Development of a non-radiolabeled glucosyltransferase activity assay for C. difficile toxin A and B using ultra performance liquid chromatography

Loughney, John W.; Lancaster, Catherine; Price, Colleen; Hoang, Van M.; Ha, Sha; Rustandi, Richard R.

doi:10.1016/j.chroma.2017.01.083

Cited by 3 publications

(4 citation statements)

References 26 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Robust inhibition of TcdA glucosyltransferase activity relies on the ability of the peptides to outcompete the TcdA GTD’s substrate, UDP-Glucose. Given TcdA’s relatively low Michaelis constant (K M ~ 4.5 μM) compared to the cellular concentration of its substrate UDP-Glucose (92 μM), it is especially important that the peptides exhibit high binding strength and selectivity to TcdA GTD (see Supplementary material for detailed analysis) 11 , 44 – 47 .…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Design of 8-mer peptides that block Clostridioides difficile toxin A in intestinal cells

Sarma,

Catella,

San Pedro

et al. 2023

Commun Biol

View full text Add to dashboard Cite

Infections by Clostridioides difficile, a bacterium that targets the large intestine (colon), impact a large number of people worldwide. Bacterial colonization is mediated by two exotoxins: toxins A and B. Short peptides that can be delivered to the gut and inhibit the biocatalytic activity of these toxins represent a promising therapeutic strategy to prevent and treat C. diff. infection. We describe an approach that combines a Peptide Binding Design (PepBD) algorithm, molecular-level simulations, a rapid screening assay to evaluate peptide:toxin binding, a primary human cell-based assay, and surface plasmon resonance (SPR) measurements to develop peptide inhibitors that block Toxin A in colon epithelial cells. One peptide, SA1, is found to block TcdA toxicity in primary-derived human colon (large intestinal) epithelial cells. SA1 binds TcdA with a KD of 56.1 ± 29.8 nM as measured by surface plasmon resonance (SPR).

show abstract

Section: Resultsmentioning

confidence: 99%

“…4b ). Beads displaying selective TcdA GTD-binding peptides displace UDP-Glucose-Fluorescein from the GTD, and thus peptide:TcdA GTD binding, can be observed as a loss of green fluorescence 44 from the TcdA binding region (Fig. 4c ).…”

Section: Resultsmentioning

confidence: 99%

Design of 8-mer peptides that block Clostridioides difficile toxin A in intestinal cells

Sarma,

Catella,

San Pedro

et al. 2023

Commun Biol

View full text Add to dashboard Cite

show abstract

“…The intensity of the halo correlates with the amount of TcdA-AF594 bound 38 (Figure 4B). Beads displaying selective TcdA GTD-binding peptides displace UDP-Glucose-Fluorescein from the GTD, and thus peptide:TcdA GTD binding, can be observed as a loss of green fluorescence 44 from the TcdA binding region (Figure 4C).…”

Section: Resultsmentioning

confidence: 99%

“…Robust inhibition of TcdA glucosyltransferase activity relies on the ability of the peptides to outcompete the TcdA GTD's substrate, UDP-Glucose. Given TcdA's relatively low Michaelis constant (KM ~ 4.5 M) compared to the cellular concentration of its substrate UDP-Glucose (92 M), it is especially important that the peptides exhibit high binding strength and selectivity to TcdA GTD (see SI Appendix for detailed analysis) 11,[44][45][46][47] .…”

Section: Bead-based Pre-screening For Tcda Bindingmentioning

confidence: 99%

Design of 8-mer Peptides that BlockClostridioides difficileToxin A in Intestinal Cells

Sarma

Catella

Pedro

et al. 2023

Preprint

View full text Add to dashboard Cite

Clostridioides difficile (C. diff.) is a bacterium that causes severe diarrhea and inflammation of the colon. The pathogenicity of C. diff. infection is derived from two major toxins, toxins A (TcdA) and B (TcdB). Peptide inhibitors that can be delivered to the gut to inactivate these toxins are an attractive therapeutic strategy. In this work, we present a new approach that combines a peptide binding design algorithm (PepBD), molecular-level simulations, rapid screening of candidate peptides for toxin binding, a primary human cell-based assay, and surface plasmon resonance (SPR) measurements to develop peptide inhibitors that block the glucosyltransferase activity of TcdA by targeting its glucosyltransferase domain (GTD). Using PepBD and explicit-solvent molecular dynamics simulations, we identified seven candidate peptides, SA1-SA7. These peptides were selected for specific TcdA GTD binding through a custom solid-phase peptide screening system, which eliminated the weaker inhibitors SA5-SA7. The efficacies of SA1-SA4 were then tested using a trans-epithelial electrical resistance (TEER) assay on monolayers of the human gut epithelial culture model. One peptide, SA1, was found to block TcdA toxicity in primary-derived human jejunum (small intestinal) and colon (large intestinal) epithelial cells. SA1 bound TcdA with a KD of 56.1 +/- 61617; 29.8 nM as measured by surface plasmon resonance (SPR).

show abstract

Development of an ADP-ribosylation assay for residual toxicity in C. difficile binary toxin CDTa using automated capillary western blot

Rustandi

Hamm

2020

Journal of Pharmaceutical and Biomedical Analysis

View full text Add to dashboard Cite

Development of a non-radiolabeled glucosyltransferase activity assay for C. difficile toxin A and B using ultra performance liquid chromatography

Cited by 3 publications

References 26 publications

Design of 8-mer peptides that block Clostridioides difficile toxin A in intestinal cells

Design of 8-mer peptides that block Clostridioides difficile toxin A in intestinal cells

Design of 8-mer Peptides that BlockClostridioides difficileToxin A in Intestinal Cells

Development of an ADP-ribosylation assay for residual toxicity in C. difficile binary toxin CDTa using automated capillary western blot

Contact Info

Product

Resources

About