Virtual Screening of IL-6 Inhibitors for Idiopathic Arthritis

Juvenile idiopathic arthritis (JIA) is a heterogeneous disease characterized by the arthritis of unknown origin and IL6 is a known target for JIA. 20 known inhibitors towards IL-6 were screened and Methotrexate (MTX) having PubChem ID: 126941 showed high binding capacity with the receptor IL-6. The similarity searching with this compound gave 269 virtual screened compounds. The said screening presented 269 possible drugs having structural similarity to Methotrexate. The docking studies of the screened drugs separated the compound having PubChem CID: 122677576 (re-rank value of -140.262). Toxicity and interaction profile validated this compound for having a better affinity with the target protein. Conclusively, this study shows that according to ADMET profile and BOILED-Egg plot, the compound (PubChem CID: 122677576) obtained from Virtual Screen could be the best drug in future during the prevention of juvenile idiopathic arthritis. In the current study, the drug CID: 122677576 is a potent candidate for treating JIA. The pharmacophore study revealed that the drug CID: 122677576 is a non-inhibitor of CYP450 microsomal enzymes and was found to be non-toxic, similar to the established drug Methotrexate (CID: 126941). It has a lower LD50 value of 2.6698mol/kg as compared to the established compound having LD50 value as 23.4955mol/kg. Moreover, the compound was found to be non-carcinogenic.


Background:
Juvenile idiopathic arthritis is a group of diseases characterized by arthritis of unknown origin, which is seen before age of 16 years [1]. It is a group of diseases that encompasses all forms of arthritis beginning before the age of 16 years and stays for more than 6 weeks. 250,000 children in the United States alone are estimated to be affected by Juvenile Idiopathic Arthritis (JIA). Short and long term disabilities are also caused by JIA. There are three major types of presentation of the term JIA, which encompasses a heterogeneous group of diseases: (a) oligo-arthritis, (b) polyarthritis and (c) systemic-onset JIA (SoJIA) [2]. One subspace, systemic JIA (sJIA), is the additional presence of exhausting fever, serositis (inflammation of the serous tissues of the body), fugitive rash, lymph adenopathy (disease of the lymph nodes, in which they are abnormal in size) and hepatosplenomegaly (a disorder where both the liver and spleen swell beyond their normal size). Osteoporosis, growth retardation, systemic amyloidosis and macrophage activation syndrome are some severe complications. These are observed more frequently in patients with the longstanding disease than in other JIA subclasses. Patients of sJIA have a range of other prominent features, which includes marked elevation of erythrocyte sedimentation rate (ESR) and C-reactive complement protein (CRP), leucocytes with high neutrophil counts ©Biomedical Informatics (2019) 122 and thrombocytosis. It has a yearly occurence of 2-20 cases per 100,000 population in countries having a high income.
IL-6 has a pleiotropic effect on inflammation, immune response, and hematopoiesis. It is a soluble mediator. Human IL-6 is composed of 212 amino acids, including a 28-amino-acid signal peptide. Its gene has been mapped to chromosome 7p21. Although the core protein is ∼20kDa, glycosylation accounts for the size of 21-26 kDa of natural IL-6. Disproportioned continual synthesis of IL-6 has a pathological impact on chronic inflammation and autoimmunity. A humanized anti-IL-6 receptor Tocilizumab is an antibody was developed for the same reason [3]. It is evident that the production of IL-6 is particularly high in sJIA. By a variant of the gene encoding IL-6 in a significant fraction of patients, it can be genetically determined. A significant increase in soluble IL-6 receptor (sIL-6R) concentrations, in addition to the increase in serum IL-6, has been determined in the JIA patients. Different levels of IL-6 correlate with the activity of disease, pattern of fever and platelet counts, which indicates an important role of IL-6 in the pathogenesis of sJIA. A humanized anti-human IL-6 receptor antibody MRA (Tocilizumab) of kappa-IgG1 subclass is developed collaboratively by Osaka University and Chugai Pharmaceutical Company Ltd (Japan). MRA is humanized as the complementary determining regions of a mouse anti-human IL-6 receptor monoclonal antibody are grafted onto human IgG1 by using recombinant DNA technology [3].

Methodology: Selection of inhibitors
For the determination of inhibitors in the present examination, the existing inhibitors of IL-6 against Juvenile Idiopathic Arthritis were chosen from various literature studies. The accessibility of preexisting inhibitors is 20, chosen to promote perceptions (Table1).

Drug-Drug comparative study:
The unnamed complex structure was redeemed from an established drug docking result and was simply imported. It was cleaned by removing all ligand constraints and eventually imported the bestposed drug and exported as best drug docked file in SDF format. Again the complex structure was retrieved from virtual docking result and the procedure was repeated. The excel sheet was prepared to check all the affinities, hydrogen interaction and high re-rank score.

Results and Discussion
The docking studies of complete pre-established 20 drugs were performed and it was found that the compound Methotrexate (CID: 126941) is the best-established compound. Table [2] as the compound having lowest energy with -105.677 as re-rank score shows the higher affinity score directed towards our target protein and has the great affinity properties like molecular weight 454.447 g/mol, 5 hydrogen bond donor and 12 hydrogen bond acceptor, topological polar surface area 211 A^2 and log value of-1.8.Thus, the compound reveals the superior inhibitory affinity over protein IL-6.The docking studies were resulted in Table 2.   Further, the similarity search for this inhibitor displayed 269 compounds. Table 3 contains the docking result of the top 10 of 269 virtual screened compounds. The compound with PubChem CID122677576 with higher affinity is selected. This compound has a molecular weight of 496.528 g/mol, 5 hydrogen bond donor and 12 hydrogen bond acceptor, a topological surface area of 208 A^2and a log P value is -0.7. Similarly, among all 269virtual screened compounds and 20 pre-established compounds, the drug with PubChem CID: 122677576 have much potential inhibition against juvenile idiopathic arthritis over the target protein IL-6.
The compound with PubChem Id: 122677576 prove more efficient than the already established drug Methotrexate and it is shown in Table 4. External ligand interactions and Protein-ligand interactions, along with total energy evidently shows the stable interactions of the compound (PDB ID: 122677576) with the target protein IL-6. Moreover, steric energy for the established compound also indicates the better stability of the compound obtained after the virtual screening studies.
Pharmacophore study is done for the better clarification of the interactive attributes of the compound, which are important for the biological functioning of that compound. The pharmacophore mapping gives spatial essential systematic features of the molecular interaction with a specific target receptor apart from the method of molecular docking. Pharmacophore studies provide accurate query on the optimum interaction with suitable target annotations and represent the aligned poses of the molecule and help us to find the high interaction mode between target protein and compound. Owing to admirable affinity and good interaction profile of virtual screened compound (PubChemCID 122677576) over the most effective pre-established compound Methotrexate (PubChem CID 126941), the study carries forwarded to the pharmacophore results. Pharmacophore mapping results in the positive intensities of electrostatics as well as varying intensities and the charges in aromatic interaction, respectively. The pharmacophoric feature includes the study of different types of interactions, such as H-bond interactions, electrostatic interactions, hydrophobic interactions, aromatic interactions, and van der Waals (vdW) interactions. These interactions are shown below.

Figure 2 represents Van der Waals interaction between residues
interactions of virtual screened compound (PubChem CID-122677576) present in the cavity of IL-6 protein. The interaction in the figure represents the residues with ligands displayed in green to be van der Waals interaction and the residues displayed in pink to be electrostatic interactions. The figure depicts four hydrogen bond interaction; two with Ser72, one with Thr120 and Ser122 residues represented by a green dotted line. Consequently, the van der Waal interactions were also shown by the residues Thr124, Asp92, Leu90, Lys45, Val93, Phe155, Trp115 and Val175 were circled with green. Furthermore, the interaction of pi-pi between the Pro121 and the compound are depicted in orange color.    ©Biomedical Informatics (2019)   The further study of bioactivity in the profile of excretion and toxicity is almost equivalent. In reference to carcinogens, they both show the same carcinogenicity. The mutagenicity of the compound can be predicted by ADMET regression toxicity study. Both the compounds in the properties of Rat Acute Toxicity are nearly equal to each other. The possibility of having higher toxicity than these two molecules is shown in Table  6. Further, the study of bioactivity in the profile of excretion and toxicity is similar.

Comparative ADMET profile study of the compounds and the control:
The comparative ADMET profile for the inhibitors was predicted based on the parameters such as Blood-Brain Barrier (BBB), Human Intestinal Absorption (HIA), AMES Toxicity and LD50. The established compound Methotrexate with PubChem CID: 126941 and the best virtual screened compound with PubChem CID-122677576 along with other top 2 compounds Salsalate having PubChemCID5161 and the compound having PubChemCID102026478 was preferred for comparative ADMET studies. These four compounds were graphically estimated using R-programming as shown in Figure 4. The parameters: BBB, HIA, AMES toxicity and LD50 procured from the admetSAR database. The compounds were tabulated according to their predicted values and properties. So, according to the graph as well as Table 7 among all the four compounds the values of BBB are similar in both the compound Methotrexate (PubChem CID 126941) and the compound (PubChem ID-102026478). Human Intestinal Absorption (HIA) value is similar in both the compound Methotrexate (PubChem CID 126941) and the compound (PubChem ID-102026478). LD50 in rat is lower in Salsalate (PubChem ID-5161). The compound with PubChem ID-122677576 has higher HIA. Methotrexate has an equivalent property in LD50 in rat and Ames toxicity in comparison with compound (PubChem Id -102026478), which also shows the regression in toxicity.

Conclusion:
Inhibition of IL-6 interactions has now surfaced as an important drug target against Juvenile Idiopathic Arthritis. We study 20 preestablished inhibitors of IL-6 which are associated with JIA using molecular docking analysis, an inhibitor of IL-6 for JIA among presently effective inhibitor Methotrexate and virtual screened compound Pub CID: 122677576. We foresee Methotrexate and CID: 122677576 are structurally cognant. However, Methotrexate is a good inhibitor, but compound 122677576 has the lowest re-rank score and can emerge as an important drug in the treatment of disease in the future ahead.