Int J Pharm Pharm Sci, Vol 11, Issue 3, 66-71Original Article


MOLECULAR DOCKING STUDY OF SIX PYRIMIDINE DERIVATIVES AS EGFR (EPIDERMAL GROWTH FACTOR RECEPTOR) AND CA IX (CARBONIC ANHYDRASE IX) INHIBITOR

SHIKHA SHARMA, V. J. SHUKLA

*Department of Pharmaceutical Chemistry, IPGT and RA, Gujarat Ayurved University, Jamnagar
Email: shikhavishishth@gmail.com

Received: 05 Dec 2018 Revised and Accepted: 26 Jan 2019

ABSTRACT

Objective: The present study was carried out to discover whether these pyrimidine derivatives have the potential to be used as epidermal growth factor receptor (EGFR) and carbonic anhydrase (CA) IX inhibitors through structure-based in silico study.

Methods: Docking was performed on 6 pyrimidine analogs; cetuximab and curcumin were taken as reference drug. The structure of the target protein retrieved from the RCSB Protein databank and the protein-ligand docking was performed using Pyrx AutoDock wizard with MGL tools 1.5.6 by using Lamarckian algorithm.

Results: All the compounds have shown lower binding energy and inhibition constant (Ki) value than reference drug cetuximab and curcumin. Out of the 6 inhibitors analyzed vkh has shown minimum binding energy against the target protein EGFR and CA IX respectively. Smaller Ki value shows stronger interaction. The scoring value of the interaction of vkh i. e-10.74 and-9.93 Kcal/mol and Ki 13.17ɳM and 53.04ɳM against the target protein EGFR and CA IX respectively while the reference drug cetuximab has shown binding energy-6.09 Kcal/mol with Ki value 34.44 µM and curcumin has shown binding energy-6.02 kcal/mol with Ki value 38.60 µM.

Conclusion: It can be concluded that the molecule vkh could have potential to be used as an EGFR inhibitor and CA IX inhibitor.

Keywords: Docking, Pyrimidine, Pyrx, Molecule

© 2019 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
DOI: http://dx.doi.org/10.22159/ijpps.2019v11i3.31183

INTRODUCTION

Pyrimidines are the most vital heterocyclic aromatic organic compound containing two nitrogen atoms at positions 1 and 3 of the six-membered ring which shows the wide range of biological activities. These are the essential constituent of all cells and thus of all living matter. DNA and RNA is the main component of the chromosome carrying genetic information contain pyrimidine base in cytosine, uracil, and thymine. These occur in nature in two forms glycosylated pyrimidines and unglycosylated pyrimidines. Unglycosylated pyrimidines such as amino acids (ecotine), Vitamines (B1), antibiotics (Becimethrin, Bleomycin) and quinazoline alkaloids while glycosylated as DNA and RNA [1]. Pyrimidine derivatives have been reported as anti-neoplastic, anti-malarial, diuretic, cardiovascular agents [2]. Condensed pyrimidine derivatives have been reported as antimicrobial [3, 4], analgesic, anti-convulsants, anti-inflammatory [5], antibacterial, antitubercular [6], antifungal [7], and anti-tumor [8] agents. Anti-breast cancer activity of some novel pyrimidine derivatives has been also reported [9]. Various activities displayed by these nitrogen-containing heterocyclic rings such as pyrimidine made it, promising structural moiety for future drug design.

Cancer is the most dreadful disease affecting the human population these days. It is characterized by abnormal growth of the cells. Different mechanisms account for the cytotoxic effect of pyrimidines, where they had been reported to act as glycogen synthase kinase (GSK) inhibitors [10], cyclin-dependent kinase (CDK) inhibitors [11], dual src/Ab1 kinase inhibitors, epidermal growth factor receptor (EGFR) inhibitors [12] and carbonic anhydrase (CA) [13]. In the present study, docking is performed against two macromolecules i.e. EGFR and CA IX. FDA approved EGFR as the successful target in colorectal cancer and various other types of cancers (as shown in table 1). The CA IX is reported to be associated with tumorigenesis being highly overexpressed in hypoxic tumors and restrictedly expressed in normal tissues. CA IX monoclonal antibody is already in Phase III clinical trials (as shown in table 1) and several small molecule inhibitors are in advanced preclinical evaluation with its overexpression in many cancer tissues and not in their normal counterparts [14]. This study includes the molecular docking study of the pyrimidine derivatives with EGFR and CA IX. All of the compounds were auto docked for the inhibition of the EGFR and CA IX. Cetuximab was taken as the reference drug in case of EGFR and curcumin for CA IX. This paper aims to elucidate the anti-cancer molecular mechanism of pyrimidines and provide the reference for its clinical application and further drug development.

Table 1: TTD ID of targeted receptor

TTD ID Target name Target type Disease Drugs
TTDS00355 Epidermal growth factor receptor Successful target Colorectal cancer, Cancers Cetuximab
TTDR00211 Carbonic anhydrase IX Clinical trial target Cancer, Discovery agent Curcumin

MATERIALS AND METHODS

Procurement of sample

The samples were procured from Saurashtra University and code name was given to each of the molecule vkb, vkc, vkd, vkf, vkg, and vkh respectively. The “IUPAC” name of samples was obtained by using ChemDraw online while the molecular properties were generated by using ACD chemsketch (freeware) 2015 2.5(as shown in table 2 and 3).

Preparation of protein

Three-dimensional structure of the protein should be retrieved from the RCSB Protein data bank (PDB); afterward the retrieved structure should be pre-processed for removal of heteroatoms then energy minimization was performed by using Argus lab and visualization was done by using UCSF Chimera 1.11.2. Ramachandran plot was generated by using Discovery studio 3.5.

Table 2: Chemical structure, code name and IUPAC name of compounds

Structure Code IUPAC Name
vkb "4-(3,4-dimethoxyphenyl)-6-methyl-N-(4,5,6,7-tetrahydro-3al5,7al5-
benzo[d]thiazol-2-yl)-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide",
vkc "4-(3-bromophenyl)-6-methyl-N-(4,5,6,7-tetrahydro-3al5,7al5-
benzo[d]thiazol-2-yl)-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide"
vkd "6-methyl-4-(3-nitrophenyl)-N-(4,5,6,7-tetrahydro-3al5,7al5-
benzo[d]thiazol-2-yl)-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide
vkf "4-(1H-indol-3-yl)-6-methyl-N-(4,5,6,7-tetrahydro-3al5,7al5-
benzo[d]thiazol-2-yl)-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide",
vkg "4-(4-hydroxyphenyl)-6-methyl-N-(4,5,6,7-tetrahydro-3al5,7al5-
benzo[d]thiazol-2-yl)-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide
vkh "4-(anthracen-9-yl)-6-methyl-N-(4,5,6,7-tetrahydro-3al5,7al5-
benzo[d]thiazol-2-yl)-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide

Preparation of ligand

Ligands can be retrieved from several databases such as ZINC, PubChem or can be sketched by applying the Chemsketch tool. Ligand 2D structures were drawn using ACD chemsketch (freeware) 2015 2.5. Chem 3D viewer was used to convert the 2D structure into 3D. The drug molecules of cetuximab and curcumin were collected in 3D SDF format from the PubChem database. The compounds were added hydrogens and energy minimized with UFF force field using conjugate gradient algorithm by open babel in pyrx. All structures were saved as the pdb file format for input to pyrx 0.8. All the ligand structures were then saved in Pdbqt file format, for input into AutoDock version. Later, all lead molecules were converted into Auto Dock Pdbqt format.

While picking out the ligand, the LIPINSKY'S RULE OF 5 should be utilized. Lipinski rule of 5 assists in discerning amongst non-drug like and drug-like candidates. It promises the high chance of success or failure due to drug-likeness for molecules abiding by with 2 or more than of the complying rules. For the choice of a ligand allowing to the LIPINSKY'S RULE: (1) Less than five hydrogen bond donors (2) Less than ten hydrogen bond acceptors (3) Molecular mass of less than 500 Da (4) High lipophilicity (not over 5) and (5) Molar refractivity should be between 40-130. The rule is important in the drug discovery process to ensure the selectivity of the compound or determine if a chemical compound has physical or chemical properties that would make it likely orally active.

Docking

Ligand was docked against the protein and the interactions were analyzed by using by pyrx 0.8. For the docking of ligands into protein active site and to estimate the binding affinities of docked compounds, an advanced molecular docking program AutoDock Vina (4) was used in this study. All computational studies were carried out using pyrx AutoDock wizard with MGL tools 1.5.6 installed in a Pentium ®Dual–Core CPU T4200 machine running on a 2.0 GHz Intel core processor with 2GB RAM by using the lamarckian algorithm. The scoring function gives the score on the basis of best-docked ligand complex is picked out.

RESULTS

EGFR and CA IX is a clinically validated target for the treatment of various types of cancer and tumors has received considerable interest from the scientist in the design and development of newer anticancer drugs. The EGFR family plays an essential role in normal organ development by mediating morphogenesis and differentiation through effects on cell proliferation, differentiation, apoptosis, invasion, and angiogenesis [15, 16]. Whereas the CA from a family of enzymes that catalyze the interconversion between carbon dioxide and water and the dissociated  ions of carbonic acid (i.e. bicarbonate and protons) and leads to regulation of tumor microenvironment. This interconversion is a reversible reaction and the enzyme catalyzes both reactions, forward and reverse. The active site of most carbonic anhydrases contains a zinc ion; they are therefore classified as metalloenzymes [17]. This study is primarily concerned with the in silico molecular docking of six pyrimidine derivatives with EFGR and CA IX proposing their role as an inhibitor in cancer studies. These compounds are tested in silico for drug-likeness and anticancer activity by docking with the protein via pyrx docking software.

Table 3: Molecular properties of the compounds

Molecular properties vkb vkc vkd vkf vkg vkh
Molecular
formula		 C21H24N4
S2O2		 C19H19Br4
OS2		 C19H19N5
O3S2		 C21H21N5
OS2		 C19H20N4
S2O2		 C27H24N4
OS4
Composition C(56.73%)
H(5.44%)
N(12.6%)
O(10.80%)
S(14.44%)

C(49.24%),

H(4.13%),

N(12.1%),

O(3.45%),

S(13.84%)
Br(17.24%)

 C(53.13%)
H(4.46%)
N (16.3%)
O(11.17%)
S(14.93%)		 C(59.55%)
H(5.00%)
N(16.5%)
O(3.78%)
S(15.14%)		 C(56.98%)
H(5.03%)
N(13.9%)
O(7.99%)
S(16.01%)		 C(66.91%)
H(4.99%)
N(11.5%)
O(3.30%)
S(13.23%)
Molar
refractivity		 119.95±
0.4 cm³		 114.94±
0.4 cm³		 113.26±
0.4 cm³		 118.70±
0.4 cm³		 108.75±
0.4 cm³		 142.34±
0.4 cm³
Molar
value		 319.9±5.0c
m³		 289.2±5.0c
m³		 287.6±5.0c
m³		 289.3±5.0
cm³		 273.3±5.0c
m³		 342.1±5.0
cm³
Parachor 927.9±6.0c
m³		 861.7±6.0c
m³		 867.7±6.0c
m³		 876.9±6.0
cm³		 825.9±6.0c
m³		 1020.5±6.0 cm³
Index of
refraction		 1.673±0.03 1.725±0.03 1.717±0.03 1.757±0.03 1.726±0.03 1.771±0.03
Surface
tension		 70.7±5.0
dyne/cm³		 78.7±5.0
dyne/cm³		 82.8±5.0
dyne/cm³		 84.4±5.0
dyne/cm³		 83.3±5.0
dyne/cm³		 79.1±5.0
dyne/cm³
Density 1.38±o.1
g/cm³		 1.60±o.1
g/cm³		 1.49±o.1
g/cm³		 1.48±o.1
g/cm³		 1.46±o.1
g/cm³		 1.41±o.1
g/cm³
Polarisabi lity 47.55±0.5
1024cm³		 45.56±0.5
10² cm³		 44.90±0.5
1024cm³		 47.06±0.5
1024cm³		 43.11±0.5
1024cm³		 56.42±0.5
1024cm³
RDBE 12 12 13 14 12 18
Monoisotr
opic mass		 444.12898
1Da		 462.01835
8Da		 429.09292
9Da		 423.11875
Da		 400.10276
6Da		 484.13915
1Da
Nominal mass 444Da 462Da 429Da 423Da 400Da 484Da
Avg. mass 444.5703Da 463.4144Da 429.5159Da 423.5543Da 400.5177Da 484.6357Da
m+ 444.12843
2Da		 462.01780
82Da		 429.09238
1Da		 423.11820
2Da		 400.10221
7Da		 484.13860
3Da
m- 444.12952
9Da		 462.01890
5Da		 429.09347
8Da		 423.11929
9Da		 400.10331
5Da		 484.1397
Da
(m+H)⁺ 445.13625
7Da		 463.25633
Da		 430.10020
6Da		 424.12602
7Da		 401.11004
2Da		 485.14642
8Da
(m+H)⁻ 445.13735
4Da		 463.2673
Da		 430.10130
3Da		 424.12712
4Da		 401.11114
Da		 485.14752
5Da
(m-H)⁺ 443.12060
7Da		 461.00998
3Da		 428.08456
6Da		 422.11037
7Da		 399.09439
2Da		 483.13077
8Da
(m-H)⁻ 443.12170
4Da		 461.01108
Da		 428.08566
3Da		 422.11147
4Da		 399.09548
9Da		 483.13187
5Da

Table 4: Results of the lipinski rule calculator

Compound Mass (Da) H bond donor H bond acceptors LOG P Molar refractivity
Vkb 444 1 6 4.7463 124.68478
Vkc 420 1 4 5.548411 117.3252
Vkd 421 1 6 4.56482 114.10259
Vkf 420 3 4 0.854811 115.55609
Vkg 394 2 5 2.0183 110.24445
Vkh 482 2 4 4.73961 138.71896

Table 5: Output of molecular docking score of ligand-EGFR with respect to minimum binding energy and inhibition constant

S. No. Name Run Minimum binding energy (Kcal/mol) Inhibition constant
1 vkb 6 -6.63 13.71 µM
2 vkc 9 -8.03 1.29µM
3 vkd 5 -7.77 2.03 µM
4 vkf 8 -8.7 419.55 ɳM
5 vkg 5 -7.7 2.26 uM
6 vkh 5 -10.75 13.17ɳM
7 Cetuximab 4 -6.09 34.44 µm

The Protein-Ligand interaction plays a significant role in structural based designing. All the compounds have shown ≥5 hydrogen bond donors, ≥10hydrogen bond acceptors, molecular mass ≥500 Da, highly lipophilic in nature and molar refractivity between 40-130 (except for vkh has shown molar refractivity of 138.71) as shown in table 4. The rule describes molecular properties imperative for drug's pharmacokinetics in the human body, including their absorption, distribution, metabolism, and excretion.

Docking studies of pyrimidine derivatives with both the receptors revealed that all the compounds have lower binding energy and inhibition constant (Ki) value than reference drugs. 3-D structure of EGFR, Ramachandran plot of EGFR and various docked conformations and active site interactions with EGFR have been shown in fig. 1 and 3-D structure of CA IX, Ramachandran plot of CA IX and various docked conformations and active site interactions with CA IX have been shown in fig. 2. Further details of important interactions in terms of energies between ligands and the proteins with EGFR and CA IX receptor with predicted Ki values are shown in the table is given in table 5 and 6.

Table 6: Output of molecular docking score of ligand-CA IX with respect to minimum binding energy and inhibition constant

S. no Name Run Minimum binding energy (Kcal/mol) Inhibition constant
1 vkb 7 -7.83 1.82 µM
2 vkc 9 -9.47 114.57 ɳM
3 vkd 9 -7.17 5.55 µM
4 vkf 5 -8.4 690.47ɳM
5 vkg 5 -9.39 131.71 ɳM
6 vkh 9 -9.93 53.04 ɳM
7 Curcumin 4 -6.02 38.60 µM

Fig. 1: (a) 3D structure of EGFR; (b) Ramachandran plot of EGFR; (c) Docking of EGFR Protein receptor with Cetuximab; (d) Docking of EGFR Protein receptor with vkb; (e) Docking of EGFR Protein receptor with vkc; (f) Docking of EGFR Protein receptor with vkd; (g) Docking of EGFR Protein receptor with vkf; (h) Docking of EGFR Protein receptor with vkg; (i) Docking of EGFR Protein receptor with vkh

DISCUSSION

The EGF is the prototype of a large family of peptide ligands that bind to cell membrane receptors and activate a myriad of intracellular signaling pathways to control tumor cell growth, proliferation, survival, metastasis, and angiogenesis [18]. Whereas CA IX enzymes catalyze a very simple physiological reaction, the interconversion between carbon dioxide and the bicarbonate ion, and are thus involved in crucial physiological processes connected with respiration and transport of CO2/bicarbonate, pH and CO2 homeostasis, electrolyte secretion in a variety of tissue/organs, biosynthetic reactions, bone resorption, calcification, tumorigenicity and many other physiologic or pathologic processes [19]. In this work, totally 6 compounds which are pyrimidine derivatives were examined for ligand-based docking. The ligands are screened for their ability to dock within the active site of the inhibitor protein. All the compounds have shown ≥5 hydrogen bond donors, ≥10hydrogen bond acceptors, molecular mass ≥500 Da, log P value ≥5 and molar refractivity between 40-130 (except for vkh has shown molar refractivity of 138.71) which means compounds have good oral bioavailability. The Ramachandran plot generated displays the dihedral angles φ and ψ for each residue in the protein that is displayed in the workspace. The plot area displays a plot of protein dihedrals for all residues in the protein. The area "green region" corresponds to "core" region representing the most favorable combinations. Ideally, 90% of the residues should be in this “core” region. After analyzing the different docking interactions of ligands, out of the 6 inhibitors analyzed vkh has shown the binding energy of-10.74 and-9.93 Kcal/mol and Ki 13.17ɳM and 53.04ɳM against the target protein EGFR and CA IX respectively. The best drug was selected, depending upon the binding energy and Ki. Smaller Ki value shows the stronger interaction [20, 21]. Out of the 6 derivatives compound, vkh shows the highest affinity towards the protein compared with the standard drug cetuximab and curcumin. Thus compound vkh may act as a better and efficient anticancer drug.

Fig. 2: (a) 3D structure of CA IX; (b) Ramachandran plot of CA IX; (c) Docking of CA IX Protein receptor with Cetuximab; (d) Docking of CA IX Protein receptor with vkb; (e) Docking of CA IX Protein receptor with vkc; (f) Docking of CA IX Protein receptor with vkd; (g) Docking of CA IX Protein receptor with vkf; (h) Docking of CA IX Protein receptor with vkg; (i) Docking of CA IX Protein receptor with vkh

 

CONCLUSION

EGFR kinase domain and CA IX have been emphasized in the majority of cancers including colorectal cancer, breast, lung, ovarian, anal cancers, and tumors etc. Owing to their dominant role in cancer, molecular docking of EGFR and CA IX against the pyrimidine molecule was carried out. Based upon the results obtained from the docking study in the present study all the compounds may be suggested as the potential therapeutic drug for the treatment of cancer. However, vkh has a strong interaction and binding with target protein as evident by docking score. Further in vitro and in vivo experimental work is required for validation of our in silico results and to generate more effective and potential drug through ligand-based drug designing approaches.

ACKNOWLEDGMENT

Last but not the least, the author expresses sincere thanks to the Institute of Post Graduate Teaching and Research in Ayurveda, Gujarat Ayurved University and Saurashtra University for their encouragement, co-operation, support, suggestions, and affection throughout my studies.

AUTHORS CONTRIBUTIONS

This work is carried out by Shikha Sharma under the valuable guidance of Dr. V. J. Shukla sir. He guided me and helped me throughout the study.

CONFLICT OF INTERESTS

All the authors declared that there is no conflict of interest

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