ABSTRACT
Anaplastic lymphoma kinase (ALK) positive non-small cell lung cancer (NSCLC) patients are mostly treated with ALK tyrosine kinase inhibitors (TKIs). Crizotinib is the first generation ALK inhibitor practiced as a primary chemo to combat cancer cells followed by second generation inhibitor ceritinib which are effective against crizotinib resistant ALK mutations. However, patients treated with these drugs invariably relapsed because of the development of new drug resistance mutations. In this study we explored the crizotinib resistance in the presence of ALK mutations L1196M and G1269A through molecular dynamics simulation studies. Further mutation specific inhibitors CID 71748211 and CID 71728095 were identified to potentially inhibit ALK with mutations L1196M and G1269A, respectively. This computational investigation in-sighted the molecular factors involved in crizotinib resistance which enhanced in the identification of new ALK drugs that brings individualized medicine to treat ALK positive NSCLC patients with specific mutations. J. Cell. Biochem. 118: 3462-3471, 2017. © 2017 Wiley Periodicals, Inc.
Subject(s)
Carcinoma, Non-Small-Cell Lung/enzymology , Drug Resistance, Neoplasm , Lung Neoplasms/enzymology , Molecular Dynamics Simulation , Mutation, Missense , Protein Kinase Inhibitors/chemistry , Pyrazoles/chemistry , Pyridines/chemistry , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Receptor Protein-Tyrosine Kinases/chemistry , Amino Acid Substitution , Anaplastic Lymphoma Kinase , Carcinoma, Non-Small-Cell Lung/diet therapy , Carcinoma, Non-Small-Cell Lung/genetics , Crizotinib , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Receptor Protein-Tyrosine Kinases/geneticsABSTRACT
BACKGROUND: Zebrafish is an important model organism for human neurobehavioural studies and compound screening for neurodegenerative disorders like Alzheimer's disease and dementia. PURPOSE: We wanted to analyse the rapid neurobehavioural effects based on acetylcholinesterase inhibitors from Tephrosia purpurea in zebrafish. METHODS: Tephrosia purpurea (a herbal neuroactive molecule) extract was analyzed for its rapid neurobehavioural effects and the corresponding psychotic twitches were quantified. The inhibitory activity of acetylcholinesterase was analysed in Zebrafish. The activity of the molecule was confirmed after column chromatography and RP-HPLC purifications. The toxicity of the molecule was also studied in developing zebrafish embryos. RESULTS: The psychotic body twitches were calculated as crude 484.67 ± 18.01 at 10 µg/mL, column purified 616 ± 9.64 at 8 µg/mL, Sep-Pak C18 712.67 ± 19.5 at 6 µg/mL, and HPLC elution showed 815 ± 14.93. The minimum inhibitory concentration of acetylcholinesterase in crude and column purified was 100 ng/mL and 5 ng/mL. The IC50 value of acetylcholinesterase inhibitor was calculated as 626ng/mL for crude (P<0.0001), 28.92 ng/mL for Sep-Pak column elution and 64 ng/mL for Donepezil. The HPLC fifth elution showed inhibition percentage as 96.97 ± 0.12. The organogenesis effects were seen in the drug concentration of 10 µg/mL. Pericardial bulging, trunk and tail flexure with heart edema and head edema were observed in the embryos at higher dose of 30- 40 µg. The LC50 value was 34.87 µg/mL. The studies showed that the inhibitory concentration of acetylcholinesterase is comparatively higher than Donepezil. CONCLUSION: Rapid behaviour based screening is an inexpesive assay to identify neuroactive small molecules. This herbal can be further used for the development of drugs for Alzheimer's disease.
