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

Tsuda, Yuko; Cygler, Mirosław; Gibbs, Bernard F.; Pędyczak, Artur; Féthière, James; Yue, Shi Yi; Konishi, Yasuo

doi:10.1021/bi00252a010

Cited by 31 publications

(21 citation statements)

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“…More recently, the incorporation of optimized, substrate-like tetrapaptides consisting exclusively of natural amino acids, as in 5, was also reported as a successful strategy to confer resistance against thrombin cleavage [75]. More potent inhibitors were obtained by replacing the active-site-binding segment by small-molecule inhibitor segments derived from the nonelectrophilic inhibitors argatroban 10 (to yield 8) [76] or NAPAP 12 [77], and from electrophilic inhibitors such as boronic acids [78], arginyl methyl ketones [79,80] and α-keto amides [81]. Furthermore, a series of bivalent thrombin inhibitors termed hirunorms was generated that contain a peptidic module that blocks the active-site in a nonsubstrate mode [82,83].…”

Section: Early Direct Thrombin Inhibitorsmentioning

confidence: 99%

Direct thrombin inhibitors – a survey of recent developments

Schwienhorst¹

2006

Cell. Mol. Life Sci.

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Thrombin is a plasma serine protease that plays a key role in coagulation and hemostasis but also in thromboembolic diseases. Direct thrombin inhibitors could, therefore, be beneficial for future anticoagulant therapy in the prophylaxis of venous and arterial thrombosis as well as myocardial infarction. However, development of direct thrombin inhibitors has brought researchers more heartache than success. The most recent setback came this year when AstraZeneca withdrew Ximelagatran, the first orally bioavailable direct thrombin inhibitor that had received regulatory approval (France, 2003), after reports of serious hepatoxicity in a fraction of patients. This review describes the status of direct thrombin inhibitors, focusing on drug candidates that are at present in clinical trials. In addition, some more recent research strategies in the design of novel direct thrombin inhibitors are discussed, which may very well contribute to future developments of potent anticoagulants.

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Section: Early Direct Thrombin Inhibitorsmentioning

confidence: 99%

Direct thrombin inhibitors – a survey of recent developments

Schwienhorst¹

2006

Cell. Mol. Life Sci.

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“…19,20 Pip has also been incorporated into several bioactive peptides and used in designing potent inhibitors. 10,21,22 Recently, a Pip-containing pentapeptide has been used to determine the thermodynamic properties of the interaction between the peptidyl prolyl cis-trans isomerase Pin1 and its substrate. 23 The azetidine ring of peptides has both down-and uppuckered conformations, although the degree of puckering is smaller than that of the pyrrolidine ring.…”

Section: Introductionmentioning

confidence: 99%

Conformational Preferences of Proline Analogues with Different Ring Size

Jhon

Kang

2007

J. Phys. Chem. B

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The conformational study on L-azetidine-2-carboxylic acid (Ac-Aze-NHMe, the Aze dipeptide) and (S)-piperidine-2-carboxylic acid (Ac-Pip-NHMe, the Pip dipeptide) is carried out using ab initio HF and density functional methods with the self-consistent reaction field method to explore the differences in conformational preferences and cis-trans isomerization for proline residue and its analogues with different ring size in the gas phase and in solution (chloroform and water). The change of ring size by deleting a CH2 group from or adding a CH2 group to the prolyl ring results the remarkable changes in backbone and ring structures compared with those of the Pro dipeptide, especially in the C'-N imide bond length and the bond angles around the N-C(alpha) bond. The four-membered azetidine ring can have either puckered structure depending on the backbone structure because of the less puckered structure. The six-membered piperidine ring can adopt chair and boat conformations, but the chair conformation is more preferred than the boat conformation. These calculated preferences for puckering are consistent with experimental results from analysis of X-ray structures of Aze- and Pip-containing peptides. On going from Pro to Aze to Pip, the axiality (i.e., a tendency to adopt the axial orientation) of the NHMe group becomes stronger, which can be ascribed to reduce the steric hindrances between 1,2-substituted Ac and NHMe groups. As the solvent polarity increases, the polyproline II-like conformation becomes more populated and the relative stability of conformation tC with a C7 hydrogen bond between C'=O of the amino group and N-H of the carboxyl group decreases for both the Aze and Pip dipeptides, as seen for the Pro dipeptide. The cis population and rotational barriers for the imide bond increase with the increase of solvent polarity for both the Aze and Pip dipeptides, as seen for the Pro dipeptide. In particular, the cis-trans isomerization proceeds in common through only the clockwise rotation with omega' approximately +120 degrees about azetyl and piperidyl peptide bonds in the gas phase and in solution, as seen for alanyl and prolyl peptide bonds. The pertinent distance d(N...H-N(NHMe)) and the pyramidality of imide nitrogen can describe the role of this hydrogen bond in stabilizing the transition state structure, but the lower rotational barriers for the Aze and Pip dipeptides than those for the Pro dipeptide, which is observed from experiments, cannot be rationalized.

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“…This is a ≈ 320‐fold improvement in the inhibition constant compared with the racemic active site inhibitor Nas‐ S,R m Adf‐iNip‐NHMe. However, this affinity was significantly lower than found for bivalent inhibitors with arginine‐derived, active‐site‐directed segments [8,11,14]. Therefore, we assume that the coupling to the linker‐exo site inhibitor portion probably induces a disturbed binding of the active site‐directed part of BZA‐1 hirulog.…”

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confidence: 77%

“…A second strategy [14] was the incorporation of an arylsulfonyl‐arginyl‐ R ‐pipecolic acid active‐site‐directed segment derived from the potent thrombin inhibitor argatroban [15], which also eliminated the scissile peptide bond after the P1‐arginyl residue. A combination of this active‐site‐directed moiety with optimized linker and FRE‐directed inhibitor segments resulted in compounds with inhibition constants similar to r‐hirudin (P613, K i = 270 f m ; [8]).…”

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confidence: 99%

Design and evaluation of novel bivalent thrombin inhibitors based on amidinophenylalanines

Steinmetzer¹,

Renatus²,

Künzel³

et al. 1999

European Journal of Biochemistry

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Two bivalent thrombin inhibitors were synthesized, which consist of a benzamidine-based active-site-blocking segment, a fibrinogen recognition exosite inhibitor and a peptidic linker connecting these fragments. BZA-1 hirulog contains an N a -(2-naphthylsulfonyl)-S-3-amidinophenylalanyl-isonipecotic acid residue connected via the carboxyl group to the linker segment. The active-site-directed moiety of BZA-2 hirulog [N a -(2-naphthylsulfonyl-glutamyl)-R-4-amidinophenylalanyl-piperidide] was coupled to the linker via the side chain of the glutamic acid. Both BZA-hirulogs contain almost identical linker-exo site inhibitor parts, except for the substitution of a glycine as the first linker residue in BZA-1 hirulog by a g-amino butyric acid in BZA-2 hirulog, thus increasing flexibility and linker length by two additional atoms. BZA-1 hirulog showed moderate potency (K i = 0.50^0.14 nm), while BZA-2 hirulog was characterized as a slow, tight binding inhibitor of thrombin (K i = 0.29^0.08 pm). The stability in human plasma of both analogs was strongly improved compared with hirulog-1. For BZA-2 hirulog a significantly reduced plasma clearance was observed after intravenous injection in rats compared with BZA-1 hirulog and hirulog-1. The X-ray structure of the BZA-2 hirulog in complex with human a-thrombin was solved and confirmed the expected bivalent binding mode.Keywords: anticoagulants; bivalent inhibitor; enzyme kinetics; hirudin; thrombin.The hirulogs [1,2] are highly potent and specific synthetic thrombin inhibitors derived from the naturally occurring 65-amino acid polypeptide hirudin, which was originally isolated from the medicinal leech [3]. Similar to hirudin, the hirulogs inhibit thrombin by a bivalent binding mode. The C-terminus of the hirulogs is almost identical to the C-terminal residues H53±65 of the desulfated r-hirudin (prefix H used for numbering based on the hirudin sequence), which bind to the fibrinogen recognition exo site (FRE) of thrombin. This FRE-directed inhibitor segment is connected by a peptidic linker to an additional inhibitor moiety, which occupies the active site of thrombin.The prototype, hirulog-1 (dPhe-Pro-Arg-Pro-(Gly) 4 -Asn-GlyAsp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-OH; K i = 2. [7,8] enhanced the affinity of the inhibitors by 1±2 orders of magnitude. However, the strongest improvements in activity were achieved by incorporation of more potent and proteolytically stable active site inhibitor segments. One strategy was the introduction of transition-state analogs containing different P1-residues, like arginyl methyl ketones [9±11], a-keto-amides [12] or boronic acid derivatives [13].A second strategy [14] was the incorporation of an arylsulfonyl-arginyl-R-pipecolic acid active-site-directed segment derived from the potent thrombin inhibitor argatroban [15], which also eliminated the scissile peptide bond after the P1-arginyl residue. A combination of this active-site-directed moiety with optimized linker and FRE-directed inhibitor segments resulted in compounds with inhibition ...

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

Cited by 31 publications

References 50 publications

Direct thrombin inhibitors – a survey of recent developments

Direct thrombin inhibitors – a survey of recent developments

Conformational Preferences of Proline Analogues with Different Ring Size

Design and evaluation of novel bivalent thrombin inhibitors based on amidinophenylalanines

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