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Recent studies of the synthetic selective inhibitors; with special reference to non-plasmin fibrinolytic enzyme, plasmin and plasma-kallikrein
THROMBOSIS RESEARCH, Supplement VIII; 131-141, 1988 0049-3848/88 $3.00 t .OO Printed in the USA. Copyright (c) 1988 Pergamon Press plc. All rights reserved.
REVIEW RECENT STUDIES OF THE SYNTHETIC SELECTIVE INHIBITORS; WITH SPECIAL REFERENCE TO NON-PLASMIN FIBRINOLYTIC ENZYME, PLASMIN AND PLASMA-KALLIKREIN
S.Okamoto,
U.Okamoto*,
A.Hijikata-Okunomiya**,
K.Wanaka and Y.Okada*
Hospital, Saiseikai Kobe Research Projects on Thrombosis and Haemostasis, Kobe 673 Japan and **School of *Kobe-Gakuin University, Kobe 651 Japan, Allied Medical Sciences, Kobe University, Kobe 654 Japan
I. INTRODUCTORY
REMARKS
In 1975, a short review was presented by Okamoto and Hijikata, in which selective as rationally to the highly trials to approach as possible (tinhibitors such as epsilon-amino-caproic acid (EACA) and tranexamic acid was very AMCHA) were described (1). The mode of action of these inhibitors unique. Fact is, years more than one decade elapsed, when it was found that they inhibit the fibrinolysis by plasmin through their binding to the lysinebinding-sites (LBS) of the heavy chain of the plasmin and plasminogen (2,3). In the following undertaking by Okamoto and his colleagues, the target the highly selective synthetic inhibitors of which were enzyme was thrombin, systematically pursued. The first step of stud&es was to examine the thrombin methyl ester inhibitory activities of the homologs of N -tosyl-arginine (TAMe), which had weak thrombin inhibitory activity. TAMe was easily hydrothis might be understood from the fact that most of the lyzed by thrombin; as it were, "subassumed the inhibitory activity, substrates to the enzyme strate inhibition". It was soon found that some chemical modifications of the tosy1 radical increased the stability of the compounds as well as inhibitory Thus, stepwise chemical modifications were activity with regard to thrombin. the compound called No. &05 was obtained, Ki conducted; as the resui&s, 4value of which, 3.7~10 M; No. 205 was chemically N -dansyl-arginine ethylpiperidine amide (4).
plasma kallikrein, words: plasmin, Key elastase, thrombin, synthetic substrate
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synthetic
inhibitor,
leukocyte
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It was noticed that No. 205 showed high toxicity (i.p. LD50 for mice cholinwas 80 mg/kg) and that inhibitory activity of the compound to plasma esterase was extremely strong (5). Results accumulated supported the possibility that the 4-ethylpiperidine radical of No. 205 assumed the toxicity of @-alanine the compound. Therefore, instead of the ethylpiperidine, methyl the non-toxic radical, was introduced into the C-terminal of Arg. The ester, for mice, 1000 mg/kg, Ki compound obtained _yas No. 329, of which i.p. LD for thrombin, cholin10 M order and the inhibitory ac so ivity to plasma was very weak. Encouraged with this finding, again stepwise chemesterase, ical modificati ns were made and finally No. 805 was obtained (6). Ki of No. -8 805 was 1.9x10 M; i.p. LD for mice, 700 mg/kg (Fig 1). No. 805 has been broadly investigated either5' experimentally and clinically. Its medical utility has been still under investigations in a large number of the clinical institutions in Japan (7-9). The mode of action of No. 805 (abbreviated as MD-805 clinically) was typically competitive inhibition; This implies that the active site of thrombin takes the structure mold of No. 805. A question may remain why No. 205 inhibits both thrombin and plasma cholinesterase while No. 805 inhibits thrombin only. Hijikata-Okunomiya et al. have bgen working on the problem mentioned above, and reported in 1987 that N -dansyl-arginine 4-phenylpiperidine amide was highly selective inhibitor of plasama cholinesterase. This finding would be one of the most successful cases in designing newly the highly selective synthetic inhibitors (10). It should be noteworthy that the background knowledge of the comparative aspects of different serine proteases has been accumulated, not only employing laboratory-experimental technique but also using the computergenerated molecular model to predict the nature of the active site as well as selective site of the enzyme concerned (11).
-N
3
COOH (2R ,4R)
FIG. I Chemical structure of
No. 805 (MD-805)
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SYNTHETIC SELECTIVE INHIBITOR
II. NON-PLASMIN
FIBRINOLYTIC
133
PROTEASE
Although the plasmin system is known to play an important role in fibrinolysis in the blood, several studies have clearly demonstrated the presence of a plasmin-independent fibrinolysis in leukocyte by Plow et al. (19751, Bilezikian and Nossel (1977) and Kopec et al. (1978) (12-14). The protease has been purified from leukocyte extracts by using sepharose chromatography, indicating an indipendent fibrinolytic pathway from plasmin system. The authors group including Nagamatsu et al. also obtained a neutral protease capable of fibrin degradation which was extracted especially from the human spleen obtained at the time of the autopsy of ca. 400 cases (15). The enzyme, spleen fibrinolytic protease (SFP), was found to be a neutral serine protease different from plasmin based on its mode of action against synthetic substrates and inhibitors. The physicochemical and enzymatic properties of the enzyme were found very similar or identical to those of leukocyte elastase (Table 1). Later an effective purific,tion method was found using a new affinity chromatography coupled with Sue-L-Tyr-D-Leu-D-Val-pNA (16). The purified preparation contained practically no chymotrypsin-like protease activity. The enzyme so purified readily degraded fibrin, fibrinogen and Val-type synthetic peptide substrates, such as Sue-L-Tyr-L-Leu-L-Val-pNA and sue-LAla-L-Tyr-L-Leu-L-Val-pNA substrates were not hydrolyzed by (17). These plasmin. The enzyme was inhibited by Sue-L-Tyr-D-Leu-D-Val-pNA competitively (Table 2) and by Boc-L-Ala-L-Tyr-L-Leu-L-Val-CH Cl irreversibly (Table 3) as reported by Tsuda et al. (18). DFP and SBTI in ibited the enzyme. Neither tAMCHA nor TLCK inhibited the enzyme. Notable was that the species difference of the non-plasmin fibrinolytic enzyme was considerably great.
2,
significance of non-plasmin fibrinolysis is still Pathophysiological obscure. However, concerning the possible role of the leukocyte and spleen in thrombolysis, we expect that the studies may provide some precise tools for fibrinolysis in a broad sense. Our further studies are now continuing the
TABLE 1 Comparative
Activities of Spleen Fibrinolytic and Leukocyte Elastase (LE)
Protease
Substrate Sue-Tyr-Leu-Val-pNA Sue-Tyr-Leu-Ile-pNA Sue-Tyr-Leu-Ala-pNA Sue-Tyr-Leu-Leu-pNA Sue-Tyr-Leu-Phe-pNA Sue-Tyr-Leu-Gly-pNA Sue-Tyr-Leu-Met-pNA Sue-Tyr-Leu-Arg-pNA
SFP 100 % 33 16 CO.2 0 0 0 0
LE -_p 100 % 23 13 CO.2 0 0 0 0
(SFP)
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TABLE 2 Reversible p3
Inhibitors of SFP
p2
Ki (mM)
p1
0.12 0.19 0.20 0.24 0.35 0.94
Sue-L-Tyr-D-Leu-D-Val-pNA LLDDDDDDLLDLDDLSubstrate:
Sue-L-Tyr-L-Leu-L-Val-pNA
TABLE 3 Irreversible
Inhibitor
Inhibitors of SFP
I4 (MxlO )
Half life (set)
0.1 0.1 0.1 0.1
31 14 23 28
Boc-Ala-Tyr-Leu-Val-CH2C1 Boc-Tyr-Leu-Val-CH2Cl Dan-Ala-Tyr-Leu-Val-CH2Cl Ac-Ala-Tyr-Leu-Val-CH2C1 Substrate:
Sue-Ala-Tyr-Leu-Val-pNA
2240 4950 3010 2480
(0.5 mM)
with the scope that non-plasmin fibrinolysis would be far more significant than ever expected, particularly in the late period of the thrombosis in vivo. An exciting task challenges us to find a new way, how to mobilize this particular enzyme in patients.
III. AMIDOLYSIS
AND FIBRINOLYSIS
WITH PLASMIN
Apart from the LBS-directed inhibitors such as EACA or t-AMCHA, the series of active-site directed inhibitors of plasmin have been studies by our (19). Based on the facts that plasmin splits mainly the C-terminal of group lysine in fibrinogen, our group first noticed N'-tosyl-lysine methyl ester (TLMe) was a very weak and relatively specific inhibitor as well as substrate on plasmin. A number of derivatives of TLMe including S-2251 and its homologs substrates were re-examined and results obtained were theoretically summarized according to the Free and Wilson method. Thus, the D-Ile-Phe-LyspNA was found to be the more sensitive substrate than its homologs (Table 4) (20). Interesting was that D-Ile was significant, which was well coincident with the theoretical prediction on the structure of suitable substrate of plasmin, proposed by Szabo et al. (21).
about
Stimulated by the presence of the sensitive substrate mentioned above, 900 kinds of new compounds were synthesized in our laboratories and
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TABLE 4 Kinetic Parameters
for the Amidolysis
Substrate D-Ile-Phe-Lys-pNA 3-MV-Phe-Lys-pNA Ile-Phe-Lys-pNA
of Substrate by Plasmin
Km (mM) 0.02 0.18 0.33
-1
kcat (s
)
10 18 19
3-MV: 3-methylvaleryl
Ki for thrombin
FIG. 2 Chemical structure of OS 153
potent and reversible inhibitors of plasmin were several kinds of selective, obtained, chemical structure of one of them called OS 153 being shown in Fig. 2. characterized with the inhibitors belong to lysine derivatives, These presence of NH2 in the chemical structure which covers the N-terminal of lysine. The radical which covers the C-terminal of lysine is much complicated when compared with pNA. The structure of such synthetic inhibitors may take the structure mold of the active site of plasmin. of t-AMCHA was compared with these of OS group When the activities plasmin inhibiotrs, remarkable differences are found as shown in Table 5. Inhibiting activities of t-AMCHA (and EACA) are characterized by its strong inhibition on fibrinolysis but neither on fibrinogenolysis nor on amidolysis While OS inhibitors inhibit nearly equally of S-series synthetic substrates. not only fibrinolysis by plasmin, but also fibrinogenolysis and also _gmidolysis by plasmin, with Ki values of very low concentration such as_+0 M. In particular, Ki value of OS 324 was found to be in the orde_g of 10 M. This value can be compared with that of MD-805, Ki of which , 10 M.
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TABLE 5 Inhibitory
Effect of t-AMCHA and OS 153 on Amidolysis, and Fibrinogenolysis by Plasmin
Amidolysis(a) Ki (M) t-AMCHA OS 153
-2 4.0X10-6 7.4x10
Fibrinolysis(b) I50 (M)
Fibrinolysis
Fibrinogenolysis(b) I50 (M)
-5 6.0~10_~ 6.1x10
-2 l.Ox10_5 2.0x10
(a) Amidolysis was measured with D-Val-Leu-Lys-pNA as substrate. (b) Fibrinolysis and Fibrinogenolysis,were measured using 0.04% and 0.2% fibrinogen, respectively.
IV. PLASMA KALLIKREIN
AND ITS HIGHLY SELECTIVE
INHIBITORS
It should be recalled that the action of aprotinin has been characterized by its potent inhibition toward glandular kallikrein and plasmin: And that the action of aprotinin toward plasma kallikrein (PK) is approximately 10 times weaker than that to glandular kallikrein. Highly selective synthetic inhibitors of PK has been first reported by the authors, very recently (22). In this review, the novel series of highly selective inhibitors celled PKSI are introduced but briefly. PKSIFig. 3 shows a chemical structure of one of the PK inhibitors. 1007 assumed a tripod structure, the basic skeleton of which was L-arginine. This structure showed trans-4-aminomethylcyclohexanecarbonyl to be at the Nterminal of the arginine molecule and 4-ethoxycarbonylanilide to be at the Cterminal position.
FIG. 3 Chemical
structure of PKSI-1007
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TABLE 6 Enzyme Inhibition
P.Kallikrein Ki (pM) PKSI-1007 (Index) PKSI-0180 (Index) PKSI-0527 (Index)
8.0
(1) 0.47
(1) 0.81
(1)
of PKSI-Inhibitors
G.Kallikrein Ki (FM) 120
Plasmin Ki (pM) 270
(15) 53 (113) >500 (>617)
(34) 20 (43) 390 (481)
Thrombin Ki (pM)
FXa Ki (pM)
>500 >500 (>63) (>63) >500 330 (>1064) (702) ,500 ,500 (>617) (>617)
Substrate: S-2302 for P.kallikrein, S-2266 for G.kallikrein, S-2251 for plasmin, S-2238 for thrombin and S-2222 for factor Xa.
The mode of the inhibition of PKSI inhibitors was competitive toward PK as well as the different enzyme examined. Inhibitory effects of PKSI inhibitors on the enzymes were measured using the synthetic chromogenic substrates, as shown in Table 6. (a) PKSI-1007 showed Ki value of 8.0 PM for PK. By Ki_ of 120 PM for glandular kallikrein, 270 uM for plasmin and more contrast, than 500 PM for thrombin and factor Xa were obtained. (b) PKSI-0180 was more inhibitor than PKSI-1007, potent Ki value for PK being 0.47 PM. (c) PKSI0527 showed that Ki value for PK was 0.81 pM, while Ki values for plasmin, glandular kallikrein, thrombin and factor Xa were 390 PM, >500)~M, >5OOpM and >500 PM, respectively. High selectivity toward PK was thus remarkably demonso far as the enzymes examined concerned. In particular, Ki values strated, plasmin, thrombin and factor Xa were of PKSI-0527 for glandular kallikrein, times larger than that for PK, as indicated by the figures in the hundreds parenthesis in Table 6. In addition, it can be noted that i.v. LD50 was over 100 mg/kg in our preliminary toxicity test, which encourages us to extend the studies toward the in vivo applications. As the preliminary test toward in vivo studies, the intact human plasma was employed and the inhibitory effect of PKSI-0180 on the bradykinin (BK) formation by kaolin as well as effect on PTT were examined. As shown in Fig. of BK formed (left ordinate) in the kaolin-activated human 4, the amount plasma decreased by increasing the concentration of PKSI-0180 (abscissa) concluded the inhibition of BK formation by PKSI-0180 in Results gradually. such a contact activated system. Fig 4 also showed that PKSI-0180 was able to prolong PTT. The right ordinate indicated the ratio of PTT with different of PKSI-0180 to PTT without inhibitor. The results concluded concentration that PK inhibitor was able to prolong PTT presumably by inhbiting PK of the contact system. the ascites of tumor bearing mice was emIn the following studies, The Sarcoma 180 was inoculated into the abdominal cavity of mice and ployed. the the ascites on the 8th day was obtained. The ascites collected underwent The samples were incuto get rid of the kininase activity. acid-treatment bated at 37OC and the aliquots taken out were measured of the amount of BK In the control aliquots, the amount of BK showed remarkable increase formed. The presence of PKSI-0527 of the final with the time course of incubation. showed the significant concentration of 10 p and 100 FM in the ascites
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I
25 PKSI-0180 (Plf)
FIG. 4 Lnhibitory effect of PKSI-0180 on bradykinin formation by kaolin and PT? BK formation: The intact human plasma containing o-phenanthroline was incubated with PKSI-0180 and kaolin (0.2 mg/ml) at 37OC for 2 min. BK levels in the plasma were measured by EIA method using a MARKIT-A-Bradykinin kit (Dainippon Pharmaceutical Co.). PTT was measured with PTT reagent purchased from Behring institute.
600 -
‘= a .
400
-
% .g
300
-
z 3 2 m
200 -
100 oI
0
I
I
10
20 Tim&in)
FIG. 5 Inhibitory effect of PKSI-0527 on BK formation in acid-treated ascites of Sarcoma 180 mice
30
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decrease of the amount of BK, indicating clearly the dose-dependency of the concentration of PKSI-0527. The PKSI-0180 showed similar effect in suppressing the BK formation in the ascites (Fig. 5). PKSI-1007, 0180 and 0527 were obtained as the highly selective inhibitors. However, possible role of PK concerning with the contact system should involve the complicated problems in health and disease. studies The present may provide some tools for the studies in this field; the correlation between coagulation, BK formation, fibrinolysis and possibly platelets aggreagation in the contact system would be extremely complicated; even so we expect that results obtained by using PKSI-inhibitors cast light on a promising perspective to us. Needless to say, the further studies are urgently required and they are now under investigation in our laboratories.
V. SIGNIFICANCE OF PROTEOLYTIC REACTION _ The Present and the Future Recent studies of the different proteases revealed the inherent ability of the enzyme to cleave their substrates with the unexpectedly high selectivity, which could be enough compared with that of the antigen-antibody reaction. In fact, such a high selectivity provided the systematic and stepwise activation of the proteases as seen in the coagulation cascade. The present paper dealt mainly with the results of our investigations conducted to provide the highly selective synthetic inhibitors which may be even the excellent tools for revealing significance of the target proteases, utilities of synthetic so final goal should be to find the clinical compounds. These studies long continued more than 40 years have been very inhibitors much encouraged by knowing the presence of natural with high selectivity just as 0.2PI. Studies of ours have been developing fortunately and selective synthetic inhibitors of trypsin are now under investigation. At the same time, I have learned from Homma that some kinds of virus including AIDS virus may step require the activation by trypsin-like protease in each multiplication (23). This reminded us the activation of the zymogen to the active enzyme by some certain proteases. The future of the investigation of proteases seems to be far more promising than our expectation, which has been cherishing in our mind.
ACKNOWLEDGEMENT in express the cordial thanks to their close co-workers The authors different institutions who contributed immensely. Dr. T. Naito and his coworkers, the institute of Showa Denko have been working closely together for Special their contributions being appreciated. the recent several years, central laboratories, and his co-workers, thanks to Dr. R. Kikumoto Mitsubishi Chemical Industries for the studies of MD-805 and its homologs. To those other close friends who supported us, we ask their pardon for missing even names due to the space allotted.
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REFFERENCES 1.
OKAMOTO,S. and HIJIKATA,A. Drug Design VI. E.J.Ariens 143-169.
Rational Approach to Proteinase Inhbitors, In: (Ed.) San Francisco: Academic Press, 1975, pp.
2.
IWAMOTO, M. Plasminogen-plasmin system.IX. Specific binding of tranexamic 573-585, 1975. 2, acid to plasmin. Thrombos.Diathes.Haemorrh.(Stuttq.)
3.
MARKUS,G., PRIORE,R.L. and WISSLER,F.C. The binding of tranexamic acid to on native (Glu) and modified (Lys) human plasminogen and its effect conformation. J.Biol.Chem. 254, 1211-1216, 1979.
4.
HIJIKATA,A., OKAMOTO,S., KIKUMOTO,R. and TAMAO,Y. Kinetic studies on the peptide selectivity of a synthetic thrombin-inhibitor using synthetic substrates. Thrombos.Haemostas.(Stuttq.) 2, 1039-1045, 1979.
5.
IKEZAWA,K., MIMURA,K., MATSUOKA,A., HIJIKATA,A. NAGANO,S., OKAMOTO,S., and TAMAO,Y. Fluorescence studies on the mode of action of two synthetic thrombin inhibitors, No.205 and No.805. Thrombos.Haemostas. 46, 45, 1981
6.
HIJIKATA,A., KIKUMOTO,R., TONOMURA,S., HARA,H., NINOMIYA,K., OKAMOTO,S., SUGANO,M. and TAMAO,Y. Potent inhibition of thrombin by the MARUYAMA,A., arginine derivative no.805. newly synthesized The importance of stereocarboxamide portion. Biochem.Biophvs.Res. of its hydrophobic structure Commun. 101, 440-446, 1981.
7.
MAEDA,K. MATSUI,N., NAKAGAWA,S., KOSHIKAWA,S., KAWAGUCHI,H., OTA,K., and SASAOKA,T. Clinical evaluation of a new thrombin inhibiHIRASAWA,Y. tor available for haemodialysis'. Proc.Eur.Dialysis Transplant.Assoc. z_O, 144-149, 1983.
a.
KUMON,K., TANAKA,K., NAKAJIMA,N., NAITO,Y. and FUJITA,T. Anticoagulation with a synthetic thrombin inhibitor after cardiovascular surgery and for treatment of disseminated intravascular coagulation. Crit.Care Med. l2, 1039-1043, 1984.
9.
YONEKAWA,Y., HANDA,H., OKAMOTO,S., KAMIJO,Y., ODA,Y., ISHIKAWA,J., TSUDA,H., SHIMIZU,Y., SATOH,M., YAMAGAMI,T., YANO,I., HORIKAWA,Y. and TSUDA,E. Treatment of cerebral infarction in the acute stage with synthetic antithrombin MD-805: Clinical study among multiple institutions. Arch.Jpn.Chir. 55, 711-722, 1986.
10. HIJIKATA-OKUNOMIYA,A., OKAMOTO,S., KIKUMOTO,R. and TAMAO,Y. Stereogeometry of the active sites of serine enzymes gathered from synthetic thrombin-inhibitors. Thrombos.Haemostas. 58, 489, 1987. 11. HIJIKATA-OKUNOMIYA,A., OKAMOTO,S., KIKUMOTO,R., TAMAO,Y., OHKUBO.K., TEZUKA,T., TONOMURA,S. and MATSUMOTO,O. Similarity and dissimilarity in the stereogeometry of the active sites of thrombin, trypsin, plasmin and glandular kallikrein. Thromb.Res. 45, 451-462, 1987. 12. Plow,E.F. and EDGINGTON,T.S. An alternative pathway for fibrinolysis. of fibrinogen by leukocyte protease at physiologic The cleavage J.Clin.Invest. 56, 30-38, 1975.
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13. BILEZIKIAN,S.B. and NOSSEL,H.L. Unique pattern of fibrinogen cleavage by human leukocyte proteases. Blood 50, 21-28, 1977. 14. KOPEC,M., WEGRZYNOWICZ,Z., ARDELT,W., TEISSEYRE,E. and LATALL0,Z.S. Effect of elastolytic enzymes from human leukocyte granules and from pig pancreas on plasminogen, fibrinogen, and fibrin. In: Progress in Chemical Fibrinolysis and Thrombolysis. J.F.Davidson, R.M.Rowan, M.M.Samama and P.C.Desnoyers (Eds.) New York: Raven Press, 3, 1978, pp. 417-425. 15. NAGAMATSUIY., ~KAM~T~,u., OKADA,N., AMEMIYA,T., HIRASE,F. and KUBOTA,A. Statistical of malignant tumor cases found in spleen characteristics fibrinolytic protease (SFP) activity distribution of 308 autopsy cases. IGAKU NO AYUMI (in Japanese)=, 273-275, 1983. 16. NAGAMATSUIY., OKAMOTO,U., TSUDA,Y. and OKADA,Y. Human leukocyte elastaselike proteinase purified by affinity chromatography with Suc-L-Tyr-D-LeuD-Val-pNA, and its identification with human spleen fibrinolytic proteinase. Thromos.Haemostas.(Stuttg.) 2, 243-247, 1984. TSUDA,Y. and OKADA,Y. A new polypeptide sub17. OKAMOTO,U., NAGAMATSU,Y., Sue-Tyr-Leu-Val-pNA, specific for spleen fibrinolytic proteinase strate, 97, 28-32, 1980. (SFP). Biochem.Biophys.Res.Commun. .18. Tsuda,Y., and OKAMOTO,U. Suynthesis of peptide OKADA,Y., NAGAMATSU,Y. chloromethyl ketones and examination of their inhibitory effects on human spleen fibrinolytic proteinase (SFP) and human leukocyte elastase (LE). Chem.Pharm.Bull. 35, 3576-3584, 1987. 19. OKAMOTO,S., OKAMOTO,U., OKADA,Y., HIJIKATA-OKUNOMIYA,A., WANAKA,K., HORIE,N. and NAITO,T. Coding, decoding and noise of proteinase: a rational approach to plasmin inhibitors. In: Fundamental and clinical fibrinolysis, F.J.Castellino, P.J.Gaffney, M.M.Samama and A.Takada (Eds.), Amsterdam-Oxford-New York: Excerpta Medica, 1987, pp.67-82. TSUDA,Y., TENO,N., WANAKA,K., SASAKI,K., HIJIKATA,A., NAITO,T. 20. OKADA,Y. Synthesis of plasmin substrates and relationship between and OKAMOTO,S. their structure and plasmin activity. Int.J.Peptide Protein Res. 22, 7985, 1986. POZSGAY,M. and ELODI,P. Investigation of the substrate21. Cs.-SZABO,G., binding site of human plasmin using tripeptidyl-p-nitroanilide substrates. Thromb.Res. 2O, 199-206, 1980. WANAKA,K., HIJIKATA-OKUNOMIYA,A., BOHGAKI,M., OKAMOTO,U., 22. OKAMOTO,S., Highly selective synthetic inhibitors and OKADA,Y. NAITO,T., HORIE,N. with regard to plasma-kallikrein activities. KININ V. (in press). Evidence of proteolytic activation 23. TASHIRO,M. and HOMMA,M. virus in mouse lung. Archives of Virology 77, 127-137, 1983.
of
Sendai
REVIEW RECENT STUDIES OF THE SYNTHETIC SELECTIVE INHIBITORS; WITH SPECIAL REFERENCE TO NON-PLASMIN FIBRINOLYTIC ENZYME, PLASMIN AND PLASMA-KALLIKREIN
S.Okamoto,
U.Okamoto*,
A.Hijikata-Okunomiya**,
K.Wanaka and Y.Okada*
Hospital, Saiseikai Kobe Research Projects on Thrombosis and Haemostasis, Kobe 673 Japan and **School of *Kobe-Gakuin University, Kobe 651 Japan, Allied Medical Sciences, Kobe University, Kobe 654 Japan
I. INTRODUCTORY
REMARKS
In 1975, a short review was presented by Okamoto and Hijikata, in which selective as rationally to the highly trials to approach as possible (tinhibitors such as epsilon-amino-caproic acid (EACA) and tranexamic acid was very AMCHA) were described (1). The mode of action of these inhibitors unique. Fact is, years more than one decade elapsed, when it was found that they inhibit the fibrinolysis by plasmin through their binding to the lysinebinding-sites (LBS) of the heavy chain of the plasmin and plasminogen (2,3). In the following undertaking by Okamoto and his colleagues, the target the highly selective synthetic inhibitors of which were enzyme was thrombin, systematically pursued. The first step of stud&es was to examine the thrombin methyl ester inhibitory activities of the homologs of N -tosyl-arginine (TAMe), which had weak thrombin inhibitory activity. TAMe was easily hydrothis might be understood from the fact that most of the lyzed by thrombin; as it were, "subassumed the inhibitory activity, substrates to the enzyme strate inhibition". It was soon found that some chemical modifications of the tosy1 radical increased the stability of the compounds as well as inhibitory Thus, stepwise chemical modifications were activity with regard to thrombin. the compound called No. &05 was obtained, Ki conducted; as the resui&s, 4value of which, 3.7~10 M; No. 205 was chemically N -dansyl-arginine ethylpiperidine amide (4).
plasma kallikrein, words: plasmin, Key elastase, thrombin, synthetic substrate
131
synthetic
inhibitor,
leukocyte
132
SYNTHETICSELECTIVE INHIBITOR
Suppl. VIII,
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It was noticed that No. 205 showed high toxicity (i.p. LD50 for mice cholinwas 80 mg/kg) and that inhibitory activity of the compound to plasma esterase was extremely strong (5). Results accumulated supported the possibility that the 4-ethylpiperidine radical of No. 205 assumed the toxicity of @-alanine the compound. Therefore, instead of the ethylpiperidine, methyl the non-toxic radical, was introduced into the C-terminal of Arg. The ester, for mice, 1000 mg/kg, Ki compound obtained _yas No. 329, of which i.p. LD for thrombin, cholin10 M order and the inhibitory ac so ivity to plasma was very weak. Encouraged with this finding, again stepwise chemesterase, ical modificati ns were made and finally No. 805 was obtained (6). Ki of No. -8 805 was 1.9x10 M; i.p. LD for mice, 700 mg/kg (Fig 1). No. 805 has been broadly investigated either5' experimentally and clinically. Its medical utility has been still under investigations in a large number of the clinical institutions in Japan (7-9). The mode of action of No. 805 (abbreviated as MD-805 clinically) was typically competitive inhibition; This implies that the active site of thrombin takes the structure mold of No. 805. A question may remain why No. 205 inhibits both thrombin and plasma cholinesterase while No. 805 inhibits thrombin only. Hijikata-Okunomiya et al. have bgen working on the problem mentioned above, and reported in 1987 that N -dansyl-arginine 4-phenylpiperidine amide was highly selective inhibitor of plasama cholinesterase. This finding would be one of the most successful cases in designing newly the highly selective synthetic inhibitors (10). It should be noteworthy that the background knowledge of the comparative aspects of different serine proteases has been accumulated, not only employing laboratory-experimental technique but also using the computergenerated molecular model to predict the nature of the active site as well as selective site of the enzyme concerned (11).
-N
3
COOH (2R ,4R)
FIG. I Chemical structure of
No. 805 (MD-805)
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SYNTHETIC SELECTIVE INHIBITOR
II. NON-PLASMIN
FIBRINOLYTIC
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PROTEASE
Although the plasmin system is known to play an important role in fibrinolysis in the blood, several studies have clearly demonstrated the presence of a plasmin-independent fibrinolysis in leukocyte by Plow et al. (19751, Bilezikian and Nossel (1977) and Kopec et al. (1978) (12-14). The protease has been purified from leukocyte extracts by using sepharose chromatography, indicating an indipendent fibrinolytic pathway from plasmin system. The authors group including Nagamatsu et al. also obtained a neutral protease capable of fibrin degradation which was extracted especially from the human spleen obtained at the time of the autopsy of ca. 400 cases (15). The enzyme, spleen fibrinolytic protease (SFP), was found to be a neutral serine protease different from plasmin based on its mode of action against synthetic substrates and inhibitors. The physicochemical and enzymatic properties of the enzyme were found very similar or identical to those of leukocyte elastase (Table 1). Later an effective purific,tion method was found using a new affinity chromatography coupled with Sue-L-Tyr-D-Leu-D-Val-pNA (16). The purified preparation contained practically no chymotrypsin-like protease activity. The enzyme so purified readily degraded fibrin, fibrinogen and Val-type synthetic peptide substrates, such as Sue-L-Tyr-L-Leu-L-Val-pNA and sue-LAla-L-Tyr-L-Leu-L-Val-pNA substrates were not hydrolyzed by (17). These plasmin. The enzyme was inhibited by Sue-L-Tyr-D-Leu-D-Val-pNA competitively (Table 2) and by Boc-L-Ala-L-Tyr-L-Leu-L-Val-CH Cl irreversibly (Table 3) as reported by Tsuda et al. (18). DFP and SBTI in ibited the enzyme. Neither tAMCHA nor TLCK inhibited the enzyme. Notable was that the species difference of the non-plasmin fibrinolytic enzyme was considerably great.
2,
significance of non-plasmin fibrinolysis is still Pathophysiological obscure. However, concerning the possible role of the leukocyte and spleen in thrombolysis, we expect that the studies may provide some precise tools for fibrinolysis in a broad sense. Our further studies are now continuing the
TABLE 1 Comparative
Activities of Spleen Fibrinolytic and Leukocyte Elastase (LE)
Protease
Substrate Sue-Tyr-Leu-Val-pNA Sue-Tyr-Leu-Ile-pNA Sue-Tyr-Leu-Ala-pNA Sue-Tyr-Leu-Leu-pNA Sue-Tyr-Leu-Phe-pNA Sue-Tyr-Leu-Gly-pNA Sue-Tyr-Leu-Met-pNA Sue-Tyr-Leu-Arg-pNA
SFP 100 % 33 16 CO.2 0 0 0 0
LE -_p 100 % 23 13 CO.2 0 0 0 0
(SFP)
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TABLE 2 Reversible p3
Inhibitors of SFP
p2
Ki (mM)
p1
0.12 0.19 0.20 0.24 0.35 0.94
Sue-L-Tyr-D-Leu-D-Val-pNA LLDDDDDDLLDLDDLSubstrate:
Sue-L-Tyr-L-Leu-L-Val-pNA
TABLE 3 Irreversible
Inhibitor
Inhibitors of SFP
I4 (MxlO )
Half life (set)
0.1 0.1 0.1 0.1
31 14 23 28
Boc-Ala-Tyr-Leu-Val-CH2C1 Boc-Tyr-Leu-Val-CH2Cl Dan-Ala-Tyr-Leu-Val-CH2Cl Ac-Ala-Tyr-Leu-Val-CH2C1 Substrate:
Sue-Ala-Tyr-Leu-Val-pNA
2240 4950 3010 2480
(0.5 mM)
with the scope that non-plasmin fibrinolysis would be far more significant than ever expected, particularly in the late period of the thrombosis in vivo. An exciting task challenges us to find a new way, how to mobilize this particular enzyme in patients.
III. AMIDOLYSIS
AND FIBRINOLYSIS
WITH PLASMIN
Apart from the LBS-directed inhibitors such as EACA or t-AMCHA, the series of active-site directed inhibitors of plasmin have been studies by our (19). Based on the facts that plasmin splits mainly the C-terminal of group lysine in fibrinogen, our group first noticed N'-tosyl-lysine methyl ester (TLMe) was a very weak and relatively specific inhibitor as well as substrate on plasmin. A number of derivatives of TLMe including S-2251 and its homologs substrates were re-examined and results obtained were theoretically summarized according to the Free and Wilson method. Thus, the D-Ile-Phe-LyspNA was found to be the more sensitive substrate than its homologs (Table 4) (20). Interesting was that D-Ile was significant, which was well coincident with the theoretical prediction on the structure of suitable substrate of plasmin, proposed by Szabo et al. (21).
about
Stimulated by the presence of the sensitive substrate mentioned above, 900 kinds of new compounds were synthesized in our laboratories and
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TABLE 4 Kinetic Parameters
for the Amidolysis
Substrate D-Ile-Phe-Lys-pNA 3-MV-Phe-Lys-pNA Ile-Phe-Lys-pNA
of Substrate by Plasmin
Km (mM) 0.02 0.18 0.33
-1
kcat (s
)
10 18 19
3-MV: 3-methylvaleryl
Ki for thrombin
FIG. 2 Chemical structure of OS 153
potent and reversible inhibitors of plasmin were several kinds of selective, obtained, chemical structure of one of them called OS 153 being shown in Fig. 2. characterized with the inhibitors belong to lysine derivatives, These presence of NH2 in the chemical structure which covers the N-terminal of lysine. The radical which covers the C-terminal of lysine is much complicated when compared with pNA. The structure of such synthetic inhibitors may take the structure mold of the active site of plasmin. of t-AMCHA was compared with these of OS group When the activities plasmin inhibiotrs, remarkable differences are found as shown in Table 5. Inhibiting activities of t-AMCHA (and EACA) are characterized by its strong inhibition on fibrinolysis but neither on fibrinogenolysis nor on amidolysis While OS inhibitors inhibit nearly equally of S-series synthetic substrates. not only fibrinolysis by plasmin, but also fibrinogenolysis and also _gmidolysis by plasmin, with Ki values of very low concentration such as_+0 M. In particular, Ki value of OS 324 was found to be in the orde_g of 10 M. This value can be compared with that of MD-805, Ki of which , 10 M.
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TABLE 5 Inhibitory
Effect of t-AMCHA and OS 153 on Amidolysis, and Fibrinogenolysis by Plasmin
Amidolysis(a) Ki (M) t-AMCHA OS 153
-2 4.0X10-6 7.4x10
Fibrinolysis(b) I50 (M)
Fibrinolysis
Fibrinogenolysis(b) I50 (M)
-5 6.0~10_~ 6.1x10
-2 l.Ox10_5 2.0x10
(a) Amidolysis was measured with D-Val-Leu-Lys-pNA as substrate. (b) Fibrinolysis and Fibrinogenolysis,were measured using 0.04% and 0.2% fibrinogen, respectively.
IV. PLASMA KALLIKREIN
AND ITS HIGHLY SELECTIVE
INHIBITORS
It should be recalled that the action of aprotinin has been characterized by its potent inhibition toward glandular kallikrein and plasmin: And that the action of aprotinin toward plasma kallikrein (PK) is approximately 10 times weaker than that to glandular kallikrein. Highly selective synthetic inhibitors of PK has been first reported by the authors, very recently (22). In this review, the novel series of highly selective inhibitors celled PKSI are introduced but briefly. PKSIFig. 3 shows a chemical structure of one of the PK inhibitors. 1007 assumed a tripod structure, the basic skeleton of which was L-arginine. This structure showed trans-4-aminomethylcyclohexanecarbonyl to be at the Nterminal of the arginine molecule and 4-ethoxycarbonylanilide to be at the Cterminal position.
FIG. 3 Chemical
structure of PKSI-1007
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TABLE 6 Enzyme Inhibition
P.Kallikrein Ki (pM) PKSI-1007 (Index) PKSI-0180 (Index) PKSI-0527 (Index)
8.0
(1) 0.47
(1) 0.81
(1)
of PKSI-Inhibitors
G.Kallikrein Ki (FM) 120
Plasmin Ki (pM) 270
(15) 53 (113) >500 (>617)
(34) 20 (43) 390 (481)
Thrombin Ki (pM)
FXa Ki (pM)
>500 >500 (>63) (>63) >500 330 (>1064) (702) ,500 ,500 (>617) (>617)
Substrate: S-2302 for P.kallikrein, S-2266 for G.kallikrein, S-2251 for plasmin, S-2238 for thrombin and S-2222 for factor Xa.
The mode of the inhibition of PKSI inhibitors was competitive toward PK as well as the different enzyme examined. Inhibitory effects of PKSI inhibitors on the enzymes were measured using the synthetic chromogenic substrates, as shown in Table 6. (a) PKSI-1007 showed Ki value of 8.0 PM for PK. By Ki_ of 120 PM for glandular kallikrein, 270 uM for plasmin and more contrast, than 500 PM for thrombin and factor Xa were obtained. (b) PKSI-0180 was more inhibitor than PKSI-1007, potent Ki value for PK being 0.47 PM. (c) PKSI0527 showed that Ki value for PK was 0.81 pM, while Ki values for plasmin, glandular kallikrein, thrombin and factor Xa were 390 PM, >500)~M, >5OOpM and >500 PM, respectively. High selectivity toward PK was thus remarkably demonso far as the enzymes examined concerned. In particular, Ki values strated, plasmin, thrombin and factor Xa were of PKSI-0527 for glandular kallikrein, times larger than that for PK, as indicated by the figures in the hundreds parenthesis in Table 6. In addition, it can be noted that i.v. LD50 was over 100 mg/kg in our preliminary toxicity test, which encourages us to extend the studies toward the in vivo applications. As the preliminary test toward in vivo studies, the intact human plasma was employed and the inhibitory effect of PKSI-0180 on the bradykinin (BK) formation by kaolin as well as effect on PTT were examined. As shown in Fig. of BK formed (left ordinate) in the kaolin-activated human 4, the amount plasma decreased by increasing the concentration of PKSI-0180 (abscissa) concluded the inhibition of BK formation by PKSI-0180 in Results gradually. such a contact activated system. Fig 4 also showed that PKSI-0180 was able to prolong PTT. The right ordinate indicated the ratio of PTT with different of PKSI-0180 to PTT without inhibitor. The results concluded concentration that PK inhibitor was able to prolong PTT presumably by inhbiting PK of the contact system. the ascites of tumor bearing mice was emIn the following studies, The Sarcoma 180 was inoculated into the abdominal cavity of mice and ployed. the the ascites on the 8th day was obtained. The ascites collected underwent The samples were incuto get rid of the kininase activity. acid-treatment bated at 37OC and the aliquots taken out were measured of the amount of BK In the control aliquots, the amount of BK showed remarkable increase formed. The presence of PKSI-0527 of the final with the time course of incubation. showed the significant concentration of 10 p and 100 FM in the ascites
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I
25 PKSI-0180 (Plf)
FIG. 4 Lnhibitory effect of PKSI-0180 on bradykinin formation by kaolin and PT? BK formation: The intact human plasma containing o-phenanthroline was incubated with PKSI-0180 and kaolin (0.2 mg/ml) at 37OC for 2 min. BK levels in the plasma were measured by EIA method using a MARKIT-A-Bradykinin kit (Dainippon Pharmaceutical Co.). PTT was measured with PTT reagent purchased from Behring institute.
600 -
‘= a .
400
-
% .g
300
-
z 3 2 m
200 -
100 oI
0
I
I
10
20 Tim&in)
FIG. 5 Inhibitory effect of PKSI-0527 on BK formation in acid-treated ascites of Sarcoma 180 mice
30
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decrease of the amount of BK, indicating clearly the dose-dependency of the concentration of PKSI-0527. The PKSI-0180 showed similar effect in suppressing the BK formation in the ascites (Fig. 5). PKSI-1007, 0180 and 0527 were obtained as the highly selective inhibitors. However, possible role of PK concerning with the contact system should involve the complicated problems in health and disease. studies The present may provide some tools for the studies in this field; the correlation between coagulation, BK formation, fibrinolysis and possibly platelets aggreagation in the contact system would be extremely complicated; even so we expect that results obtained by using PKSI-inhibitors cast light on a promising perspective to us. Needless to say, the further studies are urgently required and they are now under investigation in our laboratories.
V. SIGNIFICANCE OF PROTEOLYTIC REACTION _ The Present and the Future Recent studies of the different proteases revealed the inherent ability of the enzyme to cleave their substrates with the unexpectedly high selectivity, which could be enough compared with that of the antigen-antibody reaction. In fact, such a high selectivity provided the systematic and stepwise activation of the proteases as seen in the coagulation cascade. The present paper dealt mainly with the results of our investigations conducted to provide the highly selective synthetic inhibitors which may be even the excellent tools for revealing significance of the target proteases, utilities of synthetic so final goal should be to find the clinical compounds. These studies long continued more than 40 years have been very inhibitors much encouraged by knowing the presence of natural with high selectivity just as 0.2PI. Studies of ours have been developing fortunately and selective synthetic inhibitors of trypsin are now under investigation. At the same time, I have learned from Homma that some kinds of virus including AIDS virus may step require the activation by trypsin-like protease in each multiplication (23). This reminded us the activation of the zymogen to the active enzyme by some certain proteases. The future of the investigation of proteases seems to be far more promising than our expectation, which has been cherishing in our mind.
ACKNOWLEDGEMENT in express the cordial thanks to their close co-workers The authors different institutions who contributed immensely. Dr. T. Naito and his coworkers, the institute of Showa Denko have been working closely together for Special their contributions being appreciated. the recent several years, central laboratories, and his co-workers, thanks to Dr. R. Kikumoto Mitsubishi Chemical Industries for the studies of MD-805 and its homologs. To those other close friends who supported us, we ask their pardon for missing even names due to the space allotted.
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KUMON,K., TANAKA,K., NAKAJIMA,N., NAITO,Y. and FUJITA,T. Anticoagulation with a synthetic thrombin inhibitor after cardiovascular surgery and for treatment of disseminated intravascular coagulation. Crit.Care Med. l2, 1039-1043, 1984.
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13. BILEZIKIAN,S.B. and NOSSEL,H.L. Unique pattern of fibrinogen cleavage by human leukocyte proteases. Blood 50, 21-28, 1977. 14. KOPEC,M., WEGRZYNOWICZ,Z., ARDELT,W., TEISSEYRE,E. and LATALL0,Z.S. Effect of elastolytic enzymes from human leukocyte granules and from pig pancreas on plasminogen, fibrinogen, and fibrin. In: Progress in Chemical Fibrinolysis and Thrombolysis. J.F.Davidson, R.M.Rowan, M.M.Samama and P.C.Desnoyers (Eds.) New York: Raven Press, 3, 1978, pp. 417-425. 15. NAGAMATSUIY., ~KAM~T~,u., OKADA,N., AMEMIYA,T., HIRASE,F. and KUBOTA,A. Statistical of malignant tumor cases found in spleen characteristics fibrinolytic protease (SFP) activity distribution of 308 autopsy cases. IGAKU NO AYUMI (in Japanese)=, 273-275, 1983. 16. NAGAMATSUIY., OKAMOTO,U., TSUDA,Y. and OKADA,Y. Human leukocyte elastaselike proteinase purified by affinity chromatography with Suc-L-Tyr-D-LeuD-Val-pNA, and its identification with human spleen fibrinolytic proteinase. Thromos.Haemostas.(Stuttg.) 2, 243-247, 1984. TSUDA,Y. and OKADA,Y. A new polypeptide sub17. OKAMOTO,U., NAGAMATSU,Y., Sue-Tyr-Leu-Val-pNA, specific for spleen fibrinolytic proteinase strate, 97, 28-32, 1980. (SFP). Biochem.Biophys.Res.Commun. .18. Tsuda,Y., and OKAMOTO,U. Suynthesis of peptide OKADA,Y., NAGAMATSU,Y. chloromethyl ketones and examination of their inhibitory effects on human spleen fibrinolytic proteinase (SFP) and human leukocyte elastase (LE). Chem.Pharm.Bull. 35, 3576-3584, 1987. 19. OKAMOTO,S., OKAMOTO,U., OKADA,Y., HIJIKATA-OKUNOMIYA,A., WANAKA,K., HORIE,N. and NAITO,T. Coding, decoding and noise of proteinase: a rational approach to plasmin inhibitors. In: Fundamental and clinical fibrinolysis, F.J.Castellino, P.J.Gaffney, M.M.Samama and A.Takada (Eds.), Amsterdam-Oxford-New York: Excerpta Medica, 1987, pp.67-82. TSUDA,Y., TENO,N., WANAKA,K., SASAKI,K., HIJIKATA,A., NAITO,T. 20. OKADA,Y. Synthesis of plasmin substrates and relationship between and OKAMOTO,S. their structure and plasmin activity. Int.J.Peptide Protein Res. 22, 7985, 1986. POZSGAY,M. and ELODI,P. Investigation of the substrate21. Cs.-SZABO,G., binding site of human plasmin using tripeptidyl-p-nitroanilide substrates. Thromb.Res. 2O, 199-206, 1980. WANAKA,K., HIJIKATA-OKUNOMIYA,A., BOHGAKI,M., OKAMOTO,U., 22. OKAMOTO,S., Highly selective synthetic inhibitors and OKADA,Y. NAITO,T., HORIE,N. with regard to plasma-kallikrein activities. KININ V. (in press). Evidence of proteolytic activation 23. TASHIRO,M. and HOMMA,M. virus in mouse lung. Archives of Virology 77, 127-137, 1983.
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