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

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.


Two views, 90° apart, of the overall binding mode of 2 in carboxypeptidase B.Shown is 2 as a CPK model and carboxypeptidase B as a solvent accessible surface. Also indicated is the catalytic zinc. The anabaenopeptin C molecule fills the active site and access to the active site like a plug.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5015106&req=5

f5: Two views, 90° apart, of the overall binding mode of 2 in carboxypeptidase B.Shown is 2 as a CPK model and carboxypeptidase B as a solvent accessible surface. Also indicated is the catalytic zinc. The anabaenopeptin C molecule fills the active site and access to the active site like a plug.

Mentions: The general binding modes of 1–3 were found to be very similar and in the following discussion the complex with 2 is used as an example. The linear part of the anabaenopeptins, which mimics the carboxy-terminus of the TAFIa substrate fibrin, deeply penetrated the active site channel and bound in the carboxylate- and specificity-pockets, while the circular parts of the anabaenopeptins were positioned in the entrance of the channel and completely blocked the channel like a plug, thereby preventing other molecules from entering it (Fig. 5). Calculations with PISA33 revealed that the total surface area of 2 was 1036 Å3, while the area of the 2–CBP interface was 519 Å3. Thus, about 50% of the surface of 2 got buried upon binding, contributing to the high affinity.


Isolation, Co-Crystallization and Structure-Based Characterization of Anabaenopeptins as Highly Potent Inhibitors of Activated Thrombin Activatable Fibrinolysis Inhibitor (TAFIa)
Two views, 90° apart, of the overall binding mode of 2 in carboxypeptidase B.Shown is 2 as a CPK model and carboxypeptidase B as a solvent accessible surface. Also indicated is the catalytic zinc. The anabaenopeptin C molecule fills the active site and access to the active site like a plug.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Two views, 90° apart, of the overall binding mode of 2 in carboxypeptidase B.Shown is 2 as a CPK model and carboxypeptidase B as a solvent accessible surface. Also indicated is the catalytic zinc. The anabaenopeptin C molecule fills the active site and access to the active site like a plug.
Mentions: The general binding modes of 1–3 were found to be very similar and in the following discussion the complex with 2 is used as an example. The linear part of the anabaenopeptins, which mimics the carboxy-terminus of the TAFIa substrate fibrin, deeply penetrated the active site channel and bound in the carboxylate- and specificity-pockets, while the circular parts of the anabaenopeptins were positioned in the entrance of the channel and completely blocked the channel like a plug, thereby preventing other molecules from entering it (Fig. 5). Calculations with PISA33 revealed that the total surface area of 2 was 1036 Å3, while the area of the 2–CBP interface was 519 Å3. Thus, about 50% of the surface of 2 got buried upon binding, contributing to the high affinity.

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.