title: Time-resolved ligand exchange reactions: kinetic models for competitive inhibitors with recombinant human renin.
abstract: The on and off rate constants (kon and koff) were determined for a series of peptidomimetic, competitive inhibitors of human renin using a novel binding assay. The method entails analyzing a pair of ligand exchange reactions in which a dansylated inhibitor serves as the fluorescent probe. The first in the pair of reactions involves preincubating renin with the probe and initiating the reaction by addition of a sample inhibitor; the second reaction involves preincubating renin with the sample inhibitor and initiating the reaction by addition of probe. Both reactions yield progress curves which contain complementary information concerning the kon and koff of each ligand. The two curves are fitted simultaneously using models derived from the differential rate equations describing the ligand exchange process. The kon and koff rate constants for the probe were 6.85 x 10(6) M-1 s-1 and 2.96 x 10(-4) s-1, respectively, giving a calculated Kd of 43.2 pM. The Kd values for the inhibitor series varied over 2 orders of magnitude (27-2320 pM), while the individual kon (10(6)-10(7) M-1 s-1) and koff (10(-4)-10(-3) s-1) constants varied only over 1 order of magnitude.
authors.affiliation: Department of Inflammatory Diseases, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, Connecticut 06877-0368, USA.
authors.forename: M M
authors.initials: MM
authors.lastname: Morelock
authors.forename: C A
authors.initials: CA
authors.lastname: Pargellis
authors.forename: E T
authors.initials: ET
authors.lastname: Graham
authors.forename: D
authors.initials: D
authors.lastname: Lamarre
authors.forename: G
authors.initials: G
authors.lastname: Jung
mesh_terms: D001667:Binding, Competitive; D006801:Humans; D007700:Kinetics; D008024:Ligands; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D000092502:Renin Inhibitors
chemical_list: D008024:Ligands; D011994:Recombinant Proteins; D000092502:Renin Inhibitors