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Protein-protein interactions often involve secondary structural elements, such as helices. Protein-protein interactions within the Bcl-2 family are mediated by the helical BH3 domain of proapoptotic family members, such as Bad, Bak, Bax, or Bid. Here, we report that two 5-residue fragments located at the N- and C-termini of the 16-residue BH3 domain of Bad, respectively, serve as affinity-enhancing motifs (AEMs) for the BH3 domain. When added to the BH3 domain derived from other proapoptotic proteins such as Bak, Bax, or Bid, these AEMs significantly increased the Bcl-2-binding affinity of these BH3 peptides by promoting the helical structure. This finding may point to a new strategy for studying and mimicking helical peptide-protein interactions involved in apoptosis.

Apoptosis, a fundamental process for both human health and disease, is initiated and regulated by protein-protein interactions, notable examples of which are the interactions involving Bcl-2 and IAP protein families. This article discusses recent advances in the use of chemical approaches in discovering and studying small molecules targeted to proteins of the Bcl-2 and IAP families. These small molecules and their complexes with receptors provide the tools and model systems to probe the basic mechanism of molecule recognition underling the life and death of cells and develop novel strategies for therapeutic intervention of the dysregulated apoptotic process. The review of these studies highlights the opportunity and challenge in this emerging area of chemical and chemical biological research. [Copyright &y& Elsevier]

Bcl-2 family proteins are key regulators of programmed cell death or apoptosis that is implicated in many human diseases, particularly cancer. In recent years, they have attracted intensive interest in both basic research to understand the fundamental principles of cell survival and cell death and drug discovery to develop a new class of anticancer agents. The Bcl-2 family includes both anti- and pro-apoptotic proteins with opposing biological functions in either inhibiting or promoting cell death. High expression of anti-apoptotic members such as Bcl-2 and Bcl-xL commonly found in human cancers contributes to neoplastic cell expansion and interferes with the therapeutic action of many chemotherapeutic drugs. The functional blockade of Bcl-2 or Bcl-xL could either restore the apoptotic process in tumor cells or sensitize these tumors for chemo- and radiotherapies. This article reviews the recent progress in the design and discovery of small molecules that block the anti-apoptotic function of Bcl-2 or Bcl-xL. These chemical inhibitors are effective modulators of apoptosis and promising leads for the further development of new anticancer agents. Oncogene (2000) 19, 6627–6631. [ABSTRACT FROM AUTHOR]

Cytochrome c release is a central step in the apoptosis induced by many death stimuli. Bcl-2 plays a critical role in controlling this step. In this study, we investigated the upstream mechanism of cytochrome c release induced by ethyl 2-amino-6-bromo-4-(1-cyano-2-ethoxy2-oxoethyl)-4H-chromene-3-carboxylate (HA14-1), a recently discovered small molecule inhibitor of Bcl-2. HA14-1 was found to induce cytochrome c release from the mitochondria of intact cells but not from isolated mitochondria. Cytochrome c release from isolated mitochondria requires the presence of both HA14-1 and exogenous Ca2+. This suggests that both mitochondrial and extramitochondrial signals are important. In intact cells, treatment with HA14-1 caused Ca2+ spike, change in mitochondrial membrane potential (ΔΨm) transition, Bax translocation, and reactive oxygen species (ROS) generation prior to cytochrome c release. Pretreatment with either EGTA acetoxymethyl ester or vitamin E resuited in a significant decrease in cytochrome c release and cell death induced by HA14-1. Furthermore pretreatment with RU-360, an inhibitor of the mitochondrial Ca2+ uniporter, or with EGTA acetoxymethyl ester, but not with vitamin E, prevented the HA14-1-induced ΔΨm transition and Bax translocation. This suggests that ROS generation is an event that occurs after the ΔΨm transition and Bax translocation. Together these data demonstrate that the Ca2+ spike, mitochondrial Bcl-2 presensitization, and subsequent ΔΨm transition, Bax translocation, and ROS generation are important upstream signals for cytochrome c release upon HA14-1 stimulation. The involvement of endoplasmic reticulum and mitochondrial signals suggests both organelles are crucial for HA14-1-induced apoptosis. [ABSTRACT FROM AUTHOR]

Protein–protein interactions within the Bcl-2 family are mediated by the helical BH3 domains of pro-apoptotic family members. To study the mechanism of this BH3 domain–protein interaction, a series of cyclic lactam bridged BH3 peptide analogues were synthesized by a novel combined Fmoc/tBu/Bzl protections strategy. These peptide analogues were studied by circular dichroism spectroscopy and found to adopt highly helical structure. These helical peptides stabilized by a lactam bridge serve as useful models to analyze the structure–function relationship of the pro-apoptotic BH3 domains. Furthermore, the synthetic method for lactam bridge incorporation reported here may find application in studies of other helical structures and development of helix mimics. [Copyright &y& Elsevier]

Summary: ARTS (apoptosis-related protein in the TGF-βsignaling pathway) is a mitochondrial protein that binds XIAP (X-linked inhibitor of apoptosis protein) upon entering the cytosol, thus promoting cell death. Expression of ARTS is lost in some malignancies. Here, we show that ARTS binds to XIAP at BIR1, a domain distinct from the caspase-binding sites. Furthermore, ARTS interacts with the E3 ligase Siah-1 (seven in absentia homolog 1) to induce ubiquitination and degradation of XIAP. Cells lacking either Siah or ARTS contain higher steady-state levels of XIAP. Thus, ARTS serves as an adaptor to bridge Siah-1 to XIAP, targeting it for destruction. [ABSTRACT FROM AUTHOR]

One of the main goals in drug discovery is to identify new chemical entities that have a high likelihood of binding to the target protein to elicit the desired biological response. To this end, virtual screening is being increasingly used as a complement to high-throughput screening to improve the speed and efficiency of the drug discovery and development process. The availability of inexpensive high-performance computing platforms in recent years has transformed this field into one that is highly diverse and rapidly evolving, where large chemical databases have been successfully screened to identify hits for a wide range of targets such as Bcl-2 family proteins, G protein-coupled receptors, kinases, metalloproteins, nuclear hormone receptors, proteases and many more. [Copyright &y& Elsevier]