Transcreener Assay Helps Find New Approach to C. difficile

Clostridium difficile – One of the Deadliest Enemies in Hospitals

There are half a million reasons to consider Clostridium difficile as “Enemy Number 1” for public health. That’s because the annual cases of C. difficile, or C diff, infection now top 500,000 per year in the U.S.—resulting in more than 29,000 deaths per year. Worse yet, most C diff infections (CDIs) occur in vulnerable people: hospitalized patients, who come into contact with spores while they are receiving treatment as inpatients.

The insidious spores set off a horrific chain of events in the human gut: two virulence factors called toxin A (TcdA) and toxin B (TcdB) bind to target receptors on host cells in the gut epithelium, internalize into early endosomes which have acidic pH that triggers conformational changes in TcdA and TcdB, causing 1) unfurling, 2) punching pores in membranes, 3) release of glucosyltransferase domain (GTD) , 4) inactivation of Rho and Ras family GTPases, followed by glucosylation and apoptosis of gut epithelial cells.¹

In other words, C diff pulverizes the gut from the inside out and causes cell death that leads to debilitating, dehydrating, and unrelenting diarrhea. And for some patients, death.

Even if a CDI responds to antibiotics, more than one-third of patients who experience CDI will experience recurrent, miserable infections. The total economic toll of CDI is more than $6.3 billion per year in the United States. Despite heavy medical and economic incentive, few solutions are available: an anti-TcdB antibody therapy (bezlotoxumab) recently approved for adults receiving anti-C diff antimicrobial therapy and who are at high risk of re-infection, and still-relatively-experimental fecal microbiota transplants.²

A New Approach

Clearly, more options are needed to fight CDI. A research team from Genesis Biotechnology Group decided to look for TcdB inhibitors, using a Transcreener® Assay in a 6M compound high throughput study done in 1536-well format.³ The goal was to identify small molecules that inhibit the glucosyltransferase activity of TcdB for development as small molecule anti-virulence treatments for CDI.³ Considering the suffering that CDI causes, it is unequivocally a good goal. And fortunately, it turned out to be a good strategy.

The HTS approach allowed the interrogation of a collection of six million compounds, using the fluorescence polarization assay for UDP-glucose hydrolysis activity by the C-terminal glucosyltransferase domain of TcdB.³ Five hit structures were identified, which set the stage for a rational structure-activity relationship (SAR) approach that was augmented and informed by in silico docking studies.³

The result: design and synthesis of analogs with high potency in the single-digit nanomolar IC₅₀ range.³ Further pharmacokinetics/pharmacodynamics studies using cell-based assays (Chinese hamster ovary, or CHO cells and stability studies in presence of human or mouse liver microsomes), as well as in vivo pharmacokinetics determinations using intravenous or oral administration in CD-1 mice, yielded valuable information.³ Unfortunately, the most potent compounds also proved to have low levels of stability in mouse plasma. However, the research team reported resolve to conduct “further optimization in this series to provide highly potent TcdB inhibitors with improved stability in mouse plasma, improved pharmacokinetic profiles and in vivo activity.”

New weapons in the anti-CDI arsenal are badly needed. The ability of Transcreener technology to identify inhibitor candidates that can inform rapid, precise SAR and PK/PD studies enables hope for half a million patients who suffer from C diff each year. For every person who experiences CDI, new approaches simply can’t come soon enough.

– Robyn M. Perrin, PhD

Learn More About the Transcreener UDP² Assay

References

[1] Orrell KE, Zhang Z, Sugiman-Marangos SN, Melnyk RA. 2017. Clostridium difficile toxins A and B: Receptors, pores, and translocation into cells. Crit Rev Biochem Mol Biol. 52(4):461-473.

[2] McDonald LC, et al. 2018. Clinical Practice Guidelines for Clostridium difficile Infection in Adults and Children: 2017 Update by the Infectious Diseases Society of America (IDSA) and Society for Healthcare Epidemiology of America (SHEA. Clinical Infect. [Epub ahead of print] [3] Letourneau JJ, Stroke IL, Hilbert DW, Sturzenbecker LJ, Marinelli BA, Quintero JG, Sabalski J, Ma L, Diller DJ, Stein PD, Webb ML. 2018. Identification and initial optimization of inhibitors of Clostridium difficile (C. difficile) toxin B (TcdB). Bioorg Med Chem Lett. [Epub ahead of print]