Transferrin receptor (TfR) has been shown to be significantly overexpressed in different types of cancers. We discovered TfR as a target for gambogic acid (GA), used in traditional Chinese medicine and a previously undiscovered link between TfR and the rapid activation of apoptosis. The binding site of GA on TfR is independent of the transferrin binding site, and it appears that GA potentially inhibits TfR internalization. Down-regulation of TfR by RNA interference decreases sensitivity to GA-induced apoptosis, further supporting TfR as the primary GA receptor. In summary, GA binding to TfR induces a unique signal leading to rapid apoptosis of tumor cells. These results suggest that GA may provide an additional approach for targeting the TfR and its use in cancer therapy.rapid apoptosis ͉ caspases ͉ target identification

The structure of the ion conduction pathway or pore of voltage-gated ion channels is unknown, although the linker between the membrane spanning segments S5 and S6 has been suggested to form part of the pore in potassium channels. To test whether this region controls potassium channel conduction, a 21-amino acid segment of the S5-S6 linker was transplanted from the voltage-activated potassium channel NGK2 to another potassium channel DRK1, which has very different pore properties. In the resulting chimeric channel, the single channel conductance and blockade by external and internal tetraethylammonium (TEA) ion were characteristic of the donor NGK2 channel. Thus, this 21-amino acid segment controls the essential biophysical properties of the pore and may form the conduction pathway of these potassium channels.

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By applying a novel cell- and caspase-based HTS assay, 2-amino-3-cyano-7-(dimethylamino)-4-(3-methoxy-4,5-methylenedioxyphenyl)-4H-chromene (1a) has been identified as a potent apoptosis inducer. Compound 1a was found to induce nuclear fragmentation and PARP cleavage, as well as to arrest cells at the G(2)/M stage and to induce apoptosis as determined by the flow cytometry analysis assay in multiple human cell lines (e.g. Jurkat, T47D). Through structure-activity relationship (SAR) studies of the 4-aryl group, a 4- and 7-fold increase in potency was obtained from the screening hit 1a to the lead compounds 2-amino-4-(3-bromo-4,5-dimethoxyphenyl)-3-cyano-7-(dimethylamino)-4H-chromene (1c) and 2-amino-3-cyano-7-(dimethylamino)-4-(5-methyl-3-pyridyl)-4H-chromene (4e), with an EC(50) of 19 and 11 nM in the caspase activation assay in T47D breast cancer cells, respectively. The 2-amino-4-aryl-3-cyano-7-(dimethylamino)-4H-chromenes also were found to be highly active in the growth inhibition MTT assay, with GI(50) values in the low nanomolar range for compound 1c. Significantly, compound 1c was found to have a GI(50) value of 2 nM in the paclitaxel resistant, p-glycoprotein overexpressed, MES-SA/DX5 tumor cells. Functionally, compound 1c was found to be a potent inhibitor of tubulin polymerization and to effectively inhibit the binding of colchicine to tubulin. These results confirm that the cell-based caspase activation assay is a powerful tool for the discovery of potent apoptosis inducers and suggest that the 4-aryl-4H-chromenes have the potential to be developed into future anticancer agents.

Different types of K+ channels play important roles in many aspects of excitability. The isolation of cDNA clones from Drosophila, Aplysia, Xenopus, and mammals points to a large multigene family with several distinct members encoding K+ channels with unique electrophysiological and pharmacological properties. Given the pivotal role K+ channels play in the fine tuning of electrical properties of excitable tissues, we studied the spatial and temporal basis of K+ channel diversity. We report the isolation of two putative K+ channels that define two new subfamilies based upon amino acid sequence similarities with other known K+ channels. Northern blot and in situ hybridization studies revealed differences in the spatial and temporal expression patterns for these two new clones along with mRNAs from other K+ channel subfamilies. Two of the K+ channels studied are predominantly expressed in the brain. One of the "brain-specific" K+ channels is first expressed after about 2 weeks of postnatal cerebellar development and remains at levels about 10-fold higher in the cerebellum than in the rest of the brain.

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We have identified 5-(3-chlorothiophen-2-yl)-3-(4-trifluoromethylphenyl)-1,2,4-oxadiazole (1d) as a novel apoptosis inducer through our caspase- and cell-based high-throughput screening assay. Compound 1d has good activity against several breast and colorectal cancer cell lines but is inactive against several other cancer cell lines. In a flow cytometry assay, treatment of T47D cells with 1d resulted in arrest of cells in the G(1) phase, followed by induction of apoptosis. SAR studies of 1d showed that the 3-phenyl group can be replaced by a pyridyl group, and a substituted five-member ring in the 5-position is important for activity. 5-(3-Chlorothiophen-2-yl)-3-(5-chloropyridin-2-yl)-1,2,4-oxadiazole (4l) has been found to have in vivo activity in a MX-1 tumor model. Using a photoaffinity agent, the molecular target has been identified as TIP47, an IGF II receptor binding protein. Therefore, our cell-based chemical genetics approach for the discovery of apoptosis inducers can identify potential anticancer agents as well as their molecular targets.