Discovery of thrombin inhibitor fragments from chemical microarray screening

Letters in Drug Design and Discovery

Volumn 2, Issue 8, 2005, Pages 590-594

  Neumann, Thomas ^a   Junker, Hans Dieter ^a   Keil, Oliver ^a   Burkert, Klaus ^a   Ottleben, Holger ^a   Gamer, Jürgen ^a   Sekul, Renate ^b   Deppe, Holger ^c   Feurer, Achim ^c   Tomandl, Dirk ^d   Metz, Günther ^d  

^a Graffinity Pharmaceuticals AG   (Germany)

^b Biochemistry   (Germany)

^c Santhera Pharmaceuticals   (Germany)

^d NONE   (Germany)

Author keywords

Chemical microarrays; Fragment discovery; Non basic S1 binder; Surface plasmon resonance; Thrombin

Indexed keywords

THROMBIN INHIBITOR;

ARTICLE; CRYSTALLOGRAPHY; DRUG SCREENING; ENZYME INHIBITION; MICROARRAY ANALYSIS; PRIORITY JOURNAL; SURFACE PLASMON RESONANCE;

EID: 29044442133     PISSN: 15701808     EISSN: None     Source Type: Journal    
DOI: 10.2174/157018005774717343     Document Type: Article

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34. Pefabloc, T.H. was obtained from LOXO Ltd., 69221 Dossenheim, Germany. The Ki towards thrombin is 4nM and the inhibitor was used at a concentration of 1 μM in the competition experiment.
35. The Ki determinations were carried out at 20 °C with the fluorogenic substrate Tosyl-GPR-AMC (Bachern, Heidelberg, Germany; λexc = 370 nm, λem = 450 nm) at a thrombin concentration of 100 pM in HBS (10 mM Hepes, 150 mM NaCl, 0.005 % Tween20, pH 7.4). The substrate was added to a final concentration of 20 μM in a total assay volume of 100 μl The emission at 450 nm was monitored in 1 minute intervals for 10 minutes using a polarstar reader (BMG Laboratories, Offenburg, Germany). Initial velocities of the control and the inhibited reactions (vo and vi) were estimated in FU/min at different compound concentrations. The inhibition constants were calculated using the Michaelis-Menten equation for competitive inhibition.
36. Human alpha-thrombin has been crystallized using a hanging drop method. Co-crystals have been obtain by a backsoaking procedure in presence of the small molecule ligands. The resolution of the final structures were 1.40 Å and 1.25 Å for the complexes with 7 and 8, respectively. The structures are deposited in the PDB database (remark : PDB code will be provied ASAP).
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