Catheter ablation of atrial fibrillation is typically performed with uninterrupted anticoagulation with warfarin or interrupted non-vitamin K antagonist oral anticoagulant therapy. Uninterrupted ...anticoagulation with a non-vitamin K antagonist oral anticoagulant, such as dabigatran, may be safer; however, controlled data are lacking. We investigated the safety of uninterrupted dabigatran versus warfarin in patients undergoing ablation of atrial fibrillation.
In this randomized, open-label, multicenter, controlled trial with blinded adjudicated end-point assessments, we randomly assigned patients scheduled for catheter ablation of paroxysmal or persistent atrial fibrillation to receive either dabigatran (150 mg twice daily) or warfarin (target international normalized ratio, 2.0 to 3.0). Ablation was performed after 4 to 8 weeks of uninterrupted anticoagulation, which was continued during and for 8 weeks after ablation. The primary end point was the incidence of major bleeding events during and up to 8 weeks after ablation; secondary end points included thromboembolic and other bleeding events.
The trial enrolled 704 patients across 104 sites; 635 patients underwent ablation. Baseline characteristics were balanced between treatment groups. The incidence of major bleeding events during and up to 8 weeks after ablation was lower with dabigatran than with warfarin (5 patients 1.6% vs. 22 patients 6.9%; absolute risk difference, -5.3 percentage points; 95% confidence interval, -8.4 to -2.2; P<0.001). Dabigatran was associated with fewer periprocedural pericardial tamponades and groin hematomas than warfarin. The two treatment groups had a similar incidence of minor bleeding events. One thromboembolic event occurred in the warfarin group.
In patients undergoing ablation for atrial fibrillation, anticoagulation with uninterrupted dabigatran was associated with fewer bleeding complications than uninterrupted warfarin. (Funded by Boehringer Ingelheim; RE-CIRCUIT ClinicalTrials.gov number, NCT02348723 .).
Anti-apoptotic BCL-2 family proteins block cell death by trapping the critical α-helical BH3 domains of pro-apoptotic members in a surface groove. Cancer cells hijack this survival mechanism by ...overexpressing a spectrum of anti-apoptotic members, mounting formidable apoptotic blockades that resist chemotherapeutic treatment. Drugging the BH3-binding pockets of anti-apoptotic proteins has become a highest-priority goal, fueled by the clinical success of ABT-199, a selective BCL-2 inhibitor, in reactivating apoptosis in BCL-2-dependent cancers. BFL-1 is a BCL-2 homolog implicated in melanoma, lymphoma, and other cancers, and remains undrugged. A natural juxtaposition of two unique cysteines at the binding interface of the NOXA BH3 helix and BFL-1 pocket informed the development of stapled BH3 peptides bearing acrylamide warheads to irreversibly inhibit BFL-1 by covalent targeting. Given the frequent proximity of native cysteines to regulatory binding surfaces, covalent stapled peptide inhibitors provide a new therapeutic strategy for targeting pathologic protein interactions.
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•A unique juxtaposition of cysteines occurs in the NOXA BH3/BFL-1 interaction•Oxidizing conditions yield a disulfide bond between NOXA BH3 and BFL-1•Stapled BH3 peptides bearing electrophilic warheads selectively react with BFL-1•Covalent stapled peptide inhibitors represent a new class of protein modulators
Huhn et al. report the development of stapled peptides that covalently react with a discrete cysteine at the BH3-binding interface of anti-apoptotic BFL-1, representing a new strategy for selective covalent targeting of pathologic proteins in cancer and other diseases.
Antibodies are essential biological research tools and important therapeutic agents, but some exhibit non-specific binding to off-target proteins and other biomolecules. Such polyreactive antibodies ...compromise screening pipelines, lead to incorrect and irreproducible experimental results, and are generally intractable for clinical development. Here, we design a set of experiments using a diverse naïve synthetic camelid antibody fragment (nanobody) library to enable machine learning models to accurately assess polyreactivity from protein sequence (AUC > 0.8). Moreover, our models provide quantitative scoring metrics that predict the effect of amino acid substitutions on polyreactivity. We experimentally test our models' performance on three independent nanobody scaffolds, where over 90% of predicted substitutions successfully reduced polyreactivity. Importantly, the models allow us to diminish the polyreactivity of an angiotensin II type I receptor antagonist nanobody, without compromising its functional properties. We provide a companion web-server that offers a straightforward means of predicting polyreactivity and polyreactivity-reducing mutations for any given nanobody sequence.
Annexin A7 (ANXA7) is a member of the multifunctional calcium or phospholipid-binding annexin gene family. While low levels of ANXA7 are associated with aggressive types of cancer, the clinical ...impact of ANXA7 in prostate cancer remains unclear. Tissue microarrays (TMA) have revealed several new molecular markers in human tumors. Herein, we have identified the prognostic impact of ANXA7 in a prostate cancer using a tissue microarray containing 637 different specimens.
The patients were diagnosed with prostate cancer and long-term follow-up information on progression (median 5.3 years), tumor-specific and overall survival data (median 5.9 years) were available. Expression of Ki67, Bcl-2, p53, CD-10 (neutral endopeptidase), syndecan-1 (CD-138) and ANXA7 were analyzed by immunohistochemistry.
A bimodal distribution of ANXA7 was observed. Tumors expressing either high or no ANXA7 were found to be associated with poor prognosis. However, ANXA7 at an optimal level, in between high and no ANXA7 expression, had a better prognosis. This correlated with low Ki67, Bcl-2, p53 and high syndecan-1 which are known predictors of early recurrence. At Gleason grade 3, ANXA7 is an independent predictor of poor overall survival with a p-value of 0.003. Neoadjuvant hormonal therapy, which is known to be associated with overexpression of Bcl-2 and inhibition of Ki67 LI and CD-10, was found to be associated with under-expression of ANXA7.
The results of this TMA study identified ANXA7 as a new prognostic factor and indicates a bimodal correlation to tumor progression.
BCL-2 family proteins are high-priority cancer targets whose structures provide essential blueprints for drug design. Whereas numerous structures of anti-apoptotic BCL-2 protein complexes with ...α-helical BH3 peptides have been reported, the corresponding panel of apo structures remains incomplete. Here, we report the crystal structure of apo BFL-1 at 1.69-Å resolution, revealing similarities and key differences among unliganded anti-apoptotic proteins. Unlike all other BCL-2 proteins, apo BFL-1 contains a surface-accessible cysteine within its BH3-binding groove, allowing for selective covalent targeting by a NOXA BH3-based stapled peptide inhibitor. The crystal structure of this complex demonstrated the sulfhydryl bond and fortuitous interactions between the acrylamide-bearing moiety and a newly formed hydrophobic cavity. Comparison of the apo and BH3-liganded structures further revealed an induced conformational change. The two BFL-1 structures expand our understanding of the surface landscapes available for therapeutic targeting so that the apoptotic blockades of BFL-1-dependent cancers can be overcome.
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•The apo structure of BFL-1 reveals key differences among BCL-2 proteins•The unliganded form of BFL-1 bears a unique cysteine at its BH3-binding surface•A BFL-1/stapled peptide complex provides a blueprint for covalent targeting•The conformation of the BH3 groove is altered in response to covalent inhibition
Harvey et al. solved the structure of unliganded BFL-1, revealing distinctive features of its canonical BH3-binding groove, including a surface-exposed cysteine not found in other BCL-2 family proteins. The structure of the covalent complex between BFL-1 and a cysteine-reactive stapled peptide inhibitor provides a blueprint for selective targeting of BFL-1 in cancer.
BAX is a pro-apoptotic protein that transforms from a cytosolic monomer into a toxic oligomer that permeabilizes the mitochondrial outer membrane. How BAX monomers assemble into a higher-order ...conformation, and the structural determinants essential to membrane permeabilization, remain a mechanistic mystery. A key hurdle has been the inability to generate a homogeneous BAX oligomer (BAXO) for analysis. Here, we report the production and characterization of a full-length BAXO that recapitulates physiologic BAX activation. Multidisciplinary studies revealed striking conformational consequences of oligomerization and insight into the macromolecular structure of oligomeric BAX. Importantly, BAXO enabled the assignment of specific roles to particular residues and α helices that mediate individual steps of the BAX activation pathway, including unexpected functionalities of BAX α6 and α9 in driving membrane disruption. Our results provide the first glimpse of a full-length and functional BAXO, revealing structural requirements for the elusive execution phase of mitochondrial apoptosis.
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•Fos-12 induces homogeneous BAX oligomers that recapitulate physiologic activation•SAXS, HXMS, and crosslinking analyses reveal conformational features of BAXO•BAXO distinguishes between the structural determinants of activation and poration•BAXO uncovered roles for α6 and α9 in the execution phase of mitochondrial apoptosis
Hauseman, Harvey, et al. overcome long-standing barriers in generating a full-length, homogeneous oligomer of BAX for analysis. BAXO is a curvilinear species of 6–8 monomeric units spanning 20 nm. BAXO bridges the gap between monomeric/dimeric BAX and its macromolecular pores, providing mechanistic insights into the execution phase of mitochondrial apoptosis.
Cancer cells overexpress a diversity of anti-apoptotic BCL-2 family proteins, such as BCL-2, MCL-1, and BFL-1/A1, to enforce cellular immortality. Thus, intensive drug development efforts have ...focused on targeting this class of oncogenic proteins to overcome treatment resistance. Whereas a selective BCL-2 inhibitor has been FDA approved and several small molecule inhibitors of MCL-1 have recently entered phase I clinical testing, BFL-1/A1 remains undrugged. Here, we developed a series of stapled peptide design principles to engineer a functionally selective and cell-permeable BFL-1/A1 inhibitor that is specifically cytotoxic to BFL-1/A1-dependent human cancer cells. Because cancers harbor a diversity of resistance mechanisms and typically require multi-agent treatment, we further investigated BFL-1/A1 co-dependencies by mining a genome-scale CRISPR-Cas9 screen. We identified ataxia-telangiectasia-mutated (ATM) kinase as a BFL-1/A1 co-dependency in acute myeloid leukemia (AML), which informed the validation of BFL-1/A1 and ATM inhibitor co-treatment as a synergistic approach to subverting apoptotic resistance in cancer.
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•A stapled NOXA BH3 helix was engineered for selective BFL-1 targeting•Key design principles eliminated membrane disruption and maximized cell uptake•A lead BFL-1 inhibitor is selectively cytotoxic to BFL-1-dependent cancer•A genetic co-dependency of BFL-1 and ATM informed a synergistic treatment
Guerra et al. constructed an exquisitely selective BFL-1 inhibitor capable of covalent BFL-1 targeting and cellular penetrance without membrane disruption. Mining a genetic dependency database revealed a spectrum of BFL-1 dependency in cancer and an ATM co-dependency in AML, prompting the combination of BFL-1 and ATM inhibitors to achieve synergistic cytotoxicity.
The BCL-2 family is composed of anti- and pro-apoptotic members that respectively protect or disrupt mitochondrial integrity. Anti-apoptotic overexpression can promote oncogenesis by trapping the ...BCL-2 homology 3 (BH3) “killer domains” of pro-apoptotic proteins in a surface groove, blocking apoptosis. Groove inhibitors, such as the relatively large BCL-2 drug venetoclax (868 Da), have emerged as cancer therapies. BFL-1 remains an undrugged oncogenic protein and can cause venetoclax resistance. Having identified a unique C55 residue in the BFL-1 groove, we performed a disulfide tethering screen to determine if C55 reactivity could enable smaller molecules to block BFL-1's BH3-binding functionality. We found that a disulfide-bearing N-acetyltryptophan analog (304 Da adduct) effectively targeted BFL-1 C55 and reversed BFL-1-mediated suppression of mitochondrial apoptosis. Structural analyses implicated the conserved leucine-binding pocket of BFL-1 as the interaction site, resulting in conformational remodeling. Thus, therapeutic targeting of BFL-1 may be achievable through the design of small, cysteine-reactive drugs.
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•A disulfide tethering screen identifies small molecules that target BFL-1 C55•Structural analyses reveal the conformational consequences of disulfide formation•Lead molecule 4E14 effectively competes with BH3-binding at the BFL-1 groove•Covalent 4E14 targeting of C55 inhibits BFL-1 suppression of mitochondrial apoptosis
Harvey et al. perform a disulfide tethering screen of BFL-1, a BCL-2 protein implicated in cancer, in order to identify molecules capable of covalently targeting a unique C55 residue of the anti-apoptotic groove. 4E14 derivatization (304 Da moiety) effectively blocks BFL-1, informing the design of cysteine-reactive drugs to selectively target BFL-1.
While there are hundreds of predicted E3 ligases, characterizing their applications for targeted protein degradation has proved challenging. Here, we report a chemical biology approach to evaluate ...the ability of modified recombinant E3 ligase components to support neo-substrate degradation. Bypassing the need for specific E3 ligase binders, we use maleimide-thiol chemistry for covalent functionalization followed by E3 electroporation (COFFEE) in live cells. We demonstrate that electroporated recombinant von Hippel-Lindau (VHL) protein, covalently functionalized at its ligandable cysteine with JQ1 or dasatinib, induces degradation of BRD4 or tyrosine kinases, respectively. Furthermore, by applying COFFEE to SPSB2, a Cullin-RING ligase 5 receptor, as well as to SKP1, the adaptor protein for Cullin-RING ligase 1 F box (SCF) complexes, we validate this method as a powerful approach to define the activity of previously uncharacterized ubiquitin ligase components, and provide further evidence that not only E3 ligase receptors but also adaptors can be directly hijacked for neo-substrate degradation.
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•COFFEE tests neo-substrate degradation by chemically modified E3 ligase components•Recombinant E3s are labeled with maleimide-functionalized neo-substrate ligands•Proof-of-concept degradation by electroporated JQ1- or dasatinib-labeled VHL•SPSB2 and SKP1 can be redirected to degrade tyrosine kinases
Pinch et al. present a method to test neo-substrate degradation by recombinant E3 ligase components in live cells, called COFFEE (covalent functionalization followed by E3 electroporation). Following proof-of-concept studies with VHL, they demonstrate neo-substrate degradation by the Cullin-RING ligase receptor SPSB2, and by the adaptor protein, SKP1.
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