Design and evaluation of novel bivalent thrombin inhibitors based on amidinophenylalanines

Abstract

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 ...

Exploring ligand recognition and ion flow in comparative models of the human GABA type A receptor

“…Coordinates for the GABA molecule were obtained from the PDB dataset 1QUR [87]. The Cambridge Structural Database was consulted for coordinates for muscimol (CSD code: MUSIMO01) [10], flunitrazepam (CSD code: CAGWUC) [15] and alprazolam (CSD code: CIZQAD01) [72].…”

Direct thrombin inhibitors – a survey of recent developments

“…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].…”

Amidino-Containing Schiff Base Copper(II) and Iron(III) Chelates as a Thrombin Inhibitor

“…[15][16][17] On the other hand, many compounds having benzamidine or arginine moiety as a partial structure have been synthesized and their inhibitory effects on thrombin examined. [18][19][20][21][22][23] Argatroban (MD-805) proposed by Okamoto et al 18) is one of most potent synthetic thrombin inhibitors (K i ϭ1.9ϫ10 Ϫ8 M) and is clinically used. Complexes of human or bovine a-thrombin with synthetic arginyl peptides have been analyzed by X-ray diffraction studies.…”