4-Anilino-6-phenyl-quinoline inhibitors of mitogen activated protein kinase-activated protein kinase 2 (MK2).

A class of inhibitors of mitogen activated protein kinase-activated kinase 2 (MK2) was discovered via high-throughput screening. This compound class demonstrates activity against the enzyme with sub-microM IC(50) values, and suppresses LPS-induced TNFalpha levels in THP-1 cells. MK2 inhibition kinetic measurements indicated mixed binding approaching non-ATP competitive inhibition.

A novel and conserved pocket of human kappa-Fab fragments: design, synthesis, and verification of directed affinity ligands.

Antibodies of type IgG may be divided into two classes, called lambda or kappa, depending on the type of light chain. We have identified a conserved pocket between the two domains CH1 and CL of human IgG kappa-Fab, which is not present in the lambda type. This pocket was used as a target docking site with the purpose of exploring the possibilities of designing affinity ligands that could function as such even after immobilization to gel. The idea of the design arose mainly from the results of the saturated transfer difference (STD-NMR) screening of 46 compounds identified by means of virtual docking of 60 K diverse compounds from the Available Chemicals Directory (ACD). Surface plasmon resonance (SPR) was used as an alternative method to monitor binding in solution. A total of 24 compounds belonging to a directed library were designed, synthesized, and screened in solution. They consist essentially of an amino acid condensed to a N,N'-methylated phenyl urea. STD-NMR results suggest that a small hydrophobic side chain in the condensed amino acid promotes binding, whereas a hydroxyl-group-containing side chain implies absence of STD-NMR signals. Three compounds of the directed library were immobilized and evaluated as chromatographic probes. In one case, using D-Pro as the condensed amino acid, columns packed with ligand-coupled Sepharose (Amersham Biosciences) retained two different monoclonal samples of kappa-Fab fragments with different variable regions, whereas a sample of monoclonal lambda-Fab fragments was not retained under similar chromatographic conditions.

Rational design, synthesis, and verification of affinity ligands to a protein surface cleft.

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.

Structure-based discovery of a new affinity ligand to pancreatic alpha-amylase.

A ligand useful for affinity capture of porcine pancreatic alpha-amylase was found by virtual screening of the commercially available compound data base MDL Available Chemicals Directory. Hits from the virtual screening were investigated for binding by nuclear magnetic resonance (NMR) and surface plasmon resonance. Selected compounds were tested for inhibition of the enzyme using a NMR-based assay. One of the binders found was covalently coupled to a chromatographic resin and a column, packed with this resin, could retain alpha-amylase, which subsequently was eluted by introduction of the known inhibitor acarbose to the elution buffer.