Anti- SARS-CoV-2 main protease complex (Mpro) activity of Palmatine

The Pandemic situation caused due to SARS-CoV-2 causing Coronavirus Disease (CoVID-19) around globe. Recent, COVID-19 main protease complex (M pro ), highly modulating enzyme in SARS-CoV-2 was reported for viral replication and transcription. This multifunctionality of M pro attracts for identication of potential drug target. Considering impact, In silico analysis was performed for Palmatine alkaloid against M pro . Naturally, present in Tinospora cordifolia, found effective against Cancer, HIV, viral infections, diabetics. In methods, physico-chemical analysis by ProtParam tool and Structure of M pro was predicted by SWISS-MODEL Workspace homology modeling server. Superimposition Structure and signicant equal QMQE, QSQE values were found for eight highly similar templates. Structural assessment validation by Ramachandran plot (97.67% favoured), Local Quality estimate ratio (>0.6) and higher QMEAN score (y-axis). Further, docking was performed with validated M pro model by SwissDock server. Interaction with -8.281919 ΔG indicates reliable Interaction. Also, comparative docking reveals, most favoured Palmatine interaction. Thus, an attempt was made to nd potent inhibitor for SARS-CoV-2, as there is no promising and specic anti-viral drug or vaccine available for prevention and treatment of infections. However, In Vitro studies are required. Toxicity studies reported against Palmatine for acute effect (135 mg/kg body weight) on mouse model LD 50.


Introduction
Corona viruses are commonly a group of RNA Viruses infecting and causing diseases in birds and mammals including Humans [1]. Belongs to subfamily Orthocoronavirinae in family coronaviridae, order Nidovirales [2]. This group of viruses are Positive-sense ssRNA Genome surrounded by spherical envelope with genome size ranging from 26-to 32 kb [3,4].
Recently, new Corona virus causing threat around the globe due to Severe Acute Respiratory Syndrome Corona virus 2 (SARS-CoV-2) [5]. Humans are mostly affected by causing respiratory tract infections disease Corona virus disease 2019 identi ed as COVID-19 [5]. Upon infection, mild to death effect were widely reported in human population [6]. As on present there is no promising and speci c anti-viral drug or Vaccine available for prevention and treatment of infections cause by COVID-19 [6]. While similar variety of viruses including SARS and MERs has divesting effect on human existence during past decades [7]. Therefore, by considering present pandemic situation around the globe it was decided to develop novel and potent strategies.
In present scenario, discovery of active components from the plant and their biological function in disease control has lead to active interest in the plant products across the globe [8]. Similarly, in modern medicine many drugs are derived from plant products and their derivatives [8]. The variety of lead compounds was obtained from plant secondary metabolites for treatment of various diseases [9]. The alkaloids and avinoid are having activity as strong analgesic effect and dietary supplement respectively [8]. The most commonly used secondary metabolites artemisinin were widely used phytomedicine [10].
In present analysis, An attempt was made to evaluating Antiviral activity of Palmatine alkaloid against COVID-19 main protease complex (M pro ) of SARS-CoV-2. Basically, alkaloid was found in naturally occurring Tinospora cordifolia commonly known as 'Guduchi' [8]. It has been reported for its diverse application in the treatment of various diseases i.e. Cancer, HIV, viral infections, neurological, diabetics etc. [8].
By considering pandemic impact and medical urgency the computational analysis were performed for Anti-COVID-19 main protease complex (M pro ) of SARS-CoV-2 by potential Palmatine alkaloid molecule.
Completely characterized COVID-19 main protease complex (M pro ) is submitted in PDB [11]. It is Multifunctional and highly modulating enzyme in SARS-CoV-2 replication and transcription [11].
Therefore, identi ed COVID-19 main protease complex (M pro ) is attracting for potential drug target. Methods 1. ProtParam tool (https://web.expasy.org/protparam/) Physico-chemical parameters was analysed for given protein or user provide protein sequence, present in Swiss-Prot or TrEMBL [2]. Then FASTA Format protein Sequence of COVID-19 main protease (M pro ) was provided in one-letter Code for computing parameter.
3. SWISS-MODEL (https://swissmodel.expasy.org/) ExPASy web server Accessible fully functional protein Structure homology modelling server. It facilitates modelling service to life research trough worldwide [16].The primary amino acid FASTA Format sequence was submitted to SWISS-MODEL workspace on June 9, 2020. Then homology modelling project (COVID-19 main protease complex (M pro )) was performed.

Template Selection
The features of template-target alignments evaluate identi cation and quality of template. Then best quality templates were Consider for model building [17].

Model Building
ProMod3 predicted model on the target-template alignment [18]. The highly Conserved Coordinates between the target and the template are taken from the template to the model [18]. For Rebuilding of Insertions and deletions are remodelled using a fragment library [18]. Finally, force eld method was applied for the satisfying geometry of the predicted model [18]. PROMOD-II is used when loop modelling with ProMod3 fails, to build model [18].

Model Quality Estimation
The global and per-residue model quality has been assessed using the QMEAN scoring function [19].

Ligand Modelling
Template structure having ligands are transferred by homology to the model, after satisfying following criteria by ligands with model: (a) Biological signi cance and annotation in template library, (b) Proximity, (c) Matching, (d) Conserved bonding between target and template [16]. However, ligand fails to satisfy criteria, is not included in model [16].

Oligomeric State Conservation
To model the oligomeric target sequence, annotated quaternary structure of the template is used [20].
Including, interface conservation, structural clustering, and other template aspects to reveal a quaternary structure quality estimate (QSQE). On the basis of supervised machine learning algorithm, SVM (Support Vector Machines) method.
The QSQE score prediction ranges between zero to one, indicates expected accuracy of inter linkage of chains for a model. Higher numbers is directly proportional to reliability of model [20]. This is synergetic to GMQE score for predicting correctness of resultant model at tertiary structural level [20]. 4. PubChem (https://www.ncbi.nlm.nih.gov/pccompound) The database contains validated ful lled chemical informational data [21. Accessible to pre-cluster and cross-referenced Structure of Compound by identity and similarity groups compounds were available [21]. Palmatine was searched by selecting PubChem Compound in Drop down menu in search box of NCBI Home Page 5. SwissDock (http://www.swissdock.ch/) SwissDock is EADock DSS based docking service via ExPASy web server. It was used to predict protein ligand docking [22,23].The molecular interactions that may occur between a target protein and a small molecule were evaluated [22,23]. SwissDock docking is based on EADock DSS Software [22,23]. . Its algorithm consists of the following steps: Initially, various binding modes are created in box i.e. local docking or vicinity of all target cavities i.e. blind docking. Simultaneously, their CHARMM (https://www.charmm.org//) energies are estimated. Further, clusters computed by SwissDock were downloaded for further visualization and analysis [22] Results

Physio-chemical
The analysis of COVID-19 main protease (M pro ) protein using protparam tool provide precious information about proteins including number of amino acids, Molecular weight, Theoretical pI etc ( Table  1). The Total number of amino acids in protein 306, while Leucine ( 29 (9.5%) most frequently occurring amino acid and least one is Pyl (O) & Sec (U) 0(0.0%). Molecular weight is 33796.64 ( Table 1). The theoretical pI of protein is 5.95 (Table 10). Total numbers of negatively and positively charged residues are 26 and 22 respectably. The molecular formula is described based on atomic combination of Carbon, Hydrogen, Nitrogen and Oxygen found in protein (Table 1). Extinction coe cients (M-1 cm-1) at 280nm were 33640 and Absorption (Abs 0.1% =1 g/l) 0.995 were observed ( Table 1). The Instability index is inversely proportional to the stability of proteins, 27.65 were observed. Estimated half-life of protein was observed to be 1.9 hours (Table 1).

Model Building
The results for homology modelling project "COVID-19 main protease complex (M pro ) "submitted to SWISS-MODEL workspace on June 9, 2020, with an amino acid sequence with alignment is given in Figure 1.
The SWISS-MODEL template library for evolutionary related structures matching the target sequence in Table2. For details on the template search, were described in Materials and Methods section above.
Overall 386 templates were found to match the target sequence. This list was ltered by a heuristic down to eight Entries ( Table 2). The highly identical eight templates are listed below with complete analysis of homology modeling parameters ( Table 2). QMQE value was observed Equal (0.99) to every template (Table 2). While, QSQE value ranging from 0.90 to 0.95 ( Table 2). All the entries showing signi cant 100 identities ( Table 2). X-ray diffraction Method for modeling studies with different resolution ranging from1.6Å to 2.2Å were Considered (Table 2). Further, all proteins were observed in homo-dimer Oligomeric State ( Table 2). The pre requisite Library annotated Ligands were listed According to templates ( Table 2).

COVID-19 main protease (M pro ) structure
The individual predicted protein structure was obtained by clicking on checkbox ( Figure 3A) and can be analysed with different angle and conformational visualization models [16,17]. Similarly, Different templates structures superposition was visualized and compared in 3D viewer ( Figure 3B). Templates Selected according to target similarity coverage were found very similar and superimposed ( Figure 3B). Despite, some local dissimilarity was observed due to turns and loop region ( Figure 3B).

Local Quality Estimate and Comparison
The local quality estimate was performed between local similarity to target and Number of residue. Two chains are shown in different colours in target protein was illustrated (Figure 4). Here both the chains occurred abundantly between 0.8 to 1.0 Predicted Local similarity Score with increasing Residue Number ( Figure 4A). QMEAN score on y-axis indicating, quality of the model ( Figure 4B). The high comparison scores obtained for high-resolution crystal structures [16,17]. Comparison with Non-Redundant Set of PDB Structures was observed between 0.5 to 1.0 Normalized QMEAN Score ( Figure 4B).

SwissDock
Binding modes are scored using their FullFitness and clusteres. Avearage value of FullFitness parameter of their elements were used to Arrange clusters. [27].The molecular docking was performed with SwissDock is based on the docking software EADock DSS, whose algorithm consists of well de ned steps mentioned above in method parts [27].
The docking was performed with palmatine against COVID-19 main protease complex (M pro

Comparative Docking
For further validation of Ligand molecule, comparative docking was performed with two different Ligands namely, Gangerol and Berberine. The Overall different Docking parameters were calculated ( Table 4). The Signi cant ΔG value was observed for Palmatine among the Comparative Ligand Compounds (Table 4).

Discussion
Palmatine is an alkaloid molecule with active principle associated with variety of biological activities of Tinospora cordifolia commonly named as "Guduchi" [8,30,31,32]. T. cordifolia Belongs to family Menispermaceae family which is genetically diverse shrub, naturally found at tropical regions of the Indian subcontinent [30,31,32]. From long back it is use in traditional medicine for treatment of various disorders [31,32]. T. cordifolia exhibit variety of phytochemicals derived from the plant like alkaloids, steroids, diterpenoid lactones, aliphatics, and glycosides and mixed other compounds [32]. These compounds reported to have potential biological activities in different disease conditions attracting applicability in clinical research [8]. It is anti-viral, anti-spasmodic, anti-microbial, anti-osteoporotic, anti-in ammatory, anti-arthritic, anti-allergic, and anti-diabetic conditions. Speci cally, Palmatine has been studied for used separately or in mixture for the treatment of anti-viral, jaundice, Dysenty, hypertension, im amention and liver related diseases [8,33]. Speci cally, Palmatine was reported for suppressing Dengue and yellow fever virus in dose-dependent manner [34]. However, mode of ant-viral activity of is not yet understood.
Further, targeted molecule in present study analysed with physico-chemical properties, Reveals functional acidic environment and hydrophobic nature of protein (Table 1). The distance between different templates is proportional to the pair wise sequence similarity ( Figure 1). Highly identical score of predicts COVID-19 main protease complex (M pro ) is evolutionary conserved.The protein predictions with individual target and template protein have shown reliable similarities ( Figure 3A, B). Typically, Sequence Variability affects superposition's structure by turns and loops ( Figure 3B). In other Context, they often involved in protein function, hence, they are of crucial importance in accurate modelling [27].Structural assessment by Local estimates for model quality based on QMEAN scoring function, shown as per-residue plot ( Figure 4A) and as a global score in relation to a set of high-resolution PDB structures (Z-score) as show in Figure 4B. In present study score is associated to each residue of the model (reported on the x-axis), re ecting the expected similarity to the native structure (y-axis). Residue score < 0.6 are expected to be of low quality [16,18]. Therefore, > 0.6 score for both the chains reveals high quality of protein modelling ( Figure 4A). Further, higher QMEAN score comparison value indicates quality of the model for experimental structures [16,18] and similar results shown in Figure 4B provides reliable quality of model.
Multifunctional COVID-19 main protease complex (M pro ) of SARS-CoV-2 was found to be involved in replication and transcription of viral RNA responsible for processing of polyprotien [11]. Also, host translational Inhibition by interacting with host 40S ribosomal unit [11,35,36 ]. Also, Modulating replication and transcription of the Viral Genome [11,35,36]. Similalrly, different molecular functions were also reported Such as, Helicase, Hydrolase, Protease, hydrolase [11,35,36]. Therefore, docking studies with M pro was carried Out (Table3, Figure 5). The different docking parameter indicating the reliable and promising Ligand-Protien Interaction (Table3, Figure 5). Further, the comparative docking was performed with different ligands and M pro (Table 5). Among the three ligand-target interaction, Palmatime interacts with signi cant ΔG value (Table 5). In Conclusion, multifunctionality of COVID-19 main protease complex (M pro ) attracts for potential drug target. Therefore, an attempt was made to nd potent inhibitor against SARS-CoV-2 in present pandemic. Further, In Vitro studies are required for con rmation of inhibitor. However, toxicity studies reported against Palmatine for acute effect (135 mg/kg body weight) on mouse model LD 50 [26]. Figure 1 A predicted overview alignment for sequence similarity between the selected templates of amino acid sequence.   Comparison with Non-Redundant Set of PDB Structures.