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

Effects of Ia1 and Ia2 on Aβ degradation.A,B, Ia1 (A) and Ia2 (B) fail to activate the degradation of Aβ, in the presence or the absence of ATP, when Aβ is the sole substrate; Instead, partial inhibition is seen at higher concentrations. Data are mean±SEM for 4 independent experiment; *P<0.05. C,D, Ia1 and Ia2 strongly activate Aβ degradation when presented together with a short substrate (FRET1; 10 µM). C, Representative time-course of Aβ activation by Ia1 and Ia2. D, Results of 4 independent experiments showing results normalized to DMSO-only controls. Data are mean±SEM and are statistically different for all comparisons (P<0.01).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2668070&req=5

pone-0005274-g005: Effects of Ia1 and Ia2 on Aβ degradation.A,B, Ia1 (A) and Ia2 (B) fail to activate the degradation of Aβ, in the presence or the absence of ATP, when Aβ is the sole substrate; Instead, partial inhibition is seen at higher concentrations. Data are mean±SEM for 4 independent experiment; *P<0.05. C,D, Ia1 and Ia2 strongly activate Aβ degradation when presented together with a short substrate (FRET1; 10 µM). C, Representative time-course of Aβ activation by Ia1 and Ia2. D, Results of 4 independent experiments showing results normalized to DMSO-only controls. Data are mean±SEM and are statistically different for all comparisons (P<0.01).

Mentions: We then explored the effects of Ia1 and Ia2 on the degradation of Aβ by IDE. To the extent that the IDE-activating compounds mimic the actions of ATP, they were predicted to exert no effect or even to inhibit IDE-mediated Aβ degradation. Alternatively, however, they might prove capable of relieving the inhibitory effect of ATP on Aβ degradation (see Fig. 1A), thereby activating the protease. To explore these possibilities, we tested the effects of Ia1 and Ia2 on IDE-mediated Aβ degradation in the absence or presence of a partially inhibitory concentration of ATP (5 mM). With the exception of very high doses of Ia1 or Ia2, which elicited partial inhibition, we observed no significant effect of Ia1 and Ia2 on Aβ degradation in the presence or absence of ATP (Fig. 5A,B).


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)

Effects of Ia1 and Ia2 on Aβ degradation.A,B, Ia1 (A) and Ia2 (B) fail to activate the degradation of Aβ, in the presence or the absence of ATP, when Aβ is the sole substrate; Instead, partial inhibition is seen at higher concentrations. Data are mean±SEM for 4 independent experiment; *P<0.05. C,D, Ia1 and Ia2 strongly activate Aβ degradation when presented together with a short substrate (FRET1; 10 µM). C, Representative time-course of Aβ activation by Ia1 and Ia2. D, Results of 4 independent experiments showing results normalized to DMSO-only controls. Data are mean±SEM and are statistically different for all comparisons (P<0.01).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0005274-g005: Effects of Ia1 and Ia2 on Aβ degradation.A,B, Ia1 (A) and Ia2 (B) fail to activate the degradation of Aβ, in the presence or the absence of ATP, when Aβ is the sole substrate; Instead, partial inhibition is seen at higher concentrations. Data are mean±SEM for 4 independent experiment; *P<0.05. C,D, Ia1 and Ia2 strongly activate Aβ degradation when presented together with a short substrate (FRET1; 10 µM). C, Representative time-course of Aβ activation by Ia1 and Ia2. D, Results of 4 independent experiments showing results normalized to DMSO-only controls. Data are mean±SEM and are statistically different for all comparisons (P<0.01).
Mentions: We then explored the effects of Ia1 and Ia2 on the degradation of Aβ by IDE. To the extent that the IDE-activating compounds mimic the actions of ATP, they were predicted to exert no effect or even to inhibit IDE-mediated Aβ degradation. Alternatively, however, they might prove capable of relieving the inhibitory effect of ATP on Aβ degradation (see Fig. 1A), thereby activating the protease. To explore these possibilities, we tested the effects of Ia1 and Ia2 on IDE-mediated Aβ degradation in the absence or presence of a partially inhibitory concentration of ATP (5 mM). With the exception of very high doses of Ia1 or Ia2, which elicited partial inhibition, we observed no significant effect of Ia1 and Ia2 on Aβ degradation in the presence or absence of ATP (Fig. 5A,B).

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