FIELD OF THE INVENTION
This invention relates to a Method of Possible computational insights and a novel 5 atomic-level viewpoint
on the EGFR inhibition of Epicathechin Gallate in Lung cancer activity.
BACKGROUND OF THE INVENTION
1. INTRODUCTION
Cancer that originates in the lungs is called lung cancer. A cancerous tumour develops when the body's
10 cells grow unchecked and out of control. In the United States, lung cancer is the greatest cause of cancerrelated
death, coming in second only to colon cancer. As of 2020, 247,270 people will be diagnosed with
lung cancer, with a total of 116,930 of those cases being women and 130,340 of those being men.
Previously, research have found that lung cancer killed more people in men and women aged 40 to 60
than breast, prostate, colorectal and leukaemia combined. When screening criteria were introduced and
15 cigarette usage decreased, the death rate for lung cancer decreased by 48% among men and 23% among
women. By 2020, the number of deaths from second lung cancer is expected to reach roughly 140,730
(Figure 1). Smoking is a major risk factor for lung cancer. There is a 26 percent greater risk of developing
lung cancer in people who smoke. A family history of lung cancer, as well as exposure to hazardous
substances including polycyclic aromatic hydrocarbons and heavy metals, are all risk factors for the
20 disease.[1-5]
Lung cancer, also known as lung carcinoma (David et al., 2020), is a malignant lung cancer characterised
by uncontrolled cell growth in lung tissues. About 98–99 percent of all lung cancers are carcinomas.
SUMMARY OF THE INVENTION
The Present Invention says that in the United States, lung cancer is the biggest cause of cancer-related
25 mortality, ranking in second only to colon cancer. As of 2020, 247,270 persons had been diagnosed with
lung cancer, with a total of 116,930 of those cases being women and 130,340 of those being males. EGFR
is a type of tyrosine kinase receptor which promotes uncontrolled cell division. In some types of cancer
cells, genetic mutation (EGFR) promotes the EGFR proteins at greater levels than normal. By inhibiting
EGFR may be useful for preventing or reducing the growth of lung cancer against NSCLC. In this study
30 16 phyto-chemicals were selected from IMPPAT, a manually curated database. The crystal structure of
EGFR kinase domain (2ITY and 3WTO) was downloaded from Protein Data Bank. These derivatives’
binding affinities were predicted using Glide, which indicated that EGFR-kinase inhibitors had good
binding energies, ranging from -10.49 to -3. 643 kcal/mol. Olmutinib and Osimertinib are used as
standard. All the compounds were examined for their ADMET characteristics using SwissDock. The
Page 2 of 43
docking results were validated by molecular dynamics simulations to show the durability of hydrogen
bonding interactions. Results reveals that Epigallocatechin Gallate (EGCG) had superior docking score
when compared with both the standard drugs. The possible Mechanism of action of EGCG in treatment of
cancer by modifying cell signalling pathways is also explored.
BRIEF 5 DESCRIPTION OF THE DRAWINGS
Figure 1: A look at current data on lung cancer and Estimated number of lung cancer cases in 2018 and
2040 by region and country
Figure 2: Different Stages of Cancer
Figure 3: Stages of developing lung cancer, in summary (Modified from Rupasinghe et al., 2014)
10 Figure 4: Possible mechanisms underlying varying responses to EGFR inhibitors in NSCLC
Figure 5: Proteins used for docking (2ITY and 3W2O)
Figure 6 (A): Docking interaction on 2ITY with best phytoconstituents Epigallocatechin gallate
Figure 6 (B): Docking interaction on 2ITY with best phytoconstituents Epigallocatechin gallate
Figure 7 (A): Docking interaction on 2ITY with Cannabisin-G
15 Figure 7 (B): Docking interaction on 2ITY with Cannabisin-G
Figure 8 (A): Docking interaction on 2ITY with LicochalconeA
Figure 8 (B): Docking interaction on 2ITY with LicochalconeA
Figure 9 (A): Docking interaction on 2ITY with Osimertinib (Std. 1)
Figure 9 (B): Docking interaction on 2ITY with Osimertinib (Std. 1)
20 Figure 10 (A): Docking interaction on 2ITY with Olmutinib (Std. 2)
Figure 10 (B): Docking interaction on 2ITY with Olmutinib (Std. 2)
Figure 11 (A): Docking interaction on 3W2O with Epigallocatechingallate
Figure 11 (B): Docking interaction on 3W2O with Epigallocatechingallate
Figure 12 (A): Docking interaction on 3W2O with CANNABISIN-G
25 Figure 12 (B): Docking interaction on 3W2O with CANNABISIN-G
Figure 13 (A): Docking interaction on 3W2O with Ashwagandhanolide
Figure 13 (B): Docking interaction on 3W2O with Ashwagandhanolide
Figure 14 (A): Docking interaction on 3W2O with Damnacanthal
Figure 14 (B): Docking interaction on 3W2O with Damnacanthal
30 Figure 15 (A): Docking interaction on 3W2O with Olmutinib
Figure 15 (B): Docking interaction on 3W2O with Olmutinib
Figure 16 (A): Docking interaction on 3W2O with Osimertinib
Figure 17 (B): Docking interaction on 3W2O with Osimertinib
Figure 18: (A) Protein information of EGFR domain 3W2O (B) Ligand information (EGCG)
Page 3 of 43
Figure 19: Protein Ligand RMSD and secondary structure of protein
Figure 20: Ligand RMSF and Protein Ligand Contacts
Figure 21: Ligand-Protein contact and interaction with different amino acids
Figure 22: Ligand (EGCG) torsion profile
Figure 5 23: Ligand Properties
Figure 24: Different mechanisms of EGCG with the membrane receptors
Figure 25: Different mechanism of EGCG in ROS
DETAILED DESCRIPTION OF THE INVENTION
1.1 Normal structure and function of the lungs
10 Pneumonia is a respiratory disease that affects two sponge-like organs located in the chest. A third of the
right lung lobe is separated from the rest by a thin membrane. Both of the left lungs have two lobes.
Lungs on the left are smaller than those on the right because the heart takes up more room there. Air
enters the lungs through the mouth or nose and goes through the trachea as you inhale (windpipe). A
series of tubes called bronchial tubes emerge from the trachea and branch out into the lungs. Bronchioles
15 are tiny branches of the lungs. These little air sacs sit at the end of the bronchioles and are called alveoli.
When you breathe in, oxygen enters the bloodstream through the alveoli, and when you exhale, carbon
dioxide leaves the bloodstream. Lungs are primarily responsible for two functions: oxygen ingestion and
carbon dioxide expulsion. The cells of the bronchi and other sections of the lungs, such as the bronchioles
or alveoli, are typically where lung malignancies begin. The pleura, a thin covering of tissue that
20 surrounds the lungs, protects them. When you breathe, the lungs expand and collapse, and the pleura
helps them to move back and forth against the chest wall. The diaphragm, a dome-shaped muscle that
separates the chest from the abdomen, is located below the lungs. While breathing, the diaphragm rises
and falls, propelling air into and out of the lungs.[6-7]
1.1 Risk Factors for Lung Cancer Include:
25 (a) Smoking: the risk of lung cancer increases with the number of cigarettes you smoke each day and the
number of years you have smoked. Quitting at any age can significantly lower the risk of developing lung
cancer. (b) Exposure to second hand smoke. (c) Exposure to radon gas. (d) Exposure to carcinogens: such
as: asbestos, arsenic, chromium, and nickel. (e) Family history of lung cancer: a parent or a sibling.
1.2 Types of lung cancer
30 There are 2 main types of lung cancer and they are treated very differently. Fig 2 shows all the stages of
lung cancer.
(A) Non-small cell lung cancer (NSCLC)
NSCLC accounts for about 80% to 85% of lung cancer cases. Adenocarcinoma, squamous cell
carcinoma, and giant cell carcinoma are the three primary subtypes of NSCLC. Because their therapy and
Page 4 of 43
prognoses (outlook) are typically similar, these subtypes, which start from distinct kinds of lung cells, are
classed together as NSCLC.
Adenocarcinoma: Adenocarcinomas begin in cells that normally secrete mucus-like substances. This
type of lung cancer occurs mainly in people who currently smoke or have previously smoked, but it is
also the most common type of lung cancer seen in people who do 5 not smoke. It is more common in
women than men, and is more likely to occur in younger people than other types of lung cancer.
Adenocarcinoma is usually found in the outer parts of the lung and is more likely to be found before it has
spread. People with one type of adenocarcinoma called adenocarcinoma in situ (formerly called
bronchioloalveolar carcinoma) have a better outlook than people with other types of lung cancer.
10 Squamous cell carcinoma: Squamous cell carcinomas start in squamous cells, which are flat cells that
line the inside of the airways in the lungs. They are often linked to a history of smoking and tend to be
found in the central part of the lungs, near a main airway (bronchus).
Large cell (undifferentiated) carcinoma: Large cell carcinoma can appear in any part of the lung. It
tends to grow and spread quickly, which can make it harder to treat. A subtype of large cell carcinoma,
15 known as large cell neuroendocrine carcinoma, is a fast-growing cancer that is very similar to small cell
lung cancer.
Other subtypes: A few other subtypes of NSCLC, such as adenosquamous carcinoma and sarcomatoid
carcinoma, are much less common.
(B) Small cell lung cancer (SCLC)
20 About 10% to 15% of all lung cancers are SCLC and it is sometimes called oat cell cancer.
This type of lung cancer tends to grow and spread faster than NSCLC. About 70% of people with SCLC
will have cancer that has already spread at the time they are diagnosed. Since this cancer grows quickly, it
tends to respond well to chemotherapy and radiation therapy. Unfortunately, for most people, the cancer
will return at some point.
25 (C) Other types of lung tumors
Along with the main types of lung cancer, other tumors can occur in the lungs.
Lung carcinoid tumors: Carcinoid tumors of the lung account for fewer than 5% of lung tumors. Most
of these grow slowly. For more information about these tumors, see Lung Carcinoid Tumor.
Other lung tumors: Other types of lung cancer such as adenoid cystic carcinomas, lymphomas, and
30 sarcomas, as well as benign lung tumors such as hamartomas are rare. These are treated differently from
the more common lung cancers and are not discussed here.
Cancers that spread to the lungs: Cancers that start in other organs (such as the breast, pancreas,
kidney, or skin) can sometimes spread (metastasize) to the lungs, but these are not lung cancers. For
example, cancer that starts in the breast and spreads to the lungs is still breast cancer, not lung cancer.
Page 5 of 43
Treatment for metastatic cancer to the lungs is based on where it started (the primary cancer site).(
American Cancer Society cancer.org)[8-10]
1.3 STAGES OF LUNG CANCER:
All the four stages of lung cancers have been shown in figure 2. Stage 1: Cancer is found in the lung, but
it has not spread outside the lung, Stage 2: Cancer is found in the lung 5 and nearby lymph nodes, Stage 3:
Cancer is in the lung and lymph nodes in the middle of the chest, Stage 3A: Cancer is the lung in lymph
nodes but only on the same side of the chest where cancer first started growing, Stage 3B: Cancer has
spread to lymph nodes on the opposite side of the chest or to lymph nodes above the collarbone and Stage
4: Cancer has spread to both lungs into the area around the lungs or to distant organs. [11]
10 1.4 Development of lung cancer
The development of lung in shown in Fig 3, which is having three stages initiation, promotion and
progression.
1.5 Role of EGFR in Lung Cancer
Some cell possess protein that binds with EGFR results in alteration of cellular mechanisms that control
15 cell division. Genetic mutation in EGFR enhances its EGFR level in cells leads to rapid mitosis. Tyrosine
kinase receptors are Anti-EGFR agents cures cancers. The EGFR binds with at least 7 discrete ligands,
able to detoxify another protein for the adjacent EGFR, allowing the receptor nucleus to access.
(A) EGFR inhibitors:
Approx. 10-15% of all lung cancers responds to EGFR. Researchers target to block EGFR which
20 leads to slow down growth of lung cancerous tissues with EGFR mutations. Afatinib, Dacomitinib,
Entrectinib, Erlotinib, Gefitinib and Osimertinib are FDA approved drugs (Figure 4). [11-14].
(B) EGFR pathways
The EGFR gene is situated on human chromosome is a member of TK-I receptors family. EGFR
includes 28 exons, binds to EGFR and regulates cellular proliferation. EGFR exon 20 and 18-point
25 mutations are less predominant than at 19 and 21 deletions. EGFR-targeting drugs are gefitinib, erlotinib,
cetuximab and panitumumab. [15-18].
The most common acquired resistance mutation in NSCLC patients is the TK domain mutation (T790M).
42,43 A subset of NSCLC patients with the T790M mutation have never been treated with an EGFR-TKI.
44,45 These results indicate that the T790M mutation may be a possible target in NSCLC patients. 44 To
30 conquer drug resistance, initiatives and treatments must be developed. [18-21]
(C) PI3K/AKT/mTOR pathways
In NSCLC, the PI3K/AKT/mTOR pathway is thought to be a possible target. Cell growth,
differentiation and apoptosis, as well as gene transcription and protein synthesis, are all regulated by the
PI3K signalling cascade. [22].It has been shown that the PI3K/AKT/mTOR mechanisms activate
Page 6 of 43
upstream receptors (EGFR and PDGFR) and are mutated in many cancers, including breast, gastric
cancer and NSCLC. PIK3CA mutations have been found in approx. 4% of NSCLC tumour cells
expressing PTEN protein, that hinders the PI3K/AKT/mTOR mechanisms. [22-24]. Furthermore, several
miRNAs that target the PI3K/AKT/mTOR pathways have demonstrated antitumor activity and the
potential to reverse EGFR-TKI resistance. MiR-126, miR-203, and miR-5 34a downregulation has been
shown to restrict drug resistance via PI3K/AKT signalling pathway. [25-28]
(D) RAS–MAPK pathways
The RAS gene group includes KRAS, NRAS, and HRAS, GTPases control cell growth through various
pathways27. Some upstream receptors like EGFR and FGFR, are linked to the RAS family. Furthermore,
10 some downstream pathways (the RAS-MAPK and PI3K-AKT-mTOR pathways) are involved in cell
differentiation and survival via the RAS family. [29-30]
KRAS is the most frequently mutated RAS family gene in lung cancer. 28 KRAS mutations are found in
20-40% of lung adenocarcinomas, accounting for 20% of cases in Western countries and 10% of cases in
Asian countries. Sorafenib is an oral multi-kinase blocker that targets RAF showed promising efficacy
15 and was well tolerated in early clinical trials, with good patient consistency. 82 Other miRNAs, such as
miRNA-48a-3p and miR-193a-3p, have shown a reliable trend. [31-33]
(E) JAK–STAT pathways
STAT contains 7 proteins namely STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, STAT6, and
STAT7 that stimulate several proteins named as JAKs. 39, 40. These signals are involved in cell growth,
20 proliferation and cancer progression. STAT3 is the most studied STAT has been linked to different
cancers.[34-36]
Furthermore, in lung cancer, the STAT pathways regulate apoptotic genes, inhibit cellular proliferation,
and enhance EGFR-TKI therapeutic effectiveness. 41 Combining EGFR-TKIs or chemotherapy with
JAK/STAT pathway inhibitors has been reported to enhance antitumor efficacy and reduces drug
25 resistance. [37-39]
(F) HER3
HER3 is a member of HER group and inactive receptor. EGFR and HER4 both have multiple ligands,
whereas HER3 only has one, called heregulin (HRG) or neuregulin (NRG). When a ligand binds to the
extracellular region of EGFR or HER3/4, leads to dimerization. 49 To avoid dimerization, EGFR, HER3,
30 and HER4 are typically found as molecularly folded monomers (inactive state). Unlike other members of
HER family (EGFR, HER2, and HER4) the HER3 possess little or no TK activity. [40-44].
U3-1402 is a recently designed HER3-targeted antibody-drug conjugate that contains a complete human
HER3-targeted antibody and topoisomerase I inhibitor payload. [45]
2. Material and Methods
Page 7 of 43
2.1 Hardware and Software
Except for molecular dynamics simulations, the whole computational inquiry was carried out using
Windows 10 (64-bit) operating systems with 8 GB RAM and a 2.11GHz Intel® CoreTM i5-10210U
processor. Desmond version ran Molecular Dynamics simulations using Ubuntu 14.04.5 on a Linux
system with 16 GB RAM and a 4 GB graphics card. The Scripps Research 5 Institute's PyRx python
prescription 0.8 binary package for Windows is available for freely.
https://pyrx.sourceforge.io/downloads.
2.2 Preparation of Ligands
Chem Create was used to draw the ligand structures, and Open Babel in PyRx was used to do energy
10 reduction using UFF (universal force field). Ligands were prepared using LigPrep application (Trial
version of Schrödinger). In this process, OPLS 2005 force field was used for energy optimization for all
ligands until 0.01 Å RMSD cutoff not obtained. Tautomers and all possible ionization states were
generated using Epic at target pH of 7±2 and low energy ring conformation for each ligand was also
generated. Prepared ligands were saved as a maestro output format. Figure 5
15 2.3 Docking studies
The theoretical binding mode of synthesized compounds to EGFR using the trial version of Schrodinger’s
Glide module software. The target protein was derived from the RCSB protein data bank (PDB). Crystal's
structure of the EGFR tyrosine kinase domain with Iressa (PDB code 2ITY) at resolution 3.42 Å and
EGFR tyrosine Kinase domain T790M/L858R Mutant with TAK-285 (PDB code 3W2O) at resolution
20 2.35 Å imported into Glide software. To refine the structure minimization till RMSD constraint value of
0.3 Å was done using OPLS-2005 as force field after optimization of hydrogen bonds. Grid box
dimensions were 50 × 50 × 50 (all in Å) with a 0.375 Å grid point spacing. Protein residues that include
ligand-restricting sites are logically expected to contain certain ligands that can bind reversibly. The
correct orientation for other protein-affirming amino acid residues was provided.
25 2.4 Molecular Dynamics Simulations:
Each EGFR protein was produced using the Protein Preparation Wizard of the Schrodinger Suite 2015
and then imported as the starting structure into molecular dynamics software. One chain containing an
inhibitor had been preserved for each EGFR crystal complex with multiple chains. Desmond 4.2 was used
for each MD simulation. The TIP4P water model was used to generate a solvent system, which would
30 operate well with the OPLS force field.
All of the prepared systems were first minimised using the steepest-descent (SD) method until a gradient
threshold (25 kcal/mol/) was reached, and then connected to a Berenson thermostat with a 3000 K
reference temperature and a Berendsen barostat with a 1.01325 bar reference pressure. The particle mesh
Page 8 of 43
Ewald method was used to calculate long-range electrostatics. The coulomb interaction cutoff was set at
9.0. After equilibration, all systems began to run for 2.4 ns in the NPT ensemble.
2.5 Analysis of Simulation Data
The root-mean square deviation (RMSD), root mean square fluctuation (RMSF), and other measures for
the distance between residues and inhibitors were determined using the VMD (5 version 1.9.1) or in-house
Perl scripts after analysing the MD trajectories.
2.6 Selection of Phytoconstituents:
18 Phyto-constituents are selected on the basis of review of literature from search engines and IMPPAT
2.0 databases which provides which has been constructed via digitalization of information from more than
10 100 books on traditional Indian medicine, 7000+published research articles and other existing resources.
The Phyto-constituents are selected on the basis of their antiviral effects. This database offers the
information regarding the phytochemicals, medicinal applications, 2D and 3D chemical structures in
order to screen virtually the capacity of phytoconstituents to bind at spike protein. Table 1 shows the
phytoconstituents selected for the study with their IMPPAT-ID (https://cb.imsc.res.in/imppat/).
15
Page 9 of 43
Page 10 of 43
Table 1: Phytochemicals isolated from fruits and vegetables for lung cancer chemoprevention and their mechanisms of action (Kunnathur et al.,
2015). [46]
S.
NO.
Compound Name Biological Sources Mechanism of Action Uses
(1) Licochalcone A IMPHY004374 G. glabra Suppress cancer cell line, propagate and block cell
growth at the G2/M transition and stimulate apoptosis
Anti-inflammatory,
anticancer
(2) EGCG IMPHY011670 C. sinensis Hinder cell proliferation and apoptosis Anticancer
(3) Delphinidine IMPHY007124 S.molongena Suppresses1L-1beta activation of NF-kB Anticancer, antiinflammatory,
antioxidant.
(4) Damnacanthal IMPHY003162 M. citrifolia L. Blocking C/EBP through RNA in reduction of NAG-1. Anticancer
(5) Osimertinib IMPHY003064 E. gerardiana It binds TK blockers T790, L858, and exon19 leads to
suppression of lung cancer
Anticancer
(6) Cnnabisin G IMPHY010255 C. sativa L. Suppress the CB1 and CB2 receptors Anticancer
(7) Ashwaganhdanolide IMPHY013466 W. somnifera Inhibition in NC1 H460 lung cancer cell line Anticancer
(8) Crocetin IMPHY010351 Q. quercase Inhibition of QR2(quinone reductase) Anti-inflammatory,
anticancer, antihypertensive
(9) Oleanolic acid IMPHY011826 Phytoolacca,
Syzygium
Suppression of EGF Antiviral, antitumor
(10) Genestine IMPHY004643 Glycine max Suppression of WNT/beta-catenin and AKT signalling Anticancer
(11) Licoagrochalcone IMPHY004621 G. glabra Linn Suppression of cell line, block cell progression at the
G2/M stage and induce apoptosis
Anti-inflammatory,
anticancer
Page 11 of 43
(12) Podophyllin IMPHY011856 P. peltatum Linn It arrests mitosis at metaphase Anticancer
(13) Taraxerol IMPHY011677 T. officinale Suppression of Epstein-
(14) Olmutinib IMPHY012102 Inhibit the kinase Lung cancer
(15) Withaferina A IMPHY004118 W. somnifera Inhibition of human papilloma virus, deactivation
tumour suppresses protein p53and pr6
Anti-inflammatory
Anticancer
(16) Ursolic acid IMPHY011880 M. jalapa Mediated by cross talk between calcium and kinase Cosmetic,
antimalarial,
anticancer
(17) Withanone IMPHY005214 W. somnifera Inhibition of human papilloma virus, inactivation the
tumour suppresses protein p53and pr6
Anti-inflammatory
and anticancer
(18) ß-Sitosterol IMPHY014836 O. sativa Inhibits 59-reductase Alzheimer
3. Results
3.1 Docking results
The docking results on 2ITY are shown in table 2, 3 and 4. Docking interaction of 2ITY with some potent
compounda are Epigallocatechin gallate (Fig 6A, 6B), Cannabisin-G (Fig 7A, 7B), LicochalconeA (Fig
8A, 8B), Osimertinib (Fig 9A, 9B) and Olmutinib (Fig 5 10A, 10B). The most potent drug
Epigalocatechine is virtually docked with 2ITY protein with docking score of -7.996 kcal/mol, 12
rotatable bonds and affinities with amino acids like LEU844, ALA743VAL726, ALA722, LEU718,
CYS797, PHU794, PET743, LEU792, THR854 and ASS719. This drug has shown more prominent score
with the first standard drug Osimertinib with docking score of -7.626 kcal/mol, 11 rotatable bonds and
10 affinities with amino acids like TYR718, LEU718, PHE790, PEU794, LEU791, VAL726, LUL780,
ALA743, PET793, LEU844, THR790, THR854 and GAN790 and approx. 1.5 times than the second
standard drug Olmutinib with docking score of -4.868 kcal/mol, 8 rotatable bonds and affinities with
amino acids like VAL726, PHE723, ALA722, LEU718, LEU844, LEU792,5ALA722, LEU718,
LEU844, LEU792, PET790, PRO794, CYS797, ALA790, LEU797, THR854, THR790 and GLN791.
15 The docking results on 3W2O are shown in table 5, 6 and 7. Docking interaction of 3W2O with some
potent compounda are Epigallocatechin gallate (Fig 11A, 11B), Cannabisin-G (Fig 12A, 12B),
Ashwagandhanolide (Fig 13A, 13B), Damnacanthal (Fig 14A, 14B), Olmutinib (Fig 15A, 15B) and
Osimertinib (Fig 17A, 17B). The most potent drug Epigalocatechine is virtually docked with 3W2O
protein with docking score of -10.49 kcal/mol, 59 rotatable bonds and affinities with amino acids PE766,
20 LEU747, ALA743, LEU790, ME719, PHE723, VAL726, LEU792, CYS797, PET793, PRO794,
PNE766, LEU718, ASN842, GLN791, THR854. This drug has shown more approx. twice potent with the
first standard drug Osimertinib with docking score of -5.185 kcal/mol, 49 rotatable bonds and affinities
with amino acids like MET730, LET726, LEU797, MET760, ALA733, LET730, PET790, LUE794,
PET790, LEU744, LEU794, CYS779, VAL726, PEL703, THR854, AGN840, GLN791 and approx.
25 twice potent than the second standard drug Olmutinib with docking score of -5.419 kcal/mol, 50 rotatable
bonds and affinities with amino acids PE766, LEU747, ALA743, LEU790, ME719, PHE723, VAL726,
LEU792, CYS797, PET793, PRO794, PNE766, LEU718, LEU844, ASN842, GLN791 and THR854.
Page 12 of 43
Page 13 of 43
Table 2: Hydrophobic and polar bond interaction of selected phytoconstituents with 2ITY Protein
S. No. Compound names Hydrophobic bond Polar bond
1) Licochalcone A LEU718, LEU794, MET793, LEU792, LEU844, MET766, ALA793,
LAL726, PEL723, LEU747
THR854, THR790
2) Delphinidine ALA743, LEU844, LEU718, CYS797, PRO794, VAL726, MET766,
CYS775, LEU788, ALA743
THR790, THR854
3) Damnacanthal ALA743, LEU844, PET726, PRO794, PHE790, CYS775, VAL727,
LEU718, LEU844
THR790, THR854,
GAN790
4) Osimertinib TYR718, LEU718, PHE790, PEU794, LEU791, VAL726, LUL780,
ALA743, PET793, LEU844
THR790, THR854,
GAN790
5) Oleanolic acid LEU844, CYS797, LEU718, ALA723, PHE723, VAL726 ANS842
6) Olmutinib VAL726, PHE723, ALA722, LEU718, LEU844, LEU792,5ALA722,
LEU718, LEU844, LEU792, PET790, PRO794, CYS797, ALA790,
LEU797
THR854, THR790,
GLN791
7) Withanone ALA743, LEU844, LEU792, PET743, PHU794, PHE793, CYS797,
VAL726, LEU718, PHE743
8) Withaferina ALA743, LEU844, LEU792, PET743, PHU794, PHE793, CYS797,
VAL726, LEU718
THR854, ASN842
9) Ursolicacid ALA722, LEU718, CYS797, LEU844, MET793, LEU792, ALA743,
VAL726
THR854, SER720
10) ßsitosterol ALA743, LEU844, LEU792, PET743, PHU794, PHE793, THR854
Page 14 of 43
Table 3: Docking score and other parameters on 2ITY
S.
No.
Variant Glide
rotatable
bonds
Docking
score
Glide
ligand
efficiency
Glide
ligand
efficiency
sa
Glide
ligand
efficiency
ln
Xp
gscore
Glide
gscore
Glide
evdw
Glide
ecoul
Glide
energy
Glide
einternal
Glide
emodel
(1) Epigallocatechingallate 12 -7.996 -0.242 -0.777 -1.778 -7.996 -7.996 -28.54 -
18.427
-
46.967
0 -
69.174
11) Ashwaganhdanolide PHU794, PHE793, CYS797, VAL726, LEU718, PHE743, ALA743,
LEU844, LEU792, VAL726
THR854, GLN791,
ANS719, THR791
12) Cnnabisin G CYR801, LEU844, ALA743, CYS797, LEU718, PHE726, PHU766,
VAL726, PET743, LEU792, LEU726, PET726
THR854, GLN791,
ANS719, THR791
13) Corcetin LEU844, ALA743VAL726, ALA722, LEU718, CYS797, PHU794,
PET743, LEU792
THR854, GLN791,
ANS719, THR791
14) Epigalocatechine LEU844, ALA743VAL726, ALA722, LEU718, CYS797, PHU794,
PET743, LEU792
THR854, ASS719
15) Genestine CYR801, LEU844, ALA743, CYS797, LEU718, PHE726, PHU766,
VAL726, PET743, LEU792
THR854, ASS719
16) Licoagrochalcone LEU844, ALA743, CYS797, LEU718, PHE726, PHU766, VAL726,
PET743, LEU792,
THR854, ASS719
17) Podophyllin LEU844, CYS797, LEU718, ALA723, PHE723, VAL726 THR854, THR790,
GLN791
18) Taraxerol LEU844, ALA743VAL726, ALA722, LEU718, CYS797, PHU794,
PET743, LEU792
THR854, ASS719
Page 15 of 43
(2) Cannabisin-G 19 -7.961 -0.173 -0.62 -1.649 -7.961 -7.961 -
56.084
-
12.389
-
68.473
10.655 -
90.354
(3) Licochalcone A 8 -7.947 -0.318 -0.93 -1.884 -7.947 -7.947 -
33.017
-
12.762
-
45.779
9.37 -
57.302
(4) Delphinidine 7 -7.94 -0.361 -1.011 -1.941 -7.94 -7.94 -
27.509
-
17.079
-
44.588
0.738 -
58.391
(5) Damnacanthal 3 -7.782 -0.371 -1.022 -1.924 -7.782 -7.782 -
31.041
-4.938 -
35.979
1.403 -
46.985
(6) Osimertinib (Std. 1) 11 -7.626 -0.206 -0.687 -1.654 -7.626 -7.626 -
48.786
-9.97 -
58.756
1.983 -
83.089
(7) Licoagrochalcone 8 -6.71 -0.28 -0.806 -1.606 -6.71 -6.71 -
32.786
-10.1 -
42.886
10.827 -59.38
(8) Podophyllin 9 -6.679 -0.215 -0.677 -1.506 -6.679 -6.679 -
28.714
-
17.204
-
45.918
0 -
57.735
(9) Genestein 4 -6.669 -0.333 -0.905 -1.669 -6.669 -6.669 -
27.517
-
11.782
-
39.299
1.802 -
51.372
(10) Ashwagandhanolide 14 -6.429 -0.093 -0.382 -1.228 -6.429 -6.429 -
45.427
-7.879 -
53.307
4.959 -
64.493
(11) Crocetin 10 -5.641 -0.235 -0.678 -1.35 -5.641 -5.641 -
30.654
-7.801 -
38.456
12.438 -
41.276
(12) Olmutinib (Std. 2) 8 -4.868 -0.139 -0.455 -1.069 -4.868 -4.868 -44.5 -5.132 -
49.632
5.689 -
71.625
(13) Oleanolicacid 3 -4.851 -0.147 -0.471 -1.079 -4.851 -4.851 -
30.137
-
10.336
-
40.473
0.425 -
41.613
(14) Withaferina 5 -4.044 -0.119 -0.385 -0.894 -4.044 -4.044 -
31.455
-5.173 -
36.628
3.971 -
40.995
Page 16 of 43
(15) Ursolicacid 3 -3.626 -0.11 -0.352 -0.806 -3.626 -3.626 -26.32 -0.211 -
26.531
2.625 -
12.617
(16) Withanone 4 -3.538 -0.104 -0.337 -0.782 -3.538 -3.538 -
33.801
-2.66 -36.46 3.067 -
42.964
(17) Taraxerol 1 -3.497 -0.113 -0.354 -0.789 -3.497 -3.497 -
36.801
-0.929 -
37.731
0.325 -
45.996
(18) ß-Sitosterol 7 -3.36 -0.112 -0.348 -0.763 -3.36 -3.36 -
19.492
-8.909 -
28.401
3.559 -
34.095
Table 4: Docking score and other parameters on 2ITY Contd….
S.
No
.
Variant XP
HBond
XP
Pho
bEn
XP
PhobE
nHB
XP
LowM
W
XP
RotP
enal
XP
Lipophi
licEvdW
XP
Electro
XP Sitemap XP
Penalti
es
XP
ExposP
enal
Glid
e
conf
num
Glide
posen
um
Glide
eff state
penalty
XP
PoseR
ank
(1) Epigallocat
echingallate
-3.807 0 0 -0.426 0.309 -3.287 -1.153 -0.65 0 0.071 1 1 0 1
(2) Cannabisin-
G
-1.997 0 0 0 0.244 -5.888 -0.929 -0.156 0.142 0.623 1 4 0 1
(3) Licochalco
ne A
-1.33 0 -1 -0.372 0.354 -4.309 -0.957 -0.4 0 0.067 1 1 0 1
(4) Delphinidin
e
-2.55 0 0 -0.489 0.086 -3.78 -1.281 0 0 0.074 1 4 0 1
(5) Damnacant
hal
-1.33 0 -1 -0.5 0 -3.767 -0.37 -0.814 0 0 1 1 0 1
(6) Osimertinib -1.859 0 -1.5 0 0.315 -4.853 -0.748 -0.4 1.001 0.418 1 1 0 1
Page 17 of 43
(Std. 1)
(7) Licoagroch
alcone
-1.621 0 -0.293 -0.419 0.381 -3.665 -0.758 -0.4 0 0.064 1 1 0 1
(8) Podophylli
n
-1.764 0 -0.014 -0.059 0.137 -3.515 -1.29 -0.249 0 0.075 1 7 0 1
(9) Genestein -1.81 0 0 -0.5 0.105 -3.58 -0.884 0 0 0 1 17 0 1
(10) Ashwagand
hanolide
-1.956 0 0 0 0.08 -3.957 -0.591 -0.27 0 0.264 1 2 0 1
(11) Crocetin -0.96 0 0 -0.405 0.597 -4.317 -0.585 0 0 0.029 1 9 0 1
(12) Olmutinib
(Std. 2)
-2.529 0 0 0 0.041 -2.125 -0.795 0 0 0.396 1 1 0 1
(13) Oleanolicac
id
-0.092 0 -1.5 0 0.294 -4.576 -0.385 -0.149 1.124 0.415 1 1 0 1
(14) Withaferina -0.96 0 0 0 0.117 -2.929 -0.388 -0.255 0 0.37 1 1 0 1
(15) Ursolicacid -0.48 0 0 0 0.041 -3.236 -0.016 -0.194 0 0.259 1 16 0 1
(16) Withanone -0.48 0 0 0 0.078 -3.076 -0.199 -0.056 0 0.195 1 4 0 1
(17) Taraxerol -0.7 0 0 -0.118 0.197 -2.235 -0.668 0 0 0.164 1 1 0 1
(18) ß-Sitosterol 0 0 0 -0.084 0 -2.633 0.014 0 0 0.269 1 1 0 1
Page 18 of 43
Table 5: Hydrophobic and polar bond interaction of selected phytoconstituents with 3W2O Protein
S. No. Compound names Hydrophobic bond Polar bond
1) Licochalcone A LEU718, PRO794, MET793, LEU792, ALA743, MET790, LEU788, ILE759,
LEU747, VAL726, LEU744, ALA763, MET766
THR854, ASN842,
GLN791
2) Delphinidine PET791, LEU792, PET790, CYS772, LEU788, LEN720, PET726, ALA719,
LEN720, LES797, ALA743, VAL726, LEU718
GLN791, THR854
3) Damnacanthal ALA743, VAL726, MET790, LEU792, MET793, LEU844, LEU972,
PRO794, LEU844, LEU718
GLN718
4) Osimertinib MET730, LET726, LEU797, MET760, ALA733, LET730, PET790, LUE794,
PET790, LEU744, LEU794, CYS779, VAL726, PEL703
THR854, AGN840,
GLN791
5) Crocetin LEU719, ALA748, IE744, IE753, ALA743, MET766, LEU790, IE789,
MET790, VAL726, LEU844, LEU752, MET793, PRO794, PHE72, CYS797
GLN791
6) Oleanolic acid LEU719, ALA748, IE744, IE753, ALA743, MET766, LEU790, IE789,
MET790, VAL726,
THR854, GLN791
7) Olmutinib PE766, LEU747, ALA743, LEU790, ME719, PHE723, VAL726, LEU792,
CYS797, PET793, PRO794, PNE766, LEU718, LEU844
ASN842, GLN791,
THR854
8) Withaferina PE766, LEU747, ALA743, LEU790, ME719, PHE723, VAL726, LEU792,
CYS797, PET793, PRO794, PNE766, LEU718
GLN791, THR854
9) Ursolicacid PET744, LEU718, ALA743, LEU747, LEU748, PET790, ME719, PHE723,
VAL726, CYS797, LEU794
THR854
10) Withanone LEU844, PET744, LEU797, VAL766, ME790, ALA722, PHE723, ALA743, GLN791, THR854
Page 19 of 43
Table 6: Docking score and other parameters on 3W2O
S.
No.
Variant Glide
rotatable
bonds
Docking
score
Glide
ligand
efficiency
Glide
ligand
efficiency
sa
Glide
ligand
efficiency
ln
Xp
gscore
Glide
gscore
Glide
evdw
Glide
ecoul
Glide
energy
Glide
einternal
Glide
emodel
(1) Epigallocatechingallate 59 12 -10.49 -0.318 -1.02 -2.333 -10.49 - - -14.082 -59.81 8.069
LEU797
11) ß-Sitosterol LEU718, CYS797, PHU724, LEU792, PET790, LEU718, ALA748, LEU747,
PET726, LEU777, ALA743, MET729, PHE722 VAL726, LEU844
GLN791, THR854
12) Ashwaganhdanolide PE766, LEU747, ALA743, LEU790, ME719, PHE723, VAL726, LEU792,
CYS797, PET793, PRO794, PNE766, LEU718
GLN791, THR854
13) Cnnabisin G LEU718, CYS797, PHU724, LEU792, PET790, LEU718, ALA748, LEU747,
PET726, LEU777, ALA743, MET729, PHE722 VAL726, LEU844
GLN791, THR854
14) Epigalocatechine PE766, LEU747, ALA743, LEU790, ME719, PHE723, VAL726, LEU792,
CYS797, PET793, PRO794, PNE766, LEU718
ASN842, GLN791,
THR854
15) Genestine MET730, LET726, LEU797, MET760, ALA733, LET730, PET790, LUE794,
PET790, LEU744, LEU794, CYS779, VAL726, PEL703
ASN842, GLN791,
THR854
16) Licoagrochalcone LET726, LEU797, MET760, ALA733, LET730, PET790, LUE794, PET790,
LEU744, LEU794, CYS779, VAL726, PEL703
GLN791
17) Podophyllin PET744, LEU718, ALA743, LEU747, LEU748, PET790, ME719, PHE723,
VAL726, CYS797, LEU794
ASN842, GLN791,
THR854
18) Taraxerol LEI790, LEU726, LAL722, LUL718, ALA745, CYS797, PRO877, ALU844 THR854
Page 20 of 43
10.49 45.728
(2) Delphinidine 53 7 -8.062 -0.366 -1.027 -1.971 -8.062 -
8.062
-
26.636
-13.658 -40.294 0
(3) Cannabisin-G 61 19 -7.991 -0.174 -0.622 -1.655 -7.991 -
7.991
-
47.903
-12.543 -60.446 0
(4) Ashwagandhanolide 60 14 -7.329 -0.106 -0.436 -1.4 -7.329 -
7.329
-
45.195
-18.955 -64.15 15.471
(5) Damnacanthal 19 3 -7.227 -0.344 -0.949 -1.787 -7.227 -
7.227
-
22.112
-5.636 -27.748 0.876
(6) Licoagrochalcone 64 8 -6.868 -0.286 -0.825 -1.644 -6.868 -
6.868
-
34.715
-8.567 -43.282 4.399
(7) Crocetin 54 10 -6.4 -0.267 -0.769 -1.532 -6.4 -6.4 -
31.359
-7.223 -38.581 14.905
(8) Licochalcone A 29 8 -6.089 -0.244 -0.712 -1.443 -6.089 -
6.089
-
37.944
-2.014 -39.957 5.247
(9) Genestein 24 4 -6.079 -0.304 -0.825 -1.521 -6.079 -
6.079
-
32.604
-3.431 -36.035 5.111
(10) Olmutinib (Std. 2) 50 8 -5.419 -0.155 -0.506 -1.19 -5.419 -
5.419
-47.12 -7.855 -54.975 7.485
(11) ß-Sitosterol 48 7 -5.192 -0.173 -0.538 -1.18 -5.192 -
5.192
-29.91 0.574 -29.336 2.447
(12) Osimertinib (Std. 1) 49 11 -5.185 -0.14 -0.467 -1.124 -5.185 -
5.185
-
53.465
-3.841 -57.305 8.996
(13) Podophyllin 66 9 -5.015 -0.162 -0.508 -1.131 -5.015 -
5.015
-
38.604
-5.715 -44.319 8.621
(14) Oleanolic Acid 70 3 -4.859 -0.147 -0.472 -1.081 -4.859 - - -4.244 -43.426 2.752
Page 21 of 43
4.859 39.182
(15) Withaferina 75 5 -4.672 -0.137 -0.445 -1.032 -4.672 -
4.672
-
31.797
-5.698 -37.495 9.789
(16) Taraxerol 56 1 -4.441 -0.143 -0.45 -1.002 -4.441 -
4.441
-
36.921
0.134 -36.787 0.661
(17) Ursolic Acid 69 3 -3.75 -0.114 -0.364 -0.834 -3.75 -3.75 -
40.068
0.874 -39.194 0.804
(18) Withanone 76 4 -3.623 -0.107 -0.345 -0.8 -3.623 -
3.623
-
32.298
-1.454 -33.752 5.073
Table 7: Docking score and other parameters on 3W2O Contd….
S.
No.
Variant XP
HBond
XP
PhobEn
XP
Phob
EnHB
XP
LowMW
XP
RotPenal
XP
Lipophilic
EvdW
XP
Electro
XP
Sitemap
XP
Pen
alties
XP
Expos
Penal
Glide
conf
num
Glide
pose
num
Glide
eff
state
penalty
XP Pose
Rank
(1) Epigallocatechingallate -4.261 -0.282 0 0 0.164 -5.003 -1.056 -0.052 0 0 1 7 0 1
(2) Delphinidine -3.714 0 0 -0.489 0.086 -2.92 -1.024 0 0 0 1 3 0 1
(3) Cannabisin-G -2.602 0 0 0 0.244 -5.019 -0.941 0 0.113 0.214 1 1 0 1
(4) Ashwagandhanolide -3.952 0 0 0 0.08 -2.966 -1.422 -0.134 0 1.064 1 4 0 1
(5) Damnacanthal -1.33 -0.15 -1 -0.5 0 -3.447 -0.423 -0.657 0 0.28 1 2 0 1
(6) Licoagrochalcone -1.18 -0.275 0 -0.419 0.381 -5.011 -0.642 -0.14 0 0.418 1 1 0 1
(7) Crocetin -1.342 -0.193 0 -0.405 0.597 -4.463 -0.542 -0.245 0 0.193 1 4 0 1
(8) Licochalcone A -0.459 -0.15 0 -0.372 0.354 -5.006 -0.151 -0.304 0 0 1 7 0 1
(9) Genestein -1.458 0 0 -0.5 0.105 -3.969 -0.257 0 0 0 1 1 0 1
Page 22 of 43
(10) Olmutinib (Std. 2) -0.649 -0.175 0 0 0.294 -4.785 -0.589 -0.118 0.46 0.142 1 1 0 1
(11) ß-Sitosterol -0.03 -0.424 0 -0.118 0.197 -4.859 0.043 0 0 0 1 11 0 1
(12) Osimertinib (Std. 1) -0.343 0 0 0 0.315 -5.052 -0.288 -0.235 0 0.417 1 1 0 1
(13) Podophyllin -1.498 0 0 -0.059 0.137 -3.191 -0.429 -0.186 0 0.211 1 14 0 1
(14) Oleanolic Acid -1.18 -0.199 0 0 0.041 -3.262 -0.318 -0.078 0 0.137 1 12 0 1
(15) Withaferina -1.093 0 0 0 0.117 -3.258 -0.427 -0.189 0 0.178 1 9 0 1
(16) Taraxerol -0.488 -0.168 0 -0.078 0 -3.828 0.01 0 0 0.111 1 20 0 1
(17) Ursolic Acid -0.48 -0.182 0 0 0.041 -3.162 0.066 -0.097 0 0.065 1 14 0 1
(18) Withanone -0.48 0 0 0 0.078 -3.064 -0.109 -0.133 0 0.085 1 6 0 1
3.2 Molecular Dynamics Simulation
To validate the docking scores, the best docking model was simulated. The EGCG of 3W2O had the
highest docking score (-10.49 kcal/mol) and multiple amino acid residues were observed to interact with
3W2O. The protein-ligand complex was simulated for 50.098 ns and having 38102 atoms. RMSD and
RMSF calculations were performed to analyse the trajectory generated after simulation 5 and represented in
Fig. 19 and 20. The RMSD of the protein-ligand complex attained a value of 3.5 Å between 27 ns and a
constant value till 12 ns with an average of 3.2 Å. RMSD having mean value of 3.085 Å with standard
deviation of 0.816. Rad Gyration (rGyr) has mean value of 20.498 A0 with standard deviation of 0.129
(Fig 23).
10 Calculations of RMSF of the backbone atoms to check the mobility of residues to allow conformational
changes on forming the complex ligand were also performed. The RMSF fluctuations for the 3W2O
complex were not high, contained in the range of 0.5–5.2 Å. RMSF (Ca) having mean value of 2.176 Å
with standard deviation of 1.117 and RMSF of Side chain having mean value of 2.601 Å with standard
deviation of 1.213
15 On basis of RMSD and RMSF analyses, it can be concluded that the docked protein-ligand complex of
3W2O complex with EGCG was stable and the docking result could be validated (Fig 21). The protein
secondary structure shows 24.90% Helix, 16.29 % Strand and 41.19% Protein secondary structure
elements (SSE) (Fig 19). Lys 745 and Met 793 have shown have shown most hydrophobic bonds, Leu
788, Met 793, Arg 841 and Asp 855 had shown most hydrogen bonding, in which Asp 855 is most H20
bonded with ligand EGCG. Hbonds having mean value of 4.382 with standard deviation of 0.974.
There was no ionic interaction. The torsion profile of ligands is shown in the Fig 22. This shows that there
is no bond which can rotate with 3600 angles. The hydroxyl bonds are rotatable and shown in the figures.
4. Discussion
4.1 Possible Mechanism of Epigallocatechin gallate
25 EGCG-Mediated Regulation of Growth Factors and Signalling Pathways
Signalling pathways play an essential role in cell function by involving growth factors [92]. Numerous
TK are involved in internal signal transduction pathways through a sequence of molecular switches [48-
52]. Either, they are transmembrane receptor kinases like EGFR, \VEGFR, or cytoplasmic kinases such as
AKT, P13, RAS, and PKC [53-56]. Almost all types of cancers encounter altered signal transduction
30 including signalling molecules which lead to uncontrolled cell division. So, these signalling molecules
may be potential targets for anticancer candidates (Fig 24) [57-59].
4.2 EGCG has the affinity to bind with membrane receptors
EGCG has also been known to regulate the activities of various growth factors receptors which include
epidermal growth EGFR, VEGFR, TK and, ILGFR. It specifically binds to the platelet-derived growth
Page 23 of 43
factor receptor and interferes in signals transduction in smooth muscle cells. It also possesses ability to
induce H2O2-mediated phosphorylation of insulin receptors inhibit tyrosine proliferation. Further, the
observed elevation in laminin receptor (67LR) and Bcl-2 proteins by EGCG is critical for cancerous cells
(Fig 24).
EGCG can also target the tumour cells specifically through i 5 nduction of apoptosis via 67LR and
underlines the line of treatment that is restricted only for cancer cells. Besides, it has also been able to
bind with other proteins like IGFIR, FYN, glucose-regulated protein 78 and zeta chain-associated 70-kDa
protein to show inhibitory action in various cancer cell lines. [60].
Many studies have supported the inhibitory action of EGCG on EGFR signalling cascade and in particular
10 on EGFR tyrosine kinase activity and is well correlated with decreased expression of VEGF in cancer
cells. Hepatocyte growth factor–Hepatocyte growth factor receptor (HGF–HGFR) is responsible for
regulation of growth, movement, and invasion of cells by activating TK signal transduction pathways.
EGCG even at low concentration has been found to inhibit the HGFR phosphorylation that is associated
with decreased cell invasion.
15 EGCG has been reported to reduce the phosphorylation of JUN N-terminal kinase, JUN, MEK1, MEK2,
ERKI, ERK2, ELK1 (Ets-like protein1), and RAF1 kinases in different cell lines. [46] Similarly,
inhibition of proteasomes (20S) by EGCG followed by growth arrest suggests that the proteasome could
be a molecular target of EGCG and may contribute to the cancer preventive effects of tea. The inhibition
of the activities of MMP2 and MMP9 again suggests other targets for EGCG during metastasis and
20 angiogenesis. [47-48]. The other possible molecular targets for chemo-preventive action of EGCG include
DNA methyltransferase, dihydrofolate reductase, glucoase-6-phosphatase dehydrogenase and
gluteraldehyde-3-phosphate dehydrogenase. [61-64]
Nearly, 80–85% of the cases of lung cancer are associated with NSCLC while the rest are of SC (WHO).
Lung cancer is treated by surgical removal of affected tissue followed by radiation therapy and
25 chemotherapy. Natural compounds especially the EGCG, which is a strong antioxidant and can act in
multiple ways, could play an effective role in prevention as well as control of cancer progression.
EGCG has been reported to enhance the expression of CTR1 via non-coding RNA and Nuclear Enriched
Abundant Transcript. This indicates the possible role of non-coding RNAs in the cell growth,
differentiations, and proliferation. EGCG at a concentration ranging from 10 to 100 mM has been shown
30 to cause demethylation and increased expression of crucial genes like GAS1, TIMP4, ICAM1, and
WISP2. The demethylation and re-expression of important genes like GAS1, TIMP4, ICAM1, and
WISP2 by EGCG provide sufficient evidence that it can be used as a potential epigenetic drug. The
angiogenesis and epithelial–mesenchymal transition (EMT) are the two important key regulators of
metastasis and invasion of cancerous cells to other organs. EGCG inhibits the expressions of HIF- 1a and
Page 24 of 43
VEGF as well as the process of EMT that reduces the migration and invasion of cancer cells. It also
hinders the process of angiogenesis by inhibiting the HIF-1a and further downregulates the expressions of
VEGFR, IL-8, and CD31 that play the pivotal role in cell survival. This suggests that EGCG is an
appreciable player to curb angiogenesis and EMT crucial for migration and invasion of cancer cells. [65-
5 69].
Moreover, EGCG is also reported to suppress ku70/ku80, upregulate the level of JWA, and downregulate
the expression of topoisomerase IIa both in vitro and in vivo conditions. EGCG has also been shown to
hinder lung tumorigenesis by inhibiting the DNA methylatransferase 1(DNMT1), p-AKT, and g-H2AX.
While DNMT1 is a key enzyme that mediates DNA methylation, whereas AKT mediates signalling
10 pathways, downstream activated tyrosine kinases, and phosphatidylinositol 3-kinase. gH2AX is a
sensitive marker for DNA double-strand breaks which is the gravest form of DNA damage. Since EGCG
modulates DNA methylation and AKT mediating signalling pathways; hence, it is speculated to interfere
with a variety of biological responses, including cell growth, proliferation, and survival of tumor .Further,
EGCG has been reported to alter the modulation of spliceosomal UsnRNA at an early stage of
15 carcinogenesis, thereby adversely affecting the RNA processing which would restrict the early pre
malignant lung lesions, as spliceosomal UsnRNA are associated with cell proliferation and development.
(Table 8 and Fig 25) [70-74]
Page 25 of 43
Table 8: Mechanism of action of EGCG in management of cancer through modulating cell signalling pathways.
Pathogenesis Types of Genes Mechanism Refs.
Inflammation Necrosis factor (TNF)-
a/intercellular adhesion
molecule-1 expression
EGCG protected against tumor necrosis factor-a-mediated lung inflammation through downregulation
of oxidative stress and intercellular adhesion molecule-1 expression
75
Breast cancer Vascular endothelial
growth factor
EGCG have been proven to reduce vascular endothelial growth factor production 76
Breast cancer HIF-1a and NF?B EGCG inhibited the activation of HIF-1a and NF?B and VEGF expression 76
Laryngeal carcinoma
cells/ Colon carcinoma
cells/Cervical carcinoma
cells
Apoptosis EGCG was found to induce apoptosis in cells of the examined neoplastic cell lines in a dose-related
manner
77
Hepatocellular
carcinoma
Bcl-2 and NF-?B EGCG-induced apoptosis of cancer cells was linked with a substantial decrease in Bcl-2 and NF-?B
expression
78
Human prostate cancer p53 Epigallocatechin-3-gallate, activate p53 via acetylation at the Lys373 and Lys382 residues through
inhibiting class I HDACs
79
Lung cancer p53 EGCG play crucial role in the inhibition of anchorage-independent growth of human lung cancer cells
through upregulating p53 expression
80
Pancreatic cancer Pten EGCG is capable of decreasing proliferation and induce the apoptosis linked with the expression of
PTEN.
81
Pancreatic cancer Pten EGCG upregulate
PTEN expression and downregulate the expression of pAKT and p-mTOR
81
Pancreatic cancer PI3K/Akt/mTOR pathway EGCG subdue the expression of p-Akt and p-mTOR through PTEN to regulate the PI3K/Akt/mTOR
pathway
82
Biliary tract cancer p21 EGCG reduced the mRNA levels of various cell cycle-related genes, but enhanced the expression of
the cell cycle inhibitor p21
83
Page 26 of 43
5. Conclusion
In this study, 186 phytoconstituents and two standard drugs were attached to 2ITY and 3W2O proteins.
The phytoconstituent EGCG did well with both proteins, but it worked best with the 3W2O protein. MD
simulation studies have also shown this to be true. So, the effect of EGCG is different for each type of
NSCLC. The fact that EGCG can change whether or not EGFR is phosphorylated 5 seems to be directly
linked to the fact that it can stop the EGFR signalling pathway from working. Overall, this makes EGCG
a possible drug to help treat lung cancer.
6. References
(1) Rupasinghe, H.P.V.; Nair, S.V.G.; Robinson, R.A. Chemopreventive properties of fruit phenolic
10 compounds and their possible mode of actions. Stud. Nat. Prod. Chem. 2014, 42, 229–266.
(2) Adlercreutz, H.; Mazur, W. Phyto-oestrogens and Western diseases. Ann. Med. 1997, 29, 95–120.
(3) Kunnathur Murugesan Sakthivel, Javadi Monisha, Ajaikumar B. Kunnumakkara and
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We Claims:

1. A Method of Possible computational insights and a novel atomic-level viewpoint on the EGFR inhibition of Epicathechin Gallate in Lung cancer activity comprising the steps of:
Preparing of Ligands by Chem Create is used to draw the ligand structures, and Open Babel in PyRx is used to do energy reduction using UFF (universal force field);
using LigPrep application (Trial version of Schrödinger); In this process, OPLS 2005 force field is used for energy optimization for all ligands until 0.01 Å RMSD cutoff not obtained; generating Tautomers and all possible ionization states using Epic at target pH of 7±2 and low energy ring conformation for each ligand was also generated. Prepared ligands were saved as a maestro output format.
2. The method as claimed in claim 1, wherein the theoretical binding mode of synthesized compounds to EGFR using the trial version of Schrodinger’s Glide module software; and the target protein is derived from the RCSB protein data bank (PDB).
3. The method as claimed in claim 1, wherein Crystal's structure of the EGFR tyrosine kinase domain with Iressa (PDB code 2ITY) at resolution 3.42 Å and EGFR tyrosine Kinase domain T790M/L858R Mutant with TAK-285 (PDB code 3W2O) at resolution 2.35 Å imported into Glide software; wherein to refine the structure minimization till RMSD constraint value of 0.3 Å was done using OPLS-2005 as force field after optimization of hydrogen bonds; and Grid box dimensions are 50 × 50 × 50 (all in Å) with a 0.375 Å grid point spacing; and Protein residues that include ligand-restricting sites are logically expected to contain certain ligands that can bind reversibly; and the correct orientation for other protein-affirming amino acid residues was provided.
4. The method as claimed in claim 1, wherein Each EGFR protein is produced using the Protein Preparation Wizard of the Schrodinger Suite 2015 and then imported as the starting structure into molecular dynamics software; and One chain containing an inhibitor had been preserved for each EGFR crystal complex with multiple chains; Desmond 4.2 is used for each MD simulation; and the TIP4P water model was used to generate a solvent system, which would operate well with the OPLS force field.
5. The method as claimed in claim 1, wherein Each EGFR protein was produced using the Protein Preparation Wizard of the Schrodinger Suite 2015 and then imported as the starting structure into molecular dynamics software; and One chain containing an inhibitor had been preserved for each EGFR crystal complex with multiple chains. Desmond 4.2 is used for each MD simulation; and The TIP4P water model is used to generate a solvent system, which would operate well with the OPLS force field.
6. The method as claimed in claim 1, wherein all of the prepared systems were first minimised using the steepest-descent (SD) method until a gradient threshold (25 kcal/mol/) was reached, and then connected to a Berenson thermostat with a 3000 K reference temperature and a Berendsen barostat with a 1.01325 bar reference pressure; and the particle mesh Ewald method was used to calculate long-range electrostatics; and the coulomb interaction cutoff is set at 9.0. After equilibration, all systems began to run for 2.4 ns in the NPT ensemble.

7. The method as claimed in claim 1, wherein all of the prepared systems were first minimized using the steepest-descent (SD) method until a gradient threshold (25 kcal/mol/) is reached, and then connected to a Berenson thermostat with a 3000 K reference temperature and a Berendsen barostat with a 1.01325 bar reference pressure; and the particle mesh Ewald method was used to calculate long-range electrostatics; and the coulomb interaction cutoff was set at 9.0. After equilibration, all systems began to run for 2.4 ns in the NPT ensemble.