Small Molecules for Apoptosis Research
Table of Contents
Introduction | Apoptosis Inducers | Apoptosis Inhibitors | Determining an Effective Drug Concentration | Experimental Controls in Apoptosis Research
Regulating Apoptosis with Small Molecules
Apoptosis is a process of programmed cell death that is necessary for physiological processes such as organ development and morphogenesis, immune cell education, and the maintenance of tissue homeostasis. However, under some conditions, apoptosis can also be detrimental. For example, neuronal apoptosis is associated with neurodegenerative diseases and CD4+ T cells apoptosis is associated with HIV-1-related pathology.
Apoptosis can be induced by either intracellular or extracellular signals which activate a cascade of proteins such as the pro-apoptotic Bcl-2 family members and caspases. Effector caspases execute cell death by proteolytically cleaving proteins that are necessary for cell function. Cells undergoing apoptosis show phenotypic characteristics such as cell blebbing, shrinkage, DNA fragmentation, and chromatin condensation.
Small molecules can play an important role in defining factors that induce apoptosis, as well as differentiating the specific pathway that leads to cell death. For example, apoptosis mediated by Caspase-9 can be assessed using a fluorogenic Caspase-9 substrate that generates a fluorescent signal upon proteolytic cleavage. The involvement of other caspases as well as other proteins that mediate apoptosis can also be manipulated by small molecule agonists, antagonists, and substrates.
Apoptosis Inducers
Apoptosis inducers produce pro-apoptotic effects via different mechanisms. These include DNA cross-linking, inhibition of anti-apoptotic proteins, and activation of caspases or p53. These apoptosis inducers can target specific cellular processes to induce anti-tumor or anti-neoplastic effects.
Apoptosis-Inducing Compounds
Apoptosis Inhibitors
Blocking the progression of apoptosis frequently involves either a cell-permeable, broad-spectrum caspase or caspase subtype-selective inhibitor. For instance, if measuring cytochrome c release from mitochondria, pan-caspase inhibitors will prevent the downstream activation of the caspase cascade. While caspase-mediated cell death is blocked, cell survival may not be long term. Similarly, cells treated with caspase inhibitors and death receptor ligands (e.g. TNF-α) are diverted to other cell death pathways (i.e. necroptosis). Learn more about caspase cleavage and activation in apoptosis.
Caspase Inhibitors
How to Determine an Effective Drug Concentration
The suitable drug concentration required to initiate or inhibit an apoptotic response in the majority of targeted cells can be determined by performing a dose titration experiment. For a good starting point, consult the literature to identify potential working concentrations. It is also important to understand the relationship between concentration and exposure times. If inducing apoptosis at a low dose, or with a mild stimulus, then a longer treatment time may be necessary.
Why are Experimental Controls Important in Apoptosis Research?
All experiments measuring apoptosis should include both positive and negative controls. During data analysis, negative and positive controls are key to identify and separate healthy and apoptotic populations in experimental samples. Negative controls are also used to assess the health of vehicle-treated cells and serve as baseline measurements crucial for determining the magnitude of the apoptotic response. If cells lacking treatment with apoptosis inducers or treated with caspase inhibitors already show significant signs of cell death, then the activity and effect of the drug will be difficult to establish.
Additional Resources
- Caspase Activation & Apoptosis Poster
- Apoptosis Signaling Pathway
- The Caspase-1 Inflammasome & its Role in Autoinflammatory Diseases
- Mini-review: Caspases
- Mini-review: Apoptosis II: Beyond Cytochrome c, the Other Mitochondrial Proteins
- Apoptosis: Caspase Pathways
- Novel Caspase-8 Roles Revealed by Knockouts