Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Substrate-selective inhibitors that reprogram the activity of insulin-degrading enzyme

Nature Chemical Biology volume 15, pages 565–574 (2019)Cite this article

Subjects

Abstract

Enzymes that act on multiple substrates are common in biology but pose unique challenges as therapeutic targets. The metalloprotease insulin-degrading enzyme (IDE) modulates blood glucose levels by cleaving insulin, a hormone that promotes glucose clearance. However, IDE also degrades glucagon, a hormone that elevates glucose levels and opposes the effect of insulin. IDE inhibitors to treat diabetes, therefore, should prevent IDE-mediated insulin degradation, but not glucagon degradation, in contrast with traditional modes of enzyme inhibition. Using a high-throughput screen for non-active-site ligands, we discovered potent and highly specific small-molecule inhibitors that alter IDE’s substrate selectivity. X-ray co-crystal structures, including an IDE-ligand-glucagon ternary complex, revealed substrate-dependent interactions that enable these inhibitors to potently block insulin binding while allowing glucagon cleavage, even at saturating inhibitor concentrations. These findings suggest a path for developing IDE-targeting therapeutics, and offer a blueprint for modulating other enzymes in a substrate-selective manner to unlock their therapeutic potential.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: High-throughput screen for IDE exo-site ligands and discovery of substrate-selective IDE inhibitors.
Fig. 2: Structure-activity relationships and potency optimization of substrate-selective IDE inhibitors.
Fig. 3: Concentration-dependent substrate discrimination and metalloprotease specificity of potent substrate-selective IDE inhibitors.
Fig. 4: Structural basis for substrate-selective small-molecule inhibition of IDE.
Fig. 5: SSIs reprogram IDE’s substrate binding without inducing conformational changes or allosteric effects on the catalytic site.

Data availability

Complete results of the IDE high-throughput screens and the IDE inhibition counter-screen are available in the PubChem BioAssay database (1259348, 1259349), the IDE–37, IDE–63 and the IDE–63–glucagon X-ray structures are available in the PDB (PDB IDs 6BYZ, 6MQ3 and 6EDS, respectively).

References

  1. Mirsky, I. A. & Broh-Kahn, R. H. The inactivation of insulin by tissue extracts; the distribution and properties of insulin inactivating extracts. Arch. Biochem. 20, 1–9 (1949).

    CAS  PubMed  Google Scholar 

  2. Duckworth, W. C. & Kitabchi, A. E. Insulin and glucagon degradation by the same enzyme. Diabetes 23, 536–543 (1974).

    Article  CAS  Google Scholar 

  3. Roglic, G. & World Health Organization. Global Report on Diabetes (World Health Organization, 2016).

  4. Costes, S. & Butler, P. C. Insulin-degrading enzyme inhibition, a novel therapy for type 2 diabetes? Cell. Metab. 20, 201–203 (2014).

    Article  CAS  Google Scholar 

  5. Tang, W. J. Targeting insulin-degrading enzyme to treat type 2 diabetes mellitus. Trends Endocrinol. Metab. 27, 24–34 (2016).

    Article  CAS  Google Scholar 

  6. Duckworth, W. C., Bennett, R. G. & Hamel, F. G. Insulin degradation: progress and potential. Endocr. Rev. 19, 608–624 (1998).

    CAS  PubMed  Google Scholar 

  7. Abdul-Hay, S. O. et al. Deletion of insulin-degrading enzyme elicits antipodal, age-dependent effects on glucose and insulin tolerance. PLoS One 6, e20818 (2011).

    Article  CAS  Google Scholar 

  8. Farris, W. et al. Insulin-degrading enzyme regulates the levels of insulin, amyloid beta-protein, and the beta-amyloid precursor protein intracellular domain in vivo. Proc. Natl Acad. Sci. USA 100, 4162–4167 (2003).

    Article  CAS  Google Scholar 

  9. Villa-Perez, P. et al. Liver-specific ablation of insulin-degrading enzyme causes hepatic insulin resistance and glucose intolerance, without affecting insulin clearance in mice. Metab. Clin. Exp. 88, 1–11 (2018).

    Article  CAS  Google Scholar 

  10. Steneberg, P. et al. The type 2 diabetes-associated gene ide is required for insulin secretion and suppression of alpha-synuclein levels in beta-cells. Diabetes 62, 2004–2014 (2013).

    Article  CAS  Google Scholar 

  11. Maianti, J. P. et al. Anti-diabetic activity of insulin-degrading enzyme inhibitors mediated by multiple hormones. Nature 511, 94–98 (2014).

    Article  CAS  Google Scholar 

  12. Durham, T. B. et al. Dual exosite-binding inhibitors of insulin-degrading enzyme challenge its role as the primary mediator of insulin clearance in vivo. J. Biol. Chem. 290, 20044–20059 (2015).

    Article  CAS  Google Scholar 

  13. Ahren, B. Avoiding hypoglycemia: a key to success for glucose-lowering therapy in type 2 diabetes. Vasc. Health Risk Manag. 9, 155–163 (2013).

    Article  Google Scholar 

  14. Bennett, R. G., Duckworth, W. C. & Hamel, F. G. Degradation of amylin by insulin-degrading enzyme. J. Biol. Chem. 275, 36621–36625 (2000).

    Article  CAS  Google Scholar 

  15. Malito, E., Hulse, R. E. & Tang, W. J. Amyloid beta-degrading cryptidases: insulin degrading enzyme, presequence peptidase, and neprilysin. Cell. Mol. Life Sci. 65, 2574–2585 (2008).

    Article  CAS  Google Scholar 

  16. Unger, R. H. & Cherrington, A. D. Glucagonocentric restructuring of diabetes: a pathophysiologic and therapeutic makeover. J. Clin. Inv. 122, 4–12 (2012).

    Article  CAS  Google Scholar 

  17. Drag, M. & Salvesen, G. S. Emerging principles in protease-based drug discovery. Nat. Rev. Drug Discov. 9, 690–701 (2010).

    Article  CAS  Google Scholar 

  18. Berg, D. T., Wiley, M. R. & Grinnell, B. W. Enhanced protein C activation and inhibition of fibrinogen cleavage by a thrombin modulator. Science 273, 1389–1391 (1996).

    Article  CAS  Google Scholar 

  19. Xu, X., Chen, Z., Wang, Y., Bonewald, L. & Steffensen, B. Inhibition of MMP-2 gelatinolysis by targeting exodomain-substrate interactions. Biochem. J. 406, 147–155 (2007).

    Article  CAS  Google Scholar 

  20. Knapinska, A. M. et al. SAR studies of exosite-binding substrate-selective inhibitors of A disintegrin and metalloprotease 17 (ADAM17) and application as selective in vitro probes. J. Med. Chem. 58, 5808–5824 (2015).

    Article  CAS  Google Scholar 

  21. Madoux, F. et al. Discovery of an enzyme and substrate selective inhibitor of ADAM10 using an exosite-binding glycosylated substrate. Sci. Rep. 6, 11 (2016).

    Article  Google Scholar 

  22. Panwar, P. et al. Tanshinones that selectively block the collagenase activity of cathepsin K provide a novel class of ectosteric antiresorptive agents for bone. Br. J. Pharmacol. 175, 902–923 (2018).

    Article  CAS  Google Scholar 

  23. Leissring, M. A. et al. Designed inhibitors of insulin-degrading enzyme regulate the catabolism and activity of insulin. PLoS ONE 5, e10504 (2010).

    Article  Google Scholar 

  24. Deprez-Poulain, R. et al. Catalytic site inhibition of insulin-degrading enzyme by a small molecule induces glucose intolerance in mice. Nat. Comm. 6, 8250 (2015).

    Article  CAS  Google Scholar 

  25. Hendriks, B. S., Seidl, K. M. & Chabot, J. R. Two additive mechanisms impair the differentiation of ‘substrate-selective’ p38 inhibitors from classical p38 inhibitors in vitro. BMC Syst. Biol. 4, 23 (2010).

    Article  Google Scholar 

  26. Abdul-Hay, S. O. et al. Optimization of peptide hydroxamate inhibitors of insulin-degrading enzyme reveals marked substrate-selectivity. J. Med. Chem. 56, 2246–2255 (2013).

    Article  CAS  Google Scholar 

  27. Charton, J. et al. Imidazole-derived 2-[N-carbamoylmethyl-alkylamino]acetic acids, substrate-dependent modulators of insulin-degrading enzyme in amyloid-beta hydrolysis. Eur. J. Med. Chem. 79, 184–193 (2014).

    Article  CAS  Google Scholar 

  28. Abdul-Hay, S. O. et al. Selective targeting of extracellular insulin-degrading enzyme by quasi-irreversible thiol-modifying inhibitors. ACS Chem. Biol. 10, 2716–2724 (2015).

    Article  CAS  Google Scholar 

  29. Charton, J. et al. Structure-activity relationships of imidazole-derived 2-[N-carbamoylmethyl-alkylamino]acetic acids, dual binders of human insulin-degrading enzyme. Eur. J. Med. Chem. 90, 547–567 (2015).

    Article  CAS  Google Scholar 

  30. Busschots, K. et al. Substrate-selective inhibition of protein kinase PDK1 by small compounds that bind to the PIF-pocket allosteric docking site. Chem. Biol. 19, 1152–1163 (2012).

    Article  CAS  Google Scholar 

  31. Rettenmaier, T. J. et al. A small-molecule mimic of a peptide docking motif inhibits the protein kinase PDK1. Proc. Natl Acad. Sci. USA 111, 18590–18595 (2014).

    Article  CAS  Google Scholar 

  32. Shah, N. G. et al. Novel noncatalytic substrate-selective p38alpha-specific MAPK inhibitors with endothelial-stabilizing and anti-Inflammatory activity. J. Immunol. 198, 3296–3306 (2017).

    Article  CAS  Google Scholar 

  33. Hall, M. D. et al. Fluorescence polarization assays in high-throughput screening and drug discovery: a review. Methods Appl. Fluoresc. 4, 022001 (2016).

    Article  Google Scholar 

  34. Lowe, J. T. et al. Synthesis and profiling of a diverse collection of azetidine-based scaffolds for the development of CNS-focused lead-like libraries. J. Org. Chem. 77, 7187–7211 (2012).

    Article  CAS  Google Scholar 

  35. Malito, E. et al. Molecular bases for the recognition of short peptide substrates and cysteine-directed modifications of human insulin-degrading enzyme. Biochemistry 47, 12822–12834 (2008).

    Article  CAS  Google Scholar 

  36. Sebaugh, J. L. Guidelines for accurate EC50/IC50 estimation. Pharm. Stat. 10, 128–134 (2011).

    Article  CAS  Google Scholar 

  37. Degorce, F. et al. HTRF: A technology tailored for drug discovery—a review of theoretical aspects and recent applications. Curr. Chem. Genomics 3, 22–32 (2009).

    Article  CAS  Google Scholar 

  38. Leung, C. S., Leung, S. S., Tirado-Rives, J. & Jorgensen, W. L. Methyl effects on protein-ligand binding. J. Med. Chem. 55, 4489–4500 (2012).

    Article  CAS  Google Scholar 

  39. Shroyer, L. A. & Varandani, P. T. Purification and characterization of a rat liver cytosol neutral thiol peptidase that degrades glucagon, insulin, and isolated insulin A and B chains. Arch. Biochem. Biophys. 236, 205–219 (1985).

    Article  CAS  Google Scholar 

  40. Shen, Y., Joachimiak, A., Rosner, M. R. & Tang, W. J. Structures of human insulin-degrading enzyme reveal a new substrate recognition mechanism. Nature 443, 870–874 (2006).

    Article  CAS  Google Scholar 

  41. Leissring, M. A. & Selkoe, D. J. Structural biology: enzyme target to latch on to. Nature 443, 761–762 (2006).

    Article  CAS  Google Scholar 

  42. McCord, L. A. et al. Conformational states and recognition of amyloidogenic peptides of human insulin-degrading enzyme. Proc. Natl Acad. Sci. USA 110, 13827–13832 (2013).

    Article  CAS  Google Scholar 

  43. Song, E. S., Juliano, M. A., Juliano, L. & Hersh, L. B. Substrate activation of insulin-degrading enzyme (insulysin). A potential target for drug development. J. Biol. Chem. 278, 49789–49794 (2003).

    Article  CAS  Google Scholar 

  44. Im, H. et al. Structure of substrate-free human insulin-degrading enzyme (IDE) and biophysical analysis of ATP-induced conformational switch of IDE. J. Biol. Chem. 282, 25453–25463 (2007).

    Article  CAS  Google Scholar 

  45. Song, E. S., Rodgers, D. W. & Hersh, L. B. A monomeric variant of insulin degrading enzyme (IDE) loses its regulatory properties. PLoS One 5, e9719 (2010).

    Article  Google Scholar 

  46. Duggan, K. C. et al. (R)-Profens are substrate-selective inhibitors of endocannabinoid oxygenation by COX-2. Nat. Chem. Biol. 7, 803–809 (2011).

    Article  Google Scholar 

  47. Rose, K. et al. Insulin proteinase liberates from glucagon a fragment known to have enhanced activity against Ca2+ + Mg2+-dependent ATPase. Biochem. J. 256, 847–851 (1988).

    Article  CAS  Google Scholar 

  48. Vandenbroucke, R. E. & Libert, C. Is there new hope for therapeutic matrix metalloproteinase inhibition? Nat. Rev. Drug Discov. 13, 904–927 (2014).

    Article  CAS  Google Scholar 

  49. McMurray, J. J. Neprilysin inhibition to treat heart failure: a tale of science, serendipity, and second chances. Eur. J. Heart Fail. 17, 242–247 (2015).

    Article  CAS  Google Scholar 

  50. Zeke, A. et al. Systematic discovery of linear binding motifs targeting an ancient protein interaction surface on MAP kinases. Mol. Syst. Biol. 11, 837 (2015).

    Article  Google Scholar 

  51. Geu-Flores, F., Nour-Eldin, H. H., Nielsen, M. T. & Halkier, B. A. USER fusion: a rapid and efficient method for simultaneous fusion and cloning of multiple PCR products. Nucleic Acid. Res. 35, e55 (2007).

    Article  Google Scholar 

  52. Vonrhein, C. et al. Data processing and analysis with the autoPROC toolbox. Acta Crystallogr. D 67, 293–302 (2011).

    Article  CAS  Google Scholar 

  53. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    Article  CAS  Google Scholar 

  54. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004).

    Article  Google Scholar 

  55. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010).

    Article  CAS  Google Scholar 

  56. APEX2 v.2014.11-0 (Bruker AXS, Madison, WI, USA, 2014).

  57. Krause, L., Herbst-Irmer, R., Sheldrick, G. M. & Stalke, D. Comparison of silver and molybdenum microfocus X-ray sources for single-crystal structure determination. J. Appl. Crystallogr. 48, 3–10 (2015).

  58. Sheldrick, G. M. SHELXT—integrated space-group and crystal-structure determination. Acta Crystallogr. A 71, 3–8 (2015).

    Article  Google Scholar 

  59. Sheldrick, G. M. Crystal structure refinement with SHELXL. Acta Crystallogr. C 71, 3–8 (2015).

    Article  Google Scholar 

  60. Dolomanov, O. V., Bourhis, L. J., Gildea, R. J., Howard, J. A. K. & Puschmann, H. OLEX2: a complete structure solution, refinement and analysis program. J. App. Crystallogr. 42, 339–341 (2009).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank A. Saghatelian, S. Schreiber and M. Morningstar for helpful discussions. We are grateful to S. Trauger and J. Wang for mass spectrometry assistance. We thank J. Bittker, M. Wawer and V. Dancik for assistance with library management and analysis. We thank Z. Foda and A. Lyczek for ligand docking studies and D. Dobrovolsky for assistance with assays. We thank S.-L. Zheng for small-molecule structural determination. IDE X-ray diffraction data were collected at ALS (operated by LBNL) and NSLS2 (operated by BNL) on behalf of DOE and this is supported by DOE Office of Biological and Environmental Research (KP1605010) and NIH (R01GM105404, S10OD018483, P41GM111244). This research was supported by the NIH grant nos. R35 GM118062 (to D.R.L.), R01 EB022376 (to D.R.L.), R35 GM119437 (to M.A.S.), R56 DK106200 (to M.A.S.) and the Howard Hughes Medical Institute (to D.R.L.). The Fonds de Recherche en Santé du Québec and Alfred Bader Fund provided fellowship support to J.P.M.

Author information

Author notes

  1. These authors contributed equally: Juan Pablo Maianti, Grace A. Tan.

Authors and Affiliations

  1. Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of MIT and Harvard, Cambridge, MA, USA

    Juan Pablo Maianti & David R. Liu

  2. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA

    Juan Pablo Maianti & David R. Liu

  3. Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA

    Juan Pablo Maianti & David R. Liu

  4. Chemical Biology and Therapeutics Science, Broad Institute of MIT and Harvard, Cambridge, MA, USA

    Juan Pablo Maianti, Amedeo Vetere, Bridget K. Wagner & David R. Liu

  5. Department of Pharmacological Sciences, Stony Brook University, Stony Brook, NY, USA

    Grace A. Tan, Amie J. Welsh & Markus A. Seeliger

Authors
  1. Juan Pablo Maianti
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Grace A. Tan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Amedeo Vetere
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Amie J. Welsh
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Bridget K. Wagner
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Markus A. Seeliger
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. David R. Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.P.M. designed the exo-site screen, expressed proteins, synthesized the SSIs and ran biochemistry assays. G.A.T. co-crystallized and solved the IDE X-ray structures with A.J.W.’s assistance. A.V. optimized screen analysis. B.K.W. supervised the screen. M.A.S. and D.R.L. supervised the research program. All authors contributed to the writing of the manuscript.

Corresponding authors

Correspondence to Markus A. Seeliger or David R. Liu.

Ethics declarations

Competing interests

J.P.M. and D.R.L. are co-inventors on patents and patent applications based on this work, and are co-founders of Exo Therapeutics, a small-molecule drug discovery company.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Tables 1–9, Supplementary Figures 1–6

Reporting Summary

Supplementary Note

Synthetic protocols

Supplementary Video

Supplementary animation generated using PyMOL (1,000 frames).

Supplementary Data Set 1

Supplementary data deposited in PubMed BioAssay (numbers 1259349 and 1259348).

Supplementary Data Set 2

Supplementary reports and data provided by EuroFins (Belgium) that is summarized in Supplementary Table 6.

Supplementary Data Set 3

Nuclear Magnetic Resonance spectra (1H-, 13C-, and 19F-NMR).

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Maianti, J.P., Tan, G.A., Vetere, A. et al. Substrate-selective inhibitors that reprogram the activity of insulin-degrading enzyme. Nat Chem Biol 15, 565–574 (2019). https://doi.org/10.1038/s41589-019-0271-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41589-019-0271-0

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing