piperidines and benzamidine

Pit viper venoms contain a number of serine proteinases that exhibit one or more thrombin-like activities on fibrinogen and platelets, this being the case for the kinin-releasing and fibrinogen-clotting KN-BJ from the venom of Bothrops jararaca. A three-dimensional structural model of the KN-BJ2 serine proteinase was built by homology modeling using the snake venom plasminogen activator TSV-PA as a major template and porcine kallikrein as additional structural support. A set of intrinsic buried waters was included in the model and its behavior under dynamic conditions was molecular dynamics simulated, revealing a most interesting similarity pattern to kallikrein. The benzamidine-based thrombin inhibitors alpha-NAPAP, 3-TAPAP, and 4-TAPAP were docked into the refined model, allowing for a more insightful functional characterization of the enzyme and a better understanding of the reported comparatively low affinity of KN-BJ2 toward those inhibitors.

Topics: Amidines; Amino Acid Sequence; Animals; Benzamidines; Binding Sites; Bothrops; Crotalid Venoms; Dipeptides; Glycoproteins; Kallikreins; Models, Molecular; Molecular Sequence Data; Piperidines; Protein Conformation; Sequence Homology, Amino Acid; Serine Endopeptidases; Serine Proteinase Inhibitors; Static Electricity; Structural Homology, Protein; Thrombin; Water

Replacement of the highly basic benzamidine moiety of NAPAP by the moderately basic 1-aminoisoquinoline moiety resulted in thrombin inhibitors with improved selectivity towards trypsin and enhanced Caco-2 cell permeability.

Topics: Administration, Oral; Anticoagulants; Benzamidines; Caco-2 Cells; Dipeptides; Drug Design; Humans; Isoquinolines; Piperidines; Thrombin; Trypsin Inhibitors

In an effort to discover novel nonpeptide glycoprotein IIb/IIIa (GPIIb/IIIa, alpha IIb/beta 3) inhibitors, we investigated RGD mimetics featuring a 3-substituted benzoic acid as the core, benzamidine as the basic moiety, and a series of beta- and alpha-substituted beta-alanine derivatives as aspartic acid surrogates. It was found that the use of beta-methyl beta-alanine slightly improved the anti-aggregant potency in human platelet-rich plasma over the unsubstituted beta-alanine compound, while beta-substitution with a trifluoromethyl group resulted in considerable loss in activity. Significant enhancement (up to 100-fold) in potency was obtained when the beta-alanine was replaced with N2-substituted 1-2,3-diaminopropionic acid derivatives. Among the three types of alpha-substituents (carbamate, amide, and sulfonamide) investigated, no apparent preference was observed with respect to in vitro potency. However, alkyl groups were more favorable than arylalkyl groups (Cbz) in the carbamate analogues. We also investigated piperidine, piperazine, and N-formamidinopiperidine as replacements for the benzamidine moiety. The former two replacements led to a drop in potency while the latter replacement resulted in maintenance of activity as compared with the corresponding benzamidine analogue.

Topics: Alanine; Alkylation; Animals; Aspartic Acid; Benzamides; Benzamidines; Benzoates; Benzoic Acid; beta-Alanine; Carbamates; Esterases; Humans; Liver; Magnetic Resonance Spectroscopy; Oligopeptides; Piperazine; Piperazines; Piperidines; Platelet Aggregation Inhibitors; Platelet Glycoprotein GPIIb-IIIa Complex; Structure-Activity Relationship; Sulfonamides; Swine

The fibrinogen clotting time (FCT) is a measure of thrombin activity, and is used to evaluate the potential thrombogenicity of prothrombin complex concentrates (PCC). We have defined end points for clot formation in this test which allow the measurement in PCC of thrombin concentrations as low as 0.001 IU/ml. The FCT of thrombin and PCC samples which did not contain antithrombin III (ATIII) were the same when measured at 20 degrees C or 37 degrees C. In the presence of ATIII (0.05 or 0.25 IU/ml), samples of PCC which were known to contain thrombin showed shorter FCT at 20 degrees C than at 37 degrees C. Inclusion of both ATIII (0.25 IU/ml) and heparin (4 IU/ml) in PCC ensured the complete inactivation of endogenous thrombin.

Topics: Antithrombin III; Benzamidines; Blood Coagulation; Blood Coagulation Factors; Blood Coagulation Tests; Fibrinogen; Heparin; Hirudins; Humans; Phenylalanine; Piperidines; Reproducibility of Results; Temperature; Thrombin; Thrombosis

Well-diffracting crystals of bovine epsilon-thrombin in complex with several "non-peptidic" benzamidine and arginine-based thrombin inhibitors have been obtained by co-crystallization. The 2.3 A crystal structures of three complexes formed either with NAPAP, 4-TAPAP, or MQPA, were solved by Patterson search methods and refined to crystallographic R-values of 0.167 to 0.178. The active-site environment of thrombin is only slightly affected by binding of the different inhibitors; in particular, the exposed "60-insertion loop" essentially maintains its typical projecting structure. The D-stereoisomer of NAPAP and the L-stereoisomer of MQPA bind to thrombin with very similar conformations, as previously inferred from their binding to bovine trypsin; the arginine side-chain of the latter inserts into the specificity pocket in a "non-canonical" manner. The L-stereoisomer of 4-TAPAP, whose binding geometry towards trypsin was only poorly defined, is bound to the thrombin active-site in a compact conformation. In contrast to NAPAP, the distal p-amidino/guanidino groups of 4-TAPAP and MQPA do not interact with the carboxylate group of Asp189 in the thrombin specificity pocket in a "symmetrical" twin N-twin O manner, but through "lateral" single N-twin O contacts; in contrast to the p-amidino group of 4-TAPAP, however, the guanidyl group of MQPA packs favourably in the pocket due to an elaborate hydrogen bond network, which includes two entrapped water molecules. These thrombin structures confirm previous conclusions of the important role of the intermolecular hydrogen bonds formed with Gly216, and of the good sterical fit of the terminal bulky hydrophobic inhibitor groups with the hydrophobic aryl binding site and the S2-cavity, respectively, for tight thrombin active site binding of these non-peptidic inhibitors. These accurate crystal structures are presumed to be excellent starting points for the design and the elaboration of improved antithrombotics.

Topics: Amidines; Amino Acid Sequence; Animals; Antithrombins; Arginine; Benzamidines; Binding Sites; Cattle; Crystallography; Dipeptides; Fibrinolytic Agents; Hydrogen Bonding; Ligands; Models, Molecular; Molecular Sequence Data; Pipecolic Acids; Piperidines; Protein Conformation; Structure-Activity Relationship; Sulfonamides; Thrombin

The mode of binding of four active-site directed inhibitors to human thrombin has been determined by x-ray crystallographic analysis. The inhibitors studied are benzamidine, PPACK, NAPAP, and MD-805, of which the last three are compounds evolved specifically to inhibit thrombin. Crystal structures were determined in the presence of both the inhibitor and the undecapeptide [des-amino Asp55]hirudin(55-65) which binds distant from the active site. Despite having significantly different chemical structures, NAPAP and MD-805 bind to thrombin in a very similar "inhibitor binding mode" which is not that expected by direct analogy with the binding of substrate. Both inhibitors bind to thrombin in a similar way as to trypsin, but thrombin has an extra loop, the "Tyr-Pro-Pro-Trp loop," not present in trypsin, which gives further binding interactions and is seen to move somewhat to accommodate binding of the different inhibitors. The fact that NAPAP and MD-805 require different stereochemistry for potent inhibition is demonstrated, and its structural basis clarified. The wealth of data on analogs and variants of these lead compounds is shown to be compatible with this inhibitor binding mode.

Topics: Amino Acid Chloromethyl Ketones; Antithrombins; Arginine; Benzamidines; Binding Sites; Dipeptides; Humans; Models, Molecular; Pipecolic Acids; Piperidines; Stereoisomerism; Sulfonamides; Thrombin; Trypsin

1. v([I]) data were obtained for the hydrolysis of the chromogenic substrate H-D-Phe-L-Pip-L-Arg-pNA (S-2238) by native human thrombin in the presence of the synthetic inhibitors Benzamidine and N-dansyl-(p-guanidino)-phenylalanine-piperidide (I-2581). v([S]) data were also obtained in the absence and presence of fixed concentrations of each of the inhibitors. 2. Analysis of the kinetic data was based on the numerical fitting to rate equations of the polynomial quotient type of degree n:m using nonlinear regression methods. The discrimination between rate equations with different degrees was performed by application of the statistical F test. 3. Of eight v([I]) curves fitted, in six cases it was found that degree 1:2 was significantly better than degree 1:1 at a confidence level of 95% or higher; in no case was a significant improvement found with rate equations with a higher number of parameters. For the v([S]) data, of eleven curves fitted it was found that in nine cases degree 2:2 significantly improved degree 1:1 at confidence levels 99% and in one case at a level of 95%; no significant improvement was found with rate equations of higher degree for these data either. 4. Our findings allow us to propose that inhibition of the amidolytic activity of native human thrombin by benzamidine and I-2581 may be accounted for by mechanisms whose v([I]) rate equation will be a minimum of degree 1:2, thus implying a pure inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amides; Amidines; Benzamidines; Humans; Hydrolysis; Indicators and Reagents; Kinetics; Phenylalanine; Piperidines; Thrombin