Identification and analyses of inhibitors targeting apolipoprotein(a) kringle domains KIV-7, KIV-10, and KV provide insight into kringle domain function.

Increased plasma concentrations of lipoprotein(a) (Lp(a)) are associated with an increased risk for cardiovascular disease. Lp(a) is composed of apolipoprotein(a) (apo(a)) covalently bound to apolipoprotein B of low-density lipoprotein (LDL). Many of apo(a)'s potential pathological properties, such as inhibition of plasmin generation, have been attributed to its main structural domains, the kringles, and have been proposed to be mediated by their lysine-binding sites.

Design of Selective sPLA-X Inhibitor (-)-2-{2-[Carbamoyl-6-(trifluoromethoxy)-1-indol-1-yl]pyridine-2-yl}propanoic Acid.

A lead generation campaign identified indole-based sPLA-X inhibitors with a promising selectivity profile against other sPLA isoforms. Further optimization of sPLA selectivity and metabolic stability resulted in the design of (-)-, a novel, potent, and selective sPLA-X inhibitor with an exquisite pharmacokinetic profile characterized by high absorption and low clearance, and low toxicological risk. Compound (-)- was tested in an ApoE murine model of atherosclerosis to evaluate the effect of reversible, pharmacological sPLA-X inhibition on atherosclerosis development.

Discovery of a Series of Indole-2 Carboxamides as Selective Secreted Phospholipase A Type X (sPLA-X) Inhibitors.

In order to assess the potential of sPLA-X as a therapeutic target for atherosclerosis, novel sPLA inhibitors with improved type X selectivity are required. To achieve the objective of identifying such compounds, we embarked on a lead generation effort that resulted in the identification of a novel series of indole-2-carboxamides as selective sPLA2-X inhibitors with excellent potential for further optimization.

Methyl group reorientation under ligand binding probed by pseudocontact shifts.

Liquid-state NMR spectroscopy is a powerful technique to elucidate binding properties of ligands on proteins. Ligands binding in hydrophobic pockets are often in close proximity to methyl groups and binding can lead to subtle displacements of methyl containing side chains to accommodate the ligand. To establish whether pseudocontact shifts can be used to characterize ligand binding and the effects on methyl groups, the N-terminal domain of HSP90 was tagged with caged lanthanoid NMR probe 5 at three positions and titrated with a ligand.

Structure and biophysical characterization of the human full-length neurturin-GFRa2 complex: A role for heparan sulfate in signaling.

Neurturin (NRTN) provides trophic support to neurons and is considered a therapeutic agent for neurodegenerative diseases, such as Parkinson's disease. It binds to its co-receptor GFRa2, and the resulting NRTN-GFRa2 complex activates the transmembrane receptors rearranged during transfection (RET) or the neural cell adhesion molecule (NCAM). We report the crystal structure of NRTN, alone and in complex with GFRa2.

Pharmacokinetics and the drug-target residence time concept.

The concept of drug-target residence time has been in focus in recent drug discovery literature. However, few studies consider the combined effect of pharmacokinetics (PK) and binding kinetics (BK) on the duration of effect of a drug. Using a simple model that takes both PK and BK into account, we found that prolongation of binding owing to a long drug-target residence time can only occur when the binding dissociation is slower than the PK elimination.

The use of TrkA-PathHunter assay in high-throughput screening to identify compounds that affect nerve growth factor signaling.

The TrkA-PathHunter cell-based assay was used in high-throughput screening (HTS) to identify compounds that inhibit nerve growth factor (NGF)/TrkA signaling. The assay was conducted in a 384-well format, and typical Z' values during HTS ranged from 0.3 to 0.

Automated nano-electrospray mass spectrometry for protein-ligand screening by noncovalent interaction applied to human H-FABP and A-FABP.

A method for ligand screening by automated nano-electrospray ionization mass spectrometry (nano-ESI/MS) is described. The core of the system consisted of a chip-based platform for automated sample delivery from a 96-well plate and subsequent analysis based on noncovalent interactions. Human fatty acid binding protein, H-FABP (heart) and A-FABP (adipose), with small potential ligands was analyzed.

Combined use of NMR relaxation measurements and hydrodynamic calculations to study protein association. Evidence for tetramers of low molecular weight protein tyrosine phosphatase in solution.

We describe a novel method for determining weak association constants of oligomeric protein complexes formed transiently under equilibrium conditions. This type of equilibrium process is recognized as being biologically important, but generally hard to study. Heteronuclear spin relaxation rates measured at multiple protein concentrations are analyzed using relaxation rates predicted from hydrodynamic calculations, yielding equilibrium constants and structural characterization of the protein complexes.

Structure-based screening as applied to human FABP4: a highly efficient alternative to HTS for hit generation.

The time-limiting step in HTS often is the development of an appropriate assay. In addition, hits from HTS fairly often turn out to be false positives and generally display unfavorable properties for further development. Here we describe an alternative process for hit generation, applied to the human adipocyte fatty acid binding protein FABP4.

Intramolecular dynamics of low molecular weight protein tyrosine phosphatase in monomer-dimer equilibrium studied by NMR: a model for changes in dynamics upon target binding.

Low molecular weight protein tyrosine phosphatase (LMW-PTP) dimerizes in the phosphate-bound state in solution with a dissociation constant of K(d)=1.5(+/-0.1)mM and an off-rate on the order of 10(4)s(-1).