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

Abstract

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

"Mirror image" antagonists of thrombin-induced platelet activation based on thrombin receptor structure.

“…Both recombinant native and mutant thrombins retained the ability to bind benzamidine (Fig. 2), a reversible inhibitor of serine proteases which specifically occupies the small substrate binding pocket/S 1 subsite (25). The recombinant mutant thrombins were also able to bind and sequester the fluorogenic thrombin substrate D-Phe-Pro-Arg-AFC, thereby blocking its hydrolysis by native thrombin (data not shown).…”

“…Recombinant prothrombin production was determined by enzyme-linked immunosorbent assay (ELISA) and Western blots and the highest yielding clones were grown to confluence in a 24,000-cm2 surface cell "factory" (Nunc, Inter Med, Naperville, IL) in minimum essential medium (MEM) a-nucleoside-deficient medium with 80 nM methotrexate, 100 U/ml penicillin, 100 .g/ml streptomycin, 25 mM Hepes buffer, 5 $ig/ml vitamin K, 0.2 mg/ml proline, and 10% dialyzed bovine calf serum. When the cultures reached full confluence, all medium was removed, all growing surfaces were washed six times with phosphate-buffered saline to remove contaminating bovine prothrombin and thrombin, and cells were grown in MEM a-nucleoside-defi-cient medium containing 100 units/ml penicillin, 100 gg/ml streptomycin, 25 mM Hepes buffer, 5 jg/ml vitamin K, 0.2 mg/ml proline, 1 gg/ml insulin and 5 ytg/ml transferrin for 36-48 h. Recombinant S205A prothrombin was purified from conditioned media by anion exchange chromatography (12), diluted to -100 Ig/ml in 150 mM NaCl, 10mM Tris-HCl, pH 7.4, 0.5% polyethylene glycol (PEG) 6000, and treated for 1 h with prothrombinase complex as previously described (I13). pH was then changed to 7.0 with 1 M HCl and the S205A mutant thrombin-containing solution treated with an -1,000-fold molar excess of (p-amidinophenyl)methanesulfonyl fluoride (APMSF)' to inhibit factor Xa and any bovine thrombin that might contaminate the preparation.…”

Distinguishing the Specificities of Closely Related Proteases

“…Another important observation of the structural studies is that the major distinction between the substrate binding cleft of t-PA and u-PA occurs in the region corresponding to the aryl binding site of thrombin (2,3,36). In u-PA this pocket is partially filled by an insertion of two amino acids (threonine 97A, leucine 97B; chymotrypsin numbering) that is absent in t-PA. Consequently, the aryl biding site is significantly larger in t-PA than in u-PA.…”

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

“…Various X-ray diffraction studies [24][25][26][27][28][29][30] of thrombin-inhibitor clarified that the thrombin active site is mainly defined by the specificity S1 pocket, the hydrophobic Ppocket, and the hydrophobic D-pocket, as is shown in Figs. 1 and 2.…”

“…Complexes of human or bovine a-thrombin with synthetic arginyl peptides have been analyzed by X-ray diffraction studies. [24][25][26][27][28][29][30] The X-ray data suggested that the thrombin active site consists of the specificity (S1) pocket, the hydrophobic proximal pocket (P-pocket), and the hydrophobic distal pocket (D-pocket). The guanidino nitrogen atoms of inhibitors form a salt bridge with Asp189 (numbering of the thrombin amino acid residues is based on the chymotrypsinogen nomenclature introduced by Bode et al) 24,28) in S1 pocket, and additional favorable interactions between the inhibitor and D-pocket or P-pocket enhance the inhibitory potency.…”