In the present study we set out to discover new

In the present study we set out to discover new non-steroidal SEGRA and to analyse their mechanism of function. Towards this goal we initiated a virtual screening (VS) approach, utilizing structure-based pharmacophore modeling, in silico docking and molecular dynamics (MD) simulations. Our pharmacophore model was generated based on the bound pose of an aminopyrazole analogue bearing a terminal pyrrolidinone moiety, which was shown in vitro to display the desired biological profile (named as ‘compound 12′ in Biggadike et al [9]). The selected non-steroidal epinastine hydrochloride is accommodated in the extended binding cavity of the GR LBD which is expanded beyond the typical steroidal A-ring region. Due to the lack of the respective complex crystal structure, the x-ray structure of GR LBD with another aminopyrazole analogue bearing a terminal d-prolinamide moiety (named as ‘compound 11′ [9]; pdb: 3K23) was used as an initial template to dock the pyrrolidinone amide. The derived complex was subsequently subjected to MD simulations in order to define the essential pharmacophore features. The pharmacophore model was optimized and validated by utilizing carefully selected receptor modulators with the desired biological profile. Ambinter database (7.8 million chemical structures) was filtered based on the physicochemical properties of known SEGRAs and screened against the optimized pharmacophore model. The best fitted to the model compounds were evaluated for their binding potential to GR LBD through in silico docking by applying algorithms of increasing accuracy. Among the best ranked, 14 compounds were acquired taking into account the crucial binding contacts and the predicted ADME profile. Biological evaluation of the selected compounds showed that none of them mediate GRE-dependent, GR-mediated transactivation of gene expression while nine of them significantly repress transactivation induced by classical GR agonists. Two of these compounds (1 and 13) bind to GR, although with lower affinity compared to dexamethasone, thus leading to its nuclear translocation and to repression of NF-kB-mediated transactivation of a subset of pro-inflammatory genes in a GR-dependent manner. Our data provide evidence that compounds 1 and 13, which share a benzothiazole scaffold linked to a sulfanyl-N-phenylethyl acetamide moiety, are genuine non-steroidal SEGRA.
Materials and methods
Results
Discussion and conclusion In the present study we implemented a pharmacophore-based VS coupled with molecular docking approach in order to identify novel non-steroidal SEGRA. Our VS protocol designated a series of chemotypes as potential ligands and the biological evaluation revealed two hits (1 and 13) which share a 1,3-benzothiazole scaffold linked to a sulfanyl-N-phenylethyl acetamide moiety at position 2. The predicted binding poses of 1 and 13 lay at the extended cavity of GR LBD contacting crucial residues (Asn564, Gln570, Phe623, Arg611). Moreover, the discovered hits suggest new binding contacts at the edge of the extended cavity (Trp577 and Lys667) which could be exploited further as possible key residues in hit optimization. This specific binding motif along with their high predicted lipophilicity (clogP 1 = 5.9, clogP 13 = 5.8) could contribute significantly to their biological effectiveness. The potent role of these two residues (Trp577 and Lys667) has been further highlighted by alanine scanning MD studies. Hit 13 is predicted to bind with slightly higher affinity at GR-LDB as compared to hit 1 (Δ and Docking score), favored by the observed embracement of the naphthyl moiety into the cavity formed by the lipophilic residues of the α helices H5 and H10. Biological evaluation (overviewed in Supplementary Figure S11) revealed that the two hit compounds, 1 and 13, cannot mediate GRE-dependent transactivation, as shown for the GRE-dependent luciferase reporter and endogenous FKBP5 genes, and therefore they are not classical GCs. However, they were able to inhibit HC-mediated transactivation by binding to GR and displacement of HC. These data are in agreement with previous observations showing that the steroidal SEGRA 21 partially displaces dexamethasone from rat GR and inhibits dexamethasone-induced transactivation of a GRE-dependent reporter gene whereas it cannot activate expression of tyrosine aminotransferase (TAT), a GRE-dependent gene in rat hepatocytes [30,31]. Displacement of HC by binding of 1 and 13 to GR was supported by the fluorescence polarization data showing that compounds 1 and 13 bind to GR although with less affinity as compared to dexamethasone. The lack of binding to GR at 10 μM for seven compounds that inhibit HC-mediated transactivation may be due, at least in part, to differences in the critical conditions of each assay but it can also imply that (some of) the other compounds bind to GR at higher concentrations. However, for the biological evaluation we selected compounds 1 and 13 that bind to GR at low micromolar concentration. Moreover, binding of 1 and 13 to GR is corroborated by GR nuclear translocation data. Localization of the unliganded GR both in the cytoplasm and in the nucleus of HeLa B2 cells is in agreement with the shuttling of GR between these two cellular compartments [reviewed in 32]. Induction of GR nuclear localization by 13 and 21 to an extent similar to that observed upon treatment with dexamethasone and the nuclear localization partially induced by 1 are in agreement with the higher GR-binding affinity and the higher potency in the inhibition of hydrocortizone-mediated transactivation of 13 and 21 as compared to 1. This was also predicted by our in silico data thus enhancing the level of confidence of our theoretical calculations. Taken together, our data provide evidence for binding of 1 and 13 to GR.