Research Article INTRODUCTION. R. Priyadharsini 1 *, T. Kalaivani 1, M. Keerthana 1, U. Logeshwari 1, S. Nivedhitha 1, S. Bhuvaneswari 2 ABSTRACT

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1 Research Article In silico identification of newer potential spleen tyrosine kinase and protein arginine deiminase 4 inhibitors as potent antirheumatoid arthritic agents R. Priyadharsini 1 *, T. Kalaivani 1, M. Keerthana 1, U. Logeshwari 1, S. Nivedhitha 1, S. Bhuvaneswari 2 ABSTRACT Background: Over the past few decades, rheumatoid arthritis (RA) has become a major global health problem which accentuates the need for newer drugs. Spleen tyrosine kinase (SYK) is a cytoplasmic, non-receptor tyrosine kinase involved in signal transduction in a variety of cell types and plays an important role in uncontrolled growth of tumor cells and protein arginine deiminase 4 (PAD4), a calcium-dependent enzyme, mainly expressed in granulocytes and the only isoform localized in the cell nucleus which catalyzes the conversion of peptidyl-arginine to peptidyl-citrulline. Aim: This Prompted us to design newer YK and PAD4 inhibitors as efficient therapeutic drugs for the treatment of rheumatic arthritis. Materials and Methods: Based on the common pharmacophoric features for the inhibition of SYK and PAD4, a series of leads were designed using computational methods. A virtual library consisting of newly designed 60 molecules as SYK and PAD4 inhibitors of 30 each, has been constructed using features such as hydrogen bond acceptor, hydrogen bond donor, and hydrophobic features. The binding mechanism of newly designed ligands with target enzyme SYK and PAD4 was studied using Arguslab 4.1 and AutoDock tools Conculsion: The designed compounds were subjected and filtered by applying ADMET properties, Lipinski rule of five, molecular docking and found to be more effective SYK and PAD4inhibitors for treatment of RA. KEY WORDS: ADMET properties, Docking studies, Lipinski rule of five, Protein arginine deiminase 4 inhibitors, Rheumatoid arthritis, Spleen tyrosine kinase inhibitors INTRODUCTION Rheumatoid arthritis (RA) is a long-lasting autoimmune disorder that primarily affects joints. It typically results in warm, swollen, and painful joints. Most commonly, the wrist and hands are involved, with the same joints typically involved on both sides of the body. This may also result in a low red blood cell count, inflammation around the lungs, and inflammation around the heart. Fever and low energy may also be present. [1,2] The disease can begin at any age, but the commencement is most frequent in woman older than 65 years. [3] It has been projected that 55 70% of patients with RA have progressive disease, resulting in joint destruction, and disability. Thus, there is a need for the newer anti-ra drugs that selectively inhibit the target enzyme that is responsible for Access this article online Website: prsolutions.info ISSN: causing RA. Spleen tyrosine kinase (SYK) is a cytoplasmic, non-receptor tyrosine kinase involved in signal transduction in a variety of cell types and plays an important role in uncontrolled growth of tumor cells and Protein arginine deiminase 4 (PAD4), a calcium-dependent enzyme, mainly expressed in granulocytes and the only isoform localized in the cell nucleus which catalyzes the conversion of peptidyl-arginine to peptidylcitrulline. [4-8] Selective inhibition of SYK and PAD4 which may be promising targets for B-cell inhibition in treatment of RA. Hence, computeraided drug design approaches were used to identify potent and novel SYK and PAD4 inhibitors which can cause inhibition of SYK and PAD4. By reviewing efficient journals and research articles, common pharmacophoric features responsible for inhibiting SYK and PAD4 are identified. A series of leads is designed that selectively modulates the activity of SYK and PAD4 for exhibiting anti- RA activity. A virtual library consisting of newly designed 30 molecules each of SYK and PAD4 1 Department of Pharmaceutical Chemistry, College of Pharmacy, Madras Medical College, Chennai, Tamil Nadu, India, 2 KR College of Arts and Science, KR Nagar, Kovilpatti, India *Corresponding author:dr. R. Priyadarsini,College of Pharmacy, Madras Medical College, Chennai , Tamil Nadu, India. rpdharsinimpharm@yahoo.co.in Received on: ; Revised on: ; Accepted on: Journal of Pharmacy Research Vol 12 Issue

2 inhibitors was constructed, and docking studies were performed using molecular docking tools such as Arguslab 4.1 and AutoDock tools Drug likeliness properties were performed for all the newly designed SYK and PAD4 inhibitors using different online software such as Lipinski s rule of five, Osiris online software, and molinspiration online database. MATERIALS AND METHODS Selection of Target The targets creating the greatest enthusiasm at this time for the treatment of RA and inflammatory diseases include Janus-associated kinase, SYK, phosphodiesterase-4, Bruton s tyrosine kinase, PAD4, and phosphatidylinositol-3 kinase. [9,10] Protein selection is carried out using protein data bank (PDB).Using Q-site finder software tool, receptors with low resolution were selected and further evaluated by its resolution value (R value) and optimized using crystal ligand interaction details and found SYK and PAD4 are the best targets for the current study. Crystal structure of SYK (PDB code: 5C27) with R value-2.15 and PAD4 (PDB code: 4NFU) with R value-2.21 was selected for the docking study. Pharmacophore Identification By reviewing the efficient journals and articles, compounds containing chemical features such as hydrogen bond acceptor (HBA), hydrogen bond donor (HBD), and hydrophobic (HYP) features were used to screen knowledge database, and a virtual scaffold library consisting of newly designed 30 molecules each as SYK and PAD4 inhibitors has been constructed [Table 1]. [11] properties are filtered and considered for further molecular docking [Tables 2 and 3]. Docking Studies In the drug designing process, the molecular docking is a method which predicts the preferred orientation, the most appropriate conformation and interaction of each ligand at the active site of the target. [12] Docking studies were performed using AutoDock tools and Arguslab 4.1. For molecular docking calculation, crystal structure of protein, 5C27 for SYK, and 4NFU for PAD4 with optimal R value was selected as the targets. The binding site was identified based on the volume occupied by cocrystal ligand in the protein. The optimized ligands were docked into the active sites of the target. Based on the docking score obtained, the affinity, molecular interaction, and the hydrogen bond interaction between the ligand and the active site residue of the target protein are studied, and the best-docked poses were selected [Tables 4-7]. RESULTS AND DISCUSSION In the search of new and potent SYK and PAD4 inhibitors as anti-ra agents, a virtual scaffold library of 60 molecules was constructed using Chemsketch by reviewing efficient articles and journals and based on features such as HBA, HBD, and HYP features. Virtual Scaffold Library of SYK Inhibitors Lead Optimization The newly designed ligands were subjected to molecular docking, ADME properties, Lipinski s rule of five, and toxicity prediction. Through this, the newly generated ligands are filtered and refined that constitutes optimization of leads. From the molecular docking studies, the ligands which have best docking score were selected. During ADME investigation, the compounds were checked for low permeation across blood-brain barrier, optimal solubility, good absorption, noninhibition of enzymes, and non-hepatotoxicity. Lipinski s rule of five estimates the absorption and intestinal permeability of the compound. The toxicological properties such as teratogenicity, mutagenicity, irritant effect, and reproductive effect of designed ligands were predicted using Osiris online software. The compounds having better-estimated activity value and drug-like Journal of Pharmacy Research Vol 12 Issue

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4 Virtual Scaffold Library of PAD4 Inhibitors Journal of Pharmacy Research Vol 12 Issue

5 Lead Optimization The newly designed ligands were subjected to molecular docking, ADME properties, Lipinski s rule of five, and toxicity prediction. Through this, the newly generated ligands are filtered and refined that constitutes optimization of leads. The compounds having better-estimated activity value and drug-like properties are filtered and considered for further molecular docking. Docking Studies In the drug designing process, the molecular docking is a method which predicts the preferred orientation, the most appropriate conformation and interaction of each ligand at the active site of the target. [12] Docking studies were performed using AutoDock tools and Arguslab 4.1. Based on the docking results, all the newly designed ligands were categorized as highly active, moderately active, and low active hits as below. CONCLUSION Our in silico identification approach has revealed that all newly designed the SYK inhibitors and PAD4 inhibitors have a crucial role in the 126 Journal of Pharmacy Research Vol 12 Issue

6 inhibition of B-cells for the treatment of RA. By reviewing the literature, the important chemical features which can inhibit the activity of SYK and PAD4 were identified, and three-dimensional structural query of newer heterocyclic ligands was screened to retrieve new potent SYK inhibitors and PAD4 inhibitors. Lipinski s rule of five and ADMET properties screening assisted us to discard the non-drug like compounds. Furthermore, the screened drug-like compounds were identified and were further subjected to molecular docking study. Finally, molecular dynamics simulation Table 1: Molecular fragments used in construction of library of SYK and PAD4 inhibitors HBD HBA HYP tail Imidazole, thiadiazole Benzimidazole, aminothiazole, Phenolic OH, Aniline, Alkylamines, oxazole, guanidine, Morpholine C=O of aliphatic and aromatic amides, C=O of aromatic ketones, C=O of diamide Phenyl, biphenyl, diazole, Pyridine, Triazole, Quinazoline, tolyl, dimethylbenzene HBD: Hydrogen bond donor, HBA: Hydrogen bond acceptor, HYP: Hydrophobic, SYK: Spleen tyrosine kinase, PAD4: Protein arginine deiminase 4 Table 2: Lipinski rule of five reports for the SYK ligands COMPOUND LOG P MOL.WT TPSA nohnh non Number of rotatable bonds Number of violations SI SI SI SI SI SI SI SI SI SI SI SI SI SI SI SI SI SI SI SI SI SI SI SI SI SI SI SI SI SI SYK: Spleen tyrosine kinase Table 3: Lipinski rule of five reports for the PAD4 ligands COMPOUND LOG P MOL.WT TPSA nohnh non Number of rotatable bonds Number of violations PI PI PI PI PI PI PI PI PI PI PI PI (Cond) Journal of Pharmacy Research Vol 12 Issue

7 Table 3: (Condinude) COMPOUND LOG P MOL.WT TPSA nohnh non Number of rotatable bonds Number of violations PI PI PI PI PI PI PI PI PI PI PI PI PI PI PI PI PI PI PAD4: Protein arginine deiminase 4 Table 4: Docking scores of SYK inhibitors using Arguslab 4.1 and AutoDock tools LIGAND AUTODOCK DOCKING SCORE ARGUS DOCKING SCORE SI SI SI SI SI SI SI SI SI SI SI SI SI SI SI SI SI SI SI SI SI SI SI SI SI SI SI SI SI SI SYK: Spleen tyrosine kinase Table 5: Docking scores of PAD4 inhibitors using Arguslab 4.1 and AutoDock tools LIGAND AUTODOCK DOCKING SCORE ARGUS DOCKING SCORE PI PI PI PI PI PI PI PI PI PI PI ,4433 PI PI PI PI PI PI PI PI PI PI PI PI PI PI PI PI PI PI PI PAD4: Protein arginine deiminase 4 was employed to study the stability of docked conformation and to investigate the binding interaction in details. Hence, we propose that the final hit compounds such as SI 2, SI 3, SI 5 -SI 9, SI 13, SI 20, SI 22 -SI 25, SI 27 -SI 30 and PI 1, PI 13, PI 14, PI 16-19, PI 21 25, PI 27, PI 29, PI 30 as a possible virtual leads to design novel SYK inhibitors and PAD4 inhibitors which can be synthesized and screened for in vitro and in vivo anti- RA activity further in future. ACKNOWLEDGMENT We consider this, as an opportunity to express our sincere thanks to The Dean and Our Principal, 128 Journal of Pharmacy Research Vol 12 Issue

8 Table 6: Docking results of SYK and PAD4 inhibitors using Arguslab 4.1 Receptors Highly active (>12) Moderately active (9 12) Low active (<9) SYK inhibitors SI 26 SI 2, SI 3, SI 5 SI 9, SI 13, SI 20, SI 22 SI 25, SI 27 SI 30 SI 1, SI 4, SI 10 SI 12, SI 14 SI 19, SI 21 PAD4 inhibitors PI 17, PI 18, PI 21 PI 23 PI 25, PI 27, PI 29, PI 30 P I1 PI 16, PI 19, PI 20, PI 22, PI 26, PI 28 SYK: Spleen tyrosine kinase, PAD4: Protein arginine deiminase 4 Table 7: Docking results of SYK and PAD4 inhibitors using AutoDock tools Receptors Highly active (>8) Moderately active (7 8) Low active (<7) SYK inhibitors SI 25 SI 27, SI 29, SI 30 SI 3, SI 7, SI 8, SI 20, SI 23, SI 24, SI 28 SI 1, SI 2, SI 4 SI 6, SI 9 SI 19, SI 21, SI22. PAD4 inhibitors PI 26 PI 30 P I1, PI 13, PI 14, PI 16, PI 18, PI 19, PI 22, PI 23, PI 24 PI 2 PI 12, PI 15, PI 17, PI 20, PI 21, PI 25. SYK: Spleen tyrosine kinase, PAD4: Protein arginine deiminase 4 College of Pharmacy, Madras Medical College, Chennai REFERENCES 1. Majithia V, Geraci SA. Rheumatoid arthritis: Diagnosis and management. Am J Med 2007;120: Handout on Health: Rheumatoid Arthritis. National Institute of Arthritis and Musculoskeletal and Skin Diseases; Available from: [Last retrieved on 2015 Jul 02]. 3. Scott DL, Wolfe F, Huizinga TW. Rheumatoid arthritis. Lancet 2010;376: Jones J, Causey C, Knuckley B, Slack-Noyes JL, Thompson PR. Protein arginine deiminase 4 (PAD4): Current understanding and future therapeutic potential. Curr Opin Drug Discov Dev 2009;12: Vossenaar ER, Zendman AJ, van Venrooij WJ, Pruijn GJ. PAD, a growing family of citrullinating enzymesenes, features and involvement in disease. Bioessays 2003;25: Nakashima K, Hagiwara T, Ishigami A, Nagata S, Asaga H, Kuramoto M, et al. Molecular characterization of peptidylarginine deiminase in HL-60 cells induced by retinoic acid and 1alpha,25-dihydroxyvitamin D(3). Biol Chem 1999;274: Hagiwara T, Nakashima K, Hirano H, Senshu T, Yamada M. Deimination of arginine residues in nucleophosmin/r23 and histones in HL-60 granulocytes. Biochem Biophys Res Commun 2002;290: Nakashima K, Hagiwara T, Yamada MJ. Nuclear localization of peptidylarginine deiminase V and histone deimination in granulocytes. Biol Chem 2002;277: Reproduced with Permission from Elsevier. This Table was Published in Signal Transduction; p. 2. Available from: [Last retrieved on 2013 Jan 07]. 10. Ehrlich P. Present status of chemotherapy. Ber Dtsch Chem Ges 1909;42: Miller MW, Howe HL Jr, Kasubick RV, English AR. Synthesis of 2-[2-chloro- phenyl]-4,5-diphenyl imidazoles. J Med Chem 1970;13: Kitchen DB, Decorenz H, Fur JR, Bajorath J. Docking and scoring in virtual screening for drug discovery; methods and application. Nat Rev Drug Discov 2014;3: Journal of Pharmacy Research Vol 12 Issue