STRUCTURE-BASED MULTITARGETED MOLECULAR DOCKING ANALYSIS OF PYRAZOLE-CONDENSED HETEROCYCLICS AGAINST LUNG CANCER

Objective: The significant drawbacks of chemotherapy are that it destroys healthy cells, resulting in adverse effects. Hence, there is a need to adopt new techniques to develop cancer-specific chemicals that target the molecular pathways in a non-toxic fashion. This study aims to screen pyrazole-condensed heterocyclics for their anticancer activities and analyse their enzyme inhibitory potentials EGFR, ALK, VEGFR and TNKS receptors. Methods: The structures of the compounds were confirmed by IR, NMR and Mass spectral studies. The in silico techniques applied in this study were molecular docking and pharmacophore modeling to analyse the protein-ligand interactions, as they have a significant role in drug discovery. Drug-likeness properties were assessed by the Lipinski rule of five and ADMET properties. Anticancer activity was performed by in vitro MTT assay on lung cancer cell lines. Results: The results confirm that all the synthesised pyrazole derivatives interacted well with the selected targets showing docking scores above-5 kcal/mol. Pyrazole 2e interacted well with all the four lung cancer targets with its stable binding mode and was found to be potent as per the in vitro reports, followed by compounds 3d and 2d. Pharmacophore modeling exposed the responsible features responsible for the anticancer action. ADMET properties reported that all the compounds were found to have properties within the standard limit. The activity spectra of the pyrazoles predicted that pyrazolopyridines (2a-2e) are more effective against specific receptors such as EGFR, ALK and Tankyrase. Conclusion: Thus, this study suggests that the synthesised pyrazole derivatives can be further investigated to validate their enzyme inhibitory potentials by in vivo studies.


INTRODUCTION
Lung cancer is one of the leading causes of cancer mortality in men and women, [1,2] responsible for 1.6 million deaths. Non-small-cell lung cancers (NSCLCs), including large-cell carcinoma, adenocarcinoma, and squamous cell carcinoma, contribute approximately 80-85% of lung cancer.
The major shortcoming of lung cancer chemotherapy is that it causes damages to normal cells, causing surplus adverse effects. Therefore targeted therapies [3] are needed to target only cancer cells, avoiding injuries to the healthy cells. One of the novel methods adopted in lung cancer therapy is developing cancer-specific compounds that can attack the molecular signalling pathways, thus creating non-toxic substances. The significant targets of paramount importance for lung cancers are EGFR (Epidermal growth factor receptor) [4,5] ALK (Anaplastic lymphoma tyrosine kinase) [6,7] BRAF (v-raf murine sarcoma viral oncogene homolog B1) [8,9] VEGFR receptors (Vascular endothelial growth factor) [10,11], and Wnt signalling pathway [12].
The EGFR receptor is recognised as a significant anticancer target. It belongs to the ErbB (epidermal growth factor) receptor tyrosine kinase family and is expressed at high levels on the surface of some cancer cells. The inhibition of EGFR plays a crucial role in angiogenesis, tumour suppression, and metastasis [13].
In anaplastic non-Hodgkin's lymphoma, the ALK gene was first described as a driver mutation. Dysregulated ALK expression is now an identified driver mutation in nearly twenty different human malignancies. The dysregulated ALK expression is now recognised as the driver mutation, including 4-9% of NSCLC [14].
One of the critical mediators promoting the angiogenesis process is VEGFR, as it has a prominent role in maintaining the vascular supply within the tumour. Its increased levels are a confirmatory factor in diverse human cancers, including NSCLC [15]. The Wnt signalling pathway is another potential target for lung cancer. Effective pharmacological inhibitors of the Wnt pathway have only recently become available. The tankyrase (TNKS), a poly-ADP-ribose polymerase (PARP) enzyme, was the critical mediator of Wnt signalling by the screens for small molecular antagonists of the Wnt pathway. Hence, using the targets mentioned above as partial agonists/antagonists can show promising treatment strategies [16].
Nitrogen-containing heterocycle-pyrazole has a vital role in the development of cancer therapies. The anticancer activity of these compounds is by the inhibition of different types of proteins, receptors and enzymes, which has a crucial role in cell division. Condensed pyrazole rings such as pyrazolopyrimidines, pyrazolopyridines and pyranopyrazole are known for their anticancer properties [20], and the available drugs with these core moieties are depicted in fig. 1.
An extension of previous works on pyrazole scaffolds [21,22] and in silico studies [23,24], we have performed an analysis to screen the inhibitory potency of synthesized pyrazole fused derivatives on various targets EGFR, ALK, VEGFR and TNKS by employing molecular docking and pharmacophore modelling techniques.

MATERIALS AND METHODS
Most of the chemicals were purchased from Sigma Aldrich, and further purification was not required. Melting points was determined by the capillary method and were uncorrected. Shimadzu Perkin Ekmer 8201 Pc IR Spectrometer used in recording IR spectra (KBr pellets), and frequencies are expressed in cm -1 . Bruker Avance II, 400 NMR spectrometer, recorded NMR spectra and Shimadzu LCMS 8030, Japan Mass spectrometer recorded mass spectra.

Preparation of pyrazolopyrimidines (2a-2e) and pyrazolopyridines (3a-3e)
A solution of 0.01 mole of malonitrile/diethyl malonate and different pyrazole carbonitrile derivatives 1 (0.01 mole) was prepared in sodium ethoxide and ethanol, which was refluxed for eight hours. The solution was concentrated, and the obtained residue was filtered, washed with ice-cold water [25].

Modeling platform
In silico analysis was carried out on Maestro 11.9 (Schrödinger, 2019-4) [26]. This software package is programmed on DELL Inc.27" workstation machine running on Intel Core i7-7700 CPU@ 3.60 GHz x8, a processor with 8GB RAM and 1000 GB hard disk with Linux-x86_64 as the operating system.

Molecular docking and binding free energy calculation
Based on the literature, the selected targets for lung cancers are EGFR, ALK, VEGFR and tankyrase and their crystal structures EGFR (PDB ID: 4WKQ) [27], ALK (PDB ID: 4Z55) [28], VEGFR (PDB ID: 4AG8) [29], TNKS (PDB ID: 4W5S) [30] were availed from the protein data bank. The downloaded proteins were minimised by Protein Preparation Wizard, using the OPLS-2005 force field of Schrodinger software. The designed fused-pyrazoles were prepared by LigPrep application (Schrödinger, 2019-4) [26] and were used for docking. The minimized protein was employed to generate the grid, and the grid box was developed by applying default parameters. Glide-XP (extra precision) [31] was used for molecular docking computations. The binding free energy MMGBSA (Molecular Mechanics, Generalized Born Model and Solvent Accessibility) dGbind (kcal/mol), between the receptor and ligands, were calculated by the Prime module (Schrödinger, 2019-4) [26]. The docking scores and the 2D and 3D conformations were generated to analyse further the affinities and binding interactions of the selected ten fused-pyrazole molecules.
The docking procedure was confirmed by redocking the co-crystal ligand of the proteins into the binding sites, respectively. The docking poses of the co-crystals in XP method and experimental conditions were compared and found to be similar with RMSD, thus validating the docking results.

Pharmacophore modeling
Pharmacophore modeling was performed by Phase tool (Schrödinger, 2019-4) [26]. In this model, six pyrazoles were considered active (≥ 69 %), and four were inactive based on their percent inhibition on lung cancer cells. Common pharmacophore hypotheses (CPH) were searched, and the best CPH was selected based on the survival score until at least one hypothesis was found and scored successfully. Pharmacophore-matching tolerance was set to 2 A • .

Drug-likeness, ADMET property and prediction of activity spectral studies
The compounds were screened for drug-likeness properties by checking with the Lipinski Rule of five [32] and ADMET (Absorption, Distribution, Metabolism, Excretion and Toxicity) property prediction by the QikProp tool [26]. The features considered for ADMET studies are the following: QPlogHERG, QPPCaco Caco-2 cell permeability, QPlogKhsa, Percent Human Oral Absorption. Further, to validate them as appropriate drug candidates, an online tool, prediction of activity spectra for substances (PASS), was used, which evaluate the biological activity based on their structural data [33]. This tool gives the values for the probability of activity (Pa) and inactivity (Pi) by comparing more than 300 pharmacological effects and biochemical mechanisms of compounds.

In vitro anticancer study by MTT assay
We procured A-549 (Human small-cell lung carcinoma) cell culture from National Centre for Cell Sciences (NCCS), Pune, India. Ten compounds were incubated with different concentrations (25, 50, 100, 200 µM) to screen the cytotoxic activity of the compounds against human small-cell lung carcinoma (A-549). The cell viability was then determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) assay after 24 h of incubation. Percent inhibition was calculated from the absorbance as % growth inhibition [34].

Chemistry
The fused pyrazole derivatives were synthesized from substituted aminopyrazoles cyclising with malononitrile and diethyl malonate to yield pyrazolopyridines and pyrazolopyrimidines. IR, NMR and mass spectroscopic techniques were used to confirm the structures (table 1).

Pharmacophore hypothesis generation and modeling
The results of all featured pharmacophore hypotheses are in table 6. DHRRR_1 is having the best survival score of 5.1979 in this study, which consists of one hydrophobic group (H), one hydrogen bond donor (D), and three aromatic rings (R). The distances between the sites in the common pharmacophore hypothesis DHRRR_1 are given in fig. 6 (a-b) and table 7.

Drug-likeness, ADMET and prediction of activity spectral studies
The synthesized ten pyrazoles have good drug-likeness properties, as shown in table 8. We evaluated the physicochemical properties to fit into the Lipinski rule of five, which is a way to determine if they are orally bioavailable. The compounds have shown no violations for the Lipinski rule of 5. Their ADMET properties were analysed, and reported that all the compounds checked were found to have all the properties within the standard limit (table 8). The activity spectra for anticancer activity of the pyrazoles were predicted to find out the inhibitory effect on the particular enzymes (table 9). The compounds bearing pyrazolopyridines (2a-2e) are more effective against specific receptors such as EGFR, ALK and tankyrase.

In vitro anticancer study by MTT assay
The results of the cytotoxicity studies were presented in table 10. Compound 2e, at the highest concentration, 200 µM, exhibited the most increased activity, which was 92% cytotoxic in nature and compounds 2d and 3d showed moderate cell growth inhibition around 80%. On correlating with their docking scores, these compounds have excellently interacted with the four lung cancer targets. Thus the results interpret that the synthesized derivatives might inhibit any of the four targets discussed and exert their anticancer action. On further analysis of the top interacted pyrazole 2e, they have maximum interaction with the VEGFR receptor, which proves their mechanism.

DISCUSSION
We found that the pyrazole condensed derivatives interacted with four lung cancer targets EGFR, ALK, VEGFR and TNKS, and their cytotoxicity action was proved against lung cancer. The compound 2e was the most active in both in silico and in vitro studies, followed by 3d and 2d. Top compound 2e interacted with the VEGFR receptor excellently with stable binding mode and affinity. The best pharmacophore hypothesis, DHRRR_1 reveals the importance of the hydrogen bond donors, hydrophobic and aromatic groups essential for the anticancer action. Thus, validating the hydrogen bonds, hydrophobic groups and pi-pi interactions, which were showed by molecular docking. As per the cytotoxicity studies, the anticancer activity of the compounds 2e, 3d and 2d might be due to the introduction of electron-withdrawing fluorine and chlorine atoms in the benzene ring attached to the pyrazole ring.
Lung cancer development is stimulated by specific signaling pathways produced by receptors such as EGFR, ALK, VEGFR and TNKS. Much research has been performed to prove the anticancer efficacy of pyrazolopyrimidines on lung cancer [35], and some reported their inhibitory potentials on specific targets such as EGFR [36], VEGFR [37], tankyrase inhibitors [38] etc. We have screened the anticancer action by in vitro studies using A549 cell lines as a preliminary evaluation. Some reports are interfering in EGFR [39,40] /VEGFR [41] /ALK [42] /Wnt [43,44] /pathways inhibits the proliferation of A549 cell lines, and with this proof, we have carried the MTT assay. Cucurbitacin [39] and diazole [40] have been reported in proliferation inhibition in A549 cells by interfering EGFR signaling pathway. A study was performed to evaluate the TNKS small molecule inhibitor XAV939 on the proliferation and migration of lung adenocarcinoma A549 cells and found that XAV939 intervention inhibited A549 cell proliferation [43]. Determination of the appropriate target should be performed by analysing the enzyme antagonistic potential further, authenticating the mechanism of inhibition.

CONCLUSION
The synthesized pyrazole derivatives interacted well with the selected lung cancer targets-EGFR, ALK, VEGFR and TNKS; with their docking scores above-5 kcal/mol equivalent with their standards. The molecular interactions are based on various parameters such as glide score, binding free energy, polar interactions, hydrophobic interactions, and hydrogen bond interactions. Further, the in vitro results exhibit compounds 2e as the best anti-lung cancer agents followed by 3d and 2d, which was in agreement with their docking results. ADMET properties reported that all the compounds were found to have properties within the standard limit. The activity spectra of the pyrazoles predicted that pyrazolopyridines (2a-2e) are more effective against specific receptors such as EGFR, ALK and Tankyrase. Thus, this study suggests that the synthesized pyrazole derivatives can be further investigated to validate their enzyme inhibitory potentials by in vivo studies.