Synthesis and biological evaluation of novel, selective, nonsteroidal glucocorticoid receptor antagonists

We report the discovery of a novel class of glucocorticoid receptor (GR) antagonists based on the chromene molecular scaffold. The compounds exhibit good functional potency and an improved receptor selectivity profile for GR over other steroid receptors when compared to the classical steroidal GR-antagonist, RU-486.     

Discovery of potent and selective nonsteroidal indazolyl amide glucocorticoid receptor agonists

Modification of a phenolic lead structure based on lessons learned from increasing the potency of steroidal glucocorticoid agonists lead to the discovery of exceptionally potent, nonsteroidal, indazole GR agonists. SAR was developed to achieve good selectivity against other nuclear hormone receptors with the ultimate goal of achieving a dissociated GR agonist as measured by human in vitro assays. The specific interactions by which this class of compounds inhibits GR was elucidated by solving an X-ray co-crystal structure.     

In vitro characterization of trimegestone: a new potent and selective progestin

Trimegestone (TMG) is a novel 19-norpregnane progestin under development for hormone replacement therapy and oral contraception. The objective of the current study was to characterize the potency and steroid receptor selectivity of TMG in several in vitro assays and to compare its activity to that of medroxyprogesterone acetate (MPA). TMG and MPA had a similar competitive binding affinity for human and rabbit progesterone receptor (PR). However, TMG had a significantly higher affinity for rat PR (IC(50) = 3.3 nM) than MPA (IC(50) = 53.3 nM). In T47D cells, both compounds increased alkaline phosphatase activity and cell proliferation with comparable potencies (EC(50s) of 0.1 nM and of 0.02 nM, respectively). To further characterize the progestational activity and steroid receptor selectivity, we established an immortalized human endometrial stromal cell line (HESC-T). This cell line lacks endogenous estrogen receptor (ER) and PR but does have functional glucocorticoid receptors (GR). When ER is transiently expressed in the cells, 17beta-estradiol (E(2)) induces PR, allowing the study of PR-regulated genes. In HESC-T cells expressing exogenous ER, and therefore PR, both TMG and MPA increased HRE-tk-luciferase activity tenfold with an EC(50) of 0.2 nM. In HESC-T cells without exogenous ER, and therefore no PR, TMG did not induce HRE-tk-luciferase activity, whereas MPA induced the reporter activity with an EC(50) of about 10 nM. This MPA-induced reporter activity is believed to be mediated through GR. The steroid receptor selectivity of TMG was further evaluated using the HRE-tk-luciferase assay in the human lung carcinoma cell line A549, which contains GR but no PR. In these cells TMG had no effect on luciferase activity, whereas MPA increased the reporter activity in a dose-dependent manner with an EC(50) of approximately 30 nM. Furthermore, HRE-tk-luciferase assay in mouse fibroblast cell line L929, which expresses androgen receptor (AR) but no PR, showed that TMG had weak antiandrogenic activity whereas MPA had androgenic activity. In summary, data from several in vitro assays demonstrate that TMG is a potent progestin with a better receptor selectivity profile than MPA.     

Discovery and SAR study of novel dihydroquinoline containing glucocorticoid receptor ligands

We report the discovery of a novel class of glucocorticoid receptor (GR) ligands based on 1,2-dihydroquinoline molecular scaffold. The compounds exhibit good GR binding affinity and selectivity profile against other nuclear hormone receptors.     

Highly inducible expression from vectors containing multiple GRE''s in CHO cells overexpressing the glucocorticoid receptor.

A conditional glucocorticoid-responsive expression vector system is described for highly inducible expression of heterologous genes in mammalian cells. This host-vector system requires high level expression of the glucocorticoid receptor (GR) protein in the host cell and multiple copies of the receptor binding site within the expression vector. Transfection and selection of Chinese hamster ovary cells with expression vectors encoding the rat GR yielded cell lines which express functional receptor at high levels. Insertion of multiple copies of the MMTV enhancer (glucocorticoid responsive element, GRE) into an Adenovirus major late promoter (AdMLP) based expression vector yielded greater than 1000-fold inducible expression by dexamethasone (dex) in transient DNA transfection assays. The induced expression level was 7-fold greater than that obtained with an AdMLP based vector containing an SV40 enhancer, but lacking GRE's. Vectors containing the SV40 enhancer in combination with multiple GRE's exhibited elevated basal expression in the absence of dex, but retained inducibility in both transient assays and after integration and amplification in the CHO genome. This expression system should be of general utility for studying gene regulation and for expressing heterologous genes in a regulatable fashion.     

Discovery of betamethasone 17alpha-carbamates as dissociated glucocorticoid receptor modulators in the rat

A series of betamethasone 17alpha-carbamates were designed, synthesized, and evaluated for their ability to dissociate the two main functions of the glucocorticoid receptor, that is, transactivation and transrepression, in rat cell lines. A number of alkyl substituted betamethasone 17alpha-carbamates were identified with excellent affinity for the glucocorticoid receptor (e.g., 7, GR IC(50) 5.1 nM) and indicated dissociated profiles in functional assays of transactivation (rat tyrosine aminotransferase, TAT, and rat glutamine synthetase, GS) and transrepression (human A549 cells, MMP-1 assay). Gratifyingly, the in-vivo profile of these compounds, for example, 7, also indicated potent anti-inflammatory activity with impaired effects on glucose, insulin, triglycerides, and body weight. Taken together, these results indicate that dissociated glucocorticoid receptor modulators can be identified in rodents.     

Identification of acetylcholinesterase inhibitors using homogenous cell‐based assays in quantitative high‐throughput screening platforms

Acetylcholinesterase (AChE) is an enzyme responsible for metabolism of acetylcholine, a neurotransmitter associated with muscle movement, cognition, and other neurobiological processes. Inhibition of AChE activity can serve as a therapeutic mechanism, but also cause adverse health effects and neurotoxicity. In order to efficiently identify AChE inhibitors from large compound libraries, homogenous cell‐based assays in high‐throughput screening platforms are needed. In this study, a fluorescent method using Amplex Red (10‐acetyl‐3,7‐dihydroxyphenoxazine) and the Ellman absorbance method were both developed in a homogenous format using a human neuroblastoma cell line (SH‐SY5Y). An enzyme‐based assay using Amplex Red was also optimized and used to confirm the potential inhibitors. These three assays were used to screen 1368 compounds, which included a library of pharmacologically active compounds (LOPAC) and 88 additional compounds from the Tox21 program, at multiple concentrations in a quantitative high‐throughput screening (qHTS) format. All three assays exhibited exceptional performance characteristics including assay signal quality, precision, and reproducibility. A group of inhibitors were identified from this study, including known (e.g. physostigmine and neostigmine bromide) and potential novel AChE inhibitors (e.g. chelerythrine chloride and cilostazol). These results demonstrate that this platform is a promising means to profile large numbers of chemicals that inhibit AChE activity.     

Single-point mutation in a conserved TPR domain of Hip disrupts enhancement of glucocorticoid receptor signaling

The Hsp70-interacting protein Hip has been identified as a transient participant in the assembly of both glucocorticoid (GR) and progesterone receptor complexes. Although it has been difficult to identify a physiological role for Hip, it is believed to have intrinsic chaperoning properties and has been identified as a potential anti-apoptotic target of Granzyme B. In vitro assays have provided evidence that Hip may interact with GR complexes in an Hsp70 independent manner and can enhance the function of GR in hormone based reporter assays. In this study, a cDNA for human Hip was used in mutational analysis to map Hip function to critical structural elements. A single amino acid substitution (L211S) resulted in a loss of Hip function. This mutation also appears to disrupt the interaction of Hip with Hsp70 in vitro. Failure to recover Hip-L211S constructs in co-immunoprecipitation assays with an Hsp70 monoclonal antibody suggests that the mutation is unlikely to result in a misfolded substrate.     

2-Benzenesulfonyl-8a-benzyl-hexahydro-2H-isoquinolin-6-ones as selective glucocorticoid receptor antagonists

The 2-azadecalin ring system was evaluated as a scaffold for the preparation of glucocorticoid receptor (GR) antagonists. High affinity, selective GR antagonists were discovered based on a hypothetical binding mode related to the steroidal GR antagonist RU-43044. 2-Benzenesulfonyl substituted 8a-benzyl-hexahydro-2H-isoquinolin-6-ones exemplified by (R)-37 had low nanomolar affinity for GR with moderate functional activity (200 nM) in a reporter gene assay. These compounds were devoid of affinity for other steroidal receptors (ER, AR, MR, and PR). Analogues based on an alternative putative binding mode (CP-like) were found to be inactive.     

Parallel strategies for the preparation and selection of liver-targeted glucocorticoid receptor antagonists

Libraries of mifepristone analogs, MP-Acids, were designed and synthesized to increase the chances of identifying GR antagonists that possess liver-selective pharmacological profiles. MP-Acids were uniformly potent GR antagonists in binding and in cell-based functional assays. A high throughput pharmacokinetic selection strategy that employs the cassette dosing of MP-Acids was developed to identify liver-targeting compounds. Thus, resource-intensive in vivo assays to measure liver-selective pharmacology were enriched with GR antagonists that achieve high concentrations in the liver.     

Bendigoles D-F, bioactive sterols from the marine sponge-derived Actinomadura sp. SBMs009

Marine derived actinomycetes have become an important source of bioactive natural products. Here we report the structure and bioactivity of the bendigoles D-F (1-3), 3-keto sterols isolated from the new marine sponge derived bacterium, Actinomadura sp. SBMs009. The isolation of these compounds was guided by a novel high-content screen for NF-κB and glucocorticoid receptor (GR) activity, and cytotoxicity assays. The structures of 1-3 were determined by detailed analysis of NMR, MS, and single crystal X-ray diffraction data. Interestingly, 1 displayed cytotoxicity against the L929 (mouse fibroblast) cell line with an IC(50) approximated to 30 μM and was the most active inhibitor of GR-translocation, while 3 was the most effective inhibitor of NF-κB nuclear translocation with an IC(50) of 71 μM.     

Evaluation of cell-based assays for steroid nuclear receptors delivered by recombinant baculoviruses

The authors describe the use of modified baculoviruses containing mammalian expression cassettes (BacMam technology) in steroid nuclear receptor reporter assays designed for screening and profiling agonist and antagonist compounds. Baculo-viruses were constructed that express full-length human genes for mineralocorticoid receptor (MR), glucocorticoid receptor (GR), progesterone receptor A (PR-A), and progesterone receptor B (PR-B) from the cytomegalovirus immediate early promoter. A virus carrying the mouse mammary tumor virus-firefly luciferase (MMTV-Luc) cassette was generated to provide a suitable reporter construct. Feasibility studies with BacMam-MR in single-dose tests of 1000 compounds showed high correlation to the standard transfection-based assay results. Likewise, in dose-response experiments, BacMam-based assays for GR and PR-B produced potency and efficacy values similar to transfection assay results. At various receptor/reporter ratios, the BacMam assays showed good flexibility, demonstrating consistent signal-to-background (S/B) ratios and compound potencies. Increasing transduction time from 24 to 48 h provided no benefit, actually reducing overall assay performance as measured by S/B and Z' values. The BacMam technology was applied in studies of isoforms PR-A and PR-B, which showed similar responses to a series of agonists. Taken together, the results demonstrate the utility of steroid nuclear receptor BacMam constructs for compound screening procedures with high reproducibility, reduced turnaround time, and lower cost.     

Activity-guided screening of bioactive natural compounds implementing a new glucocorticoid-receptor-translocation assay and detection of new anti-inflammatory steroids from bacteria

Using an in vitro cell-based assay in a flow-design, we have applied activity-guided screening to search for new bioactive compounds isolated from microorganisms. A first assay employs the stable expression of nuclear factor kappa B (NF-κB) while a second assay utilizes the glucocorticoid receptor (GR) coupled to green fluorescent protein. A specialized assay was implemented for both the translocation of NF-κB and to inhibit the translocation of cytokine-mediated NF-κB. In addition, we developed in a wide palette of cell lines used for a highly specialized GR-translocation assay to detect anti-inflammatory effects. This approach demonstrates the straight-forward combination of cell-based assays arranged with an automated fluorescence microscope. This allows for the direct sorting of extracts which are acting in a pharmaceutically interesting way. Initial results using this technique have led to the detection of new anti-inflammatory steroids from bacterial crude extracts.     

Compound A, a dissociated glucocorticoid receptor modulator, inhibits T-bet (Th1) and induces GATA-3 (Th2) activity in immune cells

Background

Compound A (CpdA) is a dissociating non-steroidal glucocorticoid receptor (GR) ligand which has anti-inflammatory properties exerted by down-modulating proinflammatory gene expression. By favouring GR monomer formation, CpdA does not enhance glucocorticoid (GC) response element-driven gene expression, resulting in a reduced side effect profile as compared to GCs. Considering the importance of Th1/Th2 balance in the final outcome of immune and inflammatory responses, we analyzed how selective GR modulation differentially regulates the activity of T-bet and GATA-3, master drivers of Th1 and Th2 differentiation, respectively.

Results

Using Western analysis and reporter gene assays, we show in murine T cells that, similar to GCs, CpdA inhibits T-bet activity via a transrepressive mechanism. Different from GCs, CpdA induces GATA-3 activity by p38 MAPK-induction of GATA-3 phosphorylation and nuclear translocation. CpdA effects are reversed by the GR antagonist RU38486, proving the involvement of GR in these actions. ELISA assays demonstrate that modulation of T-bet and GATA-3 impacts on cytokine production shown by a decrease in IFN-γ and an increase in IL-5 production, respectively.

Conclusions

Taken together, through their effect favoring Th2 over Th1 responses, particular dissociated GR ligands, for which CpdA represents a paradigm, hold potential for the application in Th1-mediated immune disorders.     

Development of binding assays to screen ligands for Plasmodium falciparum thioredoxin and glutathione reductases by ultrafiltration and liquid chromatography/mass spectrometry

To identify potential lead compounds for malaria drug discovery, ultrafiltration and liquid chromatography and mass spectrometry (UF and LC/MS) based binding assays were developed for the first time for Plasmodium falciparum thioredoxin (PfTrxR) and glutathione (PfGR) reductases. In the binding assays, curcuminoids (bis-demethoxycurcumin 1, demethoxycurcumin 2, and curcumin 3) were used to study the binding affinity for PfTrxR and PfGR enzymes. The optimum binding was observed when the curcumimoids mixture (1 μM) was incubated with 1 μM PfTrxR and 0.5 μM PfGR enzymes separately for 60 min at 25 °C. The peak areas of the ligands in the chromatogram corresponding to incubation with active PfTrxR and PfGR enzymes increased by 1.6- and 2.0-fold respectively compared to the chromatogram of test compounds incubated with denatured enzymes. Further, binding assay experiments were carried out for compound 2 under non-competitive and competitive incubation conditions with 1 μM PfTrxR and 0.5 μM PfGR enzymes, separately. The binding affinity of compound 2 was higher for both the enzymes under non-competitive incubation conditions. To validate the binding assay developed, we have tested bis-2,4-dinitrophenyl sulfide (4) which is reported as an inhibitor of PfTrxR and PfGR enzymes. Compound 4 showed greater binding affinity for both enzymes under competitive incubation conditions. The relative peak area of compound 4 increased by 3.2- and 6-fold when incubated with active PfTrxR (1 μM) and PfGR (0.5 μM) enzymes respectively compared to the peak areas of the compound in control experiments. The current method developed has a potential for automated high-throughput screening to rapidly determine the binding affinity of ligands for these enzymes.