Department Of Pharmaceutical Chemistry, Madras Medical College, Chennai, Tamil Nadu, India.
Colorectal cancer (CRC) is a major global health concern, ranking as the third most commonly diagnosed cancer and the second leading cause of cancer-related mortality. Despite current therapeutic options, the prognosis remains poor, necessitating the discovery of novel, effective, and targeted treatments. The AXL tyrosine kinase receptor, overexpressed in several cancers including CRC, plays a pivotal role in tumor progression, metastasis, and resistance to apoptosis, making it a promising therapeutic target.This study aims to design and evaluate novel pyrimidine derivatives as potential AXL tyrosine kinase inhibitors using a comprehensive computational and experimental approach. A virtual library of 100 pyrimidine-based ligands was constructed using ChemSketch and subjected to molecular docking against the AXL tyrosine kinase receptor (PDB ID: 5U6C) using AutoDock Vina. Ramachandran plot analysis confirmed the stability of the target protein, and binding site prediction ensured accuracy in docking. ADMET profiling using Swiss ADME and Osiris Property Explorer indicated that selected ligands possessed favorable pharmacokinetic properties and were non-toxic. Top-performing ligands demonstrated superior binding affinity compared to the standard drug 5-fluorouracil. Hydrogen bonding and hydrophobic interactions were visualized to understand ligand-receptor binding mechanisms. Selected lead compounds are currently being synthesized for further in-vitro evaluation using the HCT-116 colorectal cancer cell line. This integrative study highlights the potential of pyrimidine derivatives as AXL tyrosine kinase inhibitors and paves the way for the development of effective, targeted therapies against colorectal cancer.
Colorectal cancer (CRC) ranks as the third most common cancer globally and is the second leading cause of cancer-related deaths (1). Despite advancements in diagnostics and treatment, the high mortality rate underscores the need for novel therapeutic agents. CRC progression is influenced by genetic mutations, chromosomal instability, and epigenetic alterations, leading to uncontrolled cell proliferation and metastasis (2). One promising therapeutic target in CRC is the AXL tyrosine kinase receptor, part of the TAM family (Tyro3, AXL, Mer). AXL is overexpressed in various cancers and promotes tumor progression, metastasis, immune evasion, and resistance to apoptosis. Its inhibition has shown potential in reducing tumor burden and enhancing anti-tumor immune responses. Studies by Xu et al. (2021) and Inoue et al. (2021) have reported promising small-molecule AXL inhibitors with significant anticancer activity (3). Pyrimidine, a versatile heterocyclic scaffold, has shown broad pharmacological activity, including anticancer effects. Min Jung Choi et al. (2018) designed aminopyrimidine derivatives targeting AXL kinase, demonstrating potent activity in sub micromolar ranges (4). Similarly, Siddharth et al. (2018) used 3D-QSAR analysis to correlate pyrimidine structural features with AXL kinase inhibition, supporting their application in drug design (5). Advancements in computer-aided drug design (CADD) enable efficient screening of compounds using docking, ADME prediction, and toxicity profiling. Elizabeth et al. (2022) highlighted the value of structure-based and ligand-based design in accelerating drug discovery (6). This study focuses on designing and synthesizing novel pyrimidine derivatives targeting AXL kinase in CRC. It integrates in silico modeling, ADMET prediction, molecular docking, synthesis, and in-vitro evaluation to develop potent, selective, and non-toxic anticancer agent.
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Bottom of Form
MATERIALS AND METHODS
Ligand preparation
ChemSketch (freeware version) (7) was used to draw and optimize 100 novel chemical structures to design a total of 100 novel ligands. The ligands were stored in mol format and further energy minimized by Chem3D (8). We converted the minimized structures into. pdbqt format for molecular docking studies using Auto Dock Tools (ADT).
Ramachandran Plot Analysis
The Ramachandran plot analysis confirmed the structural reliability of 5U6C, with most residues positioned in the favored regions, ensuring accurate docking results (9).
Receptor preparation
The crystal structures of AXL tyrosine kinase (PDB ID: 5U6C) were downloaded from the Protein Data Bank (10). These structures were selected due to their high resolution (approximately 2 Å) and determination by the reliable X-ray diffraction method. Receptors were prepared by removing water molecules, adding polar hydrogens, and assigning Kollman charges using Auto Dock Tools.
ADMET Prediction
The absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties of the ligands were evaluated using Swiss ADME (11) and OSIRIS Property Explorer (12). These tools predicted drug-likeness, bioavailability, and toxicity, ensuring the ligands adhered to Lipinski's rule of five and exhibited favorable pharmacokinetic profiles.
Molecular docking
Docking studies were performed using AutoDock Vina 1.5.6 (13). The active sites of AXL tyrosine kinase and 5U6C were defined based on their co crystallized ligands. The grid box dimensions and coordinates (x, y, z) for the docking simulations were determined using the CB-Dock server (14), which predicts the binding pockets and centers the grid box around the identified active site residues. This automated approach ensured optimal coverage of the ligand-binding site and included all key residues critical for each ligand was docked against both receptors, and binding affinities were recorded as binding energies (kcal/mol).
Visualization and Interaction Analysis
Binding interactions of the docked ligands with the receptors were analyzed using Molegro Molecular Viewer (MMV) (15). Hydrogen bonds, hydrophobic interactions, and π-π stacking interactions were identified. The top-scoring ligands were visualized and compared with standard drugs, correlating to the interaction pattern with the active site residues.
RESULTS AND DISCUSSION
Ramachandran Plot
The Ramachandran plot analysis results indicate that the selected protein, AXL tyrosine kinase with (PDB ID:5U6C), predominantly (more than 90%) have their amino-acid residues situated within the most favored region as represented in the fig no 1.
Figure No 1: Ramchandran Plot For 5U6C
Novelty Assessment
The novelty of the designed ligands was checked by using the ZINC 15 and PUBCHEM database. The outcomes were shown below.
Table No 1: Ligands Showing Novelty
|
Novel Ligands |
Already existing compounds |
|
A1,A2,A3,A4,A5,A6,A7,A8,A9,A10,A11,A12,A13,A14,A15,A16,A17,A18,A19,A20,A21,A22,A23,A25,A26,A28,A29,A30,A33,A34,A35,A36,A38,A39,A40,A41,A43,A44,A46,A47,A48,A49,A51,A52,A54,A55,A56,A57,A59,A60,A62,A63,A64,A65,A66,A67,A70,A71,A72,A73,A74,A75,A76,A78,A79,A80,A81,A82,A83,A84,A86,A87,A88,A89,A91,A92,A95,A96,A97,A99,A100,A101,A103,A104,A105,A107. |
A24,A27,A31,A32,A37,A42,A45,A50,A53,A58,A61,A68,A69,A77,A85,A90,A93,A94,A98,A102,A106,A108. |
In Silico ADMET
Based on the novelty results, the developed ligands are evaluated for the drug-likeness and toxicity property by using the Swiss ADME Online Software tool and the Osiris Property Explorer. Mutagenicity, tumorigenicity, irritant, reproductive are presented by M, T, I and R respectively.
Table No 2: ADMET Properties of Novel Ligands
|
Lig No |
M |
T |
I |
R |
Log P |
Mol Wt. |
HBD |
HBA |
Rule Of 5 |
|
1 |
No |
No |
No |
No |
2.17 |
285.36 |
1 |
3 |
0 |
|
2 |
No |
No |
No |
No |
2.26 |
234.30 |
1 |
4 |
0 |
|
3 |
No |
No |
No |
No |
1.54 |
216.24 |
1 |
4 |
0 |
|
4 |
No |
No |
No |
No |
2.06 |
241.29 |
1 |
3 |
0 |
|
5 |
No |
No |
No |
No |
1.84 |
271.34 |
1 |
3 |
0 |
|
6 |
No |
No |
No |
No |
1.64 |
220.27 |
1 |
4 |
0 |
|
7 |
No |
No |
No |
No |
1.08 |
202.21 |
1 |
4 |
0 |
|
8 |
No |
No |
No |
No |
1.71 |
227.26 |
1 |
3 |
0 |
|
9 |
No |
No |
No |
No |
1.72 |
313.37 |
1 |
4 |
0 |
|
10 |
No |
No |
No |
No |
1.55 |
262.31 |
1 |
5 |
0 |
|
11 |
No |
No |
No |
No |
1.02 |
244.25 |
1 |
5 |
0 |
|
12 |
No |
No |
No |
No |
1.68 |
269.30 |
1 |
4 |
0 |
|
13 |
No |
No |
No |
No |
1.06 |
314.36 |
2 |
4 |
0 |
|
14 |
No |
No |
No |
No |
0.94 |
263.30 |
2 |
5 |
0 |
|
15 |
No |
No |
No |
No |
0.32 |
245.24 |
2 |
5 |
0 |
|
16 |
No |
No |
No |
No |
0.88 |
270.29 |
2 |
4 |
0 |
|
17 |
No |
No |
No |
No |
2.16 |
299.35 |
1 |
4 |
0 |
|
18 |
No |
No |
No |
No |
1.61 |
248.28 |
1 |
5 |
0 |
|
19 |
No |
No |
No |
No |
1.19 |
230.22 |
1 |
5 |
0 |
|
20 |
No |
No |
No |
No |
0.98 |
255.27 |
1 |
4 |
0 |
|
21 |
No |
No |
No |
No |
2.35 |
313.37 |
1 |
4 |
0 |
|
22 |
No |
No |
No |
No |
2.43 |
262.31 |
1 |
5 |
0 |
|
23 |
No |
No |
No |
No |
1.83 |
244.25 |
1 |
5 |
0 |
|
25 |
No |
No |
No |
No |
2.62 |
325.43 |
1 |
4 |
0 |
|
26 |
No |
No |
No |
No |
2.58 |
274.36 |
0 |
4 |
0 |
|
28 |
No |
No |
No |
No |
3.02 |
339.45 |
0 |
3 |
0 |
|
29 |
No |
No |
No |
No |
3.02 |
339.45 |
0 |
3 |
0 |
|
30 |
No |
No |
No |
No |
2.61 |
288.39 |
0 |
4 |
0 |
|
33 |
No |
No |
No |
No |
2.52 |
326.42 |
2 |
5 |
0 |
|
34 |
No |
No |
No |
No |
2.47 |
275.35 |
2 |
6 |
0 |
|
35 |
No |
No |
No |
No |
1.75 |
257.29 |
1 |
5 |
0 |
|
36 |
No |
No |
No |
No |
2.24 |
282.24 |
2 |
5 |
0 |
|
38 |
No |
No |
No |
No |
1.48 |
228.25 |
1 |
4 |
0 |
|
39 |
No |
No |
No |
No |
1.56 |
219.24 |
2 |
5 |
0 |
|
40 |
No |
No |
No |
No |
1.36 |
229.24 |
1 |
5 |
0 |
|
41 |
No |
No |
No |
No |
1.46 |
220.23 |
1 |
5 |
0 |
|
43 |
No |
No |
No |
No |
1.55 |
234.28 |
1 |
4 |
0 |
|
44 |
No |
No |
No |
No |
1.98 |
293.37 |
1 |
4 |
0 |
|
46 |
No |
No |
No |
No |
0.98 |
214.22 |
1 |
4 |
0 |
|
47 |
No |
No |
No |
No |
1.03 |
205.22 |
2 |
5 |
0 |
|
48 |
No |
No |
No |
No |
1.00 |
215.21 |
1 |
5 |
0 |
|
49 |
No |
No |
No |
No |
0.89 |
206.20 |
1 |
5 |
0 |
|
51 |
No |
No |
No |
No |
1.12 |
220.25 |
1 |
4 |
0 |
|
52 |
No |
No |
No |
No |
1.44 |
279.34 |
1 |
4 |
0 |
|
54 |
No |
No |
No |
No |
0.94 |
256.26 |
1 |
5 |
0 |
|
55 |
No |
No |
No |
No |
0.88 |
247.25 |
2 |
6 |
0 |
|
56 |
No |
No |
No |
No |
0.86 |
257.25 |
1 |
6 |
0 |
|
57 |
No |
No |
No |
No |
0.96 |
248.24 |
1 |
6 |
0 |
|
59 |
No |
No |
No |
No |
0.82 |
262.29 |
1 |
5 |
0 |
|
60 |
No |
No |
No |
No |
1.34 |
321.38 |
1 |
5 |
0 |
|
62 |
No |
No |
No |
No |
0.33 |
257.25 |
2 |
5 |
0 |
|
63 |
No |
No |
No |
No |
0.38 |
248.24 |
3 |
6 |
0 |
|
64 |
No |
No |
No |
No |
0.45 |
258.24 |
2 |
6 |
0 |
|
65 |
No |
No |
No |
No |
0.32 |
249.23 |
2 |
6 |
0 |
|
66 |
No |
No |
No |
No |
0.74 |
256.26 |
2 |
4 |
0 |
|
67 |
No |
No |
No |
No |
0.02 |
263.28 |
2 |
5 |
0 |
|
70 |
No |
No |
No |
No |
1.52 |
242.23 |
1 |
5 |
0 |
|
71 |
No |
No |
No |
No |
1.10 |
233.23 |
2 |
6 |
0 |
|
72 |
No |
No |
No |
No |
0.97 |
243.22 |
1 |
6 |
0 |
|
73 |
No |
No |
No |
No |
0.81 |
234.21 |
1 |
6 |
0 |
|
74 |
No |
No |
No |
N o |
1.73 |
241.25 |
1 |
4 |
0 |
|
75 |
No |
No |
No |
No |
1.14 |
248.25 |
1 |
5 |
0 |
|
76 |
No |
No |
No |
No |
1.56 |
307.35 |
1 |
5 |
0 |
|
78 |
No |
No |
No |
No |
1.77 |
256.26 |
1 |
5 |
0 |
|
79 |
No |
No |
No |
No |
0.96 |
247.25 |
2 |
6 |
0 |
|
80 |
No |
No |
No |
No |
1.55 |
257.25 |
1 |
6 |
0 |
|
81 |
No |
No |
No |
No |
0.81 |
248.24 |
1 |
6 |
0 |
|
82 |
No |
No |
No |
No |
2.00 |
255.27 |
1 |
4 |
0 |
|
83 |
No |
No |
No |
No |
1.63 |
262.29 |
1 |
5 |
0 |
|
84 |
No |
No |
No |
No |
1.50 |
321.38 |
1 |
5 |
0 |
|
86 |
No |
No |
No |
No |
2.15 |
268.29 |
1 |
5 |
0 |
|
87 |
No |
No |
No |
No |
1.95 |
259.31 |
2 |
6 |
0 |
|
88 |
No |
No |
No |
No |
1.72 |
269.30 |
1 |
6 |
0 |
|
89 |
No |
No |
No |
No |
1.99 |
260.30 |
1 |
6 |
0 |
|
91 |
No |
No |
No |
No |
2.24 |
274.34 |
1 |
5 |
0 |
|
92 |
No |
No |
No |
No |
2.27 |
333.43 |
1 |
5 |
0 |
|
95 |
No |
No |
No |
No |
1.80 |
273.33 |
2 |
6 |
0 |
|
96 |
No |
No |
No |
No |
1.91 |
283.33 |
1 |
6 |
0 |
|
97 |
No |
No |
No |
No |
2.04 |
274.32 |
1 |
6 |
0 |
|
99 |
No |
No |
No |
No |
2.20 |
288.37 |
1 |
5 |
0 |
|
100 |
No |
No |
No |
No |
2.41 |
347.46 |
1 |
5 |
0 |
|
101 |
No |
No |
No |
No |
3.62 |
341.43 |
0 |
3 |
0 |
|
103 |
No |
No |
No |
No |
2.99 |
375.81 |
2 |
5 |
0 |
|
104 |
No |
No |
No |
No |
2.30 |
358.39 |
2 |
4 |
0 |
|
105 |
No |
No |
No |
No |
3.67 |
356.44 |
0 |
4 |
0 |
|
107 |
No |
No |
No |
No |
2.63 |
368.39 |
2 |
5 |
0 |
Molecular Docking
The ligands with good drug likeness properties and no toxicity were selected for molecular docking studies against AXL tyrosine kinase receptor (PDB ID: 5U6C).
Table No 3: Binding scores of ligands
|
S no |
Compound code |
AXL tyrosine kinase receptor 5U6C |
|
1 |
A1 |
-8.32 |
|
2 |
A2 |
-7.42 |
|
3 |
A3 |
-6.73 |
|
4 |
A4 |
-7.3 |
|
5 |
A5 |
-8.3 |
|
6 |
A6 |
-6.9 |
|
7 |
A7 |
-6.73 |
|
8 |
A8 |
-7.55 |
|
9 |
A9 |
-7.92 |
|
10 |
A10 |
-8.01 |
|
11 |
A11 |
-8.54 |
|
12 |
A12 |
-8.05 |
|
13 |
A13 |
-8.05 |
|
14 |
A14 |
-8.52 |
|
15 |
A15 |
-7.68 |
|
16 |
A16 |
-8.28 |
|
17 |
A17 |
-9.79 |
|
18 |
A18 |
-7.45 |
|
19 |
A19 |
-6.82 |
|
20 |
A20 |
-7.41 |
|
21 |
A21 |
-9.21 |
|
22 |
A22 |
-7.58 |
|
23 |
A23 |
-7.29 |
|
24 |
A25 |
-8.99 |
|
25 |
A26 |
-8.3 |
|
26 |
A28 |
-8.27 |
|
27 |
A29 |
-8.47 |
|
28 |
A30 |
-8.26 |
|
29 |
A33 |
-8.74 |
|
30 |
A34 |
-7.86 |
|
31 |
A35 |
-7.46 |
|
32 |
A36 |
-8.74 |
|
33 |
A38 |
-7.41 |
|
34 |
A39 |
-6.77 |
|
35 |
A40 |
-7.38 |
|
36 |
A41 |
-8.24 |
|
37 |
A43 |
-7.33 |
|
38 |
A44 |
-6.76 |
|
39 |
A46 |
-7.42 |
|
40 |
A47 |
-8.84 |
|
41 |
A48 |
-8.3 |
|
42 |
A49 |
-7.83 |
|
43 |
A51 |
-7.41 |
|
44 |
A52 |
-8.77 |
|
45 |
A54 |
-8.75 |
|
46 |
A55 |
-8.42 |
|
47 |
A56 |
-9.03 |
|
48 |
A57 |
-8.48 |
|
49 |
A59 |
-7.91 |
|
50 |
A60 |
-7.07 |
|
51 |
A62 |
-7.92 |
|
52 |
A63 |
-6.61 |
|
53 |
A64 |
-7.02 |
|
54 |
A65 |
-7.27 |
|
55 |
A66 |
-7.35 |
|
56 |
A67 |
-7.68 |
|
57 |
A70 |
-6.84 |
|
58 |
A71 |
-9.22 |
|
59 |
A72 |
-8.27 |
|
60 |
A73 |
-7.01 |
|
61 |
A74 |
-7.97 |
|
62 |
A75 |
-8.24 |
|
63 |
A76 |
-8.45 |
|
64 |
A77 |
-8.12 |
|
65 |
A78 |
-9.0 |
|
66 |
A79 |
-7.64 |
|
67 |
A80 |
-8.57 |
|
68 |
A81 |
-8.65 |
|
69 |
A82 |
-7.86 |
|
70 |
A83 |
-8.7 |
|
71 |
A84 |
-8.56 |
|
72 |
A86 |
-8.32 |
|
73 |
A87 |
-8.4 |
|
74 |
A88 |
-8.08 |
|
75 |
A89 |
-7.23 |
|
76 |
A91 |
-6.9 |
|
77 |
A92 |
-7.09 |
|
78 |
A95 |
-7.36 |
|
79 |
A96 |
-7.89 |
|
80 |
A97 |
-8.36 |
|
81 |
A99 |
-7.21 |
|
82 |
A100 |
-8.8 |
|
83 |
A101 |
-9.38 |
|
84 |
A103 |
-10.1 |
|
85 |
A104 |
-11.11 |
|
86 |
A105 |
-10.66 |
|
87 |
A107 |
-10.2 |
|
88 |
5 Fluoro uracil |
-7.33 |
Table No 4: Chemical structures of top-performing ligands based on docking scores
|
Lig No |
Structure |
|
A5 |
|
|
A11 |
|
|
A13 |
|
|
A16 |
|
|
A26 |
|
|
A101 |
|
|
A103 |
|
|
A104 |
|
|
A105 |
|
|
A107 |
|
|
5-Fluorouracil |
|
Table No 5: Ligand-Receptor Binding Pose Visualisations
|
AXL Tyrosine Kinase Receptor |
|
|
A5 |
|
|
A11 |
|
|
A13 |
|
|
A16 |
|
|
A26 |
|
|
A101 |
|
|
A103 |
|
|
A104 |
|
|
A105 |
|
|
A107 |
|
|
5-Fluorouracil |
|
Table No 6: Ligand-Receptor Interactions
|
Lig Code |
Hydrogen Bonding Axl Tyrosine Kinase Receptor |
|
Lig 5 |
ASP 741 |
|
Lig 11 |
Met 647, ASP 741 |
|
Lig 13 |
ASP 678, PRO 672 |
|
Lig 16 |
ASP 678, Met 674 |
|
Lig 103 |
ASP 741 |
|
Lig 104 |
Met 650, LYS 619 |
|
Lig 107 |
PRO 672 |
|
5-Fluorouracil |
LEU 724, ASP 723 |
CONCLUSION
Colorectal cancer (CRC) remains one of the most prevalent and lethal malignancies worldwide, necessitating the development of novel therapeutic strategies. AXL tyrosine kinase, a member of the TAM receptor family, has emerged as a crucial target due to its role in tumor progression, metastasis, immune evasion, and resistance to conventional therapies. The current study was designed to explore the potential of novel pyrimidine-based derivatives as AXL tyrosine kinase inhibitors using an integrated approach of computational drug design, synthesis, and biological evaluation. Through structure-based design, a virtual library of pyrimidine derivatives was developed and screened using molecular docking to identify candidates with high binding affinity for the AXL receptor. Promising ligands exhibited favorable ADMET properties and non-toxic profiles as predicted by in silico tools. Selected compounds were synthesized and are intended for further in-vitro evaluation against the HCT-116 colorectal cancer cell line.
REFERENCES
Priyadharshini R., Archana S.*, Guhan G., Hanitha Mathanke J., Department of Pharmaceutical Chemistry, Madras Medical College, Chennai, Tamil Nadu, India, Int. J. of Pharm. Sci., 2025, Vol 3, Issue 6, 4443-4459. https://doi.org/10.5281/zenodo.15745638
10.5281/zenodo.15745638
