Crystal structures of factor Xa specific inhibitors in complex with trypsin: structural grounds for inhibition of factor Xa and selectivity against thrombin

Abstract

Crystal structures of DX9065a and a related bisamidino-aryl inhibitor specific for the blood-clotting factor Xa have been solved in complex with bovine ~-trypsin to a resolution of 1.9 A. Each inhibitor exhibits an extended conformation along the active site, in contrast to the compact folded structures observed for thrombin specific inhibiturs. Few direct contacts (predominantly in the S1 pocke0 are made between trypsin and the inhibitors. Transfer of the inhibitors to the active site of factor Xa suggests a three-site interaction: salt bridge formation at the base of the primary specificity pocket, extensive hydrophobie surface burial and a weak electrostatic interaction between the distal basic component of the inhibitor and an electronegative cavity of factor Xa formed by three backbone carbonyl oxygens. Additivity of these three interactions is the basis for the observed strong inhibition of factor Xa and provides a framework for the design of novel factor Xa inhibitors. A propionic acid group of the inhibitor would clash with the thrombin specific '60-insertion loop', thus conferring selectivity against thrombin.

X-ray Structure of Active Site-inhibited Clotting Factor Xa

“…Additionally, the binding energy of the aryl-binding site interactions may contribute due to overall stiffness of DX-9065a. In any case, the movement is apparently characteristic of naphthamidine binding in trypsin-like serine proteinases since a corresponding structure was also observed in naphthamidine binding in thrombin (31) and trypsin (15). This supports the hypothesis that the energy required derives from the naphthamidine interaction itself.…”

“…We present here the crystal structure of the factor Xa⅐DX-9065a complex. The inhibitor binds in the active site in an extended conformation, which was expected from earlier studies (14,15). Both hydrophobic and electrostatic interactions characterize the complex formation, which is also accompanied by local rearrangements in the active site of fXa.…”

“…The binding in the S1 site, as expected, involves formation of a salt bridge between the naphthamidine amidino group and Asp-189. The orientation of the amidino group would allow a twin-twin arrangement (30) if Asp-189 were fixed as in the arginine-bound fXa structure (23); twin-twin geometry has been observed for naphthamidine binding in thrombin (31) and trypsin (15). Surprisingly, however, the aspartic acid side chain apparently moves, mainly by a 2 rotation of almost 90°, away from its original position, resulting in a single Asp O ␦1 -twin (naphthamidine) arrangement (Fig.…”

Catalytic Domain Structures of MT-SP1/Matriptase, a Matrix-degrading Transmembrane Serine Proteinase

“…This loop contains two of the "zymogen triad" residues, Ser 32 and His 40 , which, together with Asp 194 , would stabilize the inactive zymogen-like conformation of the proenzyme (28,29). Around Gln 38 , this loop deviates markedly from the path followed in most other chymotrypsin-like proteinases (Fig.…”

“…In addition, they can favorably interact with the overall negative potential created by the 96-, 97-, and 98-carbonyls, in full agreement with the preference of MT-SP1 for basic P4 residues. This MT-SP1 S4 subsite is reminiscent of the corresponding region of coagulation factor Xa, which has also been shown to function as a cation binding site (32). The Arg 39 (I) side chain of BPTI (see Fig.…”