| Assay Method Information | |
| | Apoptosis Inhibition Assay |
| Description: | In order to identify the target protein of tTC09 apoptosis inhibitors, we synthesized two sets of probes for target identification based on the SAR study. The first set of probes was designed for direct activity based protein profiling (ABPP) with biotin tag linked at different site. The addition of these biotin tags resulted in varying degrees of activity loss, possibly owing to steric hindrance of the bulky fragment or a change of cellular permeability (Table 2). Compounds 30 and 31 were selected as positive and negative probes for pulldown assays and identified succinate dehydrogenase subunit B (SDHB), a component of the respiratory complex II located on the mitochondrial inner membrane, as the covalent binding target. TC09 apoptosis inhibitors could stabilize the mitochondrial respiratory chain after Bim/tBid overexpression and help maintain the normal function of mitochondria. A second set of probes with a terminal alkyne group designed for click-reaction-assisted ABPP were also prepared. Most of these compounds (28, 29, 32-34) retained relatively potent activity, with EC50 values below 1 μM. Click-reaction-assisted ABPP was performed using compound 29 as a positive probe and compound 27 as a competitor; these probes also identified SDHB as the cellular target. To further verify that SDHB was indeed the molecular target, we also synthesized a derivative of compound 29 with an FITC tag for fluorescence imaging (compound 35, EC50=995 nM). The green fluorescence of compound 35 merged well with the specific red fluorescent staining of mitochondria, indicating that most of compound 35 was localized in mitochondria. |
| Affinity data for this assay | |
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