The Blood Glucose-lowering Effect of Racecadotril is not Attributable to Inhibition of Insulin-degrading Enzyme

Dear Editor,

We read with interest the article by Lee and colleagues [1], entitled Inhibition of Insulin Degrading Enzyme by Racecadotril in the Brain of Wister Rats [Lee et al. Horm Metab Res 2011; 43: 489–493]. This article concludes that racecadotril and its metabolite thiorphan exert blood glucose-lowering properties by inhibiting insulin-degrading enzyme (IDE), a zinc-metallopeptidase involved in regulating insulin levels. Our group has been attempting to develop effective inhibitors of IDE for more than a decade, so the idea that IDE could be inhibited effectively by a commercially available compound was highly surprising to us. We therefore tested whether racecadotril or thiorphan can directly inhibit IDE in vitro. To that end, we conducted dose-response curves using a range of concentrations (0.03 nM to 100 μM) of each inhibitor or, as a control, a potent IDE inhibitor developed previously by our group, IDE inhibitor 1 (Ii1) [2]. We used a well-characterized IDE activity assay [3] [4] comprised of a fluorogenic peptide substrate (FRET1) combined with purified recombinant human IDE ([Fig. 1a]). Consistent with previous findings, Ii1 potently inhibited IDE, with an IC₅₀ of 6.9±1.2 nM (k _i=4.6±0.8 nM), with maximal inhibition occurring at concentrations as low as 1 μM. In marked contrast, racecadotril and thiorphan showed no evidence of inhibition at any concentration tested, up to a maximum of 100 μM.

Fig. 1 Racecadotril and thiorphan do not inhibit IDE. a Dose-response curves for inhibition of IDE by Ii1, racecadotril, and thiorphan using the hydrolysis of FRET1 as a measure of IDE activity. b Progress curves for insulin degradation by freshly isolated endogenous rat IDE in the presence of Ii1 (100 μM), racecadotril (100 μM), thiorphan (100 μM), or vehicle (DMSO) alone. c Comparison of the insulin-degrading activity present in blood extracts isolated from wild-type (WT) mice vs. mice lacking IDE (IDE-KO). Note that virtually no insulin-degrading activity is present in blood from IDE-KO mice, indicating that IDE mediates essentially all insulin-degrading activity in blood.

Pharmacological modulators of IDE are known to be substrate selective, an unusual feature that extends even to active site-directed inhibitors such as Ii1 [4] [5]. Moreover, there could conceivably be salient differences between the recombinant human IDE grown in bacteria used in the fluorogenic activity assays and the endogenous rat IDE used in the experiments of Lee et al. implicating racecadotril and thiorphan as inhibitors of IDE [1]. We therefore conducted an additional IDE activity assay, in this case monitoring the degradation of insulin by freshly isolated endogenous rat IDE. For these experiments, IDE was isolated from blood, because it is a readily available source of freshly prepared endogenous IDE, which is abundantly expressed in erythrocytes [6]. Consistent with the results obtained with the fluorogenic peptide substrate, insulin degradation by rat IDE was not inhibited by racecadotril or thiorphan (100 μM), while being strongly inhibited by Ii1 (100 μM) ([Fig. 1b]). As an important control, insulin degradation was shown to be virtually absent in blood isolates obtained from IDE-null mice ([Fig. 1c]), indicating that essentially all the insulin-degrading activity in blood is attributable to IDE and not to other unrelated proteases.

Our results indicate that IDE is not inhibited to any appreciable extent by racecadotril or its active metabolite thiorphan. This fact is not surprising, given that IDE belongs to a superfamily of zinc-metalloproteases (Clan M16) that evolved completely independently from most other metalloproteases [7]. In the M16 clan, the active-site residues involved in zinc-binding and catalysis exhibit a motif (HxxEH) that is inverted with respect to that within conventional zinc-metalloprotease (HExxH) [7]. Moreover, recent crystal structures of IDE [8] [9] reveal a highly distinctive “clamshell”-like structure, featuring a bipartite active-site [3]. The many distinguishing structural features of IDE help to explain why thiorphan and racecadotril are so ineffective at inhibiting this protease. We conclude that the blood glucose-lowering properties of racecadotril and thiorphan are not attributable to direct inhibition of IDE, and must therefore involve other mechanisms.

^* Equal contributors.

• References

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