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 of trypsin- trypsin Bovine 9.0 X 10"9 8 formation of such a tight enzyme- ogen into the conformation of the 6.0 X 10"9 7 inhibitor complex is normally associ• active enzyme without the activation Plasmin Porcine 4.0 X 10"9 8 10 ated with i ) a perfect fit of the reac• peptide being cleaved off (Table 2) Human 2.3 X 10" 8 tive-site residue of the inhibitor into (4, 46). On the other hand, the rela• the specificity pocket of the enzyme; tively low affinity of aprotinin to it) the formation of a tetrahedral chymotrypsin in comparison to tryp• recently (45), 57% of the amino acid intermediate by an attack of the sin reflects primarily the disturbed residues of trypsin mediating the nucleophilic hydroxyl group of the fit of the Lys15-residue into the speci• contact in the complex with aprotinin serine residue of the active site of the ficity pocket of this enzyme (3). are preserved in kallikrein; especially enzyme on the carbonyl group of the In the case of the plasmin-aprotinin the residues participating in more lysine residue of the reactive peptide complex the contact regions of the than just one interaction with the bond of the inhibitor, and Hi) stabili• partners should be distinctly smaller inhibitor remain unchanged. Of the zation of the adduct by additional and/or their fit not nearly as perfect numerous interactions in the trypsin- weaker interactions in the vicinity as in the trypsin-aprotinin complex aprotinin complex about 70% also of the specificity pocket and reactive (see Table 2). The same should hold seem to be possible in the complex peptide bond, respectively (26, 27, true for the complexes formed between with porcine pancreatic kallikrein, 32). Taking into account the surplus various kallikreins and aprotinin, as especially the contacts around the of energy gained by the numerous may be deduced from the K< values reactive site peptide bond Lys15- contacts between enzyme and in• compiled in Table 3 (10, 17). In this Ala16 of the inhibitor (45). hibitor, it is not surprising that not both, respect it is interesting that, based on formation of the tetrahedral inter• the primary structure of porcine mediate and the perfect fit of the pancreatic kallikrein as elucidated Structurally homologous inhibitors—structural basis of kallikrein inhibition Another possible approach for ob• taining an insight into the structural basis of kallikrein inhibition by apro• tinin is a comparison of the inhibition specificities and reactive site se• quences of structurally homologous inhibitors (Table 4) (10, 24). From the results of the crystallographic work it is known that the amino acid resi• dues of aprotinin with the most intimate contact to trypsin in the complex are, besides Cys15 and Cys38,
TABLE 3. Dissociation constants of complexes between aprotinin and various kallikreins
Ki Kallikrein Species (mol/liter) pH
Pancreatic Porcine 1 X 10"9 8.0 Subman• dibular Porcine 1.6 X 10"9 9.0 Urinary Porcine 1.7 X 10"9 9.0 Human 1 X 10"10 8.0 Plasma Human 3 X 10"8 8.0 Porcine 1 X 10"7 7.8
ENZYME INHIBITORS OF THE KALLIKREIN AND RENIN SYSTEMS 2755 TABLE 4. Identified and mutual reactive site residues of structurally related trypsin-chymotrypsin inhibitors of the aprotinin type
Inhibition of
Positions of" the residues Tissue Plasma kalli• kalli• Inhibitor (from) P, P,' Plasmin P2 P2' P/ P.-,' P» kreins kreins
(Position") 14 15 16 17 18 19 20 38 39
Aprotinin Cys Lys- Ala Arg He He Arg Cys Arg + + + + + SAI, sea anemone Cys Arg- Ala Arg Phe Pro Arg Cys Arg + + + + +
HPIK, snail Cys Lys- Ala Ser Phe Arg Gin Cys Arg + + + + RVV II, snake venom Cys Arg-Gly His Leu Arg Arg Cys Gly + + + NNV II, snake venom Cys Lys- Ala Arg lie Arg Ser Cys Gly + + + HHV II, snake venom Cys Lys- Ala Tyr He Arg Ser Cys Gly + + + + + CTI, cow colostrum Cys Lys- Ala Ala Leu Leu Arg Cys Gin + + - - Kunitz, soybean* Tyr Arg-He Arg Phe Ile Ala + + - + +
" In aprotinin. h Structurally not homologous. + + strong, + weaker, — no inhibition;
If, on the other hand, a basic resi• are very similar, especially if the K{ The modified inhibitor was used to due is present in position 19 (see values of the complexes are so closely produce chemical mutants of apro• snake venom inhibitor HHV II), related, as in the case of porcine tissue tinin (28, 43). As expected, replace• kallikrein inhibitory activity is ex• kallikreins (Table 5) (10). ment of Lys15 by Arg via several steps hibited again. Obviously, in an apro- (cf. left and lower part of Fig. 3) did tinin-type inhibitor the basic nature The reactive site bond— not influence the inhibitory proper• of amino acid residues in one or more chemical mutants of aprotinin ties of aprotinin, whereas replace• of the positions 17, 19, and 39 is ment of the basic active site residue necessary to gain enough energy for There is general agreement that by Phe or Trp caused a significant complex formation with a kallikrein. aprotinin is a single-headed inhibitor increase in the affinity of the inhibi• In contrast, the affinity of an with the Lys15-residue in its active tory protein for chymotrypsin and aprotinin-type inhibitor to plasmin center. Location of the reactive site decrease in the affinity for trypsin. The total synthesis of aprotinin achieved recently opens a way to study
TABLE 5. Dissociation constants Kt of complexes between aprotinin-type inhibitors (cf. the influence of amino acid exchanges Table 4) and porcine tissue kallikreins on the inhibitory properties of this molecule in detail (42). Kallikrein from porcine Structurally homologous inhibitor from Pancreas Submand. gland Urine Selected aspects of Bovine lung TKI 0.8 0.9 1.0 aprotinin medication Sea anemone SAI 5-II 0.8 0.6 0.8 In view of the application of aprotinin Snake venom NNV-II 1.1 1.3 1.3 (e.g., as Trasylol, the registered trade Snake venom HHV-II 8.3 210 109 name in favor of Bayer AG, Germany) Snail HPIK 27.7 210 204 Cow colostrum CTI — in medical therapy and experimental animal studies, some of its properties Values in nanomoles per liter. -, no inhibition. are of special interest.
2756 FEDERATION PROCEEDINGS VOL. 38, NO. 13 DECEMBER 1979 Virgin inhibitor Modified inhibitor and hence blood clotting, by the amount of inhibitor administered 14 ß KS 38 (16, 39). Regarding the release of -CyLyss - LLy s - Ala Cys Çys-Lys Ala Cys- appreciable amounts of neutral leu• plasmin, pH 5 kocytic proteinases during the inflam• S-S matory response, inhibition of gran• (l)reductioTn (2)enzymic ulocytic elastase by aprotinin is also (3)CpdaseB modif. of interest (33). (5) Phe or Trp The trypsin-like acrosomal pro• (4) teinase acrosin, which assists the (4) Arg, Cpdose B, •Cys Ala- •Cys- -Cys-Arg-Ala Cys- spermatozoa to penetrate the zona trypsin complex ^ pellucida of the ovum, is also, though L s-s- (5) Phe or Trp, CpdaseA, -s-s rather weakly, inhibited by aprotinin chymotr. complex (18). In vivo application of aprotinin Des- Lys inhibitor Semisynthetic inhibitor for contraceptive purposes is hin• dered further by the high concentra• Figure 3. Modification of aprotinin by limited proteolysis. By the action of catalytic amounts of tion (about 10 mmol/liter) necessary plasmin at pH 5 a thermodynamic equilibrium is accomplished between virgin inhibitor (reactive to achieve passage of the inhibitor site peptide bond intact) and modified inhibitor (reactive site bond hydrolyzed), cf. upper part. across sperm head membranes (W.-B. Semisynthetic reactive site modified inhibitors are obtained by a series of chemical and enzymic reactions as indicated in the left and lower part of the figure: / ) selective reduction of the Cys14- Schill, G. Feifel and H. Fritz, ms in Cys38 disulfide bridge, 2 ) selective proteolytic cleavage of the Lys,5-Ala16 peptide bond, 3 ) removal preparation). Implantation of blasto• of Lys15 by treatment with carboxypeptidase B, 4 ) insertion of Arg and resynthesis of the reactive cysts into rabbit uteri could be pre• site peptide bond via the trypsin complex, 5 ) insertion of Phe or Trp and resynthesis of the reactive vented, however, in the presence of site peptide bond via chymotrypsin complex. much lower concentrations (about 1 /xmol/liter) of aprotinin probably via Aprotinin strongly inhibits both (urinary, submandibular, pancreatic) inhibition of a trypsin- or kallikrein- human cationic and anionic trypsin kallikrein—Kj values close to 0.1 like trophoblast proteinase (8, 9). but rather weakly inhibits human nmol/liter were reported for the chymotrypsin I and human pancre• corresponding complexes, cf. Tables Occurrence in bovine tissue and atic protease E or elastase; human 2 and 3—compared to the clearly possible biological function chymotrypsin II is not inhibited (11, weaker inhibition of human plasma Although detailed knowledge of the
34, 35). Tissue kallikreins and plasma kallikrein—the Kf value of this complex biochemistry, pharmacology, and kallikrein of man, pig, arid cattle are is higher than 10 nmol/liter, cf. Table mechanism of action of aprotinin has relatively strongly inhibited by apro• 3. Due to this significant difference accumulated, its physiological func• tinin, whereas the corresponding in the affinity of aprotinin to plasmin tion in the bovine organism has so far kallikreins of guinea pig and dog are and plasma kallikrein it seems possi• remained obscure (13, 48, 49). This not inhibited (47, 48). We emphasize ble to regulate the desired effect, was due, at least partly, to its wide• especially the rather strong inhibition of namely inhibition of plasmin and thus spread distribution in functionally human plasmin and human tissue fibrinolysis or of plasma kallikrein totally different organs such as lung,
Figure 4. Localization of aprotinin in mast cells as revealed by immunofluorescence (19). a) Distribution pattern of the fluorescent spots in tis• sue sections of bovine lung after treatment with aprotinin-specific antibodies (control sections treated with unspecific antibodies showed no fluorescence), b) Distribution pattern of the mast cells as revealed by metachromatic staining with toluidine blue. Magnification: x 128.
ENZYME INHIBITORS OF THE KALLIKREIN AND RENIN SYSTEMS 2757 parotid gland, lymph nodes, liver, metachromatic staining with toluidine substrate specificity of mast cell pancreas, seminal vesicles, ovary, blue or specific fluorescence. An proteinases (40) is in favor of such an heart, etc. The availability of aproti• additional indication of the presence assumption. Inhibition of the pancre• nin antibodies stimulated us to look of aprotinin in mast cells is the similar atic proteinases and of plasmin and for the origin of this inhibitor at the distribution of mast cells and aproti• plasma kallikrein should be due to the cellular level, hoping to find in this nin in the tissues of the bovine organ• relationship of all these proteinases way an indication of its biological ism; tissues known to contain high as members of the serine proteinase function. Figure 4b shows the dis• numbers of mast cells are also rich in family of enzymes. Considering the tribution of mast cells in tissue sec• aprotinin and vice versa. The ubiqui• species specificity of such proteinases, tions of bovine lung as revealed by tous occurrence of mast cells in all aprotinin-like inhibitors which do not selective staining with toluidine blue. connective tissues of the organism inhibit or only weakly interact with The similar pattern of fluorescent offers a simple explanation for the the pancreatic enzymes normally used spots obtained after treatment of occurrence of aprotinin in nearly for the search of inhibitors could be acetone-fixed tissue sections with every organ or tissue of cattle. present also in other animals and men. aprotinin-directed antibodies using The presence of aprotinin in such The discovery that aprotinin origi• the indirect immunofluorescence tech• a unique and highly specialized cell nates from tissue mast cells does, on nique (Fig. 4a) indicates strongly population as the mast cells (50) the one hand, terminate a long debate that aprotinin is localized in mast cells implies an important biological func• concerning its unusual overall dis• (19). In order to corroborate the mast tion of this molecule. Regarding our tribution throughout the bovine or• cell character of the cells showing knowledge of the biochemistry of ganism. On the other hand, we expect specific fluorescence, additional com• aprotinin, this inhibitory protein is a stimulating effect regarding the bined light and electron microscopic most likely involved in the regulation search for the biological function of studies were performed (19). The of mast cell proteinase activities either this fascinating molecule as well as the granulated cells thus identified were intra- or extracellularly. The chymo• biochemistry and pharmacology of identical with the cells showing trypsin- or trypsin- or kallikrein-like mast cell proteinases. S3
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