Designed Inhibitors of Insulin-Degrading Enzyme Regulate the Catabolism and Activity of Insulin Malcolm A. Leissring1,2,3*, Enrico Malito4, Sabrine Hedouin1, Lael Reinstatler1, Tomoko Sahara1, Samer O. Abdul-Hay1, Shakeel Choudhry1, Ghulam M. Maharvi1, Abdul H. Fauq 1 , Malwina Huzarska2, Philip S. May2, Sungwoon Choi5, Todd P. Logan 3 , Benjamin E. Turk6, Lewis C. Cantley7, Marika Manolopoulou 4 , Wei-Jen Tang 4 , Ross L. Stein5, Gregory D. Cuny5, Dennis J. Selkoe3 1 Department of Neuroscience, Mayo Clinic Florida, Jacksonville, Florida, United States of America, 2 Department of Molecular Therapeutics, The Scripps Research Institute, Scripps Florida, Jupiter, Florida, United States of America, 3 Center for Neurologic Diseases, Brigham & Women’s Hospital and Harvard Medical School, Boston, Massachusetts, United States of America, 4 Ben-May Institute for Cancer Research, University of Chicago, Chicago, Illinois, United States of America, 5 Laboratory for Drug Discovery in Neurodegeneration, Brigham & Women’s Hospital and Harvard Medical School, Cambridge, Massachusetts, United States of America, 6 Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut, United States of America, 7 Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, United States of America Abstract Background: Insulin is a vital peptide hormone that is a central regulator of glucose homeostasis, and impairments in insulin signaling cause diabetes mellitus. In principle, it should be possible to enhance the activity of insulin by inhibiting its catabolism, which is mediated primarily by insulin-degrading enzyme (IDE), a structurally and evolutionarily distinctive zinc- metalloprotease. Despite interest in pharmacological inhibition of IDE as an attractive anti-diabetic approach dating to the 1950s, potent and selective inhibitors of IDE have not yet emerged. Methodology/Principal Findings: We used a rational design approach based on analysis of combinatorial peptide mixtures and focused compound libraries to develop novel peptide hydroxamic acid inhibitors of IDE. The resulting compounds are ,106 times more potent than existing inhibitors, non-toxic, and surprisingly selective for IDE vis-a`-vis conventional zinc- metalloproteases. Crystallographic analysis of an IDE-inhibitor complex reveals a novel mode of inhibition based on stabilization of IDE’s ‘‘closed,’’ inactive conformation. We show further that pharmacological inhibition of IDE potentiates insulin signaling by a mechanism involving reduced catabolism of internalized insulin. Conclusions/Significance: The inhibitors we describe are the first to potently and selectively inhibit IDE or indeed any member of this atypical zinc-metalloprotease superfamily. The distinctive structure of IDE’s active site, and the mode of action of our inhibitors, suggests that it may be possible to develop inhibitors that cross-react minimally with conventional zinc-metalloproteases. Significantly, our results reveal that insulin signaling is normally regulated by IDE activity not only extracellularly but also within cells, supporting the longstanding view that IDE inhibitors could hold therapeutic value for the treatment of diabetes. Citation: Leissring MA, Malito E, Hedouin S, Reinstatler L, Sahara T, et al. (2010) Designed Inhibitors of Insulin-Degrading Enzyme Regulate the Catabolism and Activity of Insulin. PLoS ONE 5(5): e10504. doi:10.1371/journal.pone.0010504 Editor: Jun Liu, Johns Hopkins School of Medicine, United States of America Received February 22, 2010; Accepted April 12, 2010; Published May 7, 2010 Copyright: 2010 Leissring et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the National Institutes of Health (AG025070 and DA024888 to MAL; AG12749 to MAL and DJS; GM81539 to W-J T), the Ellison Medical Foundation (to MAL), the American Health Assistance Foundation (to EM), and by a generous donation from The Unforgettable Fund (to SH and MAL). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Leissring@mayo.edu Introduction Insulin is a tightly regulated peptide hormone that is centrally invovled in multiple vital physiological processes, ranging from energy and glucose homeostasis to memory and cognition [1,2,3]. The tertiary structure of insulin is unique among peptide hormones, being comprised of 2 peptide chains and containing 1 intra- and 2 interchain disulfide bonds, and the relative rigidity and bulk of insulin render it a poor substrate for most proteases [4]. The proteolytic degradation and inactivation of insulin is believed to be mediated primarily by insulin-degrading enzyme (IDE), a ubiquitously expressed, soluble, secreted zinc-metalloprotease [5,6]. IDE belongs to a small superfamily of zinc-metalloproteases (clan ME, family M16) that evolved independently of conventional zinc- metalloproteases [7]. Members of this superfamily are commonly referred to as ‘‘inverzincins,’’ because they feature a zinc-binding motif (HxxEH) that is inverted with respect to that within conventional zinc-metalloproteases (HExxH) [8]. Like insulin, IDE is structurally distinctive, consisting of two bowl-shaped halves connected by a flexible linker that can switch between ‘‘open’’ and ‘‘closed’’ states [9]. In its closed state, IDE completely encapsulates its substrates within an unusually large internal cavity [9] that appears remarkably well-adapted to accommodate insulin [10]. IDE degrades several other interme- PLoS ONE | www.plosone.org 1 May 2010 | Volume 5 | Issue 5 | e10504