Bcl-2 Family

Overview of Bcl-2 Family

Bcl-2 is the first discovered anti cell death gene that has been shown to prevent various stimuli induced cell apoptosis, including serum deprivation, heat shock, and chemotherapy agents. In addition, Bcl-2 can also inhibit certain forms of necrotic cell death, such as necrosis induced by hypoxia and respiratory depression.

The Bcl-2 family proteins share structural similarities: firstly, they have a hydrophobic transmembrane domain (TM) at the carbon end, which determines the subcellular localization of the protein; Secondly, they contain four conserved BH domains (BH1-BH4), with BH1 and BH2 shared by all members and involved in the dimerization of Bcl-2 family members. One of the significant features of Bcl-2 family proteins is the ability to form homodimers and heterodimers. The heterodimerization between anti apoptotic and pro apoptotic members of the Bcl-2 family proteins is believed to inhibit the biological activity of their partners, and is mediated by inserting the BH3 region of the pro apoptotic protein into a hydrophobic pocket composed of anti apoptotic proteins BH1, BH2, and BH3. Besides BH1, BH2, and BH3, BH4 is necessary for anti apoptotic activity, while BH3 is necessary for pro apoptotic activity.

Figure 1: Homology of Bcl-2 Family Proteins

Bcl-2 Family Type

According to the role of Bcl-2 family members in apoptosis, Bcl-2 family members can be divided into two categories: anti apoptotic and pro apoptotic.

1. The anti apoptotic Bcl-2 Family proteins mainly include Bcl-2, Bcl xl, Bcl-w, A1, Mcl-1, Bfl-1, etc. They contain at least two structural domains, BH1 and BH2, and have anti apoptotic effects.

2. The pro apoptotic Bcl-2 Family protein can be divided into two types based on its structural characteristics:

① The Bax subtypes mainly include Bax (Bcl-2 associated protein), Bakl (Bcl-2 antagonist killer 1), and Bok, which contain three domains: BH1, BH2, and BH3, but not the BH4 domain.

② BH3 only subtypes: mainly including Bid, Bad, Bik, Bim, Noxa, only Puma, etc., containing only the BH3 domain.

Bax and Bak are the main apoptotic molecules in the Bcl-2 family. Activated Bax and Bak are translocated to mitochondria and undergo conformational changes for oligomerization, inserting into mitochondria to form mitochondrial outer membrane pores, leading to increased mitochondrial outer membrane permeability (MOMP) and releasing many pro apoptotic factors into the cytoplasm, causing apoptosis.

Bcl-2 Family and Apoptosis

The Bcl-2 family proteins mainly exert their function of regulating cell apoptosis through self dimerization or forming heterodimers with proteins such as Bax. Bax-Bcl-2 heterodimers can counteract the anti apoptotic effect of Bcl-2 BCl-2 homodimers and promote apoptosis. The binding of Bcl-2 protein to the membrane is extremely important for its function. Experiments have shown that the anti apoptotic ability of Bcl-2 protein, which loses its membrane localization ability, is significantly weakened, and Bcl-2 proteins with different subcellular localization may participate in different apoptotic pathways in different types of cells.

1. Bcl-2 on the mitochondrial membrane plays a role in inhibiting apoptosis at least at three levels:

① Bcl-2 can alter the redox state of mitochondrial thiol groups to control their membrane potential and regulate cell apoptosis;

② Bcl-2 protein can prevent the oligomerization of Bax/BAK, thereby inhibiting the release of important proteins such as mitochondrial cytochrome C and/or AIF (apoptosis mediator), and improving the survival ability of cells;

③ Bcl-2 can prevent caspase activation, such as: caspase-2， Plays its anti apoptotic role upstream of mitochondrial cytochrome C release. Research has found that this effect is mainly achieved through CED-4. Bcl-2 can inhibit the oligomerization of CED-4 in the CED-3/CED-4 complex by directly interacting with CED-9, thereby preventing the activation of caspase.

2. Bcl-2 inhibits apoptosis by suppressing the release of Ca2+during cell death. During the process of cell apoptosis, DNA digestion requires the involvement of Ca2+- dependent endonucleases, and the Bcl-2 protein on the endoplasmic reticulum membrane can block the release of Ca2+from the endoplasmic reticulum, reducing the activity of Ca2+- dependent endonucleases and thus blocking cell apoptosis.

3. Bcl-2 protein can act on signaling molecules on mitochondria and nuclear pore complexes, controlling cellular signal transduction and prolonging cell survival. Bcl-2 protein may exert its antioxidant effect or inhibit the production of oxidative free radicals and cell apoptosis by regulating the transcription factor NF kappaB.

4. Bcl-2 can induce activation of the P53 dependent apoptotic pathway. Studies on multiple cell lines in vitro have shown that silencing Bcl-2 can generally induce P53 dependent apoptosis in colon cancer, and Bax and caspase-2 are essential mediators in the Bcl-2/P53 apoptosis pathway. Based on this, it is speculated that under physiological conditions, Bcl-2 may inhibit the function of the apoptotic precursor of P53, and under the influence of some stress factors, the activation potential of P53 will be induced. It has been confirmed that activated and stable P53 can alter the expression rate of Bcl-2, thereby inducing apoptosis.

Figure 2: Bcl-2 and cell survival and apoptosis

Bcl-2 Family and Tumors

The inhibition of programmed cell death is the most critical step in the process of tumor formation. Bcl-2 gene, as an important apoptosis inhibiting gene, breaks the self stabilizing balance mechanism of cells, preventing damaged or mutated cells from being cleared in a timely manner. Under the synergistic effect of proliferation genes and growth inhibiting genes, it develops into tumors. Bcl-2 prolongs cell lifespan and accumulates cells by inhibiting apoptosis, initiating its role in tumorigenesis. The Bcl-2 gene itself does not have strong oncogene activity, but its activity is significantly enhanced after point mutation, promoting tumor development. Somatic mutations accompanied by translocation of Bcl-2 gene can transform follicular lymphoma into diffuse large cell lymphoma. Bcl-2 can interact with vascular endothelial growth factor (VEGF) and other factors, thereby affecting tumor angiogenesis and promoting tumor formation and development. Bcl-2 is highly expressed in many human tumors, such as liver cancer, prostate cancer, colon cancer, lung cancer, stomach cancer, non Hodgkin lymphoma, neuroblastoma, breast cancer, etc. At present, it is believed that the high expression of Bcl-2 is not a characteristic of a certain tumor, but reflects a proliferative state of tumor cells.

The Bcl-2 protein prevents cell apoptosis, which is not only related to the formation of many tumors, but also plays an important role in the formation of tumor chemotherapy resistance. Experiments have shown that overexpression of Bcl-2 can make cells more effectively tolerant to chemotherapy drugs, including dexamethasone, cytarabine, methotrexate, cyclophosphamide, doxorubicin, etoposide (VP-16), camptothecin, nitrogen mustard, 5-fluorouracil, mitoxantrone, cisplatin, vincristine, and some retinoic acid compounds. The drug tolerance of tumor cells caused by elevated Bcl-2 protein levels is different from general chemical tolerance, with the main differences being: ① Bcl-2 protein cannot prevent drug accumulation in cells; ② Bcl-2 protein does not alter the degree of DNA damage induced by drugs or the repair rate of damaged DNA by cells Bcl-2 protein has no effect on nucleotide pool or cell cycle rate.

Bcl-2 Family Inhibitors

1、  Venetoclax (ABT-199; GDC-0199)：It is a BH3 mimic that can mimic the action of BH3 monofunctional proteins and bind to BCL-2 protein in a manner similar to BH3, thereby inhibiting the ability of BCL-2 to bind to BAX or BAK. Venetoclax is a highly efficient, selective, and orally effective Bcl-2 inhibitor with a Ki of less than 0.01nM.

2、  Lisaftoclax（APG-2575）：It is a dual inhibitor of Bcl-2 and Bcl xl, with IC₅₀ values of 2nM and 5.9nM for Bcl-2 and Bcl xl, respectively. By inhibiting Bcl-2 protein, the programmed cell death mechanism (apoptosis) of tumor cells is restored, thereby inducing tumor cell apoptosis and achieving the goal of treating tumors.

3、  Sonrotoclax：It is an effective oral active Bcl-2 inhibitor, indicated for chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), acute myeloid leukemia (AML), non Hodgkin lymphoma (including WM, MCL, MZL), and multiple myeloma (MM).

4、  DT2216: DT2216 is a protein hydrolysis targeted chimeric protein (PROTAC) that can effectively and selectively inhibit BCL-2 wild-type and multiple drug-resistant mutant types (including G101V, D103Y, etc.) with high efficiency. DT2216 degrades BCR-ABL protein by recruiting Von Hippel Lindau (VHL) E3 ubiquitin ligase.