RESUMEN
Starting from the weakly active dual CatS/K inhibitor 5, structure-based design supported by X-ray analysis led to the discovery of the potent and selective (>50,000-fold vs CatK) cyclopentane derivative 22 by exploiting specific ligand-receptor interactions in the S2 pocket of CatS. Changing the central cyclopentane scaffold to the analogous pyrrolidine derivative 57 decreased the enzyme as well as the cell-based activity significantly by 24- and 69-fold, respectively. The most promising scaffold identified was the readily accessible proline derivative (e.g., 79). This compound, with an appealing ligand efficiency (LE) of 0.47, included additional structural modifications binding in the S1 and S3 pockets of CatS, leading to favorable in vitro and in vivo properties. Compound 79 reduced IL-2 production in a transgenic DO10.11 mouse model of antigen presentation in a dose-dependent manner with an ED50 of 5 mg/kg.
Asunto(s)
Catepsinas/antagonistas & inhibidores , Inhibidores de Cisteína Proteinasa/síntesis química , Animales , Ciclopentanos/química , Inhibidores de Cisteína Proteinasa/farmacocinética , Humanos , Ratones , Prolina/análogos & derivados , Relación Estructura-ActividadRESUMEN
A series of amides bearing a variety of amidine head groups was investigated as BACE1 inhibitors with respect to inhibitory activity in a BACE1 enzyme as well as a cell-based assay. Determination of their basicity as well as their properties as substrates of P-glycoprotein revealed that a 2-amino-1,3-oxazine head group would be a suitable starting point for further development of brain penetrating compounds for potential Alzheimer's disease treatment.
Asunto(s)
Amidas/química , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Inhibidores de Proteasas/química , Enfermedad de Alzheimer/tratamiento farmacológico , Amidas/metabolismo , Amidas/uso terapéutico , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Ácido Aspártico Endopeptidasas/metabolismo , Sitios de Unión , Humanos , Simulación del Acoplamiento Molecular , Inhibidores de Proteasas/metabolismo , Inhibidores de Proteasas/uso terapéutico , Unión Proteica , Estructura Terciaria de Proteína , Relación Estructura-ActividadRESUMEN
The aspartic protease BACE2 is responsible for the shedding of the transmembrane protein Tmem27 from the surface of pancreatic ß-cells, which leads to inactivation of the ß-cell proliferating activity of Tmem27. This role of BACE2 in the control of ß-cell maintenance suggests BACE2 as a drug target for diabetes. Inhibition of BACE2 has recently been shown to lead to improved control of glucose homeostasis and to increased insulin levels in insulin-resistant mice. BACE2 has 52% sequence identity to the well studied Alzheimer's disease target enzyme ß-secretase (BACE1). High-resolution BACE2 structures would contribute significantly to the investigation of this enzyme as either a drug target or anti-target. Surface mutagenesis, BACE2-binding antibody Fab fragments, single-domain camelid antibody VHH fragments (Xaperones) and Fyn-kinase-derived SH3 domains (Fynomers) were used as crystallization helpers to obtain the first high-resolution structures of BACE2. Eight crystal structures in six different packing environments define an ensemble of low-energy conformations available to the enzyme. Here, the different strategies used for raising and selecting BACE2 binders for cocrystallization are described and the crystallization success, crystal quality and the time and resources needed to obtain suitable crystals are compared.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/química , Ácido Aspártico Endopeptidasas/química , Fragmentos Fab de Inmunoglobulinas/química , Células Secretoras de Insulina/enzimología , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Área Bajo la Curva , Ácido Aspártico Endopeptidasas/genética , Ácido Aspártico Endopeptidasas/metabolismo , Dominio Catalítico , Cristalización , Humanos , Fragmentos Fab de Inmunoglobulinas/metabolismo , Células Secretoras de Insulina/metabolismo , Ratones , Modelos Moleculares , Mutagénesis , Conformación Proteica , Resonancia por Plasmón de Superficie , Difracción de Rayos XRESUMEN
The cysteine protease rhodesain of Trypanosoma brucei parasites causing African sleeping sickness has emerged as a target for the development of new drug candidates. Based on a triazine nitrile moiety as electrophilic headgroup, optimization studies on the substituents for the S1, S2, and S3 pockets of the enzyme were performed using structure-based design and resulted in inhibitors with inhibition constants in the single-digit nanomolar range. Comprehensive structure-activity relationships clarified the binding preferences of the individual pockets of the active site. The S1 pocket tolerates various substituents with a preference for flexible and basic side chains. Variation of the S2 substituent led to high-affinity ligands with inhibition constants down to 2 nM for compounds bearing cyclohexyl substituents. Systematic investigations on the S3 pocket revealed its potential to achieve high activities with aromatic vectors that undergo stacking interactions with the planar peptide backbone forming part of the pocket. X-ray crystal structure analysis with the structurally related enzyme human cathepsin L confirmed the binding mode of the triazine ligand series as proposed by molecular modeling. Sub-micromolar inhibition of the proliferation of cultured parasites was achieved for ligands decorated with the best substituents identified through the optimization cycles. In cell-based assays, the introduction of a basic side chain on the inhibitors resulted in a 35-fold increase in antitrypanosomal activity. Finally, bioisosteric imidazopyridine nitriles were studied in order to prevent off-target effects with unselective nucleophiles by decreasing the inherent electrophilicity of the triazine nitrile headgroup. Using this ligand, the stabilization by intramolecular hydrogen bonding of the thioimidate intermediate, formed upon attack of the catalytic cysteine residue, compensates for the lower reactivity of the headgroup. The imidazopyridine nitrile ligand showed excellent stability toward the thiol nucleophile glutathione in a quantitative in vitro assay and fourfold lower cytotoxicity than the parent triazine nitrile.
Asunto(s)
Catepsina L/química , Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Imidazoles/farmacología , Nitrilos/farmacología , Piridinas/farmacología , Triazinas/farmacología , Trypanosoma brucei brucei/enzimología , Cristalografía por Rayos X , Inhibidores de Cisteína Proteinasa/síntesis química , Inhibidores de Cisteína Proteinasa/química , Relación Dosis-Respuesta a Droga , Humanos , Imidazoles/síntesis química , Imidazoles/química , Ligandos , Modelos Moleculares , Estructura Molecular , Nitrilos/síntesis química , Nitrilos/química , Pruebas de Sensibilidad Parasitaria , Piridinas/síntesis química , Piridinas/química , Relación Estructura-Actividad , Triazinas/síntesis química , Triazinas/química , Trypanosoma brucei brucei/efectos de los fármacosRESUMEN
An extensive fluorine scan of 1,3-oxazines revealed the power of fluorine(s) to lower the pKa and thereby dramatically change the pharmacological profile of this class of BACE1 inhibitors. The CF3 substituted oxazine 89, a potent and highly brain penetrant BACE1 inhibitor, was able to reduce significantly CSF Aß40 and 42 in rats at oral doses as low as 1 mg/kg. The effect was long lasting, showing a significant reduction of Aß40 and 42 even after 24 h. In contrast to 89, compound 1b lacking the CF3 group was virtually inactive in vivo.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Animales , Química Encefálica , Inhibidores Enzimáticos/farmacocinética , Inhibidores Enzimáticos/uso terapéutico , Femenino , Flúor/química , Humanos , Indicadores y Reactivos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Moleculares , Oxazinas/síntesis química , Oxazinas/farmacología , Ratas , Ratas Wistar , Relación Estructura-Actividad , Difracción de Rayos XRESUMEN
The serine protease chymase (EC = 3.4.21.39) is expressed in the secretory granules of mast cells, which are important in allergic reactions. Fynomers, which are binding proteins derived from the Fyn SH3 domain, were generated against human chymase to produce binding partners to facilitate crystallization, structure determination and structure-based drug discovery, and to provide inhibitors of chymase for therapeutic applications. The best Fynomer was found to bind chymase with a KD of 0.9 nM and koff of 6.6x10 (-4) s (-1) , and to selectively inhibit chymase activity with an IC 50 value of 2 nM. Three different Fynomers were co-crystallized with chymase in 6 different crystal forms overall, with diffraction quality in the range of 2.25 to 1.4 Å resolution, which is suitable for drug design efforts. The X-ray structures show that all Fynomers bind to the active site of chymase. The conserved residues Arg15-Trp16-Thr17 in the RT-loop of the chymase binding Fynomers provide a tight interaction, with Trp16 pointing deep into the S1 pocket of chymase. These results confirm the suitability of Fynomers as research tools to facilitate protein crystallization, as well as for the development of assays to investigate the biological mechanism of targets. Finally, their highly specific inhibitory activity and favorable molecular properties support the use of Fynomers as potential therapeutic agents.
Asunto(s)
Proteínas Portadoras/metabolismo , Quimasas/metabolismo , Proteínas Proto-Oncogénicas c-fyn/metabolismo , Dominios Homologos src , Secuencia de Aminoácidos , Sitios de Unión , Biocatálisis/efectos de los fármacos , Proteínas Portadoras/química , Proteínas Portadoras/genética , Dominio Catalítico , Quimasas/química , Quimasas/genética , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , Cinética , Modelos Moleculares , Biblioteca de Péptidos , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-fyn/genéticaRESUMEN
Factor Xa, a serine protease from the blood coagulation cascade, is an ideal enzyme for molecular recognition studies, as its active site is highly shape-persistent and features distinct, concave sub-pockets. We developed a family of non-peptidic, small-molecule inhibitors with a central tricyclic core orienting a neutral heterocyclic substituent into the S1 pocket and a quaternary ammonium ion into the aromatic box in the S4 pocket. The substituents were systematically varied to investigate cation-π interactions in the S4 pocket, optimal heterocyclic stacking on the flat peptide walls lining the S1 pocket, and potential water replacements in both the S1 and the S4 pockets. Structure-activity relationships were established to reveal and quantify contributions to the binding free enthalpy, resulting from single-atom replacements or positional changes in the ligands. A series of high-affinity ligands with inhibitory constants down to K(i)=2 nM were obtained and their proposed binding geometries confirmed by X-ray co-crystal structures of protein-ligand complexes.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Inhibidores del Factor Xa , Isoxazoles/síntesis química , Péptidos/química , Tiofenos/síntesis química , Agua/química , Sitios de Unión , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Factor Xa/química , Factor Xa/genética , Humanos , Isoxazoles/química , Isoxazoles/farmacología , Conformación Molecular , Serina Endopeptidasas/metabolismo , Estereoisomerismo , Termodinámica , Tiofenos/química , Tiofenos/farmacología , Tirosina/genéticaRESUMEN
In two series of small-molecule ligands, one inhibiting human cathepsinâ L (hcatL) and the other MEK1 kinase, biological affinities were found to strongly increase when an aryl ring of the inhibitors is substituted with the larger halogens Cl, Br, and I, but to decrease upon F substitution. X-ray co-crystal structure analyses revealed that the higher halides engage in halogen bonding (XB) with a backbone C=O in the S3â pocket of hcatL and in a back pocket of MEK1. While the S3â pocket is located at the surface of the enzyme, which provides a polar environment, the back pocket in MEK1 is deeply buried in the protein and is of pronounced apolar character. This study analyzes environmental effects on XB in protein-ligand complexes. It is hypothesized that energetic gains by XB are predominantly not due to water replacements but originate from direct interactions between the XB donor (Caryl-X) and the XB acceptor (C=O) in the correct geometry. New X-ray co-crystal structures in the same crystal form (space group P2(1)2(1)2(1)) were obtained for aryl chloride, bromide, and iodide ligands bound to hcatL. These high-resolution structures reveal that the backbone C=O group of Gly61 in most hcatL co-crystal structures maintains water solvation while engaging in XB. An aryl-CF3-substituted ligand of hcatL with an unexpectedly high affinity was found to adopt the same binding geometry as the aryl halides, with the CF3 group pointing to the C=O group of Gly61 in the S3â pocket. In this case, a repulsive F2C-Fâ â â O=C contact apparently is energetically overcompensated by other favorable protein-ligand contacts established by the CF3 group.
Asunto(s)
Catepsina L/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Halógenos/química , MAP Quinasa Quinasa 1/metabolismo , Dominio Catalítico , Catepsina L/antagonistas & inhibidores , Catepsina L/química , Cristalografía por Rayos X , Inhibidores Enzimáticos/farmacología , Ligandos , MAP Quinasa Quinasa 1/antagonistas & inhibidores , MAP Quinasa Quinasa 1/química , Unión Proteica , Relación Estructura-ActividadRESUMEN
A series of (3R,4R)-pyrrolidine-3,4-dicarboxylic acid amides was investigated with respect to their factor Xa inhibitory activity, selectivity, pharmacokinetic properties, and ex vivo antithrombotic activity. The clinical candidate from this series, R1663, exhibits excellent selectivity against a panel of serine proteases and good pharmacokinetic properties in rats and monkeys. A Phase I clinical study with R1663 has been finalized.
Asunto(s)
Inhibidores del Factor Xa , Pirrolidinas/farmacología , Pirrolidinas/químicaRESUMEN
Starting from a hit identified by focused screening, 3-aminopyrrolidine factor Xa inhibitors were designed. The binding mode as determined by X-ray structural analysis as well as the pharmacokinetic behaviour of selected compounds is discussed.
Asunto(s)
Aminoquinolinas/farmacología , Antitrombina III/farmacología , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Factor Xa/metabolismo , Aminoquinolinas/química , Aminoquinolinas/metabolismo , Aminoquinolinas/farmacocinética , Antitrombina III/química , Antitrombina III/metabolismo , Antitrombina III/farmacocinética , Cristalografía por Rayos X , Factor Xa/química , Humanos , Modelos Moleculares , Conformación MolecularRESUMEN
In the completion of our fluorine scan of tricyclic inhibitors to map the fluorophilicity/fluorophobicity of the thrombin active site, a series of 11 new ligands featuring alkyl, alkenyl, and fluoroalkyl groups was prepared to explore fluorine effects on binding into the hydrophobic proximal (P) pocket, lined by Tyr 60A and Trp 60D, His 57, and Leu 99. The synthesis of the tricyclic scaffolds was based on the 1,3-dipolar cycloaddition of azomethine ylides, derived from L-proline and 4-bromobenzaldehyde, with N-(4-fluorobenzyl)maleimide. Introduction of alkyl, alkenyl, and partially fluorinated alkyl residues was achieved upon substitution of a sulfonyl group by mixed Mg/Zn organometallics followed by oxidation/deoxyfluorination, as well as oxidation/reduction/deoxyfluorination sequences. In contrast, the incorporation of perfluoroalkyl groups required a stereoselective nucleophilic addition reaction at the "upper" carbonyl group of the tricycles, thereby yielding scaffolds with an additional OH, F, or OMe group, respectively. All newly prepared inhibitors showed potent biological activity, with inhibitory constants (K(i) values) in the range of 0.008-0.163 microM. The X-ray crystal structure of a protein-ligand complex revealed the exact positioning of a difluoromethyl substituent in the tight P pocket. Fluorophilic characteristics are attributed to this hydrophobic pocket, although the potency of the inhibitors was found to be modulated by steric rather than electronic factors.
Asunto(s)
Antitrombinas/química , Antitrombinas/farmacología , Flúor/química , Antitrombinas/síntesis química , Cristalografía por Rayos X , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Estructura Molecular , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectrofotometría InfrarrojaRESUMEN
Two series of tricyclic inhibitors of the serine protease thrombin, imides (+/-)-1-(+/-)-8 and lactams (+/-)-9-(+/-)-13, were analysed to evaluate contributions of orthogonal multipolar interactions with the backbone C=O moiety of Asn98 to the free enthalpy of protein-ligand complexation. The lactam derivatives are much more potent and more selective inhibitors (K(i) values between 0.065 and 0.005 microM, selectivity for thrombin over trypsin between 361- and 1609-fold) than the imide compounds (Ki values between 0.057 and 23.7 microM, selectivity for thrombin over trypsin between 3- and 67-fold). The increase in potency and selectivity is explained by the favorable occupancy of the P-pocket of thrombin by the additional isopropyl substituent in the lactam derivatives. The nature of the substituent on the benzyl ring filling the D pocket strongly influences binding potency in the imide series, with Ki values increasing in the sequence: F < OCH2O < Cl < H < OMe < OH < N(pyr)<< Br. This sequence can be explained by both steric fit and the occurrence of orthogonal multipolar interactions with the backbone C[double bond, length as m-dash]O moiety of Asn98. In contrast, the substituent on the benzyl ring hardly affects the ligand potency in the lactam series. This discrepancy was clarified by the comparison of X-ray structures solved for co-crystals of thrombin with imide and lactam ligands. Whereas the benzyl substituents in the imide inhibitors are sufficiently close (< or =3.5 Angstroms) to the C=O group of Asn98 to allow for attractive orthogonal multipolar interactions, the distances in the lactam series are too large (> or =4 Angstroms) for attractive dipolar contacts to be effective.
Asunto(s)
Imidas/química , Lactamas/química , Inhibidores de Serina Proteinasa/química , Trombina/antagonistas & inhibidores , Sitios de Unión , Cristalografía por Rayos X , Ciclización , Imidas/farmacología , Cinética , Lactamas/farmacología , Modelos Químicos , Unión Proteica , Inhibidores de Serina Proteinasa/farmacología , Trombina/química , Trombina/metabolismoRESUMEN
Carnitine palmitoyltransferases 1 and 2 (CPTs) facilitate the import of long-chain fatty acids into mitochondria. Modulation of the catalytic activity of the CPT system is currently under investigation for the development of novel drugs against diabetes mellitus. We report here the 1.6 A resolution structure of the full-length mitochondrial membrane protein CPT-2. The structure of CPT-2 in complex with the generic CPT inhibitor ST1326 ([R]-N-[tetradecylcarbamoyl]-aminocarnitine), a substrate analog mimicking palmitoylcarnitine and currently in clinical trials for diabetes mellitus treatment, was solved at 2.5 A resolution. These structures of CPT-2 provide insight into the function of residues involved in substrate binding and determination of substrate specificity, thereby facilitating the rational design of antidiabetic drugs. We identify a sequence insertion found in CPT-2 that mediates membrane localization. Mapping of mutations described for CPT-2 deficiency, a hereditary disorder of lipid metabolism, implies effects on substrate recognition and structural integrity of CPT-2.
Asunto(s)
Carnitina O-Palmitoiltransferasa/química , Cristalografía por Rayos X/métodos , Diabetes Mellitus/metabolismo , Secuencia de Aminoácidos , Animales , Betaína/análogos & derivados , Betaína/química , Sitios de Unión , Carnitina/análogos & derivados , Carnitina/química , Diabetes Mellitus/terapia , Humanos , Metabolismo de los Lípidos , Modelos Químicos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Fenotipo , Unión Proteica , Conformación Proteica , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas/química , Ratas , Especificidad por Sustrato , UltracentrifugaciónRESUMEN
The discovery of a highly potent and selective tissue factor/factor VIIa inhibitor is described. Upon oral administration of its double prodrug in the guinea pig, a dose-dependent antithrombotic effect is observed in an established model of arterial thrombosis without prolonging bleeding time. The pharmacodynamic properties of this selective inhibitor are compared to the behaviour of a mixed factor VIIa/factor Xa inhibitor.
Asunto(s)
Anticoagulantes/síntesis química , Anticoagulantes/farmacología , Inhibidores de Factor de Coagulación Sanguínea/síntesis química , Inhibidores de Factor de Coagulación Sanguínea/farmacología , Factor VIIa/antagonistas & inhibidores , Tromboplastina/antagonistas & inhibidores , Administración Oral , Animales , Tiempo de Sangría , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Cobayas , Humanos , Modelos Moleculares , Estructura Molecular , Profármacos/síntesis química , Profármacos/farmacología , Ratas , Estereoisomerismo , Relación Estructura-Actividad , Trombosis/tratamiento farmacológico , Trombosis/prevención & controlRESUMEN
We describe the structure-based design and synthesis of highly potent, orally bioavailable tissue factor/factor VIIa inhibitors which interfere with the coagulation cascade by selective inhibition of the extrinsic pathway.
Asunto(s)
Anticoagulantes/química , Anticoagulantes/farmacología , Factor VIIa/antagonistas & inhibidores , Inhibidores de Serina Proteinasa/química , Inhibidores de Serina Proteinasa/farmacología , Tromboplastina/antagonistas & inhibidores , Administración Oral , Animales , Anticoagulantes/síntesis química , Disponibilidad Biológica , Cristalografía por Rayos X , Diseño de Fármacos , Glicina/análogos & derivados , Glicina/química , Humanos , Estructura Molecular , Ratas , Inhibidores de Serina Proteinasa/síntesis químicaAsunto(s)
Factor Xa/química , Compuestos de Amonio Cuaternario/química , Sitios de Unión , Cationes/química , Simulación por Computador , Cristalografía por Rayos X , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Factor Xa/metabolismo , Ligandos , Sustancias Macromoleculares/química , Modelos Biológicos , Modelos Químicos , Modelos Moleculares , Estructura Molecular , Estereoisomerismo , Trombina/químicaRESUMEN
Proof of concept experiments have shown that tissue factor/factor VIIa inhibitors have antithrombotic activity without enhancing bleeding propensity. Starting from lead compounds generated by a biased combinatorial approach, phenylglycine amide tissue factor/factor VIIa inhibitors with low nanomolar affinity and good selectivity against other serine proteases of the coagulation cascade were designed, using the guidance of X-ray structural analysis and molecular modelling.
Asunto(s)
Factor VIIa/antagonistas & inhibidores , Fibrinolíticos/síntesis química , Glicina/análogos & derivados , Glicina/síntesis química , Glicina/farmacología , Tromboplastina/antagonistas & inhibidores , Diseño de Fármacos , Fibrinolíticos/farmacología , Humanos , Cinética , Modelos Moleculares , Estructura Molecular , Inhibidores de Serina Proteinasa/síntesis química , Relación Estructura-ActividadRESUMEN
The serine protease factor VIIa (FVIIa) in complex with its cellular cofactor tissue factor (TF) initiates the blood coagulation reactions. TF.FVIIa is also implicated in thrombosis-related disorders and constitutes an appealing therapeutic target for treatment of cardiovascular diseases. To this end, we generated the FVIIa active site inhibitor G17905, which displayed great potency toward TF.FVIIa (Ki = 0.35 +/- 0.11 nM). G17905 did not appreciably inhibit 12 of the 14 examined trypsin-like serine proteases, consistent with its TF.FVIIa-specific activity in clotting assays. The crystal structure of the FVIIa.G17905 complex provides insight into the molecular basis of the high selectivity. It shows that, compared with other serine proteases, FVIIa is uniquely equipped to accommodate conformational disturbances in the Gln217-Gly219 region caused by the ortho-hydroxy group of the inhibitor's aminobenzamidine moiety located in the S1 recognition pocket. Moreover, the structure revealed a novel, nonstandard conformation of FVIIa active site in the region of the oxyanion hole, a "flipped" Lys192-Gly193 peptide bond. Macromolecular substrate activation assays demonstrated that G17905 is a noncompetitive, slow-binding inhibitor. Nevertheless, G17905 effectively inhibited thrombus formation in a baboon arterio-venous shunt model, reducing platelet and fibrin deposition by approximately 70% at 0.4 mg/kg + 0.1 mg/kg/min infusion. Therefore, the in vitro potency of G17905, characterized by slow binding kinetics, correlated with efficacious antithrombotic activity in vivo.