Discovery and characterization of a potent and selective non-amidine inhibitor of human factor Xa.
Benzothiophene-anthranilamide 1 (3-chloro-N-[2-[[(4-fluorophenyl)amino]carbonyl]-4-methylphenyl]benzo[b]thiophene-2-carboxamide) was discovered by high throughput screening to be a highly potent and selective non-amidine inhibitor of human factor Xa with a K(i) of 15+/-4nM. Compound 1 is a selective inhibitor of human factor Xa as suggested by the K(i)((app)) determined for nine other human serine proteases and bovine trypsin. The activity of reconstituted human prothrombinase complex was inhibited by compound 1 when assayed in physiological concentrations of the substrate prothrombin. However, 27-fold higher inhibitor concentrations were needed to achieve the same level of inhibition than were required for the inhibition of free factor Xa, due in part to non-specific binding of the inhibitor to phospholipid under the assay conditions. Failure to demonstrate enzymatic cleavage of compound 1 suggests that compound 1 is solely an inhibitor rather than a substrate for factor Xa. The inhibition of factor Xa by compound 1 was reversible upon dilution of the enzyme/inhibitor mixture. Analyses of the inhibition mechanism with Dixon, Cornish-Bowden, and Lineweaver-Burk plots showed that compound 1 is a linear mixed-type inhibitor with 5-fold higher affinity for free factor Xa than the factor Xa/substrate complex. The linear mixed-type inhibition suggests that compound 1 binds to the active site region of factor Xa, but its binding cannot be fully displaced by the substrate S2222 (1:1 mixture of N-benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide and N-benzoyl-Ile-Glu(gamma-OMe)-Gly-Arg-p-nitroanilide hydrochloride). Thus, the inhibition mechanism for compound 1 is novel compared to most serine protease inhibitors including amidine-containing factor Xa inhibitors, which rely on binding to the S1 pocket of the enzyme active site. Compound 1 represents an attractive, novel structural template for further development of efficacious, safe, and potentially orally active human factor Xa inhibitors.
Berlex Biosciences, 2600 Hilltop Drive, P.O. Box 4099, Richmond, CA 94806-0099, USA. amy_liang@gerlex.com
Amy M
AM
Liang
David R
DR
Light
Monica
M
Kochanny
Galina
G
Rumennik
Lan
L
Trinh
Dao
D
Lentz
Joseph
J
Post
John
J
Morser
Michael
M
Snider
D000925:Anticoagulants / Q000494:pharmacology; D001665:Binding Sites; D004353:Drug Evaluation, Preclinical; D065427:Factor Xa Inhibitors; D006801:Humans; D010743:Phospholipids; D012697:Serine Endopeptidases / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013876:Thiophenes / Q000494:pharmacology*; D062367:ortho-Aminobenzoates / Q000494:pharmacology*
D000925:Anticoagulants; D065427:Factor Xa Inhibitors; D010743:Phospholipids; D015842:Serine Proteinase Inhibitors; D013876:Thiophenes; D062367:ortho-Aminobenzoates; C088015:benzothiophene; D012697:Serine Endopeptidases; C000219:anthranilamide