ABSTRACT
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
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.
