1Jaspamide as dual inhibitor targeting SARS CoV-2’s replication: Virtual

2screening, ADME, molecular dynamics simulations and binding free

3energies

4 Khattab Al-Khafajia, Imren Bayilb, Dunya Al-Duhaidahawic and Tugba Taskin

5 Toka,b*

7a Gaziantep University, Faculty of Arts and Sciences, Department of Chemistry, 27310,

8Gaziantep, Turkey.

9b Gaziantep University, Institute of Health Sciences, Department of Bioinformatics and

10Computational Biology, 27310, Gaziantep, Turkey.

11c University of Kufa, Pharmacy, Pharmaceutical Chemistry, AL-Najaf, Iraq.

12

13∗ To whom correspondence should be addressed:

14Tugba TASKIN TOK

15Faculty of Arts and Sciences, Department of Chemistry, Gaziantep University, 27310,

16Gaziantep, Turkey;

17Institute of Health Sciences, Department of Bioinformatics and Computational Biology,

18Gaziantep University, 27310, Gaziantep, Turkey.

19Tel: +90 342 317 2996, Fax: +90 342 360 1032;

20Email: ttaskin@gantep.edu.tr; taskin.tugba@gmail.com

1
 1Jaspamide as dual inhibitor targeting SARS CoV-2’s replication: Virtual

2screening, ADME, molecular dynamics simulations and binding free

3energies

4When disease caused by virus infection is overspread, non-surprisingly the maximum likelihood

5estimator is both dangerous and dreadful and therefore should be waged war against the pathogenic

6virus. Late last year, we watched how a SARS CoV-2 pummeled Wuhan, China at the beginning and

7then in similar fashion it strikes them worldwide. Other research groups reported novel 2′-O-

8methyltransferase (2′-O-MTase) capping system to hide its RNA of innate immune perception. Such

9nsp15 and nsp10-nsp16 can be represented as preventers innate immunity to recognized SARS-CoV-2

10viral RNA that are the crux of the synthesis and replication of SARS-CoV-2. Computational methods

11became realistic and affordable to accelerate the identification of effective treatment against SARS-

12CoV-2. Here we screened of some of natural-derived peptides against NSP15 and NSP10-NSP16 by

13utilizing the molecular docking and ADME calculations. Jaspamide passed successfully through both

14the docking screening and ADMET calculations. Posterior molecular dynamics simulation and

15MMPBSA are commonly performed to investigate the stability of protein-drug complexes, thereby we

16ran them to examine the stability complexed jaspamide with both NSP15 and NSP10-NSP16. The

17results of molecular dynamics simulations, MMPBSA calculations and interaction entropy

18estimations revealed that Jaspamide can act as dual inhibitor. The results conduce to several ways to

19target undruggable NSP16-NSP10 complex and NSP15 and creates a probable way of inhibition

20NSP16-NSP10 complex and NSP15 by jaspamide as dual inhibitor to fight SARS CoV-2.

21
22
23Keywords: Virtual Screening, Dual-target Inhibitor, SARS-CoV-2, ADME, Molecular Dynamic

24Simulations, MMPBSA, Interaction Entropy.

25

26Abbreviations: MOF: Multi-Organ Failure; MOD: Multi-Organ Dysfunction; ACE2: Angiotensin-

27Converting Enzyme II; RdRp: RNA dependent RNA polymerase; SAM: S-adenosyl-l-methionin;

2
 1HTS: High-Throughput Screening; TIP3P: Three-Point Transferable Intermolecular Potential; MD:

2Molecular Dynamics; MMPBSA: Molecular Mechanics Poisson-Boltzmann Surface Area; RMSD:

3Root Mean Square Deviation; RMSF: Root Mean Square Fluctuation; PDB: Protein Data Bank;

4ADFR: Autodock Flexible Residue; AGFR: AutoGridFr Flexible Residue.

6Introduction

7With increasing of coronavirus infections the pathogens of emergency respiratory diseases

8became challenging (Chan, Kok, et al., 2020). Coronavirus is a single-stranded RNA virus

9which can be defined as an indifferent species of animal (Chen, Liu, & Guo, 2020). This

10virus is capable of cross-species barrier and transmission to humans causing mild to severe

11illness ranging from common cold (81%) to severe illness this occurred in 14 % of cases.

12Including in of 5 percent of cases, coronavirus leads to variations in the mass, respiratory

13failure, septic shock. Besides This virus causes a multi-organ failure (MOF) or multi-organ

14dysfunction (MOD) or (Chan, Yuan, et al., 2020; N. Chen et al., 2020; Xu et al., 2020). Many

15efforts across the scientific community to develop a vaccine to stop spreading of the SARS-

16CoV-2 but the current strategies of fighting SARS-CoV-2 can be categorized into four

17distinct approaches, namely: (i) broad-spectrum antiviral drugs (Totura & Bavari, 2019); (ii)

18host cell proteases inhibitors that play a key role in the priming of the viral spike (Hasan et

19al., 2020); (iii) drugs that target host-virus interface fusing the viral S protein to angiotensin-

20converting enzyme type II receptors (ACE2) in host cells (C. Wu et al., 2020); (iv) Drugs that

21prevent proinflammatory hypercytokinemia (cytokine storm) such as IL-6 antibody, IL-1

22receptor blockers, and JAK inhibitors (D. Wu & Yang, 2020). Polyadic strategies Many

23strategies are needed to follow for inhibition of SARS-CoV-2, such as drug repositioning (Al-

24Khafaji, Al-Duhaidahawi, & Taskin Tok, 2020; Y. Zhou et al., 2020) synergistic drug

3
 1combinations, high throughput screening, inhibition of cellular autophagy, multi-target

2treatment approaches, nasal administration of drugs. The majority of repurposing endeavors

3seem to be directed towards targeting NSP5 protease (3CLpro) dependent viral replication

4machinery (Nukoolkarn, Lee, Malaisree, Aruksakulwong, & Hannongbua, 2008), S-proteins-

5driven viral host cellular entry in addition to RNA dependent RNA polymerase (RdRp)

6NSP12 that catalyze viral RNA synthesis (Hoffmann et al., 2020). SARS-COV-2 ribonucleic

7acids capped at 5' ending to hinder degradation by exoribonuclease enzyme to guarantee

8effective translation and avoid identification by the host cell inherent immune system (Chan,

9Kok, et al., 2020). Interestingly, the viral 2'-O-methyl transferase enzyme abbreviated (2'-O-

10MTase) NSP16 is RNA cap modifying protein which is activated by NSP10 (Chen et al.,

112013). The methylation phase thereby obeys an aligned sequence by which RNA cap

12guanine-N7-methyltransferase (N7-MTase)-mediated N7-guanine methylation, a cellular

13incidence which helps the NSP16 coupled to the surface of coated substrate of RNA and the

14S-adenosyl-l-methionine (SAM) methyl donors, maintaining the SAM binding motif and

15expanding the capped RNA binding groove(Decroly et al., 2011). The NSP16 /NSP10

16interface and binding RNA substrates could have enhanced therapeutic objectives than

17MTase active site for the evolution of advanced anti-SARS-CoV-2 drugs. NSP15

18(endoribonuclease) is biochemically characterized in SARS CoV-2 that RNA at uridylates at

19the 3′-position to form a 2′-3′ cyclic phosphodiester product (Bhardwaj, Sun, Holzenburg,

20Guarino, & Kao, 2006). It is important to note that the NSP15 protein starts the degradation

21of the viral dsRNA due to stop detecting the virus by the host immune system (Hackbart,

22Deng, & Baker, 2020). The crystal structure of NSP15 of SARS CoV-2 has been reported

23(Kim et al., 2020). NSP15 is promoted the endoribonuclease activity by Mn as cofactor

24(Bhardwaj, Guarino, & Kao, 2004). Targeting both of NSP10-NSP16 and NSP 15 was done

25computationally through virtual screening and MD simulations, where the obtained results

4
 1revealed that solvanol has good affinity toward NSP16 while somniferine showed affinity

2toward NSP15(Parida, Paul, & Chakravorty, 2021).

3According to the known structures of viral proteins, antiviral peptides can work as highly

4selective for their respective targets or exert broad antiviral activity (Schütz et al., 2020).

5Therefore, we here used computational tools such as molecular docking, ADME predicting

6tool, MD simulation to identify dual inhibitor for NSP10-NSP16 and NSP15 through virtual

7screening of some antiviral natural-derived peptides for fighting against SARS-CoV-2

8Material and Methods

92.1. Ligand-Protein Docking

10As many researchers over worldwide scramble to fight the SARS-CoV-2 of a drastically

11evolving paradigm in the health care industry, here we are attempt to enter the race. The

12traditional computer aided drug discovery to identifying new compounds with high affinities

13is the molecular docking simulation, i.e., a scoring sampling of docking algorithms. In our

14first step is to apply molecular docking to assess the draggability of selected 25 peptides

15against both of NSP16-NSP10 complex and NSP15 protein. These peptides were selected

16according to their antiviral activities in the referenced literatures as shown in Table 1. The

173D structures of these peptides were retrieved from Pubchem

18(https://pubchem.ncbi.nlm.nih.gov/). After downloading the peptides were processed by

19“Prepare Ligands” protocol in Discovery Studio 2019. And the crystal structures of NSP16-

20NSP10 complex and NSP15 protein were retrieved from the protein data bank (PDB ID:

216W4H (Rosas-Lemus et al., 2020a) and 7K0R (Pillon et al., 2020)), respectively. Before

22starting the docking process, the 3D structure of protein was optimized by removing water

23molecules, metals and ligand from crystal structures. Where the S-Adenosyl Methionine

24(SAM), native ligand (Aldahham et al., 2020; Rosas-Lemus et al., 2020b) was chosen as

5
 1control ligand and compare the docking score of under investigated peptides with its docking

2score. While Uridine-5’-monophosphate (U5P) was selected as reference ligand (Pillon et al.,

32020) for NSP15 and also compare its docking score with the those of selected peptides.

4The AutodockFR (ADFR) program (Ravindranath, Forli, Goodsell, Olson, & Sanner, 2015)

5and AutoGridFR (AGFR version 1.0) (Al-Khafaji & Taskin Tok, 2020b; Yunhong Wang,

6Miller, Roulston, Bixby, & Shao, 2016) programs are used in the execution of the molecular

7docking which are capable for establishing configuration file which contains the data for

8running controlled flexible docking by identifying the residues of the complex’s binding site.

9Autogrid maps estimated with a default grid map were set on the position of reference ligands

10at (40, 40, 40) box spacing of 0.375A0. ADFR's presumptive parameters enable the ligand to

11reaches buried grooves (Al-Khafaji & Taskin Tok, 2020a). ADFR uses the scoring function

12of Autodock but customized for flexible receptor (Ravindranath et al., 2015). The docking

13was implemented using default search settings (exhaustiveness set to 8). The docking

14programs is validated through redocking the reference ligand of corresponding protein. The

15high scored generated pose of reference ligand superimposed on co-crystal ligand. Molsoft

16ICM browser was used to appraising the ultimate results of docking computations.

172.2. ADME Screening

18The failures of compounds at early stage of drug discovery to be drug, it results from

19unfavorable pharmacokinetic features (James, 2010). From the above considerations,

20evaluation of pharmacokinetic properties is necessary in the early phases of drug discovery

21process, therefore, we screened the successful compounds by using Insilco ADME

22estimations (Yulan Wang et al., 2015). QikProp module usage of the Schrödinger suite has

23been launched to benchmark the ADME of some natural derived peptides (Jorgensen &

24Duffy, 2002). QikProp provides a fit of quality similar to that obtained of 95% of known

6
 1drugs. QPlogS, CIQPlogS, QPlogHERG, QPPCaco, QPlogBB, QPPMDCK, Molecular

2weight (MW), acceptor hydrogen bonding (accptHB), donor hydrogen bonding (donorHB),

3and human oral absorption (HOA), in particular, are a fundamental descriptor of

4pharmacokinetic evaluations.

52.3 Dynamics simulation

6Dynamics simulations is of vital interest to investigators in the field of computer aided drug

7discovery (Kumar et al., 2019). Thus we are using the molecular dynamics simulations to

8supply as with information about the conformational stability (Shukla, Munjal, & Singh,

92019). Like these technologies, molecular dynamics simulation consists of a set of standard

10protocols of GROMACS 2018.1 and an implementation. We implemented these protocols for

11both of NSP16-NSP10/jaspamide and NSP15/jaspamide and apo forms (Abraham et al.,

122015), with Charmm 27 force field for all atoms (Bjelkmar, Larsson, Cuendet, Hess, &

13Lindahl, 2010). For first step of molecular dynamics, we employed the Swiss PARAM to

14create the topology file of jaspamide (Zoete, Cuendet, Grosdidier, & Michielin, 2011). The

15apo and holo of NSP15 and NSP10-NSP16 were placed and centered in cubic cell unit with at

16least 0.1 nm distance from box edges. The, we chose three-point transferable intermolecular

17potential (TIP3P) that it is used as solvent for our protein-ligand complexes. And if

18complexes were charged, we neutralized the complexes through adding opposite ions either

19sodium ion or chloride ion. An appropriate way to proceed with this molecular dynamic

20simulation is to use steepest descent algorithm threshold value of 1000 kJ / mol nm for

21minimization. Both of the NVT and NPT ensembles of 0.1 ns were used with the position

22constraint on the protein molecules for controlling pressure at 1 atm and temperature at 300

23K. In next step, to assess the impact of electrostatic interactions on the diversified behaviors

24of complexes, we utilized the Particle Mesh Ewald summation (Darden, York, & Pedersen,

7
 11993). After evaluating first 10-ns MD simulation we found that our systems need less than 7

2ns to reach to equilibrium. Hence we implemented 50 ns as done by (Ancy, Sivanandam,

3Kalaivani, & Kumaradhas, 2020). The total process is performed by using restraint-free

4molecular dynamic simulation on protein molecules or ligands to assess stability to this end.

5Before examining the MD trajectories by using the RMSD, RMSF, Rg, SASA and hydrogen

6bonding, we utilized the Periodic boundary condition (PBC) refinements to optimize the

7trajectories outputs. Further, hydrogen bonding occupancy was analyzed by using

8readHBmap.py of Python software (Qu, Song, Zhu, Liu, & Zhao, 2018).

92.4 Binding free energy calculations

10Thus, a precise determination of jaspamide affinity toward NSP16-NSP10 and NSP15, along

11with a precise determination of MD analysis, the using of The Molecular Mechanics/Poisson-

12Boltzmann Surface Area (MM-PBSA) computations allow to determine the binding affinity

13(∆GBind) between protein-peptide via g_mmpbsa tool (Kumari, Kumar, Open Source Drug

14Discovery, & Lynn, 2014). This quantity is computed based on the following equations (Leão

15et al., 2020):

16∆GBind = ∆Gcomplex − ∆Greceptor − ∆Gligand (1)

17 ∆GBind = ∆H − T∆S ≈ ∆EMM + ∆Gsolv − T∆S (2)

18∆EMM = ∆Einternal + ∆Eele + ∆EvdW (3)

19∆Gsolv = ∆GPol + ∆Gnonpol (4)

20The potential energy in a vacuum and free solvent energy is expressed by ΔEMM and

21ΔGsolvation, respectively. The electrostatic (ΔEele) and van der Waals elements (ΔEvdW)

22decide the energy of the molecular dynamics (ΔEMM). Calculated energy of solvation is the

8
 1function of ΔGpol and non-polar energy ΔGnonpol. Where ΔGpol derived by using the Poisson-

2Boltzmann (PB) method, ΔGnonpol determined from the Solvent-Accessible Surface Area

3(SASA). The simplest way that incorporates the entropic contributions and MMPBSA values

4was introduced to calculate the binding energy. The entropic contribution (− T∆S) is

5estimated by using Duan et al’s approach (Al-Khafaji & Tok, 2020; Li et al., 2018) which is

6called interaction entropy IE and calculated as described in our previous work (Al-Khafaji &

7Tok, 2020).

8Result and Discussion

93.1 Docking affinity based natural-derived peptides screening

10It is worth recalling that the most urgent question about the treatment of COVID-19 is

11whether it can be available soon. The possibility of developing non-conventional drugs in

12actual situation is tantamount to being able to defeat COVID-19 effectively. Indeed, the

13current dependence on the supportive care as a kind of available treatment against COVID-19

14by giving oxygen, fluids, and using of approved antiviral drugs remdesivir, lopinavir-

15ritonavir, hydroxychloroquine- azithromycin. (Cao et al., 2020; Gautret et al., 2020; Porter &

16Jänicke, 1999; Yao et al., 2020). Current repurposed therapies fail to achieve optimum

17reperfusion in many patients. Therefore, in order to match the global search of new drug or

18vaccine with high activity. We screened some bioactive peptides from natural sources that

19have antiviral activities against dual targets: NSP10-NSP 16 and NSP15 are responsible for viral

20replication-transcription. Importantly, the structures of some targetable proteins of SARS-

21CoV-2 are recently reported thus, are amenable for molecular modeling. Encouraged with

22this and taking the advantage of a recently released crystal structure of NSP10-NSP16 and

23NSP15 from SARS-CoV-2. Virtual screening was conducted for 25-antiviral natural-derived

24peptides. The list of peptides as displayed in Table 1. With regard to docking energy, the best

9
1compounds which have higher docking score (higher than reference ligands as control) were

2between -48.438 to -31.535 kJ/mol. Also, the generated poses of docked peptides inside the

3binding grooves of NSP10-NSP16 and NSP15 presented in Figure 1.

6Figure 1: the generated poses of peptides inside the binding site of NSP10-NSP16 and

7NSP15.

8Table 1. Docking scores of the selected natural peptides against NSP10-NSP16 and NSP15.

  NSP10/ NSP16 NSP15

Docking Score RMSD (Å) Docking Score RMSD (Å)
Drugs Drugs

10
 (kJ/mol) (kJ/mol)
Kahalide F (Hamann, Otto, -48.438 2.802 Verlamelin B -32.825 2.857

Scheuer, & Dunbar, 1996)

Homophymine B (Zampella -45.480 2.905 Mirabamide B -39.142 2.847

et al., 2008)
Mirabamide A (da Mata, -45.442 2.657 Neamphamide -38.685 2.749

Mourão, Rangel, & Schwartz,

2017)
Theopapuamide B (da Mata -45.396 1.952 Homophymine B -38.126 2.367

et al., 2017)
Arenamide C (Asolkar et al., -45.032 1.969 Arenamide C -38.058 2.539

2009)
Mirabamide B(da Mata et al., -44.367 2.232 Kahalide F -37.711 2.006

2017)
Jaspamide (Anjum et al., -33.786 1.590 Mirabamide A -37.407 2.446

2016)
celebeside A (da Mata et al., -33.610 2.272 Callipeltin A -37.004 2.831

2017)
Halovir A (Rowley, Kelly, -41.865 2.250 Theopapuamide B -36.424 1.875

Kauffman, Jensen, & Fenical,

2004)
Callipeltin A (da Mata et al., -41.844 2.544 Halovir C -35.730 1.565

2017)
Neamphamide (da Mata et al., -40.631 3.099 Tyrocidine A -35.311 1.800

2017)
Halovir B (Rowley et al., -40.631 1.976 Halovir A -35.151 2.226

2004)
Halovir E (Rowley et al., -40.229 1.970 Valinomycin -34.846 2.008

2004)
Plitidepsin (Drożdżal et al., -39.949 2.810 Halovir B -34.526 2.371

2020)
Tyrocidine A (Abdalla, 2016) -39.359 1.923 Plitidepsin. -34.309 1.835

Gramicidin S (Bourinbaiar & -38.886 2.947 Dactinomycin -33.854 2.279

Lee-Huang, 1994)
Dactinomycin (Chakraborti, -38.882 2.926 Halovir E -33.549 2.724

Bheemireddy, & Srinivasan,

2020)
Halovir C (Rowley et al., -38.472 1.980 Gramicidin S -32.407 1.881

2004)
Valinomycin (Zhang, Ma, -38.288 2.247 Arenamide A -32.331 1.508

Chen, Lu, & Chen, 2020)

Verlamelin B (Youssef, -37.836 2.529 Halovir D -31.638 2.512

11
 Ashour, Singab, & Wink,

2019)
Halovir D (Rowley et al., -37.187 1.861 Jaspamide -31.037 1.584

2004)
Asperterrestide A (He et al., -27.911 1.394 Verlamelin A -30.404 2.833

2013)
Arenamide A (Dembitsky, -36.581 1.896 celebeside A -29.978 2.942

2017)
Verlamelin A (Liang, Nong, -31.911 2.371 Arenamide B -28.035 1.421

Huang, & Qi, 2017)

Arenamide B (Dembitsky, -31.535 1.274 Asperterrestide -23.034 1.518

2017)
SAM -32.786 2.032 5UMP -26.844 1.475
1

23.2 Docking validation

3To validate our docking results, we applied the redocking of the cocrystal ligand (SAM) of
4NSP10-NSP16 (6W4H) and cocrystal ligand (5UP) of NSP15 (7K0R) and grid center was
5their positions in their corresponding protein. The results obtained were in good agreement
6with the experimental poses, where showing an RMSD of 0.631 between docked SAM and
7co-crystal SAM as shown in Figure 2(A). And the results of docking validation of 5UP
8revealed that RMSD is 1.913 between cocrystal 5UP and docked 5UP as shown in Figure
92(B). RMSD values of superimposing do not exceed the 2 A0 threshold (Mascarenhas et al.,
102020).

11

12

13Figure 2: (A) overlay of co-crystal ligand SAM (Blue colored) on docked conformation
14(green colored) of the co-crystallized SAM, RMSD is 0.631 (B) overlay of co-crystal ligand

12
 15UP (yellow colored) on docked conformation (dark green colored) of the co-crystallized
25UP, RMSD is 1.913.

33.3. ADME screening

4We have screened the ADME (pharmacokinetic parameters) values for peptides with high-

5docking scores by using QikProp (Jorgensen & Duffy, 2002). The ADME values of selected

6peptides are presented in the Table 2. To assess the drug likeliness of selected peptides, we

7applied Lipinski Rule of Five. The rule proclaims that for a drug must not be more than 1 of

8the following criteria: molecular weight <750 g/mol (Berg, Tymoczko, & Stryer, 2012),

9octanol–water partition, hydrogen bond donor ≤5 coefcient <5, hydrogen bond acceptor ≤10.

10Table 2 revealed that jaspamide and arenamide A obeyed Lipinski rules. Another parameter

11is QPPCaco, it is used to assess the permeability of compounds across the gut–blood barrier.

12QPPCaco predicts the Caco2 permeability (Z. Wang, Hop, Leung, & Pang, 2000) is

13established as in-silico technique to evaluate the oral absorption and the transport mechanism

14of the drugs. Only jaspamide and arenamide A have intermediate permeability. Analysis of

15the distribution of all selected peptides assessed through blood-brain barrier (BBB)

16permeability by QPlogBB. Table 2 shows that jaspamide and arenamide A fall within the

17accepted range, which indicates these two peptides as a functioning drug. Next parameter is

18#metab, is a descriptor of metabolic reactions. #metab reflects how efficiently a drug is

19converted to metabolites after being absorbed, distributed, and excreted (Moujir et al., 2020).

20Arenamide A, Halovir C and E follow the accepted range. Predicted values for the selected

21peptides fit the lipophilicity parameter (QPlogPo/w), an important physicochemical

22characteristic for a potential drug, as it plays an important role in absorption, bioavailability,

23metabolism, excretion and toxicity (W. Zhou, Wang, Lu, & Zhang, 2016). The QPlogS

24values are within well in the accepted range except the celebeside A, Plitidepsin and

25Tyrocidine A. This indicates of the peptides have good solubility (Ramachandran, Anbumani,

13
 1Ampasala, & Venkataraman, 2020). Another important parameter is QPlogHERG, which is

2the numerical value of the estimated IC50 value when the HERG K channels are blocked,

3Jaspamide falls well within the standard ranges (Ye, Yang, Xu, Chan, & Ma, 2019). This

4indicates jaspamide safer peptide than the rest selected peptides and lesser cardiotoxicity.

5QPPCaco predicts the Caco-2 Permeability, where Caco-2 cells are a model for the gut–blood

6barrier. Evaluation of QPPCaco indicated that Jaspamide and Arenamide A felt between 25

7and 500 and show medium permeability (Muthiah, Rajendran, & Dhanaraj, 2020). In parallel,

8Jaspamide and Arenamide A show moderate value of QPPMDCK. Which predicts the

9permeability through Madin-Darby canine kidney (MDCK) monolayers and is widely used to

10make oral absorption estimates, the reason being that these cells also express transporter

11proteins but only express very low levels of metabolizing enzymes (Amoa Onguene et al.,

122014). Finally, the percentages of human oral absorption of all the Jaspamide and Arenamide

13A were 73.89560.275, respectively. These results tell us that the Jaspamide may be a safe

14potential NSP10-NSP16 and NSP15 dual inhibitor.

14
1Table 2: In silico Pharmacokinetic properties of top-docking score peptides
QPlogPo/w Percent Human #metab
Molecule mol_MW donorHB accptHB QPlogS QPlogHERG QPPCaco QPlogBB QPPMDCK Rule of Five
Oral Absorption
–2.0 – 6.5 <25 poor, 1-8
<750 –6.5 – <25 poor, >80% is high
Ranges 0.0-6.0 2.0 – 20.0 < -5 >500 –3.0 – 1.2 <4
g/mol 0.5 >500 great <25% is poor
great
Plitidepsin 1110.352 1.25 27.2 1.55 -1.994 2.067 24.539 -3.887 41.553 35.923 12 2
Dactinomycin 1255.432 2 32.5 -0.558 0.574 5.783 13.802 -3.622 23.832 1.491 13 3
Valinomycin 1111.334 1.5 22.5 4.233 -4.425 2.616 18.889 -4.072 31.720 35.885 12 3
Jaspamide 709.679 2.25 9 4.892 -5.77 -2.28 244.507 -1.821 471.788 73.895 10 2
Arenamide A 671.876 1.25 10.75 3.786 -4.372 0.706 109.194 -2.491 221.559 60.275 7 1
celebeside A 891.908 8.5 24.9 -2.712 0.99 4.885 0.001 -7.86 0.001 0 10 3
Halovir A 866.192 6 18.4 1.946 -3.518 3.973 18.738 -4.064 32.73 21.226 9 3
Halovir C 850.193 5 16.7 2.711 -6.909 2.634 7.114 -4.926 23.151 23.958 8 3
Halovir D 838.139 6 18.4 0.8 -1.914 3.901 1.087 -5.001 4.458 0 9 3
Halovir B 838.139 6 18.4 0.768 -3.535 3.367 1.949 -5.204 6.467 1.442 9 3
Halovir E 822.139 5 16.7 2.658 -3.76 3.303 1.297 -5.134 6.386 1.838 8 3
Verlamelin B 872.069 4.25 17.7 1.388 -1.157 3.957 0.518 -5.407 1.971 0 12 3
Gramicidin S 1141.461 6 22 0.865 0.362 5.579 0.264 -3.658 0.968 0 16 3
Tyrocidine A 1270.493 9 25.25 -1.504 0.635 7.394 0.014 -6.44 0.11 0 21 3

12
 13.3 Dynamic simulation

23.6.1. Conformational stability

3To understand effect of jaspamide on the dynamic behaviour of the NSP10-NSP16 complex and

4NSP15, the MD simulation was executed across four systems. The root mean square deviation

5(RMSD), the root mean square fluctuation (RMSF), radius of gyration (Rg), solvent accessible

6surface area (SASA) and Hydrogen bonding during 50 ns. The computed RMSD values for the

7NSP10-NSP16 backbone atoms after reaching equilibirum organized between 0.31-0.44 nm (Figure

83(A)) and in the same manner the RMSD values of NSP15 backbone atoms varied from 0.13 to 0.32

9(Figure 4(A)). This implies that the systems were optimally converged. Jaspamide-bound NSP10-

10NSP16 reached to the equilibrated status at ~ 5 ns while apo NSP10-NSP16 reached to equilibrium

11through 7 ns. Jaspamide-bound and jaspamide-unbound reached to equilibrium at 9 ns similarly. As

12shown in Figure 3(A), from 28 ns to 50 ns holo and apo NSP10-NSP16 fluctuated identically.

13Another observation in Figure 4(A) was seen that the holo NSP15 fluctuated less than apo NSP15

14from 14 ns to 43 ns.

153.6.2. Residual stability

16The conformational fluctuations of the NSP10-NSP16 and NSP15 were analyzed by observing the

17residual variations that caused by jaspamide. The RMSF plots of the backbone atoms of the viral

18targets and their complex with jaspamide are presented in Figure 3(B) and Figure 4(B). It is observed

19that RBD NSP1-NSP16/jaspamide (Figure 3(B)) and NSP15/jaspamide (Figure 4(b)) show similar

20fluctuations as compared to only apo forms of NSP10-NSP16 and NSP15, which implies to the

21stability of these complexes.

22

13
 1

23.6.3. Compactness analysis

3The root mean square distance between an object and the center of gravity is defined as the radius of

4gyration (Rg). The radius of gyration is a measure of the compactness of the protein structure, where

5higher Rg value is referred to as a less compact structure, and low Rg value is inferred as high

6compactness that implies more stability (Rout, Swain, & Tripathy, 2020). The measured average Rg

7values of NSP10-NSP16, NSP10-NSP16/jaspamide, NSP15/ and 5jaspamide are 2.286 ± 0.001 nm,

82.295 ± 0.001 nm, 2.373 ± 0.001 nm and 2.379 ± 0.001 nm, respectively. From Figure 3(C) and

9Figure 4(C), it is observed that there is a insignificant increase in the Rg values of NSP10-NSP16

10and NSP15 when they in japamide-bound form as compared to NSP10-NSP16 and NSP15. There is

11no significant observation in Rg changing, this suggests their stability after binding to jaspamide.

123.6.4. Solvent accessibility analysis

13SASA is a measure of the receptor exposure to the solvent environment during the simulation. The

14hydrophobic residues that got exposed to the solvent environment upon binding with the ligand

15molecules contribute to the SASA values (Rout et al., 2020). The plot of the SASA for the proteins

16and their ligand-bound form is presented in Figure 3(D) and Figure 4(D). The analysed average

17SASA values for the NSP10-NSP16, NSP10-NSP16/jaspamide, NSP15/ and 5jaspamide are 208.879

18± 0.160 nm2, 206.462 ± 0.118 nm2, 175.816 ± 0.075 nm2 and 178.575 ± 0.094 nm2, respectively.

19There is no significant change observed for the averaged SASA values of the complex as compared

20to only protein suggesting their stability after binding to the drug molecule.

213.6.5. Hydrogen bonding

22The number of hydrogen bonds formed between the protein–ligand complex is the measure of the

23binding strength of the jaspamide to the NSP10-NSP16 and NSP15. As shown in Figure 3 (G),

14
 1jaspamide established 1-4 hydrogen bonds with NSP16 of NSP10-NSP16 complex. Similarly,

2jaspamide formed 1-4 hydrogen bonds with NSP15 (Figure 4(G). The number of hydrogen bonds

3fluctuates throughout the simulation time for both NSP10-NSP16/jaspamide and NSP15/jaspamide,

4which suggests for conformational changes in the binding site of the ligand during the simulation.

5The observation from hydrogen bond analysis indicates that the complexes are stable for the

6performed simulation time. But overall, the conformational changes are limited as illustrated in

7Figure 3 (E) and (F) for the snapshots of jaspamide-unbound NSP10-NSP16 and jaspamide-bound

8NSP10-NSP16. In same way, the captured 50 snapshots from 50-ns MD simulation (Figure 3 (E) and

9(F)) reveal that the conformational changes are stable upon binding of jaspamide.

10

11Figure 3: (A) RMSD, (B) RMSF, (C) RG, (D) SASA, (E) 50 overlayed snapshots from 50 ns of apo

12NSP10-NSP16, (F) 50 overlayed snapshots from 50 ns of apo NSP10-NSP16/jaspamide and (G)

13hydrogen bonding.

15
 1

2Figure 4: (A) RMSD, (B) RMSF, (C) RG, (D) SASA, (E) 50 overlayed snapshots from 50 ns of apo

3NSP15, (F) 50 overlayed snapshots from 50 ns of apo NSP15/jaspamide and (G) hydrogen bonding.

43.4 Hydrogen Bonding Occupancy Analysis

5Binding interactions are an important key in the stability of a drugs or inhibitors in the active site of a proteins.

6To understand the inhibition mechanism of a specific enzyme, hydrogen bonds, salt bridges and π-π stacking

7interactions of ligand with crucial amino acid residues of binding pocket must be considered. In this work, we

8have analyzed the hydrogen bonding occupancy of jaspamide with the residues of NSP10-NSP16 and NSP15

9binding sites. There were two systems with 2910 trajectory frames. These systems have shown very good

10hydrogen bonding occupancy scores. The NSP10-NSP16’s residues Asn6899, Asp6912, Tyr6930, Asp6931

11Lys6933 are observed with 4.4%, 10.9%, 9.7%, 1.1% and 60.1%, respectively occupancy with the jaspamide

12as shown in Table 3. In case of NSP15/jaspamide, the Gln245, Lys290, Trp333 and Tyr343 residues

13interacted with jaspamide by hydrogen bonds with occupancy of 6.1%, 71.4%, 10.3% and 8.1%, respectively.

14These two systems involve interaction between jaspamide with the four catalytically important residues with

15large number of Hbond occupancies. (Table 4)

16
 1Table 3. Hydrogen bonding occupancies X
XPair Donor acceptor Occupancy %
ID
NSP10-NSP16/jaspamide
1 7097LIG(H41) 6912ASP(OD2) 4.6
2 7097LIG(H41) 6912ASP(OD1) 6.3
3 7097LIG(H32) 6931ASP(OD1) 1.1
4 6933LYS(HZ1) 7097LIG(O4) 8.7
5 6933LYS(HZ1) 7097LIG(O3) 51.4
6 6930TYR(HN) 7097LIG(O) 9.7
7 6899ASN(D21) 7097LIG(O2) 4.4
NSP15/jaspamide
1 347LIG(H32) 333TRP(NE1) 10.3
2 347LIG(H13) 343TYR(OH) 8.1
4 290LYS(HZ1) 347LIG(O4) 71.4
7 245GLN(E21) 347LIG(O) 6.1
2

43.5 MMPBSA and binding free energy analysis

5The molecular mechanics Poisson-Boltzmann surface area (MMPBSA) is of vital importance when

6evaluating the biophysical basis for jaspamide and modelling its inhibitory activity against NSP15

7and NSP10-NSP16. MMPBSA provides a quantity of ∆GvdW, ∆Gelec, ∆Gpol, ∆Gnonpol, and -T∆S, then

8can approximate to the total binding free energy of protein-drug complex (∆GBinding). In practical

9calculations we used the ∆GvdW, ∆Gelec values to estimate Interaction entropy (-T∆S).

10In summary, the binding components approximation of jaspamide are presented in Table 3.

11Moreover Figure 5 exhibits the thermodynamic behaviours (∆EMMPBSA and ∆GBinding values) of

12jaspamide as dual inhibitor through 50 ns. Note that positive values such as polar solvation free

13energy (∆Gpol) are disfavoured in certain circumstances (they inevitably lead to biases for negative

14values of binding energies). Unlike positive values, the evidence came from Table 3 that ∆Gelec,

15∆GvdW and ∆Gnonpol are favoured in similar way for jaspamide when it interacted with NSP15 and

16NSP10-NSP16. Actually, the value of configurational entropy (-T∆S) is more favour to be negatives

17than the positives, and this was obviously the negative value of jaspamide when it interacted with

18NSP10-NSP15, and this results is supported by previous observation (Venken et al., 2011). And the

17
 1values of -T∆S reach from zero when the time of simulations is progress, this reflects that the

2entropy of systems decrease and reach to equilibrium. Further, the estimated binding free energies

3(∆GBinding -18.935 kJ/mol and -17.257 kJ/mol of jaspamide with NSP10-NSP16 and NSP15,

4respectively) revealed nearly similar values because of the driven quantities are nearly similar.

5Notice, that the values of ΔG Binding of jaspamide are practically identical to the hydrogen bonding

6values of jaspamide.

8Figure 5: (A) The ΔG Binding and ΔE MMPBSA values of jaspamide with NSP10-NSP16, (B) The

9ΔG Binding and ΔE MMPBSA values of jaspamide with NSP15 and (C) The configurational entropy

10-TΔS of jaspamide against NSP15 and NSP10-NSP16.

11

18
 1

2Table 3: Estimated binding energies of obtained from MMPBSA and Interaction Entropy

3calculations

ΔE
∆GvdW ∆Gelec ∆Gpol ∆Gnonpol -TΔS
MMPBSA ΔG Binding
(kJ/mol) (kJ/mol) (kJ/mol) (kJ/mol) (kJ/mol
(kJ/mol) (kJ/mol)
)

-0.003
NSP16- -111.201 +/- -54.854 +/ 161.978 +/- -15.565 +/- -18.931 +/-
+/- -18.935 +/-
NSP10 28.667 21.380 37.058 2.861 24.002
0.001 24.003

-67.237 0.715
-101.491 +/- 164.794 +/- -14.243 +/- -17.972 +/-
NSP15 +/- +/- -17.257 +/-
19.516 52.197 2.317 43.884
16.779 0.3784 44.007

5Clearly, Figure 6 (A) shows that 4 residues of NSP15 and NSP10-NSP16 Asn6899, Asp6912,

6Asp6931 Lys6933 contributed in -4.432 kJ/mol, -7.268 kJ/mol, -5.730 kJ/mol and -22.284 kJ/mol

7identical if we compared the results of Hydrogen occupancy results, we found four from five

8residues are identical. Further, Figure 6 (B) exhibits orientation of jaspamide and how interacted

9with four residues of NSP10-NSP16. Next, Figure 6 (C) illustrates the contribution of NSP15’s

10binding site residues: Gln245, Lys290, Trp333 and Tyr343 in -6.232 kJ/mol, -43.209 kJ/mol, -5.675

11kJ/mol and -5.143 kJ/mol, respectively. Where these results are identical with hydrogen bonding

12occupancy. Figure 6 (D) shows how jaspamide interacted with the four residues of NSP15.

13

14

19
 1

2Figure 6: (A) residue contribution of binding energy Jaspamide/ NSP10-NSP16, (B) binding mode

3of jaspamide with NSP10-NSP16, (C) residue contribution of binding energy of Jaspamide/ NSP15

4and (D) binding mode of jaspamide with NSP10-NSP16

5Conclusion

6Indeed, we see that the easily transmission of SARS CoV-2 form one to another. To limit a

7continuum danger of SARS-CoV02 spread then necessitates stop the replication pathways of this

8virus. As a way out of this disease, the data refers to NSP15 and NSP10-NSP16 being involved in the

9replication pathways of virus in a rudimentary way, therefore we screened some of natural peptides

10which have antiviral activities against NSP15 and NSP10-NSP16. The screening of the selected

11peptides is characterized by two separate steps: the first step involves sorting of peptides based on

20
 1docking scores while the second step involves eliminating the peptides which have worse

2pharmacokinetic properties. jaspamide passed across docking screening and pharmacokinetic

3properties screening. In order to evaluate the dual inhibitory activity, we ran molecular dynamics

4simulation and MMPBSA to evaluate interaction stabilities. and free energy decomposition. RMSD,

5RMSF, Rg and SASA of proteins’ backbone revealed that jaspamide have conserved the stabilities of

6targeted proteins during 50-ns MD simulation when it compared with apo forms of proteins.

7Moreover, hydrogen bonding exhibited jaspamide has stale interaction within the active sites of

8NSP15 and NSP10-NSP16. The hydrogen bonding occupancy and residue contribution of binding

9energy results were in agreement which proved that jaspamide have stable interactions with NSP15

10and NSP10-NSP16 targets. The contribution of these results is considered as a first step for further

11investigation of jaspamide as dual inhibitor of SARS-CoV-2.

12

13Declaration of competing for interest

14The authors declared that they have no conflicts of interest in this work.
15
16

17Acknowledgments

18The numerical calculations reported were performed at TUBITAK ULAKBIM, High Performance

19and Grid Computing Centre (TRUBA resources).

20

21

22

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