MOLECULAR DESIGN AND CHARACTERIZATION OF AN ALPHA-THROMBIN INHIBITOR CONTAINING A NOVEL PI MOIETY

An inhibitor of cx-thrombin was designed on the basis of the X-ray cry stal structures of thrombin and trypsin. The design strategy employed the geometric and electrostatic differences between the specificity po ckets of the two enzymes. These differences arise due to the replaceme nt of Ser 190 in trypsin by Ala 190 in thrombin. The new inhibitor con tained a tryptophan side chain instead of the arginine side chain that is present in the prototypical thrombin inhibitors. This inhibitor ha d a K-1 value of 0.25 mu M, displayed more than 400-fold specificity f or thrombin over trypsin, and doubled the rat plasma APTT at a concent ration of 44.9 mu M. The X-ray crystal structure of the inhibitor/alph a-thrombin complex was determined. This represents the first reported three-dimensional structure of a thrombin/inhibitor complex where the specificity pocket of the enzyme is occupied by a chemical moiety othe r than a guanidino or an amidino group. As was predicted by the molecu lar model, the tryptophan side chain docks into the specificity pocket of the enzyme. This finding is in contrast with the indole binding re gion of thrombin reported earlier [Berliner, L. J., & Shen, Y. Y. L. ( 1977) Biochemistry 16, 4622-4626] The lower binding affinity of the ne w inhibitor for trypsin, compared to that for thrombin, appears to be due to (i) the extra energy required to deform the smaller specificity pocket of trypsin to accommodate the bulky indole group and (ii) the favorable electrostatic interactions of the indole group with the more hydrophobic specificity pocket of thrombin. The neutral indole group may be of pharmacological significance because the severe hypotension and respiratory distress observed following the administration of some thrombin inhibitors have been linked to the positively charged guanid ino or amidino functionalities.