A Model to Study the Inhibition of Arginase II with Noscapine & Its

Vishvakarma VK, et al., J Protein Res Bioinform 2020, 2: 008 DOI: 10.24966/PRB-1545/100008 HSOA Journal of Protein Research and Bioinformatics

Research Article

nase II-97 complex and the binding energy between 97 and arginase A Model to Study the Inhibition II was found to be negative i.e. -815.184 kcal/mol. of Arginase II with Noscapine & Keywords: Density functional theory; MM-PBSA; Molecular dynam- Its Derivatives ics simulation, Noscapine; Protein data bank. Introduction Vijay Kumar Vishvakarma1,2, Prashant Singh1* and Kamlesh Kumari3# Angina pectoris is an uncomfortable condition, like chest pain and it is due to less oxygen supply to the coronary artery [1,2]. Endothe- 1Department of Chemistry, Atma Ram Sanatan Dharma (ARSD) College, lium has an important role in deciding the role in the release of En- University of Delhi, New Delhi, India dothelial-Derived Relaxing Factor (EDRF) [3]. The major compound 2Department of Chemistry, University of Delhi, New Delhi, India Nitric oxide (NO) regulates the arterial pressure by dilating the blood vessels [4]. NO is synthesized from arginine by means of endothe- 3 Department of Zoology, Deen Dayal Upadhyaya (DDU) College, University of lial Nitric Oxide Synthase (eNOS) [5]. Many research groups have Delhi, Dwarka, Delhi, India focused to develop the bioactive compounds to alter the L-arginine #Equal Contribution metabolism in the body. Arginase-II catalyzes the degradation of arginine into ornithine and urea [6]. Arginase-II is responsible for the bioavailability of Abstract L-arginine for Nitric oxide synthase (NOS) by the mean of the com- Background and Purpose: Nitrate tolerance can be explained petition of substrate [7]. Therefore, when the activity of arginase is based on the reduction of the vessel responsiveness and the same increased, it causes diseases by reducing the amount of L-arginine is used for endogenous vasodilator Nitric Oxide (NO). There are in the body. It is needed by NOS to produce NOe [6-8]. In the last some limitations for the treatment of ischaemia, angina etc. and it few decades, researchers showed great interest in studying the role of attracted the scientists and researchers. The location of arginase II is arginase in the cure of diseases. Various arginase inhibitors have been endothelial cells in mitochondria and it is used to change the potency of endothelial nitric oxide synthase. reported and have shown potential under different pathophysiological conditions like renal injury in diabetic [9], atherosclerosis [10], erec- Experimental approach: A theoretical model has been developed tile dysfunction and pulmonary hypertension [11,12], hypertension to find the potent arginase II inhibitor. A library of noscapine (116 [13], allergic rhinitis [14] and many more. molecules) was designed and optimized using computational tools. Then, the designed molecules were docked with the arginase II Phthalideisoquinilines based alkaloids are popular molecules and (PDB: 4IXU) using iGemdock. Based on binding energy, the poten- cones under the class of isoquinoline based compounds viz. erythro tial candidate was screened. Further, absorption distribution me- and threo form. Noscapine contains isoquinoline and benzofuran ring tabolism, excretion and toxicity (ADMET) using online web-server as an active ingredient. Primarily it is used as an antitussive agent and density functional theory (DFT) study of the top four screened molecule has been studied by using Gaussian. Then, molecular dy- to suppress a cough. At present, noscapine its its derivatives are ex- namic simulation of arginase II with and without 97 was performed plored and under clinical trials for the treatment of different diseases using Gromacs. Further, the binding energy was determined using like cancer [15,16]. There is too much structural variability in noscap- MM-PBSA on Gromacs. ine, which make its use for different purposes. Conclusion: Compound no.97 showed the best binding with the There is need to find the arginase II inhibitor to control or cure an- arginase II based on docking. Further, the potency of the screened gina. The potential of the erythro form of noscapine against the argin- noscapine 97 against arginase II was compared with the reported ase-II has been investigated. In the present work, a theoretical model molecules. MD simulations showed the stable anchoring of the argi- for the inhibition of arginase-II by noscapine and its derivatives was developed. Molecular docking, density functional theory, Absorption *Corresponding author: Prashant Singh, Department of Chemistry, Atma Ram distribution metabolism, excretion and toxicity (ADMET), Molecular Sanatan Dharma (ARSD) College, University of Delhi, New Delhi, India, Tel: +91 1124113436; E-mail: [email protected] dynamic (MD) simulations along with Molecular mechanics Pois- son–Boltzmann surface area (MM-PBSA) analysis were performed Citation: Vishvakarma VK, Singh P, Kumari K (2020) A model to study the in- to find the potent arginage II inhibitor. hibition of Arginase II with Noscapine & its derivatives. J Protein Res Bioinform 2: 008. Experimental Received: January 07, 2020; Accepted: April 18, 2020; Published: April 24, 2020 This experimental work is categorized into five parts i.e. designing Copyright: © 2020 Vishvakarma VK, et al. This is an open-access article of molecules & molecular docking, ADMET studies, DFT studies, distributed under the terms of the Creative Commons Attribution License, which MD simulations along with MM-PBSA analysis. The overall experi- permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. mental approach of the work can be understood by flowchart 1. Citation: Vishvakarma VK, Singh P, Kumari K (2020) A model to study the inhibition of Arginase II with Noscapine & its derivatives. J Protein Res Bioinform 2: 008.

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Designing of molecules and molecular docking EBinding = EVDW + Hbond + Elec (1) Designing of molecules VDW - vander Waal energy; H - hydrogen bonding energy; There are two isomeric form of noscapine viz., erythro and threo bond form. Herein, only erythro-form of noscapine was considered due to Elec - electro statistic energy its high stability and biological potential. In the present work, a total The modelling of the docked poses is studied by Discovery studio of 116 molecules based on noscapine were designed as in table 1. visualizer v 3.5 [18]. ADMET properties The ADME (Absorption, Distribution, Metabolism, Excretion, and Toxicity) properties were calculated to check the better bioavailability of the proposed drug molecule. Physicochemical parameters The physiochemical properties like Log S, Solubility, number of heavy atoms, number of rotatable bonds, number H-bond acceptors,

number H-bond donors, Log Po/w, and physiochemical space for oral availability were checked by the web server (http://www.swissadme. ch/) [19]. Biological properties The biological properties like TPSA (Å²), GI absorption, BBB permeant, P-gp substrate, and CYP3A4 inhibitor were calculated by the web server (http://www.swissadme.ch/). Absorption (% ABS) of top four molecules was calculated according to the method described by Zhao et al. [20] TPSA is an important factor to give an idea for ability of drug transport and can be determined by using equation 2. The results are incorporated in table 4. Flowchart 1: The overall methodologies used in the whole work. %ABS = 109 – [0.345 × topological polar surface area (TPSA) (2) Geometry optimization of noscapines & reported molecules Other biological properties like GPCR ligand, ion channel mod- The designing of all compounds were done by using ACD ulator, kinase inhibitor, nuclear receptor ligand, protease inhibitor, Chemsketch and their optimization was done by choosing molecular and enzyme inhibitor value by using the online server molinspiration mechanics (MM2) as a force field. These optimized compounds were (www.molinspiration.com) [21]. used for docking. Toxicity Protein preparation The acute rat toxicity of the top four molecules was calculated Protein preparation was done by using Molegro Molecular Viewer using an online server GUSAR (http://www.way2drug.com/gusar/ (MMV 2.5). The following parameters were checked like flexible tor- acutoxpredict.html). The toxicity parameters like IP LD50, IV LD50, sion in compounds, missing charges, assigning of bonds, tripos type atoms and missing explicit hydrogen. The prepared protein was used Oral LD50, and SC LD50 for all four route of administration i.e., oral, for the docking analysis and MD simulation. intraperitoneal, intravenous, and subcutaneous for top four molecules were calculated. This toxicity model was based on a rat [22]. Molecular docking DFT analysis The docking of all noscapines (Table 1) and the reported molecules (Table 2) was performed using iGemdock [17] against the argi- Density functional theory (DFT) have been performed to study nase-II (PDB ID - 4IXV). This software used the generic algorithms the electrical properties of the noscapine derivative. Geometry op- for the docking. timization of the molecules were performed. Becke’s 3 parameters Docking parameters & Post Docking modeling functional Lee, Yang, Parr B3LYP/6-311++G (d, p) was used for the calculation with the Gaussian 09 [23]. Herein, the parameters for the docking are set with population size of 200 and generation of 70 along with two solutions for each. On In addition, DFT is very useful in providing chemical descriptors the basis of the above set parameters, the compounds were screened such as chemical hardness (η), chemical potential (µ), electronegativ- [17]. The top four compounds were selected by considering the low- ity (χ), softness (S), and global electrophilicity index (ω), these are est binding energy, can be determined by the equation 1- given in equation 3-7 [24].

Volume 2 • Issue 1 • 100008 J Protein Res Bioinform ISSN: 2692-1545, Open Access Journal DOI: 10.24966/PRB-1545/100008 Citation: Vishvakarma VK, Singh P, Kumari K (2020) A model to study the inhibition of Arginase II with Noscapine & its derivatives. J Protein Res Bioinform 2: 008.

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Parent compound Alkyl Group (R) Parent compound Alkyl Group (R) 1 -H 11 -COOH 21 -H 31 -COOH

2 -CH2OH 12 -CHO 22 -CH2OH 32 -CHO R 3 -CH Br 13 -COCH 23 -CH Br 33 -COCH 2 3 R 2 3 O 4 -CH Cl 14 -CH=CH 9' 24 -CH Cl 34 -CH=CH 2 2 O 2 2 N O 5 -NO2 15 -CH3 25 -NO2 35 -CH3 N O O 6 -NH 16 -OCH 26 -NH 36 -OCH3 O 2 3 O 2 O 7 -Cl 17 -OCH2CH3 27 -Cl 37 -OCH2CH3 O O O 8 -Br 18 -OH O 28 -Br 38 -OH O 9 -NHAc 19 -COBr O 29 -NHAc 39 -COBr 10 -COCl 20 -CN 30 -COCl 40 -CN Parent compound Alkyl Group (R) Parent compound Alkyl Group (R)

41 -CH2OH 51 -CHO 60 -CH2OH 70 -CHO R R 42 -CH2Br 52 -COCH3 61 -CH2Br 71 -COCH3

43 -CH2Cl 53 -CH=CH2 62 -CH2Cl 72 -CH=CH2

9' 44 -NO2 54 -CH3 63 -NO2 73 -CH3 O 9' 45 -NH 55 -OCH3 O 64 -NH 74 -OCH N 2 2 3 O N 46 -Cl 56 -OCH2CH3 O 65 -Cl 75 -OCH2CH3 O O 47 -Br 57 -OH O 66 -Br 76 -OH O 48 -NHAc 58 -COBr 67 -NHAc 77 -COBr O O O 49 -COCl 59 -CN O 68 -COCl 78 -CN O O 50 -COOH 69 -COOH Parent compound Alkyl Group (R) Parent compound Alkyl Group (R)

79 -CH2OH 89 -CHO 98 -CH2OH 108 -CHO R R 80 -CH2Br 90 -COCH3 99 -CH2Br 109 -COCH3

R R 81 -CH2Cl 91 -CH=CH2 100 -CH2Cl 110 -CH=CH2 9' 82 -NO 92 -CH 101 -NO 111 -CH O 2 3 9' O 2 3 83 -NH 93 -OCH 102 -NH 112 -OCH N 2 3 2 3 O N O 84 -Cl 94 -OCH2CH3 103 -Cl 113 -OCH2CH3 O O O 85 -Br 95 -OH O 104 -Br 114 -OH 86 -NHAc 96 -COBr 105 -NHAc 115 -COBr O O O O O 87 -COCl 97 -CN O 106 -COCl 116 -CN 88 -COOH 107 -COOH

Table 1: Libraries of the noscapine derivatives.

parameters was used in analysis [27]. The topology and coordinates (3) of the ligand were generated by SwissParam online web server [28]. The system was solvated by water in the cubic box manner taking (4) simple point charge model to develop a periodic boundary condition (PBC). Na+ and Cl- ions were used to neutralise the system. The ener- (5) gy minimization of the system was performed by applying a steepest descent algorithm with 1000 steps to release the conflict contacts. (6) MD simulations analysis were performed in two phases, (i) ensemble equilibration and (ii) MD production. The temperature and (7) pressure equilibration were performed to control the temperature and pressure of the system, the system is warmed to 300 K at 1 bar for Where IE is ionization potential and EA is electron affinity. 100 ps using leap-frog integrator for temperature coupled by modified MD Simulation Berendsen thermostat and leap-frog integrator for pressure coupled by Parrinello-Rahman. MD simulations were done taking cut off the The molecular dynamics (MD) is a technique used to study the size of 12 Å. Particle Mesh Ewald (PME) method was used for all fundamental structural response of protein with and without ligand at the nanoscale [25]. MD analysis of protein and protein-ligand com- long-range electrostatics charges. The MD production is run for 10ns plex was done by using GROMACS 5.1.4 [26]. CHARM force-field and the coordinates were recorded on an interval of 10 ps.

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S.N. Structure S.N. Structure S.N. Structure H OH O O N O I B NH2 HO H R1 N OH R2 R3 H H H HO - N I NH O O 2 NH2 H

(S)-2-amino-5-(2-iodoacetamido) ammonium 2-amino-6-borono- N6-(3-iodoprop-1-en-2-yl)lysine pentanoic acid hexanoate HN NH H NH2 HO COOH O H2N N O HN N R4 N N R5 NH2 OH R6 H H H S NO2 H Cl NH2 OH NH O OH O O

N-hydroxyarginine compound 1-(4-aminobutyl)guanidine methyl Nw-nitro-L-argininate with acetic acid (1:1) (nor-NOHA) sulfate hydrochloride

O OH OH OH N O OH R7 R8 O R9 H N NH 2 N 2 H2N H H2N

(S,E)-2-amino-4-(2-hydroxyguanidi- L-isoleucine L-leucine no)butanoic acid

O OH O OH O OH H Cl H Cl R10 R11 R12 NH NH 2 H2N 2 H2N H2N

(S)-2,5-diaminopentanoic acid L-lysine hydrochloride (S)-2-aminopentanoic acid hydrochloride O O OH HO HN R13 R14 R15 OH O H2N NH NH2

L-proline L-tryptophan L-valine Table 2: 15 reported arginase II inhibitors.

MM-PBSA analysis ε(r) - dielectric constant ρf(r) - fixed charge density MM-PBSA analysis is done after the screening and done by using k - Boltzmann constant; the trajectories obtained from the MD simulation. MM-PBSA study γ - Coefficient related to the surface tension of the solvent; for the complex between the ligand and arginase-II was studied by A- SASA the g_mmpbsa [29-30]. This method provide the change in free ener- b- fitting parameter gy for the formation of complex. Various binding free energy change p - coefficient related to pressure of the solvent were calculated for the complex with the help of the equations 8-15 V - SAV. respectively [29-30]. The ensemble hypothesis was used to calculate these all changes G = G – (G + G ) (8) binding complex protein ligand from the coordinates of corrected MD trajectories [29]. E = E + E = E + (E + E ) (9) ∆MM bonded nonbonded bonded vdW elec Results and Discussion Gsolvation = Gpolar + Gnonpolar (10) - 2 h + 4 /kT = 0 (11) Molecular docking G = G + G (12) Docking is a computational methodology to study the binding of ∇.r∇.φrnonpolar rrcavity sin r vdW fr G = A + b (13) small molecules with a receptor to form a complex to enhance or in- nonpolar hibit the biological potency of protein [31]. The total binding energy G = pV + b (14) nonpolar of all docked molecules are used to screen and the top four molecules

Gnonpolar = A + pV + GvdW (15) i.e., ligand 97, 109, 48 and 101 are mentioned in table 3 along with the 15 reported inhibitors. Ligand 97 shows the highest negative val- EMM - vacuum potential energy E – electrostatic ue against 4IXV, which is -133.413 KJ/mol. The designed top four elec molecules shows strong binding energy than the reported molecules. EvdW - van der Waals ϕ(r) - electrostatic potential The binding energy values for ligand 109, 48 and 101 are -129.175,

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-126.677 and -126.298 respectively. The highest value in reference THR-272, GLU-275 and GLY-226. From the binding cavity residues molecule is for the 1-(4-aminobutyl) guanidine sulfate (R5), it’s value analysis, it is clear that the main residues which are the part active is -110.052. Table 3 shows that noscapines can be used to inhibit the cavity are mainly composed of ASN-147, SER-155, ASN-158, ASP- function of arginase-II more effectively than the reference molecules. 200, ASP-202, GLY-161, GLU-205, THR-265, TYR-273, THR-272, The docking results of he designed noscapines are available in sup- GLU-275, ASP-256, ARG-274, GLY-227, GLY-269, ASP-223, THR- porting file. Docking data of all the designed noscapines are given in 310, ASP-317, GLN-325, ASN-313, VAL-267, VAL-268, and ILE- Supplementary information as in table Sa. 227.

Ligand EBinding EVDW EH-bonding EElect Noscapine Derivatives 97 -133.413 -93.6378 -39.7757 0 109 -129.175 -115.069 -14.1058 0

48 -126.677 -98.9796 -27.6971 0 101 -126.298 -95.8055 -28.7223 -1.7701 Reference Molecule R5 -110.052 -54.3164 -55.1636 -0.57243 R4 -92.2519 -50.6993 -42.7032 1.15061 R7 -87.7108 -46.2689 -39.0585 -2.38333 R3 -87.0116 -53.3087 -35.0249 1.32208 R6 -84.8008 -56.2746 -28.5261 0 R14 -79.4946 -54.3613 -20.653 -4.48028 R2 -73.7685 -46.8754 -27.4161 0.522978 R1 -72.7108 -45.508 -22.9159 -4.28693 R12 -67.9846 -50.7546 -17.23 0 R11 -64.3878 -34.752 -32.4239 2.78805 R8 -64.1126 -42.7747 -27.2768 5.93892 R10 -63.2022 -40.3005 -20.1627 -2.73904 R13 -63.1236 -46.367 -23.5179 6.76133 Figure 1(a-d): H-bond poses of 97, 109, 48 and 101 with amino acid of arginase-II. R9 -62.9151 -46.7523 -22.8751 6.71231 R15 -62.1513 -35.458 -29.7164 3.02315 Table 3: Docking score of the top four noscapine derivatives and reported molecules.

The configurational analysis of the active site of a protein is also done to check the active amino acid residues which bind with the noscapines. It is found that the noscapines targeted same active site of arginase II occupied by the first-generation arginase inhibitors like NOHA. It is clearly understood that noscapines can be used to inhibit arginase-II activity more effectively than the reported inhibitors. Li- gand 97 shows H-bond interaction given in figure 1a with SER-156 (2.33 Å) and ASN-158 (2.33695 Å & 1.93419 Å), ligand 109 shows H-bond interaction given in figure 1b with GLU-275 (2.57936 Å) and with TYR-273 (2.35556 Å), ligand 48 shows H-bonds interaction given in figure 1c with THR-310 (2.81668 Å), ARG-57 (2.08954 Å), GLN-325 (1.92582 Å) and ASP-317 (2.48569 Å) with and ligand 101 don’t show any H-bond interaction given in figure 1d. The contribution of amino acids of binding pockets within and around 8 Å of the ligand was also analyzed and a graph of amino acid versus total binding energy is also plotted for the ligand 97, 109, 48 and 101 (Graph 1a-d). The major amino acid contribution of Graph 1(a-d): Showing cavity residues and their negative contribution in the stabili- active cavity for ligand 97 are ASN-147, SER-155, ASN-158, ASP- zation of Ligand 97, 109, 48 and 101 respectively. 200, ASP-202, GLY-161, GLU-205 and THR-265, for ligand 109 are TYR-273, THR-272, GLU-275, ASP-256, ARG-274, GLY-227, GLY-269 and ASP-223, for ligand 48 are ARG-57, TYR-273, THR- In Graph 2, the structural properties of the arginase II 4IXV was 310, ASP-317, GLN-325, ASN-313, ARG-119, ASP-200, VAL-267 determined using the SAVES server (online) and the analysis was ex- and VAL-268 and for ligand 101 are ASP-223, ARG-274, ILE-227, plained as in Graph 2 regarding the allowed and disallowed regions.

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top four molecules are under the allowed limit. The allowed limit of no. of H-bond acceptor is 10 and donor should be 5, and here ligand 97, 109, and 48 are under the allowed limit. The physiochemical oral availability was also calculated to top four ligands and it is found that the top three under the space. ADME properties of all the designed molecules is given in Supplementary information as table Sb.

Properties 97 109 48 101 Log S -5.56 -5.58 -5.31 -5.80 Solubility Moderately Moderately Moderately Moderately

(a) Heavy atoms 40 42 40 42 Arginase-II No. of rotational bonds 5 7 7 7 Item Number of % of No. H-bond acceptors 10 10 9 12 amino-acids amino-acids Num. H-bond donors 0 0 1 0 Residues in most favoured regions 713 91.1 Log Po/w 3.90 4.00 4.00 3.18 Residues in additional allowed regions 69 8.8 Residues in generously allowed regions 1 0.1 Physiochemical space for oral availability Residues in disalllowed regions 0 0.0 Number of non-glycine and non-proline residues 783 100 Table 4: Physiochemical descriptors of top four noscapine derivatives. Number of end residues excluding glycine and proline 3 Number of glycine residues (shown as triangles) 78 Number of proline residue 54 Biological properties like % absorbance from TPSA, gastroin- Total number of residues 918 testinal (GI) absorbance, Blood-brain Barrier (BBB) permeation, permeability glycoprotein (P-gp) substrate value, cytochrome P450 (CYP3A4) inhibition value, molinspiration Log P (miLog P), globular protein-coupled receptor (GPCR) inhibition value, ion channel modulator, kinase inhibitor, nuclear receptor, protease inhibitor, and enzyme inhibition value were calculated and are given in table 5. The GI absorption value for top three ligands is found to high while for ligand 101 is low. The BBB value for all top four molecules is found negative, the P-gp is found as positive, cytochrome P450 (CYP3A4) inhibition value for top three positives, miLog P is below 5, the GPCR value is found positive for ligand 97 and 48, and for 109 and 101 is negative, ion channel modulator, kinase inhibitor, nuclear receptor ligand, protease inhibitor, and enzyme inhibitor value is negative for all, while the lowest score is for ligand 97 among top four, which shows the ligand 97 has more biological potential among the top four.

Graph 2: (a) the ramachandran plot and (b) the 3D structures analysis of Arginase-II.

Properties 97 109 48 101 TPSA (Å²) 123.27 Å² 109.83 Å² 104.79 Å² 167.33 Å² ADMET analysis %ABS 66.47185 71.10865 72.84745 51.27115 ADMET properties of the molecules are generally more useful GI absorption High High High Low when a new molecule is proposed as a potential drug [32]. Oral bio- BBB permeant No No No No availability of a drug can be explained as a part of oral drug in system- P-gp substrate Yes Yes Yes Yes atic circulation in the body. Oral absorption depends on the permea- CYP3A4 inhibitor Yes Yes Yes No bility and aqueous solubility of a molecule. It can be controlled by miLog K 3.95 4.26 3.73 4.38 molecular weight, log P, Topological polar surface area (TPSA), no. GPCR ligand 0.07 -0.01 0.02 -0.09 of rotatable bonds, log S, number of Hydrogen Bond Donors (HBD) Ion channel modulator -0.13 -0.37 -0.24 -0.38 and Acceptors (HBA) [33]. It was found that noscapine followed the Kinase inhibitor -0.26 -0.50 -0.34 -0.49 Lipinski’s “rule of five” with one violation. Although, the aqueous Nuclear receptor ligand -0.30 -0.46 -0.45 -0.52 solubility of noscapine was found to be low and should need to in- Protease inhibitor -0.31 -0.34 -0.35 -0.40 crease (Table 4). For four ligands 97, 109, 48 and 101the log P value Enzyme inhibitor -0.03 -0.20 -0.13 -0.24 are 3.9, 4, 4 and 3.18 respectively, which is under 5, the log S values Table 5: Biological of the top hit four molecules against arginase-II. are -5.56, -5.58, -5.31 and -5.80 respectively and are moderately solu- ble. The maximum no. of allowed rotatable bonds should be 9, and all

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The toxicity estimation of compounds was tested as QSAR model based on the rat toxicity, where the Lethal dose (LD) 50 were calculated for the fours of administration i.e. intraperitoneal, intravenous, oral and subcutaneous (Table 6). The ligand 97, 109, 48 and 101 all came in applicability domain of model while the subcutaneous LD50 value for the ligand 97 falls out of applicability domain. All the LD50 value for the ligand 97 is almost the lowest in all top four ligands.

Properties 97 109 48 101 Rat IP LD50 528,800 in 577,400 in AD 605,000 in AD 419,200 in AD (mg/kg) AD Rat IV LD50 30,580 in AD 36,110 in AD 31,610 in AD 33,730 in AD (mg/kg) Rat Oral LD50 1576,000 in 1202,000 in 947,900 in AD 885,300 in AD (mg/kg) AD AD Rat SC LD50 1256,000 out 645,000 in 782,300 in AD 468,400 in AD (mg/kg) of AD AD IP - Intraperi- IV - Intrave- Oral - Oral SC - Subcuta- toneal route of nous route of route of ad- neous route of Graph 3: Energies gap of orbitals of HOMO and LUMO of top four. administration administration ministration administration Note: out of AD - compound is out in AD - compound falls in appli- of the applicability domain of The frontier molecular orbital analysis was also done to study the cability domain of models models electronic distribution of the electron throughout the molecule. The Table 6: Toxicity of the top hit four molecules against arginase-II. HOMO and LUMO of ligand 97, 109, 48, and 101 are given in figure 2. The HOMO orbital for the ligand 97, is centered at the nitrogen of isoquinoline ring and the LUMO orbital is at benzofuran ring of DFT Analysis noscapine; for ligand 109, the HOMO located on isoquinoline ring and LUMO on substituent ring; for ligand 48, the HOMO located on DFT studies help to understand molecular properties and the isoquinoline ring and LUMO on benzofuran ring and for ligand 101, behavior of atoms in molecules. Hard molecules have a large HO- HOMO located on isoquinoline ring and LUMO on substituent ring. MO-LUMO gap and soft molecules showed a reverse pattern. Lesser Molecular dynamics simulations analysis the HOMO-LUMO gap means small excitation energy [34]. It was found that the HOMO-LUMO gap for the ligand 97, 109, 48 and 101 Molecular dynamic simulation is considered to be an important in singlet state is 0.23223, 0.1402, 0.15806 and 0.31534 (Graph 3), tool to study the behavior of protein as well as protein-ligand complex while in triplet state is 0.00895, 0.01272, 0.02807 and -0.01669. It in the context of structural stability. Herein, the strength, salvation, and conformational pattern are studied. MD simulation of the argin- reveals a good agreement with docking result to be molecule to be ase II with and without the screened ligand was performed using the enough hard in the binding pocket of protein. The chemical potential appropriate force field along with including the explicit solvent [37]. (µ) explains the ability of an electron to leave from the molecule in the Radius of gyration is an indicator of protein structure’s compactness equilibrium state [35]. The calculated chemical potential for ligand 97 over the timescale [38]. Rg graph showed slight unfolding in initial in singlet state and triplet state is found to be o.11611 and 0.00447 re- time frame but the difference in Rg value of arginase-II with and with- spectively. A significant decrease in chemical potential in triplet state out 97 is less than the 1.5. But, after a run of 5 ns, it again showed the stable folding and become stable as in graph 4(a). RMSD measures is observed. The chemical hardness of ligand 97, 109, 48 and 101 in the similarity in structure of the protein with and without ligand [39]. singlet state is found to be 0.11611, 0.0701, 0.07903 and 0.15767 re- RMSD of arginase II is approximately found to be the 0.15 nm and for spectively, which reveals the hardness order as 101>97>48>109. The arginase II-97 complex is 0.20 nm graph 4(b). The RMSD of arginase global softness value for ligand 97 is found to be 4.30609 and 111.731 II and arginase II-97 complex is less than the 0.2 nm and confirms the in singlet and triplet state respectively. The less hardness value and successful docking. more softness value makes molecule enough soft and polarizable and The root mean square fluctuation (RMSF) is able to compare the order is 48>109>97>101. The absolute electronegativity is the the fluctuation in the mean position of the backbone atoms of pro- ability to attract electrons towards itself in a covalent bond [35]. The tein [40]. The RMSF values for arginase-II with and without 97 is overall electronegativity of ligand 97, 109, 48 and 101 in singlet state ranges near the 0.1 nm. It showed the small fluctuations of the atom- is found to be 0.14192, 0.1518, 0.14183 and 0.16961 respectively and ic coordinates for the protein-ligand complex with reference to the it is less than one, indicate lower electron attraction power of the mol- protein -carbons. These fluctuations range from 0-6000 atoms of the proteins graph 4(c). These initial and small fluctuations also ecule. Global electrophilicity index indicates the behaviour of mole- support the successful docking of ligand in the active cavity of pro- cules to accept the electron density. [36]. The electrophilicity value in tein as the cavity amino acid residues belong to 100-300 sequences. singlet state is found to be 0.08673, 0.16436, 0.127267 and 0.091227 Number of H-bonds present between 97 and arginase II is found to 5 respectively for ligand 97, 109, 48 and 101. The values of all chemical in number. Among five three are conventional H-bonds while two are descriptors are given in table 7. non-conventional H-bonds as in as in graph 4(d) [41-45].

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C. N. 97 C. N. 109 C. N. 48 C. N. 101 SINGLET TRIPLET SINGLET TRIPLET SINGLET TRIPLET SINGLET TRIPLET LUMO+2 -0.01399 -0.01387 -0.06332 -0.02612 -0.02573 -0.02197 -0.06943 -0.10334 LUMO+1 -0.01686 -0.01399 -0.06898 -0.06332 -0.03473 -0.02573 -0.02863 -0.06943 LUMO -0.02581 -0.01686 -0.0817 -0.06898 -0.0628 -0.03473 -0.01194 -0.02863 HOMO -0.25804 -0.02581 -0.2219 -0.0817 -0.22086 -0.0628 -0.32728 -0.01194 HOMO-1 -0.26057 -0.25804 -0.2265 -0.2219 -0.22566 -0.22086 -0.33317 -0.32728 HOMO-2 -0.26867 -0.26057 -0.22818 -0.2265 -0.22587 -0.22566 -0.34232 -0.33317 L-H 0.23223 0.00895 0.1402 0.01272 0.15806 0.02807 0.31534 -0.01669 L+H -0.28385 -0.04267 -0.3036 -0.15068 -0.28366 -0.09753 -0.33922 -0.04057 ɳ 0.11611 0.00447 0.0701 0.004475 0.07903 0.014035 0.15767 0.014035 Χ 0.14192 0.02133 0.1518 0.07534 0.14183 0.048765 0.16961 0.020285 S 4.30607 111.731 7.132668 111.7318 6.326711 35.62522 3.17118 35.62522 µ -0.14193 -0.02134 -0.1518 -0.07534 -0.14183 -0.04877 -0.16961 0.02029 Ω 0.08673 0.05085 0.16436 0.634203 0.127267 0.084718 0.091227 0.014659 Table 7: Energies of various HOMO, LUMO, and chemical descriptors.

MM-PBSA analysis The MD trajectories with no PBC obtained from GROMACS was used to analyze the binding energy, solvation energy and electrostatic potential energy changes of the protein-ligand complex by g_mmpb- sa. APBS program was used by the g_mmpbsa to solve the Pois- son-Boltzmann (PB) equation. The values of binding energy, SASA, SAV, WCA, van der Waal energy, electrostatic energy and polar solvation along with the maximum possible error in energy are given in table 8. The value of binding energy for the ligand 97 was found to be -815.184 KJ/mol, which is much more negative to support the strong binding of the ligand into the active cavity of protein also show successful docking. The polar solvation energy is enough positive, SASA, van der Waal energy, and electrostatic energies are enough negative.

Graph 4: (a) Rg behavior of Arginase-II and Arginase II-97 complex, (b) RMSD behavior of Arginase-II and Arginase II-97 complex and (c) RMSF behavior of Ar- ginase-II and Arginase II-97 complex (d) H-bonding interaction of ligand along with pairs within 0.35 nm. Figure 2: a-e and g represent the frontier molecular orbital of HOMO and b-f and h represent the frontier molecular orbital of LUMO of compound 97, 109, 48, and 101 respectively. The graph between binding energy, ΔEmm, ΔGpolar, and ΔGnonpolar versus time were also plotted to check the overall response of

• Page 9 of 14 • the system, given in graph 5. The binding energy remains almost in- with arginase-II. Arginase-II inhibition has been done successfully by variant around the reported value in table 6, electrostatic potential ligand 97. This theoretical model was developed to inhibit the argin- energy shows more variation in initial time frame, but after 4 ns it also ase II can be used to check the potential of the small molecule against shows almost similar values, the polar solvation energy also initial any protein. variation up to 2 ns but after this the values are remains almost constant and non-polar solvation energy initial and final fluctuation but References remains almost invariant in range of 2-7 ns. 1. Benjamin S, Steinhorn, Loscalzo J, Michel T (2015) Nitroglycerin and Ni- tric Oxide - A Rondo of Themes in Cardiovascular Therapeutics. N Engl S.N. Type of energy Value (kJ/mol) Error (+/-) J Med 373: 277-280. 1 van der Waal energy -131.382 13.831 kJ/mol 2. Murrell W (1879) Nitroglycerin as a remedy for angina pectoris. Lancet, 2 Electrostatic energy -1135.49 50.531 kJ/mol 1: 80-81. Polar solvation 3 468.828 66.349 kJ/mol 3. Furchgott RF, Zawadzki JV (1980) The obligatory role of endothelial cells energy in the relaxation of arterial smooth muscle by acetylcholine. Nature 288: 4 SASA energy -17.139 1.489 kJ/mol 373-376. 5 SAV energy 0 0.000 kJ/mol 4. Palmer RM, Ferrige AG, Moncada S (1987) Nitric oxide release accounts 6 WCA energy 0 0.000 kJ/mol for the biological activity of endothelium-derived relaxing factor. Nature 7 Binding energy -815.184 32.836 kJ/mol 327: 524-526. Table 8: Results of MM-PBSA analysis of arginase-II-ligand 97 complex. 5. Balligand JL, Feron O, Dessy C (2009) eNOS activation by physical forc- es: from short-term regulation of contraction to chronic remodeling of cardiovascular tissues. Physiological Reviews 80: 481-534.

6. Guoyao WU, Sidney MM Jr (1998) Arginine metabolism: Nitric oxide and beyond. Biochem J 336: 1-17.

7. Morris SM (2002) Regulation of enzymes of the urea cycle and arginine metabolism. Annu Rev Nutr 22: 87-105.

8. Caldwell RB, Toque HA, Narayanan SP, Caldwell RW (2015) Arginase: an old enzyme with new tricks. Trends Pharmacol Sci 36: 395-405.

9. Morris SM, Gao T, Cooper TK, Kepka-Lenhart D, Awad AS (2011) Argin- ase-2 mediates diabetic renal injury. Diabetes 60: 3015-3022.

10. Olivon VC, Fraga-Silva RA, Segers D, Demougeot C, de Oliveira AM, et al. (2013) Arginase inhibition prevents the low shear stress-induced development of vulnerable atherosclerotic plaques in ApoE-/- mice. Atheroscle- rosis 227: 236-243.

11. Segal R, Hannan JL, Liu X, Kutlu O, Burnett AL, et al. (2012) Chronic Oral Administration of the Arginase Inhibitor 2(S)-amino-6-boronohex- anoic Acid (ABH) Improves Erectile Function in Aged Rats. Journal of Andrology 33: 1169-1175.

Graph 5: (a) ΔGbinding, (b) ΔEmm, (c) ΔGpolar and (d) ΔGnonpolar versus time of the argin- 12. Grasemann H, Dhaliwal R, Ivanovska J, Kantores C, McNamara PJ, et ase-II-ligand 97 complex. al (2015) Arginase inhibition prevents bleomycin-induced pulmonary hypertension, vascular remodeling, and collagen deposition in neonatal rat lungs. Am J Physiol Lung Cell Mol Physiol 308: 503-510.

Conclusion 13. Bagnost T, Ma L, da Silva RF, Rezakhaniha R, Houdayer C, et al. (2010) Cardiovascular effects of arginase inhibition in spontaneously hyperten- Herein, the molecular docking, DFT, ADME, MD simulations sive rats with fully developed hypertension. Cardiovasc Res 87: 569-577. and MM-PBSA are performed to study the potential of noscapines against the Arginase-II. The potential candidate has been chosen 14. Meurs H, Zaagsma J, Maarsingh H, van Duin M (2010) Use of Arginase Inhibitors in the Treatment of Asthma and Allergic Rhinitis. 20150164930 based on binding free energy value due to hydrogen bonding, and A1. US. 2010 van der Waals interaction and electrostatic interaction. Docking analysis shows that ligand 97 have shown the highest binding affin- 15. Chen X, Dang TT, Facchini PJ (2015) Noscapine comes of age. Phyto- chemistry 111: 7-13. ity with arginase-II. Subsequently, the molecular property screening of noscapines satisfied the Lipinski’s rule of five. The aqueous 16. Singh H, Singh P, Kumari K, Chandra A, Dass SK, et al. (2013) A Review solubility of noscapine derivatives was found to be less compared on Noscapine, and its Impact on Heme Metabolism. Current drug metab- to the reference molecules, and it suggests that it should need to in- olism 14: 351-360. crease. Further, MD simulation is performed to analyze the binding 17. Yang JM, Chen CC (2004) GEMDOCK: A generic evolutionary method stability of noscapine derivative in the cavity of protein. Rg, RMSD for molecular docking. Proteins: Structure, Function and Bioinformatics and RMSF results reveal that complex of ligand 97 with Argin- 55: 288-304. ase-II is highly stable. The high negative value binding energy from 18. Dassault Systèmes BIOVIA, Discovery Studio Modeling Environment, mmpbsa results clearly indicates the effective binding of ligand 97 Release 2017, San Diego: Dassault Systèmes, 2016.

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19. Daina A, Michielin O, Zoete V (2017) Swiss ADME: a free web tool to 33. Lipinski CA (2004) Lead- and drug-like compounds: the rule-of-five revo- evaluate pharmacokinetics, drug-likeness and medicinal chemistry friend- lution. Drug Discov Today Technol 1: 337–341. liness of small molecules. Scientific Reports 7: 42717. 34. Pearson RG (1986) Absolute electronegativity and hardness correlated 20. Zhao Y, Abraham MH, Lee J, Hersey A, Luscombe NC, et al (2002) with molecular orbital theory. Proc Natl Acad Sci USA 83: 8440-8441. Rate-limited steps of human oral absorption and QSAR studies. Pharm Res 19: 1446-1457. 35. Miura K, Kimata F, Watanabe R, Fukuhara C (2018) DFT Study for Sup- ported Pt Catalysts Focusing on the Chemical Potential. e-Journal of Sur- 21. http://www.molinspiration.com/ face Science and Nanotechnology 16: 209-213.

22. Lagunin A, Zakharov A, Filimonov D, Poroikov V (2011) QSAR Model- 36. Chattaraj PK, Giri S (2009) Electrophilicity index within a conceptual ling of Rat Acute Toxicity on the Basis of PASS Prediction. Mol Inform DFT framework. Annu. Rep. Prog. Chem., Sect C: Phys Chem 105: 13-39 30: 241-250. 37. Berhanu WM, Masunov AE (2014) Chapter 6 - The Atomic Level Inter- 23. Frisch MJ, Trucks GW, Schlegel HB, Scuseria GE, Robb MA, et al (2009) action of Polyphenols with the Aβ Oligomer Aggregate, A Molecular Dy- Gaussian 09, Revision A.02; Gaussian, Inc.: Wallingford, CT. namic Guidance for Rational Drug Design. Polyphenols in Human Health and Disease 1: 59-70. 24. Bourass M, Benjelloun AT, Benzakour M, Mcharfi M, Hamidi M, et al. 38. Lobanov MY, Bogatyreva NS, Galzitskaya OV (2008) Radius of gyration (2016) DFT and TD-DFT calculation of new thienopyrazine-based small as an indicator of protein structure compactness. Mol Biol 42: 623-628. molecules for organic solar cells. Chemistry Central Journal 10: 67. 39. Maiorov VN, Crippen GM (1994) Significance of root-mean-square de- 25. Vishvakarma VK, Singh P, Kumari K, Chandra R (2017) Rational Design viation in comparing three-dimensional structures of globular proteins. J of Threo as Well Erythro Noscapines, an Anticancer Drug: A Molecular Mol Biol 235: 625-634. Docking and Molecular Dynamic Approach. Biochem Pharmacol 6: 229. 40. Fuglebakk E, Echave J, Reuter N (2012) Measuring and comparing struc- 26. Spoel DVD, Lindahl E, Hess B, Groenhof G, Mark AE, et al. (2005) GRO- tural fluctuation patterns in large protein datasets. Bioinformatics 28: MACS: fast, flexible, and free. J Comput Chem 26: 1701-1718. 2431-2440. 27. Vanommeslaeghe K, Hatcher E, Acharya C, Kundu S, Zhong S, et al. 41. Prashant S, Durgesh K, Vijay K V, Parul Y, Abhilash J, et al. (2019) Com- (2010) CHARMM general force field: A force field for drug‐like mole- putational approach to study the synthesis of noscapine and potential of cules compatible with the CHARMM all‐atom additive biological force stereoisomers against nsP3 protease of CHIKV. Heliyon 5: e02795. fields. J Comput Chem 31: 671-690. 42. Vijay KV, Prashant S, Vinod K, Kamlesh K, Rajan P, et al. (2019) Pyrrolo- 28. Zoete V, Cuendet MA, Grosdidier A, Michielin O (2011) SwissParam: thiazolones as Potential Inhibitors for the nsP2B‐nsP3 Protease of Dengue a fast force field generation tool for small organic molecules. Journal of Virus and Their Mechanism of Synthesis. ChemistrySelect 4: 9410-9419. Computational Chemistry 32: 2359-2368. 43. Durgesh K, Kamlesh K, Abhilash J, Prashant S (2019) Development of a 29. Kumari R (2014) g_mmpbsa - A GROMACS tool for high-throughput theoretical model for the inhibition of nsP3 protease of Chikungunya virus MM-PBSA calculations. J Chem Inf Model 54: 1951-1962. using pyranooxazoles. Journal of Biomolecular and Structural Dynamics: 1-17. 30. Baker NA, Sept D, Joseph S, Holst MJ, McCammon AJ (2001) Electro- 44. Prashant S, Vijay KV, Nidhi S, Reetu, Kamlesh K, et al. (2019) A model to statics of nanosystems: Application to microtubules and the ribosome. Na- study the inhibition of nsP2B-nsP3 protease of dengue virus with imidaz- tional Academy of Sciences 98: 10037-10041. ole, oxazole, triazole thiadiazole and thiazolidine based scaffolds. Heliyon 5: e02124. 31. Vishvakarma VK, Patel R, Kumari K, Singh P (2017) Interaction between Bovine Serum Albumin and Gemini Surfactants using Molecular Docking 45. Durgesh K, Prashant S, Abhilash J, Vinod K, Kamlesh K, et al. (2019) A Characterization. Inf Sci Lett 3: 1-9. Theoretical Model to Study the Interaction of Erythro‐Noscapines with nsP3 protease of Chikungunya Virus Chemistry Select 4: 4892-4900. 32. https://www.cambridgemedchemconsulting.com/resources/ADME/

• Page 11 of 14 •

Supplimentory Tables

Ligand Total Energy EVDW EH-bonding EElect 51 -122.622 -100.795 -21.827 0 1 -91.0327 -77.5735 -13.4592 0 52 -114.65 -105.714 -8.93619 0 2 -109.384 -91.9854 -17.3988 0 53 -112.345 -101.326 -11.0192 0 3 -97.4526 -85.0978 -12.3548 0 54 -108.775 -91.8634 -16.9117 0 4 -117.05 -94.7327 -22.3173 0 55 -110.929 -92.4983 -18.4306 0 5 -122.715 -89.8135 -30.8757 -2.02593 56 -112.913 -102.64 -10.2734 0 6 -96.8304 -67.0809 -29.7495 0 57 -116.385 -103.495 -12.8901 0 7 -98.741 -81.2675 -17.4735 0 58 -115.883 -105.239 -10.644 0 8 -100.539 -82.5791 -17.9601 0 59 -108.636 -98.431 -10.2054 0 9 -108.578 -93.9345 -14.6432 0 60 -106.02 -92.5062 -13.5141 0 10 -107.478 -89.345 -18.1333 0 61 -105.844 -99.9673 -5.87681 0 11 -106.607 -91.8395 -14.3133 -0.45392 62 -109.801 -98.291 -11.5098 0 12 -91.975 -75.9856 -15.9894 0 63 -112.478 -88.3698 -22.5875 -1.52103 13 -112.295 -83.9152 -28.3796 0 64 -116.888 -89.5448 -27.3432 0 14 -91.8925 -91.6397 -0.2528 0 65 -116.913 -90.9412 -25.9715 0 15 -95.0031 -70.1665 -24.8366 0 66 -104.905 -93.8031 -11.1022 0 16 -104.709 -79.8509 -24.8586 0 67 -108.323 -78.5167 -29.8065 0 17 -97.5492 -87.2843 -10.2649 0 68 -122.707 -104.85 -17.8573 0 18 -97.2666 -75.1413 -22.1253 0 69 -120.698 -107.083 -13.1938 -0.42128 19 -110.528 -94.5839 -15.9438 0 70 -104.602 -88.7713 -15.8304 0 20 -103.258 -85.9429 -17.3153 0 71 -118.534 -102.436 -16.0977 0 21 -109.314 -93.1677 -16.1459 0 72 -109.145 -90.5591 -18.5855 0 22 -113.565 -87.7818 -25.7827 0 73 -100.979 -88.2069 -12.7722 0 23 -112.745 -94.6705 -18.0743 0 74 -105.235 -84.2613 -20.9739 0 24 -107.463 -104.963 -2.5 0 75 -104.475 -95.7479 -8.72682 0 25 -112.129 -72.67 -39.3759 -0.0833 76 -101.485 -87.2427 -14.242 0 26 -107.937 -83.6258 -24.3114 0 77 -110.902 -94.2911 -16.6108 0 27 -120.229 -98.5119 -21.7173 0 78 -113.671 -106.78 -6.89113 0 28 -102.892 -91.5625 -11.3293 0 79 -118.162 -91.5669 -26.5951 0 29 -115.049 -107.605 -7.44435 0 80 -111.439 -98.9876 -12.4519 0 30 -110.748 -98.2447 -12.5038 0 81 -102.866 -94.1186 -8.74766 0 31 -119.084 -98.0764 -22.1457 1.13783 82 -119.63 -74.5102 -43.7129 -1.40655 32 -114.935 -98.4504 -16.4849 0 83 -115 -87.7448 -27.255 0 33 -112.175 -99.6831 -12.4918 0 84 -111.556 -85.2251 -26.3308 0 34 -105.387 -85.7027 -19.6847 0 85 -111.459 -92.2666 -19.1923 0 35 -106.499 -88.5389 -17.9598 0 86 -113.328 -100.878 -12.4498 0 36 -110.126 -95.444 -14.6819 0 87 -108.786 -80.5001 -28.2856 0 37 -113.429 -103.794 -9.63506 0 88 -121.07 -80.7958 -40.764 0.489526 38 -117.84 -103.04 -14.7997 0 89 -112.954 -102.484 -10.4704 0 39 -107.051 -96.5513 -10.5 0 90 -113.741 -101.624 -12.1168 0 40 -111.64 -96.197 -15.4426 0 91 -108.255 -87.3362 -20.9189 0 41 -112.313 -92.4805 -19.8324 0 92 -105.69 -97.7104 -7.97946 0 42 -107.339 -99.6366 -7.70222 0 93 -115.311 -107.016 -8.29515 0 43 -104.554 -86.3956 -18.1585 0 94 -116.222 -92.1833 -24.0391 0 44 -121.294 -111.479 -9.81486 0 95 -124.896 -107.441 -17.4548 0 45 -121.13 -99.5557 -21.5738 0 96 -110.211 -103.04 -7.1707 0 46 -95.8639 -89.139 -6.72494 0 97 -133.413 -93.6378 -39.7757 0 47 -105.94 -95.7799 -10.1601 0 98 -119.453 -102.307 -17.1458 0 48 -126.677 -98.9796 -27.6971 0 99 -109.284 -102.616 -6.66826 0 49 -116.817 -107.396 -9.42086 0 100 -122.101 -107.862 -14.2386 0 50 -121.996 -91.9938 -32.3341 2.33227 101 -126.298 -95.8055 -28.7223 -1.77013

• Page 12 of 14 •

102 -105.874 -89.2876 -16.5864 0 34 -6.21 0.000318 -5.82 4.4 103 -109.055 -94.4301 -14.6251 0 35 -5.95 0.000561 -6.03 4.33 104 -114.627 -99.8451 -14.7815 0 36 -5.73 0.000965 -6.41 4.39 105 -114.19 -101.451 -12.7391 0 37 -5.97 0.000572 -6.24 4.4 106 -113.111 -104.256 -8.85546 0 38 -5.51 0.00156 -6.56 3.9 107 -115.01 -91.0375 -22.433 -1.53905 39 -6.58 0.000158 -6.45 4.21 108 -113.338 -90.8417 -22.4964 0 40 -5.6 0.00129 -6.56 4.07 109 -129.175 -115.069 -14.1058 0 41 -5.19 0.00336 -7.02 4 110 -118.448 -104.196 -14.2518 0 42 -6.49 0.00019 -6.38 4.4 111 -105.64 -93.8759 -11.7642 0 43 -6.1 0.000424 -6.23 4.38 112 -105.872 -92.0203 -13.8518 0 44 -5.72 0.00102 -6.61 3.88 113 -117.354 -93.8579 -23.4964 0 45 -5.3 0.00254 -6.79 3.79 114 -115.442 -97.6821 -17.7595 0 46 -6.24 0.000299 -5.97 4.5 115 -114.528 -104.288 -10.2402 0 47 -6.56 0.000155 -6.2 4.45 116 -119.553 -101.666 -17.887 0 48 -5.31 0.00267 -7.14 4 Table SA: Docking score of the noscapines. 49 -6.26 0.000305 -6.23 3.98 50 -4.09 0.0435 -8.43 3.86 51 -5.4 0.00207 -6.76 4 52 -5.61 0.00129 -6.69 3.95 Ligand Log S Solubility Log Kp Log P 53 -6.21 0.000318 -5.82 4.4 1 -4.14 0.0298 -6.9 3.59 54 -5.59 0.000561 -6.03 4.5 2 -3.68 0.0919 -7.71 4 55 -5.73 0.000965 -6.41 4.08 3 -4.99 0.00522 -7.07 4 56 -5.97 0.000572 -6.24 4.77 4 -4.6 0.0115 -6.92 3.86 57 -5.51 0.00156 -6.56 3.92 5 -4.22 0.0755 -7.29 3.01 58 -6.58 0.000158 -6.45 3.96 6 -3.8 0.0683 -7.47 3.31 59 -5.6 0.00129 -6.56 4.19 7 -4.74 0.00809 -6.66 3.71 60 -5.19 0.00336 -7.02 4.19 8 -5.06 0.00432 -6.89 3.82 61 -6.49 0.00019 -6.38 4.51 9 -3.81 0.0724 -7.83 3.6 62 -6.1 0.000424 -6.23 4.35 10 -4.76 0.00831 -6.92 3.65 63 -5.72 0.00102 -6.61 3.72 11 -1.88 6 -9.12 3.08 64 -5.3 0.00254 -6.79 3.82 12 -3.89 0.0056 -7.45 3.08 65 -6.24 0.000299 -5.97 4.5 13 -3.98 0.0487 -7.38 3.61 66 -5.56 0.000155 -6.2 4.58 14 -4.7 0.00886 -6.51 3.88 67 -5.31 0.00267 -7.14 3.99 15 -4.45 0.0153 -6.73 3.81 68 -6.26 0.000305 -6.23 4.22 16 -4.23 0.0264 -7.01 3.92 69 -4.09 0.0435 -8.43 3.67 17 -4.47 0.0155 -6.93 3.87 70 -5.4 0.00207 -6.76 3.87 18 -4.01 0.0424 -7.25 3.37 71 -5.91 0.00129 -6.69 4.09 19 -4.11 0.0355 -7.38 3.61 72 -6.21 0.000318 -5.82 4.37 20 -4.1 0.0346 -7.25 3.45 73 -5.95 0.000561 -6.03 4.42 21 -5.65 0.0011 -6.2 4.11 74 -5.73 0.000965 -6.41 4.37 22 -5.19 0.00336 -7.02 4.3 75 -5.97 0.000572 -6.24 4.68 23 -6.49 0.00019 -6.38 4.37 76 -5.51 0.00156 -6.56 3.86 24 -6.1 0.000424 -6.23 4.28 77 -6.58 0.000158 -6.45 4.32 25 -5.72 0.00102 -6.61 3.86 78 -5.6 0.00129 -6.56 4.15 26 -5.3 0.00254 -6.79 3.85 79 -4.73 0.0102 -7.83 4.11 27 -6.24 0.000299 -5.97 4.4 80 -7.33 0.0000315 -6.55 4.54 28 -6.56 0.000155 -6.2 4.5 81 -6.57 0.000159 -6.25 4.39 29 -5.31 0.00267 -7.14 4.04 82 -5.8 0.000915 -7 3.5 30 -6.26 0.000305 -6.23 4.11 83 -4.95 0.00579 -7.36 3.63 31 -4.9 0.0431 -8.43 3.24 84 -6.84 0.0000799 -5.74 4.49 32 -5.4 0.00207 -6.76 3.84 85 -7.48 0.00001216 -6.19 4.55 33 -5.61 0.00129 -6.69 4.09 86 -4.99 0.00624 -8.07 3.93

• Page 13 of 14 •

87 -6.88 0.0000819 -6.25 4.03 88 -3.98 0.0612 -9.03 2.95 89 -5.15 0.00389 -7.31 3.64 90 -5.58 0.0015 -7.17 3.81 91 -6.78 0.00009 -5.42 4.58 92 -6.26 0.000286 -5.86 4.37 93 -5.81 0.000842 -6.61 4.32 94 -6.3 0.000289 -6.27 4.91 95 -5.38 0.00217 -6.9 3.87 96 -7.51 0.0000218 -6.7 3.99 97 -5.56 0.00148 -6.91 3.9 98 -4.73 0.0102 -7.83 4.29 99 -7.33 0.0000315 -6.5 5.02 100 -6.57 0.000159 -6.25 4.41 101 -5.8 0.000915 -7 3.18 102 -4.95 0.00579 -7.36 3.45 103 -6.84 0.0000799 -7.36 3.45 104 -7.48 0.0000216 -6.19 4.78 105 -4.99 0.00624 -8.07 3.75 106 -6.88 0.0000819 -6.25 4.08 107 -3.98 0.0612 -9.03 3.12 108 -5.15 0.00389 -7.31 3.71 109 -5.58 0.0015 -7.17 4 110 -6.78 0.00009 -5.42 4.81 111 -6.26 0.000286 -5.86 4.64 112 -5.81 0.000842 -6.61 4.51 113 -6.3 0.0002.89 -6.27 5 114 -7.51 0.0000218 -6.7 4.03 115 -5.56 0.00148 -6.91 3.9 Table SB: ADME properties of the designed noscapines.

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