Publications by authors named "Gregory H Bird"

45 Publications

Glucose metabolism and pyruvate carboxylase enhance glutathione synthesis and restrict oxidative stress in pancreatic islets.

Cell Rep 2021 Nov;37(8):110037

Department of Cancer Biology, Dana-Farber Cancer Institute, 450 Brookline Ave., Boston, MA 02115, USA; Department of Medical Oncology, Dana-Farber Cancer Institute, Boston 02115, MA, USA; Department of Medicine, Harvard Medical School, 240 Longwood Ave., Boston, MA 02115, USA. Electronic address:

Glucose metabolism modulates the islet β cell responses to diabetogenic stress, including inflammation. Here, we probed the metabolic mechanisms that underlie the protective effect of glucose in inflammation by interrogating the metabolite profiles of primary islets from human donors and identified de novo glutathione synthesis as a prominent glucose-driven pro-survival pathway. We find that pyruvate carboxylase is required for glutathione synthesis in islets and promotes their antioxidant capacity to counter inflammation and nitrosative stress. Loss- and gain-of-function studies indicate that pyruvate carboxylase is necessary and sufficient to mediate the metabolic input from glucose into glutathione synthesis and the oxidative stress response. Altered redox metabolism and cellular capacity to replenish glutathione pools are relevant in multiple pathologies beyond obesity and diabetes. Our findings reveal a direct interplay between glucose metabolism and glutathione biosynthesis via pyruvate carboxylase. This metabolic axis may also have implications in other settings where sustaining glutathione is essential.
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http://dx.doi.org/10.1016/j.celrep.2021.110037DOI Listing
November 2021

Characterizing Native and Hydrocarbon-Stapled Enfuvirtide Conformations with Ion Mobility Mass Spectrometry and Hydrogen-Deuterium Exchange.

J Am Soc Mass Spectrom 2021 Mar 3;32(3):753-761. Epub 2021 Feb 3.

Metrology, National Research Council Canada, 1200 Montreal Road, Ottawa, Ontario K1A 0R6, Canada.

The number of approved peptide therapeutics, as well as those in development, has been increasing in recent years. Frequently, the biological activity of such peptides is elicited through the adoption of secondary structural elements upon interaction with their cellular target. However, many therapeutic peptides are unstructured in solution and accordingly exhibit a poor bioavailability due to rapid proteolysis . To combat this degradation, numerous naturally occurring peptides with therapeutic properties contain stabilizing features, such as N-to-C cyclization or disulfide bonds. Recently, hydrocarbon stapling via non-native amino acid substitution followed by ring-closing metathesis has been shown to induce a dramatic stabilization of α-helical peptides. Identifying the ideal staple location along the peptide backbone is a critical developmental step, and methods to streamline this optimization are needed. Mass spectrometry-based methods such as ion mobility (IM) and hydrogen-deuterium exchange (HDX) can detect multiple discrete peptide conformations, a significant advantage over bulk spectroscopic techniques. In this study we use IM-MS and HDX-MS to demonstrate that the native 36-residue enfuvirtide peptide is highly dynamic in solution and the conformational ensemble populated by stabilized constructs depends heavily on the staple location. Further, our measurements yielded results that correlate well with the average α-helical content measured by circular dichroism. The MS-based approaches described herein represent sensitive and potentially high-throughput methods for characterizing and identifying optimally stapled peptides.
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http://dx.doi.org/10.1021/jasms.0c00453DOI Listing
March 2021

Binding and transport of SFPQ-RNA granules by KIF5A/KLC1 motors promotes axon survival.

J Cell Biol 2021 01;220(1)

Department of Neurobiology, Harvard Medical School, Boston, MA.

Complex neural circuitry requires stable connections formed by lengthy axons. To maintain these functional circuits, fast transport delivers RNAs to distal axons where they undergo local translation. However, the mechanism that enables long-distance transport of RNA granules is not yet understood. Here, we demonstrate that a complex containing RNA and the RNA-binding protein (RBP) SFPQ interacts selectively with a tetrameric kinesin containing the adaptor KLC1 and the motor KIF5A. We show that the binding of SFPQ to the KIF5A/KLC1 motor complex is required for axon survival and is impacted by KIF5A mutations that cause Charcot-Marie Tooth (CMT) disease. Moreover, therapeutic approaches that bypass the need for local translation of SFPQ-bound proteins prevent axon degeneration in CMT models. Collectively, these observations indicate that KIF5A-mediated SFPQ-RNA granule transport may be a key function disrupted in KIF5A-linked neurologic diseases and that replacing axonally translated proteins serves as a therapeutic approach to axonal degenerative disorders.
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http://dx.doi.org/10.1083/jcb.202005051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7721913PMC
January 2021

Targeting a helix-in-groove interaction between E1 and E2 blocks ubiquitin transfer.

Nat Chem Biol 2020 11 17;16(11):1218-1226. Epub 2020 Aug 17.

Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.

The ubiquitin-proteasome system (UPS) is a highly regulated protein disposal process critical to cell survival. Inhibiting the pathway induces proteotoxic stress and can be an effective cancer treatment. The therapeutic window observed upon proteasomal blockade has motivated multiple UPS-targeting strategies, including preventing ubiquitination altogether. E1 initiates the cascade by transferring ubiquitin to E2 enzymes. A small molecule that engages the E1 ATP-binding site and derivatizes ubiquitin disrupts enzymatic activity and kills cancer cells. However, binding-site mutations cause resistance, motivating alternative approaches to block this promising target. We identified an interaction between the E2 N-terminal alpha-1 helix and a pocket within the E1 ubiquitin-fold domain as a potentially druggable site. Stapled peptides modeled after the E2 alpha-1 helix bound to the E1 groove, induced a consequential conformational change and inhibited E1 ubiquitin thiotransfer, disrupting E2 ubiquitin charging and ubiquitination of cellular proteins. Thus, we provide a blueprint for a distinct E1-targeting strategy to treat cancer.
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http://dx.doi.org/10.1038/s41589-020-0625-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7904387PMC
November 2020

Glucose-dependent partitioning of arginine to the urea cycle protects β-cells from inflammation.

Nat Metab 2020 05 11;2(5):432-446. Epub 2020 May 11.

Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA.

Chronic inflammation is linked to diverse disease processes, but the intrinsic mechanisms that determine cellular sensitivity to inflammation are incompletely understood. Here, we show the contribution of glucose metabolism to inflammation-induced changes in the survival of pancreatic islet β-cells. Using metabolomic, biochemical and functional analyses, we investigate the protective versus non-protective effects of glucose in the presence of pro-inflammatory cytokines. When protective, glucose metabolism augments anaplerotic input into the TCA cycle via pyruvate carboxylase (PC) activity, leading to increased aspartate levels. This metabolic mechanism supports the argininosuccinate shunt, which fuels ureagenesis from arginine and conversely diminishes arginine utilization for production of nitric oxide (NO), a chief mediator of inflammatory cytotoxicity. Activation of the PC-urea cycle axis is sufficient to suppress NO synthesis and shield cells from death in the context of inflammation and other stress paradigms. Overall, these studies uncover a previously unappreciated link between glucose metabolism and arginine-utilizing pathways via PC-directed ureagenesis as a protective mechanism.
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http://dx.doi.org/10.1038/s42255-020-0199-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7568475PMC
May 2020

A redox switch regulates the structure and function of anti-apoptotic BFL-1.

Nat Struct Mol Biol 2020 09 13;27(9):781-789. Epub 2020 Jul 13.

Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.

Apoptosis is regulated by BCL-2 family proteins. Anti-apoptotic members suppress cell death by deploying a surface groove to capture the critical BH3 α-helix of pro-apoptotic members. Cancer cells hijack this mechanism by overexpressing anti-apoptotic BCL-2 family proteins to enforce cellular immortality. We previously identified and harnessed a unique cysteine (C55) in the groove of anti-apoptotic BFL-1 to selectively neutralize its oncogenic activity using a covalent stapled-peptide inhibitor. Here, we find that disulfide bonding between a native cysteine pair at the groove (C55) and C-terminal α9 helix (C175) of BFL-1 operates as a redox switch to control the accessibility of the anti-apoptotic pocket. Reducing the C55-C175 disulfide triggers α9 release, which promotes mitochondrial translocation, groove exposure for BH3 interaction and inhibition of mitochondrial permeabilization by pro-apoptotic BAX. C55-C175 disulfide formation in an oxidative cellular environment abrogates the ability of BFL-1 to bind BH3 domains. Thus, we identify a mechanism of conformational control of BFL-1 by an intramolecular redox switch.
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http://dx.doi.org/10.1038/s41594-020-0458-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7544158PMC
September 2020

Identification of a Covalent Molecular Inhibitor of Anti-apoptotic BFL-1 by Disulfide Tethering.

Cell Chem Biol 2020 06 14;27(6):647-656.e6. Epub 2020 May 14.

Department of Pediatric Oncology, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA 02215, USA; Linde Program in Cancer Chemical Biology, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA 02215, USA. Electronic address:

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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http://dx.doi.org/10.1016/j.chembiol.2020.04.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7405809PMC
June 2020

Hydrocarbon-Stitched Peptide Agonists of Glucagon-Like Peptide-1 Receptor.

ACS Chem Biol 2020 06 27;15(6):1340-1348. Epub 2020 May 27.

Department of Pediatric Oncology, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, Massachusetts 02215, United States.

Glucagon-like peptide 1 (GLP-1) is a natural peptide agonist of the GLP-1 receptor (GLP-1R) found on pancreatic β-cells. Engagement of the receptor stimulates insulin release in a glucose-dependent fashion and increases β-cell mass, two ideal features for pharmacologic management of type 2 diabetes. Thus, intensive efforts have focused on developing GLP-1-based peptide agonists of GLP-1R for therapeutic application. A primary challenge has been the naturally short half-life of GLP-1 due to its rapid proteolytic degradation . Whereas mutagenesis and lipidation strategies have yielded clinical agents, we developed an alternative approach to preserving the structure and function of GLP-1 by all-hydrocarbon , + 7 stitching. This particular "stitch" is especially well-suited for reinforcing and protecting the structural fidelity of GLP-1. Lead constructs demonstrate striking proteolytic stability and potent biological activity . Thus, we report a facile approach to generating alternative GLP-1R agonists for glycemic control.
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http://dx.doi.org/10.1021/acschembio.0c00308DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7366314PMC
June 2020

MDM2 and MDM4 Are Therapeutic Vulnerabilities in Malignant Rhabdoid Tumors.

Cancer Res 2019 05 12;79(9):2404-2414. Epub 2019 Feb 12.

Department of Pediatric Oncology, Dana-Farber Cancer Institute and Division of Hematology/Oncology, Boston Children's Hospital, Boston, Massachusetts.

Malignant rhabdoid tumors (MRT) are highly aggressive pediatric cancers that respond poorly to current therapies. In this study, we screened several MRT cell lines with large-scale RNAi, CRISPR-Cas9, and small-molecule libraries to identify potential drug targets specific for these cancers. We discovered and , the canonical negative regulators of p53, as significant vulnerabilities. Using two compounds currently in clinical development, idasanutlin (MDM2-specific) and ATSP-7041 (MDM2/4-dual), we show that MRT cells were more sensitive than other p53 wild-type cancer cell lines to inhibition of MDM2 alone as well as dual inhibition of MDM2/4. These compounds caused significant upregulation of the p53 pathway in MRT cells, and sensitivity was ablated by CRISPR-Cas9-mediated inactivation of . We show that loss of SMARCB1, a subunit of the SWI/SNF (BAF) complex mutated in nearly all MRTs, sensitized cells to MDM2 and MDM2/4 inhibition by enhancing p53-mediated apoptosis. Both MDM2 and MDM2/4 inhibition slowed MRT xenograft growth , with a 5-day idasanutlin pulse causing marked regression of all xenografts, including durable complete responses in 50% of mice. Together, these studies identify a genetic connection between mutations in the SWI/SNF chromatin-remodeling complex and the tumor suppressor gene and provide preclinical evidence to support the targeting of MDM2 and MDM4 in this often-fatal pediatric cancer. SIGNIFICANCE: This study identifies two targets, MDM2 and MDM4, as vulnerabilities in a deadly pediatric cancer and provides preclinical evidence that compounds inhibiting these proteins have therapeutic potential.
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http://dx.doi.org/10.1158/0008-5472.CAN-18-3066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6497578PMC
May 2019

Precision Targeting of BFL-1/A1 and an ATM Co-dependency in Human Cancer.

Cell Rep 2018 09;24(13):3393-3403.e5

Department of Pediatric Oncology, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA 02215, USA; Linde Program in Cancer Chemical Biology, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA 02215, USA. Electronic address:

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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http://dx.doi.org/10.1016/j.celrep.2018.08.089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6365304PMC
September 2018

Genome-scale CRISPR-Cas9 screen identifies druggable dependencies in wild-type Ewing sarcoma.

J Exp Med 2018 08 25;215(8):2137-2155. Epub 2018 Jul 25.

Department of Pediatric Oncology, Dana-Farber Cancer Institute and Boston Children's Hospital, Harvard Medical School, Boston, MA

Ewing sarcoma is a pediatric cancer driven by EWS-ETS transcription factor fusion oncoproteins in an otherwise stable genomic background. The majority of tumors express wild-type , and thus, therapies targeting the p53 pathway would benefit most patients. To discover targets specific for wild-type Ewing sarcoma, we used a genome-scale CRISPR-Cas9 screening approach and identified and validated , , and as druggable dependencies. The stapled peptide inhibitor of MDM2 and MDM4, ATSP-7041, showed anti-tumor efficacy in vitro and in multiple mouse models. The USP7 inhibitor, P5091, and the Wip1/ inhibitor, GSK2830371, decreased the viability of Ewing sarcoma cells. The combination of ATSP-7041 with P5091, GSK2830371, and chemotherapeutic agents showed synergistic action on the p53 pathway. The effects of the inhibitors, including the specific USP7 inhibitor XL-188, were rescued by concurrent knockout, highlighting the essentiality of intact p53 for the observed cytotoxic activities.
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http://dx.doi.org/10.1084/jem.20171066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6080915PMC
August 2018

Dynamic Regulation of Long-Chain Fatty Acid Oxidation by a Noncanonical Interaction between the MCL-1 BH3 Helix and VLCAD.

Mol Cell 2018 03;69(5):729-743.e7

Department of Pediatric Oncology and the Linde Program in Cancer Chemical Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA. Electronic address:

MCL-1 is a BCL-2 family protein implicated in the development and chemoresistance of human cancer. Unlike its anti-apoptotic homologs, Mcl-1 deletion has profound physiologic consequences, indicative of a broader role in homeostasis. We report that the BCL-2 homology 3 (BH3) α helix of MCL-1 can directly engage very long-chain acyl-CoA dehydrogenase (VLCAD), a key enzyme of the mitochondrial fatty acid β-oxidation (FAO) pathway. Proteomic analysis confirmed that the mitochondrial matrix isoform of MCL-1 (MCL-1) interacts with VLCAD. Mcl-1 deletion, or eliminating MCL-1 alone, selectively deregulated long-chain FAO, causing increased flux through the pathway in response to nutrient deprivation. Transient elevation in MCL-1 upon serum withdrawal, a striking increase in MCL-1 BH3/VLCAD interaction upon palmitic acid titration, and direct modulation of enzymatic activity by the MCL-1 BH3 α helix are consistent with dynamic regulation. Thus, the MCL-1 BH3 interaction with VLCAD revealed a separable, gain-of-function role for MCL-1 in the regulation of lipid metabolism.
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http://dx.doi.org/10.1016/j.molcel.2018.02.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5916823PMC
March 2018

Iterative optimization yields Mcl-1-targeting stapled peptides with selective cytotoxicity to Mcl-1-dependent cancer cells.

Proc Natl Acad Sci U S A 2018 01 16;115(5):E886-E895. Epub 2018 Jan 16.

Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139;

Bcl-2 family proteins regulate apoptosis, and aberrant interactions of overexpressed antiapoptotic family members such as Mcl-1 promote cell transformation, cancer survival, and resistance to chemotherapy. Discovering potent and selective Mcl-1 inhibitors that can relieve apoptotic blockades is thus a high priority for cancer research. An attractive strategy for disabling Mcl-1 involves using designer peptides to competitively engage its binding groove, mimicking the structural mechanism of action of native sensitizer BH3-only proteins. We transformed Mcl-1-binding peptides into α-helical, cell-penetrating constructs that are selectively cytotoxic to Mcl-1-dependent cancer cells. Critical to the design of effective inhibitors was our introduction of an all-hydrocarbon cross-link or "staple" that stabilizes α-helical structure, increases target binding affinity, and independently confers binding specificity for Mcl-1 over related Bcl-2 family paralogs. Two crystal structures of complexes at 1.4 Å and 1.9 Å resolution demonstrate how the hydrophobic staple induces an unanticipated structural rearrangement in Mcl-1 upon binding. Systematic sampling of staple location and iterative optimization of peptide sequence in accordance with established design principles provided peptides that target intracellular Mcl-1. This work provides proof of concept for the development of potent, selective, and cell-permeable stapled peptides for therapeutic targeting of Mcl-1 in cancer, applying a design and validation workflow applicable to a host of challenging biomedical targets.
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http://dx.doi.org/10.1073/pnas.1712952115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5798337PMC
January 2018

Crystal Structures of Anti-apoptotic BFL-1 and Its Complex with a Covalent Stapled Peptide Inhibitor.

Structure 2018 01 21;26(1):153-160.e4. Epub 2017 Dec 21.

Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA; Linde Program in Cancer Chemical Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA. Electronic address:

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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http://dx.doi.org/10.1016/j.str.2017.11.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5947960PMC
January 2018

Paclitaxel Reduces Axonal Bclw to Initiate IPR1-Dependent Axon Degeneration.

Neuron 2017 Oct;96(2):373-386.e6

Department of Neurobiology, Harvard Medical School, Boston, MA 02115, USA; Departments of Cancer Biology and Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA. Electronic address:

Chemotherapy-induced peripheral neuropathy (CIPN) is a debilitating side effect of many cancer treatments. The hallmark of CIPN is degeneration of long axons required for transmission of sensory information; axonal degeneration causes impaired tactile sensation and persistent pain. Currently the molecular mechanisms of CIPN are not understood, and there are no available treatments. Here we show that the chemotherapeutic agent paclitaxel triggers CIPN by altering IP receptor phosphorylation and intracellular calcium flux, and activating calcium-dependent calpain proteases. Concomitantly paclitaxel impairs axonal trafficking of RNA-granules and reduces synthesis of Bclw (bcl2l2), a Bcl2 family member that binds IPR1 and restrains axon degeneration. Surprisingly, Bclw or a stapled peptide corresponding to the Bclw BH4 domain interact with axonal IPR1 and prevent paclitaxel-induced degeneration, while Bcl2 and Bclx cannot do so. Together these data identify a Bclw-IPR1-dependent cascade that causes axon degeneration and suggest that Bclw-mimetics could provide effective therapy to prevent CIPN.
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http://dx.doi.org/10.1016/j.neuron.2017.09.034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5680044PMC
October 2017

Challenges in Targeting a Basic Helix-Loop-Helix Transcription Factor with Hydrocarbon-Stapled Peptides.

ACS Chem Biol 2016 11 4;11(11):3146-3153. Epub 2016 Oct 4.

Department of Pediatric Oncology and the Linde Program in Cancer Chemical Biology, Dana-Farber Cancer Institute , Boston, Massachusetts 02215, United States.

Basic helix-loop-helix (bHLH) transcription factors play critical roles in organism development and disease by regulating cell proliferation and differentiation. Transcriptional activity, whether by bHLH homo- or heterodimerization, is dependent on protein-protein and protein-DNA interactions mediated by α-helices. Thus, α-helical decoys have been proposed as potential targeted therapies for pathologic bHLH transcription. Here, we developed a library of stabilized α-helices of OLIG2 (SAH-OLIG2) to test the capacity of hydrocarbon-stapled peptides to disrupt OLIG2 homodimerization, which drives the development and chemoresistance of glioblastoma multiforme, one of the deadliest forms of human brain cancer. Although stapling successfully reinforced the α-helical structure of bHLH constructs of varying length, sequence-specific dissociation of OLIG2 dimers from DNA was not achieved. Re-evaluation of the binding determinants for OLIG2 self-association and stability revealed an unanticipated role of the C-terminal domain. These data highlight potential pitfalls in peptide-based targeting of bHLH transcription factors given the liabilities of their positively charged amino acid sequences and multifactorial binding determinants.
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http://dx.doi.org/10.1021/acschembio.6b00465DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5534327PMC
November 2016

Selective Covalent Targeting of Anti-Apoptotic BFL-1 by Cysteine-Reactive Stapled Peptide Inhibitors.

Cell Chem Biol 2016 Sep 8;23(9):1123-1134. Epub 2016 Sep 8.

Department of Pediatric Oncology and the Linde Program in Cancer Chemical Biology, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA 02215, USA. Electronic address:

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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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5055752PMC
http://dx.doi.org/10.1016/j.chembiol.2016.07.022DOI Listing
September 2016

Biophysical determinants for cellular uptake of hydrocarbon-stapled peptide helices.

Nat Chem Biol 2016 10 22;12(10):845-52. Epub 2016 Aug 22.

Department of Pediatric Oncology, Linde Program in Cancer Chemical Biology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA.

Hydrocarbon-stapled peptides are a class of bioactive alpha-helical ligands developed to dissect and target protein interactions. While there is consensus that stapled peptides can be effective chemical tools for investigating protein regulation, their broader utility for therapeutic modulation of intracellular interactions remains an active area of study. In particular, the design principles for generating cell-permeable stapled peptides are empiric, yet consistent intracellular access is essential to in vivo application. Here, we used an unbiased statistical approach to determine which biophysical parameters dictate the uptake of stapled-peptide libraries. We found that staple placement at the amphipathic boundary combined with optimal hydrophobic and helical content are the key drivers of cellular uptake, whereas excess hydrophobicity and positive charge at isolated amino acid positions can trigger membrane lysis at elevated peptide dosing. Our results provide a design roadmap for maximizing the potential to generate cell-permeable stapled peptides with on-mechanism cellular activity.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5055751PMC
http://dx.doi.org/10.1038/nchembio.2153DOI Listing
October 2016

Allosteric inhibition of antiapoptotic MCL-1.

Nat Struct Mol Biol 2016 06 9;23(6):600-7. Epub 2016 May 9.

Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA.

MCL-1 is an antiapoptotic BCL-2 family protein that has emerged as a major pathogenic factor in human cancer. Like BCL-2, MCL-1 bears a surface groove whose function is to sequester the BH3 killer domains of proapoptotic BCL-2 family members, a mechanism harnessed by cancer cells to establish formidable apoptotic blockades. Although drugging the BH3-binding groove has been achieved for BCL-2, translating this approach to MCL-1 has been challenging. Here, we report an alternative mechanism for MCL-1 inhibition by small-molecule covalent modification of C286 at a new interaction site distant from the BH3-binding groove. Our structure-function analyses revealed that the BH3 binding capacity of MCL-1 and its suppression of BAX are impaired by molecular engagement, a phenomenon recapitulated by C286W mutagenic mimicry in vitro and in mouse cells. Thus, we characterize an allosteric mechanism for disrupting the antiapoptotic BH3 binding activity of MCL-1, informing a new strategy for disarming MCL-1 in cancer.
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http://dx.doi.org/10.1038/nsmb.3223DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4900187PMC
June 2016

Generation of multiple reporter ions from a single isobaric reagent increases multiplexing capacity for quantitative proteomics.

Anal Chem 2015 Oct 4;87(19):9855-63. Epub 2015 Sep 4.

Massachusetts General Hospital Cancer Center and Department of Medicine, Harvard Medical School , Charlestown, Massachusetts 02129, United States.

Isobaric labeling strategies for mass spectrometry-based proteomics enable multiplexed simultaneous quantification of samples and therefore substantially increase the sample throughput in proteomics. However, despite these benefits, current limits to multiplexing capacity are prohibitive for large sample sizes and impose limitations on experimental design. Here, we introduce a novel mechanism for increasing the multiplexing density of isobaric reagents. We present Combinatorial Isobaric Mass Tags (CMTs), an isobaric labeling architecture with the unique ability to generate multiple series of reporter ions simultaneously. We demonstrate that utilization of multiple reporter ion series improves multiplexing capacity of CMT with respect to a commercially available isobaric labeling reagent with preserved quantitative accuracy and depth of coverage in complex mixtures. We provide a blueprint for the realization of 16-plex reagents with 1 Da spacing between reporter ions and up to 28-plex at 6 mDa spacing using only 5 heavy isotopes per reagent. We anticipate that this improvement in multiplexing capacity will further advance the application of quantitative proteomics, particularly in high-throughput screening assays.
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http://dx.doi.org/10.1021/acs.analchem.5b02307DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4890644PMC
October 2015

Cellular Uptake and Ultrastructural Localization Underlie the Pro-apoptotic Activity of a Hydrocarbon-stapled BIM BH3 Peptide.

ACS Chem Biol 2015 Sep 21;10(9):2149-57. Epub 2015 Jul 21.

Department of Pediatric Oncology, Linde Program in Cancer Chemical Biology, Dana-Farber Cancer Institute , Boston, Massachusetts 02215, United States.

Hydrocarbon stapling has been applied to restore and stabilize the α-helical structure of bioactive peptides for biochemical, structural, cellular, and in vivo studies. The peptide sequence, in addition to the composition and location of the installed staple, can dramatically influence the properties of stapled peptides. As a result, constructs that appear similar can have distinct functions and utilities. Here, we perform a side-by-side comparison of stapled peptides modeled after the pro-apoptotic BIM BH3 helix to highlight these principles. We confirm that replacing a salt-bridge with an i, i + 4 hydrocarbon staple does not impair target binding affinity and instead can yield a biologically and pharmacologically enhanced α-helical peptide ligand. Importantly, we demonstrate by electron microscopy that the pro-apoptotic activity of a stapled BIM BH3 helix correlates with its capacity to achieve cellular uptake without membrane disruption and accumulate at the organellar site of mechanistic activity.
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http://dx.doi.org/10.1021/acschembio.5b00214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6839450PMC
September 2015

Inhibition of Pro-apoptotic BAX by a noncanonical interaction mechanism.

Mol Cell 2015 Mar 12;57(5):873-886. Epub 2015 Feb 12.

Department of Pediatric Oncology and the Linde Program in Cancer Chemical Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA. Electronic address:

BCL-2 is a negative regulator of apoptosis implicated in homeostatic and pathologic cell survival. The canonical anti-apoptotic mechanism involves entrapment of activated BAX by a groove on BCL-2, preventing BAX homo-oligomerization and mitochondrial membrane poration. The BCL-2 BH4 domain also confers anti-apoptotic functionality, but the mechanism is unknown. We find that a synthetic α-helical BH4 domain binds to BAX with nanomolar affinity and independently inhibits the conformational activation of BAX. Hydrogen-deuterium exchange mass spectrometry demonstrated that the N-terminal conformational changes in BAX induced by a triggering BIM BH3 helix were suppressed by the BCL-2 BH4 helix. Structural analyses localized the BH4 interaction site to a groove formed by residues of α1, α1-α2 loop, and α2-α3 and α5-α6 hairpins on the BAX surface. These data reveal a previously unappreciated binding site for targeted inhibition of BAX and suggest that the BCL-2 BH4 domain may participate in apoptosis blockade by a noncanonical interaction mechanism.
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http://dx.doi.org/10.1016/j.molcel.2015.01.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4384643PMC
March 2015

Phospho-BAD BH3 mimicry protects β cells and restores functional β cell mass in diabetes.

Cell Rep 2015 Feb 29;10(4):497-504. Epub 2015 Jan 29.

Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02115, USA; Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

Strategies that simultaneously enhance the survival and glucose responsiveness of insulin-producing β cells will greatly augment β cell replacement therapies in type 1 diabetes (T1D). We show that genetic and pharmacologic mimetics of the phosphorylated BCL-2 homology 3 (BH3) domain of BAD impart β-cell-autonomous protective effects in the face of stress stimuli relevant to β cell demise in T1D. Importantly, these benefits translate into improved engraftment of donor islets in transplanted diabetic mice, increased β cell viability in islet grafts, restoration of insulin release, and diabetes reversal. Survival of β cells in this setting is not merely due to the inability of phospho-BAD to suppress prosurvival BCL-2 proteins but requires its activation of the glucose-metabolizing enzyme glucokinase. Thus, BAD phospho-BH3 mimetics may prove useful in the restoration of functional β cell mass in diabetes.
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http://dx.doi.org/10.1016/j.celrep.2014.12.056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4991214PMC
February 2015

Direct inhibition of oncogenic KRAS by hydrocarbon-stapled SOS1 helices.

Proc Natl Acad Sci U S A 2015 Feb 26;112(6):1761-6. Epub 2015 Jan 26.

Department of Pediatric Oncology and the Linde Program in Cancer Chemical Biology, Dana-Farber Cancer Institute, Boston, MA 02215; and

Activating mutations in the Kirsten rat sarcoma viral oncogene homolog (KRAS) underlie the pathogenesis and chemoresistance of ∼ 30% of all human tumors, yet the development of high-affinity inhibitors that target the broad range of KRAS mutants remains a formidable challenge. Here, we report the development and validation of stabilized alpha helices of son of sevenless 1 (SAH-SOS1) as prototype therapeutics that directly inhibit wild-type and mutant forms of KRAS. SAH-SOS1 peptides bound in a sequence-specific manner to KRAS and its mutants, and dose-responsively blocked nucleotide association. Importantly, this functional binding activity correlated with SAH-SOS1 cytotoxicity in cancer cells expressing wild-type or mutant forms of KRAS. The mechanism of action of SAH-SOS1 peptides was demonstrated by sequence-specific down-regulation of the ERK-MAP kinase phosphosignaling cascade in KRAS-driven cancer cells and in a Drosophila melanogaster model of Ras85D(V12) activation. These studies provide evidence for the potential utility of SAH-SOS1 peptides in neutralizing oncogenic KRAS in human cancer.
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http://dx.doi.org/10.1073/pnas.1413185112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4330742PMC
February 2015

Stapled HIV-1 peptides recapitulate antigenic structures and engage broadly neutralizing antibodies.

Nat Struct Mol Biol 2014 Dec 24;21(12):1058-67. Epub 2014 Nov 24.

1] Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA. [2] Division of Hematology/Oncology, Boston Children's Hospital, Boston, Massachusetts, USA. [3] Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, USA.

Hydrocarbon stapling can restore bioactive α-helical structure to natural peptides, yielding research tools and prototype therapeutics to dissect and target protein interactions. Here we explore the capacity of peptide stapling to generate high-fidelity, protease-resistant mimics of antigenic structures for vaccine development. HIV-1 has been refractory to vaccine technologies thus far, although select human antibodies can broadly neutralize HIV-1 by targeting sequences of the gp41 juxtamembrane fusion apparatus. To develop candidate HIV-1 immunogens, we generated and characterized stabilized α-helices of the membrane-proximal external region (SAH-MPER) of gp41. SAH-MPER peptides were remarkably protease resistant and bound to the broadly neutralizing 4E10 and 10E8 antibodies with high affinity, recapitulating the structure of the MPER epitope when differentially engaged by the two anti-HIV Fabs. Thus, stapled peptides may provide a new opportunity to develop chemically stabilized antigens for vaccination.
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http://dx.doi.org/10.1038/nsmb.2922DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4304871PMC
December 2014

Photoreactive stapled peptides to identify and characterize BCL-2 family interaction sites by mass spectrometry.

Methods Enzymol 2014 ;544:25-48

Department of Pediatric Oncology and the Linde Program in Cancer Chemical Biology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA; Department of Pediatrics, Children's Hospital Boston, Harvard Medical School, Boston, Massachusetts, USA. Electronic address:

Protein interactions dictate a myriad of cellular activities that maintain health or cause disease. Dissecting these binding partnerships, and especially their sites of interaction, fuels the discovery of signaling pathways, disease mechanisms, and next-generation therapeutics. We previously applied all-hydrocarbon peptide stapling to chemically restore α-helical shape to bioactive motifs that become unfolded when taken out of context from native signaling proteins. For example, we developed stabilized alpha-helices of BCL-2 domains (SAHBs) to dissect and target protein interactions of the BCL-2 family, a critical network that regulates the apoptotic pathway. SAHBs are α-helical surrogates that bind both stable and transient physiologic interactors and have effectively uncovered novel sites of BCL-2 family protein interaction. To leverage stapled peptides for proteomic discovery, we describe our conversion of SAHBs into photoreactive agents that irreversibly capture their protein targets and facilitate rapid identification of the peptide helix binding sites. We envision that the development of photoreactive stapled peptides will accelerate the discovery of novel and unanticipated protein interactions and how they impact health and disease.
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http://dx.doi.org/10.1016/B978-0-12-417158-9.00002-9DOI Listing
February 2015

Mucosal delivery of a double-stapled RSV peptide prevents nasopulmonary infection.

J Clin Invest 2014 May 17;124(5):2113-24. Epub 2014 Apr 17.

Respiratory syncytial virus (RSV) infection accounts for approximately 64 million cases of respiratory disease and 200,000 deaths worldwide each year, yet no broadly effective prophylactic or treatment regimen is available. RSV deploys paired, self-associating, heptad repeat domains of its fusion protein, RSV-F, to form a fusogenic 6-helix bundle that enables the virus to penetrate the host cell membrane. Here, we developed hydrocarbon double-stapled RSV fusion peptides that exhibit stabilized α-helical structure and striking proteolytic resistance. Pretreatment with double-stapled RSV peptides that specifically bound to the RSV fusion bundle inhibited infection by both laboratory and clinical RSV isolates in cells and murine infection models. Intranasal delivery of a lead double-stapled RSV peptide effectively prevented viral infection of the nares. A chitosan-based nanoparticle preparation markedly enhanced pulmonary delivery, further preventing progression of RSV infection to the lung. Thus, our results provide a strategy for inhibiting RSV infection by mucosal and endotracheal delivery of double-stapled RSV fusion peptides.
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http://dx.doi.org/10.1172/JCI71856DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4001541PMC
May 2014

Hydrocarbon-stapled peptides: principles, practice, and progress.

J Med Chem 2014 Aug 6;57(15):6275-88. Epub 2014 Mar 6.

Department of Pediatric Oncology, Dana-Farber Cancer Institute , Boston, Massachusetts 02215, United States.

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.
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http://dx.doi.org/10.1021/jm4011675DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4136684PMC
August 2014

A phospho-BAD BH3 helix activates glucokinase by a mechanism distinct from that of allosteric activators.

Nat Struct Mol Biol 2014 Jan 8;21(1):36-42. Epub 2013 Dec 8.

1] Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA. [2] Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA.

Glucokinase (GK) is a glucose-phosphorylating enzyme that regulates insulin release and hepatic metabolism, and its loss of function is implicated in diabetes pathogenesis. GK activators (GKAs) are attractive therapeutics in diabetes; however, clinical data indicate that their benefits can be offset by hypoglycemia, owing to marked allosteric enhancement of the enzyme's glucose affinity. We show that a phosphomimetic of the BCL-2 homology 3 (BH3) α-helix derived from human BAD, a GK-binding partner, increases the enzyme catalytic rate without dramatically changing glucose affinity, thus providing a new mechanism for pharmacologic activation of GK. Remarkably, BAD BH3 phosphomimetic mediates these effects by engaging a new region near the enzyme's active site. This interaction increases insulin secretion in human islets and restores the function of naturally occurring human GK mutants at the active site. Thus, BAD phosphomimetics may serve as a new class of GKAs.
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http://dx.doi.org/10.1038/nsmb.2717DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4084830PMC
January 2014

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

Nat Chem Biol 2013 Oct 25;9(10):643-50. Epub 2013 Aug 25.

1] Division of Pediatric Hematology/Oncology, Boston Children's Hospital, Boston, Massachusetts, USA. [2] Harvard Medical School, Boston, Massachusetts, USA. [3] Pediatric Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA.

Enhancer of zeste homolog 2 (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, 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 α-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.
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http://dx.doi.org/10.1038/nchembio.1331DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3778130PMC
October 2013
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