Formation of unusual glutamate conjugates of 1-[3-(aminomethyl)phenyl]-N-[3-fluoro-2'-(methylsulfonyl)-[1,1'-biphenyl]-4-yl]-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (DPC 423) and its analogs: the role of gamma-glutamyltranspeptidase in the biotransformation of benzylamines.

The role of gamma-glutamyltranspeptidase (GGT) in transferring glutamate from endogenous glutathione (GSH) to the benzylamine moiety of a compound, such as 1-[3-(aminomethyl)phenyl]-N-[3-fluoro-2'-(methylsulfonyl)-[1,1'-biphenyl]-4-yl]-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (DPC 423), is described. Studies were performed with structurally related analogs of DPC 423 to demonstrate that this type of reaction was common to compounds possessing a benzylamine group. Synthesizing appropriate standards and confirming by liquid chromatography (LC)/mass spectroscopy and LC/NMR made unambiguous assignments of the structures of glutamate conjugates of DPC 423. The use of stable isotope-labeled GSH for metabolism studies has not been described before. In the present study, we report the novel use of deuterated GSH in conjunction with mass spectral analysis to demonstrate the glutamate transfer to the benzylamines in the presence of GGT. To further demonstrate that the alpha protons on the benzylamines and glutamate (as part of glutathione) were unaffected during the transpeptidation, these protons were replaced with deuterium. Acivicin (AT-125), a potent and selective inhibitor of GGT, was used to abolish the formation of the glutamate conjugates of DPC 423 in vitro and in vivo. This provided further evidence of the role of GGT in forming the glutamate conjugates of benzylamines. This study demonstrated conclusively that GGT was responsible for mediating the transfer of glutamic acid from GSH to the benzylamine moiety of a series of structurally related compounds.

Antagonism of CRF(2) receptors produces anxiolytic behavior in animal models of anxiety.

Two pharmacologically distinct CRF receptors are distributed in different brain regions and peripheral tissues. Studies suggest that CRF(1) receptors play an important role in mediating the anxiety provoking effects of CRF. In contrast, far less functional information is available on CRF(2) receptors. Therefore, we conducted dose response studies using antisauvagine-30 (anti-SVG-30, 0-20 microg, 20-min pretreatment, i.c.v.), a potent CRF(2) peptide antagonist, and tested rats in three models of anxiety - the conditioned freezing, the elevated plus maze, and the defensive-withdrawal test. Anti-SVG-30 produced a significant dose-dependent reduction in conditioned freezing. In the elevated plus maze test, administration of anti-SVG-30 effectively increased the number of entries and time spent in the open arms. In the defensive-withdrawal test, anti-SVG-30 treatment facilitated exploratory activity in a large illuminated open field. Thus, in all three animal models, administration of anti-SVG-30 was consistent in producing an anxiolytic-like behavioral effect. In addition, a dose of anti-SVG-30 (10 microg) that produced anxiolytic-like behavior had no significant effects on locomotor activity measured in an automated activity box. This latter finding suggests that antagonism of CRF(2) receptors is not associated with a non-specific increase in behavioral movements. These results provide evidence that, in addition to CRF(1) receptors, CRF(2) receptors may play an important role in the mediation of anxiety behavior.

Mechanism of Inhibition of beta-site amyloid precursor protein-cleaving enzyme (BACE) by a statine-based peptide.

Inhibition of beta-site amyloid precursor protein-cleaving enzyme by a statine-based inhibitor has been studied using steady state and stopped-flow methods. A slow onset rate of inhibition has been observed under steady state conditions, and a K(i) of 22 nm has been derived using progress curves analysis. Simulation of stopped-flow protein fluorescence transients provided an estimate of the K(d) for initial inhibitor binding of 660 nm. A two-step inhibition mechanism is proposed, wherein slower "tightening up" of the initial encounter complex occurs. Two hypotheses have been proposed in the literature to address the nature of the slow step in the inhibition of aspartic proteases by peptidomimetic inhibitors: a conformational change related to the "flap" movement and displacement of a catalytic water. We compared substrate and inhibitor binding rates under pre-steady-state conditions. Both ligands are likely to cause flap movement, whereas no catalytic water replacement occurs during substrate binding. Our results suggest that both ligands bind to the enzyme at a rate significantly lower than the diffusion limit, but there are additional rate limitations involved in inhibitor binding, resulting in a k(on) of 3.5 x 10(4) m(-)1 s(-)1 for the inhibitor compared with 3.5 x 10(5) m(-)1 s(-)1 for the substrate. Even though specific intermediate formation steps might be different in the productive inhibitor and substrate binding to beta-site amyloid precursor protein-cleaving enzyme, a similar final optimized conformation is achieved in both cases, as judged by the comparable free energy changes (DeltaDeltaG of 2.01 versus 1.97 kcal/mol) going from the initial to the final enzyme-inhibitor or enzyme-substrate complexes.

Disposition of glutathione conjugates in rats by a novel glutamic acid pathway: characterization of unique peptide conjugates by liquid chromatography/mass spectrometry and liquid chromatography/NMR.

With the advent of liquid chromatography/mass spectrometry and liquid chromatography/NMR, it has become easier to characterize metabolites that were once difficult to isolate and identify. These techniques have enabled us to uncover the existence of an alternate pathway for the disposition of glutathione adducts of several structurally diverse compounds. Studies were carried out using acetaminophen as a model compound to investigate the role of the glutamic acid pathway in disposition of the glutathione adducts. Although the mercapturic acid pathway was the major route of degradation of the glutathione adducts, it was found that the conjugation of the glutathione, cysteinylglycine, and cysteine adducts of acetaminophen with the gamma-carboxylic acid of the glutamic acid was both interesting and novel. The coupling of the glutathione adduct and the products from the mercapturic acid pathway with the glutamic acid led to unusual peptide conjugates. The natures of these adducts were confirmed unequivocally by comparisons with synthetic standards. This pathway (addition of glutamic acids) led to larger peptides, in contrast to the mercapturic acid pathway, in which the glutathione adducts are broken down to smaller molecules. The enzyme responsible for the addition of glutamic acid to the different elements of the mercapturic acid pathway is currently unknown. It is postulated that the gamma-carboxylic acid is activated (perhaps by ATP) before enzymatic addition to the alpha-amino group of cysteine or glutamate takes place. The discovery of these peptide conjugates of acetaminophen represents a novel disposition of glutathione adducts of compounds. The formation of such conjugates may represent yet another pathway by which drugs could produce covalent binding via their reactive intermediates.

Analysis of protein-peptide interaction by a miniaturized fluorescence polarization assay using cyclin-dependent kinase 2/cyclin E as a model system.

As a result of the increasing size of chemical libraries, more rapid and highly sensitive strategies are needed to accelerate the process of drug discovery without increasing the cost. One means of accomplishing this is to miniaturize the assays that enter high-throughput screening (HTS). Miniaturization requires an assay design that has few steps, has a large degree of separation between the signal and background, and has a low well to well signal variation. Fluorescence polarization (FP) is an assay type that, in many cases, meets all of the above requirements. FP is a homogenous method that allows interactions between molecules to be measured directly in solution. This article demonstrates the application of FP in a miniaturized HTS format, using 1536-well plates, to measure direct binding between cyclin-dependent kinase 2/cyclin E complex (CDK2/E) and an 8-mer-peptide kinase inhibitor. The data indicate that low variability and high specificity allow rapid and precise identification of antagonist compounds affecting CDK2/E-peptide interactions.

Amino acids that specify structure through hydrophobic clustering and histidine-aromatic interactions lead to biologically active peptidomimetics.

Acyclic beta-sheet structure can be nucleated in heptapeptides when the 4-(2-aminoethyl)-6-dibenzofuranpropanoic acid residue (1) is flanked in sequence by two His residues, a His residue and a hydrophobic residue or by two hydrophobic residues. Acyclic beta-sheet peptidomimetics having an appropriate sequence have sufficient structural integrity to exhibit antimicrobial activity equivalent to that of gramicidin S.