Selection and characterization of ultrahigh potency designed ankyrin repeat protein inhibitors of C. difficile toxin B
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...
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| Veröffentlicht in: | PLoS biology 2019-06, Vol.17 (6), p.e3000311-e3000311 |
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| creator | Simeon, Rudo Jiang, Mengqiu Chamoun-Emanuelli, Ana M Yu, Hua Zhang, Yongrong Meng, Ran Peng, Zeyu Jakana, Joanita Zhang, Junjie Feng, Hanping Chen, Zhilei |
| description | 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. |
| doi_str_mv | 10.1371/journal.pbio.3000311 |
| format | Article |
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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.</description><identifier>ISSN: 1545-7885</identifier><identifier>ISSN: 1544-9173</identifier><identifier>EISSN: 1545-7885</identifier><identifier>DOI: 10.1371/journal.pbio.3000311</identifier><identifier>PMID: 31233493</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Animals ; Ankyrin Repeat - genetics ; Ankyrins ; Antibiotics ; Antibodies, Monoclonal ; Authorship ; Bacterial Proteins - antagonists & inhibitors ; Bacterial Proteins - genetics ; Bacterial Proteins - metabolism ; Bacterial Proteins - physiology ; Bacterial Toxins - antagonists & inhibitors ; Bacterial Toxins - genetics ; Bacterial Toxins - metabolism ; Bends ; Biochemistry ; Biology and Life Sciences ; Biophysics ; Broadly Neutralizing Antibodies ; Caco-2 Cells ; Chemical inhibitors ; Chondroitin sulfate ; Clostridioides difficile - metabolism ; Clostridioides difficile - pathogenicity ; Clostridium difficile ; Clostridium Infections - metabolism ; Crops ; Cryoelectron Microscopy ; Dimers ; Drug development ; Electron microscopy ; Enterotoxins - metabolism ; Enzyme-linked immunosorbent assay ; Epithelial cells ; Exotoxins ; FDA approval ; Frizzled protein ; Funding ; Hospitals ; Humans ; Immunology ; In vivo methods and tests ; Medical imaging ; Medicine and Health Sciences ; Methods ; Mice ; Microbial toxins ; Microscopy ; Monoclonal antibodies ; Morbidity ; Neutralization ; Nosocomial infection ; Oligopeptides ; Pathogenesis ; Pathogenicity ; Pathogens ; pH effects ; Physical Sciences ; Physiological aspects ; Protein Engineering - methods ; Protein structure ; Proteins ; Proteoglycans ; Receptors ; Regulatory agencies ; Research and Analysis Methods ; Supervision ; Tight junctions ; Toxin A ; Toxin B ; Toxins ; Transmission electron microscopy</subject><ispartof>PLoS biology, 2019-06, Vol.17 (6), p.e3000311-e3000311</ispartof><rights>COPYRIGHT 2019 Public Library of Science</rights><rights>2019 Simeon et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2019 Simeon et al 2019 Simeon et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c656t-82d71cabe75b301ce9fdd4f464491b065f04359e4489c53f2ef78fc39523c8753</citedby><cites>FETCH-LOGICAL-c656t-82d71cabe75b301ce9fdd4f464491b065f04359e4489c53f2ef78fc39523c8753</cites><orcidid>0000-0002-8594-4232 ; 0000-0002-0244-3556</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6590788/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6590788/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,860,881,2096,2915,23845,27901,27902,53766,53768,79569,79570</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/31233493$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Simeon, Rudo</creatorcontrib><creatorcontrib>Jiang, Mengqiu</creatorcontrib><creatorcontrib>Chamoun-Emanuelli, Ana M</creatorcontrib><creatorcontrib>Yu, Hua</creatorcontrib><creatorcontrib>Zhang, Yongrong</creatorcontrib><creatorcontrib>Meng, Ran</creatorcontrib><creatorcontrib>Peng, Zeyu</creatorcontrib><creatorcontrib>Jakana, Joanita</creatorcontrib><creatorcontrib>Zhang, Junjie</creatorcontrib><creatorcontrib>Feng, Hanping</creatorcontrib><creatorcontrib>Chen, Zhilei</creatorcontrib><title>Selection and characterization of ultrahigh potency designed ankyrin repeat protein inhibitors of C. difficile toxin B</title><title>PLoS biology</title><addtitle>PLoS Biol</addtitle><description>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.</description><subject>Animals</subject><subject>Ankyrin Repeat - genetics</subject><subject>Ankyrins</subject><subject>Antibiotics</subject><subject>Antibodies, Monoclonal</subject><subject>Authorship</subject><subject>Bacterial Proteins - antagonists & inhibitors</subject><subject>Bacterial Proteins - genetics</subject><subject>Bacterial Proteins - metabolism</subject><subject>Bacterial Proteins - physiology</subject><subject>Bacterial Toxins - antagonists & inhibitors</subject><subject>Bacterial Toxins - genetics</subject><subject>Bacterial Toxins - metabolism</subject><subject>Bends</subject><subject>Biochemistry</subject><subject>Biology and Life Sciences</subject><subject>Biophysics</subject><subject>Broadly Neutralizing Antibodies</subject><subject>Caco-2 Cells</subject><subject>Chemical inhibitors</subject><subject>Chondroitin sulfate</subject><subject>Clostridioides difficile - metabolism</subject><subject>Clostridioides difficile - pathogenicity</subject><subject>Clostridium difficile</subject><subject>Clostridium Infections - metabolism</subject><subject>Crops</subject><subject>Cryoelectron Microscopy</subject><subject>Dimers</subject><subject>Drug development</subject><subject>Electron microscopy</subject><subject>Enterotoxins - metabolism</subject><subject>Enzyme-linked immunosorbent assay</subject><subject>Epithelial cells</subject><subject>Exotoxins</subject><subject>FDA approval</subject><subject>Frizzled protein</subject><subject>Funding</subject><subject>Hospitals</subject><subject>Humans</subject><subject>Immunology</subject><subject>In vivo methods and tests</subject><subject>Medical imaging</subject><subject>Medicine and Health Sciences</subject><subject>Methods</subject><subject>Mice</subject><subject>Microbial toxins</subject><subject>Microscopy</subject><subject>Monoclonal antibodies</subject><subject>Morbidity</subject><subject>Neutralization</subject><subject>Nosocomial infection</subject><subject>Oligopeptides</subject><subject>Pathogenesis</subject><subject>Pathogenicity</subject><subject>Pathogens</subject><subject>pH effects</subject><subject>Physical Sciences</subject><subject>Physiological aspects</subject><subject>Protein Engineering - methods</subject><subject>Protein structure</subject><subject>Proteins</subject><subject>Proteoglycans</subject><subject>Receptors</subject><subject>Regulatory agencies</subject><subject>Research and Analysis Methods</subject><subject>Supervision</subject><subject>Tight junctions</subject><subject>Toxin A</subject><subject>Toxin B</subject><subject>Toxins</subject><subject>Transmission electron microscopy</subject><issn>1545-7885</issn><issn>1544-9173</issn><issn>1545-7885</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2019</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>BENPR</sourceid><sourceid>DOA</sourceid><recordid>eNqVkk1v1DAQhiMEoqXwDxBE4gKHXezYTuJLpbLiY6WKShS4Wo4_Ei9ZO9hO1eXX43TTqos4wMn2-Jl3PK8ny55DsISogm83bvSW98uhMW6JAAAIwgfZMSSYLKq6Jg_v7Y-yJyFsACgKWtSPsyMEC4QwRcfZ1aXqlYjG2ZxbmYuOey6i8uYXvwk6nY999LwzbZcPLiordrlUwbRWyZTyY-eNzb0aFI_54BOQjsZ2pjHR-TDlr5a5NFobYXqVR3edgHdPs0ea90E9m9eT7NuH919XnxbnFx_Xq7PzhShJGRd1ISsoeKMq0iAAhaJaSqxxiTGFDSiJBhgRqjCuqSBIF0pXtRaIkgKJuiLoJHu51x16F9hsWWBFQeqKEljhRKz3hHR8wwZvttzvmOOG3QScbxn30YheMUI5lyWviIAYQ0CpqDAuhMQCSFRhmbRO52pjs1VSKJuc6w9ED2-s6VjrrlhJKEj_lARezwLe_RxViGxrglB9z61y4_RuXFKAaFkk9NUf6N-7m6mWpwaM1S7VFZMoOyOUAEIwmMq-OaCEs1Fdx5aPIbD15Zf_YD__O3vx_ZDFe1Z4F4JX-s42CNg08Lf9sWng2TzwKe3Ffcvvkm4nHP0Gacn8Dw</recordid><startdate>20190601</startdate><enddate>20190601</enddate><creator>Simeon, Rudo</creator><creator>Jiang, Mengqiu</creator><creator>Chamoun-Emanuelli, Ana M</creator><creator>Yu, Hua</creator><creator>Zhang, Yongrong</creator><creator>Meng, Ran</creator><creator>Peng, Zeyu</creator><creator>Jakana, Joanita</creator><creator>Zhang, Junjie</creator><creator>Feng, Hanping</creator><creator>Chen, Zhilei</creator><general>Public Library of Science</general><general>Public Library of Science (PLoS)</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>IOV</scope><scope>ISN</scope><scope>ISR</scope><scope>3V.</scope><scope>7QG</scope><scope>7QL</scope><scope>7SN</scope><scope>7SS</scope><scope>7T5</scope><scope>7TK</scope><scope>7TM</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8FD</scope><scope>8FE</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>ATCPS</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>LK8</scope><scope>M0S</scope><scope>M1P</scope><scope>M7N</scope><scope>M7P</scope><scope>P64</scope><scope>PATMY</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>PYCSY</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope><scope>DOA</scope><scope>CZG</scope><orcidid>https://orcid.org/0000-0002-8594-4232</orcidid><orcidid>https://orcid.org/0000-0002-0244-3556</orcidid></search><sort><creationdate>20190601</creationdate><title>Selection and characterization of ultrahigh potency designed ankyrin repeat protein inhibitors of C. difficile toxin B</title><author>Simeon, Rudo ; Jiang, Mengqiu ; Chamoun-Emanuelli, Ana M ; Yu, Hua ; Zhang, Yongrong ; Meng, Ran ; Peng, Zeyu ; Jakana, Joanita ; Zhang, Junjie ; Feng, Hanping ; Chen, Zhilei</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c656t-82d71cabe75b301ce9fdd4f464491b065f04359e4489c53f2ef78fc39523c8753</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2019</creationdate><topic>Animals</topic><topic>Ankyrin Repeat - 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metabolism</topic><topic>Enzyme-linked immunosorbent assay</topic><topic>Epithelial cells</topic><topic>Exotoxins</topic><topic>FDA approval</topic><topic>Frizzled protein</topic><topic>Funding</topic><topic>Hospitals</topic><topic>Humans</topic><topic>Immunology</topic><topic>In vivo methods and tests</topic><topic>Medical imaging</topic><topic>Medicine and Health Sciences</topic><topic>Methods</topic><topic>Mice</topic><topic>Microbial toxins</topic><topic>Microscopy</topic><topic>Monoclonal antibodies</topic><topic>Morbidity</topic><topic>Neutralization</topic><topic>Nosocomial infection</topic><topic>Oligopeptides</topic><topic>Pathogenesis</topic><topic>Pathogenicity</topic><topic>Pathogens</topic><topic>pH effects</topic><topic>Physical Sciences</topic><topic>Physiological aspects</topic><topic>Protein Engineering - methods</topic><topic>Protein structure</topic><topic>Proteins</topic><topic>Proteoglycans</topic><topic>Receptors</topic><topic>Regulatory agencies</topic><topic>Research and Analysis Methods</topic><topic>Supervision</topic><topic>Tight junctions</topic><topic>Toxin A</topic><topic>Toxin B</topic><topic>Toxins</topic><topic>Transmission electron microscopy</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Simeon, Rudo</creatorcontrib><creatorcontrib>Jiang, Mengqiu</creatorcontrib><creatorcontrib>Chamoun-Emanuelli, Ana M</creatorcontrib><creatorcontrib>Yu, Hua</creatorcontrib><creatorcontrib>Zhang, Yongrong</creatorcontrib><creatorcontrib>Meng, Ran</creatorcontrib><creatorcontrib>Peng, Zeyu</creatorcontrib><creatorcontrib>Jakana, Joanita</creatorcontrib><creatorcontrib>Zhang, Junjie</creatorcontrib><creatorcontrib>Feng, Hanping</creatorcontrib><creatorcontrib>Chen, Zhilei</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Gale In Context: Opposing Viewpoints</collection><collection>Gale In Context: Canada</collection><collection>Gale In Context: Science</collection><collection>ProQuest Central (Corporate)</collection><collection>Animal Behavior Abstracts</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Ecology Abstracts</collection><collection>Entomology Abstracts (Full archive)</collection><collection>Immunology Abstracts</collection><collection>Neurosciences Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Medical Database (Alumni Edition)</collection><collection>Technology Research Database</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest One Sustainability</collection><collection>ProQuest Central UK/Ireland</collection><collection>Agricultural & Environmental Science Collection</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Natural Science Collection</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central Korea</collection><collection>Engineering Research Database</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>ProQuest Biological Science Collection</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biological Science Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Environmental Science Database</collection><collection>Publicly Available Content Database</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central China</collection><collection>Environmental Science Collection</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><collection>PLoS Biology</collection><jtitle>PLoS biology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Simeon, Rudo</au><au>Jiang, Mengqiu</au><au>Chamoun-Emanuelli, Ana M</au><au>Yu, Hua</au><au>Zhang, Yongrong</au><au>Meng, Ran</au><au>Peng, Zeyu</au><au>Jakana, Joanita</au><au>Zhang, Junjie</au><au>Feng, Hanping</au><au>Chen, Zhilei</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Selection and characterization of ultrahigh potency designed ankyrin repeat protein inhibitors of C. difficile toxin B</atitle><jtitle>PLoS biology</jtitle><addtitle>PLoS Biol</addtitle><date>2019-06-01</date><risdate>2019</risdate><volume>17</volume><issue>6</issue><spage>e3000311</spage><epage>e3000311</epage><pages>e3000311-e3000311</pages><issn>1545-7885</issn><issn>1544-9173</issn><eissn>1545-7885</eissn><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.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>31233493</pmid><doi>10.1371/journal.pbio.3000311</doi><orcidid>https://orcid.org/0000-0002-8594-4232</orcidid><orcidid>https://orcid.org/0000-0002-0244-3556</orcidid><oa>free_for_read</oa></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 1545-7885 |
| ispartof | PLoS biology, 2019-06, Vol.17 (6), p.e3000311-e3000311 |
| issn | 1545-7885 1544-9173 1545-7885 |
| language | eng |
| recordid | cdi_plos_journals_2258795174 |
| source | MEDLINE; DOAJ Directory of Open Access Journals; EZB-FREE-00999 freely available EZB journals; PubMed Central; Public Library of Science (PLoS) |
| subjects | Animals Ankyrin Repeat - genetics Ankyrins Antibiotics Antibodies, Monoclonal Authorship Bacterial Proteins - antagonists & inhibitors Bacterial Proteins - genetics Bacterial Proteins - metabolism Bacterial Proteins - physiology Bacterial Toxins - antagonists & inhibitors Bacterial Toxins - genetics Bacterial Toxins - metabolism Bends Biochemistry Biology and Life Sciences Biophysics Broadly Neutralizing Antibodies Caco-2 Cells Chemical inhibitors Chondroitin sulfate Clostridioides difficile - metabolism Clostridioides difficile - pathogenicity Clostridium difficile Clostridium Infections - metabolism Crops Cryoelectron Microscopy Dimers Drug development Electron microscopy Enterotoxins - metabolism Enzyme-linked immunosorbent assay Epithelial cells Exotoxins FDA approval Frizzled protein Funding Hospitals Humans Immunology In vivo methods and tests Medical imaging Medicine and Health Sciences Methods Mice Microbial toxins Microscopy Monoclonal antibodies Morbidity Neutralization Nosocomial infection Oligopeptides Pathogenesis Pathogenicity Pathogens pH effects Physical Sciences Physiological aspects Protein Engineering - methods Protein structure Proteins Proteoglycans Receptors Regulatory agencies Research and Analysis Methods Supervision Tight junctions Toxin A Toxin B Toxins Transmission electron microscopy |
| title | Selection and characterization of ultrahigh potency designed ankyrin repeat protein inhibitors of C. difficile toxin B |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-02-15T09%3A32%3A21IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-gale_plos_&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Selection%20and%20characterization%20of%20ultrahigh%20potency%20designed%20ankyrin%20repeat%20protein%20inhibitors%20of%20C.%20difficile%20toxin%20B&rft.jtitle=PLoS%20biology&rft.au=Simeon,%20Rudo&rft.date=2019-06-01&rft.volume=17&rft.issue=6&rft.spage=e3000311&rft.epage=e3000311&rft.pages=e3000311-e3000311&rft.issn=1545-7885&rft.eissn=1545-7885&rft_id=info:doi/10.1371/journal.pbio.3000311&rft_dat=%3Cgale_plos_%3EA595055408%3C/gale_plos_%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=2258795174&rft_id=info:pmid/31233493&rft_galeid=A595055408&rft_doaj_id=oai_doaj_org_article_59aad6a75c1441099c7442cd4c0d374d&rfr_iscdi=true |