Project

Medicinal chemistry; Drug discovery; Bacterial toxins; Clostridium difficile

Lead
Lay summary
Bacteria rely on a host of different virulence factors to colonize the host and create pathogenesis. For some infections, therapeutic strategies targeting virulence factors such as toxins or adhesins have the advantage of not putting selective pressure on the organism, and thereby avoiding resistance problems. In recent years, the precise mechanism of pathogenesis and the crucial virulence factor for colonization of several important infections has been uncovered. This understanding provides fertile grounds for chemists to devise innovative strategies to prevent pathogenesis. Clostridium difficile is a so-called “superbug” that is the cause of serious infections, especially in hospitalized patients. When the healthy gut flora of a patient is depleted, C. difficile can colonize the colon and cause a series of life-threatening diseases. Recently, emerging hypervirulent strains of C. difficile are causing more frequent and severe diseases. The current treatment for severe C. difficile infection (CDI) is vancomycin, but is plagued by high relapse rates and the threat of resistance against this last-resort antibiotic. A newly approved antibiotic for CDI, fidaxomicin, does not improve relapse rates in common hypervirulent strains. Therefore, there is a need for new therapy that would ideally not put selective pressure on the bacteria. Two toxins (TcdA, TcdB) secreted by the bacteria in the colon are responsible for the pathogenesis. Thus, there is an opportunity to manage the disease by targeting the toxins in the colon rather than killing the bacteria. TcdA and TcdB are closely related large multi-domain proteins. The “warhead” of the toxin is a glucosyltransferase that inactivates important proteins in the cell cytosol. The remainder of the toxin is responsible for cell-binding, translocation and auto-processing, and is essential for the glucosyltransferase to reach the cytosol. In this project we aim at interfering with the uptake mechanism of the "warhead" by using small molecules. This research could provide an important therapeutic alternative to antibiotics that would not be susceptible to resistance problems.

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Last update: 21.02.2013

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Clostridium difficile is a bacterium that causes serious nosocomial infection in patients under antibiotic treatment, because this so-called “superbug” is resistant to a number of broad-spectrum antibiotics. When the healthy gut flora of a patient is depleted, C. difficile can colonize the colon and cause a series of life-threatening diseases. Recently emerging hypervirulent strains of C. difficile are causing more frequent and severe diseases. The current treatment for severe C. difficile infection (CDI) is vancomycin, but is plagued by high relapse rates and the threat of resistance against this last-resort antibiotic. A newly approved antibiotic for CDI, fidaxomicin, does not improve relapse rates in common hypervirulent strains. Therefore, there is a need for new therapy that would ideally not put selective pressure on the bacteria. Two toxins (TcdA, TcdB) secreted by the bacteria in the colon are responsible for the pathogenesis. Therefore, there is an opportunity to manage the disease by targeting the toxins in the colon rather than killing the bacteria. TcdA and TcdB are closely related large multi-domain proteins. The “warhead” of the toxin is a glucosyltransferase that inactivates important proteins in the cell cytosol. The rest of the toxin is responsible for cell-binding, translocation and auto-processing, and is essential for the glucosyltransferase to reach the cytosol. An auto-processing step triggered by the intracellular co-factor inositol hexakisphosphate (IP6) is required to release the glucosyltransferase in the cytosol. The cysteine protease domain (CPD) responsible for this cleavage binds to IP6, which then stabilizes the active conformation of the enzyme and results in cleavage.Since the glucosyltransferase itself is innocuous to cells because it is incapable of reaching the cytosol, we propose to trigger prematurely the auto-processing of the toxin in the colon lumen, and therefore inactivate the toxin. This constitutes a novel and unexplored strategy for targeting bacterial toxins. IP6 is a strictly intracellular co-factor because of its insolubility in the high concentrations of calcium found outside the cells. Therefore, IP6 itself cannot be used for this purpose. We propose to synthesize analogs of IP6 that are soluble in the presence of calcium and retain the ability to induce the toxin auto-cleavage. The specific aims of this proposal are:Aim 1: The design, synthesis, and characterization of IP6 analogs soluble in high calcium concentrations.Aim 2: The in vitro evaluation of the candidates’ activity against the CPD of the toxin. The results of this aim will inform the design of further analogs in a feedback loop.Aim 3: Further in vitro evaluation of selected candidates will be performed. In addition, the candidates will be evaluated in a cytotoxicity inhibition assay in cell cultures. The compounds’ toxicity to colon cancer cells will also be evaluated.