Monday, May 29, 2023
5/29/2023
Abstract: Human gut infections by Clostridioides (Clostridium) difficile (here, C.diff.) are the most lethal urgent threat in the 2019 CDC Antibiotic Resistance Threats Report. Excess healthcare costs from these infections have been estimated to be over $5 billion annually. Antibiotic resistance has elicited an insightful RFA RA18-725, `Generating new insights and mechanistic understanding of antibiotic resistance'. C.diff. infections (CDI) typically arise following treatment of other clinical disorders with antibiotics. Antibiotic therapy disrupts normal gut microbiota, allowing C.diff. to proliferate and to repopulate the gut following treatment. Additional antibiotic therapy to treat CDI prevents return of normal gut microbiota, leading to recurrent infections in over 20% of patients. C.diff. has acquired resistance to several common antibiotics, compounding its therapy. Recent clinical guidelines (2018) for C.diff. infections are oral vancomycin for patients in shock, hypotension, ileus or megacolon. Fecal transplant is recommended for nonresponsive infections following vancomycin treatment. mAb therapies have been FDA- approved, but are not recommended. Despite these therapies, C. difficile causes an estimated 224,000 infections and 13,000 deaths per year (CDC in 2017). Gut epithelial cell cytotoxicity results from C.diff. production of secreted toxins, primarily TcdA and TcdB (Tcds). Tcds are processed in gut cells to form active UDP-glucosyl transferases that glucosylate cytoskeletal-regulating Rho, Rac and Cdc42 GTP-binding proteins on specific threonines. Loss of cytoskeletal integrity causes severe colitis and can have a fatal outcome. Anti-toxin immunity is a historic approach to prevent host damage from circulating bacterial toxins. We propose that small molecule, tight-binding inhibitors targeting C.diff. Tcds can prevent the morbidity and mortality from gut toxins in C.diff. infections. Our transition state analog approach uses kinetic isotope effects and quantum chemistry to solve transition state structures of Tcds. Solving the first transition state structures of G-protein glucosyltransferases, and developing the first transition state analog of any UDP-sugar transferase is innovative. Electrostatic potential models of Tcd transition states will guide the design and synthesis of transition state analogs. Lead transition state analog candidates will be elaborated by cycles of crystallography and chemical design. Candidate compounds and crystal structures of Tcd complexes have been obtained in preliminary studies. Inhibitors will be characterized against Tcds in human cells and in mouse models. Agents to prevent tissue damage from C.diff. infections, without disruption of the gut microbiome or pressure for microbial resistance have important medical relevance. Inhibition of Tcds in gut epithelial cells places no selective pressure for antibiotic or anti-toxin resistance on C.diff. or on the gut microbiome, while protecting the gut by neutralizing Tcds. Mechanistically, this approach is innovative in recapitulating vaccine-based antibody neutralization of toxins using the powerful approach of transition state analogs.
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