Abstract 2438: Identification of the mitochondrial binding site on the amino-terminal end of hexokinase II

Abstract Hexokinase II is responsible for the first step in the glycolysis pathway. Hexokinase II adds a phosphate onto the glucose molecule so it can proceed down the pathway to give the cancer cell energy for its continuous growth. Tumor cells overexpress the hexokinase II enzyme. In fact, it is the overexpression of the hexokinase II enzyme that makes the diagnosis of cancer possible when imaged by positron emission tomography (PET). Hexokinase II binds to the voltage-dependent anion channel (VDAC) on the mitochondrial membrane and when it is attached to the mitochondrial membrane it is blocking a major pathway for cell death. Thus, hexokinase II is responsible for two characteristics of cancer cells, rapid tumor growth and difficulty to kill. Therefore the overall goal of our laboratory is to identify novel compounds with the ability to displace or translocate hexokinase II from the mitochondrial membrane to the cytoplasm. To attain our goal, we propose to use the crystal structure of hexokinase II to create a molecular model of the enzyme. However the amino acids responsible for binding to VDAC are not known. Therefore, we made a series of truncations and point mutations to identify the binding site to VDAC. Truncations of the first 10 and 20 amino acids indicated that important amino acids for binding were located within the first 10 amino acids. We made a series of single, double and triple point mutations within the first 10 amino acids. Immunofluorescence and immunoblot data has confirmed that mutating the fifth amino acid from histidine to proline completely abolished binding to the mitochondrial membrane. To prepare hexokinase II for crystallization, we have cloned the enzyme downstream of a thrombin cleavage site and His-tag. We were able to purify hexokinase II using a cobalt column, then eluting hexokinase II using thrombin to cleave at the thrombin cleavage site. Purified hexokinase II is isolated after removal of the thrombin using Protein A beads. After the crystal structure of hexokinase II is obtained, we will use the University of Florida Structure-based Molecular Docking Core facility to identify compounds predicted to fit into the VDAC binding site. The compounds will be characterized to validate a mechanism-of-action of translocating hexokinase II from the mitochondrial membrane to the cytoplasm, blocking glycolysis and decreasing ATP synthesis. The positive compounds will be analyzed to determine the best strategy for moving our studies closer to clinical application in cancer patients. Citation Format: Nadezda Bryan, Michelle M. Hwang, Kevin P. Raisch. Identification of the mitochondrial binding site on the amino-terminal end of hexokinase II. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2438. doi:10.1158/1538-7445.AM2015-2438