Exploring the natural chemiome to target interleukin-6 receptor (IL-6R) cytokines: an atomic scale investigation for novel rheumatoid arthritis drug discovery

Natural compounds are a gold mine for treating rheumatoid arthritis (RA). The etiology of this disease is linked to inflammation, where cytokines play an important role. Strategies have been drafted for targeting cytokines as a therapeutic option in patients with RA. Inhibiting cytokines with natural compounds has become a major focus for the development of drugs to treat RA. Here, a structure-based drug design approach was employed to identify novel leads to target the interleukin 6 receptor (IL-6R). A total of 48,531 compounds of natural origin were screened. Two of these compounds were shortlisted for molecular docking simulation and tested for inhibiting gp130 dimerization in human macrophages. The results show that Lead5 (CID5329098) significantly inhibited the release of gp130 in a dose-dependent manner, similar to the inhibitory effect of LMT-28 (p<0.005). This study provides an atomic scale outcome of a single natural compound that can be developed into a RA drug.


INTRODUCTION
Rheumatoid arthritis (RA) is a chronic inflammatory disease primarily associated with the joints that leads to cartilage and bone damage (Aletaha et al., 2010;Firestein, 2003).Inflammation is a part of the natural defense mechanism, but any anomaly can lead to various pathologies including RA (Khandpur et al., 2013;Lazzerini et al., 2014).Over the last decade, researchers have investigated the role of cytokines in the etiology of RA and have subsequently opted for cytokines as a therapeutic target (Burmester, Feist, Dörner, 2014;Siebert et al., 2015).Inhibiting cytokines has become a major focus in the development of new drugs to treat RA (Wu et al., 2016;Zhou et al., 2014).
In our study, we used the natural chemiome, as in interbioscreen (IBS) database of 48,531 compounds, known to be the world's largest collection of natural compounds, their derivatives, and mimetics.The database was used to target the crystallographic structures of the IL-6R extracellular domain (PDBID:1N26) (Varghese et al., 2002).In-silico techniques were used for an atomic scale investigation into possible IL-6R bDMARDs; virtual screening and molecular docking simulations were conducted to find a lead compound for in vitro analysis of its anti-inflammatory activity.

Preparation of the proteins and the natural chemiome dataset
The atomic coordinates for the IL-6R structure to develop novel bDMRDs were taken from the Protein Databank (PDB ID: 1N26).The coordinates were energy minimized using the Swiss PDB viewer (SPDBv).The root mean square deviation was monitored using the GROMOS96 43B1 force field (Van Gunsteren, 1996).A total of 45,000 natural compounds from the IBS database were used to target the IL-6R extracellular domain.

Virtual screening drug likeliness prediction
The ArgusLab suite was used to virtually screen the 48,531 compounds (Mark, 2010).Less than 1% (323) of the compounds were shortlisted based on their binding energy (∆G) calculations.A value of −8 Kcal/mol was set as the cut off to get the initial subset of compounds.The selected compounds were further limited by subjecting them to rules set by Lipinski (2004).The Lipinski rule of five (RO5) parameters gave us ten compounds for further analysis.

Molecular docking analysis
A structure-based drug designing method was used, and the AutoDock 4.2 tool was employed for the molecular docking study (Morris et al., 2009).This tool calculates energy values by classifying energies as internal energy and torsional free energy.Internal energy is the sum of desolvation energy, hydrogen bonding energy, van der Walls energy, and electrostatic energy.Lamarckian genetic algorithm (GA) default parameters were used to calculate ∆G for each shortlisted compound.A grid box (40×40×40 A°) was built around the IL-6R extracellular domains.The energy values generated and the binding mode with IL-6R were used to limit the list to two compounds.

Molecular visualization & molecular docking analysis
The two complexes were studied using the Pymol visualization tool (DeLano, 2002) and Discovery Studio (Studio, 2013).

Cell culture
THP-1 monocytes were procured from the American Type Culture Collection (Manassas, VA, USA) and cultured in RPMI (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA) and 100 U/ml pen-strep (Gibco).The cells were grown under standard culture conditions at 37°C and 5% CO 2 in a humidified incubator.Monocytes for macrophage differentiation were cultured with 100 ng/ ml macrophage colony stimulating factor (CSF).Stock solutions of 100 mM of the compounds were prepared in RPMI and diluted to different concentrations.Cells were stimulated with 10 ng/ml lipopolysaccharide (LPS) (Sigma-Aldrich) to stimulate TNF-α production.

Cell proliferation assay
The MTT assay is a standard test to measure cell viability and is based on the conversion of MTT to formazan crystals by active mitochondrial dehydrogenases.Briefly, a cell suspension containing about 3 × 10 4 cells was plated into each well of a 96-well plate and allowed to attach for 24 h.Lead 3 and Lead 5 prepared in DMEM were added to the wells at concentrations of 10, 20, 40, and 60, 80, and 100 μM for 24 h.After treatment, 100 μl (0.1 mg/ml) of MTT (Sigma Aldrich) solution was added to each well (dissolved in PBS).After a 4 h incubation at 37 °C in the dark, the solution was removed and 100 µl DMSO was added to solubilize the formazan.Absorbance was measured at 570 nm using an automated microplate reader (Biotek Instruments, Winooski, VT, USA).The results are depicted as percentages relative to the controls.The percentage proliferation inhibition rate was calculated as=(1 -ODsample/ODcontrol) × 100%.
gp130 Enzyme-linked immunosorbent assay (ELISA) The supernatant was harvested following Lead 3 and Lead 5 stimulation of human macrophages with LPS.gp130 concentrations (BD Pharmingen, San Diego, CA, USA) were determined by ELISA, following the manufacturer's instructions.Absorbance was read at 450 nM on an ELISA plate reader (Biotek) using the in-built software program.

Statistical analysis
The statistical analysis was carried out by one-way analysis of variance, and p-values of 0.01-0.001were considered significant.Data are expressed as mean ± SD of three independent experiments.

Interleukin-6/JAK/STAT pathway inhibition by IBS database
Chemiomes are comprised of natural compounds, their derivatives, and mimetics that inhibit the IL-6/ JAK/STAT pathway by inhibiting the IL-6R (Figure 1).IL-6R in complex with IL-6 results in homodimerization of gp130 and signal transduction through the JAK/ STAT pathway.Thus, blocking formation of the IL-6 and IL-6R complex by targeting the IL-6R extracellular domains resulted in a novel IL-6R inhibitor by virtual screening, drug-likeliness, docking and molecular dynamics simulation studies.Figure 2 depicts this methodology.Virtual screening helped limit the number of compounds from the 48,531 natural products to 323 using a limiting bias of ∆G of −8 Kcal/mol.Figure 3 shows the X-ray crystallographic structure of one of the natural compounds with IL-6R.

Drug likeliness feature of the top compounds
To limit the focus on compounds that could be promising for further development, we checked each compound for drug-likeliness.Drug-likeliness of the shortlisted compounds was defined by mutagenic and carcinogenic properties, RO5, and total polar surface area.The RO5 properties included the number of hydrogen bond donors (HBD), number of hydrogen bond acceptors (HBA), molecular weight (MW), and the octanol/water partition coefficient (logP).The permissible range is HBD≤5, HBA≤10, MW≤500 Dalton and clog p≤5.Table I shows the drug-likeliness properties of the top five compounds.The drug-likeliness values of our top compounds are values expected of typical drugs.

Intricate atomic scale details of the interactions in the top five lead compounds
The AutoDock tool was used for the molecular docking simulations, and the top binding poses based on ∆G were taken for further analysis.Each binding pose was studied using Discovery Studio, and the default parameters were used to calculate all possible interactions.The results generated are shown in Table II, where the binding energy, binding pocket, and number of hydrogen bonds formed are listed.The lead1-IL-6R complex had a binding energy of −4.43 kcal/mol and their interaction is shown in Figure 4, where lead1 formed three conventional hydrogen bonds with LYS154, PHE155, and GLU114 in the IL-6R GTP binding sites.The binding pocket of lead1 was comprised of the following amino acids LYS154, PHE155, GLY116, SER152, TRP115, GLN99, SER101, CYS113, GLU114, VAL112, SER156, LEU100, and CYS157.The binding energy was third least among five, and the number of interactions was the least among all shortlisted compounds.

Inhibiting dimerization of gp130 by human macrophages
gp130 is a key factor in a variety of inflammatory diseases.We evaluated its expression in the culture supernatants of human macrophages (which constitute the major cytokine-producing cells in highly relevant inflammatory disorders) treated with non-cytotoxic doses of Lead3 (CID176870) and Lead5 (CID5329098), considering the role of IL-6 and IL-6R regulating gp130 dimerization, to determine the suitable dosage of Lead3 and Lead5 compounds that result in less cytotoxicity.Macrophages (obtained with treatment of THP-1 monocytes with CSF) were treated with different concentrations of these compounds (10-200 μM) for 24 h, and viability was measured by the MTT assay.The results demonstrated that treatment with concentrations such as 10-100 µM exhibited the least cytotoxic effects.To test the effect of these lead compounds, macrophages were treated with the indicated concentrations of 0.001-100 µM of these two compounds with LMT-28, a selective inhibitor of IL-6R.Treating LPS stimulated macrophages with Lead5 (CID5329098) significantly inhibited release of gp130 in a dose dependent manner; similar to the inhibitory effect of LMT-28 (Figure 5).Thus, these data suggest a role inhibiting IL-6R that, in turn, downregulates gp130 and, hence, could be used a therapeutic agent to target RA.

CONCLUSION
Computer aided drug design was used to explore the natural chemiome for treating RA.The compounds have long has been described as a gold mine for arthritis treatment and studies have explored their ability to inhibit cytokines.Natural compounds have long been reported to have anti-rheumatoid effects, and this has given us an opportunity to look into them as IL-6R inhibitors.The molecular docking simulations studies revealed potential lead compounds, and some of these compounds are showing great potential as drugs for treatment of RA in preliminary in vitro investigations.

FIGURE 3 -
FIGURE 3 -The original crystallographic structure of IL-6R used for the study.

FIGURE 2 -
FIGURE 2 -Method used for screening of natural compounds against IL-6R.

FIGURE 1 -
FIGURE 1 -Protein sub networks of IL-6R with other important proteins involved in arthritis.

FIGURE 4 -
FIGURE 4 -Two dimensional representations of the top five compounds showing the physical interaction with the binding site of IL-6R.

FIGURE 5 -
FIGURE 5 -Effect of top two lead compounds on gp130 dimerization in macrophages.Cells treated with indicated concentrations of these two compounds LMT-28 or left untreated.The culture supernatants were then removed and assayed for gp130 by ELISA.Data represented as mean ±SD of results obtained from at least three independent experiments.**p < 0.01; ***p < 0.001 compared to control.

TABLE I -
Details of the top five compounds

TABLE II -
Auto Dock analysis of top five lead natural products