Apoptosis is a major form of programmed cell death that multicellular organisms utilize to maintain tissue homeostasis and to eliminate unwanted or damaged cells. It plays a critical role in development, immune responses and many other physiological events. In mammals, a crucial apoptotic pathway is the mitochondrial cytochrome c-mediated caspase activation pathway. In this pathway, various apoptotic stimuli induce cytochrome c release from mitochondria. Released cytochrome c binds to and activates the essential mediator Apaf-1 to trigger assembly of a multimeric protein complex, the apoptosome, which in turn activates the downstream molecular executioners of apoptosis, caspases. Deregulation of the cytochrome c apoptotic pathway can lead to diseases such as cancer, immune disorders, and neurodegenerative diseases. Conversely, targeting apoptotic components by both enhancing and inhibiting apoptosis represents important therapeutic approaches to treat various human diseases. Several promising targeted cancer therapies under clinical trials are designed to specifically activate the cytochrome c apoptotic pathway. On the other hand, inhibition of this pathway should also be effective in treating symptoms with pathologically enhanced apoptosis such as neurodegeneration and ischemic injuries. Importantly, inhibition of this pathway is also a promising approach for reducing adverse side effects of radiation cancer therapy, which acts on many solid tumors via non-apoptotic mechanisms but induces massive apoptosis in normal tissues especially in the hematopoietic system and gastrointestinal tract, thus results in severe and occasionally fatal damage to patients. In this grant, we will perform high throughput screening (HTS) to identify Apaf-1-inhibitory compounds from various chemical libraries, and subsequently to characterize the mechanism of action of the identified inhibitors and their potential in protecting radiation damage. To date, we have succeeded in developing and optimizing an Apaf-1 functional assay for HTS. As a validation of this HTS assay, we have screened a collection of drug-like chemical compounds and obtained multiple positive hits that inhibit Apaf-1-mediated caspase activation. This progress should pave the way for us to achieve the eventual goals of this research grant in a timely fashion, within a two-year period supported by the ARRA fund. In these two years, we will screen for additional inhibitory compounds from a distinct chemical library containing large number of natural product-like synthetic molecules that more likely target Apaf-1 via different mechanisms. We will also determine the mechanisms of action of the identified small molecule inhibitors for Apaf-1 and inhibitors for other components in the Apaf-1 pathway. For this mechanistic investigation, both in vitro biochemical studies and analyses using specific cell models will be conducted to gain insights into the molecular basis of the identified inhibitors and their potential effect in protecting apoptotic damage associated with radiation cancer therapy. The identified Apaf-1 inhibitory compounds will be promising drug leads for treating diseases such as cancer and cerebral/myocardial ischemic injuries. They will also be important pharmacological tools for studying mechanisms and roles of cytochrome c-mediated apoptosis in various physiological and disease processes.
The goal of this research proposal is to identify small molecule inhibitors for Apaf-1 by high throughput chemical screening, and to further determine the mechanism of action and potential therapeutic value of the identified inhibitors in vitro and in specific cell models. The identified Apaf-1 small molecule inhibitors will be promising drug leads in protecting cancer patients from severe damage associated with radiation cancer therapy, as well as in treating other diseases such as cerebral/myocardial ischemic injuries. These compounds will also be important pharmacological tools for studying mechanisms and roles of cytochrome c-mediated apoptosis in various physiological and disease processes.
| Feldman, Taya; Kabaleeswaran, Venkataraman; Jang, Se Bok et al. (2012) A class of allosteric caspase inhibitors identified by high-throughput screening. Mol Cell 47:585-95 |
