Thrombin active site inhibitors

Das, Jagabandhu; Kimball, S. David

doi:10.1016/0968-0896(95)00104-o

Cited by 88 publications

(37 citation statements)

References 67 publications

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“…This is in contrast with the results obtained for low molecular weight inhibitors, where it is paradigmatic that the introduction of a positive charge at P1 position improves binding, but strongly reduces selectivity~Cleason, 1994; Das & Kimball, 1995;Sanderson & Naylor-Olsen, 1998!. Taken together, these results are in keeping with our proposal~De Filippis et al, 1998!…”

Section: Thrombin Inhibitioncontrasting

confidence: 57%

“…Protease selectivity: The issue of protease selectivity is of crucial importance in the design of thrombin inhibitors, because inhibition of other physiologically relevant serine-proteases, also including those of the fibrinolytic pathway~tPA and plasmin!, can compromise their clinical use~Tapparelli et al, 1993;Cleason, 1994;Das & Kimball, 1995;Fenton et al, 1998;Sanderson & Naylor-Olsen, 1998! and has been recognized as the major determinant of severe hypotension and respiratory depression observed in vivo~Haupt-mann & Markwardt, 1992!.…”

Section: Thrombin Inhibitionmentioning

confidence: 99%

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Incorporation of noncoded amino acids into the N‐terminal domain 1‐47 of hirudin yields a highly potent and selective thrombin inhibitor

et al. 1999

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Abstract:Hirudin is an anticoagulant polypeptide isolated from a medicinal leech that inhibits thrombin with extraordinary potencỹ K d ϭ 0.2-1.0 pM! and selectivity. Hirudin is composed of a compact N-terminal region~residues 1-47, cross-linked by three disulfide bridges! that binds to the active site of thrombin, and a flexible C-terminal tail~residues 48-64! that interacts with the exosite I of the enzyme. To minimize the sequence of hirudin able to bind thrombin and also to improve its therapeutic profile, several N-terminal fragments have been prepared as potential anticoagulants. However, the practical use of these fragments has been impaired by their relatively poor affinity for the enzyme, as given by the increased value of the dissociation constant~K d ! of the corresponding thrombin complexes~K d ϭ 30-400 nM!. The aim of the present study is to obtain a derivative of the N-terminal domain 1-47 of hirudin displaying enhanced inhibitory potency for thrombin compared to the natural product. In this view, we have synthesized an analogue of fragment 1-47 of hirudin HM2 in which Val1 has been replaced by tert-butylglycine, Ser2 by Arg, and Tyr3 by b-naphthylalanine, to give the BugArgNal analogue. The results of chemical and conformational characterization indicate that the synthetic peptide is able to fold efficiently with the correct disulfide topology~Cys6-Cys14, Cys16-Cys28, Cys22-Cys37!, while retaining the conformational properties of the natural fragment. Thrombin inhibition data indicate that the effects of amino acid replacements are perfectly additive if compared to the singly substituted analogues~De Filippis V, Quarzago D, Vindigni A, Di Cera E, Fontana A, 1998, Biochemistry 37:13507-13515!, yielding a molecule that inhibits the fast or slow form of thrombin by 2,670-and 6,818-fold more effectively than the natural fragment, and that binds exclusively at the active site of the enzyme with an affinity~K d,fast ϭ 15.4 pM, K d,slow ϭ 220 pM! comparable to that of full-length hirudin~K d,fast ϭ 0.2 pM, K d,slow ϭ 5.5 pM!. Moreover, BugArgNal displays absolute selectivity for thrombin over the other physiologically important serine proteases trypsin, plasmin, factor Xa, and tissue plasminogen activator, up to the highest concentration of inhibitor tested~10 mM!.Keywords: anticoagulant peptides; hirudin; noncoded amino acids; peptide synthesis; thrombin Thrombin is a serine protease that plays a pivotal role in maintaining the intricate balance between hemostasis and thrombolysis Davie et al., 1991!, and for this reason it remains the primary target of antithrombotic therapy~Fenton et al., 1998!. Among natural anticoagulants, hirudin is the most potent and specific inhibitor of thrombin~Markwardt, 1994!. Hirudin is a polypeptidẽ ;7,000 Da! isolated from the salivary secretion of the medicinal leech and is composed of a compact N-terminal region~residues 1-47! cross-linked by three disulfide bridges and a flexible negatively charged C-terminal tail~48-64!~Folkers et al. Abbreviations: Standard three-lett...

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Section: Thrombin Inhibitioncontrasting

confidence: 57%

Section: Thrombin Inhibitionmentioning

confidence: 99%

Incorporation of noncoded amino acids into the N‐terminal domain 1‐47 of hirudin yields a highly potent and selective thrombin inhibitor

et al. 1999

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“…These alternatives include trifluoromethyl ketones, ␣-ketoesters and amides, ␣-diketones and ␣-ketoheterocycles. 18,19 Along separate lines, the finding that organoboronic acids could inhibit serine proteases through the formation of a boronate ester with the active site serine led to the development of potent peptidylboronate elastase 20 and thrombin 21 inhibitors. Many other strategies for acylating or alkylating the active site serine of serine proteases have been described (see Ref.…”

Section: B Covalent Serine Protease Inhibitorsmentioning

confidence: 98%

Small, noncovalent serine protease inhibitors

Sanderson

1999

Med. Res. Rev.

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“…In general, the antithrombotic strategies are based on the inhibition of platelet aggregation and adhesion by blocking the primary stimulus caused by several agonists like thrombin, ADP, collagen etc. Inhibition of an agonist function by anticoagulant materials prevents the blood clotting and maintains its fluidity (Ojima et al, 1995;Das et al, 1995). The 65 amino acids peptide hirudin, secreted from the salivary gland of medicinal leech (Owen et al, 1988), as well as the synthetic 20 amino acids peptide hirulog-1 (Witting et al, 1992) are directacting potent inhibitors of thrombin.…”

Section: Introductionmentioning

confidence: 99%

“…In general a long-term therapy for prevention of myocardial infarction requires oral dosing of anticoagulant, so the trend is to search for small peptides or mimetics to be used as antithrombotic drugs. Furthermore, other non-peptidic molecules, like warfarin (a coumarin derivative) and aspirin are indirect-acting anticoagulants and they are currently been used for preventing myocardial infarction (Das et al, 1995;Weiss et al, 1967;Baenziger et al, 1977).…”

Section: Introductionmentioning

confidence: 99%

Synthesis of salicyl-peptides and their effect on human platelet aggregationin vitro

Stavropoulos

Magafa

Liakopoulou-Kyriakides

et al. 1997

Amino Acids

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Summary.A series of leucine dipeptide amides containing at their N-terminal amino group the salicyl-residue [(o)-RO-C<,H4-CO-, where R=H or CH3CO] have been synthesized by conventional solution techniques and tested for their inhibitory activity on human platelet aggregation in vitro induced by collagen, ADP or adrenaline. The salicyl-peptide (o)-HO-C6H4-CO-LeuAsp(OBzl)-NH2 was found to exert strong inhibitory activity on platelet aggregation induced by collagen with an ICs0 value 4.5raM. The corresponding dipetide H2N-Leu-Asp(OBzl)-NH 2 was also examined and was found to be less active, indicating that the presence of the lipophilic fi-benzyl ester group in combination with the salicyl group enhance the inhibitory activity. All the other salicyl-peptides examined either didn't show any inhibitory or aggregatory activity or a slight inhibition at the concentration of 9-10 raM.

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Thrombin active site inhibitors

Cited by 88 publications

References 67 publications

Incorporation of noncoded amino acids into the N‐terminal domain 1‐47 of hirudin yields a highly potent and selective thrombin inhibitor

Incorporation of noncoded amino acids into the N‐terminal domain 1‐47 of hirudin yields a highly potent and selective thrombin inhibitor

Small, noncovalent serine protease inhibitors

Synthesis of salicyl-peptides and their effect on human platelet aggregationin vitro

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