{"corpus_id":195356417,"paper_sha":"a246edeef5b507bf5bf01b62662d60536662130e","doi":"10.1371/journal.pbio.3000311","arxiv_id":null,"pmid":31233493,"pmcid":"6590788","mag_id":2952637656,"dblp_id":null,"acl_id":null,"title":"Selection and characterization of ultrahigh potency designed ankyrin repeat protein inhibitors of C. difficile toxin B","year":2019,"publication_date":"2019-06-01","venue":"PLoS Biology","journal":{"name":"PLoS Biology","pages":null,"volume":"17"},"journal_issn":null,"journal_title":null,"publication_types":["JournalArticle"],"pubmed_pub_types":["Journal Article","Research Support, N.I.H., Extramural","Research Support, Non-U.S. Gov't"],"s2_fields_of_study":["Biology","Medicine","Chemistry"],"reference_count":80,"citation_count":30,"influential_citation_count":0,"is_open_access":true,"arxiv_categories":null,"arxiv_license":null,"arxiv_journal_ref":null,"mesh_headings":[{"d":"Animals","mj":false,"ui":"D000818"},{"d":"Ankyrin Repeat","mj":false,"qs":[{"q":"genetics","mj":false,"ui":"Q000235"}],"ui":"D017089"},{"d":"Antibodies, Monoclonal","mj":false,"ui":"D000911"},{"d":"Bacterial Proteins","mj":false,"qs":[{"q":"antagonists & inhibitors","mj":true,"ui":"Q000037"},{"q":"genetics","mj":false,"ui":"Q000235"},{"q":"metabolism","mj":false,"ui":"Q000378"},{"q":"physiology","mj":true,"ui":"Q000502"}],"ui":"D001426"},{"d":"Bacterial Toxins","mj":false,"qs":[{"q":"antagonists & inhibitors","mj":true,"ui":"Q000037"},{"q":"genetics","mj":false,"ui":"Q000235"},{"q":"metabolism","mj":false,"ui":"Q000378"}],"ui":"D001427"},{"d":"Broadly Neutralizing Antibodies","mj":false,"ui":"D000080908"},{"d":"Caco-2 Cells","mj":false,"ui":"D018938"},{"d":"Clostridioides difficile","mj":false,"qs":[{"q":"metabolism","mj":false,"ui":"Q000378"},{"q":"pathogenicity","mj":false,"ui":"Q000472"}],"ui":"D016360"},{"d":"Clostridium Infections","mj":false,"qs":[{"q":"metabolism","mj":true,"ui":"Q000378"}],"ui":"D003015"},{"d":"Cryoelectron Microscopy","mj":false,"ui":"D020285"},{"d":"Enterotoxins","mj":false,"qs":[{"q":"metabolism","mj":false,"ui":"Q000378"}],"ui":"D004768"},{"d":"Humans","mj":false,"ui":"D006801"},{"d":"Mice","mj":false,"ui":"D051379"},{"d":"Protein Engineering","mj":false,"qs":[{"q":"methods","mj":false,"ui":"Q000379"}],"ui":"D015202"}],"chemicals":[{"n":"Antibodies, Monoclonal","ui":"D000911","reg":"0"},{"n":"Bacterial Proteins","ui":"D001426","reg":"0"},{"n":"Bacterial Toxins","ui":"D001427","reg":"0"},{"n":"Broadly Neutralizing Antibodies","ui":"D000080908","reg":"0"},{"n":"Enterotoxins","ui":"D004768","reg":"0"},{"n":"toxB protein, Clostridium difficile","ui":"C031815","reg":"0"},{"n":"bezlotoxumab","ui":"C000613978","reg":"4H5YMK1H2E"}],"comments_corrections":null,"source_flags":5,"s2_open_access_pdf_url":"https://storage.googleapis.com/plos-corpus-prod/10.1371/journal.pbio.3000311/2/pbio.3000311.pdf?X-Goog-Algorithm=GOOG4-RSA-SHA256&X-Goog-Credential=wombat-sa%40plos-prod.iam.gserviceaccount.com%2F20210223%2Fauto%2Fstorage%2Fgoog4_request&X-Goog-Date=20210223T051129Z&X-Goog-Expires=3600&X-Goog-SignedHeaders=host&X-Goog-Signature=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","s2_open_access_landing_url":"https://www.semanticscholar.org/paper/a246edeef5b507bf5bf01b62662d60536662130e","s2_open_access_license":"CCBY","s2_open_access_status":"GOLD","pmc_open_access_pdf_url":null,"pmc_open_access_landing_url":null,"pmc_open_access_license":null,"pmc_open_access_status":null,"unpaywall_open_access_pdf_url":null,"unpaywall_open_access_landing_url":null,"unpaywall_open_access_license":null,"unpaywall_open_access_status":null,"abstract":"Clostridium difficile infection (CDI) is a major nosocomial disease associated with significant morbidity and mortality. The pathology of CDI stems primarily from the 2 C. difficile–secreted exotoxins—toxin A (TcdA) and toxin B (TcdB)—that disrupt the tight junctions between epithelial cells leading to the loss of colonic epithelial barrier function. Here, we report the engineering of a series of monomeric and dimeric designed ankyrin repeat proteins (DARPins) for the neutralization of TcdB. The best dimeric DARPin, DLD-4, inhibited TcdB with a half maximal effective concentration (EC50) of 4 pM in vitro, representing an approximately 330-fold higher potency than the Food and Drug Administration (FDA)-approved anti-TcdB monoclonal antibody bezlotoxumab in the same assay. DLD-4 also protected mice from a toxin challenge in vivo. Cryo-electron microscopy (cryo-EM) studies revealed that the 2 constituent DARPins of DLD-4–1.4E and U3—bind the central and C-terminal regions of the delivery domain of TcdB. Competitive enzyme-linked immunosorbent assay (ELISA) studies showed that the DARPins 1.4E and U3 interfere with the interaction between TcdB and its receptors chondroitin sulfate proteoglycan 4 (CSPG4) and frizzled class receptor 2 (FZD2), respectively. Our cryo-EM studies revealed a new conformation of TcdB (both apo- and DARPin-bound at pH 7.4) in which the combined repetitive oligopeptides (CROPS) domain points away from the delivery domain. This conformation of the CROPS domain is in stark contrast to that seen in the negative-stain electron microscopy (EM) structure of TcdA and TcdB at the same pH, in which the CROPS domain bends toward and “kisses” the delivery domain. The ultrapotent anti-TcdB molecules from this study serve as candidate starting points for CDI drug development and provide new biological tools for studying the pathogenicity of C. difficile. The structural insights regarding both the “native” conformation of TcdB and the putative sites of TcdB interaction with the FZD2 receptor, in particular, should help accelerate the development of next-generation anti–C. difficile toxin therapeutics.","claims":[{"public_id":"cl_2a9e1519ade7e5b934ba89105c4ac353","status":"active","text":"A series of monomeric and dimeric designed ankyrin repeat proteins were engineered for neutralization of C. difficile toxin B.","confidence":0.98,"contributors":[{"id":1,"public_id":"12632b8b5f","public_label":"Anonymous (12632b8b5f)","roles":["extraction"],"url":"https://sah.borca.ai/u/12632b8b5f"}],"url":"https://sah.borca.ai/claims/cl_2a9e1519ade7e5b934ba89105c4ac353"},{"public_id":"cl_29420dd59c568994a9cbffed2cb215f2","status":"active","text":"Cryo-electron microscopy revealed a new pH 7.4 conformation of C. difficile toxin B in which the CROPS domain points away from the delivery domain.","confidence":0.92,"contributors":[{"id":1,"public_id":"12632b8b5f","public_label":"Anonymous (12632b8b5f)","roles":["extraction"],"url":"https://sah.borca.ai/u/12632b8b5f"}],"url":"https://sah.borca.ai/claims/cl_29420dd59c568994a9cbffed2cb215f2"},{"public_id":"cl_63e0f36f8a31b6ba5d7d160f9fb4116f","status":"active","text":"DLD-4 protected mice from toxin challenge in vivo.","confidence":0.97,"contributors":[{"id":1,"public_id":"12632b8b5f","public_label":"Anonymous (12632b8b5f)","roles":["extraction"],"url":"https://sah.borca.ai/u/12632b8b5f"}],"url":"https://sah.borca.ai/claims/cl_63e0f36f8a31b6ba5d7d160f9fb4116f"},{"public_id":"cl_cb3d75118063346ba078b3d65c0814a9","status":"active","text":"The best dimeric designed ankyrin repeat protein, DLD-4, inhibited C. difficile toxin B with an in vitro EC50 of 4 pM, about 330-fold more potent than bezlotoxumab in the same assay.","confidence":0.99,"contributors":[{"id":1,"public_id":"12632b8b5f","public_label":"Anonymous (12632b8b5f)","roles":["extraction"],"url":"https://sah.borca.ai/u/12632b8b5f"}],"url":"https://sah.borca.ai/claims/cl_cb3d75118063346ba078b3d65c0814a9"},{"public_id":"cl_b0d31fccf9af341dc59a2dcfe9d29e68","status":"active","text":"The constituent designed ankyrin repeat proteins 1.4E and U3 bind the central and C-terminal regions of the delivery domain of C. difficile toxin B, respectively, and interfere with receptor interactions involving CSPG4 and FZD2.","confidence":0.95,"contributors":[{"id":1,"public_id":"12632b8b5f","public_label":"Anonymous (12632b8b5f)","roles":["extraction"],"url":"https://sah.borca.ai/u/12632b8b5f"}],"url":"https://sah.borca.ai/claims/cl_b0d31fccf9af341dc59a2dcfe9d29e68"}],"concepts":[{"public_id":"co_0d9e287a9cb9ef5401fd0ddd5d045cbb","status":"active","name":"U3","description":"A designed ankyrin repeat protein constituent of the DLD-4 dimer that binds the C-terminal region of the toxin delivery domain.","types":["protein 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interaction.","types":["protein domain"],"aliases":["delivery domain"],"contributors":[{"id":1,"public_id":"12632b8b5f","public_label":"Anonymous (12632b8b5f)","roles":["extraction"],"url":"https://sah.borca.ai/u/12632b8b5f"}],"url":"https://sah.borca.ai/concepts/co_7f2a3132176b54e9a9bf4513c1aa053a"},{"public_id":"co_86eceb3e8ad890f2fd8dbe694e3fb60c","status":"active","name":"toxin challenge","description":"An in vivo exposure experiment used to test whether an inhibitor protects animals from toxin effects.","types":["experimental assay"],"aliases":[],"contributors":[{"id":1,"public_id":"12632b8b5f","public_label":"Anonymous (12632b8b5f)","roles":["extraction"],"url":"https://sah.borca.ai/u/12632b8b5f"}],"url":"https://sah.borca.ai/concepts/co_86eceb3e8ad890f2fd8dbe694e3fb60c"},{"public_id":"co_893f16febe1e4d111cfa01096b561ef7","status":"active","name":"cryo-electron microscopy","description":"A structural technique used to determine the conformation of toxin-bound and apo toxin 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as inhibitory molecules against a bacterial toxin.","types":["protein scaffold","therapeutic candidate"],"aliases":["DARPins"],"contributors":[{"id":1,"public_id":"12632b8b5f","public_label":"Anonymous (12632b8b5f)","roles":["extraction"],"url":"https://sah.borca.ai/u/12632b8b5f"}],"url":"https://sah.borca.ai/concepts/co_b4a287ac0a1233870a6bdbae817443fa"},{"public_id":"co_eb547fbf8f6b99bab53b063679ae0607","status":"active","name":"bezlotoxumab","description":"An FDA-approved monoclonal antibody used here as a comparator anti-TcdB therapeutic.","types":["monoclonal antibody","baseline comparator"],"aliases":[],"contributors":[{"id":1,"public_id":"12632b8b5f","public_label":"Anonymous (12632b8b5f)","roles":["extraction"],"url":"https://sah.borca.ai/u/12632b8b5f"}],"url":"https://sah.borca.ai/concepts/co_eb547fbf8f6b99bab53b063679ae0607"},{"public_id":"co_ec68f6084326f9770f48ce7b5e8d0b83","status":"active","name":"1.4E","description":"A designed ankyrin repeat protein constituent of the 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