Selection and characterization of ultrahigh potency designed ankyrin repeat protein inhibitors of < i > C < /i > . < i > difficile < /i > toxin B

by Rudo Simeon, Mengqiu Jiang, Ana M. Chamoun-Emanuelli, Hua Yu, Yongrong Zhang, Ran Meng, Zeyu Peng, Joanita Jakana, Junjie Zhang, Hanping Feng, Zhilei ChenClostridium difficile infection (CDI) is a major nosocomial disease associated with significant morbidity and mortality. The pathology of CDI stems primarily from the 2C.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 ne utralization 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 friz zled class receptor 2 (FZD2), respectively. Our cryo-EM studies revealed a new conformation of TcdB (both...
Source: PLoS Biology: Archived Table of Contents - Category: Biology Authors: Source Type: research