Rational Modification of Semaxanib and Sunitinib for Developing a Tumor Growth Inhibitor Targeting ATP Binding Site of Tyrosine Kinase
Jagroop Kaur, Baljit Kaur, Palwinder Singh
PII: S0960-894X(17)31159-9
DOI: https://doi.org/10.1016/j.bmcl.2017.11.049
Reference: BMCL 25455
To appear in: Bioorganic & Medicinal Chemistry Letters
Received Date: 26 June 2017
Revised Date: 26 November 2017
Accepted Date: 27 November 2017
Please cite this article as: Kaur, J., Kaur, B., Singh, P., Rational Modification of Semaxanib and Sunitinib for Developing a Tumor Growth Inhibitor Targeting ATP Binding Site of Tyrosine Kinase, Bioorganic & Medicinal Chemistry Letters (2017), doi: https://doi.org/10.1016/j.bmcl.2017.11.049
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Analysis of the crystal structure of tyrosine kinase in complexation with an ATP analogue, supplemented with the molecular docking studies of semaxanib and sunitinib in the ATP binding site of the enzyme enabled us to make design of a series of tyrosine kinase inhibitors. The combination of pyrrole and indolinone in one molecule and placement of appropriate substituent thereof made the molecule compatible for the hydrophobic sub-pocket of the enzyme. Screening of the compounds over 60 cell line panel of human tumor cell lines identified compound 3a that exhibited GI50 35 nM and 63 nM against MCF7 and MDA-MB-468 cell lines of breast cancer.
Cancer continues to be one of the major health problems and a leading cause of human suffering and deaths worldwide.1 Population growth, increasing life expectancy and adoption of cancer associated lifestyle such as smoking are some of the specific grounds for the growing burden of cancer especially in the economically developed/developing countries. Among the various cancers, breast cancer is the most commonly diagnosed cancer in women as it rarely occurs in men. After lung cancer, breast cancer is the second leading cause of cancer deaths in women and as per the current records, in U.S. alone, 41,070 (40610 women and 460 men) people are estimated to die of breast cancer in 2017.2 Besides the other remedial measures, chemotherapy is widely used for the treatment of majority of cancers3 and a number of chemotherapeutic drugs such as taxol,4 vinblastine,5 vincristine,6 etoposide,7 camptothecin,8 mitoxantrone,9 5-fluorouracil10 and cisplatin11 are in the clinical use. These drugs target cancer associated enzymes and signaling pathways. However, the economical availability and the associated side effects of these drugs are the major bottlenecks that hamper their practical applications.12
The signaling pathways are the critical cellular links wherein the tyrosine kinases play a pivotal role in post-translational modifications and hence in maintaining normal cellular communication.13 Nonetheless, effected by the mutations, epidermal growth factor receptors (EGFR) and insulin growth factor receptors (IGFR); the activation of tyrosine kinases (TK) alters the signaling pathways and obstructs the regular cell functions like cell division, growth and normal cell death.
Consequently, the role of tyrosine kinases are implicated in the breast cancer, prostate cancer, non-small cell lung cancer and bladder cancer making tyrosine kinases as the potential targets of anti-cancer drugs.The availability of the crystal structure of tyrosine kinase and the analysis of its ATP binding site by making use of molecular modeling studies helped to a large extent in the design of TK inhibitors.15,16 Further exploration of the ATP binding site of IGFR-tyrosine kinase provided insight to the mode of interaction between ATP and TK. A number of H-bond interactions between the OH of sugar, N/NH of adenine and the amino acid residues were observed in the crystal coordinates of TK in complex with ATP analogue (Figure 1). The sugar and adenine template of ATP analogue were placed in the hydrophobic region constituted by L1002, V1010 and F1044 (Figure 1). Additionally, the interactions of TK inhibitors like sunitinib and semaxanib in the ATP binding site of the enzyme were also examined. It was observed that the polar region of sunitinib interacts through H- bonds whereas the hydrophobic region of both sunitinib and semaxanib is placed in the hydrophobic pocket of the enzyme (Figure 2). However, the large hydrophobic space in the active site of TK constituted by Val, Phe, and Leu residues remains unoccupied. Therefore, it is worthwhile to design TK inhibitors with large hydrophobic region so that they exhibit better interactions in the hydrophobic pocket of the enzyme. Advantageously, the hydrophobic interactions impart better reversibility to the enzyme–ligand complexation in comparison to the polar interactions.
Figure 1. (A) ATP analogue in the crystal coordinates of tyrosine kinase (phosphorylated insulin receptor, pdb ID 1IR3) showing H-bond interactions (pink dotted lines) with the amino acid residues, (B) ATP analogue docked in the ATP binding site of tyrosine kinase (phosphorylated insulin receptor, pdb ID 1IR3). It is apparent that the adenine and sugar units are placed in the hydrophobic pocket made of L1002, V1010 and F1044, (C) 2D view of ATP analogue docked in tyrosine kinase binding site.
The strategy of combining two or more biologically active moieties for the design of new drugs (hybrid molecules) has resulted into the development of some lead molecules with remarkable biological activity.17 In the present study, taking into consideration the biological importance of pyrrole18-21 and indole22 heterocycles; particularly, in making part of anti-cancer drugs such as semaxanib and sunitinib,23-26 we designed the conjugates of N-substituted pyrrole and indolin-2-one (3, Chart 1) and screened the molecules for tumor growth inhibition activity. It is worth to mention that semaxanib (1, chart 1) is at 100 oC for 1h to procure compound 3e.
Additionally, some polar interactions through the Cl were observed. Most remarkably, the indolinone moiety of compound 3a was placed more close to the Mg2+ in comparison to the similar placement of sunitinib and semaxanib (Figure 2, D, E, F). The synthesis of the compounds was accomplished by starting with N-substitution of pyrrole-2-carboxaldehye followed by Compound 3e was then treated with benzoyl chloride in the presence of NaH in DMF/THF for 30 min (0 oC- 25 oC) to synthesize compound 3f. Using the same reaction sequence and the reaction conditions as for the preparation of 3f; compounds 3g-i were synthesized by reacting 3e with 4-chlorobenzoyl chloride, 4-toulenesulfonyl chloride and 5-chlorothiophene-2-carbonyl chloride, respectively (Scheme 2). The Z-Configuration at the bridged C=C bond of compounds 3a-i was established with the help of NMR experiments (Figure 3; details of experiments for configuration assignment in the supporting information).
Figure 2. Semaxanib (A, D), Sunitinib (B, E) and Compound 3a (C, F) docked in the ATP binding site of tyrosine kinase (phosphorylated insulin receptor, pdb ID 1IR3) showing H-bonding interactions (pink dotted lines) with the ATP binding site residues. In the 2D view (D, E, F), red dotted lines are close distances between Mg atoms and drugs.
Scheme 2
Compounds 3a-c and 3e-i were tested for their tumor growth inhibitory activity over 60 human cancer cell lines at NCI, Bathesda, USA. Compounds were preliminary studied at 10-5 M concentration. During the single dose assay, the percent growth of tumor cells was recorded in the presence of compounds. In this primary assay, minimum growth of the tumor cells was observed in the presence of compound 3a. Hence compound 3a was further tested for tumor growth inhibitory screening by taking five different concentrations of the compound (10-4 to 10-8 M). The 50% growth inhibition (GI50) concentration, concentration causing total growth inhibition (TGI) and concentration causing.
Figure 3. Structure of compound 3a as elucidated from NMR experiments showing Z-configuration across C6 = C9. Hydrogens attached to carbons 1, 2, 3, 4; 11, 12, 13; 17, 18, 19, 20, 21 and 24, 25, 26 exhibit TOCSY within the respective groups. 50% cells death (LC50) over 60 human cancer cell lines for compound 3a are given in Table 1. The MID values (i.e mean graph midpoint) for GI50, TGI, and LC50 of compound 3a were 10.96, 75.85, and 93.32 µM, respectively.
In parallel to the design of the molecules as TK inhibitors and the expression of TK in breast cancer; compound 3a showed remarkable tumor growth inhibitory activity over breast cancer cell lines MCF7 and MDA-MB-468. It showed GI50 35 nM and 63 nM over MCF7 and MDA-MB-468 cell lines, respectively. Moreover, 3a exhibited GI50 0.54, 0.74 and 0.12 µM over ovarian cancer cell lines IGROV1, OVCAR-3 and OVCAR-4. Apart from these cell lines, compound 3a also exhibited appreciable tumor growth inhibitory activity against certain cell lines of leukemia, colon cancer, non-small cell lung cancer and renal cancer (Table 2). Remarkably, compound 3a exhibited very low toxicity to the normal cells (LC50 >100 µM).
Therefore, from a series of rationally designed pyrrole – indole hybrids, we were able to identify a highly potent molecule that was capable to inhibit the growth of breast cancer cells. Compound 3a exhibited GI50 for breast cancer cells in the nM range; 35 nM and 63 nM, respectively for MCF7 and MDA-MB- 468 cell lines. Further studies on compound 3a using the animal models and the compound – TK interaction studies are underway.
Acknowledgments
Financial assistance in the form of sponsoring research projects by DST, New Delhi and CSIR, New Delhi is gratefully acknowledged. JK acknowledge DST, New Delhi for fellowship. University Grants Commission, New Delhi is acknowledged for grant under University with potential for Excellence to Guru Nanak Dev University. NCI Bethesda USA is gratefully acknowledged for anti-cancer studies.
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