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Characterization of the Annonaceous acetogenin, annonacinone, a natural product inhibitor of plasminogen activator inhibitor-1

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ABSTRACT

Plasminogen activator inhibitor-1 (PAI-1) is the main inhibitor of the tissue type and urokinase type plasminogen activators. High levels of PAI-1 are correlated with an increased risk of thrombotic events and several other pathologies. Despite several compounds with in vitro activity being developed, none of them are currently in clinical use. In this study, we evaluated a novel PAI-1 inhibitor, annonacinone, a natural product from the Annonaceous acetogenins group. Annonacinone was identified in a chromogenic screening assay and was more potent than tiplaxtinin. Annonacinone showed high potency ex vivo on thromboelastography and was able to potentiate the thrombolytic effect of tPA in vivo in a murine model. SDS-PAGE showed that annonacinone inhibited formation of PAI-1/tPA complex via enhancement of the substrate pathway. Mutagenesis and molecular dynamics allowed us to identify annonacinone binding site close to helix D and E and β-sheets 2A.

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Elucidation of Annonacinone mechanism of action by SDS-PAGE.Influence of annonacinone on the PAI-1/tPA interaction on SDS-PAGE. PAI-1 (4 μL, 1 μM) was pre-incubated with 16 μL containing indicated concentrations of annonacinone (2% DMSO) for 30 min at 37 °C. Human tPA (4 μL, 0.5 μM) was then added to the mixture and incubated for 15 min at 37 °C. Reaction was stopped by addition of 4X SDS-PAGE loading buffer followed by immediate boiling. Samples were then analyzed on SDS-PAGE followed by silver staining. The margins indicate (left) molecular weights (×103 Da) of the markers (M), (right) the migration positions of relevant species, and (bottom) the experimental conditions.
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f3: Elucidation of Annonacinone mechanism of action by SDS-PAGE.Influence of annonacinone on the PAI-1/tPA interaction on SDS-PAGE. PAI-1 (4 μL, 1 μM) was pre-incubated with 16 μL containing indicated concentrations of annonacinone (2% DMSO) for 30 min at 37 °C. Human tPA (4 μL, 0.5 μM) was then added to the mixture and incubated for 15 min at 37 °C. Reaction was stopped by addition of 4X SDS-PAGE loading buffer followed by immediate boiling. Samples were then analyzed on SDS-PAGE followed by silver staining. The margins indicate (left) molecular weights (×103 Da) of the markers (M), (right) the migration positions of relevant species, and (bottom) the experimental conditions.

Mentions: The influence of annonacinone on PAI-1/tPA covalent complex formation was also assessed on SDS-PAGE. A concentration-dependent inhibition of PAI-1/tPA complex formation was observed in the presence of annonacinone (Fig. 3). The increase of cleaved PAI-1 and free tPA levels indicates that annonacinone inhibits PAI-1 dependant tPA inhibition probably by interfering with RCL insertion into β-sheet A and thus favouring the reaction between PAI-1 and tPA to proceed through the substrate pathway. Annonacinone also induced a dose-dependent enhancement of the intensity of free PAI-1 that may also result from allosteric modification of PAI-1 impairing its reaction with tPA.


Characterization of the Annonaceous acetogenin, annonacinone, a natural product inhibitor of plasminogen activator inhibitor-1
Elucidation of Annonacinone mechanism of action by SDS-PAGE.Influence of annonacinone on the PAI-1/tPA interaction on SDS-PAGE. PAI-1 (4 μL, 1 μM) was pre-incubated with 16 μL containing indicated concentrations of annonacinone (2% DMSO) for 30 min at 37 °C. Human tPA (4 μL, 0.5 μM) was then added to the mixture and incubated for 15 min at 37 °C. Reaction was stopped by addition of 4X SDS-PAGE loading buffer followed by immediate boiling. Samples were then analyzed on SDS-PAGE followed by silver staining. The margins indicate (left) molecular weights (×103 Da) of the markers (M), (right) the migration positions of relevant species, and (bottom) the experimental conditions.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5120274&req=5

f3: Elucidation of Annonacinone mechanism of action by SDS-PAGE.Influence of annonacinone on the PAI-1/tPA interaction on SDS-PAGE. PAI-1 (4 μL, 1 μM) was pre-incubated with 16 μL containing indicated concentrations of annonacinone (2% DMSO) for 30 min at 37 °C. Human tPA (4 μL, 0.5 μM) was then added to the mixture and incubated for 15 min at 37 °C. Reaction was stopped by addition of 4X SDS-PAGE loading buffer followed by immediate boiling. Samples were then analyzed on SDS-PAGE followed by silver staining. The margins indicate (left) molecular weights (×103 Da) of the markers (M), (right) the migration positions of relevant species, and (bottom) the experimental conditions.
Mentions: The influence of annonacinone on PAI-1/tPA covalent complex formation was also assessed on SDS-PAGE. A concentration-dependent inhibition of PAI-1/tPA complex formation was observed in the presence of annonacinone (Fig. 3). The increase of cleaved PAI-1 and free tPA levels indicates that annonacinone inhibits PAI-1 dependant tPA inhibition probably by interfering with RCL insertion into β-sheet A and thus favouring the reaction between PAI-1 and tPA to proceed through the substrate pathway. Annonacinone also induced a dose-dependent enhancement of the intensity of free PAI-1 that may also result from allosteric modification of PAI-1 impairing its reaction with tPA.

View Article: PubMed Central - PubMed

ABSTRACT

Plasminogen activator inhibitor-1 (PAI-1) is the main inhibitor of the tissue type and urokinase type plasminogen activators. High levels of PAI-1 are correlated with an increased risk of thrombotic events and several other pathologies. Despite several compounds with in vitro activity being developed, none of them are currently in clinical use. In this study, we evaluated a novel PAI-1 inhibitor, annonacinone, a natural product from the Annonaceous acetogenins group. Annonacinone was identified in a chromogenic screening assay and was more potent than tiplaxtinin. Annonacinone showed high potency ex vivo on thromboelastography and was able to potentiate the thrombolytic effect of tPA in vivo in a murine model. SDS-PAGE showed that annonacinone inhibited formation of PAI-1/tPA complex via enhancement of the substrate pathway. Mutagenesis and molecular dynamics allowed us to identify annonacinone binding site close to helix D and E and β-sheets 2A.

No MeSH data available.


Related in: MedlinePlus