A Computationally Designed Inhibitor of an Epstein-Barr Viral Bcl-2 Protein Induces Apoptosis in Infected Cells

Because apoptosis of infected cells can limit virus production and spread, some viruses have co-opted prosurvival genes from the host. This includes the Epstein-Barr virus (EBV) gene BHRF1, a homolog of human Bcl-2 proteins that block apoptosis and are associated with cancer. Computational design and experimental optimization were used to generate a novel protein called BINDI that binds BHRF1 with picomolar affinity. BINDI recognizes the hydrophobic cleft of BHRF1 in a manner similar to other Bcl-2 protein interactions but makes many additional contacts to achieve exceptional affinity and specificity. BINDI induces apoptosis in EBV-infected cancer lines, and when delivered with an antibody-targeted intracellular delivery carrier, BINDI suppressed tumor growth and extended survival in a xenograft disease model of EBV-positive human lymphoma. High-specificity-designed proteins that selectively kill target cells may provide an advantage over the toxic compounds used in current generation antibody-drug conjugates.

[1]  D. Baker,et al.  Elicitation of structure-specific antibodies by epitope scaffolds , 2010, Proceedings of the National Academy of Sciences.

[2]  P. Stayton,et al.  Diblock copolymers with tunable pH transitions for gene delivery. , 2012, Biomaterials.

[3]  J. Teruya-Feldstein,et al.  The Combination of the Proteasome Inhibitor Bortezomib and the Bcl-2 Antisense Molecule Oblimersen Sensitizes Human B-Cell Lymphomas to Cyclophosphamide , 2006, Clinical Cancer Research.

[4]  David Baker,et al.  Proof of principle for epitope-focused vaccine design , 2014, Nature.

[5]  J. Martinou,et al.  Mitochondria in apoptosis: Bcl-2 family members and mitochondrial dynamics. , 2011, Developmental cell.

[6]  Jens Meiler,et al.  ROSETTA3: an object-oriented software suite for the simulation and design of macromolecules. , 2011, Methods in enzymology.

[7]  Nathan A. Baker,et al.  Electrostatics of nanosystems: Application to microtubules and the ribosome , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[8]  Daniel W. Kulp,et al.  Generalized Fragment Picking in Rosetta: Design, Protocols and Applications , 2011, PloS one.

[9]  Jens Meiler,et al.  RosettaScripts: A Scripting Language Interface to the Rosetta Macromolecular Modeling Suite , 2011, PloS one.

[10]  Andrew I. Bell,et al.  An Epstein-Barr Virus Anti-Apoptotic Protein Constitutively Expressed in Transformed Cells and Implicated in Burkitt Lymphomagenesis: The Wp/BHRF1 Link , 2009, PLoS pathogens.

[11]  Thomas L. Madden,et al.  Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. , 1997, Nucleic acids research.

[12]  D. Benoit,et al.  Intracellular delivery of a proapoptotic peptide via conjugation to a RAFT synthesized endosomolytic polymer. , 2010, Molecular pharmaceutics.

[13]  Amy E Keating,et al.  Peptide ligands for pro-survival protein Bfl-1 from computationally guided library screening. , 2013, ACS chemical biology.

[14]  David Baker,et al.  Protein Structure Prediction Using Rosetta , 2004, Numerical Computer Methods, Part D.

[15]  Erinna F. Lee,et al.  Conformational Changes in Bcl-2 Pro-survival Proteins Determine Their Capacity to Bind Ligands* , 2009, The Journal of Biological Chemistry.

[16]  W. Hammerschmidt,et al.  Epstein-Barr Virus Provides a New Paradigm: A Requirement for the Immediate Inhibition of Apoptosis , 2005, PLoS biology.

[17]  Rainer Spang,et al.  Different Patterns of Epstein-Barr Virus Latency in Endemic Burkitt Lymphoma (BL) Lead to Distinct Variants within the BL-Associated Gene Expression Signature , 2012, Journal of Virology.

[18]  Pavol Skubák,et al.  Direct incorporation of experimental phase information in model refinement. , 2004, Acta crystallographica. Section D, Biological crystallography.

[19]  P. Colman,et al.  Structural Basis for Apoptosis Inhibition by Epstein-Barr Virus BHRF1 , 2010, PLoS pathogens.

[20]  M. Dimopoulos,et al.  The Combination of the Proteasome Inhibitor Bortezomib with Doxorubicin and Dexamethasone (PAD Regimen) as Front-Line Therapy In Newly Diagnosed, High-Risk Multiple Myeloma: Results of a Phase II Prospective Multicenter Study , 2010 .

[21]  M. Allday,et al.  Epstein-Barr Virus-Induced Resistance to Drugs That Activate the Mitotic Spindle Assembly Checkpoint in Burkitt's Lymphoma Cells , 2006, Journal of Virology.

[22]  L. Stamatatos,et al.  Computational design of epitope-scaffolds allows induction of antibodies specific for a poorly immunogenic HIV vaccine epitope. , 2010, Structure.

[23]  P. Nordlund,et al.  Structural basis for recognition by an in vitro evolved affibody , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[24]  G. Gobe,et al.  Cycloheximide‐induced apoptosis in Burkitt lymphoma (BJA‐B) cells with and without Epstein‐Barr virus infection , 1995, Immunology and cell biology.

[25]  D. Baker,et al.  Design of a Novel Globular Protein Fold with Atomic-Level Accuracy , 2003, Science.

[26]  Kevin Cowtan,et al.  research papers Acta Crystallographica Section D Biological , 2005 .

[27]  Crystallization and preliminary X-ray characterization of Epstein-Barr virus BHRF1 in complex with a benzoylurea peptidomimetic. , 2012, Acta crystallographica. Section F, Structural biology and crystallization communications.

[28]  Timothy A. Whitehead,et al.  Optimization of affinity, specificity and function of designed influenza inhibitors using deep sequencing , 2012, Nature Biotechnology.

[29]  D. Hoover,et al.  DNAWorks: an automated method for designing oligonucleotides for PCR-based gene synthesis. , 2002, Nucleic acids research.

[30]  S. Pun,et al.  Intracellular delivery and trafficking dynamics of a lymphoma-targeting antibody-polymer conjugate. , 2012, Molecular pharmaceutics.

[31]  Christopher M. MacDermaid,et al.  Computational design of a protein crystal , 2012, Proceedings of the National Academy of Sciences.

[32]  P. Marrack,et al.  The Epstein–Barr virus Bcl-2 homolog, BHRF1, blocks apoptosis by binding to a limited amount of Bim , 2009, Proceedings of the National Academy of Sciences.

[33]  T. Lindahl,et al.  Epstein-Barr virus DNA in human lymphoid cell lines: in vitro conversion. , 1976, Virology.

[34]  Timothy A. Whitehead,et al.  Computational Design of Proteins Targeting the Conserved Stem Region of Influenza Hemagglutinin , 2011, Science.

[35]  C. Tse,et al.  ABT-263: a potent and orally bioavailable Bcl-2 family inhibitor. , 2008, Cancer research.

[36]  D T Jones,et al.  Protein secondary structure prediction based on position-specific scoring matrices. , 1999, Journal of molecular biology.

[37]  J. Chin,et al.  Design and Evolution of a Miniature Bcl-2 Binding Protein. , 2001, Angewandte Chemie.

[38]  Douglas M. Fowler,et al.  Enrich: software for analysis of protein function by enrichment and depletion of variants , 2011, Bioinform..

[39]  K Dane Wittrup,et al.  Isolating and engineering human antibodies using yeast surface display , 2006, Nature Protocols.

[40]  Paul H. Johnson,et al.  pH-responsive polymeric micelle carriers for siRNA drugs. , 2010, Biomacromolecules.

[41]  D. Baker,et al.  Computational design of a protein-based enzyme inhibitor. , 2013, Journal of molecular biology.

[42]  D. Baker,et al.  Principles for designing ideal protein structures , 2012, Nature.

[43]  A. Keating,et al.  Determinants of BH3 binding specificity for Mcl-1 versus Bcl-xL. , 2010, Journal of molecular biology.

[44]  Andrew J. Wilson,et al.  Inhibition of α-helix-mediated protein-protein interactions using designed molecules. , 2013, Nature chemistry.

[45]  Adrien Treuille,et al.  Predicting protein structures with a multiplayer online game , 2010, Nature.

[46]  A. Schepartz,et al.  Paralog-selective ligands for bcl-2 proteins. , 2005, Journal of the American Chemical Society.

[47]  Brian J. Smith,et al.  Structure-guided design of a selective BCL-X(L) inhibitor. , 2013, Nature chemical biology.

[48]  Z. Otwinowski,et al.  [20] Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[49]  Emiko Fire,et al.  Mcl‐1–Bim complexes accommodate surprising point mutations via minor structural changes , 2010, Protein science : a publication of the Protein Society.

[50]  Randy J. Read,et al.  Phaser crystallographic software , 2007, Journal of applied crystallography.

[51]  M. Rowe,et al.  Epstein-Barr virus-coded BHRF1 protein, a viral homologue of Bcl-2, protects human B cells from programmed cell death. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[52]  S. Maruo,et al.  Epstein-Barr Virus-Encoded Bcl-2 Homologue Functions as a Survival Factor in Wp-Restricted Burkitt Lymphoma Cell Line P3HR-1 , 2009, Journal of Virology.

[53]  P. Forterre,et al.  Acidianus filamentous virus 1 coat proteins display a helical fold spanning the filamentous archaeal viruses lineage , 2009, Proceedings of the National Academy of Sciences.