ABSTRACT
[4-(4-Methyl-2-(4-(trifluoromethyl)phenyl)thiazole-5-yl)pyrimidine-2-amine] (JNJ-2482272), under investigation as an anti-inflammatory agent, was orally administered to rats once daily at 60 mg/kg for 6 consecutive days. Despite high plasma exposure after single administration (Cmax of 7.1 µM), JNJ-2482272 had plasma concentrations beneath the lower limit of quantification (3 ng/ml) after 6 consecutive days of dosing. To determine if JNJ-2482272 is an autoinducer in rats, plated rat hepatocytes were treated with JNJ-2482272 for 2 days. The major hydroxylated metabolites of JNJ-2482272 were isolated and characterized by mass spectrometry and NMR analyses. Compared with the vehicle-treated cells, a concentration-dependent increase was observed in the formation of phase I- and II-mediated metabolites coinciding with greater expression of cytochrome P450s (P450s) and UDP-glucuronosyltransferases (UGTs) in rat hepatocytes. CYP1A1, CYP1A2, CYP1B1, and UGT1A6 transcripts were predominantly induced, suggesting that JNJ-2482272 is an activator of the aryl hydrocarbon receptor (AhR). In a human AhR reporter assay, JNJ-2482272 demonstrated potent AhR activation with an EC50 value of 0.768 nM, a potency more comparable to the strong AhR activator and toxin 2,3,7,8-tetrachloro-dibenzodioxin than to weaker AhR activators 3-methylcholanthrene, ß-naphthoflavone, and omeprazole. In plated human hepatocytes, JNJ-2482272 induced CYP1A1 gene expression with an EC50 of 20.4 nM and increased CYP1A activity >50-fold from basal levels. In human recombinant P450s, JNJ-2482272 was exclusively metabolized by the CYP1 family of enzymes and most rapidly by CYP1A1. The summation of these in vitro findings bridges the in vivo conclusion that JNJ-2482272 is a strong autoinducer in rats and potentially in humans through potent AhR activation. SIGNIFICANCE STATEMENT: Drugs that induce their own metabolism (autoinducers) can lack sustained exposures for pharmacology and safety assessment hindering their development. JNJ-2482272 is demonstrated herein as a strong aryl hydrocarbon receptor (AhR) activator and CYP1A autoinducer, explaining its near complete loss of exposure after repeat administration in rat, which is likely translatable to human (if progressed further) considering its nanomolar potency comparable to "classical" AhR ligands like 2,3,7,8-tetrachloro-dibenzo-dioxin despite bearing a "nonclassical" drug structure.
Subject(s)
Cytochrome P-450 CYP1A1 , Receptors, Aryl Hydrocarbon , Amines , Animals , Cytochrome P-450 CYP1A1/metabolism , Humans , Pyrimidines/pharmacology , Rats , Receptors, Aryl Hydrocarbon/metabolism , Thiazoles/pharmacologyABSTRACT
Excitatory amino acid transporters (EAATs) represent a protein family that is an emerging drug target with great therapeutic potential for managing central nervous system disorders characterized by dysregulation of glutamatergic neurotransmission. As such, it is of significant interest to discover selective modulators of EAAT2 function. Here, we applied computational methods to identify specific EAAT2 inhibitors. Utilizing a homology model of human EAAT2, we identified a binding pocket at the interface of the transport and trimerization domain. We next conducted a high-throughput virtual screen against this site and identified a selective class of EAAT2 inhibitors that were tested in glutamate uptake and whole-cell electrophysiology assays. These compounds represent potentially useful pharmacological tools suitable for further exploration of the therapeutic potential of EAAT2 and may provide molecular insights into mechanisms of allosteric modulation for glutamate transporters.
Subject(s)
Amino Acid Transport System X-AG/antagonists & inhibitors , Binding Sites/drug effects , Central Nervous System Diseases/drug therapy , Excitatory Amino Acid Transporter 2/antagonists & inhibitors , Amino Acid Transport System X-AG/chemistry , Amino Acid Transport System X-AG/genetics , Animals , Binding Sites/genetics , Biological Transport/drug effects , Central Nervous System Diseases/genetics , Central Nervous System Diseases/pathology , Computational Biology , Excitatory Amino Acid Transporter 2/chemistry , Excitatory Amino Acid Transporter 2/genetics , Humans , Protein Binding/drug effects , Synaptic Transmission/drug effects , User-Computer InterfaceABSTRACT
At the onset of a drug discovery program, the goal is to identify novel compounds with appropriate chemical features that can be taken forward as lead series. Here, we describe three prospective case studies, Bruton Tyrosine Kinase (BTK), RAR-Related Orphan Receptor γ t (RORγt), and Human Leukocyte Antigen DR isotype (HLA-DR) to illustrate the positive impact of high throughput virtual screening (HTVS) on the successful identification of novel chemical series. Each case represents a project with a varying degree of difficulty due to the amount of structural and ligand information available internally or in the public domain to utilize in the virtual screens. We show that HTVS can be effectively employed to identify a diverse set of potent hits for each protein system even when the gold standard, high resolution structural data or ligand binding data for benchmarking, is not available.
Subject(s)
Drug Evaluation, Preclinical/methods , High-Throughput Screening Assays/methods , Agammaglobulinaemia Tyrosine Kinase/antagonists & inhibitors , Agammaglobulinaemia Tyrosine Kinase/chemistry , Drug Industry , HLA-DR Antigens/chemistry , HLA-DR Antigens/metabolism , Humans , Models, Molecular , Orphan Nuclear Receptors/chemistry , Orphan Nuclear Receptors/metabolism , Protein Conformation , Protein Kinase Inhibitors/pharmacology , Time Factors , User-Computer InterfaceABSTRACT
We have developed a workflow to extract, separate, and semi-quantify bioactive oxysterols from mouse colon tissues and fecal matters using solid- and liquid-phase extractions, enzymatic and chemical modifications, and stable-isotope dilution LC/MS/MS. The method was applied to a dextran sodium sulphate (DSS)-induced mouse colitis model, which revealed that one particular dihydroxycholesterol (diOHC), 7α,25-diOHC, was significantly elevated in both colon tissue and fecal matters of mice with colitis compared to that in naïve mice. The extent of 7α,25-diOHC elevation was positively correlated with colitis severity.
Subject(s)
Colitis/chemically induced , Colon/chemistry , Disease Models, Animal , Oxysterols/isolation & purification , Animals , Chromatography, Liquid , Colon/pathology , Dextran Sulfate , Female , Mice , Mice, Inbred C57BL , Oxysterols/chemistry , Tandem Mass SpectrometryABSTRACT
Here, we report a high-throughput virtual screening (HTVS) study using phosphoinositide 3-kinase (both PI3Kγ and PI3Kδ). Our initial HTVS results of the Janssen corporate database identified small focused libraries with hit rates at 50% inhibition showing a 50-fold increase over those from a HTS (high-throughput screen). Further, applying constraints based on "chemically intuitive" hydrogen bonds and/or positional requirements resulted in a substantial improvement in the hit rates (versus no constraints) and reduced docking time. While we find that docking scoring functions are not capable of providing a reliable relative ranking of a set of compounds, a prioritization of groups of compounds (e.g., low, medium, and high) does emerge, which allows for the chemistry efforts to be quickly focused on the most viable candidates. Thus, this illustrates that it is not always necessary to have a high correlation between a computational score and the experimental data to impact the drug discovery process.
Subject(s)
Enzyme Inhibitors/pharmacology , Isoenzymes/antagonists & inhibitors , Phosphoinositide-3 Kinase Inhibitors , Drug Design , High-Throughput Screening Assays , Molecular Docking Simulation , Prospective StudiesABSTRACT
We report a Cu(I)-catalyzed azide-alkyne-allyl halide three-component reaction for a one-pot synthesis of 1,4-disubstituted 5-allyl-1,2,3-triazoles. The allyl moiety provides not only the electrophile but also a coordinating ligand to Cu, which is essential for the reaction to occur under mild conditions. A concise synthesis of a potential drug candidate 1 is realized based on this key reaction.
ABSTRACT
The preclinical characterization of novel phenyl(piperazin-1-yl)methanones that are histamine H3 receptor antagonists is described. The compounds described are high affinity histamine H3 antagonists. Optimization of the physical properties of these histamine H3 antagonists led to the discovery of several promising lead compounds, and extensive preclinical profiling aided in the identification of compounds with optimal duration of action for wake promoting activity. This led to the discovery of two development candidates for Phase I and Phase II clinical trials.
ABSTRACT
In this paper we report the development of chromatographic methods for the separation of 8 biologically active hydroxycholesterols (OHC's) which include the single-hydroxyl addition species 7α-OHC, 7ß-OHC, 25-OHC and 27- OHC, together with the double-hydroxyl addition species 7α, 25-OHC, 7ß, 25-OHC, 7α, 27-OHC, and 7ß, 27-OHC. Four complementary techniques were employed (gas chromatography, normal phase and reversed phase high performance liquid chromatography, and supercritical fluid chromatography), and for each of the techniques, an optimized method for the separation of all eight compounds in a mixture is presented.
Subject(s)
Chromatography, Supercritical Fluid , Hydroxycholesterols/isolation & purification , Chromatography, Gas , Chromatography, High Pressure Liquid , Molecular ConformationABSTRACT
CuI/L5 (N,N'-dimethylethylenediamine) proves to be an efficient catalyst system for the amination of arylhalides with guanidines. The same catalyst system is then successfully applied to the one-step synthesis of 1-H-2-amino-benzimidazoles through tandem aminations of 1,2-dihaloarenes in modest yields. This methodology is also applicable for the preparation of 1-H or 1-substutituted 2-aryl- or 2-alkyl-benzimidazoles.
Subject(s)
Amidines/chemistry , Benzimidazoles/chemical synthesis , Copper/chemistry , Guanidine/chemistry , Hydrocarbons, Halogenated/chemistry , Iodides/chemistry , Amination , Benzimidazoles/chemistry , Catalysis , Ethylenediamines/chemistry , Molecular Structure , StereoisomerismABSTRACT
The synthesis and structure-activity relationships of a series of novel phenoxyphenyl diamine derivatives with affinity for both the histamine H(3) receptor and the serotonin transporter is described.
Subject(s)
Diamines/chemistry , Histamine Antagonists/chemistry , Histamine Antagonists/pharmacology , Receptors, Histamine H3/drug effects , Selective Serotonin Reuptake Inhibitors/chemistry , Selective Serotonin Reuptake Inhibitors/pharmacology , Histamine Antagonists/chemical synthesis , Humans , Molecular Structure , Receptors, Histamine H3/chemistry , Selective Serotonin Reuptake Inhibitors/chemical synthesis , Structure-Activity RelationshipABSTRACT
A series of novel 4-aryl-1,2,3,4-tetrahydroisoquinoline-based histamine H(3) ligands that also have serotonin reuptake transporter inhibitor activity is described. The synthesis, in vitro biological data, and select pharmacokinetic data for these novel compounds are discussed.
Subject(s)
Histamine Antagonists/chemical synthesis , Histamine Antagonists/pharmacology , Receptors, Histamine H3/drug effects , Selective Serotonin Reuptake Inhibitors/chemical synthesis , Selective Serotonin Reuptake Inhibitors/pharmacology , Tetrahydroisoquinolines/chemical synthesis , Tetrahydroisoquinolines/pharmacology , Animals , Crystallography, X-Ray , Dopamine Plasma Membrane Transport Proteins/drug effects , Dopamine Plasma Membrane Transport Proteins/metabolism , Half-Life , Humans , Indicators and Reagents , Models, Molecular , Molecular Conformation , Norepinephrine/metabolism , Rats , Serotonin Plasma Membrane Transport Proteins/drug effects , Serotonin Plasma Membrane Transport Proteins/metabolism , Selective Serotonin Reuptake Inhibitors/pharmacokinetics , Structure-Activity Relationship , Tetrahydroisoquinolines/pharmacokineticsABSTRACT
In supercritical fluid chromatography, the partial phase separation that occurs in the dead volume between automatic backpressure regulator and collection vessels causes significant peak tailing and delayed arrival of compounds to the collectors. As a result, when two peaks are barely baseline separated, it becomes very difficult to correctly set fraction collection triggers, which in turn results in fractions being collected with lower purity and lower yield. The problem can be solved with a simple addition of a four-port, two-position switching valve between UV detector and automatic back pressure regulator. The valve acts as a timed gate to release each peak out of the back pressure regulator and into the collection vessel, while at the same time it holds later peaks inside the UV detector and the column in a closed loop.
Subject(s)
Chromatography, Liquid/methods , Carbon Dioxide/analysis , Chromatography, High Pressure Liquid , Indicators and Reagents , Methanol , Solvents , Spectrophotometry, UltravioletABSTRACT
An automated, accurate and reliable way of acquiring and processing flow injection data for exact mass measurement using a bench-top electrospray ionization time-of-flight (ESI-TOF) mass spectrometer is described. Using Visual Basic programs, individual scans were selected objectively with restrictions on ion counts per second for both the compound of interest and the mass reference peaks. The selected "good scans" were then subjected to two different data-processing schemes ("combine-then-center" and "center-then-average"), and the results were compared at various ion count limit settings. It was found that, in general, the average of mass values from individual scans is more accurate than the centroid mass value of the combined (same) scans. In order to acquire a large number of good scans in one injection (to increase the sampling size for statistically valid averaging), an on-line dilution chamber was added to slow down the typically rapid mass chromatographic peak decay in flow-injection analysis. This simple addition worked well in automation without the need for manual sample dilution. In addition, by dissolving the reference compound directly into the mobile phase, manual syringe filling can be eliminated. Twenty-seven samples were analyzed with the new acquisition and process routines in positive electrospray ionization mode. For the best method found, the percentage of samples with RMS error less than 5 ppm was 100% with repetitive injection data (6 injections per sample), and 95% with single injection data. Afterwards, 31 other test samples were run (with MW ranging from 310 to 3493 Da, 21 samples in ESI+ and 10 in ESI- mode) and processed with similar parameters and 100% of them were mass-calculated to RMS error less than 5 ppm also.
Subject(s)
Spectrometry, Mass, Electrospray Ionization/methods , Automation/methods , Molecular Weight , Research Design , Sensitivity and SpecificityABSTRACT
Specific sequences in heparin are responsible for its modulation of the biological activity of proteins. As part of a program to characterize heparin-peptide and heparin-protein binding, we are studying the interaction of chemically discrete heparin-derived oligosaccharides with peptides and proteins. We report here the isolation and characterization, by one- and two-dimensional 1H NMR spectroscopies, of ten hexasaccharides, one pentasaccharide, and one octasaccharide serine that were isolated from depolymerized porcine intestinal mucosal heparin. Hexasaccharides were chosen for study because they fall within the size range, typically tetra- to decasaccharide in length, of heparin sequences that modulate the activity of proteins. The depolymerization reaction was catalyzed by heparinase I (EC 4.2.2.7) in the presence of histamine, which binds site specifically to heparin. Histamine increases both the rate and extent of heparinase I-catalyzed depolymerization of heparin. It is proposed that oligosaccharides produced by heparinase I-catalyzed depolymerization can inhibit the enzyme by binding to the imidazolium group of histidine-203, which together with cysteine-135 forms the catalytic domain of heparinase I. The increased rate and extent of depolymerization are attributed to competitive binding of the oligosaccharides by histamine.