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Isolation, Co-Crystallization and Structure-Based Characterization of Anabaenopeptins as Highly Potent Inhibitors of Activated Thrombin Activatable Fibrinolysis Inhibitor (TAFIa)

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ABSTRACT

Mature thrombin activatable fibrinolysis inhibitor (TAFIa) is a carboxypeptidase that stabilizes fibrin clots by removing C-terminal arginines and lysines from partially degraded fibrin. Inhibition of TAFIa stimulates the degradation of fibrin clots and may help to prevent thrombosis. Applying a lead finding approach based on literature-mining, we discovered that anabaenopeptins, cyclic peptides produced by cyanobacteria, were potent inhibitors of TAFIa with IC50 values as low as 1.5 nM. We describe the isolation and structure elucidation of 20 anabaenopeptins, including 13 novel congeners, as well as their pronounced structure-activity relationships (SAR) with respect to inhibition of TAFIa. Crystal structures of the anabaenopeptins B, C and F bound to the surrogate protease carboxypeptidase B revealed the binding modes of these large (~850 Da) compounds in detail and explained the observed SAR, i.e. the strong dependence of the potency on a basic (Arg, Lys) exocyclic residue that addressed the S1’ binding pocket, and a broad tolerance towards substitutions in the pentacyclic ring that acted as a plug of the active site.

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Structural formulae of the anabaenopeptins B, C, and F.
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f1: Structural formulae of the anabaenopeptins B, C, and F.

Mentions: To test the hypothesis that stimulation of fibrinolysis via TAFIa inhibition is associated with a lower risk of bleeding compared to established anticoagulants, we aimed at discovering small molecule inhibitors of TAFIa as novel antithrombotic agents. Purified natural products with elucidated structures were included in this search due to their proven track record as a source of leads and drugs78. A literature mining approach alerted us of the activity of anabaenopeptins against carboxypeptidase A9101112, an enzyme that is closely related to TAFIa. Anabaenopeptins are bioactive peptides, produced by cyanobacteria e.g. during algal blooms11. They are cyclic hexapeptides produced by non-ribosomal peptide synthetases (Fig. 1)1314151617. Their chemical scaffold, first described in 19951819, is characterized by a conserved D-lysine residue at position 2 that spans a pentacycle through an isopeptide bond; the pseudo C-terminal residue is linked to the ε-amino function of lysine-2 via an ureido bond. Following the central concept of chemical genetics that similar receptors bind similar ligands20, the activity of anabaenopeptins against carboxypeptidase A motivated their test against TAFIa. As reported in a recent paper, a surprisingly potent, single digit nanomolar inhibition of TAFIa by anabaenopeptins was observed, inspiring the structure-based design and synthesis of truncated small molecule analogs21.


Isolation, Co-Crystallization and Structure-Based Characterization of Anabaenopeptins as Highly Potent Inhibitors of Activated Thrombin Activatable Fibrinolysis Inhibitor (TAFIa)
Structural formulae of the anabaenopeptins B, C, and F.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5015106&req=5

f1: Structural formulae of the anabaenopeptins B, C, and F.
Mentions: To test the hypothesis that stimulation of fibrinolysis via TAFIa inhibition is associated with a lower risk of bleeding compared to established anticoagulants, we aimed at discovering small molecule inhibitors of TAFIa as novel antithrombotic agents. Purified natural products with elucidated structures were included in this search due to their proven track record as a source of leads and drugs78. A literature mining approach alerted us of the activity of anabaenopeptins against carboxypeptidase A9101112, an enzyme that is closely related to TAFIa. Anabaenopeptins are bioactive peptides, produced by cyanobacteria e.g. during algal blooms11. They are cyclic hexapeptides produced by non-ribosomal peptide synthetases (Fig. 1)1314151617. Their chemical scaffold, first described in 19951819, is characterized by a conserved D-lysine residue at position 2 that spans a pentacycle through an isopeptide bond; the pseudo C-terminal residue is linked to the ε-amino function of lysine-2 via an ureido bond. Following the central concept of chemical genetics that similar receptors bind similar ligands20, the activity of anabaenopeptins against carboxypeptidase A motivated their test against TAFIa. As reported in a recent paper, a surprisingly potent, single digit nanomolar inhibition of TAFIa by anabaenopeptins was observed, inspiring the structure-based design and synthesis of truncated small molecule analogs21.

View Article: PubMed Central - PubMed

ABSTRACT

Mature thrombin activatable fibrinolysis inhibitor (TAFIa) is a carboxypeptidase that stabilizes fibrin clots by removing C-terminal arginines and lysines from partially degraded fibrin. Inhibition of TAFIa stimulates the degradation of fibrin clots and may help to prevent thrombosis. Applying a lead finding approach based on literature-mining, we discovered that anabaenopeptins, cyclic peptides produced by cyanobacteria, were potent inhibitors of TAFIa with IC50 values as low as 1.5 nM. We describe the isolation and structure elucidation of 20 anabaenopeptins, including 13 novel congeners, as well as their pronounced structure-activity relationships (SAR) with respect to inhibition of TAFIa. Crystal structures of the anabaenopeptins B, C and F bound to the surrogate protease carboxypeptidase B revealed the binding modes of these large (~850 Da) compounds in detail and explained the observed SAR, i.e. the strong dependence of the potency on a basic (Arg, Lys) exocyclic residue that addressed the S1’ binding pocket, and a broad tolerance towards substitutions in the pentacyclic ring that acted as a plug of the active site.

No MeSH data available.