title: Structures of potent selective peptide mimetics bound to carboxypeptidase B.
abstract: This article reports the crystal structures of inhibitors of the functional form of thrombin-activatable fibrinolysis inhibitor (TAFIa). In vivo experiments indicate that selective inhibitors of TAFIa would be useful in the treatment of heart attacks. Since TAFIa rapidly degrades in solution, the homologous protein porcine pancreatic carboxypeptidase B (pp-CpB) was used in these crystallography studies. Both TAFIa and pp-CpB are zinc-based exopeptidases that are specific for basic residues. The final development candidate, BX 528, is a potent inhibitor of TAFIa (2 nM) and has almost no measurable effect on the major selectivity target, carboxypeptidase N. BX 528 was designed to mimic the tripeptide Phe-Val-Lys. A sulfonamide replaces the Phe-Val amide bond and a phosphinate connects the Val and Lys groups. The phosphinate also chelates the active-site zinc. The electrostatic interactions with the protein mimic those of the natural substrate. The primary amine in BX 528 forms a salt bridge to Asp255 at the base of the S1' pocket. The carboxylic acid interacts with Arg145 and the sulfonamide is hydrogen bonded to Arg71. Isopropyl and phenyl groups replace the side chains of Val and Phe, respectively. A series of structures are presented here that illustrate the evolution of BX 528 from thiol-based inhibitors that mimic a free C-terminal arginine. The first step in development was the replacement of the thiol with a phosphinate. This caused a precipitous drop in binding affinity. Potency was reclaimed by extending the inhibitors into the downstream binding sites for the natural substrate.
authors.affiliation: Bayer HealthCare Pharmaceuticals, 2600 Hilltop Drive, PO Box 4099, Richmond, California 94804-0099, USA.
authors.forename: Marc
authors.initials: M
authors.lastname: Adler
authors.forename: Brad
authors.initials: B
authors.lastname: Buckman
authors.forename: Judi
authors.initials: J
authors.lastname: Bryant
authors.forename: Zheng
authors.initials: Z
authors.lastname: Chang
authors.forename: Kieu
authors.initials: K
authors.lastname: Chu
authors.forename: Kumar
authors.initials: K
authors.lastname: Emayan
authors.forename: Paul
authors.initials: P
authors.lastname: Hrvatin
authors.forename: Imadul
authors.initials: I
authors.lastname: Islam
authors.forename: John
authors.initials: J
authors.lastname: Morser
authors.forename: Drew
authors.initials: D
authors.lastname: Sukovich
authors.forename: Christopher
authors.initials: C
authors.lastname: West
authors.forename: Shendong
authors.initials: S
authors.lastname: Yuan
authors.forename: Marc
authors.initials: M
authors.lastname: Whitlow
mesh_terms: D001665:Binding Sites; D043424:Carboxypeptidase B / Q000737:chemistry / Q000378:metabolism*; D025901:Carboxypeptidase B2 / Q000737:chemistry; D018360:Crystallography, X-Ray / Q000379:methods; D006801:Humans; D006860:Hydrogen Bonding; D008958:Models, Molecular; D015394:Molecular Structure; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D010455:Peptides; D043424:Carboxypeptidase B; D025901:Carboxypeptidase B2