In silico screening of Pueraria tuberosa (PTY-2) for targeting COVID-19 by countering dual targets Mpro and TMPRSS2

Abstract COVID-19 pandemic was started in Wuhan city of China in December 2019; immensely affected global population. Herein, an effort was made to identify potential inhibitors from active phytochemicals of Pueraria tuberosa (PTY-2) via molecular docking study. Our study showed five potential inhibitors (Robinin, Genistin, Daidzin, Hydroxytuberosone, Tuberostan) against Mpro and five inhibitors (Robinin, Anhydrotuberosin, Daidzin, Hydroxytuberosone, Stigmasterol) against TMPRSS2. Out of these, Robinin, Daidzin and Hydroxytuberosone were common inhibitors for Mpro and TMPRSS2. Among these, Robinin showed the highest binding affinity, therefore, tested for MD simulation runs and found stable. ADMET analysis revealed the best-docked compounds are safe and follow the Lipinski Rule of Five. Thus, it could be suggested that phytochemicals of PTY-2 could serve as potential inhibitors for COVID-19 targets. Communicated by Ramaswamy H. Sarma Highlights Application of active phytoconstituents of Pueraria tuberosa (PTY-2) for the repurposing in the management of COVID-19. Promising effect of Robinin as a multifocal inhibitor of virus-host interaction including main protease (Mpro) and TMPRSS2 with highest binding energy through molecular docking and molecular dynamics simulations studies. Robinin acts as common inhibitor against Mpro and TMPRSS2.


Introduction
On December 31, 2019, China informed the World Health Organization (WHO) regarding a cluster of cases of atypical pneumonia in Wuhan city in Hubei Province, now known as COVID-19 or coronavirus disease 2019. As of this paper is being drafted, COVID-19 globally has effected 116,061,296 cases, including 2,580,050 deaths as of 7:43 pm CEST, February 28th, 2021, as per the WHO situation report (WHO, 2020a), these figures on the COVID-19 spread and related casualties are rapidly becoming out-dated, suggesting extreme and rapid transmission rate. On January 31, 2020, the WHO declared it a public health emergency of international concern and pandemic on March 12, 2020 (Cascella et al., 2020). As for India, it once hold the largest number of confirmed cases in Asia (Times of India, 2020), and globally had recorded the second-highest number of confirmed cases after the US (Johns Hopkins Coronavirus Resource Center, 2020). The causative agent is known by many names, such as Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), 2019 novel coronavirus (2019-nCoV), and is responsible for COVID-19 that belongs to the Sarbecovirus subgenus, genus Betacoronavirus, family Coronaviridae. It has 2020; Zhu et al., 2020), respectively, which has affected the human population from time to time. Be it SARS, MERS (Middle East Respiratory Syndrome), which affects the respiratory tract, leading to severe difficulty in breathing and fever, cough, dyspnoea, renal system failure, respiratory system and even death if not controlled. The recent genomic study, including a group of patients with atypical pneumonia from the Wuhan region, has 89% nucleotide in a similar context to bat SARS-like-CoVZXC21 and 82% with SARS-CoV-2. The single-stranded RNA genome contains 29891 nucleotides, encoding 9860 amino acids (Chan et al., 2020).
SARS-CoV-2 is a single-stranded RNA virus that belongs to b-coronavirus of the family Coronaviridae. It is the seventh strain of viruses that have four structural proteins, namely the nucleocapsid (N), the encasing envelope (E), the membrane (M) and the spike (S). The nucleocapsid forms the genetic core, encased in a ball formed from the envelope and the membrane. The spike protein that sticks out forming club-shaped protrusion from the ball mimics a crown or the sun's corona, hence the name CORONAVIRUS. There are three significant steps involved in damaging the host respiratory system by the SARS-CoV-2 viruses which are: 1. Binding of the viruses to the host membrane. 2. Fusion and transmission of the viruses. 3. Manipulation of the host immune system. SARS-CoV-2 embraces more than 30,000 nucleotides containing replicase gene at pp1a and pp1b (polyprotein) (L. Zhang et al., 2020) essential for replication and rapid transmission, a 38-KDa enzyme known as main protease (M pro ) or 3 C-like main protease(3CLM pro )  digest the protein on at least eleven conserved sites causing proteolysis and releasing functional viral pp1a and pp1b that helps in the formation of non-structural proteins required for viral translation (Shree et al., 2020) signifying the role of M pro in viruses life cycle and serving as a curious target for an antiviral drug.
A study done by Shulla and colleagues identified that trans membrane serine proteases are somehow related to respiratory infections of viral origin. The proteases expedite the process of viral entry into the pulmonary region (Shulla et al., 2011). Recently a study performed by Hoffman et al. (2020) observed that TMPRSS2 (a member of the serine protease transmembrane family type II) initiate the cleavage of the coronavirus fusion Spike glycoprotein. TMPRSS2 facilitates the merging of viruses and host cell membrane, thereby easing the entry of SARS-CoV-2 in the lungs, suggesting that blocking TMPRSS2 may provide a treatment option by blocking the tissue tropism of the virus and thereby preventing further extension and complications. Many in silico study on Mpro and TMPRSS2 has been going on against COVID-19 targets using commercially available drugs. In our work, we have focused on searching naturally available phytoconstituents from medicinal plants' treasure against these targets.
Natural products have provided an answer for various ailments from time to time, including respiratory system disorders. Ayurveda has always viewed the human being as a sacred entity and has used a holistic approach in treating the disorder and working on creating a balanced environment within the body to prevent any disease or disorder. Pueraria tuberosa (Roxb. ex Willd.) DC. is a perennial herb commonly known as 'vidarikanda' distributed in the tropical parts of India. The tubers of Pueraria have abundant flavonoids and isoflavones namely puerarin (8.31%), daidzein(1.70%), genistein(1.37%), robinin, daidzin, genistin, tuberostan, tuberosin, 4 0 -methoxypuerarin, quercetin, hydroxytuberosone, biochanin A, biochanin B, irisolidon, glycoside (C-glycoside 4 0 ,6-diacetyl), puerarone and tectoridin (Maji et al., 2014;Rastogi et al., 2013). Reported pharmacological activities of vidarikand includes its anti-inflammatory (Y. Tripathi et al., 2013), antioxidant (Nagwani & Tripathi, 2010;Y. Tripathi et al., 2013), antidiabetic properties (Srivastava et al., 2017(Srivastava et al., , 2018, immunomodulatory (Sawale et al., 2013;Shukla et al., 2017) and nootropic activity ('NOPR: Nootropic activity of tuber extract of Pueraria tuberosa (roxb) ', n.d.). In vivo and in vitro studies offered various bioactive phytochemicals in Pueraria tuberosa, mostly isoflavonoids support immune-boosting activity. In one of the recent studies aqueous extract of tuber amplified the phagocytic ability of macrophages and enhanced the level of IgG and IgA antibodies in serum sample of mice (Sawale et al., 2013). In numerous other studies upturn antibody production, phagocytosis suppression of delayed-type hypersensitivity reaction, hindering the production of TNF-alpha, NF-kB, MIP-2 and the expression of iNOS, COX-2 and CRP (Cooke et al., 2006;Maji et al., 2014;R. Zhang et al., 1997). Vidarikand or PTY-2 helps reduce the symptoms of the Flu. In the ancient textbooks of Ayurveda, Flu or influenza is known as Vata Shleshmika Jwara, a viral infection of the upper respiratory tract. According to Ayurveda, Vata, Pitta and Kapha dosha go out of balance during the seasonal changes, resulting in Flu. Pueraria tuberosa lessens the symptoms of Flu and fights against seasonal changes due to its Rasayana (rejuvenating) properties (Jackson III & AHS, 2020;Tsang et al., 2017), suggesting PTY-2 as a potent immunomodulator which can be repurposed for treatment against COVID-19.
In the current work, we have tested the bioactive phytochemicals of PTY-2 for identifying their potential inhibitors for SARS-CoV-2 main protease (M pro ) and Transmembrane protein receptor TMPRSS2 for the treatment of COVID-19 infection through molecular docking approach (Figure 1). The docked compounds' stability with the macromolecular targets were assessed by molecular dynamics simulation studies and drug-likeness, ADMET profile analysis were simultaneously carried out.

Protein preparation
Three dimensional structure of SARS-CoV-2 main protease (M pro ) (PDB ID: 7BQY) was retrieved from RCSB Protein Data Bank (https://www.rcsb.org/). M pro has one chain of total 306 amino acids with a resolution of 1.7 Ð. Preparation of proteins was done with the help of 'Prepare protein' protocol of BIOVIA Discovery studio 4.5 (DS 4.5) at physiological pH 7.4 and water molecules, other heteroatoms were removed from the structure.

Ligands selection
For identification of potential inhibitors, total 25 active phytochemicals gathered from LC-MS analysis of Pueraria tuberosa (PTY-2) (Table S1), were taken from PubChem database (https://pubchem.ncbi.nlm.nih.gov/) in 2D SDF format and conversion of all small molecules in 3D PDB format, geometry minimization was done via DS 4.5.

Homology modelling
The crystal structure of TMPRSS2 has not been resolved yet, which is found to be the entry point for coronavirus spike protein. There is a need to build a model for which we have used SWISSMODEL server for homology modelling. Target sequence of TMPRSS2 has been downloaded from Uniprot database (https://www.uniprot.org/) with Uniprot ID O15393. Template search for the target sequence has been done using SWISS MODEL server (https://swissmodel.expasy.org/ interactive) (Schwede et al., 2003) and found 50 templates (https://swissmodel.expasy.org/interactive/ZnD5rx/templates/). Out of these we have select template of Serine protease hepsin (PDB ID: 5CE1) having 33.82% sequence identity, 38% sequence similarity and 0.53 GMQE value. Alignment of target-template sequence and model building was done through this server. Energy minimization of created model was done through 'clean geometry' protocol of DS 4.5. Additionally 'prepare protein' protocol of DS 4.5 was used for created model preparation at physiological pH 7.4. Further verification of created homology model was carried out using web-based tools ProSA (Wiederstein & Sippl, 2007) and PROCHECK (Laskowski et al., 1993) to evaluate the quality of homology model structure.

Molecular docking
Selected compounds from PTY-2 were docked with SARS-CoV-2 main protease (PDB ID: 7BQY) and homology Model 01 for TMPRSS2 using Autodock Vina-based YASARA software (Krieger et al., 2014;Trott & Olson, 2010). For complete molecular docking study, prepared ligand files and receptors were used to set the target and play macro. A command 'Macro file dockrun_mcr' was used to estimate interaction energy between receptor and selected ligands independently. Using YASARA, 25 VINA docking runs of the ligand object 2 to the receptor object 1 was performed. Further docked complexes were saved in PDB file format for 2D-3D interactive visualization study. The result log files sorted based of binding energy [kcal/mol] and dissociation constant [pM]. The compound having more positive binding energies indicates stronger binding, and negative energies denote no binding.

Molecular dynamic simulations
In recent times, MD simulations have been a remarkable approach for identifying protein-ligand stability, structural transformations, binding energies change in complexes and so on. We have also examined the binding stability of the compound Robinin with the selected two receptors M pro and TMPRSS2 of SARS-CoV-2 for 20 ns simulation period. For this purpose, the Gromacs simulation package was employed using Gromos54A7 force field for the calculation of simulation parameters (Berendsen et al., 1995). PRODRG webs server was utilized for generating ligand topology (van Aalten et al., 1996). The simulation setup was prepared using SPC water model for aqueous environment and 0.15 M salt concentration for proper electrostatic distribution in a cubic simulation box. Charge neutralization was done by adding counter ions. For energy minimization, 50,000 steps of steepest descent algorithm were executed with Verlet cut-off scheme to calculate the neighbouring interactions. Further, equilibration process under NPT and NVT conditions for 1 ns was done Parrinello-Rahman and V-rescale methods for pressure and temperature coupling respectively. LINCS algorithm was used for calculating bond parameters (Hess et al., 1997). Particle Mesh Ewald (PME) for long-range electrostatics with Fourier spacing of 0.16 was used for production MD. Finally, production MD run for all systems was performed in periodic boundary conditions for 20 ns each. Further, MD analysis consisting Root Mean Square Fluctuation (RMSF), Root Mean Square Deviation (RMSD), Radius of Gyration (Rg) of C-a atoms were done using gmx rms, rmsf and gyrate commands. For binding energy calculation, g_mmpbsa tool was implemented (Hou et al., 2011).

Homology modelling
Out of 50 templates for target sequences we selected template of Serine protease hepsin with PDB ID: 5CE1 for homology modelling. The target-template sequence alignment for Model 01 and Serine protease hepsin was performed using SWISSMODEL server. The generated model 01 has sequence identity of 33.82% while sequence similarity was 38% with Serine protease hepsin ( Figure 2).
It has been reported that homology modelling shares 30% or more of amino acid sequence identity between target and template sequence considered to be successful and reliable (Xiang, 2006). Using homology modelling server, structural alignment of target structure with the 3D structure of Serine protease hepsin template was executed using Chimera 1.14 visualizer, suggesting resemblance between the target and the template (Figure 3).
Further the verification of our homology modelling (model 01) of TMPRSS2 was done using a web-based version of Protein analysis tool ProSA which is used to calculate the zscore that indicates overall model quality, correlating model score to all the experimentally available scores on PDB website. Accordingly, the z-score of our homology model 01 was À8.67, which is in range of all PDB conformations (Figure 4(a)). Further, amino acid positions depending upon its relative local energy were articulated by the energy plot, where negative values concur with the accuracy of the structure (Figure 4(b)). Additionally, a web-based tool PROCHECK was used to generate Ramachandran plot for evaluating the energetically allowed regions of our generated model (Figure 4(c)). 84.6% of the residues were found in most favoured regions, 14.7% residues are present in additionally allowed regions and 0.3% of the remaining residues are present in disallowed regions, indicating good stereochemical quality of model 01 of TMPRSS2.

Molecular docking
Based on YASARA scoring system, molecular docking study identifies seven different phytochemicals from PTY-2 with high binding affinity with viral and host macromolecular targets. Table 1 shows the list of the active phytochemicals which showed significant binding affinity (>8.0 kcal/mol) with SARS-CoV-2 main protease (M pro ) and Transmembrane receptor TMPRSS2.

Potential inhibitors for SARS-CoV-2 main protease (M pro )
Molecular docking study revealed that out of 25 active phytochemicals of PTY-2, five phytochemicals namely Robinin, Genistin, Daidzin, Hydroxytuberosone and Tuberostan have highest binding affinity with M pro . Robinin was found to have highest binding affinity of 8.87 kcal/mol. Robinin is a Kaempferol derived glycosyloxyflavone and dihydroxyflavone, reported to have antibacterial and antifungal activity (Rosu et al., 2012). Robinin formed numerous conventional and carbon hydrogen bonding with the residues Glu 288, Lys 5, Lys 137, Asp 197, Arg 131, Asn 238, Thr 199 and Tyr 237, two p-alkyl interactions were formed with the residues Leu 286 and Leu 287, a p-anion bond was formed with Asp 289 and remaining residues showed few van der Waals interactions ( Figure 5(a,b)). Genistin was found to be second inhibitor of M pro with binding energy 8.18 kcal/mol. It is an isoflavone derived from Genistein, reported to have antiviral activity (Donovan et al., 2009) and estrogenic activity (Allred et al., 2001). Molecular docking study showed Genistin forming different ligand-protein interactions, including conventional and p-donor hydrogen bonding with the residues Gln 192, Glu 166, His 41 and Gln 189, p-sulfur and p-alkyl interactions was formed with Met 165, p-p T-shaped interaction was formed with the residue His 41 and the residue Thr 26 showed unfavourable donordonor and unfavourable acceptor-acceptor interaction and multiple van der Waals interactions were formed with the receptor protein ( Figure 5(c,d)). In continuation to this, Daidzin was found to be the third inhibitor of M pro with binding affinity 8.15 kcal/mol. Daidzin is an isoflavone glycoside derived from Daidzein with tumour preventive and antidipsotropic activity (Keung & Vallee, 1998). Different interactions shown by Daidzin which includes conventional, carbon and p-donor hydrogen bonding with the residues Gln 192, His 41, Glu 166 and Gln 189, p-alkyl interaction was formed with the residues Pro 168 and Met 165, a p-p T-shaped interaction was formed with the residue His 41, a p-sulfur interaction was made with the residue Met 165 and many van der Waals interactions were indicated by remaining residues ( Figure 5(e,f)). Fourth inhibitor was Hydroxytuberosone with binding affinity 8.09 kcal/mol. Interactions shown by Hydroxytuberosone involves conventional hydrogen bonds with residues Asp 295, Gln 110 and Thr 111, p-alkyl interactions with Tyr 154 and Arg 298 and van der Waals interactions showed by remaining residues (Figure 5(g,h)). Tuberostan was reported to be the fifth inhibitor for M pro with predicted binding energy 8.01 kcal/ mol. It showed conventional hydrogen bonding with the residue Arg 131, a p-anion interaction was formed with the residue Asp 289, alkyl and p-alkyl interactions with the residues Leu 286, Tyr 239, Leu 272 and Leu 287 and few van der Waals interactions were also formed with the receptor protein ( Figure 5(i,j)). It was deduced from the molecular docking study that significant binding energies of Robinin, Genistin, Daidzein, Hydroxytuberosone and Tuberostan could block the active site of M pro enzyme.

Potentials inhibitors for TMPRSS2
Robinin bound to our model 01 with binding affinity of 8.84 kcal/mol. It formed many conventional and carbon hydrogen bonding with the residues Val 280, Gln 438, Gly 464, Ser 436, His 296 and Glu 389, amide-p stacked bonds with the residues Trp 461 and Cys 437 and multiple van der Waals interactions were formed with the receptor protein ( Figure 6(a,b)). Anhydrotuberosin was found to be second   (Figure 6(c,d)). Daidzein was found to be third, having binding energy of 8.24 kcal/mol. It showed conventional hydrogen bonding with the residues Glu 289, Asn 193 and Arg 182, a p-cation bond with Arg 240, p-p stacked and p-p T-shaped interactions with the residues Phe 357 and Phe 194, p-alkyl interaction were formed with the residues Ala 243, Pro 288 and Ile 242 and some van der Waals interactions were also formed by remaining residues (Figure 6(e,f)). Fourth inhibitor was found to be Hydroxytuberosone with binding energy 8.03 kcal/mol. It    showed a p-p stacked interaction with the residue Phe 194, a p-alkyl interaction was formed with the residue Ile 242 and remaining residues formed van der Waals interactions ( Figure  6(g,h)). Stigmasterol was found to be the fifth inhibitor, having binding affinity of 8.01 kcal/mol. It is a steroid that maintains the cell membranes' structure and physiology (Ferrer et al., 2017), reported to have anti-angiogenic, anti-inflammatory and anti-cancerous activity (Kangsamaksin et al., 2017). It formed a p-donor hydrogen bond with Phe 357, alkyl and p-alkyl interactions with residues Ala 243, Pro 288, Ile 242 and Phe 194, and many van der Waals interactions were also formed by remaining residues (Figure 6(i,j)). Strong binding affinities of Robinin, Anhydrotuberosin, Daidzin, Hydroxytuberosone and Stigmasterol with TMPRSS2 could block its active sites to reactivate SARS-CoV-2 for reattaching ACE2, thus preventing COVID-19 infection.
As per molecular docking study, from YASARA scoring system, it had been observed that Robinin, Genistin, Daidzin, Hydroxytuberosone, Tuberostan, Anhydrotuberosin and Stigmasterol from PTY-2, can act as probable inhibitors of SARS-CoV-2 M pro and TMPRSS2. Structures of best docked phytochemicals were shown in Figure 7.

Molecular dynamics simulations
To further analyze the stability of binding, interactions of the best-docked compound, that is, ROBININ, we performed MD simulations for two setups upto 150 ns. These complexes are formed by the compound Robinin with proteins Mpro and TMPRSS. We have also calculated binding energy for the complexes from simulation trajectories.

M pro -Robinin
The docked complex of main protease with high scoring and best interacting compound Robinin was investigated for its binding stability and found to be stable upto 150 ns with minor fluctuations within the range of 0.2-0.4 nm. As shown in Figure 8, the RMSD plot showed quite stable trend with an average value of approx. 0.32 nm. The slight fluctuations were observed near 11-14 ns, 150-180 ns and in last 10 ns with minimum varying values 0.3-0.36 nm. Similar trends were observed with radius of gyration where the average value of Rg was 2.19 nm. A few fluctuations were seen in the Rg value upto 80 ns and afterward, the plot became stable at 2.18 nm. However, the RMSF plot showed little fluctuations in the favourable range. The catalytic site residues, including His41 and Cys145 are found to be least fluctuating throughout the simulation period. Further, for the last 20 ns of simulation trajectory, the binding energy was also observed to correlate with other parameters (Figure 9(A)). The average binding energy was À31.9 ± 27.2 kJ/mol for the protein-ligand complex.

TMPRSS2-Robinin
The complex of serine protease TMPRSS2 with Robinin was simulated till 150 ns using GROMOS54a7 forcefield. As shown in Figure 10, the complex has experienced very little fluctuations and has gained an average of RMSD 0.43 nm. In terms of compactness, the radius of gyration of the protein upon binding with complex is reduced (average Rg is 2.14 nm) which shows attained stability by the protein with inhibitor. Also, the RMSF plot was observed to be varying between 0.2 to 0.5 nm throughout the simulation. Lastly, the binding energy values were calculated and found to be in decreasing trend for the last 20 ns (Figure 9(B)). However, there were heavy fluctuations observed in last 8 ns time of simulation. Overall, the binding energy for last 20 ns was calculated to be À50.4 ± 33.6 kJ/mol.

Drug likeness and ADMET prediction
Drug-likeness of our best-docked compounds was predicted using Lipinski rule of five which states for any ligand to be considered as drug-like, a molecule should follow five parameters: molecular weight <500 Dalton, number of H-bond donors <5, number of H-bond acceptors <10, LogP <5 and molar refractivity between 40-130. It should follow 2 or more of its constraints; consequently, all of our best-docked compounds from our study followed this rule and subsequently deliberated as drug-like compounds. Further, admertSAR server was used for the prediction of the ADMET profile of the best-docked compounds. Moreover, our compounds were found to have good optimal oral bioavailability, human intestinal absorption, Caco-2 permeability, non-carcinogenic effect (within the reference range), considered safe and harmless ( Table 2).
The current rising scenario of COVID-19 pandemic demands its resolution to save the global population. At present, both individual drugs and in combination of antimalarial, antiviral and corticosteroids are being utilized as a treatment strategy in the modern medicine. In recent time WHO is recommending Dexamethasone (WHO, 2020b). Glenmark an Indian-based pharmaceutical has introduced antiviral drug Favipiravir ('Glenmark launches Covid-19 drug at Rs 103 per tablet -India News -Hindustan Times', 2020) for treating mild to moderate COVID-19 patient has got regulatory approval for Phase III assessment. Drug repurposing will save the time and in silico analysis of the natural phytoconstituents is one of the initial and crucial steps in this direction. M pro and TMPRSS2 are shown to be chief targets for curbing COVID-19 infection. In the contemporary work, molecular docking analysis revealed seven different active phytochemicals from PTY-2 (Robinin, Genistin, Daidzin, Hydroxytuberosone, Tuberostan, Anhydrotuberosin and Stigmasterol) as potential inhibitors of M pro and TMPRSS2. Robinin, Daidzin and Hydroxytuberosone act as common inhibitor for M pro and TMPRSS2. From these, Robinin has significant binding affinity for Mpro and TMPRSS2 and showed structural stability, thereby acting as a dual target inhibitor against COVID-19. Overall, the analyzed active phytoconstituents of PTY-2 attacks on Mpro prevent its attachment to host receptor ACE2 and prevent its attachment to TMPRSS2 which activates SARS-CoV-2 for reattachment to ACE2 responsible for COVID-19 infection. These active phytochemicals were portrayed as drug-like compounds as per Lipinski rule and exhibited safe ADMET properties, supporting developing more efficient and potent COVID-19 inhibitors.

Conclusion
In current investigation, the active phytoconstituents of Ayurvedic medicinal plant Pueraria tuberosa (PTY-2) have multitargeted potency to counteract the infection of COVID-19. Our study identified seven potential inhibitors (Robinin, Genistin, Daidzin, Hydroxytuberosone, Tuberostan, Anhydrotuberosin and Stigmasterol) against COVID-19 molecular targets. From these Robinin, Daidzin and Hydroxytuberosone were found to be common inhibitors for M pro and TMPRSS2. Robinin with the highest binding affinity for M pro and TMPRSS2 acts as a common inhibitor. During trajectory analysis, Robinin showed significant stability with M pro and TMPRSS2, thus reducing the strength of the SARS-CoV-2 virus against the host. It also has a drug-like property with a harmless ADMET profile suggesting in development of improved potent COVID-19 inhibitors.

Disclosure statement
Authors declare no conflict of interest.

Funding
The author(s) received no financial support for the research, authorship, and/or publication of this article.