Development of plasma kallikrein selective inhibitors

During the course of the development of active center-directed plasmin inhi bitors, it was found that N-(trans-4-aminomethylcyclohexanecarbonyl)-lysine -4-methoxycarbonylanilide inhibited plasma kallikrein more potently than ot her enzymes such as plasmin, urokinase, and thrombin although the inhibitor y activity was not as potent and enzyme selectivity not as high. Based on s tudies of structure-activity relationship we designed and synthesized the p lasma kallikrein selective inhibitor, N-(trans-4-aminomethylcyclohexanecarb onyl)-phenylalanine-4-carboxymethyl-anilide (Tra-Phe-APAA). Tra-Phe-APAA in hibited plasma kallikrein with a K-i value of 0.81 mu M, while it inhibited glandular kallikrein, plasmin, urokinase, tissue plasminogen activator fac tor Xa, factor XIIa, and thrombin with K-i values of > 500, 390 200 > 500 > 500 > 500, and > 500 mu M, respectively. We designated Tra-Phe-APAA as PKS I-527. Using PKSI-527 as an affinity ligand, we synthesized a new affinity gel (PKSI-Toyopearl) and employed it for the rapid purification of plasma k allikrein from human plasma. Human plasma activated with kaolin after acid treatment was applied To a PKSI-527-Toyopearl column. Adsorbed protein was eluted with 50 mM glycine-hydrochloric acid buffer (pH 3.0). Plasma kallikr ein was purified 181-fold with a yield of 85% from the kaolin-activated pla sma. (C) 1999 John Wiley & Sons, Inc.