Molecular Docking Studies of Phytocompounds from Withania Sominifera Against IL6 and JAK3 Targets in Rheumatoid Arthritis

Rheumatoid arthritis is an auto immune disorder and inammatory that decreases the life expectancy and it is characterized by persistent joint pain. This disease is not curable but can be maintained. People have been using a plant known as Withania somnifera or Indian Ginseng for its medicinal properties. The phytocompounds of this plant shows anti-inammatory potentials against Rheumatoid Arthritis cytokine pathogenesis. In our study we are going to use IL6 and JAK3 protein as targets for docking and compounds of Withania somnifera as ligands.


Introduction
Rheumatoid arthritis (RA) is a life-long autoimmune illness characterized by chronic in ammation that causing joint pain and destroying cartilage and bone [1]. 1-3 % of the population is affected with this disorder all over the globe. Women over the age of 55 are 2-3 times more prone than men to Rheumatoid Arthritis. RA is symptomized by swelling, persisting pain, stiffness, Tenderness and ares or excaherbation especially in synovial joints (Knee, Shoulders, Elbows, Wrist, knuckles and feet) in the body. Synovial joints are present as a connection between the bones/cartilage that move against each other; normally 1-3 cells thick layer present at the site of synovial joint where synovial cavity is found. Synovial cavity is lled with a thick, dense, and viscous uid known as Synovia or synovial uid, that acts as a lubricant in bones friction or movement [7].
Synovial membrane or layer is hypertrophied in RA, and layer becomes (8-10 cells) thick joint space reduces because of that [2]. In this disorder, synovial area is heavily penetrated with own immune cells and in ammatory cells including lymphocytes (T and B cells) neutrophils, mast cells, eosinophils, and macrophages). New blood vessels are formed near the in amed area. Because of this auto-immune mechanism, cartilage and bones are destroyed and eroded by the hypertrophied synovium and it leads deformity and dysfunction of bones and cartilage [8]. No permanent treatment is available, although many disease-modifying anti-rheumatic drugs (DMARDs), steroidal drugs, and non-steroidal antiin ammatory drugs are available in the market with severe to low side -effects and high cost [9].
India and its neighbouring countries (China,Bangladesh,Nepal and sri Lanka) have been using plants since ages for its surprising medicinal properties and therapeutic effects [3]. And "Withania somnifera" is one of the most used medicinal plants in India, Sri-Lanka and Bangladesh for its therapeutic potential. Withania somnifera is also known as the Indian Ginseng in local language and Ashwagandha in Hindi. It belongs to the family Solanaceae and found in hot and dried region areas. It is a shrub plant [10]. And has many medicinal properties including Anti-in ammatory, Anti-bacterial, Aphrodisiac, Anti-cancer, Adaptogenic, Anti-depression, and Anti-stress properties. Dried roots have been using for therapeutic use, studies show that leaves also have medicinal effects [4]. This plant has many phyto-compounds including Phenolics, Alkaloids, Steroidal and other compounds [5]. The disease "Rheumatoid Arthritis" is characterized by In ammation and several cytokine pathways I/II on the molecular level [6]. So, we chose this Withania's 18 compounds for its anti-in ammatory potential as ligands. And Interleukin-6(IL6) and Page 3/9 JAK3 as the target proteins involved in Rheumatoid Arthritis Pathogenesis. So, we used 18 ligands for molecular docking (This is a blind docking) against IL6 and JAK3 targets in Rheumatoid Arthritis.

Materials And Methods
Protein Preparation and Retrieval IL6[11] and JAK3[12] Protein's crystallographic structures were taken from RCSB Protein Data Bank (https://www.rcsb.org/) in pdb format. IL6 PDB ID is (1ALU) and JAK3 ID is (3LXK). IL6 is of 186 sequence length and JAK3 is of 327 sequence length. Both of the protein were edited suitably by using Molegro Molecular Viewer software by removing ligands and water molecules. For visualization, we used chimera software.

Ligand Preparation
Compounds we had selected as ligands were retrieved from (https://pubchem.ncbi.nlm.nih.gov/) by using their pubchem ID. And then we used Open Babel software to convert ligands from sdf format to mol2 format because the online docking software that we were using only accept in pdb or mol2 format.
Compounds names and their pubchem ID are given in Table.1. Withasomniferol C also showed high binding energy in range of -8.7 to -7.7 with Interleukin-6 target. Compounds Chlorogenic Acid, Quercetin, and Kaempferol also showed high binding energy in range of -8.1 to -7.8 with JAK3 target. More than 20 different poses were obtained for every complex structure out of which top energy level complex is selected due to high binding a nity along with more hydrogen bonds. The binding energy along with no of hydrogen bonds, binding pockets and Intra-residue H-bonds Distance is shown in Table.2 and Table.3 of medicinal compound complexes with target Interleukin-6 and JAK3.  Complex of target ligand docking