Small molecule inhibitors of the Bcl-XL/Bak interaction activate the cell death pathway


The removal of unneeded or damaged cells by apoptosis is essential for the maintenance of cellular homeostasis and the prevention of cancer. Apoptosis also plays a critical role in cancer therapy, as many cytotoxic agents used in chemotherapy act by promoting apoptotic cell death. Indeed, insensitivity of tumor cells to radiation or chemotherapy can often be attributed to defects in apoptotic signaling pathways. Therefore, compounds that can stimulate apoptosis might become powerful new anticancer therapeutics.

Attractive therapeutic target: Members of the Bcl-2 protein family are central regulators of apoptosis, making disrupting the interaction between Bcl-2 and its pro-apoptotic binding partners an attractive therapeutic target. There are two pan Bcl-2 small molecule inhibitors in clinical phase II trials (Ascenta Therapeutics; Gemin X), and several in pre-clinical stage development (Abbott) for both hematological and solid tumors.

Screen for small molecule activators of apoptosis: Bax and Bak, two proapoptotic members of the family permeablize the outer mitochondrial membrane and trigger the apoptosis cascade. Bcl-2, Bcl-XL and other anti-apoptotic proteins bind to the BH3 domain of Bax and Bak, reducing their activity and preventing apoptosis. Dr. Yuan’s laboratory developed an in vitro screen to discover small molecules that disrupt binding of Bcl-XL to Bak’s BH3 domain. In the screen, a fluorescently tagged Bak-derived peptide is bound to the recombinant Bcl-XL. Screen hits (termed BH3 inhibitors) displaced this peptide and caused a change in fluorescence.

BH3 inhibitors (BH3Is) block Bcl-XL/Bak interaction and induce cell death: The secondary screening efforts summarized below validated the cellular target and physiological effects of the screen hits. Taken together these findings imply that discovered BH3 inhibitors are a new class of highly selective small molecules that can induce apoptosis in cancerous cells.

• BH3 inhibitors are selective: In vitro binding assays demonstrated that the small molecule screen hits specifically disrupted interactions between the BH3 domain and anti-apoptotic Bcl-2 family proteins, and did not disrupt non-specific protein-protein interactions.

• BH3 inhibitors increase cell death in cancer-like cells: An increased level of apoptosis was observed in cultured human cells upon treatment with BH3Is. Remarkably, cells pre-treated with a caspase inhibitor, which lead to decreased apoptosis (a hallmark of cancer cells), were also susceptible to the BH3Is.

• BH3 inhibitors disrupt Bax/Bcl-XL interaction in vivo: Fluorescently tagged proteins, Bax-YFP and Bcl-XL-CFP were co-expressed and their in vivo interaction was confirmed using FRET, an optical microscopy technique that measures protein-protein binding. Upon addition of the BH3Is, the FRET signal diminished indicating that Bax, the proapoptotic protein, was not bound to Bcl- XL and could trigger cell death.


Therapeutics: This invention addresses the need for selective and efficient inhibitors of BH3 domain-mediated heterodimerization of Bcl-2 family proteins. Treatment with small molecule BH3 inhibitors disrupts its interaction with Bax and Bak and enables them to trigger the cell death pathway.

The exciting prospects of clinical manipulation of the Bcl-XL/Bak interaction may provide a novel therapeutic strategy for the treatment of cancers and autoimmune diseases:

• Cancer therapy: Anti-apoptotic Bcl-2 proteins are over-expressed in a number of tumors, including hematological cancers (leukemias), and several types of solid cancer, including prostate, breast and lung cancers. In addition, they are frequently associated with resistance to conventional chemo- and radiotherapy. The BH3 inhibitors discovered in the Yuan laboratory can be used alone or in combination with already existing therapeutic agents.

• Rheumatoid arthritis and other diseases: Recent reports indicate that BH3-only members of the Bcl-2 family can be immunomodulators and play a role in the development of rheumatoid arthritis. BH3 inhibitors can potentially become therapeutics against this chronic and widespread disease.

Screen to identify new BH3 inhibitors: The high-throughput screening method developed by the inventors is simple, cheap and efficient. Secondary screening validated the activity and specificity of the small molecule screen hits. This screening method could be used to screen a larger library and discover new small molecule BH3 inhibitors.

Additional Information:

Intellectual Property Status: Issued U.S. patent nos.: 6,706,766; 7,390,836

Intellectual Property Status: Patent(s) Pending