Gregory H. Bird

BAX activation is initiated at a novel interaction site

BAX is a pro-apoptotic protein of the BCL-2 family that is stationed in the cytosol until activated by a diversity of stress stimuli to induce cell death. Anti-apoptotic proteins such as BCL-2 counteract BAX-mediated cell death. Although an interaction site that confers survival functionality has been defined for anti-apoptotic proteins, an activation site has not been identified for BAX, rendering its explicit trigger mechanism unknown. We previously developed stabilized α-helix of BCL-2 domains (SAHBs) that directly initiate BAX-mediated mitochondrial apoptosis. Here we demonstrate by NMR analysis that BIM SAHB binds BAX at an interaction site that is distinct from the canonical binding groove characterized for anti-apoptotic proteins. The specificity of the human BIM-SAHB–BAX interaction is highlighted by point mutagenesis that disrupts functional activity, confirming that BAX activation is initiated at this novel structural location. Thus, we have now defined a BAX interaction site for direct activation, establishing a new target for therapeutic modulation of apoptosis.

Hydrocarbon-Stapled Peptides: Principles, Practice, and Progress

Protein structure underlies essential biological processes and provides a blueprint for molecular mimicry that drives drug discovery. Although small molecules represent the lion’s share of agents that target proteins for therapeutic benefit, there remains no substitute for the natural properties of proteins and their peptide subunits in the majority of biological contexts. The peptide α-helix represents a common structural motif that mediates communication between signaling proteins. Because peptides can lose their shape when taken out of context, developing chemical interventions to stabilize their bioactive structure remains an active area of research. The all-hydrocarbon staple has emerged as one such solution, conferring α-helical structure, protease resistance, cellular penetrance, and biological activity upon successful incorporation of a series of design and application principles. Here, we describe our more than decade-long experience in developing stapled peptides as biomedical research tools and prototype therapeutics, highlighting lessons learned, pitfalls to avoid, and keys to success.

Dual role of proapoptotic BAD in insulin secretion and beta cell survival

The proapoptotic BCL-2 family member BAD resides in a glucokinase-containing complex that regulates glucose-driven mitochondrial respiration. Here, we present genetic evidence of a physiologic role for BAD in glucose-stimulated insulin secretion by beta cells. This novel function of BAD is specifically dependent upon the phosphorylation of its BH3 sequence, previously defined as an essential death domain. We highlight the pharmacologic relevance of phosphorylated BAD BH3 by using cell-permeable, hydrocarbon-stapled BAD BH3 helices that target glucokinase, restore glucose-driven mitochondrial respiration and correct the insulin secretory response in Bad-deficient islets. Our studies uncover an alternative target and function for the BAD BH3 domain and emphasize the therapeutic potential of phosphorylated BAD BH3 mimetics in selectively restoring beta cell function. Furthermore, we show that BAD regulates the physiologic adaptation of beta cell mass during high-fat feeding. Our findings provide genetic proof of the bifunctional activities of BAD in both beta cell survival and insulin secretion.

Targeted disruption of the EZH2-EED complex inhibits EZH2-dependent cancer.

Enhancer of zeste homolog2 (EZH2) is the histone lysine N-methyltransferase component of the Polycomb repressive complex 2 (PRC2), which in conjunction with embryonic ectoderm development (EED) and suppressor of zeste 12 homolog (SUZ12), regulates cell lineage determination and homeostasis. Enzymatic hyperactivity has been linked to aberrant repression of tumor suppressor genes in diverse cancers. Here, we report the development of stabilized alpha-helix of EZH2 (SAH-EZH2) peptides that selectively inhibit H3 Lys27 trimethylation by dose-responsively disrupting the EZH2/EED complex and reducing EZH2 protein levels, a mechanism distinct from that reported for small molecule EZH2 inhibitors targeting the enzyme catalytic domain. MLL-AF9 leukemia cells, which are dependent on PRC2, undergo growth arrest and monocyte/macrophage differentiation upon treatment with SAH-EZH2, consistent with observed changes in expression of PRC2-regulated, lineage-specific marker genes. Thus, by dissociating the EZH2/EED complex, we pharmacologically modulate an epigenetic “writer” and suppress PRC2-dependent cancer cell growth.

BH3-Triggered Structural Reorganization Drives the Activation of Proapoptotic BAX

BAX is a proapoptotic BCL-2 family member that lies dormant in the cytosol until converted into a killer protein in response to cellular stress. Having recently identified the elusive trigger site for direct BAX activation, we now delineate by NMR and biochemical methods the essential allosteric conformational changes that transform ligand-triggered BAX into a fully activated monomer capable of propagating its own activation. Upon BAX engagement by a triggering BH3 helix, the unstructured loop between α helices 1 and 2 is displaced, the carboxy-terminal helix 9 is mobilized for membrane translocation, and the exposed BAX BH3 domain propagates the death signal through an autoactivating interaction with the trigger site of inactive BAX monomers. Our structure-activity analysis of this seminal apoptotic process reveals pharmacologic opportunities to modulate cell death by interceding at key steps of the BAX activation pathway.

Hydrocarbon double-stapling remedies the proteolytic instability of a lengthy peptide therapeutic

The pharmacologic utility of lengthy peptides can be hindered by loss of bioactive structure and rapid proteolysis, which limits bioavailability. For example, enfuvirtide (Fuzeon, T20, DP178), a 36-amino acid peptide that inhibits human immunodeficiency virus type 1 (HIV-1) infection by effectively targeting the viral fusion apparatus, has been relegated to a salvage treatment option mostly due to poor in vivo stability and lack of oral bioavailability. To overcome the proteolytic shortcomings of long peptides as therapeutics, we examined the biophysical, biological, and pharmacologic impact of inserting all-hydrocarbon staples into an HIV-1 fusion inhibitor. We find that peptide double-stapling confers striking protease resistance that translates into markedly improved pharmacokinetic properties, including oral absorption. We determined that the hydrocarbon staples create a proteolytic shield by combining reinforcement of overall α-helical structure, which slows the kinetics of proteolysis, with complete blockade of peptide cleavage at constrained sites in the immediate vicinity of the staple. Importantly, double-stapling also optimizes the antiviral activity of HIV-1 fusion peptides and the antiproteolytic feature extends to other therapeutic peptide templates, such as the diabetes drug exenatide (Byetta). Thus, hydrocarbon double-stapling may unlock the therapeutic potential of natural bioactive polypeptides by transforming them into structurally fortified agents with enhanced bioavailability.

A stapled BIM peptide overcomes apoptotic resistance in hematologic cancers

Cancer cells subvert the natural balance between cellular life and death, achieving immortality through pathologic enforcement of survival pathways and blockade of cell death mechanisms. Pro-apoptotic BCL-2 family proteins are frequently disarmed in relapsed and refractory cancer through genetic deletion or interaction-based neutralization by overexpressed antiapoptotic proteins, resulting in resistance to chemotherapy and radiation treatments. New pharmacologic strategies are urgently needed to overcome these formidable apoptotic blockades. We harnessed the natural killing activity of BCL-2-interacting mediator of cell death (BIM), which contains one of the most potent BH3 death domains of the BCL-2 protein family, to restore BH3-dependent cell death in resistant hematologic cancers. A hydrocarbon-stapled peptide modeled after the BIM BH3 helix broadly targeted BCL-2 family proteins with high affinity, blocked inhibitory antiapoptotic interactions, directly triggered proapoptotic activity, and induced dose-responsive and BH3 sequence-specific cell death of hematologic cancer cells. The therapeutic potential of stapled BIM BH3 was highlighted by the selective activation of cell death in the aberrant lymphoid infiltrates of mice reconstituted with BIM-deficient bone marrow and in a human AML xenograft model. Thus, we found that broad and multimodal targeting of the BCL-2 family pathway can overcome pathologic barriers to cell death.

Targeted Disruption of the BCL9/β-Catenin Complex Inhibits Oncogenic Wnt Signaling

Blocking BCL9/β-catenin interaction with a stapled peptide inhibits Wnt-dependent transcription and suppresses growth and metastasis in colon cancer and multiple myeloma. Stapling Down Oncogenic Wnt Signaling The Wnt signaling pathway plays ancient and essential roles—it’s required for embryonic development in all animals and for key functions in adult tissues. Dysregulation of the pathway, however, underlies multiple human cancers. The development of Wnt pathway inhibitors has received considerable attention, but to be useful, such inhibitors must not disrupt vital pathway functions. To address this issue, Takada and colleagues now target an interaction between two Wnt pathway proteins, one of which (BCL9) is highly expressed in tumors but not in the cells of tumor origin. Wnt signaling ultimately increases nuclear levels of the transcriptional activator β-catenin, which promotes the expression of genes involved in cell survival and division. Certain coactivators, including BCL9, can form a complex with β-catenin and increase such gene expression. Takada et al. aimed to disrupt the BCL9–β-catenin interaction with a structured peptide mimicking the BCL9 binding interface. BCL9 binds to a site on β-catenin that differs from those of other binding partners; contact occurs via an α-helical domain of BCL9. The authors stabilized peptides representing that domain by using hydrocarbon stapling, in which chemical restraints reinforce the α-helical structure. These peptides, unlike the unmodified version, were taken up by cancer cells. Additionally, one stabilized α helix of BCL9 (SAH-BCL9) bound β-catenin, selectively dissociating BCL9/β-catenin complexes and inhibiting Wnt-dependent transcription. SAH-BCL9, but not a mutant control peptide, reduced the proliferation of Wnt-dependent colorectal cancer and multiple myeloma cell lines. (SAH-BCL9 did not affect cell lines that do not express BCL9 or depend on Wnt signaling.) Furthermore, in mouse xenograft models of Wnt-driven colon cancer and multiple myeloma, SAH-BCL9 suppressed tumor growth, invasion into nearby tissues, and metastasis, as well as local formation of new blood vessels, in an apparently nontoxic manner. Thus, targeting the BCL9–β-catenin interaction may represent a useful approach for treating Wnt-dependent cancers. Additional experiments will be required to further optimize the drug-like properties of SAH-BCL9. Deregulated Wnt/β-catenin signaling underlies the pathogenesis of a broad range of human cancers, yet the development of targeted therapies to disrupt the resulting aberrant transcription has proved difficult because the pathway comprises large protein interaction surfaces and regulates many homeostatic functions. Therefore, we have directed our efforts toward blocking the interaction of β-catenin with B cell lymphoma 9 (BCL9), a co-activator for β-catenin–mediated transcription that is highly expressed in tumors but not in the cells of origin. BCL9 drives β-catenin signaling through direct binding mediated by its α-helical homology domain 2. We developed a stabilized α helix of BCL9 (SAH-BCL9), which we show targets β-catenin, dissociates native β-catenin/BCL9 complexes, selectively suppresses Wnt transcription, and exhibits mechanism-based antitumor effects. SAH-BCL9 also suppresses tumor growth, angiogenesis, invasion, and metastasis in mouse xenograft models of Colo320 colorectal carcinoma and INA-6 multiple myeloma. By inhibiting the BCL9–β-catenin interaction and selectively suppressing oncogenic Wnt transcription, SAH-BCL9 may serve as a prototype therapeutic agent for cancers driven by deregulated Wnt signaling.

RNA polymerase II carboxy-terminal domain phosphorylation is required for cotranscriptional pre-mRNA splicing and 3'-end formation.

ABSTRACT We investigated the role of RNA polymerase II (pol II) carboxy-terminal domain (CTD) phosphorylation in pre-mRNA processing coupled and uncoupled from transcription in Xenopus oocytes. Inhibition of CTD phosphorylation by the kinase inhibitors 5,6-dichloro-1β-d-ribofuranosyl-benzimidazole and H8 blocked transcription-coupled splicing and poly(A) site cleavage. These experiments suggest that pol II CTD phosphorylation is required for efficient pre-mRNA splicing and 3′-end formation in vivo. In contrast, processing of injected pre-mRNA was unaffected by either kinase inhibitors or α-amanitin-induced depletion of pol II. pol II therefore does not appear to participate directly in posttranscriptional processing, at least in frog oocytes. Together these experiments show that the influence of the phosphorylated CTD on pre-mRNA splicing and 3′-end processing is mediated by transcriptional coupling.

Direct activation of full-length proapoptotic BAK

Proapoptotic B-cell lymphoma 2 (BCL-2) antagonist/killer (BAK) and BCL-2–associated X (BAX) form toxic mitochondrial pores in response to cellular stress. Whereas BAX resides predominantly in the cytosol, BAK is constitutively localized to the outer mitochondrial membrane. Select BCL-2 homology domain 3 (BH3) helices activate BAX directly by engaging an α1/α6 trigger site. The inability to express full-length BAK has hampered full dissection of its activation mechanism. Here, we report the production of full-length, monomeric BAK by mutagenesis-based solubilization of its C-terminal α-helical surface. Recombinant BAK autotranslocates to mitochondria but only releases cytochrome c upon BH3 triggering. A direct activation mechanism was explicitly demonstrated using a liposomal system that recapitulates BAK-mediated release upon addition of BH3 ligands. Photoreactive BH3 helices mapped both triggering and autointeractions to the canonical BH3-binding pocket of BAK, whereas the same ligands crosslinked to the α1/α6 site of BAX. Thus, activation of both BAK and BAX is initiated by direct BH3–interaction but at distinct trigger sites. These structural and biochemical insights provide opportunities for developing proapoptotic agents that activate the death pathway through direct but differential engagement of BAK and BAX.