Hexokinase II Redistribution Assay

• Designed to assay compounds for their ability to release Hexokinase II from mitochondria
• Coupled to EGFP for easy monitoring of the cellular translocation event
• Robust cell-based assay for use in high content analysis and fluorescence microscope applications

• Biologically relevant data: Compounds tested in a cellular environment
• Validated: Functionally tested cells provided with an optimized assay protocol
• Easy to use: Just plate cells, add compounds, and image

Hexokinase II (HKII) mediates the first step of glycolysis, by phosphorylation of glucose to glucose 6-phosphate. It has been known for many years that tumor cells to a large extent depend on glycolysis to produce ATP. This is partly due to overexpression of HKII, which is predominantly localized to mitochondria through binding to the voltage-dependent anion channel (VDAC). This localization enables HKII to get preferential access to mitochondrial ATP and thereby create an increase in the rate of glycolysis, known as aerobic glycolysis. In addition, binding of HKII to VDAC is believed to prevent binding of Bax to mitochondria and thereby block release of cytochrome C, resulting in decreased sensitivity to apoptosis. Thus, compounds that cause dissociation of HKII from mitochondria have potential to act directly as anti-cancer agents or to potentiate the effect of chemotherapeutic agents and thereby be useful in anti-cancer treatment.

Figure 1. Translocation of HKII-EGFP in response to clotrimazole. Cells were treated with 100 µM clotrimazole (right panel) or untreated (DMSO control, left panel). Arrows indicate localization of HKII-EGFP in mitochondria detected by the image analysis algorithm.

Figure 2: Clotrimazole concentration response curve in the HKII Redistribution® assay. Concentration response was measured in 9 point half log dilution series of clotrimazole (n=8). Cells were then fixed and the mitochondria to cytoplasm translocation was measured using the Cellomics ArrayScan V^TI Reader and the SpotDetector V3 BioApplication. % activity was calculated relative to the positive (100 µM clotrimazole) and negative control (0.25% DMSO). The EC₅₀ value of clotrimazole in the assay is approximately 15 µM.