Cancer remains the primary cause of death second to cardiovascular diseases. Current drive in cancer research is towards the development of drugs, which targets the cell cycle progression. Among of the key class of the protein type of targets, which are known to be cell cycle regulators, is the Cyclin Dependent Kinases (CDKs). CDKs have attracted major attention in oncology. In couple of years, Cyclins and Cyclin Dependent Kinases are most extensively studied targets for cancer chemotherapy because the tumor cells exhibit false checkpoints and can proliferate even if the genome is compromised.
The activity of CDKs is regulated through subunits, which are known as cyclins whose level normally oscillates throughout cell cycle. Inappropriate activation of CDK protein kinases occurs through over-expression of subunits (mainly Cyclin D and E). Cyclin Dependent Kinases (CDKs) ensures the tight regulation of the cell cycle execution by mediating phosphorylation of cellular proteins. Mis-regulation of the CDK2 activity by cellular & external factors leads to many diseases like cancer and inflammatory diseases.
Ultimate goal of the undertaken project is to Design, Develop and Identify small molecule inhibitor of CDK2 with use of luminescence and fluorescence based biochemical assays and cell based cytotoxicity and apoptosis assays.
Study design
Co-Expression and Purification of recombinant kinases/cyclin in insect cells and bacteria
Establishment and validation of fluorescent and luminescent read-out based kinase assays [i.e. BIOCHEMICAL ASSAYS}
Establishment and validation of cell based apoptosis and cytotoxicity assays
Synthesis and Acquisition of kinase inhibitors (small molecule and peptide) ,
Screening of compounds against selective panel of kinases (list of kinases which are validated drug target for cancers)
Screening of compounds for apoptosis and cytotoxicity assays [i.e. CELL BASED ASSAYS]
Data collection (variables, predictors and confounders), Measures (detection and quantification): Statistical considerations (sample size and data analysis): Interventions
Derivation of IC50 values of compounds
Derivation of EC50 values of compounds
Study of Structure Activity Relationships among the similar pharmacophores (compounds with common structural moiety) from biochemical assays
SAR from Cell based assays
Refinement and comments to improve selectivity and potency for specific kinases for selected hits (compounds which scores better IC50 in biochemical/cell based assay)
Experimental Techniques:
Recombinat DNA tech , Insect cell culture , Human cancer cell lines, Baculovirus , RT-PCR, Cloning , Ligation, Lysis and Affinity chromatography purification.
Cell culture of MCF-7, HeLa, HL60 , HEK293T cell lines.
Apoptosis and Cytotoxicty assay. Ganpat Vidyanagar and IIAR , Koba-Gandhinagar
Friday, April 20, 2007
Subscribe to:
Posts (Atom)
