Journal of Biomolecular Screening
17(10) 1348 –1361
© 2012 Society for Laboratory
Automation and Screening
According to the Aβ hypothesis, Aβ peptides contribute to
Alzheimer disease (AD) by serving as a source of neurotoxic-
ity. It remains unclear whether neurotoxicity stems from accu-
mulation of the monomeric, oligomeric (ADDLs), and/or
fibrillar forms of Aβ.1 Independent of the neurotoxic species,
strategies aimed at reducing Aβ levels hold promise for the
treatment of AD. Because biosynthetic and clearance activities
regulate steady-state Aβ levels, either or both activities could
be manipulated to reduce Aβ. Although most research on Aβ
has centered on its production (i.e., the α, β, and γ secretases),
several mechanisms are known to clear Aβ.2
One prominent Aβ clearance mechanism involves pro-
teolytic cleavage of the Aβ monomer, which eliminates its
ability to form higher order structures. The insulin-degrading
enzyme (IDE; EC 184.108.40.206) is one of several Aβ-cleaving
enzymes; others include neprilysin, plasmin, matrix metal-
loprotease-9, angiotensin-converting enzyme, and endothe-
lin-converting enzyme.2 In animal models, IDE deficiency
correlates with increased Aβ levels and increased risk of
AD, whereas IDE overexpression appears to protect against
AD.3,4 Genetic linkage and biochemical analyses also
strongly support a connection between IDE and AD.5,6 IDE
also cleaves other small molecules (e.g., insulin, glucagon,
and amylin) that can adopt β secondary structure and form
IDE belongs to the M16A family of zinc-dependent
metalloproteases, which are evolutionarily widespread and
highly conserved in sequence and structure.8,9 An inverted
zinc-binding motif is characteristic of this family. The
recent elucidation of several M16A enzyme structures,
including that of human IDE, pitrilysin (Escherichia coli),
and the related M16C enzyme PreP1 (Arabidopsis thali-
ana), reveals a structure resembling that of a clamshell in
which substrates form β-sheet contacts with β-strands of
IDE.8,9 Members of the M16A family can recognize each
others’ substrates as evident by the ability of mammalian
kday et al.Journal of Biomolecular Screening
1The University of Georgia, Athens, GA, USA
*These authors contributed equally to this study.
Received Jan 15, 2012, and in revised form Apr 20, 2012. Accepted for
publication May 24, 2012.
Supplementary material for this article is available on the Journal of
Biomolecular Screening Web site at http://jbx.sagepub.com/supplemental.
Walter K. Schmidt, A416, Fred Davison Life Sciences Building, 120 East
Green Street, Athens, GA 30602, USA
Cell-Permeable, Small-Molecule Activators
of the Insulin-Degrading Enzyme
Sayali S. Kukday1*, Surya P. Manandhar1*, Marissa C. Ludley1,
Mary E. Burriss1, Benjamin J. Alper1, and Walter K. Schmidt1
The insulin-degrading enzyme (IDE) cleaves numerous small peptides, including biologically active hormones and disease-
related peptides. The propensity of IDE to degrade neurotoxic Aβ peptides marks IDE as a potential therapeutic target for
Alzheimer disease. Using a synthetic reporter based on the yeast a-factor mating pheromone precursor, which is cleaved by
multiple IDE orthologs, we identified seven small molecules that stimulate rat IDE activity in vitro. Half-maximal activation
of IDE by the compounds is observed in vitro in the range of 43 to 198 µM. All compounds decrease the Km of IDE. Four
compounds activate IDE in the presence of the competing substrate insulin, which disproportionately inhibits IDE activity.
Two compounds stimulate rat IDE activity in a cell-based assay, indicating that they are cell permeable. The compounds
demonstrate specificity for rat IDE since they do not enhance the activities of IDE orthologs, including human IDE, and they
appear specific for a-factor–based reporters since they do not enhance rat IDE-mediated cleavage of Aβ-based reporters.
Our results suggest that IDE activators function in the context of specific enzyme-substrate pairs, indicating that the choice
of substrate must be considered in addition to target validation in IDE activator screens.
Aβ, IDE, protease, activator, Alzheimer disease
by guest on October 19, 2015jbx.sagepub.com Downloaded from
Kukday et al.
IDE and pitrilysin to substitute for the yeast M16A enzymes
Axl1p and Ste23p in production of the yeast a-factor mating
pheromone10,11 and the ability of Ste23p and pitrilysin to
The proposed physiological role of IDE in Aβ clearance
has led to the hypothesis that its hyperactivation could be
therapeutically beneficial. Studies of IDE involving under-
and overexpression in transgenic mice support this hypoth-
esis.3,4 Small-molecule activators of IDE can thus be
considered potential therapeutic agents, and several such
molecules have been reported.14,15 Nucleotide triphosphates
(i.e., adenosine triphosphate [ATP]) activate IDE to cleave
certain small substrates other than Aβ. This effect occurs at
ATP concentrations (i.e., 0.1–1 mM) much higher than that
thought to exist physiologically in the extracellular environ-
ment where Aβ is supposedly proteolyzed by IDE (i.e.,
5–50 nM) but within the range expected intracellularly (4–5
mM).16,17 ATP-dependent enhancement of IDE activity does
not involve ATP hydrolysis; the effect is also buffer depen-
dent.14 High-throughput screening (HTS) has identified two
additional small-molecule activators of IDE.15 Curiously,
these molecules enhance IDE-mediated cleavage of an Aβ
reporter only when the synthetic non-Aβ HTS substrate is
also present. The synthetic compound suramin has also
been indicated as an IDE activator.18 To date, data pertain-
ing to its effectiveness as an IDE activator or AD therapeu-
tic have not been released.
In this study, we report seven chemical activators of IDE
that were identified by HTS from a pharmacophore-rich
small-molecule library provided by the National Institutes
of Health (NIH) Developmental Therapeutics Program. We
demonstrate the ability of identified compounds to enhance
the activity of rat IDE toward both synthetic and natural
peptides based on the yeast a-factor mating pheromone. We
also describe a novel internally quenched Aβ-based reporter
useful for direct measurement of Aβ-cleaving activity. The
results we present in this study collectively demonstrate the
existence of cell-permeable, substrate- and species-specific
activators of IDE.
Materials and Methods
Two synthetic substrates were used to monitor the in vitro
activities of the IDE orthologs evaluated in this study. One
was an internally quenched, fluorogenic peptide Abz-
SEKKDNYIIKGV-NitroY-OH (AnaSpec, Inc., San Jose,
CA; CHI Scientific, Inc., Maynard, MA), where Abz is
aminobenzoic acid and NitroY is 3-nitro-tyrosine. The pep-
tide is based on the sequence flanking the M16A cleavage
site found in the yeast a-factor precursor. It was used for the
activator screen and for monitoring activities since it is
recognized by multiple IDE orthologs, including rat IDE,
yeast Ste23p, worm IDE, bovine trypsin, and pronase E;
human IDE does not recognize this substrate. The second
IDE substrate was the internally quenched, fluorogenic
EDANSDVGSNK-OH (CHI Scientific), where KDABCYL is
ε-DABCYL-L-lysine and EEDANS is EDANS-L-glutamate.
The peptide, based on the Aβ1–28 sequence, was used for
monitoring activities of rat, human, and worm IDE. Powder
forms of both peptides were resuspended in DMSO
(10 mM) and stored at –80 °C. Diluted 2× working stock
solutions (100 µM) were heated to 65 °C for 3 min and then
cooled to room temperature prior to use. Product formation
was measured using a Bio-Tek Synergy microplate reader
(Bio-Tek, Winooski, VT) equipped with 320/420-nm and
320/485-nm excitation/emission filter sets, respectively.
The substrates used to monitor activities of yeast Rce1p
and yeast Ste24p were internally quenched, fluorogenic,
farnesylated peptide substrates that are based on the K-Ras
C-terminus.19,20 Product formation for these substrates was
measured using a 320/420-nm excitation/emission filter set.
Recombinant Enzymes and Other Reagents
Plasmids encoding rat and yeast Ste23p have been
described.13,21 The plasmid encoding human IDE was con-
structed by amplifying the human IDE open reading frame
from plasmid IDE-pSRα using oligos designed to encode a
6X-His tag at the N-terminus and restriction sites for sub-
cloning the PCR product into the XbaI and NotI sites of
pET30b(+) (Novagen, Madison, WI).22 The plasmid encod-
ing Caenorhabditis elegans IDE was constructed by ampli-
fying the cDNA sequence of F44E7.4 from the RB1 cDNA
library by PCR and subcloning into the XbaI and NotI sites
of pET30b(+).23 Recombinant rat, human, C. elegans, and
yeast IDE were inducibly expressed in BL21 (DE3) E. coli
and recovered by immobilized nickel affinity chromatogra-
phy essentially as previously described.13,21 Purified IDE
was stored at –80 °C as 1 mg/mL aliquots in storage buffer
(50 mM HEPES, 140 mM NaCl, 20% glycerol [pH 7.4] or
25 mM KPi, 200 mM NaCl, 20% glycerol [pH 7.6]).
Membranes containing yeast Rce1p or yeast Ste24p activ-
ity were prepared as previously described.20 Bovine trypsin,
pronase E, proteinase K, ATP, bovine serum albumin
(BSA), and human recombinant insulin were all from
Sigma-Aldrich (St. Louis, MO). Ia1 and Ia2 were from Key
Organics (London, UK).
The Diversity Set compound library was obtained through
the NIH Developmental Therapeutics Program (DTP).24 This
compound library contains 1981 compounds with unique
pharmacophore characteristics that were reduced from a
parent set of more than 70 000 compounds with the use of the
by guest on October 19, 2015jbx.sagepub.comDownloaded from
Journal of Biomolecular Screening 17(10)
Formation, Secondary Pathology, and Premature Death. Neu-
ron 2003, 40(6), 1087–1093.
5. Kim, M.; Hersh, L.B.; Leissring, M.A.; Ingelsson, M.; Matsui,
T.; Farris, W.; Lu, A.; Hyman, B.T.; Selkoe, D.J.; Bertram,
L.; Tanzi, R.E. Decreased Catalytic Activity of the Insulin
Degrading Enzyme in Chromosome 10–Linked Alzheimer’s
Disease Families. J. Biol. Chem. 2007, 282(11), 7825–7832.
6. Miller, B.C.; Eckman, E.A.; Sambamurti, K.; Dobbs, N.;
Chow, K.M.; Eckman, C.B.; Hersh, L.B.; Thiele, D.L.
Amyloid-Beta Peptide Levels in Brain Are Inversely Correlated
with Insulysin Activity Levels In Vivo. Proc. Natl. Acad. Sci.
U. S. A. 2003, 100(10), 6221–6226.
7. Becker, A.B.; Roth, R.A. Insulysin and Pitrilysin: Insulin-
Degrading Enzymes of Mammals and Bacteria. Methods
Enzymol. 1995, 248, 693–703.
8. Johnson, K.A.; Bhushan, S.; Stahl, A.; Hallberg, B.M.; Frohn,
A.; Glaser, E.; Eneqvist, T. The Closed Structure of Prese-
quence Protease PreP Forms a Unique 10,000 Angstroms3
Chamber for Proteolysis. Embo J. 2006, 25(9), 1977–1986.
9. Shen, Y.; Joachimiak, A.; Rosner, M.R.; Tang, W.J. Structures
of Human Insulin-Degrading Enzyme Reveal a New Substrate
Recognition Mechanism. Nature 2006, 443(7113), 870–874.
10. Kim, S.; Lapham, A.; Freedman, C.; Reed, T.; Schmidt, W.
Yeast as a Tractable Genetic System for Functional Studies of
the Insulin-Degrading Enzyme. J. Biol. Chem. 2005, 280(30),
11. Alper, B.J.; Nienow, T.E.; Schmidt, W.K. A Common Genetic
System for Functional Studies of Pitrilysin and Related M16A
Proteases. Biochem. J. 2006, 398(1), 145–152.
12. Cornista, J.; Ikeuchi, S.; Haruki, M.; Kohara, A.; Takano,
K.; Morikawa, M.; Kanaya, S. Cleavage of Various Peptides
with Pitrilysin from Escherichia coli: Kinetic Analyses Using
Beta-Endorphin and Its Derivatives. Biosci. Biotechnol. Bio-
chem. 2004, 68(10), 2128–2137.
13. Alper, B.; Rowse, J.; Schmidt, W. Yeast Ste23p Shares
Functional Similarities with Mammalian Insulin-Degrading
Enzymes. Yeast 2009, 26(11), 595–610.
14. Song, E.S.; Juliano, M.A.; Juliano, L.; Fried, M.G.; Wagner,
S.L.; Hersh, L.B. ATP Effects on Insulin-Degrading Enzyme
Are Mediated Primarily through Its Triphosphate Moiety.
J. Biol. Chem. 2004, 279(52), 54216–54220.
15. Cabrol, C.; Huzarska, M.A.; Dinolfo, C.; Rodriguez, M.C.;
Reinstatler, L.; Ni, J.; Yeh, L.A.; Cuny, G.D.; Stein, R.L.;
Selkoe, D.J.; Leissring, M.A. Small-Molecule Activators of
Insulin-Degrading Enzyme Discovered through High-Through-
put Compound Screening. PLoS ONE 2009, 4(4), e5274.
16. Farias, M., III; Gorman, M.W.; Savage, M.V.; Feigl, E.O.
Plasma ATP during Exercise: Possible Role in Regulation of
Coronary Blood Flow. Am. J. Physiol. Heart Circ. Physiol.
2005, 288(4), H1586–H1590.
17. Kwak, J.; Wang, M.H.; Hwang, S.W.; Kim, T.Y.; Lee, S.Y.;
Oh, U. Intracellular ATP Increases Capsaicin-Activated Chan-
nel Activity by Interacting with Nucleotide-Binding Domains.
J. Neurosci. 2000, 20(22), 8298–8304.
18. Adessi, C.; Enderle, T.; Grueninger, F.; Roth, D. Acti-
vator for Insulin Degrading Enzyme. 2006. U.S. Patent
19. Hollander, I.; Frommer, E.; Mallon, R. Human Ras-Converting
Enzyme (hRCE1) Endoproteolytic Activity on K-Ras-Derived
Peptides. Anal. Biochem. 2000, 286(1), 129–137.
20. Porter, S.B.; Hildebrandt, E.R.; Breevoort, S.R.; Mokry, D.Z.;
Dore, T.M.; Schmidt, W.K. Inhibition of the CaaX Proteases
Rce1p and Ste24p by Peptidyl (Acyloxy)methyl Ketones. Bio-
chim. Biophys. Acta 2007, 1773(6), 853–862.
21. Alper, B.; Schmidt, W. A Capillary Electrophoresis Method
for Evaluation of Aβ Proteolysis In Vitro. J. Neurosci. Meth-
ods 2009, 178, 40–45.
22. Affholter, J.A.; Hsieh, C.L.; Francke, U.; Roth, R.A. Insulin-
Degrading Enzyme: Stable Expression of the Human Com-
plementary DNA, Characterization of Its Protein Product, and
Chromosomal Mapping of the Human and Mouse Genes. Mol.
Endocrinol. 1990, 4(8), 1125–1135.
23. Barstead, R.J.; Waterston, R.H. The Basal Component of
the Nematode Dense-Body Is Vinculin. J. Biol. Chem. 1989,
24. Holbeck, S.L. Update on NCI In Vitro Drug Screen Utilities.
Eur. J. Cancer 2004, 40(6), 785–793.
25. Goode, D.R.; Totten, R.K.; Heeres, J.T.; Hergenrother, P.J.
Identification of Promiscuous Small Molecule Activators in
High-Throughput Enzyme Activation Screens. J. Med. Chem.
2008, 51(8), 2346–2349.
26. Chan, R.K.; Otte, C.A. Isolation and Genetic Analysis of Sac-
charomyces cerevisiae Mutants Supersensitive to G1 Arrest
by a Factor and Alpha Factor Pheromones. Mol. Cell. Biol.
1982, 2(1), 11–20.
27. Michaelis, S.; Herskowitz, I. The a-Factor Pheromone of
Saccharomyces cerevisiae Is Essential for Mating. Mol. Cell.
Biol. 1988, 8(3), 1309–1318.
28. Adames, N.; Blundell, K.; Ashby, M.N.; Boone, C. Role of
Yeast Insulin-Degrading Enzyme Homologs in Propheromone
Processing and Bud Site Selection. Science 1995, 270, 464–
29. Elble, R. A Simple and Efficient Procedure for Transformation
of Yeasts. BioTechniques 1992, 13, 18–20.
30. Sikorski, R.S.; Hieter, P. A System of Shuttle Vectors and Yeast
Host Strains Designed for Efficient Manipulation of DNA in Sac-
charomyces cerevisiae. Genetics 1989, 122(1), 19–27.
31. Chen, P.; Sapperstein, S.K.; Choi, J.D.; Michaelis, S. Bio-
genesis of the Saccharomyces cerevisiae Mating Pheromone
a-Factor. J. Cell. Biol. 1997, 136(2), 251–269.
32. Lipinski, C.; Lombardo, F.; Dominy, B.; Feeney, P. Experi-
mental and Computational Approaches to Estimate Solubility
and Permeability in Drug Discovery and Development Set-
tings. Adv. Drug Del. Rev. 1997, 23, 3–25.
33. Tam, A.; Schmidt, W.K.; Michaelis, S. The Multispanning
Membrane Protein Ste24p Catalyzes CAAX Proteolysis and
NH2-Terminal Processing of the Yeast a-Factor Precursor.
J. Biol. Chem. 2001, 276(50), 46798–46806.
by guest on October 19, 2015jbx.sagepub.comDownloaded from
Kukday et al.
34. Im, H.; Manolopoulou, M.; Malito, E.; Shen, Y.; Zhao, J.;
Neant-Fery, M.; Sun, C.Y.; Meredith, S.C.; Sisodia, S.S.;
Leissring, M.A.; Tang, W.J. Structure of Substrate-Free
Human Insulin-Degrading Enzyme (IDE) and Biophysical
Analysis of ATP-Induced Conformational Switch of IDE.
J. Biol. Chem. 2007, 282(35), 25453–25463.
35. Kurochkin, I.V. Amyloidogenic Determinant as a Substrate
Recognition Motif of Insulin-Degrading Enzyme. FEBS Lett.
1998, 427(2), 153–156.
36. Selkoe, D.J. Clearing the Brain’s Amyloid Cobwebs. Neuron
2001, 32(2), 177–180.
37. Kaur, M.; Reed, E.; Sartor, O.; Dahut, W.; Figg, W.D. Sura-
min’s Development: What Did We Learn? Invest. New Drugs
2002, 20(2), 209–219.
38. McCain, D.F.; Wu, L.; Nickel, P.; Kassack, M.U.; Kreimeyer,
A.; Gagliardi, A.; Collins, D.C.; Zhang, Z.Y. Suramin Deriva-
tives as Inhibitors and Activators of Protein-Tyrosine Phos-
phatases. J. Biol. Chem. 2004, 279(15), 14713–14725.
39. Zagorski, M.; Yang, J.; Shao, H.; Ma, K.; Zeng, H.; Hong,
A. Methodological and Chemical Factors Affecting Amyloid
Beta Peptide Amyloidogenicity. Methods Enzymol. 1999, 309,
40. Leissring, M.A.; Lu, A.; Condron, M.M.; Teplow, D.B.; Stein,
R.L.; Farris, W.; Selkoe, D.J. Kinetics of Amyloid Beta–Protein
Degradation Determined by Novel Fluorescence- and Fluores-
cence Polarization–Based Assays. J. Biol. Chem. 2003, 278(39),
41. Zorn, J.A.; Wells, J.A. Turning Enzymes ON with Small Mol-
ecules. Nat. Chem. Biol. 2010, 6(3), 179–187.
by guest on October 19, 2015jbx.sagepub.comDownloaded from