Purpose: Overexpression of Bcl-2 protein has been observed in more than 80% of B-cell lymphomas, including diffuse large cell lymphoma (DLCL), the most common subtype of non-Hodgkin's lymphoma. We have previously employed the natural product (−)-gossypol to test its therapeutic potential as a small-molecule inhibitor of Bcl-2 for the treatment of B-cell lymphomas. Experimental Design: Recently, we have used a structure-based strategy to design a new class of potent small-molecule inhibitor acting on Bcl-2. One such lead compound is the benzenesulfonyl derivative TW-37, which was designed to target the BH3-binding groove in Bcl-2 where proapoptotic Bcl-2 proteins, such as Bak, Bax, Bid, and Bim bind. Results: In our fluorescence polarization–based binding assays using recombinant Bcl-2, Bcl-X_L, and Mcl-1 proteins, TW-37 binds to Bcl-2, Bcl-X_L, and Mcl-1 with K_i values of 290, 1,110 and 260 nmol/L, respectively. Hence, TW-37 is a potent inhibitor of Bcl-2 and has >3-fold selectivity over Bcl-X_L. In vitro, TW-37 showed significant antiproliferative effect in a de novo chemoresistant WSU-DLCL₂ lymphoma cell line and primary cells obtained from a lymphoma patient with no effect on normal peripheral blood lymphocytes. Coimmunoprecipitation experiments showed that TW-37 disrupted heterodimer formation between Bax or truncated-Bid and antiapoptotic proteins in the order Mcl-1 > Bcl-2 >> Bcl-X_L. As expected, TW-37 caused apoptotic death. Pre-exposure of lymphoma cells to TW-37 significantly enhanced the killing effect of cyclophosphamide-doxorubicin-vincristine-prednisone (CHOP) regimen. The maximum tolerated dose of TW-37 in severe combined immunodeficient (SCID) mice was 40 mg/kg for three i.v. injections when given alone and 20 mg/kg, ×3 when given in combination with CHOP. Using WSU-DLCL₂-SCID mouse xenograft model, the addition of TW-37 to CHOP resulted in more complete tumor inhibition compared with either CHOP or TW-37 alone. Conclusions: We conclude that the administration of TW-37, as a potent Bcl-2 and Mcl-1 inhibitor, to standard chemotherapy may prove an effective strategy in the treatment of B-cell lymphoma.

A critical regulator of the apoptotic machinery is the Bcl‐2 family proteins whose over expression confers a protective effect on malignant cells against death signals of apoptosis. Cancer cells that are resistant to various anti‐cancer drugs and treatment regimen are found to over express these Bcl‐2 proteins such as Bcl‐2, Bcl‐X_L, Mcl‐1, Bcl‐w, and A1/Bfl1. In recent years there has been an exponential growth in the identification as well as synthesis of non‐peptidic cell permeable small‐molecule inhibitors (SMIs) of protein–protein interaction. The focus of this article is on inhibitors of anti‐apoptotic protein Bcl‐2. This review summarizes an up to date knowledge of the available SMIs, their mode of action as well as their current status in preclinical as well as clinical development. J. Cell. Physiol. 218: 13–21, 2009.

Overexpression of Bcl-2/Bcl-XL protein has been observed in more than 80% of B-cell lymphomas. Diffuse large cell lymphoma (DLCL) is the most common subtype of non-Hodgkin's lymphoma. (−)-Gossypol, a natural product isolated from cottonseeds, was discovered as a potent small-molecule inhibitor of Bcl-2 and Bcl-XL proteins, with a Ki value in the nanomole per liter range for both. In vitro, (−)-gossypol showed significant growth inhibition effect against WSU-DLCL2 lymphoma cell line and fresh cells obtained from a lymphoma patient with no effect on normal peripheral blood lymphocytes. As expected (−)-gossypol induced complete cytochrome c release from mitochondria, increased caspases-3 and -9 activity, and caused apoptotic death without affecting protein levels of Bcl-2, Bcl-XL, Bax, and Bak. The addition of cyclophosphamide-Adriamycin-vincristine-prednisolone (CHOP) regimen to lymphoma cells preexposed to (−)-gossypol enhanced killing significantly. The maximum tolerated dose of (−)-gossypol in severe combined immunodeficient (SCID) mice was 40 mg/kg for three i.v. injections when given alone and 20 mg/kg × 3 when given in combination with CHOP. Using WSU-DLCL2-SCID mouse xenograft model, the tumor growth inhibition, the tumor growth delay, and the log10 kill of mice treated with (−)-gossypol + CHOP were better than CHOP or (−)-gossypol alone. We conclude that adding Bcl-2/Bcl-XL small-molecule inhibitor to standard chemotherapy may prove an effective strategy in lymphoma therapy.

Elevated expression of anti-apoptotic Bcl-2 family proteins have been linked to a poor survival rate of patients with Follicular Lymphoma (FL). This prompted us to evaluate a very potent non-peptidic Small-Molecule Inhibitor (SMI) targeting Bcl-2 family proteins, Apogossypolone (ApoG2) using follicular small cleaved cell lymphoma cell line (WSU-FSCCL) and cell isolated from lymphoma patients. ApoG2 inhibited the growth of WSU-FSCCL significantly with a 50% growth inhibition of cells (IC₅₀) of 109 nM and decreased cell number of fresh lymphoma cells. ApoG2 activated caspases-9, -3, and -8, and the cleavage of Poly (ADP-ribose) polymerase (PARP) and Apoptosis Inducing Factor (AIF). In the WSU-FSCCL-SCID xenograft model, ApoG2 showed a significant anti-lymphoma effect, with %ILS of 84% in the intravenous and 63% in intraperitoneal treated mice. These studies suggest that ApoG2 can be an effective therapeutic agent against FL.