title: Rational design, synthesis, and verification of affinity ligands to a protein surface cleft.
abstract: The structure-based design, synthesis, and screening of a glucuronic acid scaffold library of affinity ligands directed toward the catalytic cleft on porcine pancreas alpha-amylase are presented. The design was based on the simulated docking to the enzyme active site of 53 aryl glycosides from the Available Chemicals Directory (ACD) selected by in silico screening. Twenty-three compounds were selected for synthesis and screened in solution for binding toward alpha-amylase using nuclear magnetic resonance techniques. The designed molecules include a handle outside of the binding site to allow their attachment to various surfaces with minimal loss of binding activity. After initial screening in solution, one affinity ligand was selected, immobilized to Sepharose (Amersham Biosciences), and evaluated as a chromatographic probe. A column packed with ligand-coupled Sepharose specifically retained the enzyme, which could be eluted by a known inhibitor.
authors.affiliation: Amersham Biosciences, Björkgatan 30, S-751 84 Uppsala, Sweden.
authors.forename: Herbert
authors.initials: H
authors.lastname: Baumann
authors.forename: Sara
authors.initials: S
authors.lastname: Ohrman
authors.forename: Yasuro
authors.initials: Y
authors.lastname: Shinohara
authors.forename: Oguz
authors.initials: O
authors.lastname: Ersoy
authors.forename: Devapriya
authors.initials: D
authors.lastname: Choudhury
authors.forename: Andreas
authors.initials: A
authors.lastname: Axén
authors.forename: Ulf
authors.initials: U
authors.lastname: Tedebark
authors.forename: Enrique
authors.initials: E
authors.lastname: Carredano
mesh_terms: D000818:Animals; D020134:Catalytic Domain / Q000502:physiology*; D020723:Glucuronic Acid / Q000378:metabolism*; D008024:Ligands; D013552:Swine / Q000378:metabolism; D000516:alpha-Amylases / Q000235:genetics / Q000378:metabolism*
chemical_list: D008024:Ligands; D020723:Glucuronic Acid; D000516:alpha-Amylases