Cancer is a class of diseases or disorders characterized by uncontrolled division of cells and the ability of these cells to spread, either by direct growth into adjacent tissue through invasion, or by implantation into distant sites by metastasis (where cancer cells were transported through the bloodstream or lymphatic system). Cancer may affect people at all ages, but risk tends to increase with age. It is one of principal causes of death in developed countries.Carcinogenesis, which means the initiation or generation of cancer, is the process of derangement of the rate of cell division due to damage to DNA^[1].

Chronic myelogenous (or myeloid) leukemia (CML), also known as chronic granulocytic leukemia (CGL), is a form of leukemia characterized by the increased and unregulated growth of predominantly myeloid cells in the bone marrow and the accumulation of these cells in the blood. CML is a clonal bone marrow stem cell disorder in which proliferation of mature granulocytes (neutrophils, eosinophils, and basophils) and their precursors is the main finding. It is a type of myeloproliferative disease associated with a characteristic chromosomal translocation called the Philadelphia chromosome^[2].

Historically, it has been treated with chemotherapy, interferon and bone marrow transplantation, although targeted therapies introduced at the beginning of the 21st century have radically changed the management of CML.

Proto oncogenes were genes which promote cell growth and mitosis, a process of cell division, and tumor suppressor genes discourage cell growth, or temporarily halt cell division in order to carry out DNA repair. Typically, a series of several mutations to these genes were required before a normal cell transforms into a cancer cell.Proto-oncogenes promote cell growth through a variety of ways. Many can produce hormones, a "chemical messenger" between cells which encourage mitosis, the effect of which depends on the signal transduction of the receiving tissue or cells. Some were responsible for the signal transduction system and signal receptors in cells and tissues themselves, thus controlling the sensitivity to such hormones. They often produce mitogens, or were involved in transcription of DNA in protein synthesis, which creates the proteins and enzymes responsible for producing the products and biochemical cells use and interact with.

Mutations in proto-oncogenes can modify their expression and function, increasing the amount or activity of the product protein. When this happens, they become oncogenes, and thus, cells have a higher chance to divide excessively and uncontrollably. The chance of cancer cannot be reduced by removing proto-oncogenes from the genome as they were critical for growth, repair and homeostasis of the body. It is only when they become mutated that the signals for growth become excessive.

CML was the first malignancy to be linked to a clear genetic abnormality, the chromosomal translocation known as the Philadelphia chromosome. This chromosomal abnormality is so named because it was first discovered and described in 1960 by two scientists from Philadelphia, Pennsylvania. In this translocation, parts of two chromosomes (the 9th and 22nd by conventional karyotypic numbering) switch places. As a result, part of the BCR ("breakpoint cluster region") gene from chromosome 22 is fused with the ABL gene on chromosome 9^[3]. This abnormal "fusion" gene generates a protein of p210 or sometimes p185 weight (p is a weight measure of cellular proteins in kDa). Because ABL carries a domain that can add phosphate groups to tyrosine residues (a tyrosine kinase), the bcr-abl fusion gene product is also a tyrosine kinase.

CML is treated with inhibitors of tyrosine kinase, the first of which was imatinibmesylate (marketed as Gleevec or Glivec; previously known as STI-571). In the past, antimetabolites (e.g. cytarabine, hydroxyurea), alkylating agents, interferon alfa 2b, and steroids were used, but these drugs have been replaced by imatinib^[4,5]. Imatinib was approved by the United States FDA in 2001 and specifically targets Bcr/abl, the constitutively activated tyrosine kinase fusion protein caused by the Philadelphia chromosome translocation. It is better tolerated and more effective than previous therapies. Bone marrow transplantation was also used as initial treatment for CML in younger patients before the advent of imatinib, and while it can often be curative, there was a high rate of transplant-related mortality. The transplant-related mortality rate in the present is now less than 5%. A number of newer drugs are being used to treat the minority of patients who develop imatinib resistance. However trials, such as SPIRIT 2, are also underway to evaluate these newer drugs as 'upfront' therapy for patients with newly diagnosed chronic phase CML.

Imatinib is a 2-phenylaminopyrimidine derivative that functions as a specific inhibitor of a number of tyrosine kinase enzymes. It occupies the TK active site, leading to a decrease in activity.

To overcome imatinib resistance and to increase responsiveness of TK inhibitors, two novel agents have been developed^[6,7]. The first, dasatinib, is a TK inhibitor that blocks several oncogenic proteins and has been approved by the US FDA to treat CML patients who are either resistant to or intolerant of imatinib in 2007. Another TK inhibitor, nilotinib, is also approved by the US FDA for the same indication. Nilotinib is designed to bind more tightly than imatinib to the Bcr-Abl abnormal fusion protein responsible for chronic myeloid leukemia. Dasatanib and nilotinib failed to overcome the Imatinib resistance caused by the T315I mutation.

Most anticancer drugs are cytotoxic agents intended to selectively kill enough cancer cells that our immune system can mop up the remainder. Imatinib and other tyrosine kinase inhibitors are neat because their primary action is not to kill cancer cells, but rather to suppress their growth by selectively inhibiting the tyrosine kinases responsible for their existence^[8]. Imatinib is particularly good at shutting down BCR-Abl, making it a near-miracle drug for those with CML.

In the present study, we report few analoguesof imatinib that show higher affinity andbetter interaction with the Bcr-Abloncoprotein. Theseresults have been confirmed by the fitness scoresobtained from GOLD docking software.

MATERIALS AND METHODS:

Selection of target protein

Bcr-Abloncoprotein was retrieved from the RCSB Protein Data Bank (http://www.rcsb.org/pdb/) with PDB Id - 1K1F. Protein reports were obtained from RCSB Protein Data Bank (http://www.rcsb.org/pdb/) with crystallographic resolution of 2.20 A^o. Active site identification is primarily done by using online tools like RCSB Ligand Explorer and PDBsum.

Selection of Lead moiety and designing of ligands

A lead compound in drug discovery is a chemical compound that has pharmacological or biological activity and whose chemical structure is used as a starting point for modifications in order to improve potency, selectivity, pharmacokinetic properties.

CML is treated with inhibitors of tyrosine kinase, the first of which was imatinib. In the past, antimetabolites (e.g. cytarabine, hydroxyurea), alkylating agents, and interferon-α 2b, and steroids were used, but these drugs have been replaced by imatinib. Imatinib was approved by the United States FDA in 2001. Imatinib is particularly good at shutting down Bcr-Abl, making it a near-miracle drug for those with CML. so imatinib itself is taken as lead moiety.

Design of Ligands

30 ligands were designed from the Lead compound by modifying the non pharmacophoric parts like R1, R2, and R3. All the ligands were designed by using Chemsketch. These ligands were designed according to the SAR properties of tyrosine kinase inhibitors.

Major considerations during designing of ligands were given to increase the hydrophobic character of the molecule which was shown in Fig: 1. Thus modifications of R₃ in all ligands increase the hydrophobicity by the presence of higher N-ring structures like Pyrrolidine, Piperidine, indoline and hydroxylic, amine substitutions were made at R₂. For some ligands at R₁ introduction of –OH increases its Hydrophilic nature. Modifications were given in the Table: 1

Fig: 1: positions for the modifications of imitanib

Table: 1: newly designed imantinib analogs

S.N	R₁	R₂	R₃
1	CH₃	OH	1-chloro-4-methyl benzene
2	OH	CH₂OH	3-methyl phenol
3	CH₃	C₂H₄NH₂	3-methyl pyridine
4	H	C₂H₄NH₂	3-methyl phenol
5	CH₃	C₂H₅	3-methyl pyridine
6	H	CH₂OH	3-methyl phenol
7	NH₂	CH₃	3-methyl pyridine
8	CH₃	OH	3-methyl pyridine
9	CH₃	H	3-methyl phenol
10	OH	C₂H₅	3-methyl phenol
11	CH₃	CH₂OH	3-methyl pyridine
12	NH₂	CH₃	3-methyl pyridine
13	CH₃	CH₂OH	1-methyl piperidine
14	OH	H	1-methyl piperidine
15	CH₃	OH	1-methyl piperidine
16	CH₃	H	1-methylpyrrolidine
17	CH₃	C₂H₄NH₂	1-methyl pyrrolidine
18	CH₃	NH₂	1-methyl pyrrolidine
19	OH	C₂H₄NH₂	1-methyl piperidine
20	OH	OH	1-methyl piperidine
21	CH₂OH	CH₂OH	1-methyl-2,3,4,5,6,7-hexahydro-1 Hindole
22	CH₂OH	CH₃	1-methyl pyrrolidine
23	CH₂OH	H	1-methyl-2,3,4,5,6,7-hexahydro-1 H indole
24	CH₂OH	NH₂	1-methyl-2,3,4,5,6,7-hexahydro-1 H indole
25	CH₂OH	C₂H₄NH₂	1-methyl-2,3,4,5,6,7-hexahydro-1 H indole
26	CH₂OH	C ₂H₅	1-methyl-2,3,4,5,6,7-hexahydro-1 H indole
27	CH₃	C₂H₅	1-methyl pyrrolidine
28	H	H	3-methyl phenol
29	OH	NH₂	1-methyl piperidine
30	H	NH₂	3-methyl phenol

Energy minimization of protein and ligands

Energy minimization of protein and ligand is performed by using Hyperchem 8.0.

Protein - The protein Bcr-Abl oncogene (ID -1K1F) downloaded from PDB was prepared for docking by deleting all hetero atoms, ligands and water molecules and optimized by minimization of energy by using Hyperchem 8.0. Parameters were maintained as RMS Gradient-0.01 k.Cal/Mol, CHARMm- 27 force field with Polak-Ribiere (conjugate gradient) minimizer, in In Vacuoconditions^[9].

Ligands - Ligands designed by using Chemsketch were optimized by using Hyperchem 8.0 and parameters were maintained as RMS Gradient 0.01 k.Cal/Mol, OPLS force field, in vacco media and Polak-Ribiereminimizer. Later, all the optimized ligands were saved in .MOL format.

Molecular Properties

Molecular properties like surface area, volume, hydration energy, log p, mass, refractivity, polarizability, were studied by using Hyperchem 8.0, for all the newly designed 30 ligands and listed in Table: 2.

Toxicity studies

Toxicity profiles of ligands were studied by using an online tool, Osiris property explorer^[10]. Toxicity parameters like mutagenicity, carcinogenicity, irritancy and teratogenicity of the ligands were predicted.

Docking:

All the 30 ligands, including imatinib were docked with Bcr-Abl oncogene in the GOLD version 3.01. GOLD (Genetic Optimization for Ligand Docking) uses a genetic algorithm to explore wide range of ligand conformational flexibility and rotational flexibility of selected receptor hydrogen^[11]. The docking orientations were ranked based on a molecular mechanics–like scoring function known as Gold score. Leucine 59 of chain- A is set as active site residue. In the aspect of producing binding energy estimates, the Gold score function appears to be more reliable than the Chemscore function. The compound binding site was defined within a 10 Å radius around the binding sites of protein. Standard default parameter settings were used to evaluate the protein-ligand interactions^{[12, 13]}.

RESULTS AND DISCUSSIONS:

Toxicity profile

Toxicity profiles of ligands are studied by using an online tool, Osiris property explorer. By employing the various toxicity problems like mutagenicity, carcinogenicity, irritancy and teratogenicity of the designed set of 30 ligands and the results are tabulatedin the Table: 3

Nearly 20 ligands are not showing any carcinogenicity. Imatinib is not showing any teratogenicity and carcinogenicity. All the ligands other than 14^th ligand are showing irritancy. Ligands like 1, 3, 5, 8, 10, 11, 14, 19, 20, 22, 23, 25, and 26 are not showing any mutagenicity, carcinogenicity, teratogenicity but they are having irritant nature. Only 14^thligand is having teratogenicity. 18, 24, 29, 30 ligands are showing partial irritancy, ligand 4 is having mutagenicity and irritancy.Ligands like 2, 6, 12, 13, 18, 21, 24, 29, and 30 are showing mutagenicity, carcinogenicity, irritancy and teratogenicity.

Table: 2: molecular properties of the designed imatinib analogs

LIGAND	SURFACE AREA	VOLUME	HYDRATION ENERGY	LOGP	REFRACTIVITY	POLARIZABILITY	MASS
IMATINIB	699.40	1437.35	-8.52	-1.16	158.86	56.68	493.61
1	727.19	1472.35	-10.95	0.19	163.39	58.12	529.04
2	734.13	1522.69	-23.09	-0.51	165.84	59.30	540.62
3	728.04	1525.74	-12.46	-0.85	166.05	56.87	522.65
4	736.41	1519.33	-20.30	-0.37	166.63	59.38	523.64
5	700.29	1468.57	-8.10	0.64	162.46	58.52	507.64
6	697.63	1448.75	-21.20	0.34	159.78	56.83	510.60
7	717.84	1476.95	-14.79	0.76	163.43	58.03	508.62
8	665.00	11407.93	-14.55	-0.93	155.18	55.49	495.58
9	699.23	1429.06	-15.17	0.43	158.68	56.19	494.60
10	720.19	1495.47	-19.38	-0.27	169.03	58.67	524.62
11	693.00	1459.94	-12.40	-0.76	159.64	57.32	509.61
12	659.74	1422.52	-12.78	-1.62	157.13	157.13	157.13
13	763.50	1564.57	-10.55	o.25	163.94	59.96	529.69
14	658.93	1419.18	-11.32	-0.84	141.68	54.45	487.60
15	702.06	1462.22	-12.74	0.49	153.83	56.29	501.63
16	667.34	1399.71	-7.22	-0.06	147.75	53.81	471.61
17	745.69	1539.19	-9.69	-0.49	161.00	58.84	514.67
18	425885.50	1051.44	16118.39	-0.41	152.24	55.17	486.62
19	723.14	1548.53	-15.52	-1.27	162.92	59.47	530.67
20	666.28	1432.59	-18.25	-0.69	151.15	55.09	503.60
21	773.76	1644.22	-13.55	0.32	172.27	63.49	571.72
22	714.32	1469.66	-8.43	-0.70	154.82	56.29	501.63
23	702.08	1560.86	-11.59	o.03	165.87	61.02	541.70
24	716.31	1605.85	-13.08	-0.32	170.35	62.37	556.71
25	781.53	1702.73	-14.05	-0.40	179.11	66.04	584.76
26	782.79	1668.47	-8.51	0.73	175.91	64.69	569.75
27	739.26	1500.00	-4.19	0.64	157.79	57.48	499.66
28	659.40	1378.78	-17.63	0.06	153.38	54.36	480.57
29	651.95	1443.65	-17.10	-1.19	154.16	55.80	502.62
30	667.43	1411.07	-21.95	-0.29	157.87	55.71	495.58

Table: 3: Toxicity profile of 30 ligands using Osiris property explorer

LIGAND	MUTAGENICITY	CARCINOGENICITY	IRRITANCY	TERATOGENICITY
Imatinib	YES	NO	YES	NO
1	NO	NO	YES	NO
2	YES	YES	YES	NO
3	NO	NO	YES	NO
4	YES	NO	YES	NO
5	NO	PARTIAL	YES	NO
6	YES	YES	YES	NO
7	YES	NO	YES	NO
8	NO	NO	YES	NO
9	YES	NO	YES	NO
10	NO	NO	YES	NO
11	NO	NO	YES	NO
12	YES	YES	YES	NO
13	YES	YES	YES	NO
14	NO	NO	NO	YES
15	YES	NO	YES	NO
16	YES	NO	YES	NO
17	YES	NO	YES	NO
18	YES	YES	YES	PATIAL
19	NO	NO	YES	NO
20	NO	NO	YES	NO
21	YES	YES	YES	NO
22	NO	NO	YES	NO
23	NO	NO	YES	NO
24	YES	YES	YES	PARTIAL
25	NO	NO	YES	NO
26	NO	NO	YES	NO
27	YES	NO	YES	NO
28	YES	NO	YES	NO
29	YES	YES	YES	PARTIAL
30	YES	YES	YES	PARTIAL

Table: 4: GOLD Docking: fitness scores of imatiniband its analogs with BCR-ABL Oncoprotein

Ligand	Fitness	S(hbext)	S(vdwext)	S(hbint)	S(vdwint)
4	30.19	0.02	52.75	0.00	-42.36
6	27.60	0.53	46.50	0.00	-36.87
28	24.92	1.90	42.63	0.00	-35.60
30	24.78	0.25	43.77	0.00	-35.65
7	22.80	2.29	41.52	0.00	-36.58
9	22.36	0.62	41.06	0.00	-34.72
27	13.58	6.00	42.01	0.00	-50.18
14	12.30	5.89	39.09	0.00	-47.33
26	12.38	4.73	48.49	0.00	-59.02
Imatinib	12.23	5.26	42.92	0.00	-52.05
1	12.18	3.18	43.47	0.00	-50.77
21	11.61	0.00	46.28	0.00	-52.02
2	11.22	8.75	40.36	0.00	-53.03
23	10.98	2.00	42.40	0.00	-49.32
16	10.72	5.43	40.86	0.00	-50.89
20	10.55	5.19	39.43	0.00	-48.86
15	9.14	5.92	40.77	0.00	-52.84
17	7.88	6.28	43.43	0.00	-58.12
8	7.79	5.35	30.71	0.00	-50.79
11	7.49	1.97	41.17	0.00	-51.08
5	7.45	4.55	41.78	0.00	-54.54
10	6.53	5.30	39.38	0.00	-52.9
25	6.24	0.00	46.68	0.00	-57.94
3	5.64	2.81	41.29	0.00	-53.95
24	5.49	1.49	41.14	0.00	-52.56
22	5.26	0.57	40.08	0.00	-50.42
18	4.93	0.00	38.94	0.00	-58.48
29	2.98	1.01	40.14	0.00	-53.22
12	2.08	4.69	38.84	0.00	-56.01
13	1.25	3.87	39.46	0.00	-56.89
19	0.73	4.97	37.96	0.00	-56.43

Docking studies

Protein - ligand interactions are studied by using GOLD 3.01 docking software. The Gold score function is a molecular mechanics-like function with four terms:

GOLD Fitness = Shb_ext + 1.375(Svdw_ext )+Shb_int + S(vdw_int)

Where, Shb_ext is the protein–ligand hydrogen-bond score and Svdw_ext is the protein-ligand van der Waals score. Shb_int is the contribution to the Fitness due to intramolecular hydrogen bonds in the ligand; this term is switched off in all calculations presented in this work: Svdw_int is the contribution due to intramolecular strain in the ligand. The analogue which is having the highest fitness score is having the highest binding affinity.

Docking results are tabulated in the Table: 4. The analogue which is having the highest fitness score is having the highest binding affinity.

From the table, ligand 4 is having the best Gold Fitness score of 30.19 showing interactions with amino acid ILE 57 at distance of 1.551 A⁰, andAla13at 1.479 A⁰and ligand 6 with fitness of 27.60 having 4 hydrogen bonds with the receptor (Gln 14: 1.978, Ile57: 2.205, Arg 55: 2.181, Glu 52: 2.704).Binding modes of ligands 4 and 6 are visualized in Fig: 2 and 3. 12 of 30 ligands are having better Gold Fitness score when compare to that of imatinib (Gold Fitness score is -12.23), a standard marketed drug. 1, 14, 26 ligands are having dock score near to imatinib and modifications to these ligands may result in better ligands than imatinib. Ligands like 18, 29, 12, 13, and 19 are having the least Dock scores.

Fig: 2: ligand 4 binding interactions with the target

Fig: 3:ligand 6 binding interactions with the target

CONCLUSION:

Present study was conducted to design and identify the potent Tyrosine kinase inhibitors for the treatment of Chronicmyelogenous (or myeloid) leukemia (CML) using InSilico tools and techniques. The interactions between Bcr-Abl Oncogene and the ligands were studied by using GOLD 3.01 docking protocol. Based on Gold Fitness score docking results were analyzed. The results were compared to imatinib to find out the best ligand which can inhibit Bcr-Abl oncogene (tyrosine kinase).

The overall review of results concludes that ligands 14, 26, and 1 have shown better properties when compared to all other ligands with no toxic profile. These ligands have also shown the highest dock-scores when compared to imatinib and other analogues. Ligand 4 is having the highest docking score compared to all other ligands but it had shown mutagenicity and irritancy. Further development and synthesis of these ligands may lead to be as better drugs for blocking Bcr-Abl oncogene in treatment of Chronic myeloid leukemia (CML).

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Received on 08.12.2011 Modified on 22.12.2011

Asian J. Research Chem. 5(1): January 2012; Page 153-158