Bcl-2 inhibits apoptosis induced by a wide variety of stimuli. In contrast, the Bcl-2 homologue, Bax, antagonizes Bcl-2's death protecting function. Bcl-2 forms protein-protein homodimers with itself and heterodimers with Bax, and previous experiments have shown that point mutations in Bcl-2 can abrogate Bax binding while leaving homodimerization intact. These mutagenesis results can be interpreted to suggest that Bcl-2 has separate binding sites that are responsible for homodimer and heterodimer formation. Results from yeast two-hybrid studies have also suggested that homodimerization and heterodimerization reflect distinct modes of interaction. However, using quantitative plate binding assays, we now show that Bax as well as peptides derived from the BH3 domains of Bax and Bak block both Bcl-2/ Bax binding and Bcl-2/Bcl-2 binding. Similar assays demonstrate that Bcl-x L can form both homodimers and heterodimers and that these interactions are also inhibited by Bax and the BH3-derived peptides. These results demonstrate that the same binding motifs are responsible for both homodimerization and heterodimerization of Bcl-2 family members.

Potent adenosine hA2A receptor antagonists are often accompanied by poor aqueous solubility, which presents issues for drug development. Herein we describe the early exploration of the structure-activity relationships of a lead pyrimidin-4-yl acetamide series to provide potent and selective 2-amino-N-pyrimidin-4-yl acetamides as hA2A receptor antagonists with excellent aqueous solubility. In addition, this series of compounds has demonstrated good bioavailability and in vivo efficacy in a rodent model of Parkinson's disease, despite having reduced potency for the rat A2A receptor versus the human A2A receptor.

A series of oxamyl dipeptides were optimized for pan caspase inhibition, anti-apoptotic cellular activity and in vivo efficacy. This structure-activity relationship study focused on the P4 oxamides and warhead moieties. Primarily on the basis of in vitro data, inhibitors were selected for study in a murine model of alpha-Fas-induced liver injury. IDN-6556 (1) was further profiled in additional in vivo models and pharmacokinetic studies. This first-in-class caspase inhibitor is now the subject of two Phase II clinical trials, evaluating its safety and efficacy for use in liver disease.

Bad, an inducer of programmed cell death, was recently isolated from a mouse cDNA library by its ability to bind to the anti-apoptotic protein BCL-2. Sequence analysis suggested that Bad was a member of the BCL-2 gene family that encodes both inducers and inhibitors of programmed cell death. To further analyze the role of BAD in the network of homo-and heterodimers formed by the BCL-2 family, we have cloned the human homologue of BAD and assessed its biological activity and its interactions with wild type and mutant BCL-2 family proteins. Our results indicate that the human BAD protein, like its mouse homologue, is able to induce apoptosis when transfected into mammalian cells. Furthermore, in yeast two-hybrid assays as well as quantitative in vitro interaction assays, human Bad interacted with BCL-2 and BCL-X L . Sequence alignments of human BAD revealed the presence of a BH-3 homology domain as seen in other BCL-2 family proteins. Peptides derived from this domain were able to completely inhibit the dimerization of BAD with BCL-X L . Thus, as previously shown for BAX, BAK, BCL-2, and BCL-X L , the BH3 domain of BAD is required for its dimerization with other BCL-2 family proteins. BAD was further analyzed for its ability to bind to various mutants of BCL-2 and BCL-X L that have lost the ability to bind BAX and BAK, some of which retain biological activity and some of which do not. Surprisingly, all of the mutated BCL-2 and BCL-X L proteins analyzed strongly interacted with human BAD. Our data thus indicate that mutations in BCL-2 and BCL-X L can differentially affect the heterodimeric binding of different death-promoting proteins and have implications concerning the relationship between heterodimerization and biological activity.The BCL-2 family of proteins consists of inhibitors and inducers of programmed cell death or apoptosis (1-3). Inhibitors include the BCL-2 (4, 5) and BCL-X L proteins (6) and inducers include BAX, BAK, and BCL-X S (6 -10). These proteins have been shown to form a network of homo-and heterodimers (11). A number of studies suggest that dimer formation is essential for the biological activity of these molecules. For example, mutagenesis data have demonstrated a correlation between BCL-2 activity and the ability to form heterodimers with BAX (12). However, other experiments with BCL-X L suggested that dimerization with Bax was not necessary for biological activity (13).Bad was originally cloned from mouse cDNA by its ability to bind to BCL-2, both in yeast two-hybrid interactions and by direct biochemical interaction (14). It was subsequently shown to interact more strongly with BCL-X L than with BCL-2, and in functional studies it antagonized the protective effect of BCL-X L . To date, the human homologue has not been reported. The sequences of the BCL-2 family proteins show several regions of clustered conserved residues, termed by some investigators BH-1 to BH-4 domains (12,15,16). The crystal and NMR structures of BCL-X L show a potential binding pocket on the surface of the molecule f...