Discovery of novel non-steroidal selective glucocorticoid receptor modulators by structure- and IGN-based virtual screening, structural optimization, and biological evaluation

Synthetic glucocorticoids (GCs) have been widely used in the treatment of a broad range of inflammatory diseases, but their clinic use is limited by undesired side effects such as metabolic disorders, osteoporosis, skin and muscle atrophies, mood disorders and hypothalamic-pituitary-adrenal (HPA) axis suppression. Selective glucocorticoid receptor modulators (SGRMs) are expected to have promising anti-inflammatory efficacy but with fewer side effects caused by GCs. Here, we reported HT-15, a prospective SGRM discovered by structure-based virtual screening (VS) and bioassays. HT-15 can selectively act on the NF-κB/AP1-mediated transrepression function of glucocorticoid receptor (GR) and repress the expression of pro-inflammation cytokines (i.e., IL-1β, IL-6, COX-2, and CCL-2) as effectively as dexamethasone (Dex). Compared with Dex, HT-15 shows less transactivation potency that is associated with the main adverse effects of synthetic GCs, and no cross activities with other nuclear receptors. Furthermore, HT-15 exhibits very weak inhibition on the ratio of OPG/RANKL. Therefore, it may reduce the side effects induced by normal GCs. The bioactive compound HT-15 can serve as a starting point for the development of novel therapeutics for high dose or long-term anti-inflammatory treatment.

Selective glucocorticoid receptor modulation: New directions with non-steroidal scaffolds.

JTP-117968, a novel selective glucocorticoid receptor modulator, exhibits improved transrepression/transactivation dissociation

(A) Introduction: Because of the central role of androgen receptor (AR) signaling in prostate cancer (PC), AR inhibition is the primary modality of initial PC treatment. While AR-targeted therapies are often initially effective in castration-resistant prostate cancer (CRPC), disease progression is common. AR and glucocorticoid receptor (GR) are structurally similar nuclear receptors, bind the same DNA response elements, and regulate common genes. Previous work from our lab demonstrated that GR expression is upregulated following AR antagonism, GR expression decreased the effectiveness of AR blockade, and inhibiting GR activity reversed these effects. However, the only FDA-approved GR antagonist, mifepristone, is not GR-specific and has significant drug-drug interactions; therefore, we report our initial data with a new generation of selective GR-antagonists (SGRMs) for PC treatment using models of CRPC. (B) Experimental Procedures: The LAPC4, CWR-22Rv1 and VCAP PC cell lines were utilized. GR target gene expression analysis was performed at various time-points by qRT-PCR and protein immunoblots subsequent to treatment with AR agonist (R1881, 1nM), AR antagonist (enzalutamide, 10 μM), GR agonist (dexamethasone, 100nM) and the new SGRMs CORT108297 and CORT118335(1μM). Quantification of cell numbers in vitro in response to these treatments was determined by trypan blue exclusion/live cell counting and the CellTiter Glo 2.0 ATP-luciferase assay. For in vivo studies, PC cells were injected subcutaneously into male nude mice and grown to a predetermined tumor size, mice were then castrated, and fifteen days later treated with mifepristone (12mg/kg), SGRM (16mg/kg, 20mg/kg), or vehicle by daily intra-peritoneal injection to assess time to CRPC. (C) Results: AR antagonism with concurrent GR activation resulted in increased expression of canonical AR/GR-target genes and SGRMs (1μM) effectively inhibited expression of glucocorticoid-mediated genes at the RNA and protein levels. SGRMs did not affect AR-mediated gene expression. When AR was antagonized, activated GR promoted cell survival, which was inhibited by SGRM treatment to varying degrees. Preliminary studies support the hypothesis that these novel SGRMs delay CRPC tumor xenograft progression in vivo. (D) Conclusion: In vitro, new generation SGRMs inhibit GR-mediated, but not AR-selective gene expression and result in decreased tumor cell number. SGRM administration to nude mice bearing PC xenografts can delay castration-resistant tumor growth. Therefore, new SGRMs may be an effective therapy for CRPC treatment worthy of further exploration. Studies are ongoing to determine how SGRMs modulate GR target gene expression globally and whether they decrease expression of tumorigenic pro-survival genes in vivo. Citation Format: Jacob Kach, Phillip Selman, Diana C. West, Donald J. Vander Griend, Suzanne D. Conzen, Russell Z. Szmulewitz. Novel selective glucocorticoid receptor modulators (SGRMs) in castration-resistant prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 334.

New selective glucocorticoid receptor modulators reverse amyloid-β peptide–induced hippocampus toxicity

BackgroundLong-term treatment with glucocorticoids (GCs) plays an important role in the management of arthritis patients, although the efficacy/safety balance is unfavorable. Alternatives with less (severe) adverse effects but with good efficacy are needed. Selective GC receptor modulators (SGRMs) are designed to engage the GC receptor with dissociative characteristics: transactivation of genes, which is mainly responsible for unwanted effects, is less strong while trans-repression of genes, reducing inflammation, is maintained. It is expected that SGRMs thus have a better efficacy/safety balance than GCs. A systematic review providing an overview of the evidence in arthritis is lacking.ObjectiveTo systematically review the current literature on efficacy and safety of oral SGRMs in comparison to GCs in arthritis.MethodsA search was performed in Medline, Embase and the Cochrane Library, from inception dates of databases until May 2017. Experimental studies involving animal arthritis models or human material of arthritis patients, as well as clinical studies in arthritis patients were included, provided they reported original data. All types of arthritis were included. Data was extracted on the SGRM studied and on the GC used as reference standard; the design or setting of the study was extracted as well as the efficacy and safety results.ResultsA total of 207 articles was retrieved of which 17 articles were eligible for our analysis. Two studies concerned randomized controlled trials (RCT), five studies were pre-clinical studies using human material, and 10 studies involved pre-clinical animal models (acute and/or chronic arthritis induced in mice or rats). PF-04171327, the only compound investigated in a clinical trial setting, had a better efficacy/safety balance compared to GCs: better clinical anti-inflammatory efficacy and similar safety.ConclusionStudies assessing both efficacy and safety of SGRMs are scarce. There is limited evidence for dissociation of anti-inflammatory and metabolic effects of the SGRMs studied. Development of many SGRMs is haltered in a preclinical phase. One SGRM showed a better clinical efficacy/safety balance.

Glucocorticoids are steroid hormones that regulate inflammation, growth, metabolism, and apoptosis via their cognate receptor, the glucocorticoid receptor (GR). GR, acting mainly as a transcription factor, activates or represses the expression of a large number of target genes, among them, many genes of anti-inflammatory and pro-inflammatory molecules, respectively. Transrepression activity of glucocorticoids also accounts for their anti-inflammatory activity, rendering them the most widely prescribed drug in medicine. However, chronic and high-dose use of glucocorticoids is accompanied with many undesirable side effects, attributed predominantly to GR transactivation activity. Thus, there is a high need for selective GR agonist, capable of dissociating transrepression from transactivation activity. Protopanaxadiol and protopanaxatriol are triterpenoids that share structural and functional similarities with glucocorticoids. The molecular mechanism of their actions is unclear. In this study applying induced-fit docking analysis, luciferase assay, immunofluorescence, and Western blot analysis, we showed that protopanaxadiol and more effectively protopanaxatriol are capable of binding to GR to activate its nuclear translocation, and to suppress the nuclear factor-kappa beta activity in GR-positive HeLa and HEK293 cells, but not in GR-low level COS-7 cells. Interestingly, no transactivation activity was observed, whereas suppression of the dexamethasone-induced transactivation of GR and induction of apoptosis in HeLa and HepG2 cells were observed. Thus, our results indicate that protopanaxadiol and protopanaxatriol could be considered as potent and selective GR agonist.

ObjectiveIn ovarian cancer (OvCa), tumor cell high glucocorticoid receptor (GR) has been associated with poor patient prognosis. In vitro, GR activation inhibits chemotherapy-induced OvCa cell death in association with transcriptional upregulation of genes encoding anti-apoptotic proteins. A recent randomized phase II study demonstrated improvement in progression-free survival (PFS) for heavily pre-treated OvCa patients randomized to receive therapy with a selective GR modulator (SGRM) plus chemotherapy compared to chemotherapy alone. We hypothesized that SGRM therapy would improve carboplatin response in OvCa patient-derived xenograft (PDX).MethodsSix high-grade serous (HGS) OvCa PDX models expressing GR mRNA (NR3C1) and protein were treated with chemotherapy +/− SGRM. Tumor size was measured longitudinally by peritoneal transcutaneous ultrasonography.ResultsOne of the 6 GR-positive PDX models showed a significant improvement in PFS with the addition of a SGRM. Interestingly, the single model with an improved PFS was least carboplatin sensitive. Possible explanations for the modest SGRM activity include the high carboplatin sensitivity of 5 of the PDX tumors and the potential that SGRMs activate the tumor invasive immune cells in patients (absent from immunocompromised mice). The level of tumor GR protein expression alone appears insufficient for predicting SGRM response.ConclusionThe significant improvement in PFS shown in 1 of the 6 models after treatment with a SGRM plus chemotherapy underscores the need to determine predictive biomarkers for SGRM therapy in HGS OvCa and to better identify patient subgroups that are most likely to benefit from adding GR modulation to chemotherapy.

Glucocorticoids (GCs) have been used for decades in the treatment of chronic inflammatory and autoimmune diseases, thanks to their powerful anti‐inflammatory properties. However, long term treatment can lead to deleterious side effects, and some patients also experience resistance to their therapeutic effects. GCs act through the glucocorticoid receptor (GR) to regulate transcription both positively and negatively. Negative regulation of transcription involves a process known as transrepression, in which ligand‐activated GR impairs transcriptional activation by nuclear factor κB (NF‐κB) and other transcription factors. It is widely believed that transrepression accounts for most of the anti‐inflammatory effects of GCs, whereas the activation of transcription (transactivation) is responsible for most side effects of GCs. Based on this principle, several pharmaceutical companies are trying to identify selective GR modulators (SGRMs) that preferentially induce transrepression rather than transactivation. Such compounds are predicted to retain the anti‐inflammatory properties of classical GCs but cause fewer side effects. There are several problems with a dogma that equates anti‐inflammatory effects of GR with transcriptional repression. One is that GCs have long been known to destabilise many pro-inflammatory mRNAs, and this property is not explained by the transrepression model. Another issue is that GCs induce the expression of many factors with powerful anti-inflammatory effects. One of these is dual specificity phosphatase 1 (DUSP1), an enzyme that dephosphorylates and inactivates mitogen‐activated protein kinases. Studies of the Dusp1‐/‐ mouse have underlined the importance of the phosphatase in the antiinflammatory response to GCs. In this work, I investigated the role of DUSP1 in (1) the post‐transcriptional regulation of pro‐inflammatory mRNA stability by GCs and (2) the anti‐inflammatory actions of SGRMs. 1‐ The classical dexamethasone (dex) was shown to upregulate DUSP1 in mouse macrophages, and to inhibit the expression of cyclooxygenase 2 (COX‐2) in a manner that was partially dependent on DUSP1. Dex destabilised COX‐2 and interleukin 1α mRNAs, and this post‐transcriptional effect appeared to require DUSP1. 2‐ Two SGRMs were characterised and shown to preferentially mediate transrepression rather than transactivation. However, they were capable of inducing the expression of DUSP1 in several different cellular systems, and their capacity to inhibit the expression of COX‐2 was correlated with DUSP1 induction. Finally, several of the anti‐inflammatory effects of the SGRMs were found significantly impaired in mouse macrophages lacking DUSP1.

<div>AbstractPurpose:<p>Chemotherapy resistance remains a major problem in many solid tumors, including breast, ovarian, and pancreatic cancer. Glucocorticoids are one potential driver of chemotherapy resistance as they can mediate tumor progression via induction of cell-survival pathways. We investigated whether combining the selective glucocorticoid receptor (GR) modulator relacorilant with taxanes can enhance antitumor activity.</p>Patients and Methods:<p>The effect of relacorilant on paclitaxel efficacy was assessed in OVCAR5 cells <i>in vitro</i> and in the MIA PaCa-2 xenograft. A phase 1 study of patients with advanced solid tumors was conducted to determine the recommended phase 2 dose of relacorilant + nab-paclitaxel.</p>Results:<p>In OVCAR5 cells, relacorilant reversed the deleterious effects of glucocorticoids on paclitaxel efficacy (<i>P</i> < 0.001). Compared with paclitaxel alone, relacorilant + paclitaxel reduced tumor growth and slowed time to progression in xenograft models (both <i>P</i> < 0.0001). In the heavily pretreated phase 1 population [median (range) of prior regimens: 3 (1–8), prior taxane in 75.3% (55/73)], 33% (19/57) of response-evaluable patients achieved durable disease control (≥16 weeks) with relacorilant + nab-paclitaxel and 28.6% (12/42) experienced longer duration of benefit than on prior taxane (up to 6.4×). The most common dose-limiting toxicity of the combination was neutropenia, which was manageable with prophylactic G-CSF. Clinical benefit with relacorilant + nab-paclitaxel was also associated with GR-regulated transcript-level changes in a panel of GR-controlled genes.</p>Conclusions:<p>The observed preclinical, clinical, and GR-specific pharmacodynamic responses demonstrate that selective GR modulation with relacorilant combined with nab-paclitaxel may promote chemotherapy response and is tolerable. Further evaluation of this combination in tumor types responsive to taxanes is ongoing.</p></div>

<div>AbstractPurpose:<p>Chemotherapy resistance remains a major problem in many solid tumors, including breast, ovarian, and pancreatic cancer. Glucocorticoids are one potential driver of chemotherapy resistance as they can mediate tumor progression via induction of cell-survival pathways. We investigated whether combining the selective glucocorticoid receptor (GR) modulator relacorilant with taxanes can enhance antitumor activity.</p>Patients and Methods:<p>The effect of relacorilant on paclitaxel efficacy was assessed in OVCAR5 cells <i>in vitro</i> and in the MIA PaCa-2 xenograft. A phase 1 study of patients with advanced solid tumors was conducted to determine the recommended phase 2 dose of relacorilant + nab-paclitaxel.</p>Results:<p>In OVCAR5 cells, relacorilant reversed the deleterious effects of glucocorticoids on paclitaxel efficacy (<i>P</i> < 0.001). Compared with paclitaxel alone, relacorilant + paclitaxel reduced tumor growth and slowed time to progression in xenograft models (both <i>P</i> < 0.0001). In the heavily pretreated phase 1 population [median (range) of prior regimens: 3 (1–8), prior taxane in 75.3% (55/73)], 33% (19/57) of response-evaluable patients achieved durable disease control (≥16 weeks) with relacorilant + nab-paclitaxel and 28.6% (12/42) experienced longer duration of benefit than on prior taxane (up to 6.4×). The most common dose-limiting toxicity of the combination was neutropenia, which was manageable with prophylactic G-CSF. Clinical benefit with relacorilant + nab-paclitaxel was also associated with GR-regulated transcript-level changes in a panel of GR-controlled genes.</p>Conclusions:<p>The observed preclinical, clinical, and GR-specific pharmacodynamic responses demonstrate that selective GR modulation with relacorilant combined with nab-paclitaxel may promote chemotherapy response and is tolerable. Further evaluation of this combination in tumor types responsive to taxanes is ongoing.</p></div>

Glucocorticoids (GCs) are a cornerstone in the treatment of lymphoid malignancies such as multiple myeloma (MM) and acute lymphoblastic leukemia (ALL). Yet, prolonged GC use is hampered by deleterious GC-related side effects and the emergence of GC resistance. To tackle and overcome these GC-related problems, the applicability of selective glucocorticoid receptor agonists and modulators was studied, in search of fewer side-effects and at least equal therapeutic efficacy as classic GCs. Compound A (CpdA) is a prototypical example of such a selective glucocorticoid receptor modulator and does not support GR-mediated transactivation. Here, we examined whether the combination of CpdA with the classic GC dexamethasone (Dex) may improve GC responsiveness of MM and ALL cell lines. We find that the combination of Dex and CpdA does not substantially enhance GC-mediated cell killing. In line, several apoptosis hallmarks, such as caspase 3/7 activity, PARP cleavage and the levels of cleaved-caspase 3 remain unchanged upon combining Dex with CpdA. Moreover, we monitor no additional inhibition of cell proliferation and the homologous downregulation of GR is not counteracted by the combination of Dex and CpdA. In addition, CpdA is unable to modulate Dex-liganded GR transactivation and transrepression, yet, Dex-mediated transrepression is also aberrant in these lymphoid cell lines. Together, transrepression-favoring compounds, alone or combined with GCs, do not seem a valid strategy in the treatment of lymphoid malignancies.

Selective glucocorticoid receptor modulators (SGRMs), which can dissociate the transactivation from the transrepression of the glucocorticoid receptor (GR), are regarded as very promising therapeutics for inflammatory and autoimmune diseases. We previously discovered a SGRM HP-19 based on the passive antagonistic conformation of GR and bioassays. In this study, we further analyzed the dynamic changes of the passive antagonistic state upon the binding of HP-19 and designed and synthesized 62 N-acyl-6-sulfonamide-tetrahydroquinoline derivatives by structural optimization of HP-19. Therein, compound B53 exhibits the best transrepression activity (IC50 NF-κB = 0.009 ± 0.001 μM) comparable with dexamethasone (IC50 NF-κB = 0.005 ± 0.001 μM) and no transactivation activity. B53 can efficiently reduce the expression of inflammatory factors IL-6, IL-1β, TNF-α, and so on and makes a milder adverse effect and is highly specific to GR. Furthermore, B53 is able to significantly relieve dermatitis on a mouse model via oral drug intervention.

Our understanding of the actions of glucocorticoids has greatly increased in the last few years. The various genomic and non-genomic mechanisms of glucocorticoid action provide interesting and sometimes very advanced starting points for the development of optimised glucocorticoids and glucocorticoid receptor ligands. SEGRAs, NO-glucocorticoids and long-circulating liposomal glucocorticods are examples to be mentioned in this regard. Selective glucocorticoid receptor agonists (SEGRAs): Currently glucocorticoid receptor ligands are being developed that cause predominantly transrepression but not transactivation. These drugs are called selective glucocorticoid receptor agonists (SEGRAs) and are thought to have an improved safety/efficacy profile. In vivo investigations and clinical trials will have to define whether SEGRAs will, as ‘improved glucocorticoids’, enter clinical medicine in the near future. NO-glucocorticoids: Recent experimental observations prompt the assessment of the clinical impact of another new class of glucocorticoid drugs, NO-glucocorticoids, which are able to release low levels of nitric oxide. They have been shown to be endowed with enhanced anti-inflammatory properties and reduced side effects. The prototype of these new steroids, 21-NO-prednisolone, is much more potent than prednisolone in models of acute and chronic inflammation but does not activate primary osteoclast activity (whereas prednisolone does). However, more studies are needed to confirm that NO-glucocorticoids will be effective as anti-inflammatory agents in clinical practice. Long-circulating liposomal glucocorticoids: The anti-inflammatory effectiveness of glucocorticoids can be improved by the additional benefits of the non-genomic actions of high glucocorticoid concentrations. On this basis, the successful use of long-circulating liposomal glucocorticoids has recently been reported which accumulate at sites of inflammation. This leads to very high glucocorticoid concentrations at e.g. the inflamed joint (but accompanied by low plasma concentrations with perhaps a lower rate of side effects) which is the key factor explaining the observed strong therapeutic effect. Conclusions: These new GCR-ligands and the administration of liposomes are very promising approaches that will hopefully soon be available in clinical practice to improve the benefit/risk ratio and well-being of patients being treated. Another intriguing issue is the study of membrane bound glucocorticoid receptors (mGCR). These surface receptors are suggested to mediate rapid nongenomic effects and have been found to be upregulated in active rheumatoid arthritis. The number of monocytes expressing mGCR is significantly correlated with disease activity. Therefore, drugs binding selectively to the mGCR may in future also prove to be of therapeutic value but the functions of mGCR must first be investigated in detail.

The development of selective GR agonist and modulators (SEGRAMs) aimed to minimize the adverse effects of chronic glucocorticoid treatment (e.g., hyperglycemia and osteoporosis) by separating the transactivation and transrepression activities of the glucocorticoid receptor (GR). Herein we report the pharmacologic profile of clinical candidate GRM-01, a novel, orally available, non-steroidal SEGRAM. In vitro GR, progesterone receptor (PR), and mineralocorticoid receptor (MR) binding and reporter gene assays were conducted to determine GRM-01 potency and selectivity. Anti-inflammatory effects were investigated in vitro using functional assays in rat and human whole blood, human lung cells, and primary fibroblast-like synoviocytes from human donors with rheumatoid arthritis. In vitro assays measured tyrosine aminotransferase [TAT] activity in human hepatocytes and osteoprotegerin release from human osteoblasts as markers of glucose and bone metabolism, respectively. In vivo studies examined the effect of GRM-01 on biomarkers in a rat model of inflammation and on cortisol levels in Cynomolgus monkeys. Animal pharmacokinetics (PK) for GRM-01 were determined and used to predict its human PK. GRM-01 is a potent and selective ligand of human GR versus human PR and MR (inhibition constant = 12 vs. 3,700 and >10,000nM, respectively). GRM-01 displayed partial induction (transactivation) at the GR (half-maximal effective concentration [EC50] = 60.2 nM, efficacy 31.8%) versus prednisolone (EC50 = 24.3 nM, efficacy 80.5%). GRM-01 demonstrated anti-inflammatory efficacy, inhibiting tumor necrosis factor-α and interferon-γ release in whole blood assays, and interleukin-6 release in cellular assays. GRM-01 weakly increased TAT activity in HepG2 cells (efficacy 14.0% vs. 92.4% with prednisolone) and partially inhibited osteoprotegerin release in MG-63 cells (by 58% vs. 100%). In vivo, GRM-01 dose-dependently reduced rat ankle swelling, had anti-nociceptive effects, and did not increase blood glucose. In Cynomolgus monkeys, GRM-01 dose-dependently reduced plasma cortisol. Animal PK found that GRM-01 had high oral bioavailability, generally low clearance, and good tissue partitioning. The predicted human total plasma clearance of GRM-01 was 0.25mL/min/kg, volume of distribution 2.124L/kg, and half-life ∼98h. GRM-01 displays a favorable preclinical pharmacologic profile consistent with a SEGRAM, and based on this is currently in Phase 1 development.