Kallikrein inhibitors1 HANS FRITZ, EDWIN FINK AND ERNST TRUSCHEIT* Abteilung für Klinische Chemie und Klinische Biochemie in der Chirurgischen Klinik der Universität München, D-8000 München 2, Germany and *Bayer AG, Pharma Forschungszentrum, 5600 Wuppertal 1, Germany SPECIFIC AND NONSPECIFIC ABSTRACT KININOGENASES So far the CÏ inactivator, a2-macroglobulin, antithrombin III (in the presence of heparin), A common feature of trypsin and and a,-antitrypsin have been identified as inhibitors of plasma kallikrein; a,-antitrypsin trypsin-like enzymes with broader reacts slowly also with tissue kallikreins. Of the various naturally occurring kallikrein substrate specificity, e.g., plasmin and inhibitors the basic trypsin-kallikrein inhibitor of bovine organs, aprotinin (the active sperm acrosin, is the cleavage of substance of Trasylol®), has attained by far the most interest. This inhibitor, which is arginyl- and lysyl-peptide bonds; produced by mast cells, has unusual properties due to its compact tertiary structure. that is, as nonspecific kininogenases Additional topics of aprotinin and structurally related inhibitors discussed are the mecha• they normally also liberate bradykinin nism of enzyme-inhibitor complex formation, the production of chemical mutants of aprotinin, the structural basis of kallikrein inhibition, and selected aspects regarding apro• from the kininogens, the natural tinin medication.—Fritz, H., E. Fink and E. Truscheit. Kallikrein inhibitors. Federation substrates of the specific kinino• Proc. 38: 2753-2759, 1979. genases or kallikreins. The kalli• kreins are characterized, on the other hand, by their limited substrate ously in therapeutical dosage (5, 31). endothelial system (2, 37). Irreversi• specificity: Tissue kallikreins (Mr ~ 25,000-35,000) from submandib• Inhibition of plasma kallikrein by ble inhibition of proteinases by the ular glands, pancreas, urine (kidney), ^-antitrypsin proceeds too slowly to other plasmatic inhibitors occurs by colon, etc. react preferably with low be observed under in vivo condi• formation of a covalent bond between molecular weight kininogen yielding tions (14). the serine residue of the active site of kallidin (13), whereas plasma kal• So far, of all plasmatic inhibitors the proteinase and an amino acid likrein has two preferential biological only ^-antitrypsin has been un• residue near the C-terminal region of substrates, high molecular kininogen equivocally identified as an inhibitor the inhibitor after cleavage of a pro- and the Hageman factor (36). of tissue kallikreins; it is a progressive, teinase-sensitive bond of the in• slowly reacting inhibitor (14). In hibitor (6, 21, 38). PLASMATIC INHIBITORS addition, the presence of a fast react• ing inhibitor of tissue kallikreins in OF KALLIKREIN THE KALLIKREIN INHIBITOR human plasma has also been reported Both plasma kallikrein and high (13). Tissue kallikreins, to our present FROM BOVINE ORGANS molecular weight kininogen are in• knowledge, act primarily as kinin- Inhibitors of plasma and/or tissue volved in the solid phase activation generating enzymes. kallikreins are produced by various of the intrinsic blood clotting cascade Inhibition by the plasmatic in• plants like potatoes and peanuts (15), (36). The significance of simultaneous hibitors is under in vivo conditions by animal tissues like kidneys (20), kinin liberation is not yet fully under• irreversible. In the case of a2-macro- and by bacteria such as the microbial stood. The relationship of plasma globulin the proteinases are en• peptides antipain and leupeptin (15). kallikrein to the clotting factors is trapped after cleavage of a suitable The inhibitor, however, that has outlined also by its inhibitory speci• peptide bond of the inhibitor (41); in attained by far the most interest of ficity (see Table 1): Plasma kallikrein this cage the enzyme is still accessible biochemists, pharmacologists, and exhibits highest affinity to the in• to lower molecular weight substrates activator of the CI esterases, but and inhibitors but normally not to the inhibition by a2-macroglobulin also natural higher molecular weight sub• 1 occurs under in vivo conditions ( 1,22, strates (41). The plasma kallikrein- From the American Society for Pharma• cology and Experimental Therapeutics Sym• 36, 41). Antithrombin III reacts 02-macroglobulin complex is one of the posium Enzyme Inhibitors of the Kallikrein and relatively slowly with plasma kal• few exceptions; it still reacts with Renin Systems presented at the 63rd Annual likrein; complex formation is ob• kininogens (36). In the form of the Meeting of the Federation of American served only when the concentration Societies for Experimental Biology, Dallas, a2-niacroglobulin complex the pro• of the CI inactivator is low and Texas, April 4, 1979. teinase is rapidly eliminated from the 2 Dedicated to Prof. Dr. E. Auhagen on the heparin is administered simultane• circulation by cells of the reticulo• occasion of his 75th birthday. 0014-9446/79/0038-2753/$01.25. © FASEB 2753 TABLE 1. Inhibitors of plasma kallikrein of physiological or medical relevance is the main reason for the unusual (preferentially inhibited enzymes in italics) stability of aprotinin against dena- turation by high temperature, acid, Inhibits plasma kallikrein and: (36) In human plasma alkali, organic solvents, or proteolytic CÎ inactivator Cls, CIr, plasmin degradation (30). The high dipole character of the aprotinin molecule cr -Microglobulin Neutral and acidic proteases from leukocytes and lysosomes," pancre• 2 is due to concentration of negatively atic proteases,0 bacterial and mold proteases; plasmin charged residues near the bottom of Antithrombin III -I- heparin Thrombin, factor Xa\ IXa, XIa, XIla, XIIafrag, Vila; CIs; plasmin the pear. r 1 orAntitrypsin Neutral proteases from leukocytes and lysosomes, pancreatic proteases,* tissue kallikreins, plasmin Mechanism of complex formation Aprotinin from bovine lung Tissue kallikreins, plasmin, trypsin, chymotrypsin The region of the inhibitor molecule that is in close contact with the enzyme " K las ta se. cathepsin G, collagenase, cathespin B and D, e.g. 6 Chymotrypsin, trypsin. ' Slowly reacting with plasma and tissue kallikreins or plasmin, therefore probably without physiological relevance in the trypsin-inhibitor complex is regarding the in vivo inhibition of these proteases. indicated by the hatched area at the physicians is the kallikrein inhibitor Figure 1. Tertiary structure of the aprotinin molecule as revealed by X-ray crystallographic from bovine organs, aprotinin or studies (26). The polypeptide chain, consisting of 58 amino acid residues cross-linked by three Trasylol®, see Table 1. It was dis• disulfide bridges, is folded in such a way that a pear-shaped molecule results. The reactive peptide 15 16 covered 50 years ago by Frey, Kraut bond Lys -Ala is localized at the top of the pear. The region of the inhibitor molecule that is in and Werle as "kallikrein inactivator" close contact with the enzyme in the trypsin-inhibitor complex is indicated by the hatched area. in bovine lymph nodes and, 6 years later, independently by Kunitz and Northrop as trypsin inhibitor in bovine pancreas (13, 48, 49). Properties and structure The following properties of aprotinin are especially striking. 1 ) It forms equimolar and reversible complexes with trypsins, chymotrypsins, plas- mins, and plasma as well as glandular kallikreins of various species (13, 48, 49). 2) Its high stability is due to the low molecular weight of 6,500 daltons and an unusually rigid tertiary struc• ture (7). 3 ) Its high basicity (the iso• electric point is close to 10.5) is re• sponsible for the marked affinity of this protein to negatively charged groups, causing adsorption of the inhibitor to mucoprotein layers (23) and fixation to the brush border membrane of the kidney after thera• peutical administration (29). 4) It exhibits low toxicity and antigenicity (13,48,49). The structure of the aprotinin molecule is shown in Fig. 1 (7, 49): 58 amino acid residues are aligned to a single polypeptide chain, which is cross-linked by three disulfide bridges. The polypeptide chain is folded in such a way that a pear-shaped mole• cule results with the hydrophobic residues concentrated in the interior of the pear and the hydrophilic residues exposed to the surrounding medium. This arrangement leads to a very compact tertiary structure, which 2754 FEDERATION PROCEEDINGS VOL. 38, NO. 13 DECEMBER 1979 TABLE 2. Dissociation constants (K) top of the pear (26): 12 residues of reactive site residue of the inhibitor t aprotinin, including its reactive site (Lys in Fig. 1 and 2) into the speci• of complexes between aprotinin and peptide bond Lysl5-Ala16, are in• ficity pocket of the enzyme are various partners volved. All these contacts (11-13 absolutely necessary for complex Kn/.ymc/ hydrogen bonds, a salt bridge, more formation. In fact, the enzymatically partner Species Ki (mol/liter) pH than 200 Van der Waals interactions) inactive anhydro-trypsin forms also a Trypsin Bovine 6.0 X IO"14 8 contribute to the free energy of 19 tight complex with aprotinin (see Anhydro- kcal/mol, the driving force of the Table 2) (25, 32). 13 trypsin Bovine <3.0 X 10" 8 complex formation, resulting in an The energy gained by the contact Tryp- extremely low dissociation constant, of the complementary regions is even sinogen Bovine 1.8 X 10"8 8 14 Ki of 6 X 10" mol/liter (32). high enough to enable the aprotinin Chymo• As schematically outlined in Fig. 2, to push the conformation