Small Molecule Inhibitor Screen Reveals Calcium Channel Signaling as a Mechanistic Mediator of Clostridium difficile TcdB-Induced Necrosis

ACS Chem Biol. 2020 May 15;15(5):1212-1221. doi: 10.1021/acschembio.9b00906. Epub 2020 Jan 14.


Clostridioides difficile is the leading cause of nosocomial diarrhea in the United States. The primary virulence factors are two homologous glucosyltransferase toxins, TcdA and TcdB, that inactivate host Rho-family GTPases. The glucosyltransferase activity has been linked to a "cytopathic" disruption of the actin cytoskeleton and contributes to the disruption of tight junctions and the production of pro-inflammatory cytokines. TcdB is also a potent cytotoxin that causes epithelium necrotic damage through an NADPH oxidase (NOX)-dependent mechanism. We conducted a small molecule screen to identify compounds that confer protection against TcdB-induced necrosis. We identified an enrichment of "hit compounds" with a dihydropyridine (DHP) core which led to the discovery of a key early stage calcium signal that serves as a mechanistic link between TcdB-induced NOX activation and reactive oxygen species (ROS) production. Disruption of TcdB-induced calcium signaling (with both DHP and non-DHP molecules) is sufficient to ablate ROS production and prevent subsequent necrosis in cells and in a mouse model of intoxication.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Actin Cytoskeleton / metabolism
  • Animals
  • Anti-Infective Agents / chemistry*
  • Anti-Infective Agents / pharmacology
  • Bacterial Toxins / metabolism
  • Calcium Channel Blockers / chemistry*
  • Calcium Channel Blockers / pharmacology
  • Calcium Channels / metabolism*
  • Calcium Signaling / drug effects*
  • Clostridioides difficile / drug effects*
  • Cytokines / metabolism
  • Dihydropyridines / chemistry*
  • Dihydropyridines / pharmacology
  • Dose-Response Relationship, Drug
  • Drug Evaluation, Preclinical
  • Glucosyltransferases / metabolism
  • Humans
  • Kinetics
  • Mice
  • NADPH Oxidases / metabolism
  • Necrosis / chemically induced
  • Necrosis / prevention & control*
  • Reactive Oxygen Species / metabolism
  • Virulence Factors / metabolism


  • Anti-Infective Agents
  • Bacterial Toxins
  • Calcium Channel Blockers
  • Calcium Channels
  • Cytokines
  • Dihydropyridines
  • Reactive Oxygen Species
  • Virulence Factors
  • 1,4-dihydropyridine
  • NADPH Oxidases
  • Glucosyltransferases