The long-term goal of this proposal is to develop small molecule antagonists for an oncogenic potassium channel KCNB (TASK3). Specific antagonists for KCNB will have therapeutic potentials for the treatment of cancer. The Specific Aims are: (1). Develop a high throughput assay (PITS) to screen for compounds that inhibit KCNB channel activity. A high throughput assay will be developed using a membrane potential sensitive dye on FLIPR (Fluorescence Imaging Plate Reader). This assay will be used to screen a library of 1 million single synthetic compounds. Compounds that cause changes in fluorescence signal in comparison with untreated cells will be identified and their activity confirmed. (2). Verify the effects of compounds on KCNB channel activity using electrophysiology. The activity of candidate compounds indicated by the HTS will be tested using a voltage clamp assay to directly measure effect of the compounds on KCNB current. The specificity of the compounds will be determined by testing their effects on related K+ channels (e.g., TREK, TASK1). (3). Use combinatorial chemistry approach to improve the potency and selectivity of the compounds. Once lead compounds are identified, combinatorial chemistry will be used to modify the structures of the existing compounds. The newly synthesized compounds will be tested initially by FLIPR followed by electrophysiology and the compounds with the best potency and specificity will be selected. (4). Test the effects of compounds on cell proliferation and tumor formation. The effect of the compounds on cell proliferation will be tested using cell lines over-expressing KCNB. Xenograft tumor models in mice will be used to test whether the compounds have any effect on tumor growth in tumors over-expressing KCNB. Currently, no specific blockers for the two-pore family of K+ channels have been developed. Therefore, the compounds derived from this proposal could be useful tools for studying the physiological functions of these channels. More importantly, these compounds should inhibit growth of certain human carcinomas and therefore would represent a new family of anti-cancer therapeutics.
Thesaurus Terms: antineoplastic, drug design /synthesis /production, ion channel blocker, potassium channel cell proliferation, combinatorial chemistry, fluorescent dye /probe, high throughput technology, membrane potential Xenopus oocyte, athymic mouse, cell line, electrophysiology, laboratory mouse, neoplasm /cancer transplantation, voltage /patch clamp
