De novo design of potent inhibitors of clostridial family toxins
<i>Clostridioides difficile</i> remains a leading cause of hospital-acquired infections, with its primary virulence factor, toxin B (TcdB), responsible for severe colitis and recurrent disease. The closely related toxin, TcsL, from <i>Paeniclostridium sordellii</i>, causes a rarer but often fatal toxic shock syndrome, particularly in gynecological and obstetric contexts. We report the de novo design of small protein minibinders that directly neutralize TcdB and TcsL by preventing their entry into host cells. Using deep learning and Rosetta-based approaches, we generated high-affinity minibinders that protect cells from intoxication with picomolar potency and, in the case of TcsL, prolonged survival following lethal toxin challenge in mice. The designed proteins against TcdB demonstrate exceptional stability in proteolytic and acidic environments, making them well-suited for oral delivery-a valuable feature for treating <i>C. difficile</i> infections localized to the gastrointestinal tract. For TcsL, potent inhibitors were identified from 48 initial designs and 48 optimized designs, highlighting the potential of computational design for rapidly developing countermeasures against life-threatening bacterial toxins.
Authors
- Jacob M. Berman (ORCID: https://orcid.org/0000-0001-6454-4996)
- Robert J. Ragotte (ORCID: https://orcid.org/0000-0002-6463-1595)
- Brian Coventry (ORCID: https://orcid.org/0000-0002-6910-6255)
- Sean Miletic (ORCID: https://orcid.org/0000-0002-2732-8626)
- Connor Weidle (ORCID: https://orcid.org/0000-0002-9354-2276)
- Roman A. Melnyk (ORCID: https://orcid.org/0000-0002-7187-2362)
- Daniel Schramek (ORCID: https://orcid.org/0000-0001-9977-2104)
- Huazhu Liang (ORCID: https://orcid.org/0000-0001-7290-7769)
- Roger Palou
- Greg L. Beilhartz (ORCID: https://orcid.org/0000-0002-8142-9444)
- Andrew J. Borst (ORCID: https://orcid.org/0000-0003-4297-7824)
- John L. Rubinstein (ORCID: https://orcid.org/0000-0003-0566-2209)
- David A. Baker (ORCID: https://orcid.org/0000-0001-7896-6217)
- Mike Tyers (ORCID: https://orcid.org/0000-0002-9713-9994)
- Matthias Glögl (ORCID: https://orcid.org/0000-0001-7011-8184)
- John Kit Chung Tam (ORCID: https://orcid.org/0000-0002-6269-3653)
- Kenneth D. Carr (ORCID: https://orcid.org/0009-0005-1929-3952)
- Xinru Wang (ORCID: https://orcid.org/0000-0001-5994-707X)
- Zhijie Li (ORCID: https://orcid.org/0009-0002-4794-8475)
Institutions
- Mount Sinai Hospital (CA)
- University of Toronto (CA)
- University of Washington (US)
- Lunenfeld-Tanenbaum Research Institute (CA)
- Sinai Hospital (US)
- Hospital for Sick Children (CA)
Publication Details
- Journal
- Proceedings of the National Academy of Sciences
- Published
- 2025-09-22
- DOI
- https://doi.org/10.1073/pnas.2509329122
- Citations
- 3
- Primary Topic
- Clostridium difficile and Clostridium perfringens research
- Type
- article
- Field-Weighted Citation Impact
- 4.19