Identification Of Novel Agonists Research Articles

Ligand-dependent modulation of MR1-restricted antigen presentation

Mucosal-associated invariant T (MAIT) cells are innate T cells recognising intermediates of the vitamin B2 biosynthetic pathway presented by the monomorphic MR1 molecule. We have demonstrated that in addition to their cytolytic activity important in antimicrobial defence, MAIT cells have also immune-modulatory functions, enhancing DC maturation, in vitro and in vivo. Furthermore, through an in silico and in vitro screening of commercial libraries, we have identified novel compounds capable of activating MAIT cells and the first compounds that inhibits of MAIT cell activation through downregulation of MR1 expression. Identification of novel agonists and antagonists of MAIT cell function paves the way for potentially harnessing MAIT cell function in vivo .

Identification of novel agonists by high-throughput screening and molecular modelling of human constitutive androstane receptor isoform 3.

Prediction of drug interactions, based on the induction of drug disposition, calls for the identification of chemicals, which activate xenosensing nuclear receptors. Constitutive androstane receptor (CAR) is one of the major human xenosensors; however, the constitutive activity of its reference variant CAR1 in immortalized cell lines complicates the identification of agonists. The exclusively ligand-dependent isoform CAR3 represents an obvious alternative for screening of CAR agonists. As CAR3 is even more abundant in human liver than CAR1, identification of its agonists is also of pharmacological value in its own right. We here established a cellular high-throughput screening assay for CAR3 to identify ligands of this isoform and to analyse its suitability for identifying CAR ligands in general. Proof-of-concept screening of 2054 drug-like compounds at 10µM resulted in the identification of novel CAR3 agonists. The CAR3 assay proved to detect the previously described CAR1 ligands in the screened libraries. However, we failed to detect CAR3-selective compounds, as the four novel agonists, which were selected for further investigations, all proved to activate CAR1 in different cellular and in vitro assays. In primary human hepatocytes, the compounds preferentially induced the expression of the prototypical CAR target gene CYP2B6. Failure to identify CAR3-selective compounds was investigated by molecular modelling, which showed that the isoform-specific insertion of five amino acids did not impact on the ligand binding pocket but only on heterodimerization with retinoid X receptor. In conclusion, we demonstrate here the usability of CAR3 for screening compound libraries for the presence of CAR agonists.

Identification of novel agonists and antagonists of the ecdysone receptor by virtual screening

Insect growth is regulated by the steroid hormone 20-hydroxyecdysone (20E), which works via the ecdysone receptor (EcR). To identify biologically active and novel ecdysone agonists/antagonists, ligand/structure-based virtual screening combined with pharmacophore modeling and molecular docking was performed to identify novel nonsteroidal lead compounds. Nine molecules were screened and selected for an in vitro cell-based reporter bioassay. The results showed that VS-006 and VS-009 exhibited antagonistic activity in S2 cells, whereas only VS-006 exhibited antagonistic activity in Bm5 cells. Molecular dynamic simulation of VS-006 complexed with the ligand binding domain of EcR validated the binding stability of VS-006 and highlighted the key residues for further lead optimization.

Ligand fishing using new chitosan based functionalized Androgen Receptor magnetic particles

Superparamagnetic nanoparticles with chemically modified chitosan has been proposed as a potential support for the immobilization of the androgen receptor (AR). The study involved comparison of different AR carriers like commercially available magnetic beads coated with silica (BcMag) and chitosan coated nanoparticles with different amount of amino groups. The immobilization was carried out through covalent immobilization of the AR through the terminal amino group or through available carboxylic acids. The initial characterization of the AR coated magnetic beads was carried out with dihydrotestosterone, a known AR ligand. Subsequently, chitosan modified nanporticles with long-distanced primary amino groups (Fe3O4CS-(NH2)3) (upto 8.34mM/g) were used for further study to isolate known AR ligands (bicalutamide, flutamide, hydroxyflutamide and levonogestrel) from a mixture of tested compounds in ammonium acetate buffer [10mM, pH 7.4]. The results showed that the selected nanoparticles are a promising semi-quantitative tool for the identification of high affinity compounds to AR and might be of special importance in the identification of novel agonists or antiandrogens.

Quantitative analysis of G-protein-coupled receptor internalization using DnaE intein-based assay.

G-protein-coupled receptors (GPCRs), the largest family of cell surface receptors, are involved in many physiological processes. They represent highly important therapeutic targets for drug discovery. Currently, there are numerous cell-based assays developed for the pharmacological profiling of GPCRs and the identification of novel agonists and antagonists. However, the development of new, faster, easier, and more cost-effective approaches to detect GPCR activity remains highly desirable. β-arrestin-dependent internalization has been demonstrated to be a common mechanism for most GPCRs. Here we describe a novel assay for quantitative analysis of GPCR internalization based on DnaE intein-mediated reconstitution of fragmented Renilla luciferase or Firefly luciferase when activated GPCRs interact with β-arrestin2 or Rab5. Further validation, using functionally divergent GPCRs, showed that EC50 values obtained for the known agonists and antagonists were in close agreement with the results of previous reports. This suggests that this assay is sensitive enough to permit quantification of GPCR internalization. Compared with conventional assays, this novel assay system is cost-effective, rapid, and easy to manipulate. These advantages may allow this assay to be used universally as afunctional cell-based system for GPCR characterization and in the screening process of drug discovery.

Identification of novel agonists of the integrin CD11b/CD18

We report the identification of novel small molecule agonists of integrin CD11b/CD18, which increased, in a dose-dependent manner, the adhesion of the integrin CD11b/CD18 expressing cells to two physiologically relevant ligands: Fibrinogen and iC3b. Compound 6 showed an ex vivo EC50 of 10.5μM and in vitro selectivity for binding to the recombinant αA-domain of CD11b/CD18. In silico docking experiments suggest that the compounds recognized a hydrophobic cleft in the ligand-binding αA-domain, implying an allosteric mechanism of modulation of integrin affinity by this novel compound.

Identification of a novel PPARα‐interacting protein

PPARα (peroxisome proliferator‐activated receptor α) is a ligand‐activated transcription factor involved in the regulation of fatty acid β‐oxidation. It is mainly expressed in tissues with elevated energy metabolism like liver, heart, kidney, skeletal muscle and brown fat. Upon ligand binding, PPARα heterodimerizes with the retinoid X receptor α (RXRα). The PPARα‐RXRα complex binds to specific PPAR response elements (PPRE) in the promoter regions of target genes enhancing their transcription. PPARα is activated by fibrates, synthetic compounds that induce peroxisome proliferation in rodents and are used as hypolipidemic drugs in humans. Many studies aim at the identification of novel agonists and cofactors that can modulate PPARα activity. We identified ACOX1 (Acyl Coenzyme A Oxidase) as a PPARα‐interacting protein by pull‐down using PPARα‐GST fusion proteins and 35S‐labeled‐in vitro translated ACOX1. ACOX1 is able to increase the transcriptional activity of PPARα in transient transfection experiments in U2OS (human osteosarcoma) cells. ACOX1 is the first and rate‐limiting enzyme of the peroxisomal fatty acid β‐oxidation and its expression is induced by PPARα. These results suggest the possibility of a novel regulatory loop between PPARα and a metabolic enzyme.Supported by the Intramural Research Program of NIDDK, NIH.

A Synthetic Peptidoglycan Fragment as a Competitive Inhibitor of the Melanization Cascade

Melanin synthesis is essential for defense and development but must be tightly controlled because systemic hyperactivation of the prophenoloxidase and excessive melanin synthesis are deleterious to the hosts. The melanization cascade of the arthropods can be activated by bacterial lysine-peptidoglycan (PGN), diaminopimelic acid (DAP)-PGN, or fungal beta-1,3-glucan. The molecular mechanism of how DAP- or Lys-PGN induces melanin synthesis and which molecules are involved in distinguishing these PGNs are not known. The identification of PGN derivatives that can work as inhibitors of the melanization cascade and the characterization of PGN recognition molecules will provide important information to clarify how the melanization is regulated and controlled. Here, we report that a novel synthetic Lys-PGN fragment ((GlcNAc-MurNAc-L-Ala-D-isoGln-L-Lys-D-Ala)2, T-4P2) functions as a competitive inhibitor of the natural PGN-induced melanization reaction. By using a T-4P2-coupled column, we purified the Tenebrio molitor PGN recognition protein (Tm-PGRP) without causing activation of the prophenoloxidase. The purified Tm-PGRP recognized both Lys- and DAP-PGN. In vitro reconstitution experiments showed that Tm-PGRP functions as a common recognition molecule of Lys- and DAP-PGN-dependent melanization cascades.

A cell-based microarrayed compound screening format for identifying agonists of G-protein-coupled receptors

The identification of agonist and antagonist leads for G-protein-coupled receptors (GPCRs) is of critical importance to the pharmaceutical and biotechnology industries. We report on the utilization of a novel, high-density, well-less screening platform known as microarrayed compound screening (μARCS) that tests 8640 compounds in the footprint of a standard microtiter plate for the identification of novel agonists for a specific G-protein-coupled receptor. Although receptors coupled to the Gα q protein can readily be assessed by fluorescence-based Ca 2+ release measurements, many GPCRs that are coupled to Gα s or Gα i/o proteins are not amenable to functional evaluation in such a high-throughput manner. In this study, the human dopamine D 4.4 receptor, which normally couples through the Gα i/o protein to inhibit adenylate cyclase and to reduce levels of intracellular cAMP, was coupled to intracellular Ca 2+ release by stably coexpressing this receptor with a chimeric G αqo5 protein in HEK-293 cells. In μARCS format, the cells expressing D 4.4 receptor and Gα qo5 protein were preloaded with fluo-4, cast into a 1% agarose gel, placed above the compound sheets, and imaged successively using a ViewLux charge-coupled device imaging system. Dopamine and other agonists evoked an increase in fluorescence response that appeared as bright spots in a time- and concentration-dependent manner. Utilizing this technology, a library of 260,000 compounds was rapidly screened and led to the identification of several novel agonists. These agonists were further characterized using a fluorometric imaging plate reader assay. Excellent confirmation rates coupled with enhanced efficiency and throughput enable μARCS to serve as an alternative platform for the screening and identification of novel GPCR agonists.

Research agenda for pediatric gastroenterology, hepatology and nutrition: molecular basis of gastrointestinal diseases. Report of the North American Society for Pediatric Gastroenterology, Hepatology and Nutrition for the Children's Digestive Health and Nutrition Foundation.

Research agenda for pediatric gastroenterology, hepatology and nutrition: molecular basis of gastrointestinal diseases. Report of the North American Society for Pediatric Gastroenterology, Hepatology and Nutrition for the Children's Digestive Health and Nutrition Foundation.