Application of Eukaryotic Elongation Factor-2 Kinase (eEF-2K) for Cancer Therapy: Expression, Purification, and High-Throughput Inhibitor Screening.
Protein kinases have emerged as an important class of therapeutic targets, as they are known to be involved in pathological pathways linked to numerous human disorders. Major efforts to discover kinase inhibitors in both academia and pharmaceutical companies have centered on the development of robust assays and cost-effective approaches to isolate them. Drug discovery procedures often start with hit identification for lead development, by screening a library of chemicals using an appropriate assay in a high-throughput manner. Considering limitations unique to each assay technique and screening capability, intelligent integration of various assay schemes and level of throughput, in addition to the choice of chemical libraries, is the key to success of this initial step. Here, we describe the purification of the protein kinase, eEF-2K, and the utilization of three biochemical assays in the course of identifying small molecules that block its enzymatic reaction.
Division of Medicinal Chemistry, College of Pharmacy, The University of Texas at Austin, Austin, TX, 78712, USA.
Clint D J
CD
Tavares
Texas Screening Alliance for Cancer Therapeutics, The University of Texas at Austin, Austin, TX, 78712, USA.
Ashwini K
AK
Devkota
Division of Medicinal Chemistry, College of Pharmacy, The University of Texas at Austin, Austin, TX, 78712, USA. dalbykn@me.com.|Texas Screening Alliance for Cancer Therapeutics, The University of Texas at Austin, Austin, TX, 78712, USA. dalbykn@me.com.
Kevin N
KN
Dalby
Texas Screening Alliance for Cancer Therapeutics, The University of Texas at Austin, Austin, TX, 78712, USA. euncho@austin.utexas.edu.|College of Pharmacy, The University of Texas at Austin, Austin, TX, 78712, USA. euncho@austin.utexas.edu.
Eun Jeong
EJ
Cho
D000970:Antineoplastic Agents / Q000302:isolation & purification / Q000494:pharmacology*; D054736:Elongation Factor 2 Kinase / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000302:isolation & purification; D005470:Fluorometry / Q000379:methods*; D005720:Gamma Rays; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D007202:Indicators and Reagents; D008163:Luminescent Measurements / Q000379:methods*; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000302:isolation & purification; D009369:Neoplasms / Q000188:drug therapy* / Q000201:enzymology; D010761:Phosphorus Radioisotopes / Q000032:analysis; D047428:Protein Kinase Inhibitors / Q000302:isolation & purification / Q000494:pharmacology*; D011874:Radiometry / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012588:Scintillation Counting
D000970:Antineoplastic Agents; D007202:Indicators and Reagents; D009363:Neoplasm Proteins; D010761:Phosphorus Radioisotopes; D047428:Protein Kinase Inhibitors; D011993:Recombinant Fusion Proteins; D054736:Elongation Factor 2 Kinase
Expression; Fluorescence; High-throughput screen; Kinase; Luminescence; Purification; Radioactive; eEF-2K