Proposed cation‐π mediated binding by factor Xa: a novel enzymatic mechanism for molecular recognition

Lin, Zhaolan; Johnson, Michael E.

doi:10.1016/0014-5793(95)00811-m

Cited by 44 publications

(37 citation statements)

References 37 publications

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“…(ii) the overall inhibitor conformation proposed by Lin and Johnson [13] is much closer to that which we see. Due to a slight inclination of the napthalene group, however, the pyrrolidinyl group does not extend as far along the enzyme surface.…”

Section: Discussionsupporting

confidence: 81%

“…Structure-activity relationships for DX9065a have shown that the distal basic moiety plays a major role in the inhibition of factor Xa, whilst the propionic acid group is responsible for its lack of activity against thrombin [10]. Molecular modelling has attributed the basicity requirement variously to interaction with factor Xa Glu-97 [10], the carbonyl group of Glu-97 [12] or to a novel cationqr interaction with the aromatic rings of Trp-215, Tyr-99 and Phe-174 [13]. Modelling studies have further proposed that selectivity against thrombin is due to electrostatic repulsion between the side chain of thrombin in factor Xa) and the propionic moiety [10].…”

Section: Discussionmentioning

confidence: 99%

“…Daiichi compound DX9065a ((+)-2-[4-[((3S)-l-acetimidoyl-3-pyrrodinyl)oxy]phenyl]-3-(7-amidino-2-napthyl)propionic acid) exhibits a K~ of 41 nM for factor Xa, no activity against thrombin and displays some oral activity [11]. The inhibitor has been modelled to the active sites of factor Xa, thrombin and human trypsin [10,[12][13]. In each case, the models suggest an extended conformation of the inhibitor.…”

Section: Introductionmentioning

confidence: 99%

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Crystal structures of factor Xa specific inhibitors in complex with trypsin: structural grounds for inhibition of factor Xa and selectivity against thrombin

1995

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Crystal structures of DX9065a and a related bisamidino-aryl inhibitor specific for the blood-clotting factor Xa have been solved in complex with bovine ~-trypsin to a resolution of 1.9 A. Each inhibitor exhibits an extended conformation along the active site, in contrast to the compact folded structures observed for thrombin specific inhibiturs. Few direct contacts (predominantly in the S1 pocke0 are made between trypsin and the inhibitors. Transfer of the inhibitors to the active site of factor Xa suggests a three-site interaction: salt bridge formation at the base of the primary specificity pocket, extensive hydrophobie surface burial and a weak electrostatic interaction between the distal basic component of the inhibitor and an electronegative cavity of factor Xa formed by three backbone carbonyl oxygens. Additivity of these three interactions is the basis for the observed strong inhibition of factor Xa and provides a framework for the design of novel factor Xa inhibitors. A propionic acid group of the inhibitor would clash with the thrombin specific '60-insertion loop', thus conferring selectivity against thrombin.

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Section: Discussionsupporting

confidence: 81%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Crystal structures of factor Xa specific inhibitors in complex with trypsin: structural grounds for inhibition of factor Xa and selectivity against thrombin

1995

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“…We present here the crystal structure of the factor Xa⅐DX-9065a complex. The inhibitor binds in the active site in an extended conformation, which was expected from earlier studies (14,15). Both hydrophobic and electrostatic interactions characterize the complex formation, which is also accompanied by local rearrangements in the active site of fXa.…”

mentioning

confidence: 53%

X-ray Structure of Active Site-inhibited Clotting Factor Xa

Brandstetter

Kühne

Bode

et al. 1996

Journal of Biological Chemistry

275

160

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The 3.0-Å resolution x-ray structure of human des-Glacoagulation factor Xa (fXa) has been determined in complex with the synthetic inhibitor DX-9065a. The binding geometry is characterized primarily by two interaction sites: the naphthamidine group is fixed in the S1 pocket by a typical salt bridge to Asp-189, while the pyrrolidine ring binds in the unique aryl-binding site (S4) of fXa. Unlike the large majority of inhibitor complexes with serine proteinases, Gly-216 (S3) does not contribute to hydrogen bond formation. In contrast to typical thrombin binding modes, the S2 site of fXa cannot be used by DX-9065a since it is blocked by Tyr-99, and the arylbinding site (S4) of fXa is lined by carbonyl oxygen atoms that can accommodate positive charges. This has implications for natural substrate recognition as well as for drug design.Hemostasis is the blood clotting process that, when functioning properly, occurs when an injury to the vasculature leads to a series of vasculomotor and cellular reactions and the activation of the blood coagulation cascade. The latter process is initiated via the extrinsic pathway, leading first to thrombin activation and then massively amplified thrombin activation due to the positive feedback of the intrinsic pathway (1). Both extrinsic and intrinsic pathways merge at the factor X activation step. An imbalance between these clotting processes, clotting inactivation processes (protein C inactivation of hemostasis cofactors), and thrombolytic processes (tissue plasminogen activator, plasminogen) can lead to thrombotic or bleeding disorders. Antithrombotics include inhibitors of thrombin, factor Xa and factor IXa, factors involved in both the extrinsic and intrinsic pathways (2). As evidence accumulates that thrombin has other important functions in cellular (3, 4) and neurological (5-10) processes, new synthetic anticoagulants increasingly target factor Xa. Daiichi published the first tight binding (K i ϭ 41 nM), specific inhibitor of fXa, 1 DX-9065a (11-13). We present here the crystal structure of the factor Xa⅐DX-9065a complex. The inhibitor binds in the active site in an extended conformation, which was expected from earlier studies (14, 15). Both hydrophobic and electrostatic interactions characterize the complex formation, which is also accompanied by local rearrangements in the active site of fXa. Considering these subtle interactions and the unpredictable ligand-induced motions involved in binding, there is clearly a need for a series of fXa-ligand complex structures to provide adequate information for structure-based drug design and an understanding of how fXa recognizes physiological substrates.EXPERIMENTAL PROCEDURES fX was isolated from human plasma; des-Gla-fX was produced via chymotryptic cleavage (removing amino acids L1-L44; chymotrypsinogen numbering is used for the catalytic domain; the sequential fX numbering, which is used for the light chain, will be indicated by the prefix "L"). Subsequently, fX was activated with the purified factor X activator from Russell's vip...

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“…Cation-IT interactions have been considered in such diverse systems as acetylcholine receptors (nicotinic, muscarinic, and ACh esterase), K+ channels, the cyclase enzymes of steroid biosynthesis, and enzymes that catalyze methylation reactions involving S-adenosylmethionine (1). Cation-Ir interactions have also been invoked to rationalize specific drug-receptor interactions (8)(9)(10)(11) …”

Section: Introductionmentioning

confidence: 99%

Cation-pi interactions in aromatics of biological and medicinal interest: electrostatic potential surfaces as a useful qualitative guide.

Mecozzi

West²,

Dougherty³

1996

Proc. Natl. Acad. Sci. U.S.A.

578

554

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The cation-t interaction is an important, general force for molecular recognition in biological receptors. Through the sidechains of aromatic amino acids, novel binding sites for cationic ligands such as acetylcholine can be constructed. We report here a number of calculations on prototypical cation-7r systems, emphasizing structures of relevance to biological receptors and prototypical heterocycles of the type often of importance in medicinal chemistry. Trends in the data can be rationalized using a relatively simple model that emphasizes the electrostatic component of the cation-ir interaction. In particular, plots of the electrostatic potential surfaces of the relevant aromatics provide useful guidelines for predicting cation-7r interactions in new systems.of relevance to biological receptors and prototype heterocycles of the type often of importance in medicinal chemistry. We find that all the trends in this series are qualitatively reproduced by considering only the electrostatic potential energy surface of the aromatic in the absence of a cation, consistent with the electrostatic model. In addition, the current model successfully rationalizes observations concerning the relative frequency of different aromatic amino acids at biological cation-Ir sites. We also show that the major trends of the ab initio surfaces are reproduced using the much less costly AM1 method, greatly expanding the range of applicability of the method.In recent years, studies of model systems and the analysis of biological macromolecular structures have established the importance of the cation-rr interaction as a force for molecular recognition in aqueous media (1). Appropriately designed cyclophane receptors serve as powerful, general hosts for quaternary ammonium, sulfonium, and guanidinium cations, in large part because of the cation-IT interaction (2-4). In the gas phase, the binding of simple cations to benzene and related structures has been shown to be quite substantial, comparable even to cation-water interactions (5). In addition, a large amount of evidence has now been developed that establishes cation-IT interactions as important in a number of biological binding sites for cations (1,6,7). Cation-IT interactions have been considered in such diverse systems as acetylcholine receptors (nicotinic, muscarinic, and ACh esterase), K+ channels, the cyclase enzymes of steroid biosynthesis, and enzymes that catalyze methylation reactions involving S-adenosylmethionine (1). Cation-Ir interactions have also been invoked to rationalize specific drug-receptor interactions (8)(9)(10)(11)

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Proposed cation‐π mediated binding by factor Xa: a novel enzymatic mechanism for molecular recognition

Cited by 44 publications

References 37 publications

Crystal structures of factor Xa specific inhibitors in complex with trypsin: structural grounds for inhibition of factor Xa and selectivity against thrombin

Crystal structures of factor Xa specific inhibitors in complex with trypsin: structural grounds for inhibition of factor Xa and selectivity against thrombin

X-ray Structure of Active Site-inhibited Clotting Factor Xa

Cation-pi interactions in aromatics of biological and medicinal interest: electrostatic potential surfaces as a useful qualitative guide.

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