the anti-viral assay and the cell suspension were passed into the BSL-3 facility.

Antiviral assays

Assays were performed applying high-throughput methods in 384-well plates [45, 46].

A549 nanoluc reporter virus (NLRV) assay

The day of each assay, a pre-titered aliquot of virus (USA_WA1/2020 modified to express nanoluciferase, received from R. Baric at UNC) [47] was removed from the freezer (-80°C) and allowed to thaw to room temperature in a biological safety cabinet. A working stock of SARS CoV-2 nanoluciferase reporter virus (NLRV) was diluted 1000-fold in media containing 160,000 A549 cells per mL resulting in an MOI of approximately 0.02. Cells and virus were stirred at 200 RPM for at least 10 minutes. A 25 μL aliquot of virus-inoculated cells (4000 A549 cells/well) was added to each well in columns 3–24 of the assay plates. The wells in columns 23–24 did not contain test compounds, only virus-infected cells for the 0% inhibition controls. Prior to virus inoculation, a 25 μL aliquot of cells was added to columns 1–2 of each plate for the cell-only 100% inhibition control (no virus, no compound). After incubating plates at 37°C/5% CO₂ and 90% humidity for 72 hours, 30 μL of NanoGlo (Promega #N1620) was added to each well according to the manufacturer’s instructions with the following modifications: the substrate was diluted 1/500 and plates were incubated for 20 minutes prior to the read. Plates were sealed with a clear cover and surface decontaminated prior to removal from the hood. Luminescence was read using a BMG CLARIOstar plate reader (bottom read) to measure luciferase activity as an index of virus titer.

Cytotoxicity assay

Compound cytotoxicity was assessed in a BSL-2 counter screen as follows. Host cells in media were added in 25 μL aliquots (4,000 cells/well) to each well of assay ready plates prepared with test compounds as described above. Cells only (100% viability) and cells treated with hyamine (Sigma cat# 1622) at 100 μM final concentration (0% viability) served as the high and low signal controls, respectively, for cytotoxic effect in the assay. DMSO was maintained at a constant concentration for all wells as dictated by the dilution factor of stock test compound concentrations. After incubating plates at 37°C/5% CO2 and 90% humidity for 72 hours, 30 μL Cell Titer-Glo (Promega) was added to each well. Luminescence was read using a BMG PHERAstar plate reader following incubation at room temperature for 10 minutes to measure cell viability.

Data analysis

For all assays the raw data from plate readers were imported into ActivityBase v.9.7 (IDBS) where values were associated with compound IDs and test concentrations.

Raw signal values were converted to % inhibition by the following formula:

% inhibition = 100 x abs(test compound value–mean value infected cell controls)/abs(mean value uninfected cell controls–mean value infected cell controls).

For the cell viability assay measuring compound cytotoxicity, % cell viability was calculated as follows:

% viability = 100 x (test compound value—mean low signal control)/(mean high signal control–mean low signal control).

IC₅₀ and CC₅₀ values were calculated from a four-parameter logistic fit of data using the Xlfit module of ActivityBase.

Curated inhibition and cell viability data from Southern Research formatted in MS Excel were imported into OriginPro, Version 2023 (OriginLab Corporation, Northampton, MA, USA). Data plots were analyzed using non-linear curve fitting (Category = Pharmacology; Function = Dose Response).

Matrix assay compound preparation

MAP30 and Dexamethasone, and MAP30 and Indomethacin, were tested in a dual combination design in the A549 NLRV assay.

Each combination consisted of seven concentrations of one compound (cmpd B) across a section of the plate with seven concentrations of the second compound (cmpd A) arranged down the same section of the plate. Serial dilutions were performed to produce a 6X concentrations such that a 5 μL transfer into the assay resulted in the final concentrations indicated (30 μL total assay volume). The anti-viral and cytotoxicity assays were performed in parallel.

Concentration response graphs of compound combinations were produced in GraphPad Prism software package.

For synergy analysis, reduced data with associated concentrations and compound ids were imported into https://synergyfinder.fimm.fi/ which applies models for detection of synergistic interactions. The zero-interaction potency (ZIP) model was applied [48].

Results and discussion

Protein production and characterization

MAP30 and variants were all well expressed in E. coli as soluble proteins and purified by conventional chromatography (S1A Fig). Similarly, for the Momordin proteins, although the expression levels were lower. MAP30 is a very soluble monomeric protein as established by analytical ultracentrifugation (S1B Fig) and all other proteins were also judged monomeric based on their gel filtration behavior. MAP30 and Momordin are α-helix and β-sheet containing proteins with similar structures (Fig 1A and 1B) and this is reflected by qualitatively similar far-UV circular dichroic spectra derived from secondary structure (S1C Fig). MAP30 is a stable protein which was required for structure determinations by NMR where protein is incubated at 40–42°C for prolonged time periods. The direct thermal stability was determined by helix-coil transition monitored by far-UV circular dichroism; an apparent melting temperate (T_m) of 70.5°C was determined which was likely underestimated due to temperature induced irreversible denaturation (S1D Fig) Although MAP30 would not be classified as a thermostable protein based on the determined T_m, the protein has higher than average stability which would be an asset for any potential biomedical formulation [49]. Momordin is also relatively thermostable but less so than MAP30 (S1D Fig).

MAP30 does not interact with ACE2

As indicated above, specific peptides from MAP30 and Momordin have been shown to reduce hypertension in rats, and to inhibit angiotensin-converting enzyme (ACE) [35], a homologue of ACE2, the main receptor for SARS-CoV-2 [50]. We therefore initially considered the possibility that MAP30 might also interact with ACE2 and block viral entry. Our preliminary in silico docking attempts indicated high surface complementarity between two MAP30 monomers and a model of the ACE2 dimer (S2A Fig), with the ACE inhibitory peptides of the proteins reasonably well positioned in the catalytic clefts, further suggesting that MAP30 and Momordin might function as inhibitors of viral entry.

To test for such an interaction between MAP30 and ACE2 we used a recombinant form of human ACE2 (APN01) which we were assaying at the time for tolerability as a potential Covid-19 therapeutic [51]. We first determined the self-association state of each protein by sedimentation equilibrium analysis and found MAP30 to be monomeric and ACE2 to be dimeric (S2B Fig). The proteins were then mixed and assayed by both gel filtration and Ni-Sepharose chromatography, but no evidence of an interaction was observed (S2C and S2D Fig). This indicated that intact MAP30 does not interact with ACE2 and likely would not function as an inhibitor of viral entry.

A549 NLRV assays

The results reported below derive from assays performed over a two-year period beginning with the start of the SARS-CoV-2 pandemic. Due to risk and urgency at the time the viral inhibition assays were performed at Southern Research in Birmingham, AL. Over that period, all the assays were replicated at least once, and for wild-type MAP30 numerous times as it was present in every assay. The replicate assays were performed with independent preparations of the proteins and assayed months apart with independent preparations of the cells.

Inhibition of viral replication was tested in a nanoluciferase reporter virus (NLRV) assay using A549 cells expressing ACE2. Both the reporter virus and the cells had been provided to Southern Research by the laboratory of Ralph Baric (UNC) specifically to assay potential SARS-CoV-2 replication inhibitors. A549 cells are commonly used to model the alveolar Type II pulmonary epithelium. They have been used to study various conditions including cancer, allergies, asthma, and respiratory infections. A549 are adenocarcinomic human alveolar basal epithelial cells and therefore high in the expression of LRP1 (https://www.proteinatlas.org/ENSG00000123384-LRP1/tissue), the putative receptor for MAP30 [52]. It was a concern that A549 cells might take up MAP30 merely because they are transformed [14]. However, MAP30 has been tested in a wide range of human cancer cells including T-cell lymphoma, T-cell leukemia, glioblastoma, and breast, lung, liver, cervical, bladder, gastric, ovarian, and kidney cancer cells [12–14, 18, 20, 53, 54], suggesting that the A549 cells might still be a useful model, particularly for virally infected cells.

Antiviral activity of MAP30 and Momordin

Using the A549 cells we assayed MAP30 and Momordin, each with and without a C-terminally appended Tat cell-penetration peptide, for inhibition of viral replication. There was clear viral inhibition (overall mean IC₅₀ = 5.7 μg/ml or ~ 0.2 μM) with little cytotoxicity (overall mean CC₅₀ = 72 μg/ml or ~ 2.4 μM) (Fig 2; Table 1, Assay 1). These values are in the ranges observed previously, 0.2–10 μM and 3–30 μM for 50% inhibition and toxicity, respectively, for Type 1 RIPs [6]. The IC₅₀ values also compare favorably with Chloroquine in this system (mean IC₅₀ = 4 μM). Interestingly, there was no substantial difference between MAP30 and Momordin, with or without the Tat-CPP. This result suggested that the catalytic (RNA N-glycosylase) activity common to both MAP30 and Momordin might be the essential property. This conclusion was strengthened by the observation that almost all of the 17 residues within 4.5 Å of the catalytic residue Y70, in both proteins, are identical and that the residues Y70, Y109, E158, and R161 responsible for this activity in MAP30 [10], and Y70, Y111, E160, and R163 in Momordin [55], are in the same relative positions. It also suggested that cell entry was mediated by factors other than the Tat-CPP, perhaps LRP1 as proposed recently [52].

Fig 2 — Viral inhibition and cell viability as a function of concentration of MAP30 and Momordin, either without (A and B) or with (C and D) a C-terminally appended Tat cell penetration peptide, as assessed in the A549 NLRV assay. The results from this and other assays are summarized in Table 1.

Table 1. Viral inhibition and cytotoxicity values of MAP30 and Momordin.

Assay^a	Test item	IC₅₀ (μg/ml)^b	CC₅₀ (μg/ml)^c	SI^d
1	MAP30	6.6	100.6	15.2
	MAP30-Tat	5.8	72.6	12.5
	Momordin	5.1	59.3	11.6
	Momordin-Tat	5.3	55.6	10.5
	Chloroquine^e	1.27	>10	>7.9
2 ^f	MAP30	5.9	50.8	8.6
	MAP30 x Dexamethasone^g	9.5	192.7	20.3
	MAP30 x Indomethacin^g	6.5	53.9	8.3
	Chloroquine	1.07	>10	>9.3
3	MAP30	9.6	96.8	10.1
	MAP30.Y70A	>1099	>1099	1.0
	Chloroquine	0.86	>10	>11.6
4	MAP30	7.4	101.7	13.7
	MAP30.K171A, K215A	33.0	318.6	9.7
	Chloroquine	1.37	>10	>7.3

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^a All are A549 human lung cancer cell nanoluciferase reporter virus (NLRV) assays.

^b-d IC₅₀, 50% inhibitory concentration; CC₅₀, 50% cytotoxicity concentration; SI, selectivity index, SI = CC₅₀/IC₅₀. The IC₅₀ and CC₅₀ values given in the text for Assay 1 are the respective overall means for the four proteins.

^e Three reference compounds (Calpain inhibitor IV, Remdesivir, and Chloroquine) were present in all assays. Chloroquine gave the most consistent data: IC₅₀ = 4.1 ± 0.93 μM; CC₅₀ >30.00 μM; n = 7.

^f Matrix format (protein concentration range, 0–80 μg/ml) x (drug concentration range, 0–10 μg/ml).

^g At highest drug concentration (10 μg/ml).

MAP30 and Momordin show no synergy with Dexamethasone and Indomethacin

With the observation that MAP30 inhibited SARS-CoV-2 as effectively as Momordin, either one with or without a Tat-CPP, we asked if MAP30 might synergize with Dexamethasone. In parallel, we also tested Indomethacin as we have previously observed that its inhibition of HIV-1 is also potentiated by MAP30 [20]. Other than a modest increase in the selectivity index value due to additivity with Dexamethasone, no synergy was observed with either compound, either due to differences between HIV-1 and SARS-CoV-2, or differences between the MT-4 lymphocytes used in the earlier study and the A549 lung cells used here (Table 1, Assay 2).

Mutational analyses of MAP30 active- and ribosome-binding sites

We also tested whether the RNA N-glycosylase activity is the essential property of MAP30 responsible for viral inhibition by mutating Y70 in the catalytic site. A MAP30.Y70A mutant was tested in the A549 NLRV assay and found to be completely inactive, showing that this activity is central to SARS-CoV-2 inhibition (Fig 3; Table 1, Assay 3).

Fig 3 — Assessment of SARS-CoV-2 inhibition by MAP30, MAP30.Y70A, and MAP30.K171, K215 in the A549 NLRV assay. The Y70A active-site mutation abrogates both viral inhibition and cytotoxicity. The MAP30.K171, K215 ribosome-binding-site mutations result in an increase of both the IC₅₀ and CC₅₀ values, while the selectivity index (SI = CC₅₀/IC₅₀) remains the same.

Having observed relatively potent viral inhibition, we next sought ways to increase the selectivity index. Ricin A-chain protein synthesis inhibition involves its binding to the ribosomal stalk P-proteins followed by depurination of A-4324 in the SRL of the 28S rRNA, and this binding was shown to involve Ricin-A residues R193 and R235 [28, 56]. To reduce or eliminate MAP30 toxicity due to ribosomal inactivation, while retaining the RNA N-glycosylase activity and potentially restricting depurination to viral targets, such as points on the viral genome, we tested the corresponding MAP30.K171, K215 construct. Disappointingly, the effect of this double mutation was to shift both the IC₅₀ and CC₅₀ to higher values with the result that there was essentially no change in the selectivity index (Fig 3; Table 1, Assay 4). As we subsequently found that in Momordin the corresponding residues are acidic, it appeared that the Ricin paradigm may not strictly apply.

Structural comparison of MAP30 and Momordin

In the viral inhibition assays described above, we compared MAP30 and Momordin because it would provide a natural experiment that might lead to insights into how differences between the two structures can lead to any differences in their selective indices, and how the latter may be increased. However, as MAP30 and Momordin consistently had essentially identical IC₅₀ and CC₅₀ values in the A549 NLRV assays, and as mutation to Ala of residues K171 and K215 in the putative ribosome binding site was ineffective at increasing the selectivity index of MAP30, we sought to better understand their structures.

Inspection of a set of RIPs varying in sequence identity from ca. 23–60% compared to MAP30, and rendered as either hydrophobicity or charge surfaces, shows them to be different in several ways. For example, the active-site side of MAP30 appears overall to be slightly less hydrophobic and more acidic than that of Momordin (Fig 4A–4C, 4D–4F). When the first structure of MAP30 was determined by NMR (PDB: 1D8V), titration with the HIV-1 LTR identified residues around the active-site involved in DNA binding [10]. Interestingly, titration with Mn⁺² and Zn⁺² ions, previously found to be important in buffers for MAP30 and other RIPs to function, led to chemical shift perturbations that indicated the existence of two distinct binding regions for these ions, one on either side of the active-site [10]. However, comparison of the MAP30 and Momordin structures here shows that while both proteins have the DNA and Zn⁺² binding sites, Momordin does not have either of the D43 or E89 residues constituting part of the Mn⁺² binding site, although a third residue (R46), which might contribute to the coordination of the ion, is present in both (Fig 1; Fig 4D–4F). In fact, comparison of the other structures in S1 Table shows that all but Saporin do not have the Mn⁺² binding site.

Fig 4 — (A-C) MAP30, TCS, and Momordin represented as hydrophobicity surfaces (blue, charged; orange, hydrophobic; white, neutral), (D-F) as charge surfaces (blue, basic; red, acidic; white, neutral), and (G-I) with residues scored as important by Evolutionary Trace shown in red. PDB codes and percent sequence identity to MAP30 as indicated. Ricin (PDB: 1APG, 30.5%) and Saporin (PDB: 1QI7, 23.5%) gave similar patterns (not shown). The binding groove with the catalytic site at the center is located between the two arrows in (A). The regions of the previously identified Mn⁺² and Zn⁺² binding areas are indicated in (D).

MAP30 and Momordin active-site faces

To assess the conservation of residues in and around the RIP active-sites we employed the Evolutionary Trace method which combines phylogenetic and entropic information from multiple sequence alignments to provide a measure of their relative importance [57]. Thus, while the overall sequence identity of MAP30 and Momordin is 54.5%, the top 25% of important residues are 87.6% identical, and they are located closely around the catalytic site (Fig 4G–4I). Neither D43 nor E89 were ranked amongst these residues, suggesting that these two residues and the Mn⁺² site may not play a role in all MAP30 reactions.

Manual fitting of the SRL (PDB: 430D) into the active-site of Momordin (PDB: 1AHA), with the extra-helical adenine base in the loop aligned to the protein-bound base, and the RNA loop rotated to minimize clashes with the protein, gave a visually good fit (S3 Fig). In this placement, the loop extends perpendicularly outward from the active-site without approaching either the Mn⁺² and Zn⁺² sites, suggesting that, unlike with the HIV-LTR DNA, these ion binding sites do not play a role in depurination of the RNA, and providing at least a partial explanation for the absence of a difference between the activities of MAP30 and Momordin.

MAP30 and Momordin active-site-distal faces

MAP30 and Momordin also differ in their active-site-distal surfaces, for example Momordin is more acidic here than MAP30 (Fig 5D and 5E), where, by analogy to TCS [29], they probably interact with the ribosomal P-proteins prior to depurination of A-4324 in the SRL. The X-ray crystal structure of the c11-P peptide (SDDDMGFGLFD) bound to TCS (PDB: 2JDL) when aligned to the crystal structures of MAP30 (PDB: 1CF5) and Momordin (PDB: 1AHA) suggests that the peptide probably binds into a hydrophobic groove in these RIP proteins in a similar manner (Fig 5A–5C). Indeed, for the three modeled complexes, the interaction areas and free energies are all very similar (S2 Table). In MAP30 the acidic N-terminal end of the peptide appears to interact with a basic patch, whereas in Momordin this patch is acidic (Fig 5D and 5E). Highlighting the residues identified with Evolutionary Trace as above (Fig 5G–5I) suggests that the hydrophobic groove is important, potentially so for binding to the ribosomal P-proteins. To investigate this further we performed a molecular dynamics simulation of the binding of the c11-P peptide to both MAP30 and Momordin (S4 Fig).

Molecular dynamics simulations of MAP30 and Momordin interaction with ribosomal P-protein peptide

In MAP30, the main residue predicted to engage the peptide is Y164, interacting with F10 in replicate simulations 69–72% of the time (S4A and S4B Fig). This involves a π-π face-to-edge interaction. A second set of (charge) interactions involves K215 interacting with D11 and the peptide C-terminus. A third (polar) interaction is between N234 and the L9 carbonyl of the peptide. A fourth set of (charge) interactions involves R167 and K171 with D2, D3, and D4 at the N-terminal end of the peptide.

In Momordin, the main residue predicted to engage the peptide is Y166, interacting with F10 in replicate simulations 37–94% of the time (S4C and S4D Fig). This involves a π-π face-to-edge interaction. A second set of (charge) interactions involves N218, K219, and K231 with D11 and the peptide C-terminus. A third (polar) interaction occurs between S235 and the L9 carbonyl of the peptide. The predicted interactions between MAP30 R167 and K171 and the peptide N-terminal residues D2, D3 and D4 cannot occur in Momordin as the corresponding residues are Q169 and E173. MAP30 R167 and K171, and Momordin Q169 and E173, are also not included in the top 25% important residues, suggesting that they may not always play a significant role in how these RIPs engage the ribosome.

In general, as is evident in the two animations (S1 and S2 Movies), the interaction of the c11-P peptide with Momordin is likely more dynamic than with MAP30, particularly at the N-terminal end which even engages intermittently with N33 over 20 Å away. However, the hydrophobic interaction of the peptide C-terminal region with both proteins appears to be quite stable. These observations of similar interactions at the peptide-binding-site, together with the similarities observed at the active-site, likely explain why the activities of MAP30 and Momordin in the A549 NLRV assays are essentially identical, despite an overall sequence identity of ca. 54% and a clear difference in the Mn⁺² binding site.

While mutation of MAP30 Y164 (or Momordin Y166) may decrease interaction with the ribosome, it is unlikely to increase the selectivity index. This is suggested by the observation above that the MAP30.K215A mutation did not increase the selectivity index. Also, stability calculations predict that mutating MAP30 Y164 to any of the other 20 amino acids is substantially more disruptive than mutating K215 to any other residue, probably due to the buried location of the former. As Y164 in MAP30 is close to R161, which is directly involved in catalysis, mutating Y164 is likely to be deleterious. Similarly, so for Momordin. For this reason, we considered where else MAP30 and Momordin might exert their antiviral effects.

A potential new point of SARS-CoV-2 inhibition

RIPs, in addition to their specific RNA N-glycosylase activity on ribosomes, also act to a lesser degree on non-ribosomal RNA and DNA. One such activity depends on the 5’ m⁷Gppp cap structure and can lead to cis-depurination throughout the mRNA and may be responsible for the antiviral effects of some RIPs (reviewed in [58]). The positive-sense, single-stranded genome of SARS-CoV-2 (NC_045512.2) has a 5’ cap [59] and 77 GAGA motifs out of which four are in regions predicted [41] to form stable hairpins (S5A Fig; S3 Table). These four hairpins are predicted to be more stable than the mean of the 73 others, and No. 2 is significantly more stable (-11.34 kcal/mol vs -4.0 ± 2.1 kcal/mol, n = 73) and it has the highest frequency in its ensemble. The No. 2 hairpin also stands out for other reasons. It has two overlapping copies of the GAGA motif of which the first has 6 nucleotides (CGAGAG) in common with the SRL, and it is this one that is clearly in the predicted loop. Hairpin No. 2, like No. 3, also has the first adenine of the GAGA motif, where the depurination occurs, on the stem-distal side of the loop where it would be readily accessible to depurination, however, it was unclear how representative these 2D predicted loops are, as that of the SRL clearly bears no resemblance to its X-ray crystal structure. To gain a better sense of the hairpins, we performed 3D structure predictions for hairpins No. 2 and No. 3 [42]. Mutual alignment and visual inspection suggest that the 3D predicted structures for hairpin No. 2 more closely resemble the SRL than do those for No. 3 (S5B Fig). Manual docking shows that hairpins related to No. 2 might engage with MAP30 and Momordin as the broad groove in which the active site is located provides some leeway in binding (S3 Fig).

The four hairpins are in ORF1ab with hairpin No. 1 in the coding region for Nsp2, which impairs microRNA-induced silencing by human cells [60] and interferon production [61]. Hairpin No. 2 is in the coding region for Nsp3, an essential component of the viral replication/transcription complex [62]. As it is located just upstream of the coding region for Mpro, the main viral protease, it may affect expression of that as well. Hairpins No. 3 and No. 4 are in the coding region for Nsp13, a helicase that unwinds dsRNA and dsDNA in the 5’-3’ direction [63]. There is by now a wealth of information about the many ways in which RIPs, particularly Ricin but also others such as MAP30, affect cells and inhibit viruses. MAP30 and Momordin could conceivably also inhibit SARS-CoV-2 replication by depurinating the genome at these points, particularly at hairpin No. 2.

Vero E6 CPE assays

In parallel with the above, MAP30 and Momordin were also tested for SARS-CoV-2 inhibition in a Vero E6 cytopathic effect (CPE) reduction assay in a cell line expressing ACE2. No viral inhibition was observed until cytotoxicity occurred, which was at approximately the same concentration as in the A549 NLRV assay. The same result was obtained when a Tat cell-penetration peptide was appended to both proteins, and no synergy was observed with either Dexamethasone or Indomethacin in matrix assays (not shown). These observations suggest that SARS-CoV-2 inhibition by RIPs may be cell line specific and that perhaps in Vero cells some factor involved in viral replication is different from that A549 cells.

Conclusions

RIPs have a long and notable history as promising antiviral agents. To our knowledge, this is the first report that RIPs such as MAP30 and Momordin inhibit SARS-CoV-2. For both proteins, the inhibition and cytotoxicity values, ca. 0.2 and 2 μM, respectively, are in the ranges previously reported for other RIPs and viruses. MAP30 and Momordin consistently have essentially identical inhibitory activities on SARS-CoV-2 despite distinct differences in their structures at both their active-sites and ribosome-binding-regions. It is not clear from the structures how to substantially improve their selectivity indices. Our structural analyses point to sites on the viral genome where these RIPs may also exert their effects.

Supporting information

S1 Fig. MAP30 and Momordin proteins.

(A) SDS-PAGE of MAP30 and Momordin, with and without a C-terminally appended Tat cell penetration peptide, after the final step of column chromatography purification. Standards (Lanes 1 and 6), MAP30 (Lanes 2, 3, 4, and 7), MAP30-Tat (Lane 8), MAP30.Y70A (Lanes 9–12, column fractions), MAP30.K171A, K215A (Lanes 13–16, column fractions), Momordin (Lanes 17 and 18), Momordin-Tat (Lanes 19–22, column fractions). (B) Sedimentation equilibrium analytical ultracentrifugation of MAP30 showing that the protein is monomeric with no tendency for self-association and with a mass corresponding to that predicted from sequence (Fig 1C). (C) Far-UV circular dichroic spectra of MAP30 and Momordin. (D) Thermal denaturation of MAP30 and Momordin monitored by helix–coil transitions at 222 nm. The denaturation of both proteins was irreversible.

(PDF)

Click here for additional data file.^{(169.1KB, pdf)}

S2 Fig. MAP30 and ACE2 do not interact.

(A) Model of how MAP30 might engage ACE2. Two copies of MAP30 (PDB: 1D8V) fitted onto the ACE2 dimer (PDB: 6M18). Top view (left), and side view (right). Note the apparent shape and charge complementarity. (B) Sedimentation equilibrium analysis of MAP30 (left), and rhACE2 (APN01) (right). MAP30 is monomeric and ACE2 is dimeric. (C) Assay for interaction between MAP30 and soluble rhACE2. Separation of MAP30 and ACE2 by Superdex 200 (left), and Ni-Sepharose (right), chromatography. (D) Analysis by SDS-PAGE of fractions from Superdex 200 (left), and Ni-Sepharose (right), chromatography. There is no evidence of an interaction between MAP30 and ACE2.

(PDF)

Click here for additional data file.^{(195.8KB, pdf)}

S3 Fig. Model of MAP30-SRL interaction.

(A) Ribbon diagram of MAP30 (PDB: 1D8V) with the residues in the active site colored green, and those involved in Mn⁺² and Zn⁺² binding colored yellow and pink, respectively. (B) Ribbon diagram of MAP30 with the SRL (PDB: 430D) modeled to align the adenine in the GAGA motif to overlap with the bound adenine in Momordin (PDB: 1AHA) and to avoid clashes with MAP30 (C). (D-F) Orthogonal views of the MAP30-SRL complex model.

(PDF)

Click here for additional data file.^{(368.9KB, pdf)}

S4 Fig. Interaction of the c11-P peptide with MAP30 and Momordin as modeled by molecular dynamics simulation.

(A and C) Interaction fractions of the peptide with MAP30 and Momordin residues, respectively. (B and D) Schematics of peptide atom interactions with MAP30 and Momordin residues, respectively. Note the large contribution of the hydrophobic interaction of F10 in the peptide with Y164 in MAP30 (occurring 72% of the time during the 250 ns simulation) and to a lesser extent with Y166 in Momordin (37%). Note also, the charge interactions of D2, D3, and D4 in the N-terminal region of the peptide with R167 and K171, and of D11 and the C-terminus of the peptide with K215, in MAP30. Similar interactions occur between the C-terminal region of the peptide, but not the N-terminal region, and Momordin.

(PDF)

Click here for additional data file.^{(297.2KB, pdf)}

S5 Fig. Points on SARS-CoV-2 genome potentially susceptible to RIP inhibition.

(A) 2D structure predictions for the SRL and the four stem-loop structures in the SARS-CoV-2 genome with the GAGA RIP recognition motif located in the loop. In stem-loops No. 2 and No. 3, the adenine in the GAGA motif where depurination might occur is in a similar position distal to the stem (indicated by an arrow). (B) Ribbon diagrams of (left) the SRL X-ray crystal structure (PDB: 430D), and (right) the top five 3D structure predictions for stem-loops No. 2 and No. 3 in (A). The adenine in the GAGA loop where depurination may occur is highlighted in magenta in each case. (C) Alignment of predicted Hairpin No. 2 with the SRL, with the respective first adenine bases in the common CGAGAG motif shown as sticks colored by heteroatom. The bases are extrahelical and essentially superimposable.

(PDF)

Click here for additional data file.^{(193KB, pdf)}

S1 Table. MAP30 and Momordin Mn⁺² binding sites.

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Click here for additional data file.^{(64.1KB, pdf)}

S2 Table. Binding parameters of c11-P peptide.

(PDF)

Click here for additional data file.^{(65.1KB, pdf)}

S3 Table. Predicted RNA hairpin folding parameters.

(PDF)

Click here for additional data file.^{(65.7KB, pdf)}

S1 Movie. Molecular dynamics simulation of MAP30 interaction with c11-P peptide.

(MOV)

Click here for additional data file.^{(13.7MB, mov)}

S2 Movie. Molecular dynamics simulation of Momordin interaction with c11-P peptide.

(MOV)

Click here for additional data file.^{(10.8MB, mov)}

Acknowledgments

All viral inhibition assays were performed at Southern Research, Birmingham, AL. APN01 (soluble recombinant human ACE2) was provided by Apeiron Biologics.

Data Availability

All assay data are available in the Dryad Repository (DOI: 10.5061/dryad.z08kprrj4).

Funding Statement

This research was supported by the Intramural Research Program of the NIH National Institute of Arthritis and Musculoskeletal and Skin Diseases.

References

1.Puri M, Kaur I, Kanwar RK, Gupta RC, Chauhan A, Kanwar JR. Ribosome inactivating proteins (RIPs) from Momordica charantia for anti viral therapy. Curr Mol Med. 2009;9(9):1080–94. doi: 10.2174/156652409789839071 [DOI] [PubMed] [Google Scholar]
2.Puri M, Kaur I, Perugini MA, Gupta RC. Ribosome-inactivating proteins: current status and biomedical applications. Drug Discov Today. 2012;17(13–14):774–83. doi: 10.1016/j.drudis.2012.03.007 [DOI] [PubMed] [Google Scholar]
3.Walsh MJ, Dodd JE, Hautbergue GM. Ribosome-inactivating proteins: potent poisons and molecular tools. Virulence. 2013;4(8):774–84. doi: 10.4161/viru.26399 [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Zhu F, Zhou YK, Ji ZL, Chen XR. The Plant Ribosome-Inactivating Proteins Play Important Roles in Defense against Pathogens and Insect Pest Attacks. Front Plant Sci. 2018;9. [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Lu JQ, Zhu ZN, Zheng YT, Shaw PC. Engineering of Ribosome-inactivating Proteins for Improving Pharmacological Properties. Toxins (Basel). 2020;12(3). doi: 10.3390/toxins12030167 [DOI] [PMC free article] [PubMed] [Google Scholar]
6.Citores L, Iglesias R, Ferreras JM. Antiviral Activity of Ribosome-Inactivating Proteins. Toxins (Basel). 2021;13(2). doi: 10.3390/toxins13020080 [DOI] [PMC free article] [PubMed] [Google Scholar]
7.Basch E, Gabardi S, Ulbricht C. Bitter melon (Momordica charantia): a review of efficacy and safety. Am J Health Syst Pharm. 2003;60(4):356–9. doi: 10.1093/ajhp/60.4.356 [DOI] [PubMed] [Google Scholar]
8.Arslan I, Akgul H, Kara M. Saporin, a Polynucleotide-Adenosine Nucleosidase, May Be an Efficacious Therapeutic Agent for SARS-CoV-2 Infection. SLAS Discov. 2021;26(3):330–5. doi: 10.1177/2472555220970911 [DOI] [PMC free article] [PubMed] [Google Scholar]
9.Hassan Y, Ogg S, Ge H. Novel Binding Mechanisms of Fusion Broad Range Anti-Infective Protein Ricin A Chain Mutant-Pokeweed Antiviral Protein 1 (RTAM-PAP1) against SARS-CoV-2 Key Proteins in Silico. Toxins (Basel). 2020;12(9). doi: 10.3390/toxins12090602 [DOI] [PMC free article] [PubMed] [Google Scholar]
10.Wang YX, Neamati N, Jacob J, Palmer I, Stahl SJ, Kaufman JD, et al. Solution structure of anti-HIV-1 and anti-tumor protein MAP30: structural insights into its multiple functions. Cell. 1999;99(4):433–42. doi: 10.1016/s0092-8674(00)81529-9 [DOI] [PubMed] [Google Scholar]
11.Bolognesi A, Bortolotti M, Maiello S, Battelli MG, Polito L. Ribosome-Inactivating Proteins from Plants: A Historical Overview. Molecules. 2016;21(12). doi: 10.3390/molecules21121627 [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Lee-Huang S, Huang PL, Nara PL, Chen HC, Kung HF, Huang P, et al. MAP 30: a new inhibitor of HIV-1 infection and replication. FEBS Lett. 1990;272(1–2):12–8. doi: 10.1016/0014-5793(90)80438-o [DOI] [PubMed] [Google Scholar]
13.Huang PL, Sun YT, Chen HC, Kung HF, Huang PL, Lee-Huang S. Proteolytic fragments of anti-HIV and anti-tumor proteins MAP30 and GAP31 are biologically active. Biochem Bioph Res Co. 1999;262(3):615–23. doi: 10.1006/bbrc.1999.1213 [DOI] [PubMed] [Google Scholar]
14.Fan X, He L, Meng Y, Li G, Li L, Meng Y. Alpha-MMC and MAP30, two ribosome-inactivating proteins extracted from Momordica charantia, induce cell cycle arrest and apoptosis in A549 human lung carcinoma cells. Mol Med Rep. 2015;11(5):3553–8. [DOI] [PubMed] [Google Scholar]
15.Yang GL, Li SM, Wang SZ. Research progress in enzyme activity and pharmacological effects of ribosome-inactivity protein in bitter melon. Toxin Rev. 2016;35(3–4):128–32. [Google Scholar]
16.Dandawate PR, Subramaniam D, Padhye SB, Anant S. Bitter melon: a panacea for inflammation and cancer. Chin J Nat Med. 2016;14(2):81–100. doi: 10.1016/S1875-5364(16)60002-X [DOI] [PMC free article] [PubMed] [Google Scholar]
17.Fang EF, Froetscher L, Scheibye-Knudsen M, Bohr VA, Wong JH, Ng TB. Emerging Antitumor Activities of the Bitter Melon (Momordica charantia). Curr Protein Pept Sci. 2019;20(3):296–301. doi: 10.2174/1389203719666180622095800 [DOI] [PMC free article] [PubMed] [Google Scholar]
18.Chan DW, Yung MM, Chan YS, Xuan Y, Yang H, Xu D, et al. MAP30 protein from Momordica charantia is therapeutic and has synergic activity with cisplatin against ovarian cancer in vivo by altering metabolism and inducing ferroptosis. Pharmacol Res. 2020;161:105157. doi: 10.1016/j.phrs.2020.105157 [DOI] [PubMed] [Google Scholar]
19.Omokhua-Uyi AG, Van Staden J. Phytomedicinal relevance of South African Cucurbitaceae species and their safety assessment: A review. J Ethnopharmacol. 2020;259:112967. doi: 10.1016/j.jep.2020.112967 [DOI] [PubMed] [Google Scholar]
20.Bourinbaiar AS, Leehuang S. Potentiation of Anti-Hiv Activity of Antiinflammatory Drugs, Dexamethasone and Indomethacin, by Map30, the Antiviral Agent from Bitter-Melon. Biochem Bioph Res Co. 1995;208(2):779–85. [DOI] [PubMed] [Google Scholar]
21.Bourinbaiar AS, Lee-Huang S. The non-steroidal anti-inflammatory drug, indomethacin, as an inhibitor of HIV replication. FEBS Lett. 1995;360(1):85–8. doi: 10.1016/0014-5793(95)00057-g [DOI] [PubMed] [Google Scholar]
22.Robert F, Roman W, Bramoulle A, Fellmann C, Roulston A, Shustik C, et al. Translation initiation factor eIF4F modifies the dexamethasone response in multiple myeloma. Proc Natl Acad Sci U S A. 2014;111(37):13421–6. doi: 10.1073/pnas.1402650111 [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Group RC, Horby P, Lim WS, Emberson JR, Mafham M, Bell JL, et al. Dexamethasone in Hospitalized Patients with Covid-19. N Engl J Med. 2021;384(8):693–704. doi: 10.1056/NEJMoa2021436 [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Ravichandran R, Mohan SK, Sukumaran SK, Kamaraj D, Daivasuga SS, Ravi S, et al. An open label randomized clinical trial of Indomethacin for mild and moderate hospitalised Covid-19 patients. Sci Rep. 2022;12(1):6413. doi: 10.1038/s41598-022-10370-1 [DOI] [PMC free article] [PubMed] [Google Scholar]
25.Grela P, Szajwaj M, Horbowicz-Drozdzal P, Tchorzewski M. How Ricin Damages the Ribosome. Toxins. 2019;11(5). doi: 10.3390/toxins11050241 [DOI] [PMC free article] [PubMed] [Google Scholar]
26.Lord MJ, Jolliffe NA, Marsden CJ, Pateman CS, Smith DC, Spooner RA, et al. Ricin. Mechanisms of cytotoxicity. Toxicol Rev. 2003;22(1):53–64. doi: 10.2165/00139709-200322010-00006 [DOI] [PubMed] [Google Scholar]
27.Putnam CD, Tainer JA. The food of sweet and bitter fancy. Nat Struct Biol. 2000;7(1):17–8. doi: 10.1038/71211 [DOI] [PubMed] [Google Scholar]
28.Jetzt AE, Li XP, Tumer NE, Cohick WS. Toxicity of ricin A chain is reduced in mammalian cells by inhibiting its interaction with the ribosome. Toxicol Appl Pharmacol. 2016;310:120–8. doi: 10.1016/j.taap.2016.09.004 [DOI] [PMC free article] [PubMed] [Google Scholar]
29.Too PH, Ma MK, Mak AN, Wong YT, Tung CK, Zhu G, et al. The C-terminal fragment of the ribosomal P protein complexed to trichosanthin reveals the interaction between the ribosome-inactivating protein and the ribosome. Nucleic Acids Res. 2009;37(2):602–10. doi: 10.1093/nar/gkn922 [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Barbieri L, Gorini P, Valbonesi P, Castiglioni P, Stirpe F. Unexpected activity of saporins. Nature. 1994;372(6507):624. doi: 10.1038/372624a0 [DOI] [PubMed] [Google Scholar]
31.Barbieri L, Valbonesi P, Bonora E, Gorini P, Bolognesi A, Stirpe F. Polynucleotide:adenosine glycosidase activity of ribosome-inactivating proteins: effect on DNA, RNA and poly(A). Nucleic Acids Res. 1997;25(3):518–22. doi: 10.1093/nar/25.3.518 [DOI] [PMC free article] [PubMed] [Google Scholar]
32.Alexandersen S, Chamings A, Bhatta TR. SARS-CoV-2 genomic and subgenomic RNAs in diagnostic samples are not an indicator of active replication. Nat Commun. 2020;11(1):6059. doi: 10.1038/s41467-020-19883-7 [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Lee-Huang S, Huang PL, Chen HC, Huang PL, Bourinbaiar A, Huang HI, et al. Anti-HIV and anti-tumor activities of recombinant MAP30 from bitter melon. Gene. 1995;161(2):151–6. doi: 10.1016/0378-1119(95)00186-a [DOI] [PubMed] [Google Scholar]
34.Meng Y, Liu S, Li J, Meng Y, Zhao X. Preparation of an antitumor and antivirus agent: chemical modification of alpha-MMC and MAP30 from Momordica Charantia L. with covalent conjugation of polyethyelene glycol. Int J Nanomedicine. 2012;7:3133–42. [DOI] [PMC free article] [PubMed] [Google Scholar]
35.Priyanto AD, Doerksen RJ, Chang CI, Sung WC, Widjanarko SB, Kusnadi J, et al. Screening, discovery, and characterization of angiotensin-I converting enzyme inhibitory peptides derived from proteolytic hydrolysate of bitter melon seed proteins. J Proteomics. 2015;128:424–35. doi: 10.1016/j.jprot.2015.08.018 [DOI] [PubMed] [Google Scholar]
36.Lee-Huang S, Huang PL, Huang PL. The Discovery of MAP30 and Elucidation of its Medicinal Activities. In: Fang EF, Ng TB, editors. Antitumor Potential and Other Emerging Medicinal Properties of Natural Compounds. Dordrecht: Springer Science+Business Media; 2013. p. 117–26. [Google Scholar]
37.Banks JL, Beard HS, Cao Y, Cho AE, Damm W, Farid R, et al. Integrated Modeling Program, Applied Chemical Theory (IMPACT). J Comput Chem. 2005;26(16):1752–80. doi: 10.1002/jcc.20292 [DOI] [PMC free article] [PubMed] [Google Scholar]
38.Humphrey W, Dalke A, Schulten K. VMD: visual molecular dynamics. J Mol Graph. 1996;14(1):33–8, 27–8. doi: 10.1016/0263-7855(96)00018-5 [DOI] [PubMed] [Google Scholar]
39.Krissinel E, Henrick K. Inference of macromolecular assemblies from crystalline state. J Mol Biol. 2007;372(3):774–97. doi: 10.1016/j.jmb.2007.05.022 [DOI] [PubMed] [Google Scholar]
40.Schymkowitz J, Borg J, Stricher F, Nys R, Rousseau F, Serrano L. The FoldX web server: an online force field. Nucleic Acids Res. 2005;33(Web Server issue):W382–8. doi: 10.1093/nar/gki387 [DOI] [PMC free article] [PubMed] [Google Scholar]
41.Gruber AR, Lorenz R, Bernhart SH, Neubock R, Hofacker IL. The Vienna RNA websuite. Nucleic Acids Res. 2008;36(Web Server issue):W70-4. doi: 10.1093/nar/gkn188 [DOI] [PMC free article] [PubMed] [Google Scholar]
42.Wang J, Wang J, Huang Y, Xiao Y. 3dRNA v2.0: An Updated Web Server for RNA 3D Structure Prediction. Int J Mol Sci. 2019;20(17). doi: 10.3390/ijms20174116 [DOI] [PMC free article] [PubMed] [Google Scholar]
43.Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, et al. UCSF Chimera—a visualization system for exploratory research and analysis. J Comput Chem. 2004;25(13):1605–12. doi: 10.1002/jcc.20084 [DOI] [PubMed] [Google Scholar]
44.Li WH, Moore MJ, Vasilieva N, Sui JH, Wong SK, Berne MA, et al. Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature. 2003;426(6965):450–4. doi: 10.1038/nature02145 [DOI] [PMC free article] [PubMed] [Google Scholar]
45.Maddox CB, Rasmussen L, White EL. Adapting Cell-Based Assays to the High Throughput Screening Platform: Problems Encountered and Lessons Learned. JALA Charlottesv Va. 2008;13(3):168–73. doi: 10.1016/j.jala.2008.02.002 [DOI] [PMC free article] [PubMed] [Google Scholar]
46.Severson WE, Shindo N, Sosa M, Fletcher T, 3rd, White EL, Ananthan S, et al. Development and validation of a high-throughput screen for inhibitors of SARS CoV and its application in screening of a 100,000-compound library. J Biomol Screen. 2007;12(1):33–40. doi: 10.1177/1087057106296688 [DOI] [PMC free article] [PubMed] [Google Scholar]
47.Hou YJ, Okuda K, Edwards CE, Martinez DR, Asakura T, Dinnon KH, 3rd, et al. SARS-CoV-2 Reverse Genetics Reveals a Variable Infection Gradient in the Respiratory Tract. Cell. 2020;182(2):429–46 e14. doi: 10.1016/j.cell.2020.05.042 [DOI] [PMC free article] [PubMed] [Google Scholar]
48.Yadav B, Wennerberg K, Aittokallio T, Tang J. Searching for Drug Synergy in Complex Dose-Response Landscapes Using an Interaction Potency Model. Comput Struct Biotechnol J. 2015;13:504–13. doi: 10.1016/j.csbj.2015.09.001 [DOI] [PMC free article] [PubMed] [Google Scholar]
49.Nikam R, Kulandaisamy A, Harini K, Sharma D, Gromiha MM. ProThermDB: thermodynamic database for proteins and mutants revisited after 15 years. Nucleic Acids Research. 2021;49(D1):D420–D4. doi: 10.1093/nar/gkaa1035 [DOI] [PMC free article] [PubMed] [Google Scholar]
50.Letko M, Marzi A, Munster V. Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses. Nat Microbiol. 2020;5(4):562–9. doi: 10.1038/s41564-020-0688-y [DOI] [PMC free article] [PubMed] [Google Scholar]
51.Shoemaker RH, Panettieri RA Jr., Libutti SK, Hochster HS, Watts NR, Wingfield PT, et al. Development of an aerosol intervention for COVID-19 disease: Tolerability of soluble ACE2 (APN01) administered via nebulizer. PLoS One. 2022;17(7):e0271066. doi: 10.1371/journal.pone.0271066 [DOI] [PMC free article] [PubMed] [Google Scholar]
52.Liu B, Zhang Z, Lu S, He Q, Deng N, Meng H, et al. In-silico analysis of ligand-receptor binding patterns of alpha-MMC, TCS and MAP30 protein to LRP1 receptor. J Mol Graph Model. 2020;98:107619. [DOI] [PubMed] [Google Scholar]
53.Lv Q, Yang XZ, Fu LY, Lu YT, Lu YH, Zhao J, et al. Recombinant expression and purification of a MAP30-cell penetrating peptide fusion protein with higher anti-tumor bioactivity. Protein Expr Purif. 2015;111:9–17. doi: 10.1016/j.pep.2015.03.008 [DOI] [PubMed] [Google Scholar]
54.Jiang Y, Miao J, Wang D, Zhou J, Liu B, Jiao F, et al. MAP30 promotes apoptosis of U251 and U87 cells by suppressing the LGR5 and Wnt/beta-catenin signaling pathway, and enhancing Smac expression. Oncol Lett. 2018;15(4):5833–40. [DOI] [PMC free article] [PubMed] [Google Scholar]
55.Husain J, Tickle IJ, Wood SP. Crystal structure of momordin, a type I ribosome inactivating protein from the seeds of Momordica charantia. FEBS Lett. 1994;342(2):154–8. doi: 10.1016/0014-5793(94)80491-5 [DOI] [PubMed] [Google Scholar]
56.Li XP, Kahn PC, Kahn JN, Grela P, Tumer NE. Arginine residues on the opposite side of the active site stimulate the catalysis of ribosome depurination by ricin A chain by interacting with the P-protein stalk. J Biol Chem. 2013;288(42):30270–84. doi: 10.1074/jbc.M113.510966 [DOI] [PMC free article] [PubMed] [Google Scholar]
57.Mihalek I, Res I, Lichtarge O. A family of evolution-entropy hybrid methods for ranking protein residues by importance. J Mol Biol. 2004;336(5):1265–82. doi: 10.1016/j.jmb.2003.12.078 [DOI] [PubMed] [Google Scholar]
58.Hartley MR. Enzymatic Activities of Ribosome-Inactivating Proteins. In: Lord JM, Hartley MR, editors. Toxic Plant Proteins. Plant Cell Monographs. 18. Heidelberg, Germany: Springer; 2010. p. 41–54. [Google Scholar]
59.Park GJ, Osinski A, Hernandez G, Eitson JL, Majumdar A, Tonelli M, et al. The mechanism of RNA capping by SARS-CoV-2. Nature. 2022;609(7928):793–800. doi: 10.1038/s41586-022-05185-z [DOI] [PMC free article] [PubMed] [Google Scholar]
60.Zou L, Moch C, Graille M, Chapat C. The SARS-CoV-2 protein NSP2 impairs the silencing capacity of the human 4EHP-GIGYF2 complex. iScience. 2022;25(7):104646. doi: 10.1016/j.isci.2022.104646 [DOI] [PMC free article] [PubMed] [Google Scholar]
61.Xu Z, Choi JH, Dai DL, Luo J, Ladak RJ, Li Q, et al. SARS-CoV-2 impairs interferon production via NSP2-induced repression of mRNA translation. Proc Natl Acad Sci U S A. 2022;119(32):e2204539119. doi: 10.1073/pnas.2204539119 [DOI] [PMC free article] [PubMed] [Google Scholar]
62.Lei J, Kusov Y, Hilgenfeld R. Nsp3 of coronaviruses: Structures and functions of a large multi-domain protein. Antiviral Res. 2018;149:58–74. doi: 10.1016/j.antiviral.2017.11.001 [DOI] [PMC free article] [PubMed] [Google Scholar]
63.Newman JA, Douangamath A, Yadzani S, Yosaatmadja Y, Aimon A, Brandao-Neto J, et al. Structure, mechanism and crystallographic fragment screening of the SARS-CoV-2 NSP13 helicase. Nat Commun. 2021;12(1):4848. doi: 10.1038/s41467-021-25166-6 [DOI] [PMC free article] [PubMed] [Google Scholar]

PLoS One. doi: 10.1371/journal.pone.0286370.r001

Decision Letter 0

Israel Silman

27 Feb 2023

PONE-D-23-02011The ribosome-inactivating protein MAP30 inhibits SARS-CoV-2PLOS ONE

Dear Dr. Watts,

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Reviewer #1: In the manuscript The ribosome-inactivating protein MAP30 inhibits SARS-CoV-2 Watts et al. tested the anti-SARS-CoV-2 activities of the MAP30 protein, which is a well-studied ribosome-inactivating protein, and its close homolog, Momordin. Overall, the authors have presented a comprehensive work of scientific values. However, there are major concerns which the authors should address before the paper can be accepted.

(1) The authors should justify the use of A549-ACE2 instead of other non-cancerous ACE2-expressing cell lines for antiviral experiments. A549 has been found to be sensitive to MAP30 treatment (see Mol Med Rep 2015, 11(5):3553-3558 and other articles). From the data it seems that cell viability and antiviral effect are (reversely) correlated with each other (Fig 2). While MAP30 mutants reduced cytotoxicity, antiviral effect was also abolished. Further, the authors also mentioned (without data shown) that the protein exhibited no effect on Vero E6 CPE assay. All these suggested a possibility, which is of concern, that the reduced viral growth may be simply due to sub-optimal cellular conditions in the presence of MAP30.

(2) Further to (1), despite the authors have showed no interaction between ACE2 and MAP30, there seems a gap between the presented data and the conclusion that the observed antiviral activity was due to RNA N-glycosidase activity. At least a single-cycle time-of-addition assay should be performed for further support.

(3) Structural comparison between MAP30 and momordin, and MAP30/momordin binding to C-11 peptide are themselves interesting. However, they currently look like a standalone portion in the manuscript. It is suggested that the authors rewrite the relevant paragraphs with more emphasis on how their findings and observations are related to anti-SARS-CoV-2 activity.

(4) Paragraph 429-447 suggested that MAP30 may depurinate the viral RNA at hairpin regions. This proposition is exciting. However the evidence presented in the manuscript was weak. The authors could have further demonstrated how the viral RNA hairpin resembles the SRL (e.g. by structural alignment). Besides, Fig S3 only shows how SRL is docked to MAP30. The authors’ claim can be further supported by docking the hairpin to MAP30. Experimental evidence would also be necessary (e.g., can the authors perform a depurination assay using the SARS-CoV-2 hairpin?)

Minor points:

Line 247: Clarify the sentence “Compounds were run in anti-viral and cytotoxicity assays when tested.”

Throughout the manuscript: Not appropriate to treat amino acids as proper nouns

Reviewer #2: RIPs have long been recognized as antiviral proteins in both plants and animals, but the mechanism responsible for this activity continues to be the subject of intense research today. In this paper, the authors mainly report that MAP30 and momordin inhibit SARS-CoV-2 replication in human A549 lung cells. This in itself is an important finding but in addition the authors try to explain how these RIPs carry out this inhibition. They study if MAP30 could interact with ACE2 and block virus entry, then by mutating key residues of MAP30 show the involvement of its RNA N-glycosylase activity and consider the possibility that RIPs could act on viral RNA by showing points on the viral genome for potential inhibition. An interesting structural comparison (of the ribosomal binding sites, ion binding sites…) of MAP30 and momordin is also reported in this work. Overall, the authors contribute to the elucidation of the structure-activity relationships of RIPs and demonstrate the potential of RIPs for the treatment of virus-related diseases.

The studies are designed and conducted in a logical manner and all conclusions match the data presented. The images and the results in general are well presented. However, I found it difficult to read the manuscript in the section "results and discussion". The headings of the subsections do not always specify the content and in some cases the subsections are too long. I recommend making more sections indicating what is to be studied in each case.

Minor comments

-Most of the work in the manuscript has been performed for both MAP30 and momordin and clearly momordin also inhibits SARS-CoV-2 replication. However, only the antiviral activity of MAP30 appears in the title “The ribosome-inactivating protein MAP30 inhibits SARS-CoV-2”. The authors should explain this.

-Line 53: RIPs are rRNA N-glycosylases (EC 3.2.2.22) that catalyze the elimination of a specific adenine (A4324 in rat ribosomes, or the equivalent in other organisms) located in the sarcin-ricin loop (SRL) of animal 28S ribosomal RNA.

Lines 53, 32, 55, 70, 315, 329, 338, 430 Change glycosidase by glycosylase.

-Line 311- . Looking at the values shown on line 311 for "viral inhibition (mean IC50 =5.7...) with low cytotoxicity (mean CC50=72...)" they do not match the values shown in Table 1. Are the values in the text for MAP30, momordin or with the TAT peptides? Are the values in Table 1, mean values or how were the values obtained? Table 1 should be better explained.

-I recommend including Figure S5 in the manuscript to illustrate the potential points of SARS-CoV-2 inhibition.

-Legend of Fig S3. Model of MAP30-SRL interaction. (D-E) Orthogonal views of the MAP30-SRL complex. Panel F should be mentioned in the legend.

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PLoS One. 2023 Jun 29;18(6):e0286370. doi: 10.1371/journal.pone.0286370.r002

Author response to Decision Letter 0

30 Mar 2023

Response To Reviewers

Before addressing the Reviewer’s concerns, we would like to thank them for their thoughtful comments and helpful recommendations. We hope that the detailed explanations below, and the changes made to the manuscript, will address all their concerns.

The Reviewer raises an important issue. In fact, they raise two broad but interrelated matters; first, the justification for using the A549-ACE2 cells instead of another cell line, and second, the validity of the results eventually obtained from that assay. We have discussed these same questions amongst ourselves at considerable length. We have also had discussions with cell biologist colleagues and with the original developers of the two assays used. There were several considerations, and we will here attempt to address them, not in order of importance but rather for clarity of explanation. We will begin with a brief historical perspective.

In the early days of the pandemic, when the world was in state of panic, there was very little information about the virus, and all possible ways to contain it were being considered. Remarkably, this state of urgency has now been almost forgotten just three years later. As we had previously found that MAP30 potently inhibited HIV-1, and then solved its structure, and therefore knew how to express and purify it, we considered testing if it might also inhibit SARS-CoV-2. The opportunities for testing at that time were extremely limited, even at the NIH. BSL3 facilities and personnel training could not just be implemented immediately, and so we evaluated three NIH-validated testing facilities, in parallel. One proved unable, the second after a long delay produced false negative results, and the third rose to the occasion. That third provider was Southern Research, and with neither time nor facilities to isolate our own cell lines and devise new assays we, like many others, employed the assays that Southern could provide. Even then, Southern struggled early on but quickly established the high-throughput screening capacity that we later found gave impressively reproducible results. Much later, the second provider was also able to obtain positive results with another unrelated and highly effective SARS-CoV-2 inhibitor that we had developed in parallel (PMID: 35816490). These results confirmed exactly those of Southern, giving us even greater confidence in the data from this provider.

Initially, we employed solely the Vero E6 CPE reduction assay. Only later did we turn to the A549 NLRV assay and do almost everything all over again. The reason for switching from the Vero assays to the A549 assays was that we observed no viral inhibition with the former, except what was merely due to a decrease in viability. We will return to this point in greater detail further below. In an earlier version of the manuscript, we detailed all the Vero results, and all the A549 results, both in equal detail, but then decided to omit the Vero results as they were all “negative”. We did however briefly mention the Vero assay results at the end of the submitted manuscript for the purpose of full disclosure. Quite aside from the MAP30 and Momordin results described in the manuscript, we also assayed numerous peptides from them, both linear and specifically designed cyclic ones. As described in the manuscript, peptides from both MAP30 and Momordin had previously been shown by others to inhibit ACE2, and our molecular modeling attempts in the early days of the pandemic, as the first spike and receptor binding domain (RBD) structures were coming out, suggested that the peptides might block viral entry. All these peptides gave negative results and, much like the full-length-RIP Vero assay results, they are not included in the final manuscript. Thus, a far larger number of potential inhibitors were assayed than are discussed in the submitted manuscript.

The Vero assays were attractive to us because, as the Reviewer points out, a non-cancerous cell line would be desirable in some ways. Unfortunately, essentially all assays at facilities such as Southern are performed with common immortal cell lines such as Hela, HepG-2, HEK293, etc. This is for practical reasons including maintenance and reproducibility over long periods. Also, in a brief survey of the literature, we have found over 20 different human cancer cell lines used to study MAP30 and other RIPs, illustrating that this is an accepted way to study these proteins. The only non-cancerous cells that we have found used with RIPs in the literature are human spermatocytes. Unlike the A549 human lung cells which are cancerous, the Vero African green monkey kidney cells are considered continuous, i.e., they can be passaged without becoming senescent. Vero cells are aneuploid and can, after hundreds of passages, produce cancer-like nodules in nude mice that then recede again. In this limited sense the Vero cell assays might have been preferable to A549 cell assays, but unfortunately, they did not “work”. By this we mean that the inhibition curve was simply the inverse of the viability curve as shown in Fig. 1 (below). This result was obtained numerous times and eventually led us to use the A549 assay. It is worth noting that quite aside from their aneuploid state, the Vero cells are still not quite normal cells because, just like the A549 cells, they are engineered to express ACE2.

Fig. 1. Test of MAP30 and MAP30-Tat in the Vero E6 CPE reduction assay. Shown is a cropped screen shot from one Southern Research activity report. As can be seen, the percent inhibition curve starts to rise at about 100 µg/ml of RIP as loss of cell viability takes over. This response was highly reproducible for both MAP30 and Momordin. We would consider this an example of the situation the Reviewer describes when they suggest that the antiviral effect may simply be due to sub-optimal cellular conditions. This response is quite distinct from that obtained with the A549-NLRV assay where viral inhibition occurs at concentrations one log lower than cytotoxicity (Fig. 2 in the manuscript).

The Reviewer points out that the A549 cells have been found to be sensitive to MAP30 and other RIPs. This is true and we were quite aware of it. It is often thought that MAP30, and likely other Type I RIPs (i.e., without benefit of the B-chain), cannot enter normal cells, but that it can enter cancerous and virally infected cells as the cell membranes have been altered. This makes at least some sense as some RIPs are considered protective antivirals. While the details of this are still far from sorted out in the literature, the premise that normal cells are resistant to MAP30, while virally infected ones are more susceptible, would be of great value were it to be used as an antiviral agent.

This brings us back to the Reviewer’s second question, about the validity of the A549 assay results. Whereas in the Vero assays, where the inhibition curve was the inverse of the viability curve, in the A549 assays there is a log difference between the IC50 and CC50, i.e., a selectivity index (SI) of 10. At cross-over the viral inhibition is ca. 80%. We obtained this result regardless of RIP type, Tat-CPP addition, presence of Dexamethasone or Indomethacin, or mutations to the ribosome interacting face. In some assays we observed a slightly higher Hill slope, but this was not sufficiently high or reproducible to be reported. While the limited selectivity index, and our inability to increase it, was a disappointment, it should be born in mind that some cancer therapeutics began with selectivity indices of 2.

We hope that we have justified the use of the A549 assays to the Reviewer, and that we have convinced them of the validity of the results. It does not seem to us that all these details need to be included in the manuscript, aside from what is already provided in the Results and Discussion. Mentioning at the end that the Vero CPE assay was not suitable for our purpose may save other researchers from trying to use it in this way. Alternatively, they may wish to determine why MAP30 and Momordin are cytotoxic to Vero cells (suggesting they do enter the cells and inactivate the ribosomes) yet they do not inactivate the virus. In other words, what is it about Vero (aneuploid monkey) cells that apparently is somewhat protective of SARS-CoV-2 against RIPs? This is a fascinating question for another study, and another group.

We did not wish to imply that a lack of interaction between MAP30 and ACE2 leads to the conclusion that viral inhibition is due its RNA N-glycosidase activity. As summarized in the Introduction, a previous report that peptides from both MAP30 and Mormordin inhibit ACE2 suggested to us that there might be an interaction between MAP30 and ACE2, and our initial molecular modeling efforts supported this. We merely eliminated that as a possible cause of viral inhibition. That the RNA N-glycosidase activity may be involved in viral inhibition has long precedent in the literature, and it was confirmed by our mutagenesis of the MAP30 active site. However, where that activity occurs is unknown. It likely takes place at several sites, including the ribosome and the viral genome. As a typical eukaryotic cell has some 10 million ribosomes, but certainly not that many viral genomes, the cell can potentially recover after viral inactivation. Our A549 NLRV assays provide clear and highly reproducible evidence of viral inhibition by these RIPs. Unlike a small molecule protease inhibitor or capsid assembly inhibitor, RIPs exert their effects at numerous points (summarized in the Introduction (Lines 84-94)). Therefore, we do not think that the proposed time-of-addition assay would resolve these points. We have however inquired with Southern, and it is not an assay that they provide, though they indicated that it might be developed as a custom service.

The Reviewer raises two related issues; the seeming standalone nature of the molecular dynamics simulation, and the how the results from that pertain to anti-SARS-CoV-2 activity. As indicated in the text, comparing two different RIPs provided a natural experiment to see which inhibited SARS-CoV-2 better. This might indicate which one to pursue further, and perhaps also how to increase the therapeutic index of that one. To our surprise, both proteins had essentially identical activities despite an almost 50% difference in sequence, distinct structural differences in the vicinity of the active site, distinct structural differences in the ribosome binding site, and no change in the selectivity index upon mutation of residues in the latter previously identified as important in ribosome binding in another RIP (Ricin). All these observations of the static structures lead us to consider them dynamically, however, that may not have been clear in the text, giving the impression that the simulation is standalone. The molecular dynamics simulation demonstrated similarly stable interactions with the ribosomal c11-P peptide despite differences in the details of binding, particularly at the N-terminal end of the peptide. In other words, the simulation indicated two different, partially overlapping, sets of interactions between the peptide and the two proteins (each different from that with TCS, as described in the literature). This would not have been learned from a static analysis. We have now added several section headings to the Results and Discussion that make these different points more obvious and why we therefore performed a molecular dynamics simulation (Lines 309, 324, 333, 373, 390, and 404).

Regarding the Reviewer’s second point, about how the results pertain to anti-SARS-CoV-2 activity. The generic name ‘Ribosome Inactivating Protein’ implies that this activity is what causes viral inhibition. Yet, surprisingly, even two simultaneous rational mutations in the ribosome binding site had relatively little effect on inhibition. This was in part explained by the molecular dynamics simulations. Instead, the most significant change in activity (complete inactivation) was caused by mutation of the active site, not the ribosome binding site. As stated in the manuscript, we therefore considered where else the N-glycosidase activity might inhibit the virus, and based on sequence analysis and molecular modeling, we predict that it may be the viral RNA genome. Again, this shows how the molecular dynamics simulations are related to understanding the anti-SARS-CoV-2 activity of these RIPs.

Due to revisions, paragraph 429-447 is now 438-456. The objective of our study was to determine if MAP30 and Momordin can inhibit SARS-CoV-2. To this end we made six different proteins, assayed them in two cell lines, tested for interactions with ACE2, tested for synergy with two drugs, and performed two molecular dynamics simulations. We have clearly shown that both proteins can inhibit SARS-CoV-2 (SI = 10). Given that viral inhibition was distinct from toxicity, we then also considered where this inhibition might occur. Based on sequence analysis and molecular modeling we propose specific hairpins in the viral genome.

While the Reviewer thinks this is exciting, they feel that we did not make a strong case and note that Fig S3 only shows how the SRL is docked to MAP30. That is because this figure only serves to compare the MAP30 and Momordin active-site faces, and their interactions with the SRL. At this point in the Results and Discussion we have not yet introduced the reader to the concept of RIPs potentially targeting the viral genome. That is illustrated in Fig S5. However, we have taken the Reviewer’s advice and performed a structural alignment of Hairpin No. 2 with the SRL. That is now shown in Fig S5, C. As in the X-ray crystal structure of the SRL (PDB: 430D), the first adenine base in the shared CGAGAG motif in the loop is flipped out into an extrahelical position where it would be susceptible to enzymatic depurination. This high structural similarity is striking and suggests that MAP30 may also depurinate this hairpin in the viral genome. We consider this a prediction rather than a conclusion, and something that others may want to investigate. That is why we have put it in the Supplementary Information. Note: in addition to the 3dRNA structure predictions described in the manuscript, we have now also performed a more focused prediction on Hairpin No. 2 with FARFAR2. The results are essentially the same, giving us further confidence in our prediction (not shown).

Minor points:

Line 247: Clarify the sentence “Compounds were run in anti-viral and cytotoxicity assays when tested.”

We merely intended to say that the inhibition and viability assays were performed at the same time, rather than at different times. We have changed the wording and moved the sentence to the end of the paragraph, where it should logically be (Line 248-249).

Throughout the manuscript: Not appropriate to treat amino acids as proper nouns

This has now been corrected throughout the manuscript (Lines 30, 31, 32, 33, and 34).

First, we wish to thank the Reviewer for their thoughtful comments and helpful recommendations. We hope that the detailed explanations below, and the changes made to the manuscript, will address all their concerns.

The Reviewer has made a good recommendation about the need for more section headings. We were too familiar with the text and could no longer see it from a new reader’s point of view. We have now added more section headings in the Results and Discussion (Lines 309, 324, 333, 373, 390, and 404).

Minor comments

The Reviewer is right, we only mentioned MAP30 in the title. This came about because MAP30 was the protein foremost in our minds, as we have studied it the longest, determined the first structure, and demonstrated its efficacy against HIV. We have now included both proteins in the title (Line 2).

Glycosylase (EC 3.2) is a broad term that comprises two types of glycosidases: O-(and S-) glycosidases (EC 3.2.1) and N-glycosidases (EC 3.2.2). Therefore N-glycosidase is the more specific term. RIPs hydrolyse the glycosidic bond between a nitrogenous base and a sugar, therefore the term N-glycosidase is more appropriate. Also, the term glycosidase has long been used for RIPs (PMID: 10571185, PMID: 7568024, PMID: 7527556, PMID: 3288622, PMID: 3226909, PMID: 3036799, PMID: 9659388, PMID: 3276522, PMID: 2642481, PMID: 15123667, PMID: 26008228, PMID: 35969718, PMID: 9403966). However, we have made the requested changes throughout the text (Lines 32, 53, 55, 70, 317, 334, 343, and 439).

The Reviewer raises two points; that the inhibition values in the text and Table 1 do not agree, and that Table 1 should be better explained. The Reviewer is right, the values given in the text (in the section now titled “Antiviral activity of MAP30 and Momordin”) do not match those in Table 1. They are average values, as was indicated, and as explained here. Altogether, we performed almost forty assays (not counting of course the internal replicates, typically duplicates but in some instances quadruplicates, nor the 8-fold internal multiplicity of each of the eight synergy assays). Half of these involved the A549 NLRV assay, and half the Vero E6 CPE assay. As it turned out that the Vero assay was not suitable for our purpose, we mention it only briefly at the end of the Results and Discussion. As the 6 proteins were prepared at different times over the course of the two-year study, they were assayed in different combinations at different times. All assays were replicated at least once, sometimes months apart, but MAP30 was present in all runs. The results of one run where MAP30 and Momordin, each with and without a Tat-CPP, were assayed side-by-side is shown in Fig. 1 (below). It illustrates the remarkably similar activities of the four proteins. To reduce the many assays to a comprehensible level, we grouped them by type as “Assay 1”, “Assay 2”, etc. in Table 1, although in addition to the assays shown below the proteins were also assayed in other runs. The respective IC50 and CC50 values for the four proteins are so similar in all the assays that we just give the overall means for these four proteins in the text which the Reviewer refers to. To make this clearer for readers, we have now changed the text to "There was clear viral inhibition (overall mean IC50 = 5.7 µg/ml or ~ 0.2 µM) with little cytotoxicity (overall mean CC50 = 72 µg/ml or ~ 2.4 µM) (Fig 2; Table 1, Assay 1)" (Lines 311-313). This is now also clarified in Table 1, footnote 2, “The IC50 and CC50 values given in the text for Assay 1 are the respective overall means for the four proteins”.

Fig. 1. Test of MAP30 and Momordin, with and without a Tat-CPP, in the A549-NLRV assay. Shown is a cropped screen shot from one Southern Research activity report. As can be seen, the four proteins have remarkably similar percent inhibition and viability values.

-I recommend including Figure S5 in the manuscript to illustrate the potential points of SARS-CoV-2 inhibition.

Both this Reviewer and the other Reviewer find this observation interesting. We initially also considered including this figure in the main manuscript, but we would prefer to leave it in the Supplementary Information as it is a prediction rather than a conclusion, and something that others may want to investigate. That is why we have put it in the Supplementary Information. Note: in addition to the 3dRNA structure predictions described in the manuscript, we have now also performed a more focused prediction on Hairpin No. 2 with FARFAR2. The results are essentially the same, giving us further confidence in our prediction (not shown).

-Legend of Fig S3. Model of MAP30-SRL interaction. (D-E) Orthogonal views of the MAP30-SRL complex. Panel F should be mentioned in the legend.

This was merely a typographical error; we intended to write (D-F), not (D-E). We have now corrected this.

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PLoS One. doi: 10.1371/journal.pone.0286370.r003

Decision Letter 1

Israel Silman

28 Apr 2023

PONE-D-23-02011R1The ribosome-inactivating proteins MAP30 and Momordin inhibit SARS-CoV-2PLOS ONE

Dear Dr. Watts,

Please submit your revised manuscript by Jun 12 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. In your revised manuscript please address the minor clarification requested by Reviewer 1.

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Reviewer #2: All comments have been addressed

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6. Review Comments to the Author

Reviewer #1: The authors have provided responses to my first concern about the use of A549 cells from a retrospective (historical) manner. They have also included now in the revised manuscript more molecular simulation experiments which were placed in the Supplementary section. The authors also provided explanations on concerns about data discrepancy both in the responses to my concern and to another reviewer's questions. Overall the paper is now improved in terms of its scientific basis and presentation.

I still recommend that the authors make the justification on the use of A549 clearer in the manuscript, so as to help the audience understand the seemingly contradictory results in A549 and Vero experiments.

Reviewer #2: (No Response)

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PLoS One. 2023 Jun 29;18(6):e0286370. doi: 10.1371/journal.pone.0286370.r004

Author response to Decision Letter 1

3 May 2023

Response to Reviewers:

Reviewer #1: The authors have provided responses to my first concern about the use of A549 cells from a retrospective (historical) manner. They have also included now in the revised manuscript more molecular simulation experiments which were placed in the Supplementary section. The authors also provided explanations on concerns about data discrepancy both in the responses to my concern and to another reviewer's questions. Overall, the paper is now improved in terms of its scientific basis and presentation.

The Reviewer has requested that we justify our choice of assay, and to explain our results more clearly.

The assays were performed with both A549 and Vero cells. Both cell lines were being used at Southern Research to assay potential SARS-CoV-2 inhibitors. The A549 NLRV assay was expected to provide information about replication inhibition. The Vero CPE assay was expected to provide information on overall viral inhibition, due to both extracellular (entry) and intracellular (replication) inhibition.

The results obtained with the A549 and Vero cell assays are not contradictory. Our data show a ten-fold difference between the 50% viral-inhibitory and cell-toxicity concentrations with the A549 cells and no difference with the Vero cells. Why the Vero cells did not “work” in this situation is something that remains to be determined. Our gel-filtration, affinity chromatography, and analytical ultracentrifugation results suggest that viral entry inhibition by blocking of the ACE2 receptor was not a factor. That the toxicity concentration for the A549 and Vero cells was very similar suggests that MAP30 entered both cells equally, and that something involved in viral replication is different in the Vero cells.

Our personal communications regarding this matter with Dr. Paige Vinson, Director of High-Throughput Screening at Southern Research, and with Drs. Timothy Sheahan and Ralph Baric at the University of North Carolina (the Baric lab is expert in this field and had provided both the A549 cells expressing ACE-2 and the USA_WA1/2020 nanoluciferase reporter construct to Southern Research to assay potential SARS-CoV-2 replication inhibitors), provided no explanation for why the Vero assay did not work. It may just be that human lung cells are more appropriate than monkey kidney cells in this case. Other cells, such as osteoclasts, keratinocytes, retinal ganglia, or follicular cells, may also prove unsuitable for assaying SARS-CoV-2 replication inhibition. A549 cells are commonly used to model the alveolar Type II pulmonary epithelium. They have been used to study various conditions including cancer, allergies, asthma, and respiratory infections. The A549 cell line has been approved by the FDA for use in a variety of applications, including in vivo testing of drugs and other therapeutics for use in clinical trials. We therefore think that the A549 assay data are relevant. As with many potential therapeutics, it is the differential between on and off-target effects that is of interest. Clearly, RIPs are not yet ready to be used as therapeutics, but our results demonstrate viral inhibition in a relevant cell type. When the molecular details of what is going on in the Vero cells is known, viral inhibition by RIPs will be better understood.

To provide more information to readers on how the cells were chosen we have added the following text at line 331.

“Both the reporter virus and the cells had been provided to Southern Research by the laboratory of Ralph Baric (UNC) specifically to assay potential SARS-CoV-2 replication inhibitors. A549 cells are commonly used to model the alveolar Type II pulmonary epithelium. They have been used to study various conditions including cancer, allergies, asthma, and respiratory infections.”

To clarify the results from the Vero assays we have added the following text at line 568.

“These observations suggest that SARS-CoV-2 inhibition by RIPs may be cell line specific and that perhaps in Vero cells some factor involved in viral replication is different from that A549 cells.”

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PLoS One. doi: 10.1371/journal.pone.0286370.r005

Decision Letter 2

Israel Silman

16 May 2023

The ribosome-inactivating proteins MAP30 and Momordin inhibit SARS-CoV-2

PONE-D-23-02011R2

Dear Dr. Watts,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Israel Silman

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PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0286370.r006

Acceptance letter

Israel Silman

22 Jun 2023

PONE-D-23-02011R2

The ribosome-inactivating proteins MAP30 and Momordin inhibit SARS-CoV-2

Dear Dr. Watts:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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Thank you for submitting your work to PLOS ONE and supporting open access.

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PLOS ONE Editorial Office Staff

on behalf of

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. MAP30 and Momordin proteins.

(PDF)

Click here for additional data file.^{(169.1KB, pdf)}

S2 Fig. MAP30 and ACE2 do not interact.

(PDF)

Click here for additional data file.^{(195.8KB, pdf)}

S3 Fig. Model of MAP30-SRL interaction.

(PDF)

Click here for additional data file.^{(368.9KB, pdf)}

S4 Fig. Interaction of the c11-P peptide with MAP30 and Momordin as modeled by molecular dynamics simulation.

(PDF)

Click here for additional data file.^{(297.2KB, pdf)}

S5 Fig. Points on SARS-CoV-2 genome potentially susceptible to RIP inhibition.

(PDF)

Click here for additional data file.^{(193KB, pdf)}

S1 Table. MAP30 and Momordin Mn⁺² binding sites.

(PDF)

Click here for additional data file.^{(64.1KB, pdf)}

S2 Table. Binding parameters of c11-P peptide.

(PDF)

Click here for additional data file.^{(65.1KB, pdf)}

S3 Table. Predicted RNA hairpin folding parameters.

(PDF)

Click here for additional data file.^{(65.7KB, pdf)}

S1 Movie. Molecular dynamics simulation of MAP30 interaction with c11-P peptide.

(MOV)

Click here for additional data file.^{(13.7MB, mov)}

S2 Movie. Molecular dynamics simulation of Momordin interaction with c11-P peptide.

(MOV)

Click here for additional data file.^{(10.8MB, mov)}

Attachment

Submitted filename: Response to Reviewers.docx

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Submitted filename: Response to Reviewers.docx

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Data Availability Statement

All assay data are available in the Dryad Repository (DOI: 10.5061/dryad.z08kprrj4).

[pone.0286370.ref001] 1.Puri M, Kaur I, Kanwar RK, Gupta RC, Chauhan A, Kanwar JR. Ribosome inactivating proteins (RIPs) from Momordica charantia for anti viral therapy. Curr Mol Med. 2009;9(9):1080–94. doi: 10.2174/156652409789839071 [DOI] [PubMed] [Google Scholar]

[pone.0286370.ref002] 2.Puri M, Kaur I, Perugini MA, Gupta RC. Ribosome-inactivating proteins: current status and biomedical applications. Drug Discov Today. 2012;17(13–14):774–83. doi: 10.1016/j.drudis.2012.03.007 [DOI] [PubMed] [Google Scholar]

[pone.0286370.ref003] 3.Walsh MJ, Dodd JE, Hautbergue GM. Ribosome-inactivating proteins: potent poisons and molecular tools. Virulence. 2013;4(8):774–84. doi: 10.4161/viru.26399 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0286370.ref004] 4.Zhu F, Zhou YK, Ji ZL, Chen XR. The Plant Ribosome-Inactivating Proteins Play Important Roles in Defense against Pathogens and Insect Pest Attacks. Front Plant Sci. 2018;9. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0286370.ref005] 5.Lu JQ, Zhu ZN, Zheng YT, Shaw PC. Engineering of Ribosome-inactivating Proteins for Improving Pharmacological Properties. Toxins (Basel). 2020;12(3). doi: 10.3390/toxins12030167 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0286370.ref006] 6.Citores L, Iglesias R, Ferreras JM. Antiviral Activity of Ribosome-Inactivating Proteins. Toxins (Basel). 2021;13(2). doi: 10.3390/toxins13020080 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0286370.ref007] 7.Basch E, Gabardi S, Ulbricht C. Bitter melon (Momordica charantia): a review of efficacy and safety. Am J Health Syst Pharm. 2003;60(4):356–9. doi: 10.1093/ajhp/60.4.356 [DOI] [PubMed] [Google Scholar]

[pone.0286370.ref008] 8.Arslan I, Akgul H, Kara M. Saporin, a Polynucleotide-Adenosine Nucleosidase, May Be an Efficacious Therapeutic Agent for SARS-CoV-2 Infection. SLAS Discov. 2021;26(3):330–5. doi: 10.1177/2472555220970911 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0286370.ref009] 9.Hassan Y, Ogg S, Ge H. Novel Binding Mechanisms of Fusion Broad Range Anti-Infective Protein Ricin A Chain Mutant-Pokeweed Antiviral Protein 1 (RTAM-PAP1) against SARS-CoV-2 Key Proteins in Silico. Toxins (Basel). 2020;12(9). doi: 10.3390/toxins12090602 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0286370.ref010] 10.Wang YX, Neamati N, Jacob J, Palmer I, Stahl SJ, Kaufman JD, et al. Solution structure of anti-HIV-1 and anti-tumor protein MAP30: structural insights into its multiple functions. Cell. 1999;99(4):433–42. doi: 10.1016/s0092-8674(00)81529-9 [DOI] [PubMed] [Google Scholar]

[pone.0286370.ref011] 11.Bolognesi A, Bortolotti M, Maiello S, Battelli MG, Polito L. Ribosome-Inactivating Proteins from Plants: A Historical Overview. Molecules. 2016;21(12). doi: 10.3390/molecules21121627 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0286370.ref012] 12.Lee-Huang S, Huang PL, Nara PL, Chen HC, Kung HF, Huang P, et al. MAP 30: a new inhibitor of HIV-1 infection and replication. FEBS Lett. 1990;272(1–2):12–8. doi: 10.1016/0014-5793(90)80438-o [DOI] [PubMed] [Google Scholar]

[pone.0286370.ref013] 13.Huang PL, Sun YT, Chen HC, Kung HF, Huang PL, Lee-Huang S. Proteolytic fragments of anti-HIV and anti-tumor proteins MAP30 and GAP31 are biologically active. Biochem Bioph Res Co. 1999;262(3):615–23. doi: 10.1006/bbrc.1999.1213 [DOI] [PubMed] [Google Scholar]

[pone.0286370.ref014] 14.Fan X, He L, Meng Y, Li G, Li L, Meng Y. Alpha-MMC and MAP30, two ribosome-inactivating proteins extracted from Momordica charantia, induce cell cycle arrest and apoptosis in A549 human lung carcinoma cells. Mol Med Rep. 2015;11(5):3553–8. [DOI] [PubMed] [Google Scholar]

[pone.0286370.ref015] 15.Yang GL, Li SM, Wang SZ. Research progress in enzyme activity and pharmacological effects of ribosome-inactivity protein in bitter melon. Toxin Rev. 2016;35(3–4):128–32. [Google Scholar]

[pone.0286370.ref016] 16.Dandawate PR, Subramaniam D, Padhye SB, Anant S. Bitter melon: a panacea for inflammation and cancer. Chin J Nat Med. 2016;14(2):81–100. doi: 10.1016/S1875-5364(16)60002-X [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0286370.ref017] 17.Fang EF, Froetscher L, Scheibye-Knudsen M, Bohr VA, Wong JH, Ng TB. Emerging Antitumor Activities of the Bitter Melon (Momordica charantia). Curr Protein Pept Sci. 2019;20(3):296–301. doi: 10.2174/1389203719666180622095800 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0286370.ref018] 18.Chan DW, Yung MM, Chan YS, Xuan Y, Yang H, Xu D, et al. MAP30 protein from Momordica charantia is therapeutic and has synergic activity with cisplatin against ovarian cancer in vivo by altering metabolism and inducing ferroptosis. Pharmacol Res. 2020;161:105157. doi: 10.1016/j.phrs.2020.105157 [DOI] [PubMed] [Google Scholar]

[pone.0286370.ref019] 19.Omokhua-Uyi AG, Van Staden J. Phytomedicinal relevance of South African Cucurbitaceae species and their safety assessment: A review. J Ethnopharmacol. 2020;259:112967. doi: 10.1016/j.jep.2020.112967 [DOI] [PubMed] [Google Scholar]

[pone.0286370.ref020] 20.Bourinbaiar AS, Leehuang S. Potentiation of Anti-Hiv Activity of Antiinflammatory Drugs, Dexamethasone and Indomethacin, by Map30, the Antiviral Agent from Bitter-Melon. Biochem Bioph Res Co. 1995;208(2):779–85. [DOI] [PubMed] [Google Scholar]

[pone.0286370.ref021] 21.Bourinbaiar AS, Lee-Huang S. The non-steroidal anti-inflammatory drug, indomethacin, as an inhibitor of HIV replication. FEBS Lett. 1995;360(1):85–8. doi: 10.1016/0014-5793(95)00057-g [DOI] [PubMed] [Google Scholar]

[pone.0286370.ref022] 22.Robert F, Roman W, Bramoulle A, Fellmann C, Roulston A, Shustik C, et al. Translation initiation factor eIF4F modifies the dexamethasone response in multiple myeloma. Proc Natl Acad Sci U S A. 2014;111(37):13421–6. doi: 10.1073/pnas.1402650111 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0286370.ref023] 23.Group RC, Horby P, Lim WS, Emberson JR, Mafham M, Bell JL, et al. Dexamethasone in Hospitalized Patients with Covid-19. N Engl J Med. 2021;384(8):693–704. doi: 10.1056/NEJMoa2021436 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0286370.ref024] 24.Ravichandran R, Mohan SK, Sukumaran SK, Kamaraj D, Daivasuga SS, Ravi S, et al. An open label randomized clinical trial of Indomethacin for mild and moderate hospitalised Covid-19 patients. Sci Rep. 2022;12(1):6413. doi: 10.1038/s41598-022-10370-1 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0286370.ref025] 25.Grela P, Szajwaj M, Horbowicz-Drozdzal P, Tchorzewski M. How Ricin Damages the Ribosome. Toxins. 2019;11(5). doi: 10.3390/toxins11050241 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0286370.ref026] 26.Lord MJ, Jolliffe NA, Marsden CJ, Pateman CS, Smith DC, Spooner RA, et al. Ricin. Mechanisms of cytotoxicity. Toxicol Rev. 2003;22(1):53–64. doi: 10.2165/00139709-200322010-00006 [DOI] [PubMed] [Google Scholar]

[pone.0286370.ref027] 27.Putnam CD, Tainer JA. The food of sweet and bitter fancy. Nat Struct Biol. 2000;7(1):17–8. doi: 10.1038/71211 [DOI] [PubMed] [Google Scholar]

[pone.0286370.ref028] 28.Jetzt AE, Li XP, Tumer NE, Cohick WS. Toxicity of ricin A chain is reduced in mammalian cells by inhibiting its interaction with the ribosome. Toxicol Appl Pharmacol. 2016;310:120–8. doi: 10.1016/j.taap.2016.09.004 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0286370.ref029] 29.Too PH, Ma MK, Mak AN, Wong YT, Tung CK, Zhu G, et al. The C-terminal fragment of the ribosomal P protein complexed to trichosanthin reveals the interaction between the ribosome-inactivating protein and the ribosome. Nucleic Acids Res. 2009;37(2):602–10. doi: 10.1093/nar/gkn922 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0286370.ref030] 30.Barbieri L, Gorini P, Valbonesi P, Castiglioni P, Stirpe F. Unexpected activity of saporins. Nature. 1994;372(6507):624. doi: 10.1038/372624a0 [DOI] [PubMed] [Google Scholar]

[pone.0286370.ref031] 31.Barbieri L, Valbonesi P, Bonora E, Gorini P, Bolognesi A, Stirpe F. Polynucleotide:adenosine glycosidase activity of ribosome-inactivating proteins: effect on DNA, RNA and poly(A). Nucleic Acids Res. 1997;25(3):518–22. doi: 10.1093/nar/25.3.518 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0286370.ref032] 32.Alexandersen S, Chamings A, Bhatta TR. SARS-CoV-2 genomic and subgenomic RNAs in diagnostic samples are not an indicator of active replication. Nat Commun. 2020;11(1):6059. doi: 10.1038/s41467-020-19883-7 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0286370.ref033] 33.Lee-Huang S, Huang PL, Chen HC, Huang PL, Bourinbaiar A, Huang HI, et al. Anti-HIV and anti-tumor activities of recombinant MAP30 from bitter melon. Gene. 1995;161(2):151–6. doi: 10.1016/0378-1119(95)00186-a [DOI] [PubMed] [Google Scholar]

[pone.0286370.ref034] 34.Meng Y, Liu S, Li J, Meng Y, Zhao X. Preparation of an antitumor and antivirus agent: chemical modification of alpha-MMC and MAP30 from Momordica Charantia L. with covalent conjugation of polyethyelene glycol. Int J Nanomedicine. 2012;7:3133–42. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0286370.ref035] 35.Priyanto AD, Doerksen RJ, Chang CI, Sung WC, Widjanarko SB, Kusnadi J, et al. Screening, discovery, and characterization of angiotensin-I converting enzyme inhibitory peptides derived from proteolytic hydrolysate of bitter melon seed proteins. J Proteomics. 2015;128:424–35. doi: 10.1016/j.jprot.2015.08.018 [DOI] [PubMed] [Google Scholar]

[pone.0286370.ref036] 36.Lee-Huang S, Huang PL, Huang PL. The Discovery of MAP30 and Elucidation of its Medicinal Activities. In: Fang EF, Ng TB, editors. Antitumor Potential and Other Emerging Medicinal Properties of Natural Compounds. Dordrecht: Springer Science+Business Media; 2013. p. 117–26. [Google Scholar]

[pone.0286370.ref037] 37.Banks JL, Beard HS, Cao Y, Cho AE, Damm W, Farid R, et al. Integrated Modeling Program, Applied Chemical Theory (IMPACT). J Comput Chem. 2005;26(16):1752–80. doi: 10.1002/jcc.20292 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0286370.ref038] 38.Humphrey W, Dalke A, Schulten K. VMD: visual molecular dynamics. J Mol Graph. 1996;14(1):33–8, 27–8. doi: 10.1016/0263-7855(96)00018-5 [DOI] [PubMed] [Google Scholar]

[pone.0286370.ref039] 39.Krissinel E, Henrick K. Inference of macromolecular assemblies from crystalline state. J Mol Biol. 2007;372(3):774–97. doi: 10.1016/j.jmb.2007.05.022 [DOI] [PubMed] [Google Scholar]

[pone.0286370.ref040] 40.Schymkowitz J, Borg J, Stricher F, Nys R, Rousseau F, Serrano L. The FoldX web server: an online force field. Nucleic Acids Res. 2005;33(Web Server issue):W382–8. doi: 10.1093/nar/gki387 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0286370.ref041] 41.Gruber AR, Lorenz R, Bernhart SH, Neubock R, Hofacker IL. The Vienna RNA websuite. Nucleic Acids Res. 2008;36(Web Server issue):W70-4. doi: 10.1093/nar/gkn188 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0286370.ref042] 42.Wang J, Wang J, Huang Y, Xiao Y. 3dRNA v2.0: An Updated Web Server for RNA 3D Structure Prediction. Int J Mol Sci. 2019;20(17). doi: 10.3390/ijms20174116 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0286370.ref043] 43.Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, et al. UCSF Chimera—a visualization system for exploratory research and analysis. J Comput Chem. 2004;25(13):1605–12. doi: 10.1002/jcc.20084 [DOI] [PubMed] [Google Scholar]

[pone.0286370.ref044] 44.Li WH, Moore MJ, Vasilieva N, Sui JH, Wong SK, Berne MA, et al. Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature. 2003;426(6965):450–4. doi: 10.1038/nature02145 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0286370.ref045] 45.Maddox CB, Rasmussen L, White EL. Adapting Cell-Based Assays to the High Throughput Screening Platform: Problems Encountered and Lessons Learned. JALA Charlottesv Va. 2008;13(3):168–73. doi: 10.1016/j.jala.2008.02.002 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0286370.ref046] 46.Severson WE, Shindo N, Sosa M, Fletcher T, 3rd, White EL, Ananthan S, et al. Development and validation of a high-throughput screen for inhibitors of SARS CoV and its application in screening of a 100,000-compound library. J Biomol Screen. 2007;12(1):33–40. doi: 10.1177/1087057106296688 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0286370.ref047] 47.Hou YJ, Okuda K, Edwards CE, Martinez DR, Asakura T, Dinnon KH, 3rd, et al. SARS-CoV-2 Reverse Genetics Reveals a Variable Infection Gradient in the Respiratory Tract. Cell. 2020;182(2):429–46 e14. doi: 10.1016/j.cell.2020.05.042 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0286370.ref048] 48.Yadav B, Wennerberg K, Aittokallio T, Tang J. Searching for Drug Synergy in Complex Dose-Response Landscapes Using an Interaction Potency Model. Comput Struct Biotechnol J. 2015;13:504–13. doi: 10.1016/j.csbj.2015.09.001 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0286370.ref049] 49.Nikam R, Kulandaisamy A, Harini K, Sharma D, Gromiha MM. ProThermDB: thermodynamic database for proteins and mutants revisited after 15 years. Nucleic Acids Research. 2021;49(D1):D420–D4. doi: 10.1093/nar/gkaa1035 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0286370.ref050] 50.Letko M, Marzi A, Munster V. Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses. Nat Microbiol. 2020;5(4):562–9. doi: 10.1038/s41564-020-0688-y [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0286370.ref051] 51.Shoemaker RH, Panettieri RA Jr., Libutti SK, Hochster HS, Watts NR, Wingfield PT, et al. Development of an aerosol intervention for COVID-19 disease: Tolerability of soluble ACE2 (APN01) administered via nebulizer. PLoS One. 2022;17(7):e0271066. doi: 10.1371/journal.pone.0271066 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0286370.ref052] 52.Liu B, Zhang Z, Lu S, He Q, Deng N, Meng H, et al. In-silico analysis of ligand-receptor binding patterns of alpha-MMC, TCS and MAP30 protein to LRP1 receptor. J Mol Graph Model. 2020;98:107619. [DOI] [PubMed] [Google Scholar]

[pone.0286370.ref053] 53.Lv Q, Yang XZ, Fu LY, Lu YT, Lu YH, Zhao J, et al. Recombinant expression and purification of a MAP30-cell penetrating peptide fusion protein with higher anti-tumor bioactivity. Protein Expr Purif. 2015;111:9–17. doi: 10.1016/j.pep.2015.03.008 [DOI] [PubMed] [Google Scholar]

[pone.0286370.ref054] 54.Jiang Y, Miao J, Wang D, Zhou J, Liu B, Jiao F, et al. MAP30 promotes apoptosis of U251 and U87 cells by suppressing the LGR5 and Wnt/beta-catenin signaling pathway, and enhancing Smac expression. Oncol Lett. 2018;15(4):5833–40. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0286370.ref055] 55.Husain J, Tickle IJ, Wood SP. Crystal structure of momordin, a type I ribosome inactivating protein from the seeds of Momordica charantia. FEBS Lett. 1994;342(2):154–8. doi: 10.1016/0014-5793(94)80491-5 [DOI] [PubMed] [Google Scholar]

[pone.0286370.ref056] 56.Li XP, Kahn PC, Kahn JN, Grela P, Tumer NE. Arginine residues on the opposite side of the active site stimulate the catalysis of ribosome depurination by ricin A chain by interacting with the P-protein stalk. J Biol Chem. 2013;288(42):30270–84. doi: 10.1074/jbc.M113.510966 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0286370.ref057] 57.Mihalek I, Res I, Lichtarge O. A family of evolution-entropy hybrid methods for ranking protein residues by importance. J Mol Biol. 2004;336(5):1265–82. doi: 10.1016/j.jmb.2003.12.078 [DOI] [PubMed] [Google Scholar]

[pone.0286370.ref058] 58.Hartley MR. Enzymatic Activities of Ribosome-Inactivating Proteins. In: Lord JM, Hartley MR, editors. Toxic Plant Proteins. Plant Cell Monographs. 18. Heidelberg, Germany: Springer; 2010. p. 41–54. [Google Scholar]

[pone.0286370.ref059] 59.Park GJ, Osinski A, Hernandez G, Eitson JL, Majumdar A, Tonelli M, et al. The mechanism of RNA capping by SARS-CoV-2. Nature. 2022;609(7928):793–800. doi: 10.1038/s41586-022-05185-z [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0286370.ref060] 60.Zou L, Moch C, Graille M, Chapat C. The SARS-CoV-2 protein NSP2 impairs the silencing capacity of the human 4EHP-GIGYF2 complex. iScience. 2022;25(7):104646. doi: 10.1016/j.isci.2022.104646 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0286370.ref061] 61.Xu Z, Choi JH, Dai DL, Luo J, Ladak RJ, Li Q, et al. SARS-CoV-2 impairs interferon production via NSP2-induced repression of mRNA translation. Proc Natl Acad Sci U S A. 2022;119(32):e2204539119. doi: 10.1073/pnas.2204539119 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0286370.ref062] 62.Lei J, Kusov Y, Hilgenfeld R. Nsp3 of coronaviruses: Structures and functions of a large multi-domain protein. Antiviral Res. 2018;149:58–74. doi: 10.1016/j.antiviral.2017.11.001 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0286370.ref063] 63.Newman JA, Douangamath A, Yadzani S, Yosaatmadja Y, Aimon A, Brandao-Neto J, et al. Structure, mechanism and crystallographic fragment screening of the SARS-CoV-2 NSP13 helicase. Nat Commun. 2021;12(1):4848. doi: 10.1038/s41467-021-25166-6 [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

The ribosome-inactivating proteins MAP30 and Momordin inhibit SARS-CoV-2

Norman R Watts

Elif Eren

Ira Palmer

Paul L Huang

Philip Lin Huang

Robert H Shoemaker

Sylvia Lee-Huang

Paul T Wingfield

Roles

Abstract

Introduction

Fig 1. Structure comparison of MAP30 and Momordin.

Materials and methods

Recombinant protein production

Analytical centrifugation

Circular dichroism

MAP30 interaction with soluble ACE2

Molecular dynamics simulations

Model assessment

Cell culture

Sample preparation

Antiviral assays

A549 nanoluc reporter virus (NLRV) assay

Cytotoxicity assay

Data analysis

Matrix assay compound preparation

Results and discussion

Protein production and characterization

MAP30 does not interact with ACE2

A549 NLRV assays

Antiviral activity of MAP30 and Momordin

Fig 2. SARS-CoV-2 inhibition by MAP30 and Momordin.

Table 1. Viral inhibition and cytotoxicity values of MAP30 and Momordin.

MAP30 and Momordin show no synergy with Dexamethasone and Indomethacin

Mutational analyses of MAP30 active- and ribosome-binding sites

Fig 3. Mutation of MAP30.

Structural comparison of MAP30 and Momordin

Fig 4. Comparison of MAP30 and Momordin active-site faces.

MAP30 and Momordin active-site faces

MAP30 and Momordin active-site-distal faces

Fig 5. Comparison of MAP30 and Momordin ribosome-binding-site faces.

Molecular dynamics simulations of MAP30 and Momordin interaction with ribosomal P-protein peptide

A potential new point of SARS-CoV-2 inhibition

Vero E6 CPE assays

Conclusions

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Israel Silman

Roles

Author response to Decision Letter 0

Decision Letter 1

Israel Silman

Roles

Author response to Decision Letter 1

Decision Letter 2

Israel Silman

Roles

Acceptance letter

Israel Silman

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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RESOURCES

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