Bcl-2, Bcl-XL, and Mcl-1 are anti-apoptotic proteins that block activation of the mitochondria! pathway of apoptosis by sequestering BH3-only proteins and preventing mitochondrial outer membrane permeabilization. Mcl-1 acts as an important survival factor in a number of hematologic malignancies, including Multiple Myeloma, Chronic Lymphocytic Leukemia, and Acute Myelogenous Leukemia, suggesting that specific inhibitors of Mcl-1 anti-apoptosis functions could be of great therapeutic value. We will present fluorescence polarization and molecular data to demonstrate that Mcl-1 interacts with the family of BH3-only proteins in a pattern that is distinct from Bcl-XL, suggesting that the structure of the Bcl-2 homology (BH) 1-3 domain hydrophobic binding groove of Mcl-1 has a distinct structure. These data support the hypothesis that it will be possible to identify low molecular weight compounds that specifically inhibit Mcl-1, thereby promoting the apoptosis of tumor cells. The goal of this proposal is to develop a peptide competition assay based on fluorescence polarization technology that will be developed into a high-throughput screen to identify inhibitors of Mcl-1. Molecular and biologic assays will be described that will confirm the target specificity and biologic effects of lead compounds.
Specific Aim #1 : To use a peptide competition binding assay to verify the fluorescence polarization (FP) binding data, and to validate the approach for the high-throughput assay.
Specific Aim #2 : To reconfigure the FP assay to a 96-well format, and to perform preliminary experiments to demonstrate its utility.
Specific Aim #3 : To develop biologic assays to determine if compounds identified in the high-throughput screen actually target Mcl-1 and induce apoptosis in a mechanistically predictable manner. ? ?
