First and foremost, these compounds constitute the first potent and selective inhibitors of IDE or, indeed, of any member of the inverzincin superfamily of zinc-metalloproteases [16]. Given the longstanding interest in IDE in general, and the predicted therapeutic value of IDE inhibitors in particular, why has their development proved so elusive for so long? The answer can be traced to the distinctive structure of IDE’s active site, which in turn reflects the separate evolutionary origins of this protease superfamily [7]. As documented by earlier studies [9,18] and the present work, IDE’s active site is bipartite, consisting of two distinct domains contained within the C- and N-terminal halves of the protease. The active site becomes fully formed only when the protease is in the closed conformation, and it is disrupted completely upon transition to the open conformation. These very large conformational changes occurring during the catalytic cycle of IDE essentially render its active site a ‘‘moving target,’’ one that cannot easily be stably occupied by small molecules, even those containing a strong zinc-binding moiety. As our co-crystal structure reveals, the potency of Ii1 can be traced to its unique ability to interact simultaneously with both the N- and C-terminal portions of the active site. In so doing, Ii1 appears to ‘‘lock’’ the protease in the closed, inactive conformation—a feature that is likely to be indispensable for effective IDE inhibitors. Second, these IDE inhibitors grant several new insights into the enzymology of this poorly understood protease. A particularly puzzling property is the substrate-dependence of Ki values for inhibition of IDE by Ii1, wherein smaller substrates show lower Ki values than larger substrates. These two categories of substrate have in fact been shown to exhibit strikingly different behaviors in multiple contexts. For example, the hydrolysis of short sub- strates—but not longer ones—can be profoundly activated by ATP and other nucleotide polyphosphates [30,31], inorganic triphosphate [30,31], as well as by structurally unrelated drug-like molecules [23]. In terms of the differences in Ki values, we speculate that larger substrates may be more capable than smaller ones of effecting the transition between the closed and open configurations, resulting in an increased off rate for the inhibitor. It may also be that the inhibitor can be trapped inside the internal chamber only in the case of smaller substrates [23]. Alternatively, given that 2 residues within Ii1 protrude into the internal chamber, it may be that larger substrates sterically block a subset of binding modes of the inhibitor. In this context, it is relevant to note that larger substrates are known to interact with an exosite present inside the catalytic chamber but opposite to the active site [9]. It is conceivable that larger substrates utilize this exosite as a point of leverage for larger substrates that normally helps position the substrate into the active site, but which may at the same time have the effect of pushing out an inhibitor. The finding that Hill slopes were consistently ,1 is also noteworthy. Given that IDE normally exists as a homodimer [32,33], this may be explained by intersubunit interactions, wherein the closing of one subunit, for example, by binding to inhibitor, favors the opening of the other subunit. Consistent with this idea, Song et al. recently reported [34] that a homodimerization-deficient IDE mutant exhibits markedly different enzymological properties than normal homodimeric IDE. For example, this mutant is not activated by polyphosphates or by other substrates [34]. Together with our own findings, these results suggest that intersubunit interactions powerfully influence the activity and substrate selectivity of IDE, possibly by influencing the transitions between the closed and open configurations of each subunit. Yet another interesting enzymological point emerges from the dose-response studies: the range of doses tested overlapped the nominal enzyme concentration (1 nM); nevertheless, the sigmoidal shape of the corresponding dose-response curves implies a much Inhibitors of IDE smaller amount of active enzyme. Consistent with the conclusions of previous studies [9,31], this result suggests that the vast majority of the protease is normally in the closed, inactive configuration. The finding that only a small fraction of IDE molecules are normally active at any one time may also help to account for IDE’s ability to be profoundly activated by multiple ligands [23,30,31,35]. Finally, the observation that Ii1 exhibits a purely competitive mode of inhibition is notable, because it suggests zinc- binding may not be the sole determinant of inhibitor potency; instead, this finding reinforces the idea that the inhibitory power of Ii1 may be better explained by its ability to stabilize the closed, inactive conformation, by virtue of joint interactions with both the N- and C-terminal domains of the protease. A third significant implication of our study relates to the degree to which Ii1 is selective for IDE vis-a`-vis other zinc-metallopro- teases (,4 orders of magnitude), which is especially impressive given their peptidic nature. While we emphasize that we cannot exclude the possibility that these compounds cross-react with other zinc-metalloproteases we did not test, it is significant to note that IDE was not inhibited at all even by broad-spectrum hydroxamic acid inhibitors of conventional zinc-metalloproteases (Table S2). These twin findings strongly suggest that it may be possible to develop highly selective IDE inhibitors, even inhibitors containing the potent hydroxamic acid moiety. In this context, it is notable that hydroxamic acids were once considered to be attractive candidates for several therapeutic applications and, indeed, continue to be tested in human trials [36]; however, as a general class, hydroxamic acid protease inhibitors fell out of favor due to a series of disappointing clinical results [37], which are commonly attributed to an innate lack of selectivity of the hydroxamic acid moiety. The remarkable degree of selectivity observed for Ii1 supports the alternative interpretation that the aforementioned clinical failures might instead be attributed to liabilities inherent in the targets of the tested compounds—more specifically, to the high degree of structural relatedness and sheer number of conventional zinc-metalloproteases present in higher mammals. Given the marked evolutionary and structural divergence of the inverzincin superfamily, and the low number of its membership, we speculate that it may be feasible to develop hydroxamate inhibitors of IDE with far fewer off-target effects. We emphasize, however, that it should also be possible to develop effective IDE inhibitors containing alternative zinc-binding moieties. Fourth, and perhaps most significantly from a biomedical perspective, the IDE inhibitors we developed show novel and potentially medicinally valuable effects on insulin signaling. Whereas IDE inhibitors have long been predicted to slow the catabolism of circulating insulin [38], we show here that IDE inhibitors also potentiate the action of insulin subsequent to its binding to the IR. Notably, the latter effect occurs on a time scale that is inconsistent with effects on extracellular insulin catabolism, but instead appears to be attributable to decreased degradation of internalized insulin, which is normally rapidly catabolized [29]. Thus, IDE inhibitors with appropriate pharmacokinetic properties may be useful in combating diabetes by virtue of both ‘‘insulin sparing’’ and ‘‘insulin sensitizing’’ effects. It is worth noting here that the wisdom of inhibiting IDE has been called into question by results from mice genetically deleted for the IDE gene [5]. In particular, IDE knockout mice have themselves been shown to develop a diabetic phenotype [5]. However, it is critical to recognize that this phenotype is a compensatory change that emerges only as a secondary consequence of chronic hyperinsu- linemia (S. Abdul-Hay, A. DelleDonne, L. Li, J. Zhao & M. Leissring, unpublished observations; see also [5]). In contrast to the PLoS ONE | www.plosone.org 7 May 2010 | Volume 5 | Issue 5 | e10504