Limits...
Small-molecule activators of insulin-degrading enzyme discovered through high-throughput compound screening.

Cabrol C, Huzarska MA, Dinolfo C, Rodriguez MC, Reinstatler L, Ni J, Yeh LA, Cuny GD, Stein RL, Selkoe DJ, Leissring MA - PLoS ONE (2009)

Bottom Line: Both compounds were found to interfere with the crosslinking of a photoaffinity ATP analogue to IDE, suggesting that they interact with a bona fide ATP-binding domain within IDE.These novel activators help to establish the putative ATP-binding domain as a key modulator of IDE proteolytic activity and offer new insights into the modulatory action of ATP.Several larger lessons abstracted from this screen will help inform the design of future screening campaigns and facilitate the eventual development of IDE activators with therapeutic utility.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Mayo Clinic, Jacksonville, Florida, United States of America.

ABSTRACT

Background: Hypocatabolism of the amyloid beta-protein (Abeta) by insulin-degrading enzyme (IDE) is implicated in the pathogenesis of Alzheimer disease (AD), making pharmacological activation of IDE an attractive therapeutic strategy. However, it has not been established whether the proteolytic activity of IDE can be enhanced by drug-like compounds.

Methodology/principal findings: Based on the finding that ATP and other nucleotide polyphosphates modulate IDE activity at physiological concentrations, we conducted parallel high-throughput screening campaigns in the absence or presence of ATP and identified two compounds--designated Ia1 and Ia2--that significantly stimulate IDE proteolytic activity. Both compounds were found to interfere with the crosslinking of a photoaffinity ATP analogue to IDE, suggesting that they interact with a bona fide ATP-binding domain within IDE. Unexpectedly, we observed highly synergistic activation effects when the activity of Ia1 or Ia2 was tested in the presence of ATP, a finding that has implications for the mechanisms underlying ATP-mediated activation of IDE. Notably, Ia1 and Ia2 activated the degradation of Abeta by approximately 700% and approximately 400%, respectively, albeit only when Abeta was presented in a mixture also containing shorter substrates.

Conclusions/significance: This study describes the first examples of synthetic small-molecule activators of IDE, showing that pharmacological activation of this important protease with drug-like compounds is achievable. These novel activators help to establish the putative ATP-binding domain as a key modulator of IDE proteolytic activity and offer new insights into the modulatory action of ATP. Several larger lessons abstracted from this screen will help inform the design of future screening campaigns and facilitate the eventual development of IDE activators with therapeutic utility.

Show MeSH

Related in: MedlinePlus

Ia1, Ia2 and ATP interact with a common domain within IDE.A, Ia1 and Ia2 disrupt the crosslinking of recombinant IDE with a photoaffinity ATP analogue. DMSO concentrations were maintained constant in all conditions; CTL = DMSO only; ATP = 10 mM ATP. B, Activation of FRET1 hydrolysis by Ia1 can be competed away with increasing doses of Ia2. Data are mean±SEM for 5 independent experiments normalized to control wells containing DMSO only. All data are statistically different from controls (P<0.05).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2668070&req=5

pone-0005274-g003: Ia1, Ia2 and ATP interact with a common domain within IDE.A, Ia1 and Ia2 disrupt the crosslinking of recombinant IDE with a photoaffinity ATP analogue. DMSO concentrations were maintained constant in all conditions; CTL = DMSO only; ATP = 10 mM ATP. B, Activation of FRET1 hydrolysis by Ia1 can be competed away with increasing doses of Ia2. Data are mean±SEM for 5 independent experiments normalized to control wells containing DMSO only. All data are statistically different from controls (P<0.05).

Mentions: Although Ia1 and Ia2 and its variants show no obvious structural (Fig. S3) or chemical (Table S1) relationship to ATP or other nucleotide polyphosphates, their similar size and common ability to activate IDE suggested that they may exert their effects by interacting with a common domain within IDE. To investigate this hypothesis, we tested the ability of Ia1 and Ia2 to interfere with the binding of ATP, as determined from the labeling of recombinant IDE with a biotinylated photoaffinity ATP analogue, 8-azidoadenosine 5′-triphosphate 2′,3′-biotin-LC-hydrazone (azido-ATP-biotin). Under conditions that produced substantial photocrosslinking of IDE, Ia1 and Ia2 both interfered with azido-ATP-biotin labeling in a dose-dependent manner, an effect that was also produced by excess unlabeled ATP, as expected (Fig. 3A). To investigate the interaction of Ia1 and Ia2 with one another, we tested and confirmed the ability of Ia2 to interfere with the more pronounced activation effect produced by Ia1 (Fig. 3B). Together, these results support the idea that Ia1 and Ia2 both exert their effects through interaction with a common domain within IDE that also binds to ATP.


Small-molecule activators of insulin-degrading enzyme discovered through high-throughput compound screening.

Cabrol C, Huzarska MA, Dinolfo C, Rodriguez MC, Reinstatler L, Ni J, Yeh LA, Cuny GD, Stein RL, Selkoe DJ, Leissring MA - PLoS ONE (2009)

Ia1, Ia2 and ATP interact with a common domain within IDE.A, Ia1 and Ia2 disrupt the crosslinking of recombinant IDE with a photoaffinity ATP analogue. DMSO concentrations were maintained constant in all conditions; CTL = DMSO only; ATP = 10 mM ATP. B, Activation of FRET1 hydrolysis by Ia1 can be competed away with increasing doses of Ia2. Data are mean±SEM for 5 independent experiments normalized to control wells containing DMSO only. All data are statistically different from controls (P<0.05).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2668070&req=5

pone-0005274-g003: Ia1, Ia2 and ATP interact with a common domain within IDE.A, Ia1 and Ia2 disrupt the crosslinking of recombinant IDE with a photoaffinity ATP analogue. DMSO concentrations were maintained constant in all conditions; CTL = DMSO only; ATP = 10 mM ATP. B, Activation of FRET1 hydrolysis by Ia1 can be competed away with increasing doses of Ia2. Data are mean±SEM for 5 independent experiments normalized to control wells containing DMSO only. All data are statistically different from controls (P<0.05).
Mentions: Although Ia1 and Ia2 and its variants show no obvious structural (Fig. S3) or chemical (Table S1) relationship to ATP or other nucleotide polyphosphates, their similar size and common ability to activate IDE suggested that they may exert their effects by interacting with a common domain within IDE. To investigate this hypothesis, we tested the ability of Ia1 and Ia2 to interfere with the binding of ATP, as determined from the labeling of recombinant IDE with a biotinylated photoaffinity ATP analogue, 8-azidoadenosine 5′-triphosphate 2′,3′-biotin-LC-hydrazone (azido-ATP-biotin). Under conditions that produced substantial photocrosslinking of IDE, Ia1 and Ia2 both interfered with azido-ATP-biotin labeling in a dose-dependent manner, an effect that was also produced by excess unlabeled ATP, as expected (Fig. 3A). To investigate the interaction of Ia1 and Ia2 with one another, we tested and confirmed the ability of Ia2 to interfere with the more pronounced activation effect produced by Ia1 (Fig. 3B). Together, these results support the idea that Ia1 and Ia2 both exert their effects through interaction with a common domain within IDE that also binds to ATP.

Bottom Line: Both compounds were found to interfere with the crosslinking of a photoaffinity ATP analogue to IDE, suggesting that they interact with a bona fide ATP-binding domain within IDE.These novel activators help to establish the putative ATP-binding domain as a key modulator of IDE proteolytic activity and offer new insights into the modulatory action of ATP.Several larger lessons abstracted from this screen will help inform the design of future screening campaigns and facilitate the eventual development of IDE activators with therapeutic utility.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Mayo Clinic, Jacksonville, Florida, United States of America.

ABSTRACT

Background: Hypocatabolism of the amyloid beta-protein (Abeta) by insulin-degrading enzyme (IDE) is implicated in the pathogenesis of Alzheimer disease (AD), making pharmacological activation of IDE an attractive therapeutic strategy. However, it has not been established whether the proteolytic activity of IDE can be enhanced by drug-like compounds.

Methodology/principal findings: Based on the finding that ATP and other nucleotide polyphosphates modulate IDE activity at physiological concentrations, we conducted parallel high-throughput screening campaigns in the absence or presence of ATP and identified two compounds--designated Ia1 and Ia2--that significantly stimulate IDE proteolytic activity. Both compounds were found to interfere with the crosslinking of a photoaffinity ATP analogue to IDE, suggesting that they interact with a bona fide ATP-binding domain within IDE. Unexpectedly, we observed highly synergistic activation effects when the activity of Ia1 or Ia2 was tested in the presence of ATP, a finding that has implications for the mechanisms underlying ATP-mediated activation of IDE. Notably, Ia1 and Ia2 activated the degradation of Abeta by approximately 700% and approximately 400%, respectively, albeit only when Abeta was presented in a mixture also containing shorter substrates.

Conclusions/significance: This study describes the first examples of synthetic small-molecule activators of IDE, showing that pharmacological activation of this important protease with drug-like compounds is achievable. These novel activators help to establish the putative ATP-binding domain as a key modulator of IDE proteolytic activity and offer new insights into the modulatory action of ATP. Several larger lessons abstracted from this screen will help inform the design of future screening campaigns and facilitate the eventual development of IDE activators with therapeutic utility.

Show MeSH
Related in: MedlinePlus