Advanced oxidation of caffeine in water: on-line and real-time monitoring by electrospray ionization mass spectrometry

High performance liquid chromatography (HPLC), ultraviolet spectroscopy (UV), and total organic carbon (TOC) analyses show that caffeine is quickly and completely degraded underthe oxidative conditions of the UV/H2O2,TiO2/ UV, and Fenton systems but that the organic carbon content of the solution decreases much more slowly. Continuous on-line and real-time monitoring by electrospray ionization mass (ESI-MS) and tandem mass spectrometric experiments (ESI-MS/MS) as well as high accuracy MS measurements and gas chromatography-mass spectrometry analysis show that caffeine is first oxidized to N-dimethylparabanic acid likely via initial OH insertion to the C4=C8 caffeine double bond. A second degradation intermediate, di(N-hidroxymethyl)parabanic acid, has been identified by ESI-MS and characterized by ESI-MS/MS and high accuracy mass measurements. This polar and likely relatively unstable compound, which is not detected by off-line GC-MS analysis, is likely formed via further oxidation of N-dimethylparabanic acid at both of its N-methyl groups and constitutes an unprecedented intermediate in the degradation of caffeine.     

Protein Contacts and Ligand Binding in the Inward‐Facing Model of Human P‐Glycoprotein

The primary aim of this work was to analyze the contacts between residues in the nucleotide binding domains (NBDs) and at the interface between the transmembrane domains (TMDs) and the NBDs in the inward‐open homology model of human P‐glycoprotein (P‐gp). The analysis revealed communication nets through hydrogen bonding in the NBD and at the NBD–TMD interface of each half involving residues from the adenosine triphosphate (ATP) motifs and the coupling helices of the intracellular loops. Similar networks have been identified in P‐gp conformations generated by molecular dynamics simulation. Differences have been recorded in the networking between both halves of P‐gp. Many of the residue contacts have also been observed in the X‐ray crystal structures of other ATP binding cassette (ABC) transporters, which confirms their validity. Next, possible binding pockets involving residues of importance for the TMD–NBD communication were identified. By studying these pockets, binding sites were suggested for rhodamine 123 (R‐site) and prazosin (regulatory site) at the NBD–TMD interface that agreed with the experimental data on their location. Additionally, one more R‐site in the protein cavity was proposed, in accordance with the available biochemical data. Together with the previously suggested Hoechst 33342 site (H‐site), all sites were interpreted with respect to their effects on the protein ATPase activity, in correspondence with the experimental observations. Several residues involved in key contacts in the P‐gp NBDs were proposed for further targeted mutagenesis experiments.     

Identification of putative binding sites of P-glycoprotein based on its homology model

A homology model of P-glycoprotein based on the crystal structure of the multidrug transporter Sav1866 is developed, incorporated into a membrane environment, and optimized. The resulting model is analyzed in relation to the functional state and potential binding sites. The comparison of modeled distances to distances reported in experimental studies between particular residues suggests that the model corresponds most closely to the first ATP hydrolysis step of the protein transport cycle. Comparison to the protein 3D structure confirms this suggestion. Using SiteID and Site Finder programs three membrane related binding regions are identified: a region at the interface between the membrane and cytosol and two regions located in the transmembrane domains. The regions contain binding pockets of different size, orientation, and amino acids. A binding pocket located inside the membrane cavity is also identified. The pockets are analyzed in relation to amino acids shown experimentally to influence the protein function. The results suggest that the protein has multiple binding sites and may bind and/or release substrates in multiple pathways.     

Inhibition of UDP-glucuronosyltransferase by 5'-O-amino acid and oligopeptide derivatives of uridine: structure-activity relationships

The inhibitory effect of a series of 5'-O-amino acid and oligopeptide derivatives of uridine on rat liver UDP-glucuronosyltransferase (UGT) activities was investigated using two assay systems. A quantitative structure-activity relationship (QSAR) study was performed. The compounds include a lipophilic residue linked to the nucleoside by a variable spacer. Moreover, half of the derivatives have two spacers linked to the uridine moiety. Compound 1, a serine derivative of isopropylideneuridine, was found to be the most potent inhibitor of both 4-nitrophenol (4-NP) and phenolphthalein (PPh) glucuronidation, with an I50 of 0.45 mM and 0.22 mM, respectively. Kinetic studies with this substance revealed a mixed type of inhibition towards 4-NP and UDP-glucuronic acid, with apparent Ki values of 150 microM and 120 microM, respectively. The dipeptide derivatives 11-14 exhibited a low activity against 4-NP conjugation. However, a marked suppression of PPh glucuronidation was found with compounds 11 and 13. Generally, compounds with two spacers are more inhibitory against the UGT activities studied. The QSAR analysis outlined the significance of the spacers with a minimum length of 5 atoms and lipophilic residues linked to them for the inhibitory effect of the compounds. The most significant contribution to this effect is given by the six-atom spacer for both 4-NP and PPh substrates. 4-NP converting UGT isoforms seem to respond more specifically to the inhibitors: a five-atom for the first and six-atom for the second spacer enhance binding to both 4-NP and PPh conjugating isoenzymes, while a long second spacer contributes to inhibitor binding to UGT isoforms only converting PPH.     

In Silico Identification of Multi-Target Ligands as Promising Hit Compounds for Neurodegenerative Diseases Drug Development

The conventional treatment of neurodegenerative diseases (NDDs) is based on the “one molecule—one target” paradigm. To combat the multifactorial nature of NDDs, the focus is now shifted toward the development of small-molecule-based compounds that can modulate more than one protein target, known as “multi-target-directed ligands” (MTDLs), while having low affinity for proteins that are irrelevant for the therapy. The in silico approaches have demonstrated a potential to be a suitable tool for the identification of MTDLs as promising drug candidates with reduction in cost and time for research and development. In this study more than 650,000 compounds were screened by a series of in silico approaches to identify drug-like compounds with predicted activity simultaneously towards three important proteins in the NDDs symptomatic treatment: acetylcholinesterase (AChE), histone deacetylase 2 (HDAC2), and monoamine oxidase B (MAO-B). The compounds with affinities below 5.0 µM for all studied targets were additionally filtered to remove known non-specifically binding or unstable compounds. The selected four hits underwent subsequent refinement through in silico blood-brain barrier penetration estimation, safety evaluation, and molecular dynamics simulations resulting in two hit compounds that constitute a rational basis for further development of multi-target active compounds against NDDs.     

Structural and Dynamical Insight into PPARγ Antagonism: In Silico Study of the Ligand-Receptor Interactions of Non-Covalent Antagonists

The structural and dynamical properties of the peroxisome proliferator-activated receptor γ (PPARγ) nuclear receptor have been broadly studied in its agonist state but little is known about the key features required for the receptor antagonistic activity. Here we report a series of molecular dynamics (MD) simulations in combination with free energy estimation of the recently discovered class of non-covalent PPARγ antagonists. Their binding modes and dynamical behavior are described in details. Two key interactions have been detected within the cavity between helices H3, H11 and the activation helix H12, as well as with H12. The strength of the ligand-amino acid residues interactions has been analyzed in relation to the specificity of the ligand dynamical and antagonistic features. According to our results, the PPARγ activation helix does not undergo dramatic conformational changes, as seen in other nuclear receptors, but rather perturbations that occur through a significant ligand-induced reshaping of the ligand-receptor and the receptor-coactivator binding pockets. The H12 residue Tyr473 and the charge clamp residue Glu471 play a central role for the receptor transformations. Our results also demonstrate that MD can be a helpful tool for the compound phenotype characterization (full agonists, partial agonists or antagonists) when insufficient experimental data are available.     

Interactions of the Multidrug Resistance Modulators Tariquidar and Elacridar and their Analogues with P‐glycoprotein

Tariquidar and elacridar are among the most potent inhibitors of the multidrug resistance transporter P‐glycoprotein (P‐gp), but how they interact with the protein is yet unknown. In this work, we describe a possible way in which these inhibitors interact with P‐gp. We rely on structure–activity relationship analysis of a small group of tariquidar and elacridar analogues that was purposefully selected, designed, and tested. Structural modifications of the compounds relate to the presence or absence of functional groups in the tariquidar and elacridar scaffolds. The activity of the compounds was evaluated by their effects on the accumulation of P‐gp substrates rhodamine 123 and Hoechst 33342 in resistant tumor cells. The data allow estimation of the ability of the compounds to interact with the experimentally proposed R‐ and H‐sites to which rhodamine 123 and Hoechst 33342 bind, respectively. Using an inward‐facing homology model of human P‐gp based on the crystallographic structure of mouse P‐gp, we demonstrate that these binding sites may overlap with the binding sites of the QZ59 ligands co‐crystallized with mouse P‐gp. Based on this SAR analysis, and using flexible alignment and docking, we propose possible binding modes for tariquidar and elacridar. Our results suggest the possibility for the studied compounds to bind to sites that coincide or overlap with the binding sites of rhodamine 123 and Hoechst 33342. These results contribute to further understanding of structure–function relationships of P‐gp and can help in the design of selective and potent P‐gp inhibitors with potential clinical use.     

Combined Pharmacophore Modeling,Docking, and 3D QSAR Studies of ABCB1 and ABCC1 Transporter Inhibitors

Quinazolinones, indolo‐ and pyrrolopyrimidines with inhibitory effects toward ABCB1 (P‐gp) and ABCC1 (MRP1) transporters were studied by pharmacophore modeling, docking, and 3D QSAR to describe the binding preferences of the proteins. The pharmacophore overlays between dual and/or highly selective inhibitors point to binding sites of different topology and physiochemical properties for MRP1 and P‐gp. Docking of selective inhibitors into the P‐gp binding cavity by the use of a structural model based on the recently resolved P‐gp structure confirms the P‐gp pharmacophore features identified, and reveals the interactions of some functional groups and atoms in the structures with particular protein residues. The 3D QSAR analysis of the dual‐effect inhibitors allows satisfactory prediction of the selectivity index of the compounds and outlines electrostatics as most important for selectivity. The results from the combined modeling approach complement each other and could improve our understanding of the protein–ligand interactions involved, and could aid in the development of highly selective and potent inhibitors of P‐gp and MRP1.     

Molecular modeling of phenothiazines and related drugs as multidrug resistance modifiers: a comparative molecular field analysis study

A set of 40 phenothiazines, thioxanthenes, and structurally related drugs with multidrug resistance modulating activity in tumor cells in vitro were selected from literature data and subjected to three-dimensional quantitative structure-activity relationship study using comparative molecular field analysis (CoMFA). More than 350 CoMFA models were derived and evaluated using steric, electrostatic, and hydrophobic fields alone and in combination. Four alignment strategies based on selected atom pairs or field fit alignment were compared. Several training and test sets were analyzed for both neutral and protonated drug forms separately. Each chemical class was trained and tested individually, and finally the classes were combined together into integrated models. All models obtained were statistically significant and most of them highly predictive. All fields contributed to MDR reversing activity, and hydrophobic fields improved the correlative and predictive power of the models in all cases. The results point to the role of hydrophobicity as a space-directed molecular property to explain differences in anti-MDR activity of the drugs studied.