Ryoji Kikumoto

Potent inhibition of thrombin by the newly synthesized arginine derivative No. 805. The importance of stereo-structure of its hydrophobic carboxamide portion

Abstract Four stereoisomers of 4-methyl-1-[N 2 -(3-methyl-1,2,3,4-tetrahydro-8-quinolinesulfonyl)-L-arginyl]-2-piperidinecarboxylic acid were synthesized and examined for the inhibitory effect on thrombin. The inhibitory potency varied largely with the stereo-configuration of the 4-methyl-2-piperidinecarboxylic acid portion. The (2R, 4R)-isomer was the most potent inhibitor with a Ki of 0.019 μM, while the (2R, 4S) and (2S, 4R)-isomers showed the values of Ki 0.24 and 1.9 μM, respectively. The least potent inhibitor, (2S, 4S)-isomer, showed a Ki of 280 μM which is approximately 15,000 times that of (2R, 4R)-isomer.

A new series of synthetic thrombin-inhibitors (OM-inhibitors) having extremely potent and selective action.

Abstract The studies in searching for potent and highly selective synthetic inhibitors to thrombin led the authors to find a new series of the thrombin-inhibitors which belong to N α -naphthalenesulfonyl-L-arginine derivatives; values of I 50 of these potent inhibitors are found in the range from 0.03 μM to 2 μM, when fibrinogen (3 μM) or N α -benzoyl-L-phenylalanyl-L-valyl-L-arginine p-nitroanilide (100 μM) is used as substrate. These potent thrombin-inhibitors are tentatively called OM-inhibitors according to their code name in our laboratories.

In vitro and in vivo studies of a new series of synthetic thrombin-inhibitors (OM-inhibitors)

Abstract To evaluate the thrombin-inhibitors (OM-inhibitors) synthetized by the authors, their actions in vitro and in vivo have been investigated. Results obtained are summarized as follows: 1. (i) The inhibitory action of the OM-inhibitor (OM-205) is highly selective to thrombin when compared with trypsin, plasmin or reptilase. 2. (ii) The mode of inhibition of the OM-inhibitor (OM-46) to thrombin is competitive in the hydrolysis of Nα-benzoyl-DL-arginine p-nitroanilide. 3. (iii) The decreasing effects of thrombin infusion in rabbits on plasma fibrinogen content and platelets in number are remarkably blocked by the presence of the OM-inhibitor (OM-189) in the blood, which suggests that the OM-inhibitors, very effective in vitro, possess the anti-thrombin activity also in vivo.

SIMILARITY AND DISSIMILARITY IN THE STEREOGEOMETRY OF THE ACTIVE SITES OF THROMBIN, TRYPSIN, PLASMIN AND GLANDULAR KALLIKREIN

The relationship between chemical modifications of arginine derivatives and inhibitory activity to trypsin, plasmin and glandular kallikrein was investigated comparing with that of thrombin and concluded as follows: The hydrophobic binding pocket, which has been reported previously to be stereogeometrically very similar in trypsin and thrombin, corresponded to the length of ethylpiperidine. Concerning the site (termed the P site) next to the hydrophobic binding pocket, there were large differences in stereogeometry between trypsin and thrombin; the binding site of trypsin extended further to allow propyl and phenyl group attached to piperidine, while that of thrombin would be much narrower and unable to allow them. The P sites of plasmin and glandular kallikrein resembled that of trypsin in being able to allow phenyl group. To substantialize the hydrophobic binding pocket and the P site, a (2R, 4R)-MQPA-trypsin complex model was generated using the results of X-ray crystallography of (2R, 4R)-MQPA and BPTI-trypsin complex by calculation to minimize van der Waals contacts, and it was of great use for understanding the geometry of the active sites of trypsin, thrombin, plasmin and glandular kallikrein.

Mechanisms of the selective inhibition of thrombin, factor xa, plasmin and trypsin

Abstract The three-dimensional structures of bovine trypsin-inhibitor complexes were determined by X-ray analysis. The selective inhibition of thrombin, factor Xa, plasmin and trypsin exhibited by arginine and lysine derivatives was clearly explained based on the structures and the homology in the amino acid sequences of these enzymes. The differences in the sequences at the positions corresponding to I1e-63, Leu-99 and Ser-190 of trypsin were shown to give each enzyme different binding affinity toward the inhibitors and result in the selective inhibition. This study provides design strategies for enzyme specific inhibitors and suggestions for site-directed mutagenesis experiments.