ABSTRACT
Clostridioides difficile causes life-threatening diarrhea and is one of the leading causes of nosocomial infections. During infection, C. difficile releases two gut-damaging toxins, TcdA and TcdB, which are the primary determinants of disease pathogenesis and are important therapeutic targets. Once in the cytosol of mammalian cells, TcdA and TcdB use UDP-glucose to glucosylate host Rho GTPases, which leads to cytoskeletal changes that result in a loss of intestinal integrity. Isofagomine inhibits TcdA and TcdB as a mimic of the glucocation transition state of the glucosyltransferase reaction. However, sequence variants of TcdA and TcdB across the clades of infective C. difficile continue to be identified, and therefore, evaluation of isofagomine inhibition against multiple toxin variants is required. Here, we show that isofagomine inhibits the glucosyltransferase domain of multiple TcdB variants and protects TcdB-induced cell rounding of the most common full-length toxin variants. Furthermore, we demonstrate that isofagomine protects against C. difficile-induced mortality in two murine models of C. difficile infection. Isofagomine treatment of mouse C. difficile infection also permitted the recovery of the gastrointestinal microbiota, an important barrier to preventing recurring C. difficile infection. The broad specificity of isofagomine supports its potential as a prophylactic to protect against C. difficile-induced morbidity and mortality.
Subject(s)
Bacterial Toxins , Boron Compounds , Clostridioides difficile , Imino Pyranoses , Animals , Mice , Bacterial Toxins/genetics , Enterotoxins , Clostridioides difficile/genetics , Bacterial Proteins/genetics , Glucosyltransferases/genetics , MammalsABSTRACT
Homozygous 5'-methylthioadenosine phosphorylase (MTAP) deletions occur in approximately 15% of human cancers. Co-deletion of MTAP and methionine adenosyltransferase 2 alpha (MAT2a) induces a synthetic lethal phenotype involving protein arginine methyltransferase 5 (PRMT5) inhibition. MAT2a inhibitors are now in clinical trials for genotypic MTAP-/- cancers, however the MTAP-/- genotype represents fewer than 2% of human colorectal cancers (CRCs), limiting the utility of MAT2a inhibitors in these and other MTAP+/+ cancers. Methylthio-DADMe-immucillin-A (MTDIA) is a picomolar transition state analog inhibitor of MTAP that renders cells enzymatically MTAP-deficient to induce the MTAP-/- phenotype. Here, we demonstrate that MTDIA and MAT2a inhibitor AG-270 combination therapy mimics synthetic lethality in MTAP+/+ CRC cell lines with similar effects in mouse xenografts and without adverse histology on normal tissues. Combination treatment is synergistic with a 104-fold increase in drug potency for inhibition of CRC cell growth in culture. Combined MTDIA and AG-270 decreases S-adenosyl-L-methionine and increases 5'-methylthioadenosine in cells. The increased intracellular methylthioadenosine:S-adenosyl-L-methionine ratio inhibits PRMT5 activity, leading to cellular arrest and apoptotic cell death by causing MDM4 alternative splicing and p53 activation. Combination MTDIA and AG-270 treatment differs from direct inhibition of PRMT5 by GSK3326595 by avoiding toxicity caused by cell death in the normal gut epithelium induced by the PRMT5 inhibitor. The combination of MTAP and MAT2a inhibitors expands this synthetic lethal approach to include MTAP+/+ cancers, especially the remaining 98% of CRCs without the MTAP-/- genotype.
Subject(s)
Deoxyadenosines , Methionine Adenosyltransferase , Neoplasms , Protein-Arginine N-Methyltransferases , Purine-Nucleoside Phosphorylase , S-Adenosylmethionine , Animals , Humans , Mice , Cell Line, Tumor , Cell Proliferation/drug effects , Deoxyadenosines/antagonists & inhibitors , Deoxyadenosines/genetics , Deoxyadenosines/metabolism , Drug Synergism , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/therapeutic use , Methionine Adenosyltransferase/antagonists & inhibitors , Methionine Adenosyltransferase/genetics , Methionine Adenosyltransferase/metabolism , Neoplasms/genetics , Neoplasms/physiopathology , Neoplasms/therapy , Protein-Arginine N-Methyltransferases/antagonists & inhibitors , Protein-Arginine N-Methyltransferases/metabolism , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Purine-Nucleoside Phosphorylase/genetics , Purine-Nucleoside Phosphorylase/metabolism , Pyrrolidines/pharmacology , Pyrrolidines/therapeutic use , S-Adenosylmethionine/metabolismABSTRACT
Clostridioides difficile causes life-threatening diarrhea and is the leading cause of healthcare associated bacterial infections in the United States. During infection, C. difficile releases the gut-damaging toxins, TcdA and TcdB, the primary determinants of disease pathogenesis and are therefore therapeutic targets. TcdA and TcdB contain a glycosyltransferase domain that uses UDP-glucose to glycosylate host Rho GTPases, causing cytoskeletal changes that result in a loss of intestinal integrity. Isofagomine inhibits TcdA and TcdB as a mimic of the oxocarbenium ion transition state of the glycosyltransferase reaction. However, sequence variants of TcdA and TcdB across the clades of infective C. difficile continue to be identified and therefore, evaluation of isofagomine inhibition against multiple toxin variants are required. Here we show that Isofagomine inhibits the glycosyltransferase activity of multiple TcdB variants and also protects TcdB toxin-induced cell rounding of the most common full-length toxin variants. Further, isofagomine protects against C. difficile induced mortality in two murine models of C. difficile infection. Isofagomine treatment of mouse C. difficile infection permitted recovery of the gastrointestinal microbiota, an important barrier to prevent recurring C. difficile infection. The broad specificity of isofagomine supports its potential as a prophylactic to protect against C. difficile induced morbidity and mortality.