Apoptosis, or programmed cell death, is a crucial process in maintaining cellular homeostasis and eliminating damaged or unwanted cells. Dysregulation of apoptosis pathways is associated with various diseases, including cancer, autoimmune disorders, and neurodegenerative diseases. Targeting key components of the apoptosis machinery provides a promising approach for therapeutic development, aiming to either promote or inhibit cell death depending on the disease context.
1. IAPs (Inhibitor of Apoptosis Proteins): In the IAP Fluorescence Polarization (FP) Assay, when excited by polarized light, the free fluorescent molecule tumbles rapidly in solution, resulting in depolarized emission. However, upon binding to the larger IAP target, the rotation slows, causing the emitted light to remain more polarized. This shift in polarization provides a quantitative measure of binding interaction strength.
2. Bcl-2 Family: In the BCL-2 HTRF (Homogeneous Time-Resolved Fluorescence) Assay, BCL-2 proteins labeled with a His tag can bind to their corresponding fluorescently labeled peptide substrates. When this binding occurs, and a detection reagent like streptavidin-Tb is added, a high HTRF signal is generated. The strength of the HTRF signal reflects the interaction between BCL-2 and its substrate, enabling quantification of binding affinity and the impact of potential inhibitors.
3. Caspase Family: In the caspase Fluorescence Intensity (FI) Assay, a short peptide substrate containing a specific recognition sequence for active caspases is labeled with a fluorescent tag. When the caspase enzyme cleaves this peptide at the recognition site, the fluorescent tag is released, causing an increase in fluorescence that can be quantified. Higher fluorescence values correlate with greater caspase activity, providing a direct measure of the enzyme’s efficiency in cleaving its target substrate.
| Target | Synonyms | Assay Type | Reference Compound | Assay Format |
|---|---|---|---|---|
| XIAP | API3, BIRC4, IAP3 | Binding | Birinapant | FP |
| cIAP-1 | API1, MIHB, RNF48 | Binding | Birinapant | FP |
| cIAP-2 | API2, MIHC, RNF49 | Binding | Birinapant | FP |
| MCL-1 | BCL2L3 | Binding | A-1210477 | HTRF |
| BCL-2 | / | Binding | ABT-737 | HTRF |
| BCL-2 | / | Binding | ABT-737 | SPR |
| BCL-xl | BCL2L, BCLX | Binding | ABT-737 | HTRF |
| BCL2L2 | BCLW, KIAA0271 | Binding | Gambogic | HTRF |
| BCL2A1 | BCL2L5, BFL1, GRS, HBPA1 | Binding | Gambogic | HTRF |
| BCL2L10 | Bcl-B, BCLB, BOO, DIVA, B2L10 | Binding | Gambogic | HTRF |
| BCL6 | BCL5, LAZ3, ZBTB27, ZNF51 | Binding | BI-3812 | HTRF |
| Caspase 1 | CASP1, IL1BC, IL1BCE | Activity | Belnacasan | FI |
| Caspase 2 | CASP2, ICH1, NEDD2 | Activity | Ac-DEVD-CHO | FI |
| Caspase 3 | CASP3, CPP32 | Activity | Ac-DEVD-CHO | FI |
| Caspase 4 | CASP4, ICH2 | Activity | Belnacasan | FI |
| Caspase 6 | CASP6, MCH2 | Activity | Ac-DEVD-CHO | FI |
| Caspase 7 | CASP7, MCH3 | Activity | Ac-DEVD-CHO | FI |
| Caspase 8 | CASP8, MCH5 | Activity | Ac-DEVD-CHO | FI |
| Caspase 10 | CASP10, MCH4 | Activity | Ac-DEVD-CHO | FI |
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