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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.

No MeSH data available.


General structure of anabaenopeptins, and structure of residues HPhe, HTyr, PNV, PNL and Cl-HTyr.R1–R6 indicate side chains of amino acids. R2 is a Lys residue in all anabaenopeptins.
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f3: General structure of anabaenopeptins, and structure of residues HPhe, HTyr, PNV, PNL and Cl-HTyr.R1–R6 indicate side chains of amino acids. R2 is a Lys residue in all anabaenopeptins.

Mentions: The structures of the anabaenopeptins were elucidated by the analyses of 2D-NMR experiments that comprised DQF-COSY, TOCSY, ROESY, HSQC, and HMBC spectra. The correlations that have been used for the identification of single amino acids and for the sequence assignment of analogue 4 are depicted in Fig. 2 as an example. The general structure of all analogues is depicted in Fig. 3, while the corresponding amino acid residues are decoded in Table 1. A complete assignment of proton and carbon chemical shifts is given in the Supporting Information.


Isolation, Co-Crystallization and Structure-Based Characterization of Anabaenopeptins as Highly Potent Inhibitors of Activated Thrombin Activatable Fibrinolysis Inhibitor (TAFIa)
General structure of anabaenopeptins, and structure of residues HPhe, HTyr, PNV, PNL and Cl-HTyr.R1–R6 indicate side chains of amino acids. R2 is a Lys residue in all anabaenopeptins.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: General structure of anabaenopeptins, and structure of residues HPhe, HTyr, PNV, PNL and Cl-HTyr.R1–R6 indicate side chains of amino acids. R2 is a Lys residue in all anabaenopeptins.
Mentions: The structures of the anabaenopeptins were elucidated by the analyses of 2D-NMR experiments that comprised DQF-COSY, TOCSY, ROESY, HSQC, and HMBC spectra. The correlations that have been used for the identification of single amino acids and for the sequence assignment of analogue 4 are depicted in Fig. 2 as an example. The general structure of all analogues is depicted in Fig. 3, while the corresponding amino acid residues are decoded in Table 1. A complete assignment of proton and carbon chemical shifts is given in the Supporting Information.

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.