Inhibition of SARS-CoV-2 M pro with Vitamin C, L-Arginine and a Vitamin C/L-Arginine Combination

Background : Drug resistance is a critical problem in health care that affects therapy outcomes and requires new approaches to drug design. SARS-CoV-2 M pro mutations are of concern as they can potentially reduce therapeutic efficacy. Viral infections are amongst the many disorders for which nutraceuticals have been employed as an adjunct therapy. The aim of this study was to examine the potential in vitro activity of L-arginine and vitamin C against SARS-CoV-2 M pro . Methods : The M pro inhibition assay was developed by cloning, expression, purification, and characterization of M pro . Selected compounds were then screened for protease inhibition. Results : L-arginine was found to be active against SARS-CoV-2 M pro , while a vitamin C/L-arginine combination had a synergistic antiviral action against M pro . These findings confirm the results of our previous in silico repurposing study that showed L-arginine and vitamin C were potential M pro inhibitors. Moreover, they suggest a possible molecular mechanism to explain the beneficial effect of arginine in COVID patients. Conclusions : The findings of the current study are important because they help to identify COVID-19 treatments that are efficient, inexpensive, and have a favorable safety profile. The results of this study also suggest a possible adjuvant nutritional strategy for COVID-19 that could be used in conjunction with pharmacological agents.


Introduction
The SARS-CoV-2 virus quickly spread around the world and was classified by the World Health Organization on March 11, 2020 as the second pandemic of the 21st century [1].SARS-CoV-2 illness is frequently accompanied by unremitting fever, hypoxemic respiratory failure, systemic complications, encephalopathy, delirium, and thromboembolic events [2][3][4].Following infection with SARS-CoV-2, patients with severe and critical illnesses suffer frequent neurological complications [4].The blood-brain barrier (BBB) is a critical interface that regulates the entry of circulating molecules into the central nervous system (CNS).The BBB is therefore essential for the treatment of viruses that can infect the CNS, such as SARS-CoV-2 [5].
Nutraceuticals, phytochemicals from medicinal plants, and dietary supplements have been used as adjunct therapies for many diseases, including viral infections.The use of adjunct antiviral therapy may be beneficial in the treatment and prophylaxis of COVID-19 [6].
Arginine is a natural molecule that crosses the BBB through a transporter with specificity for amino acid analogs that possess cationic terminal guanidine groups, such as those contained in L-arginine [7].Recently, it was shown that amino acids can improve immunity and shorten disease length in patients with COVID-19 [8].In a randomized clinical trial of adults with severe COVID-19, Larginine plus standard care significantly reduced the need for respiratory support and reduced the length of hospitalization.Large doses (1.66 g) of L-arginine were given orally twice per day for the entire hospitalization period [8].The molecular mechanisms that underlie the significant modulating effect of arginine are still to be clarified.
The main protease of SARS-CoV-2, M pro (also called 3CLpro), is an important drug target due to its crucial role in the life cycle of the virus.M pro is one of the best characterized drug targets in coronaviruses that lack homologous human protease, thus making it one of the most attractive SARS-CoV-2 drug targets [9].
We previously proposed a simple theoretical criterion for rapid virtual screening of molecular libraries for candidate inhibitors of M pro [10].After initial in silico screen- ing of Drugspace using EIIP/AQVN filter [11], followed by further filtering of drugs by virtual ligand-based screening and molecular adhesion, we identified arginine as a candidate M pro inhibitor [10].In another computer-based study, arginine was identified amongst the 20 amino acids as the best inhibitor of the main protease in SARS-CoV-2 [12].In our earlier in silico work, we also proposed vitamin C as a candidate M pro inhibitor.It was shown in another study that vitamin C inhibits SARS-CoV-2 3CLpro in vitro [13].Ascorbic acid, or vitamin C, is an important water-soluble nutrient.It is produced by plants and by virtually all animals, with the exception of primates, most bats, guinea pigs, and a few rodents which, as a consequence, require vitamin C in their diet [14].Some of the important effects of vitamin C following viral infection are reduced pro-inflammatory response, improved epithelial barrier function, enhanced alveolar fluid clearance, antiviral activity, and immune system stimulation.Vitamin C is also a crucial factor in the production of type I interferons during the antiviral immune response, and acts as an inactivating agent for RNA and DNA viruses [15].
Due to its antioxidant, anti-inflammatory, and immunomodulatory properties, vitamin C is a possible ther-apeutic option for the prevention and treatment of COVID-19 infection, as well as a possible adjuvant therapy for COVID-19 critical care [16].
The results of the present study showed that arginine has inhibitory activity against M pro in vitro.Furthermore, a vitamin C/arginine combination was found to have synergistic inhibitory activity in vitro.

Chemicals
The antibiotic Kanamycin was purchased from Invitrogen, catalog number: 11815024, Carlsbad, CA, USA.
Various components for media preparation (agar, peptone and tryptone) were purchased from Torlak, Belgrade, Serbia.Other substances were ordered from Centrohem, Belgrade, Serbia.

M pro Gene
The gene for M pro was ordered from Addgene and cloned into the pETM33 vector with N-terminal GST and His-tag (Fig. 1, Ref. [17]).
The M pro gene was cloned using NcoI and EcoRI restriction enzymes.Chimera: His-GST-HRV_3C-MP has 1656 bp with a Mr of 62.3 kDa.Recommended expression conditions when using E. coli BL21 DE3 gold cells are growth at 37 °C, induction with IPTG at a final concentration of 1 mM, and expression for 16 h at 18 °C.E. coli STAR strain was used for intracellular expression, while the DH5α strain was used for storage and propagation of plasmids.

Cell Lysis
Collected cells were resuspended in 5 mL of lysis buffer composed of 50 mM Na-phosphate buffer, 300 mM NaCl and 10 mM imidazole (pH 7.5).Samples were sonicated on ice.Aliquots were taken before induction (0 h) and after expression (24 h).Sonication was performed using an ultrasound probe, 10 times for 10 seconds each, with a 20 second pause in between.After lysis, the mix was centrifuged for 5 min at 13,000 rpm and the supernatant then passed through a sterile 0.22 µm filter.

Purification
M pro (25 mL) was purified by HPLC with a 5 mL Ni-NTA FF Sepharose column.The lysis buffer described above was used for column equilibration, and the same buffer with a gradient from 10 mM to 300 mM imidazole was then used for protein elution.Absorbance at 280 nm was monitored and fractions were examined by sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) electrophoresis.Samples (17 mL) were dialyzed against 50 mM Tris-HCl buffer with 150 mM NaCl, pH 7.5.The purified enzyme was stored in 50% glycerol at -20 °C.

Measurement of Activity
Changes in fluorescence were monitored every 135 s for 45 min at an excitation wavelength of 485 nm and an emission wavelength of 535 nm.The total volume of reaction mixture was 200 µL and the reaction buffer was 20 mM Tris with 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA) and 1 mM dithiothreitol (DTT) (pH 7.3).Five µL of enzyme and 1 µL of fluorescent substrate dissolved in dimetilsulfoksid (DMSO) were added so that the final concentration was 5 µM.L-arginine and vitamin C were tested as possible inhibitors.These were dissolved in 20 mM Tris (pH 7.3) and the pH adjusted to 7.3 if necessary.Final inhibitor concentrations used in the experiments were 2 mM, 10 mM, 20 mM, 50 mM, 100 mM, 150 mM, and 200 mM.Buffer was used as a blank, and buffer plus substrate was used as a control.

M pro Inhibition with Vitamin C, Arginine, and Vitamin C/Arginine Combination
Figs. 2,3 show the inhibition of M pro with vitamin C and arginine, respectively.The addition of 75 mM arginine to 20 mM (3.52 mg/mL) vitamin C increased the level of inhibition from 44% to 61%, while the addition of 75 mM arginine to 50 mM (8.81 mg/mL) vitamin C increased inhibition from 67 to 82% (Table 1).Therefore, vitamin C and the amino acid arginine have an additive inhibitory effect on the in vitro proteolytic activity of the M pro protease from the SARS-CoV-2 virus (Table 1).

Inhibition of M pro by Vitamin C (IC 20 mM)
It could be seen from the obtained results that around 44% inhibition was achieved using 20 mM vitamin C concentration (Fig. 2), while in the case of arginin 39% of inhibition was achieved at 75 mM concentration (Fig. 3).
When both compounds were added at the same con-  centration's inhibition was increased to 61% showing an additive inhibitory effect on the in vitro proteolytic activity of the M pro protease from the SARS-CoV-2 virus (Table 1).
Additive inhibitory effect was achieved also using 50 mM vitamin C in combination with 75 mM argining giving 82% of M pro protease decrease in activity.

Discussion
Drug resistance is a critical problem in health care that affects therapy outcomes and requires new drug design approaches.SARS-CoV-2 mutations are therefore of great concern as they could lead to drug resistance.The SARS-CoV-2 main protease (M pro ) is one of the most attractive drug targets.However, more than 19,000 mutations encompassing 282 amino acid positions have already been reported in M pro .These "hotspots" could change the M pro structure and activity and potentially reduce any therapeutic effects against this protease [18].Safe and inexpensive treatments for SARS-CoV-2 prevention and as adjunct therapy to pharmacological agents are therefore urgently required during the current pandemic.
Nutraceuticals are defined as any food or food component that provides medical or health benefits, including the prevention and treatment of disease [19].These may have potential therapeutic efficacy in the fight against the SARS-CoV-2/COVID-19 pandemic.In a randomized clin-ical trial of adults with severe COVID-19, L-arginine given with standard care was found to significantly reduce the need for respiratory support and the length of hospitalization (NCT04637906, registration date November 20, 2020; [8]).Recently, the L-Arginine and Vitamin C improves Long-COVID (LINCOLN) survey found that L-arginine when taken with vitamin C improved long-COVID, thus demonstrating for the first time the beneficial effect of this combination.The survey was completed by 1390 patients who were divided into two groups: those who received Larginine plus vitamin C, and those who received a multivitamin combination.According to the survey, supplementation with L-arginine plus vitamin C had beneficial effects for long-COVID patients, with less symptoms and significantly lower effort perception [20].
The important roles of amino acids, including arginine, in immune responses were recently reviewed [21].L-arginine is converted in the body to nitric oxide, which has been suggested as a therapeutic option for COVID-19.Nitric oxide was shown to be an effective antiviral against SARS-CoV in vitro, as well as in vivo by inhalation of very low concentrations in a small clinical trial [22].Although some published studies have linked arginine supplementation with increased nitric oxide production, other reports claim that acute L-arginine supplementation does not increase nitric oxide production in healthy subjects.The molecular mechanisms that underlie this important modulating effect of arginine therefore remain to be clarified [23].
The focus of the current study was to investigate the action of a non-toxic, natural amino acid against the M pro of SARS-CoV-2.In our previous in silico study we proposed that arginine inhibits SARS-CoV-2 M pro .This was confirmed experimentally in the present study, and was not unexpected since amino acid building blocks are often found in drugs and in drug candidates whose molecular targets bind naturally to amino acids or peptide structures.Many antiviral enzymatic inhibitors incorporate amino acid motifs or are themselves amino acids, which is also common with protease inhibitors [24].
We examined the inhibition of M pro by arginine in vitro, as well as the effect of a vitamin C/arginine combination.Arginine was found to inhibit SARS-CoV-2 M pro in vitro, while co-administration of arginine and vitamin C exerted synergistic M pro inhibition due to complementary antiviral action.In our previous work we proposed that vitamin C binds to the catalytic site and L-arginine to the allosteric site [10].Vitamin C has antiviral, antioxidant, anti-inflammatory, and immunomodulatory effects, thereby making it a potential medical treatment for COVID-19.Indeed, several randomized controlled trials have evaluated intravenous vitamin C monotherapy in patients with COVID-19 [25,26].The current level of evidence from these trials suggests that intervention with intravenous vitamin C may improve oxygenation parameters, lower inflammatory markers, shorten hospital stays, and lower the mortality rate, especially in the most severely ill patients.Oral vitamin C supplementation at high doses may also improve the rate of recovery in less severe cases.No adverse outcomes have been reported in published trials [27].To validate our M pro assay, we first examined the inhibitory effect of vitamin C on M pro in vitro [12].The previously reported inhibitory activity of vitamin C against M pro was confirmed in our study.A recent study found that SARS-CoV-2 positivity and infection severity were linked to lower levels of protective Bifidobacterium genera and to lower bacterial diversity [28].Furthermore, the addition of ascorbic acid was shown to significantly promote the growth of B. bifidum [29] and could therefore confer a protective effect to SARS-CoV-2-positive patients.Based on these findings, vitamin C could also have additional protective effects for COVID patients.
The synergistic antiviral action of arginine/vitamin C against SARS-CoV-2 M pro shown in the present study may be a consequence of blocking multiple sites on one target.This strategy was previously shown to improve therapeutic efficacy [30].Furthermore, drug combination therapy was suggested as a promising strategy to extend the lifespan of antimicrobials that must be carefully selected to minimize the evolution of resistance [31].
The inflammation triggered by oxidative stress is the cause of many chronic diseases.Oxidative stress is characterized by increased production of free oxygen radicals and represents one of the basic pathological processes of atherosclerosis.It is also closely related to endothelial dysfunction and promotes a vascular inflammatory response [32].The correlation observed between COVID-19 and atherosclerosis suggests that effort should be directed towards cardioprotection [33].A previous study reported that supplemental L-arginine and vitamin C could be antiatherogenic, as observed by the modulation of endothelial dysfunction biomarkers [34].Based on these findings, the vitamin C/arginine combination could also have a cardioprotective effect in COVID patients, in addition to the direct antiviral effect suggested by our study.
The findings of our study are significant in at least two respects.First, we demonstrated that arginine exerts inhibitory action against SARS-CoV-2 M pro , and that coadministration with vitamin C exerts a synergistic antiviral action against M pro .These observations shed light on the potential molecular mechanism underlying the signif-icant modulating effect of arginine against SARS-CoV-2.Second, we confirmed the findings of our previous in silico drug repurposing study that identified L-arginine and vitamin C as candidate M pro inhibitors from amongst 1490 approved drugs in Drugbank.In that study, we used VS protocol with sequential filters based on both long-range and short-range interactions to select candidate SARS-CoV-2 M pro inhibitors.
The results of the current study are important in the search for effective, safe and affordable therapeutics against COVID-19.Our findings could also help to develop an effective nutritional strategy to fight infectious diseases.

Conclusions
Drug resistance is an important issue in health care that affects therapeutic results and necessitates novel drug design approaches.Nutraceuticals are an interesting treatment option for COVID-19.This study examined the potential antiviral activity of L-arginine and vitamin C in vitro.The experimental results showed that arginine inhibits SARS-CoV-2 M pro , and that a vitamin C/arginine combination has synergistic antiviral action against M pro .These findings confirm the results of our previous in silico drug repurposing study.
The results of the current study suggest a potential dietary approach to COVID-19, in addition to pharmaceutical treatments.This is important because it is vital to develop COVID-19 therapies that are effective, affordable, and have a favorable safety profile.Furthermore, our findings might help to develop an effective nutritional approach for the prevention and treatment of infectious diseases, thereby reducing the burden on communities and healthcare systems.

Table 1 . M pro inhibition with vitamin C, arginine, and vitamin C/arginine.
±14.82