title: How a β-D-glucoside side chain enhances binding affinity to thrombin of inhibitors bearing 2-chlorothiophene as P1 moiety: crystallography, fragment deconstruction study, and evaluation of antithrombotic properties.
abstract: The β-d-glucose-containing compound 3, bearing 2-chlorothiophene and 1-isopropylpiperidine moieties as binders of the S1 and S4 pockets, respectively, proved to be potent competitive inhibitor of factor Xa (fXa, Ki = 0.090 nM) and thrombin (fIIa, Ki = 100 nM). The potency of 3 increases, over the parent compound 1, against fIIa (110-fold), much more than against fXa (7-fold). Experimental deconstruction of 3 into smaller fragments revealed a binding cooperativity of the P3/P4 and propylene-linked β-d-glucose fragments, stronger in fIIa (15.5 kJ·mol(-1)) than in fXa (2.8 kJ·mol(-1)). The crystal structure of human fIIa in complex with 3 revealed a binding mode including a strong H-bond network between the glucose O1', O3', and O5' and two critical residues, namely R221a and K224, belonging to the Na(+)-binding site which may allosterically perturb the specificity sites. The potential of 3 as antithrombotic agent was supported by its ability to inhibit thrombin generation and to stimulate fibrinolysis at submicromolar concentration.
authors.affiliation: Institute of Crystallography, Consiglio Nazionale delle Ricerche , Via Amendola 122/o, 70126 Bari, Italy.
authors.forename: Benny D
authors.initials: BD
authors.lastname: Belviso
authors.forename: Rocco
authors.initials: R
authors.lastname: Caliandro
authors.forename: Modesto
authors.initials: M
authors.lastname: de Candia
authors.forename: Giorgia
authors.initials: G
authors.lastname: Zaetta
authors.forename: Gianfranco
authors.initials: G
authors.lastname: Lopopolo
authors.forename: Francesca
authors.initials: F
authors.lastname: Incampo
authors.forename: Mario
authors.initials: M
authors.lastname: Colucci
authors.forename: Cosimo D
authors.initials: CD
authors.lastname: Altomare
mesh_terms: D000925:Anticoagulants / Q000737:chemistry / Q000494:pharmacology; D000991:Antithrombins / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D001665:Binding Sites; D060326:Chemistry Techniques, Synthetic; D018360:Crystallography, X-Ray; D004305:Dose-Response Relationship, Drug; D065427:Factor Xa Inhibitors / Q000737:chemistry / Q000494:pharmacology; D005337:Fibrin / Q000378:metabolism; D005342:Fibrinolysis / Q000187:drug effects; D005960:Glucosides / Q000737:chemistry*; D006801:Humans; D006860:Hydrogen Bonding; D008958:Models, Molecular; D013329:Structure-Activity Relationship; D013876:Thiophenes / Q000737:chemistry*; D013917:Thrombin / Q000737:chemistry / Q000378:metabolism
chemical_list: D000925:Anticoagulants; D000991:Antithrombins; D065427:Factor Xa Inhibitors; D005960:Glucosides; D013876:Thiophenes; D005337:Fibrin; D013917:Thrombin