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A structure-based virtual screening (SBVS) was conducted on a ligand-supported homology model of the human histamine H4 receptor (hH4R). More than 8.7 million 3D structures derived from different vendor databases were investigated by docking to the hH4R binding site using FlexX. A total of 255 selected compounds were tested by radioligand binding assay and 16 of them possessed significant [(3)H]histamine displacement. Several novel scaffolds were identified that can be used to develop selective H4 ligands in the future. As far as we know, this is the first SBVS reported on H4R, representing one of the largest virtual screens validated by the biological evaluation of the virtual hits.

The theory and practice of proton microspeciation based on NMR-pH titrations are surveyed. Principles of bi-, tri-, tetra-, and n-protic microequilibrium systems are discussed. Evaluation methods are exemplified by case studies on bi- and tetraprotic biomolecules. Selection criteria and properties of 'reporter' NMR nuclei are described. Literature data on complete microspeciations of small ligands and site-specific basicity characterizations of peptides and proteins are critically reviewed.

Rotamers of N-acetyl-L-cysteine (NAC, the most popular mucolytic drug) are characterized in terms of populations, site- and conformer-specific acid-base properties, reducing strength, and molecular pharmacology. A new, general relationship between the bulk- and rotamer-specific basicities is introduced. NAC at high pH predominantly exists in a trans thiolate-carboxylate rotameric form, whereas protonation promotes the occurrence of intramolecular hydrogen bond-forming isomers. Distribution curves of the rotamers are depicted as a function of pH. Rotamer-dependent thiolate basicities differ by up to 0.5 log k units. Carboxylate basicities show slight conformation-dependence only. The membrane-penetrating capabilities from various compartments of the body are assessed on the basis of the pH-dependent charge of the molecule. The thiol-disulfide half-cell potential is calculated, using the correlation between the thiolate basicity and oxidizability. The oxidation-reduction properties of NAC are compared to those of other biological thiols in their definite microscopic forms. The pharmacokinetic behavior is interpreted in terms of the physicochemical parameters, providing molecular/submolecular explanation for several therapeutic properties of NAC.

The site-specific basicities of imatinib (Gleevec, a new signal transduction inhibitor drug of chronic myeloid leukemia) and two of its fragment compounds were quantitated in terms of protonation macroconstants, microconstants, and group constants by NMR-pH and pH-potentiometric titrations. Sequential protonation of imatinib follows the N(34), N(11), N(31), N(13) order, in which N(11) and N(31) show commensurable basicity, but negligible intramolecular interaction. Fragment compounds include two "halves" of imatinib, and their moiety-specific basicities confirm the NMR-based protonation sequence of the parent compound. NMR-pH profiles, macro- and/or microscopic protonation schemes, and species-specific distribution diagrams are presented. On the basis of these data, imatinib is shown to be predominantly neutral, monocationic, and tricationic at intestinal, blood, and gastric pH, respectively. The molecular hypotheses on imatinib binding to the Bcr-Abl oncogene fusion protein are interpreted at the site-specific level in view of the moiety basicities of imatinib.

The time-related metabolic responses to l-arginine (ARG)-induced exocrine pancreatic toxicity were investigated using single ip doses of 1,000 and 4,000 mg/kg body weight over a 7 day experimental period in male Sprague-Dawley rats. Sequential timed urine and plasma samples were analyzed using high resolution (1)H NMR spectroscopy together with complementary clinical chemistry and histopathology analyses. Principal components analysis (PCA) and orthogonal projection on latent structures discriminant analysis (O-PLS-DA) were utilized to analyze the (1)H NMR data and to extract and identify candidate biomarkers and to construct metabolic trajectories post ARG administration. Low doses of ARG resulted in virtually no histopathological damage and distinct reversible metabolic response trajectories. High doses of ARG caused pancreatic acinar degeneration and necrosis and characteristic metabolic trajectory profiles with several distinct phases. The initial trajectory phase (0-8 h) involved changes in the urea cycle and transamination indicating a homeostatic response to detoxify excess ammonia generated from ARG catabolism. By 48 h, there was a notable enhancement of the excretion of the gut microbial metabolites, phenylacetylglycine (PAG), 4-cresol-glucuronide and 4-cresol-sulfate, suggesting that compromised pancreatic function impacts on the activity of the gut microbiota giving potential rise to a novel class of surrogate extragenomic biomarkers of pancreatic injury. The implied compromise of microbiotal function may also contribute to secondary hepatic and pancreatic toxic responses. We show here for the first time the value of metabonomic studies in investigating metabolic disruption due to experimental pancreatitis. The variety of observed systemic responses suggests that this approach may be of general value in the assessment of other animal models or human pancreatitis.

The first complete microequilibrium analysis of a tetrabasic acid is presented. A total of 18 protonation microconstants of glutathione disulfide (GSSG) were determined, 16 of them quantitate the overlapping equilibria in acidic medium, where 4 carboxylate sites protonate. The related, pH-dependent concentrations of 16 coexisting microspecies were also obtained, including the 10 different ones and their 6 identical twins of symmetry origin. The GSSG acid-base chemistry in basic medium is also characterized. It is shown for tetra-and further multibasic acids that the number of traditional microconstants mandatorily exceeds that of the pieces of independent information available by any state-of-the-art methodology, making the complete microequilibrium resolution not only highly complex but theoretically impossible on the basis of hitherto reported principles. The GSSG microspeciation was made feasible here by introducing cumulative microconstants, a new equilibrium parameter, and the invariance of the interactivity coefficient, a plausible simplifying principle, on the experimental basis of 1 H NMR-pH titrations in H 2 O/D 2 O 9/1 media. The methods and results were verified by corresponding data of GSSG-(glycyl)-diethyl-ester, a model compound of reduced microequilibrium complexity. The obtained log k U ) 2.44 and log k Y ) 3.59 "core" microconstants of the respective glutamyl and glycyl carboxylates of GSSG are close to those of the sulfhydryl (GSH) form of glutathione. The GSSG intermoiety interactions were found to be weak, apparently of Coulombic nature. The pH-dependent distribution of the 18 microspecies is depicted.