nterference with NFkB and AP-1, GC-bound GR also interacts with other transcription factors involved in immune responses like IRF-3, NFAT, STATs, GATA-3, T-bet and CREB. Of note, a recent publication indicates that a large number of genes involved in GC-induced side effects is regulated via a functional negative GRE in their Go-6983 promoter region. Furthermore, a series of studies has shown that even subtle changes in the GR-ligand may alter conformation of the ligand-receptor complex with consequences for co-factor recruitment and hence for function of the receptor. Here we describe a novel non-steroidal, low-molecular weight GR ligand, Org 214007-0, the design of which was based on interference with the loop between helix 11 and helix 12 at the ligand-GR binding site. In comparison to prednisolone Org 214007-0 shows an overall improved therapeutic index in vitro as well as in vivo. Most importantly Org 214007-0 displays sustained full anti-inflammatory activity in vivo in mice, without adverse effects on plasma glucose levels and hepatic glucose metabolism. detail in reference) was based on both structural rationale and optimization of this class of tetracyclic compounds via molecular profiling, as will be described below. The structural rationale was based on GR mutation studies and ligand-based structure-activity relationships reviewed by Lusher et al. These studies indicate that the loop between helix-11 and helix-12 of GR represents a hotspot that is crucial to the agonism/antagonism balance in GR, amongst others through modification of co-activator binding. In particular, modified binding of the third LXXLL motif in coactivators as Steroid Receptor Co-activator -1 and Transcriptional Intermediary Factor 2 by conformational changes of this loop has been desribed. The strategy in the design of Org 214007-0 was therefore to interfere with this hotspot region, potentially inducing a conformational change of GR that differs from that induced by prednisolone and modifying co-activator binding. In vitro profiling Org 214007-0 was found to bind to GR as efficient as prednisolone . For a full molecular profiling of Org 2140070 and establishing whether changes in the Org 214007-0-induced GR conformation would specifically affect the expression of a selection of GR targetgenes in vitro, the following studies were performed in which Org 214007-0 was compared to prednisolone. GR-selectivity of Org 
214007-0, either as agonist or antagonist, was evaluated by testing the compound in CHO cell lines, cotransfected with one of the human nuclear receptors and their respective reporter construct. In comparison to prednisolone, Org 214007-0 showed a strong potency but a clearly lower efficacy in the GRE-mediated induction of gene expression in the human GR transfected cell line. No agonistic activity of Org 214007-0 for other human steroid receptors was detected. Org 214007-0 only possessed minor antagonistic activity for the human progestagen and mineralocorticoid receptor, as shown in Results Design of Org 214007-0 and co-factor recruitment Org 214007-0, -N–4-methyl-1,2,3-thiadiazole-5-carboxamide], the structure of which is shown in tory activity of 10884520 Org 214007-0 in comparison to prednisolone was quantified in a stably human GR transfected cell line stimulated to elicit an inflammatory response. Org 214007-0 was shown to inhibit this response with a potency comparable to that of prednisolone, 9874164 while its efficacy was somewhat less compared to prednisolone .
