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Selection and characterization of ultrahigh potency designed ankyrin repeat protein inhibitors of C. difficile toxin B.
PLoS Biol 2019; 17(6):e3000311PB

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.

Authors+Show Affiliations

Department of Microbial Pathogenesis and Immunology, Texas A&M University Health Science Center, College Station, Texas, United States of America.Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas, United States of America.Department of Microbial Pathogenesis and Immunology, Texas A&M University Health Science Center, College Station, Texas, United States of America.Department of Microbial Pathogenesis, University of Maryland Dental School, Baltimore, Maryland, United Sates of America.Department of Microbial Pathogenesis, University of Maryland Dental School, Baltimore, Maryland, United Sates of America.Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas, United States of America.Department of Microbial Pathogenesis and Immunology, Texas A&M University Health Science Center, College Station, Texas, United States of America.National Center for Macromolecular Imaging, Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas, United States of America.Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas, United States of America.Department of Microbial Pathogenesis, University of Maryland Dental School, Baltimore, Maryland, United Sates of America.Department of Microbial Pathogenesis and Immunology, Texas A&M University Health Science Center, College Station, Texas, United States of America.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

31233493

Citation

Simeon, Rudo, et al. "Selection and Characterization of Ultrahigh Potency Designed Ankyrin Repeat Protein Inhibitors of C. Difficile Toxin B." PLoS Biology, vol. 17, no. 6, 2019, pp. e3000311.
Simeon R, Jiang M, Chamoun-Emanuelli AM, et al. Selection and characterization of ultrahigh potency designed ankyrin repeat protein inhibitors of C. difficile toxin B. PLoS Biol. 2019;17(6):e3000311.
Simeon, R., Jiang, M., Chamoun-Emanuelli, A. M., Yu, H., Zhang, Y., Meng, R., ... Chen, Z. (2019). Selection and characterization of ultrahigh potency designed ankyrin repeat protein inhibitors of C. difficile toxin B. PLoS Biology, 17(6), pp. e3000311. doi:10.1371/journal.pbio.3000311.
Simeon R, et al. Selection and Characterization of Ultrahigh Potency Designed Ankyrin Repeat Protein Inhibitors of C. Difficile Toxin B. PLoS Biol. 2019;17(6):e3000311. PubMed PMID: 31233493.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Selection and characterization of ultrahigh potency designed ankyrin repeat protein inhibitors of C. difficile toxin B. AU - Simeon,Rudo, AU - Jiang,Mengqiu, AU - Chamoun-Emanuelli,Ana M, AU - Yu,Hua, AU - Zhang,Yongrong, AU - Meng,Ran, AU - Peng,Zeyu, AU - Jakana,Joanita, AU - Zhang,Junjie, AU - Feng,Hanping, AU - Chen,Zhilei, Y1 - 2019/06/24/ PY - 2018/05/21/received PY - 2019/05/20/accepted PY - 2019/6/25/entrez PY - 2019/6/25/pubmed PY - 2019/6/25/medline SP - e3000311 EP - e3000311 JF - PLoS biology JO - PLoS Biol. VL - 17 IS - 6 N2 - 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. SN - 1545-7885 UR - https://www.unboundmedicine.com/medline/citation/31233493/Selection_and_characterization_of_ultrahigh_potency_designed_ankyrin_repeat_protein_inhibitors_of_C._difficile_toxin_B L2 - http://dx.plos.org/10.1371/journal.pbio.3000311 DB - PRIME DP - Unbound Medicine ER -