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Application Note - Nuclear Receptor; Profiling Ligands Detection of multiple nuclear receptor–coregulator interactions in a single sample using MARCoNI PamChip technology René Houtman, Dirk Pijnenburg, Diana Melchers & Rob Ruijtenbeek PamGene International B. V., ’s Hertogenbosch, The Netherlands Study Design Here, we present our next generation of high-content MARCoNI chips with a set of 155 immobilised peptides on each PamChip® array, which represent coregulatorderived motifs. Per array, the nuclear receptor/ligand conditions can be varied and in vitro interaction data for all 155 motifs are obtained within one hour. The principle and an example of nuclear receptor binding onto coregulator peptides immobilized on the PamChip® arrays are shown in figure 1. In figure 2 we show examples of the modulation of 15 different nuclear receptor ligand binding domains (Invitrogen) with their natural or synthetic ligands. Key Findings NR-coregulator interaction profiles were performed on 15 different NRs on 155 known NR coregulator proteins harboring either LXXLL (in coactivators) or LXXXIXXXL (in corepressors) motifs. Each of the NRs shows a different and specific modulation by its natural/synthetic ligand (figure 2). “Author Quote” An obvious application of this NR-coregulator interaction profiling platform is the screening at high definition of novel NR and their NR (ant)agonists. Background Cofactor recruitment is a crucial regulatory step in nuclear receptor (NR) signal transduction. The pathways towards gene expression involve ligand-dependent and independent interactions between NR and coregulator (CoR) proteins. In view of drug development, profiling these NR-CoR interactions is of importance to understand mechanisms of drug action, to steer drug specificity, to understand putative adverse drug effects (on- versus off target), and to tailor the pharmacotherapies. NR + CoReg NR + CoReg NR-CoReg NR-CoReg nd ligad n liga n ligad n liga 1 1 NR-ligand + CoReg NR-ligand + CoReg 2 NR-ligand-CoReg 2 NR-ligand-CoReg NR control control 2 d NR + Ligand + ligand + ligand 1 2 1 2 Figure 1: Schematic overview of1 Nuclear 2Receptor (NR)1and Coregulator proteins binding as function of the ligand. The blue arrow (1) shows the effect of the ligand as an antagonist on a coregulator protein whereas the red arrow (2) shows the effect of the ligand as an agonist on a coregulator protein. ER ERα +17--estradiol ERβ +17--estradiol AR- T877A Dihydroxytestosterone GR Dexamethasone PR Progesterone LXR LXRβ +T0901317 LXRα +T0901317 PPAR PPAR +WY14643 PPAR + rosiglitazone PPAR + GW501516 RXRα Methoprene acid FXR GW4064 ERR ER +4-OHT ER+4-OHT ER+XCT79 Figure 2: The modulation of 15 different nuclear receptors are shows as function of their synthetic of natural ligands across 155 coregulator proteins on the PamChip array. The modulation is the difference in binding of the ligand binding domain (LBD) of the nuclear receptor and the LBDligand complex. References: Houtman R., 2nd Benelux Nuclear Receptor Meeting, Netherlands 11-2009 Conclusion MARCoNI technology, using dynamic PamChip® peptide microarrays, provides a powerful method for profiling of endogenous and ligand-dependent nuclear receptor-coregulator interactions. ©2013 PamGene International B.V. All rights reserved Application note: #201015 (10-2010) Contact us: Wolvenhoek 10, 5211 HH Den Bosch, the Netherlands +31 73 615 80 80, [email protected]