Construction of a novel quinoxaline as a new class of Nrf2 activator

Background The transcription factor Nuclear factor erythroid-2-related factor 2 (NRF2) and its principal repressive regulator, Kelch-like ECH-associated protein 1 (KEAP1), are perilous in the regulation of inflammation, as well as maintenance of homeostasis. Thus, NRF2 activation is involved in cytoprotection against many inflammatory disorders. N′-Nicotinoylquinoxaline-2-carbohdyrazide (NQC) was structurally designed by the combination of important pharmacophoric features of bioactive compounds reported in the literature. Methods NQC was synthesised and characterised using spectroscopic techniques. The compound was tested for its anti-inflammatory effect using Lipopolysaccharide from Escherichia coli (LPSEc) induced inflammation in mouse macrophages (RAW 264.7 cells). The effect of NQC on inflammatory cytokines was measured using enzyme-linked immune sorbent assay (ELISA). The Nrf2 activity of the compound NQC was determined using ‘Keap1:Nrf2 Inhibitor Screening Assay Kit’. To obtain the insights on NQC’s activity on Nrf2, molecular docking studies were performed using Schrödinger suite. The metabolic stability of NQC was determined using mouse, rat and human microsomes. Results NQC was found to be non-toxic at the dose of 50 µM on RAW 264.7 cells. NQC showed potent anti-inflammatory effect in an in vitro model of LPSEc stimulated murine macrophages (RAW 264.7 cells) with an IC50 value 26.13 ± 1.17 µM. NQC dose-dependently down-regulated the pro-inflammatory cytokines [interleukin (IL)-1β (13.27 ± 2.37 μM), IL-6 (10.13 ± 0.58 μM) and tumor necrosis factor (TNF)-α] (14.41 ± 1.83 μM); and inflammatory mediator, prostaglandin E2 (PGE2) with IC50 values, 15.23 ± 0.91 µM. Molecular docking studies confirmed the favourable binding of NQC at Kelch domain of Keap-1. It disrupts the Nrf2 interaction with kelch domain of keap 1 and its IC50 value was 4.21 ± 0.89 µM. The metabolic stability studies of NQC in human, rat and mouse liver microsomes revealed that it is quite stable with half-life values; 63.30 ± 1.73, 52.23 ± 0.81, 24.55 ± 1.13 min; microsomal intrinsic clearance values; 1.14 ± 0.31, 1.39 ± 0.87 and 2.96 ± 0.34 µL/min/g liver; respectively. It is observed that rat has comparable metabolic profile with human, thus, rat could be used as an in vivo model for prediction of pharmacokinetics and metabolism profiles of NQC in human. Conclusion NQC is a new class of NRF2 activator with potent in vitro anti-inflammatory activity and good metabolic stability.


Introduction
Lead generation is one of the key challenge in drug discovery and development process, and it is the search of chemical compounds that will be therapeutically effective against a disease. Fragment-based drug discovery (FBDD) is a technique often used to generate structures known as 'leads' [1]. From the literature search, a number of key structures were discovered to be potent anti-inflammatory agents. This includes quinoxaline [2], hydrazine [3] and pyridine. Thus, in this study, we aim to synthesised these fragments into a lead and to investigate its antiinflammatory activity via Nrf2 activation.
Quinoxaline, fused ring of benzene and pyrazine, is one of the important class of heterocyclic compound having diverse biological activities [4][5][6][7][8][9][10] and present as integral part of diverse bioactive compounds and pharmaceuticals [11][12][13][14][15]. Hydrazines are nitrogen-nitrogen bond containing compounds which exhibit remarkable biological activities [16][17][18][19]. Pyridines possess many biological activities and present as an integral part of many medicinal compounds [20]. Quinoxalines substituted at second position are reported to possess remarkable biological activities [21]. Therefore, combining quinoxaline, hydrazine and pyridine moieties into one molecule as represented in Fig. 1 was believed to be a potential template for the synthesis of novel class of bioactive compounds.
Many reports in the literature demonstrated Nuclear factor erythroid 2 -related factor 2 (Nrf2) activation contributes to diverse biological activities by regulating the Kelch-like ECH-associated protein (Keap1)/Nrf2 signaling pathway [22][23][24]. Therefore Nrf2 activation has emerged as an attractive therapeutic approach to develop new classes of drugs as therapeutic treatment for a myriad of diseases and this includes inflammation, chronic multiple sclerosis, kidney disease, pulmonary fibrosis, cancer and chronic obstructive pulmonary disease (COPD) [25][26][27][28][29][30][31][32]. In literature, two well-studied Nrf2 activators were reported to be sulforaphane (a derived isothiocyanate from broccoli; and dimethyl fumarate, a new drug for the treatment of multiple sclerosis (Fig. 2). However, these two produce side effects due to the presence of strong electrophilic functional groups and hence covalently react with other proteins [33].
Another natural Nrf2 activator is oleanic triterpenoid bardoxolone imidazole. It was withdrawn from phase III clinical trials for patients diagnosed with type 2 diabetes and chronic kidney disease because of adverse cardiovascular events [34] that might be due to its covalent binding with key residues in protein. From these observations, it is postulated that the development of reversible covalent and non-covalent Nrf2 activators would be an ideal strategy to develop selective and safe Nrf2 activators [24,[35][36][37][38]. The above said efforts resulted in five series of compounds; tetrahydroisoquinoline, carbazone, naphthalene, thiopyrimidine and urea derivatives; that shown to activate Nrf2 whose representative structures were shown in Fig. 3.
It is noted that the proposed template and reported Nrf2 activators have few structural similarities. Therefore, in this study we synthesised the template compound, tested its efficacy in reversing the nitric oxide (NO) production, levels of pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α), level of inflammatory mediator (Prostaglandin E 2 (PGE 2 )) in LPS stimulated RAW 264.7 cells. Its' efficacy in disrupting the interaction between kelch domain of Keap 1 and Nrf2 was determined and in silico molecular docking studies were performed to acquire insights on its molecular interactions at the interface of Nrf2/Keap1. The metabolic stability profile of the compound was determined using liver microsomes (human, rat and mouse). We thought of proceeding with the synthesis of analogues only if the template compound showed promising anti-inflammatory and metabolic stability and therefore, the analogues were not synthesised.  Fig. 4.

Cell culture
Murine macrophages, cell line: RAW 264.7 was procured from ATCC, USA. The macrophage cells were grown and cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine serum (FBS) and 1% PenStrep and incubated in a 5% CO 2 at 37 °C incubator. Cells from passages 10 to 20 were used for subsequent experiments.

Cell viability
A calorimetric agent (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) (MTT) was used to as an assay to determine cell viability after being exposed to NQC. RAW 264.7 cells were seeded in a 96-well microplate at 3 × 10 5 cells/well and were allowed to grow and adhere for 24 h in a 37 °C, 5% CO 2 incubator. NQC was dissolved in DMSO and diluted with PBS to produce the required test concentrations (100, 50, 25, 12.5, 6.25 and 3.125 µM). The negative control is 0.1% (v/v) DMSO, which is the maximum effective concentration of DMSO in a well. The cells were treated with either NQC or 0.1% (v/v) DMSO for 4 h followed by 1 µg/mL LPSEc and incubated further for 20 h. MTT solution was added at 100 µL per well under dark condition and the cells were incubated further for 4 h. Upon completion of incubation, the contents in all wells were aspirated and DMSO was added at 100 µL per well. The absorbance was measured The cells without any treatment was used as a control. MTT assay was performed in triplicate. The percent cell viability is calculated using the following equation:

Determination of nitric oxide (NO)
RAW 264.7 cells were seeded in a 96-well microplate at 3 × 10 5 cells/well with complete media. The cells were allowed grow and adhere for 24 h. The cells were treated with NQC (50, 25, 12.5, 6.25 and 3.125 µM) and negative control (0.1% v/v DMSO in water) for 4 h prior to LPSEc (1 µg/mL) and allowed to incubate for 20 h at 37 °C, 5% CO 2 incubator. Nitrite exist as a stable metabolite of nitric oxide, thus nitrite present in the supernatant was quantitatively measured as a chemical marker of nitric oxide (NO) production using Griess reagent (Promega, USA; 0.1% naphthylethylenediamine dihydrochloride and 1% sulfanilamide in 2.5% phosphoric acid). This assay was carried out following the manufacturer's protocol where 100 µL of cell supernatant sample was incubated with 100 µL of Griess reagent at room temperature for 10 min and the absorbance at 540 nm was measured using Molecular Devices Spectramax M3 Multi-Mode microplate reader.

Percent Cell Viabilty =
Absorbance of Vehicle or TD − 1 treated group well Absorbance of Control well ×100.
A standard curve of sodium nitrite was plotted and used as a reference for extrapolation to quantify the amount of nitrite present.

Determination of IL-1β, IL-6, PGE 2-and TNF-α
Production of IL-1β, IL-6, PGE 2− and TNF-α in RAW 264.7 cells were quantitatively measured using an enzyme-linked immunosorbent assay (ELISA). The cells were incubated with 0.1% DMSO or NQC for 4 h, then stimulated with LPSEc, 1 µg/mL, for 20 h. The production of pro-inflammatory cytokines (IL-1β, IL-6) and TNF-α were determined using commercial ELISA kit (RayBio) according to the manufacturer's instructions in product insert. The absorbance was measured at 540 nm.
All the experiments were performed in triplicate.

Nrf2 activation assay
The Nrf2 activation by NQC was determined using 'Keap1:Nrf2 Inhibitor Screening Assay Kit' (BPS Bioscience, USA) following the instructions in product insert. The NQC was dissolved in DMSO and diluted with assay buffer provided in the kit to produce the concentrations range, 100 µM to 1 nM. The fluorescence of the solutions in each well of 96-well plate was measured at λ ex 485 nm and λ em 530 nm. The dose-response curve was constructed plotting percent reduction in Kepa1-Nrf2 binding activity versus log concentration of NQC. From dose-response curve IC 50 value was calculated.

Molecular docking studies
To acquire molecular insights on the binding mode of NQC in Keap-1 binding site, molecular docking studies were carried out. Schrödinger small-molecule drug discovery suite 2018-2 was used to perform in silico docking studies. The crystal structure (PDB code: 5CGJ) used was sourced from 'Research Collaboratory for Structural Bioinformatics Protein Data Bank' (PDB) (http://www. pdb.org). The 'protein preparation wizard' was used to prepare the protein for molecular docking studies. This was followed by grid generation using 'receptor grid generation wizard' with default settings. The chemical structure of NQC was sketched in Maestro and prepared for docking using 'ligprep' wizard. Docking between the low energy conformation of NQC onto the binding site was performed by selecting the extra precision (XP) mode where protein-ligand structural motifs and water desolvation energy are incorporated to account for scoring function of binding free energy. The binding poses and affinity was further confirmed where "Induced Fit Docking' (IFD) module was applied to NQC into the binding site. IFD module is docking protocol "Glide" and "Prime" where ligand flexibility and receptor flexibility are accounted for. GlideSP feature generates softened-potential docking protocol and was used to generate an initial number of 20 poses in the primary stage of IFD. Residues of the protein below 5.0 Å of ligand for each pose were refined. Docking of ligands from this is run again using GlideSP to generate poses. The top-ranked dock poses' binding free energy (ΔG°) were calculated using the module Prime/Molecular Mechanics-Generalized Born Surface Area (MM-GBSA) with default settings.

Metabolic stability assessment of NQC
A stock solution (SS) of NQC at 1 mM was prepared using 100% dimethyl sulfoxide. Working solution of 10 μM was further prepared by dilution of SS with 25% ACN and 50 mM tris-HCl buffer (pH 7.4). In a 96-well plate, NQC (effective concentration:1 µM) was incubated with phosphate buffer (pH7.4) and 0.5 mg/mL of liver microsomes (human, rat and mouse) at 37 °C in an incubation. Each NQC incubation with liver microsomes was performed in triplicates. The microsomal metabolic reaction was initiated by the addition of co-factor, NADPH (5 mM). Samples following the allotted time-point was drawn and quenched into a solution of acetonitrile containing 50 ng/mL internal standard. The sample was analysed by a developed HPLC method that has been validated for lower limit of quantification, linearity, precision, selectivity and accuracy according to FDA guidance [39].
Half-life of NQC was calculated using the equation: The in vitro microsomal intrinsic clearance (CL int ) was calculated based on the equation below:

Statistical analysis
The results were analysed and data are reported as mean ± standard deviation. The significance of the values was determined using student t test. Statistical significant difference was defined as P-value less than 0.05 (P < 0.05). All the statistical analyses were performed using Micosoft Excel.

Characterisation of NQC
NQC was successfully synthesised and the chemical compound was spectroscopically elucidated using 1 H Nuclear Magnetic Resonance (NMR), 13 C Nuclear Magnetic Resonance, Fourier transform-infrared spectroscopy (FT-IR), and mass spectroscopy (MS). The overall schematic representation is as shown in Fig. 3. The target compound was synthesised and tested for purity by thin layer chromatography (TLC) using hexane and ethyl acetate (1:1) as the solvent system, and the R f value calculated was 0.32. Structure of the compound was elucidated and confirmed using 1 H NMR, 13 Cl int = 0.693 in vitro t (1/2) × ml incubation mg microsome × 52.5 mg microsome g liver × 52.5 mg liver kg bw .

Cell viability
The main purpose of this assay is to determine the NQC concentration range which is non-toxic to RAW 264.7 cells. The cytotoxicity effects of NQC (at a range of concentration) against RAW 264.7 cells stimulated with LPSEc was performed using calorimetric MTT assay. Based on the results depicted in Fig. 5a, NQC do not show cytotoxic effects on RAW 264.7 cells at the tested concentrations of 3.125-50 µM (P > 0.05), while NQC at 100 µM shows significant toxicity (P < 0.05). Therefore, based on these results, NQC with concentrations between 3.125 and 50 µM was used for the subsequent experiments.

Effect of NQC on NO production
NO production as an inflammatory mediator in the cell culture medium of LPSEc stimulated RAW 264.7 cells is deduced using the Griess reaction [40]. The results are shown in Fig. 5b. The concentration of NO increased significantly in the LPSEc stimulated RAW 264.7 cells (LPSEc + DMSO) (P < 0.05) when compared to normal control (no LPSEc + DMSO). This shows that NO production was induced by LPSEc in RAW 264.7 cells. NQC between 3.125 and 50 µM significantly reversed the production of NO in LPSEc induced RAW 264.7 cells in a dose-dependent manner (P < 0.05). It was reported that NO is involved in the regulation of multistage processes found in inflammation-particularly in the initial stages of inflammatory cells transmigrating to inflammation sites [41]. Therefore, NQC is found to be potent anti-inflammatory agent with an IC 50 value of 26.13 ± 1.17 µM.

Effect of NQC on pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) and PGE 2
Inflammatory mediators and cytokines highly mediates inflammatory response especially in the initial stages of inflammation. These includes PGE 2 , NO; and proinflammatory cytokines namely IL-1β, IL-6 and TNF-α [42]. Subsequently, these cytokines will be responsible as upstream mediators where other inflammatory cytokines will be further stimulated and emitted, and ultimately leads to critical clinical symptoms of pain and its related immune disorders [43]. Therefore, the effects of NQC on inhibiting the production of IL-1β, IL-6, PGE 2-and TNF-α in the LPSEc stimulated RAW 264.7 cells were quantitatively measured using ELISA. As depicted in Fig. 5c- In the current study, it was found that NQC dramatically reduced the high levels of inflammatory mediators, NO radicals and PGE 2 , and pro-inflammatory cytokines of IL-1β, IL-6 and TNF-α stimulated by LPSEc (Fig. 1bf ). In conclusion, it is believed that the NQC exerts desirable anti-inflammatory activity via suppression of excessive inflammatory mediators and inflammatory factors produced during inflammation.

Effect of NQC on Nrf2 activation
Nrf2 is a transcription factor protein that contributes to the anti-inflammatory process by playing the role as an upstream regulator through binding with antioxidant response element (ARE); and is responsible in recruiting inflammatory cells and regulating gene expression. Anti-inflammatory gene expression and inhibition of inflammatory progression is regulated by Keap1/Nrf2/ ARE signaling pathway. Under normal homeostasis conditions, Nrf2 remains ubiquitously bounded to the cytoskeletal protein Keap1. Due to Nrf2 activation, an anti-inflammatory response ensues. Thus, we determined the activity of NQC in inhibiting Nrf2-Keap1 interaction. The NQC showed dose-dependent activity in inhibiting Nrf2-Keap1interaction as shown in Fig. 2 and its IC 50 value was 4.21 ± 0.89 µM.

Molecular docking studies
The primary docking of NQC with the binding pocket of 5CGJ was performed using Glide XP protocol where the flexibility of both ligand and receptor were disregarded. Here, favorable binding score of NQC at − 4.806 kcal/ mol and ligand efficiency of − 0.218 kcal/mol were observed. Induced Fit Docking protocol was performed for NQC into the binding pocket where the introduction of residue flexibility is below 5 Å from the ligand. The IFD protocol presented highly negative IFD score (− 647.102 kcal/mol) indicating it is a favorable binding energy; and highly negative XP G score (− 7.051 kcal/ mol). The binding free energy of the NQC was computed using MM-GBSA approach to further confirm it's binding. The binding free energies of NQC was − 47.801 kcal/ mol. The collective results give a confidence that NQC binds strongly in the binding site of Nrf2/Keap1 interface, which could be responsible for inhibition of Nrf2/ Keap1 interaction. The 3D-and 2D-interaction diagram of NQC in the binding site of Nrf2/Keap1 interface is shown in Fig. 6b

Metabolic stability studies
A calibration curve of peak area against the nominal concentrations of NQC was prepared using linear leastsquare regression model. The lower limit of quantitation (LLOQ) was determined to be 0.01 µM. The signal:noise ratio was found to be greater than 10. The linearity of the assay was r > 0.995 when assessed on the concentration range of 0.01-2.00 µM which is above the desired level of > 0.990. The assessment on intra-and inter-day precision and accuracy were carried out on three successive days using three concentration (0.02, 0.5 and 1.6 µM) where the intra-and inter-day precisions (RSD %) was less than 9.00% and the accuracy (RE %) fell in the range of − 2.12 to 5.23%. The determination of NQC extraction recovery at three concentrations, 0.02, 0.5 and 1.6 µM was carried out and was achieved by comparing the peak area of extracted analyte in six replications (n = 6) with blank samples of post-extraction. The mean recovery was observed to be > 85.95%. The residues with light green shade denote the amino acids that form hydrophobic interactions with TD-1. c The 3D-interaction diagram showing interaction between TD-1 and key amino acid residues of binding pocket of 5CGJ In vitro microsomal metabolic stability assay using human, rat and mouse liver microsomes were performed to estimate half-life and microsomal intrinsic clearance of NQC at an effective concentration of 1 µM. The ratio of NQC to internal standard over time for human, rat and mouse liver microsomes are shown in Fig. 7 and the coefficient of correlations were more than 0.97. The observe in vitro T 1/2 (63.30 ± 1.73, 52.33 ± 0.81 and 24.55 ± 0.34 min in human, rat and mouse respectively) as shown in Table 1, indicated that NQC presented faster clearance in mouse liver microsomes upon stimulation by the co-factor, NADPH. Whereas NQC presented moderate microsomal metabolism in rat and human liver microsomes. Correspondingly, the observed in vitro CL int (1.14 ± 0.31, 1.39 ± 0.87 and 2.96 ± 0.34 µL/min/g liver in human, rat and mouse respectively) revealed that NQC displayed moderate stability (< 5 mL/min/g liver) in human, rat and mouse. From this in vitro assay, rat seems to have a closer value to human than that of mouse. From these findings, rat would be a suitable animal model for in vivo pharmacokinetics and metabolism of NQC in human.