Surface plasmon resonance (SPR) technology has emerged as a new and powerful technique to investigate the interaction between low-molecular-weight molecules and target proteins. In the present work, the authors assemble from a large compound collection a library of 2226 molecules (fragments having low molecular weights between 100 and 300 Da) to screen them for binding to chymase, a serine protease. Both the active chymase and a zymogen-like form of the protein were used in parallel to distinguish between specific and unspecific binding. The relative ligand-binding activity of the immobilized protein was periodically measured with a reference compound. The screening experiments were performed at 25 °C at a fragment concentration of 200 µM in the presence of 2% DMSO. Applying the filter cascade, affinity-selectivity-competition (competition with reference compounds and cross-competition with fragments), 80 compounds show up as positive screening hits. Competition experiments between fragments show that they bind to different parts of the active site. Of 36 fragments cocrystallized for X-ray studies, 12 could be located in the active site of the protein. These results validate the authors' library and demonstrate that the application of SPR technology as a filter in fragment screening can be achieved successfully. (Journal of Biomolecular Screening. 2009:337-349)

BACE1 is a novel type I transmembrane aspartyl protease that has been implicated in the pathogenesis of Alzheimer's disease. Cleavage of the amyloid precursor protein by the beta-secretase, BACE1, is the first step in the production of the Abeta peptide and is a prime target for therapeutic intervention. Using circular dichroism, we reveal that the secondary structure of BACE1 in a membrane environment is significantly different from what was determined from the previously resolved crystal structure, and, we provide the first evidence that show differences in stability between the active (pH 4.8) and inactive (pH 7.4) forms of BACE1. In this study we have also examined Ca(2+) binding to BACE1, the effect of this binding on the secondary and tertiary structural characteristics of BACE1, and the influence of this binding on the specific activity of the purified protein. Circular dichroism and endogenous tryptophan fluorescence measurements demonstrated that the secondary and tertiary structures, respectively, are sensitive to increasing concentrations of Ca(2+). Isothermal titration calorimetry was then used to characterize the Ca(2+)-BACE1 interaction in more detail. Our results suggest that there is a high affinity of binding (k(d) = 2.0 x microM) between Ca(2+) and BACE1 and that the binding process was exothermic (DeltaH= -3.5 kcal/mol). We also could demonstrate that low concentrations of Ca(2+) (microM range) significantly increased the proteolytic activity of BACE1. Collectively, these results identify a direct interaction between BACE1 and Ca(2+) and suggest that under physiological conditions, the function(s) of BACE1 must also be influenced by Ca(2+).

No abstract

Carnitine palmitoyl transferase 2 (CPT-2) is a key enzyme in the mitochondrial fatty acid metabolism. The active site is comprised of a Y-shaped tunnel with distinct binding sites for the substrate acylcarnitine and the cofactor CoA. We investigated the thermodynamics of binding of four inhibitors directed against either the CoA or the acylcarnitine binding sites using isothermal titration calorimetry (ITC). CPT-2 is a monotopic membrane protein and was solubilized by β-octylglucoside (β-OG) above its critical micellar concentration (CMC) to perform inhibitor titrations in solutions containing detergent micelles. The CMC of β-OG in the presence of inhibitors was measured with ITC and small variations were observed. The inhibitors bound to rat CPT-2 (rCPT-2) with 1:1 stoichiometry and the dissociation constants were in the range of KD = 2–20 μM. New X-ray structures and docking models of rCPT-2 in complex with inhibitors enable an analysis of the thermodynamic data in the context of the interaction observed for the individual binding sites of the ligands. For all ligands the binding enthalpy was exothermic, and enthalpy as well as entropy contributed to the binding reaction, with the exception of ST1326 for which binding was solely enthalpy-driven. The substrate analog ST1326 binds to the acylcarnitine binding site and a heat capacity change close to zero suggests a balance of electrostatic and hydrophobic interactions. An excellent correlation of the thermodynamic (ITC) and structural (X-ray crystallography, models) data was observed suggesting that ITC measurements provide valuable information for optimizing inhibitor binding in drug discovery.

No abstract