Death by Design:
Synthesis of Purified
Death Domain Peptides

By Martin Carroll, M.D.

Almost 20 years ago, two research groups cloned the genes in the t(14;18) translocation associated with follicular lymphoma. One of the groups noted in the abstract of their paper that, “The probe appears to identify bcl-2, a gene locus on chromosome 18 (band q21) that is unrelated to known oncogenes and may be important in the pathogenesis of B-cell neoplasms with this translocation.” (Science. 1984; 226:1097-9) What the researchers didn’t realize is that cloning of bcl2 would lead to a revolutionary new understanding of cell biology. It is now known that bcl2 is one member of a family of proteins that regulate cell survival. Some family members, such as bcl2, when inappropriately expressed in follicular lymphoma, act to prevent cell death. Other members, more recently identified, act to promote cell death. In fact, in any given cell, it is the relative ratio of “death-promoting” vs “death-defying” bcl2 family members that determines cellular fate.

One of the laboratories that cloned the t(14;18) translocation was the laboratory of Dr. Stanley Korsmeyer. Dr. Korsmeyer has spent the last twenty years studying bcl2 proteins in order to understand their role in tumorigenesis and whether understanding these complex proteins could improve therapy for malignant diseases. Dr. Loren D. Walensky delivered the latest update from Dr. Korsmeyer’s laboratory today at the Plenary Session. The laboratory has solved a challenging biochemical dilemma. The critical domains within the bcl2 family member are designated by the nomenclature “bcl-2 homology” or BH domains. Some family members which promote cell death contain only the so-called BH3 domain. It has been a dream of pharmaceutical companies to utilize purified BH3 domains as therapeutics to augment the death promoting activities of chemotherapy. Although some molecular strategies have been used to demonstrate the potential of this approach, the hunt has been hampered by the lack of stable BH3 domain peptides. Because of their structure, BH3 domain peptides are generally not stable in solution. Dr. Korsmeyer’s group has now solved this chemical problem by chemically tieing together or crosslinking the critical structural domains of BH3 compounds. These crosslinked alpha helical domain structures or SAHB’s turn out to have the predicted effects of binding to other BH3 domains and inducing the release of cytochrome c from purified mitochondria, a critical initiating event in programmed cell death. Furthermore, as the authors state, “SAHBs activate apoptosis in cultured B-,Tand mixed lineage leukemia cells.” (Walensky et al, Blood 102(11), 2003). These findings confirm that the crosslinked SAHB molecules have the expected functions of BH3 domains and introduce a powerful new tool to the laboratory for studying exactly how BH3 domains function. But there are still challenges ahead.

The question now becomes whether SAHB’s will make possible the pharmaceutical industry’s dream of a compound or compounds that will enhance the therapeutic efficacy of known chemotherapeutic drugs. It is unclear if SAHB’s themselves will be pharmacologically stable enough to use as drugs, however it should be straightforward enough to develop more stable mimetics of these domains. The larger issue will be whether these compounds will have a therapeutic index. All cells in the body contain bcl2 family members and it is unclear if tumors will be more susceptible to the effects of BH3 domains than normal cells. Dr. Walensky presented preliminary results that SAHB’s can be delivered to mice without apparent toxic effect. Furthermore, the SAHB’s decreased the leukemic burden in a murine xenograft model of AML, suggesting that there may be a therapeutic index for SAHB’s. The group is proceeding with more detailed work to optimize delivery of the compound and see if we can deliver a designer death to the door of AML cells.