Inhibition of bovine and human thrombins by derivatives of benzamidine

Stürzebecher, Jörg; Walsmann, P; Voigt, B.; Wagner, G.

doi:10.1016/0049-3848(84)90302-5

Cited by 38 publications

(18 citation statements)

References 10 publications

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“…As noted from the benzamidine-binding isotherm for m-IV IIa or mzIIa-Fl (Fig. 3, Inset), all of the derivatives saturated (i.e., reached a maximum change in 2T) between 1.5 and 2.0 mM benzamidine, which agrees somewhat with the Ks (1.4mM) for bovine c~-thrombin (Sturzebecher et al, 1984). The KD values found in this work were 1.0 + 0.1 mM for both a-thrombin and meizothrombin(desF1).…”

Section: Benzamidine Binding To Labeled Thrombinssupporting

confidence: 75%

“…In order to probe other conformational properties of the mzIIa-Fl active site we examined the effects of binding the reversible thrombin inhibitor benzamidine, which binds in the basic specificity pocket of the active site (Sturzebecher et al, 1984;Brandstetter et aL, 1992). Some examples of the changes in the ESR spectra of labeled o~-thrombin and meizothrombin(desF1) derivatives are shown in Figs.…”

Section: Benzamidine Binding To Labeled Thrombinsmentioning

confidence: 99%

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Comparison of the active-site conformations of bovine α-thrombin and meizothrombin(desF1) by electron spin resonance

Boxrud

Berliner

1996

J Protein Chem

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The active site of the prothrombin activation intermediate meizothrombin(desF1) was probed using several fluorosulfonylphenyl spin labels specific for the active serine hydroxyl of serine proteases. The mobilities of the thrombin species inhibited with the nitroxide spin labels m-IV [4-(2,2,6,6-tetramethyl-piperidine-1-oxyl) -m-(fluorosulfonyl)benzamide] and m-V [3-(2,2,5,5-tetramethyl-pyrrolidine-1-oxyl) -m-(fluorosulfonyl)benzamide], which are sensitive to differences between alpha- and gamma-thrombin, were quite similar to that of alpha-thrombin. That is, no major conformational differences between meizothrombin(desF1) and alpha-thrombin were observed in this region of the extended active site. On the other hand, p-IV [4-(2,2,6,6-tetramethyl piperidine-1-oxyl)-p-(fluorosulfonyl)benzamide], p-V [3-(2,2,5,5-tetramethylpyrrolidine-1-oxyl) -p-(fluorosulfonyl)benzamide], and m-VII [N-[m- (fluorosulfonyl)phenyl]-4-N-(2,2,6,6-tetramethyl- piperidine-1-oxyl)urea], which probe an apolar binding region of bovine thrombin, exhibited large differences in mobility between alpha-thrombin and meizothrombin(desF1). The conformational consequences of indole binding to spin-labeled thrombin species demonstrated that both species also possess an indole-binding site. However, the nitroxide mobility changes upon indole binding to the spin-labeled protein derivative were somewhat different between the two thrombin species under study. In addition, the effects of the benzamidine binding were quite similar for each labeled protein. Thus is appears that, while both species posses a fully functional active site, the region in meizothrombin(desF1) probed by spin labels p-IV, p-V, and m-VII, which corresponds to the apolar binding region, differs in conformation from alpha-thrombin.

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Section: Benzamidine Binding To Labeled Thrombinssupporting

confidence: 75%

Section: Benzamidine Binding To Labeled Thrombinsmentioning

confidence: 99%

Comparison of the active-site conformations of bovine α-thrombin and meizothrombin(desF1) by electron spin resonance

Boxrud

Berliner

1996

J Protein Chem

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“…In fact, the active site blocking moiety, D-Phe-Pro-Arg-Pro, of the bivalent inhibitors was hydrolyzed slowly at Arg-Pro peptide bond (DiMaio et al, 1990;Witting et al, 1992a;Szewczuk et al,1993). The arginine and benzamidine-based thrombin inhibitors such as MD-805, NAPAP, and TAPAP are other types of active site inhibitors (Okamoto et al, 1980(Okamoto et al, , 1981Kikumoto et al, 1980aKikumoto et al, ,b, 1984Stiirzebecher et al, 1983Stiirzebecher et al, , 1984. The characteristic sequence of these compounds is hydrophobic-Arg (or benzamidine analogs)-piperidine (or analogs).…”

mentioning

confidence: 99%

Design of Potent Bivalent Thrombin Inhibitors Based on Hirudin Sequence: Incorporation of Nonsubstrate-Type Active Site Inhibitors

Tsuda¹,

Cygler²,

Gibbs³

et al. 1994

Biochemistry

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Hirudin from medicinal leech is the most potent and specific thrombin inhibitor from medicinal leech with a K, value of 2.2 x M. It consists of an active site blocking moiety, hirudin'-48, a fibrinogen-recognition exo-site binding moiety, hir~din~~-~~, and a linker, hir~din~~-~~, connecting these inhibitor moieties. Synthetic inhibitors were designed based on the C-terminal portion of hirudin. The bulky active site blocking moiety, was replaced by small nonsubstrate-type active site inhibitors of thrombin, e.g., dansyl-Arg-(D-pipecolic acid). The linker moiety was replaced by o-amino acids of (12-aminododecanoic acid)-(4-aminobutyric acid), and hirudi~P-~~ was used as a fibrinogen-recognition exo-site binding moiety in most of the inhibitors. The crystal structure of the inhibitor in complex with human a-thrombin showed that dansyl, Arg, and D-pipecolic acid of the active site blocking moiety occupy S3, S1, and S2 subsites of thrombin, respectively, and were therefore designated as P3, P1, and P2 residues.The use of dansyl-Arg-(D-pipecolic acid) improved the affinity (KJ of the inhibitor 10-100-fold (down to 1.70 x lo-" M) compared to that of the similar compounds having D-Phe-Pro-Arg as their substratetype inhibitor moiety (K = 10-9-10-10 M). The linker connected to P2 residue eliminated the scissile peptide bond. The inhibitor was also stable against human plasma proteases. Further inhibitor design revealed that the toxic dansyl group could be replaced by 4-tert-butylbenzenesulfonyl group and 1-or 2-naphthalenesulfonyl group for in vivo studies. In addition, the replacement of hir~din~~-~~ with [TY~~~,-

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“…(Received April 4/June 11,1990) -EJB 90 0374 The X-ray crystal structure of the trypsin complex formed with N"-(2-naphthyl-sulphonyl-glycyl)-~~-pamidinophenylalanyl-piperidine (NAPAP) was determined with X-ray data to 0.18-nm resolution and crystallographically refined. NAPAP binds into the active site of trypsin in a quite compact form: the pamidinophenylalanine moiety of the D-stereoisomer binds into the specificity pocket; the glycyl group is hydrogen bonded with Gly216; the naphthyl group stands perpendicular to the indole moiety of Trp215; the piperidine ring is tightly packed between this naphthyl moiety and His.57; in consequence the carboxy-terminal amido bond of NAPAP is located in such a way that it is not susceptible to the active-site Ser195.…”

mentioning

confidence: 99%

Geometry of binding of the benzamidine‐ and arginine‐based inhibitors N^α‐(2‐naphthyl‐sulphonyl‐glycyl)‐dl‐p‐amidinophenylalanyl‐piperidine (NAPAP) and (2R,4R)‐4‐methyl‐1‐[N^α‐(3‐methyl‐1,2,3,4‐tetrahydro‐8‐quinolinesulphonyl)‐l‐arginyl]‐2‐piperidine carboxylic acid (MQPA) to human α‐thrombin

Bode

Turk

Stürzebecher

1990

European Journal of Biochemistry

125

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The X-ray crystal structure of the trypsin complex formed with N"-(2-naphthyl-sulphonyl-glycyl)-~~-pamidinophenylalanyl-piperidine (NAPAP) was determined with X-ray data to 0.18-nm resolution and crystallographically refined. NAPAP binds into the active site of trypsin in a quite compact form: the pamidinophenylalanine moiety of the D-stereoisomer binds into the specificity pocket; the glycyl group is hydrogen bonded with Gly216; the naphthyl group stands perpendicular to the indole moiety of Trp215; the piperidine ring is tightly packed between this naphthyl moiety and His.57; in consequence the carboxy-terminal amido bond of NAPAP is located in such a way that it is not susceptible to the active-site perfectly into the much more restricted active site of thrombin. The accommodation of the 5'-aryl moieties in the 'aryl-binding site' and of the piperidine rings in the S2 subsite of thrombin are particularly favourable. The preference of thrombin for distinctly substituted piperidine derivatives and its generally higher (compared with trypsin) affinity for benzamidine and arginine-based inhibitors can be accounted for by these thrombin inhibitor models.Thrombin, a trypsin-like serine proteinase, plays a central role in thrombosis and hemostasis. a-Thrombin converts fibrinogen into fibrin which forms part of the blood clot. It has also been implicated in platelet activation, activates other blood coagulation factors and interacts with various cells. The formation of clots in various thrombotic diseases has been attributed to the action of thrombin (see [l]). In cases of thrombotic disorder the quick supplementation with selective thrombin inhibitors (besides antithrombin 111) might offer an attractive means of therapy. Besides hirudin, an extremely potent and specific protein inhibitor from the medical leech [2 -61, various synthetic thrombin inhibitors have been foundCarrespondence to

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Inhibition of bovine and human thrombins by derivatives of benzamidine

Cited by 38 publications

References 10 publications

Comparison of the active-site conformations of bovine α-thrombin and meizothrombin(desF1) by electron spin resonance

Comparison of the active-site conformations of bovine α-thrombin and meizothrombin(desF1) by electron spin resonance

Design of Potent Bivalent Thrombin Inhibitors Based on Hirudin Sequence: Incorporation of Nonsubstrate-Type Active Site Inhibitors

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