title: Species specificity of amidine-based urokinase inhibitors.
abstract: Inhibition of the proteolytic activity of urokinase has been shown to inhibit the progression of tumors in rodent models and is being investigated for use in human disease. Understanding the rodent/human species-specificity of urokinase inhibitors is therefore critical for interpretation of rodent cancer progression models that use these inhibitors. We report here studies with a panel of 11 diverse urokinase inhibitors in both human and mouse enzymatic assays. Inhibitors such as amiloride, B428, and naphthamidine, that occupy only the S1 subsite pocket were found to be nearly equipotent between the human and the murine enzymes. Inhibitors that access additional, more distal, pockets were significantly more potent against the human enzyme but there was no corresponding potency increase against the murine enzyme. X-ray crystallographic structures of these compounds bound to the serine protease domain of human urokinase were solved and examined in order to explain the human/mouse potency differences. The differences in inhibitor potency could be attributed to four amino acid residues that differ between murine and human urokinases: 60, 99, 146, and 192. These residues are Asp, His, Ser, and Gln in human and Gln, Tyr, Glu, and Lys in mouse, respectively. Compounds bearing a cationic group that interacts with residue 60 will preferentially bind to the human enzyme because of favorable electrostatic interactions. The hydrogen bonding to residue 192 and steric considerations with residues 99 and 146 also contribute to the species specificity. The nonparallel human/mouse enzyme inhibition observations were extended to a cell-culture assay of urokinase-activated plasminogen-mediated fibronectin degradation with analogous results. These studies will aid the interpretation of in vivo evaluation of urokinase inhibitors.
authors.affiliation: Department of Cancer Research, Abbott Laboratories, Abbott Park, Illinois 60064-6117, USA. vered.klinghofer@abbott.com
authors.forename: V
authors.initials: V
authors.lastname: Klinghofer
authors.forename: K
authors.initials: K
authors.lastname: Stewart
authors.forename: T
authors.initials: T
authors.lastname: McGonigal
authors.forename: R
authors.initials: R
authors.lastname: Smith
authors.forename: A
authors.initials: A
authors.lastname: Sarthy
authors.forename: V
authors.initials: V
authors.lastname: Nienaber
authors.forename: C
authors.initials: C
authors.lastname: Butler
authors.forename: S
authors.initials: S
authors.lastname: Dorwin
authors.forename: P
authors.initials: P
authors.lastname: Richardson
authors.forename: M
authors.initials: M
authors.lastname: Weitzberg
authors.forename: M
authors.initials: M
authors.lastname: Wendt
authors.forename: T
authors.initials: T
authors.lastname: Rockway
authors.forename: X
authors.initials: X
authors.lastname: Zhao
authors.forename: K I
authors.initials: KI
authors.lastname: Hulkower
authors.forename: V L
authors.initials: VL
authors.lastname: Giranda
mesh_terms: D000578:Amidines / Q000138:chemical synthesis / Q000737:chemistry*; D000584:Amiloride / Q000138:chemical synthesis; D000595:Amino Acid Sequence; D000818:Animals; D000970:Antineoplastic Agents / Q000138:chemical synthesis; D001665:Binding Sites; D001798:Blood Proteins; D018827:Carcinoma, Lewis Lung; D018360:Crystallography, X-Ray; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009281:Naphthalenes / Q000138:chemical synthesis; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D015842:Serine Proteinase Inhibitors / Q000138:chemical synthesis / Q000737:chemistry*; D013045:Species Specificity; D013876:Thiophenes / Q000138:chemical synthesis; D014407:Tumor Cells, Cultured; D014568:Urokinase-Type Plasminogen Activator / Q000037:antagonists & inhibitors*
chemical_list: C097398:4-iodine-benzo(b)thiophene-2-carboxamidine; D000578:Amidines; D000970:Antineoplastic Agents; D001798:Blood Proteins; D009281:Naphthalenes; D015842:Serine Proteinase Inhibitors; D013876:Thiophenes; C031220:urokinase inhibitor; C018609:beta-naphthamidine; D000584:Amiloride; D014568:Urokinase-Type Plasminogen Activator