Technical CommentsInfectious Disease

Comment on “A small-molecule antivirulence agent for treating Clostridium difficile infection”

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Science Translational Medicine  21 Dec 2016:
Vol. 8, Issue 370, pp. 370tc2
DOI: 10.1126/scitranslmed.aad8926

Abstract

New insights into the mechanism of action of ebselen, a small-molecule antivirulence agent that reduces disease pathology in a mouse model of Clostridium difficile infection, suggest a different molecular target may be responsible for its efficacy.

In their recent paper (1), Bender et al. identified a small molecule, called ebselen, that inhibited the autoprocessing activity of the major virulence factors toxins A and B (TcdA and TcdB) from Clostridium difficile. They showed that ebselen prevented toxin-induced cell damage and reduced disease pathology in an in vivo model of C. difficile infection. Because ebselen was identified using the isolated autoprocessing domain (APD) as the target in a small-molecule screen, combined with the fact that ebselen was able to prevent autoprocessing both in vitro and in vivo, it was reasonably assumed that the mechanism by which ebselen provided protection from toxin was through the inhibition of toxin autoprocessing. Here, we propose that ebselen—though indeed an inhibitor of autoprocessing—is unlikely to exert its protective effects through this particular mechanism, based on the finding that it retains the ability to inhibit cellular intoxication by a form of TcdB lacking a functional APD. We show that ebselen inhibits an entirely different process that is required for toxin action, and provide a simple explanation for the discrepancy in results, which is related to the unique chemistry of ebselen itself.

Ebselen came to our attention a year ago during the follow-up of a high-throughput phenotypic screen that we conducted to identify small molecules that protected against TcdB-induced cell rounding (2). Ebselen, which ranked high among the hits that protected cells from TcdB, was among a dozen or so hits that underwent further characterization to elucidate their mechanism of action. A key triaging step that we used in our follow-up cascade to help narrow down the mechanism of action of hits was to test whether compounds that inhibited cell rounding also inhibited the high-dose TcdB necrosis mechanism of toxicity. Toxin-induced necrosis, which manifests independently of autoprocessing and glucosyltransferase activities (3) (Fig. 1A), can be exploited as a means to help narrow down the mechanism of action of a given inhibitor of cell rounding, because only those molecules that block receptor binding or pore formation (or possibly a host factor involved in mediating necrosis) are expected to inhibit necrosis. For instance, lysosomotropic compounds, such as quinacrine, which neutralize endosomal pH and prevent toxin pore formation and translocation into the cytosol, inhibit both toxin-induced cell rounding and necrosis phenotypes (Fig. 1B). Ebselen did not inhibit toxin-induced necrosis (Fig. 1B), indicating that it was not preventing receptor binding or pore formation but rather was involved in blocking either autoprocessing activity, glucosyltransferase activity, or, potentially, a host factor implicated in cell rounding.

Fig. 1. Characterization of the mechanism of neutralization of TcdB by the small-molecule ebselen.

All methods used to characterize ebselen were published previously (2). (A) Domain architecture of TcdB with approximate domain boundaries indicated above and positions of native Cys residues [C] indicated within each domain. The catalytic cysteine C698 within the APD is denoted with an asterisk. The domains required for the cell-rounding and necrosis phenotypes are indicated. (B) Inhibition of TcdB-induced necrosis by quinacrine and ebselen. Human IMR-90 cells were treated with 1 nM TcdB in the absence or presence of increasing amounts of quinacrine and ebselen. Using the luminescent CellTiter-Glo indicator, the percent inhibition of necrosis was calculated relative to untreated cells. Quinacrine, which inhibits endosomal acidification and, thus, pore formation (2), dose-dependently inhibits necrosis, whereas ebselen shows no inhibition of necrosis up to 100 μM, suggesting that it inhibits either the GTD or the APD. Results ± SEM are the average of three independent experiments. (C) Left: Kinetic cell-rounding assay using 0.5 pM wild-type TcdB or Cys-less TcdB. Human IMR-90 cells were challenged with 0.5 pM of either wild-type or Cys-less toxin, and the cell-rounding index was determined by Cellomics at 1, 2, 3, 4, and 5 hours after toxin addition. Right: Protection from toxin-induced cell rounding at 4 hours. (D) Inhibition of TcdB-induced cell rounding using 10 pM wild-type or Cys-less TcdB at 3 hours. Cell rounding was quantified using Cellomics as previously described (2). (E) Inhibition of TcdB-induced cell rounding by EC99 values of each toxin: 0.5 pM wild-type or 10 pM Cys-less TcdB at 3 hours. In all cases, ebselen inhibited intoxication of wild-type and Cys-less TcdB equally, indicating that ebselen does not inhibit intoxication through covalent modification of a cysteine residue in TcdB. Results ± SEM are the average of five independent experiments. (F) Ebselen dose-dependently inhibited glucosyltransferase activity in a scintillation proximity–based assay developed previously (2). (G) In the absence of DTT (left), ebselen inhibits inositol hexakisphosphate (IP6)–induced autoprocessing activity, whereas in the presence of 5 mM DTT (right), no inhibition of autoprocessing was observed. Gels are representative of five independent experiments.

We approached further elucidation of the mechanism of inhibition of ebselen cautiously, mindful of its well-documented propensity for reacting with and labeling cysteine residues in various proteins (4). An important tool that we had at our disposal, originally developed for site-specific labeling purposes, was a construct of TcdB in which all nine native cysteine sites, including the APD active-site cysteine C698, were mutated. We reasoned that we could use the “Cys-less” form of TcdB to determine, first, whether the mechanism of inhibition for ebselen was through APD inhibition and, second, whether it involved covalent modification of any of the native cysteine residues. To evaluate ebselen inhibition of Cys-less TcdB and wild-type TcdB, we varied both toxin concentration and time of incubation with cells to establish multiple conditions where these toxins induced equivalent cell rounding. A time course of toxin-induced cell rounding by wild-type and Cys-less TcdB at a matched dose (0.5 pM) is shown Fig. 1C (left). As expected for a toxin lacking a functional APD, the kinetics of inducing cell rounding were slightly delayed compared to wild-type TcdB; however, by 4 hours, the cytopathic effects between the two toxins were indistinguishable (Fig. 1C). We then tested the ability of ebselen to inhibit wild-type TcdB and Cys-less TcdB at 4 hours (Fig. 1C, right) and found that ebselen was equipotent on both toxins. To confirm that these conditions were not unique, we tested inhibition under two additional conditions: against both toxins at 10 pM at 3 hours (Fig. 1D) and against each toxin at their respective EC99 concentrations (concentrations of ebselen that results in 99% inhibition of toxin-induced cell rounding) at 3 hours (Fig. 1E). In both cases, as mentioned above, ebselen showed equivalent protection against both wild-type TcdB and Cys-less TcdB. In addition to ruling out that ebselen was neutralizing toxin function through covalent labeling of cysteine residues, this key finding indicated that ebselen was not protecting cells from TcdB by inactivating the APD active-site cysteine, thus eliminating an entire domain from further analysis.

With the APD ruled out, we then turned our attention to the glucosyltransferase domain (GTD) of TcdB as a potential target of ebselen, the inhibition of which would be expected to inhibit cell rounding but not necrosis. We found that ebselen dose-dependently inhibited the ability of the GTD to transfer a glucose from UDP (uridine 5′-diphosphate)–glucose to human Rac1 (Fig. 1F). Upon further examination of the mode of inhibition of GTD by ebselen, we discovered that ebselen was indirectly inhibiting GTD by covalently labeling Rac1 at three positions (Cys105, Cys157, and Cys178), through which either steric or structural effects prevented GTD-mediated glucose transfer onto Thr37 of Rac1 (fig. S1).

These findings led us to conclude that ebselen was protecting cells from TcdB-induced cell rounding, not by inhibiting APD activity, but rather by preventing GTD-mediated effects. Thus, it was with interest that we read the study by Bender et al. reporting that ebselen was an inhibitor of toxin autoprocessing (1) and, furthermore, that it was not an inhibitor of the GTD activity of TcdB. To reconcile these conflicting findings, we evaluated all aspects of the two studies to find any differences between our methodologies that might account for the discrepancies in our results. The answer turned out to be that the reducing agent dithiothreitol (DTT), which removes ebselen from Cys side chains, was included by Bender et al. in their assay of GTD activity, but not in their assay of APD activity (1). To demonstrate this, we tested inhibition of APD by ebselen in the absence and presence of DTT and saw that inclusion of DTT completely abrogated inhibition by ebselen (Fig. 1G). Similarly, addition of DTT to ebselen-labeled Rac1 relieved the inhibition of GTD activity (fig. S1). Thus, in the absence of DTT, ebselen can covalently inhibit both the APD and the GTD; however, as outlined above, on the basis of its ability to protect cells from intoxication by wild-type and Cys-less TcdB equally, it is not the APD inhibition that is responsible for the protective effects of ebselen.

These findings agree with previous in vitro and in vivo studies, which suggested that APD activity contributes to, but is not essential for, toxin action, whereas GTD activity is essential for toxin activity (58). Nevertheless, given its unique chemistry and known polypharmacology, we cannot rule out the possibility that inhibition of other targets, including APD, contributes to the observed efficacy of ebselen. Although our data point to GTD inhibition as a potential mechanism of action, it will be very difficult to demonstrate this conclusively in vivo. As Bender et al. suggested in their discussion (1), additional studies using C. difficile strains that have been engineered to produce catalytically inactive forms of the enzyme domains are needed to help resolve both the role of different domains in pathogenesis and the mechanism of action of certain experimental drug leads.

SUPPLEMENTARY MATERIAL

www.sciencetranslationalmedicine.org/cgi/content/full/8/370/370tc2/DC1

Materials and Methods

Fig. S1. Mechanism of inhibition of TcdB glucosyltransferase activity by ebselen.

REFERENCES AND NOTES

  1. Acknowledgments: This work was supported by Natural Sciences and Engineering Research Council of Canada and Canadian Institutes of Health Research (CIHR) grants to R.A.M. G.L.B. is a recipient of a postdoctoral fellowship from CIHR.
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