The effect of AZD9567 vs. prednisolone on glycaemic control in patients with type 2 diabetes mellitus: Results from a phase 2a clinical trial.

AIMS: Corticosteroids are the treatment of choice for many inflammatory diseases but often lead to adverse effects, including hyperglycaemia. This study investigated the mechanisms driving differential effects on glucose control for AZD9567, an oral nonsteroidal selective glucocorticoid receptor modulator vs. prednisolone in 46 patients with type 2 diabetes mellitus. METHODS: In this randomized, double-blind, 2-way cross-over study (NCT04556760), participants received either AZD9567 72 mg and prednisolone 40 mg daily (cohort 1); AZD9567 40 mg and prednisolone 20 mg daily (cohort 2); or placebo and prednisolone 5 mg daily (cohort 3). Treatment duration was 3 days with a 3-week washout between treatment periods. Glycaemic control was assessed after a standardized meal and with continuous glucose monitoring. RESULTS: A significant difference between AZD9567 and prednisolone in favour of AZD9567 was observed for the change from baseline to Day 4 glucose excursions postmeal in cohort 1 (glucose area under the curve from 0 to 4 h -4.54%; 95% confidence interval [CI]: -8.88, -0.01; P = .049), but not in cohort 2 (-5.77%; 95% CI: -20.92, 12.29; P = .435). In cohort 1, significant differences between AZD9567 and prednisolone were also seen for the change from baseline to day 4 in insulin and glucagon secretion postmeal (P < .001 and P = .005, respectively) and change from baseline to Day 4 in GLP-1 response (P = .022). Significant differences between AZD9567 and prednisolone for 24-h glucose control were observed for both cohort 1 (-1.507 mmol/L; 95% CI: -2.0820, -0.9314; P < .001) and cohort 2 (-1.110 mmol/L; 95% CI -1.7257, -0.4941; P < .001). CONCLUSION: AZD9567 significantly reduced treatment-induced hyperglycaemia compared with prednisolone.

AZD9567 versus prednisolone in patients with active rheumatoid arthritis: A phase IIa, randomized, double-blind, efficacy, and safety study.

Oral corticosteroid use is limited by side effects, some caused by off-target actions on the mineralocorticoid receptor that disrupt electrolyte balance. AZD9567 is a selective, nonsteroidal glucocorticoid receptor modulator. The efficacy, safety, and tolerability of AZD9567 and prednisolone were assessed in a phase IIa study. Anti-inflammatory mechanism of action was also evaluated in vitro in monocytes from healthy donors. In this randomized, double-blind, parallel-group, multicenter study, patients with active rheumatoid arthritis were randomized 1:1 to AZD9567 40 mg or prednisolone 20 mg once daily orally for 14 days. The primary end point was change from baseline in DAS28-CRP at day 15. Secondary end points included components of DAS28-CRP, American College of Rheumatology (ACR) response criteria (ACR20, ACR50, and ACR70), and safety end points, including serum electrolytes. Overall, 21 patients were randomized to AZD9567 (n = 11) or prednisolone (n = 10), and all completed the study. As anticipated, AZD9567 had a similar efficacy profile to prednisolone, with no clinically meaningful (i.e., >1.0) difference in change from baseline to day 15 in DAS28-CRP between AZD9567 and prednisolone (least-squares mean difference: 0.47, 95% confidence interval: -0.49 to 1.43). Similar results were observed for the secondary efficacy end points. In vitro transcriptomic analysis showed that anti-inflammatory responses were similar for AZD9567, prednisolone, and dexamethasone. Unlike prednisolone, AZD9567 had no effect on the serum sodium:potassium ratio. The safety profile was not different from that of prednisolone. Larger studies of longer duration are required to determine whether AZD9567 40 mg may in the future be an alternative to prednisolone in patients with inflammatory disease.

Quaternary glucocorticoid receptor structure highlights allosteric interdomain communication.

The glucocorticoid receptor (GR) is a ligand-activated transcription factor that binds DNA and assembles co-regulator complexes to regulate gene transcription. GR agonists are widely prescribed to people with inflammatory and autoimmune diseases. Here we present high-resolution, multidomain structures of GR in complex with ligand, DNA and co-regulator peptide. The structures reveal how the receptor forms an asymmetric dimer on the DNA and provide a detailed view of the domain interactions within and across the two monomers. Hydrogen-deuterium exchange and DNA-binding experiments demonstrate that ligand-dependent structural changes are communicated across the different domains in the full-length receptor. This study demonstrates how GR forms a distinct architecture on DNA and how signal transmission can be modulated by the ligand pharmacophore, provides a platform to build a new level of understanding of how receptor modifications can drive disease progression and offers key insight for future drug design.

Designing Selective Drug-like Molecular Glues for the Glucocorticoid Receptor/14-3-3 Protein-Protein Interaction.

The ubiquitously expressed glucocorticoid receptor (GR) is a nuclear receptor that controls a broad range of biological processes and is activated by steroidal glucocorticoids such as hydrocortisone or dexamethasone. Glucocorticoids are used to treat a wide variety of conditions, from inflammation to cancer but suffer from a range of side effects that motivate the search for safer GR modulators. GR is also regulated outside the steroid-binding site through protein-protein interactions (PPIs) with 14-3-3 adapter proteins. Manipulation of these PPIs will provide insights into noncanonical GR signaling as well as a new level of control over GR activity. We report the first molecular glues that selectively stabilize the 14-3-3/GR PPI using the related nuclear receptor estrogen receptor α (ERα) as a selectivity target to drive design. These 14-3-3/GR PPI stabilizers can be used to dissect noncanonical GR signaling and enable the development of novel atypical GR modulators.

Unraveling the Allosteric Cross-Talk between the Coactivator Peptide and the Ligand-Binding Site in the Glucocorticoid Receptor.

The glucocorticoid receptor (GR) is a nuclear receptor that controls critical biological processes by regulating the transcription of specific genes. There is a known allosteric cross-talk between the ligand and coregulator binding sites within the GR ligand-binding domain that is crucial for the control of the functional response. However, the molecular mechanisms underlying such an allosteric control remain elusive. Here, molecular dynamics (MD) simulations, bioinformatic analysis, and biophysical measurements are integrated to capture the structural and dynamic features of the allosteric cross-talk within the GR. We identified a network of evolutionarily conserved residues that enables the allosteric signal transduction, in agreement with experimental data. MD simulations clarify how such a network is dynamically interconnected and offer a mechanistic explanation of how different peptides affect the intensity of the allosteric signal. This study provides useful insights to elucidate the GR allosteric regulation, ultimately providing a foundation for designing novel drugs.

Glucocorticoid receptor Thr524 phosphorylation by MINK1 induces interactions with 14-3-3 protein regulators.

The glucocorticoid receptor (GR) is a ligand-dependent transcription factor that plays a central role in inflammation. The GR activity is also modulated via protein-protein interactions, including binding of 14-3-3 proteins induced by GR phosphorylation. However, the specific phosphorylation sites on the GR that trigger these interactions and their functional consequences are less clear. Hence, we sought to examine this system in more detail. We used phosphorylated GR peptides, biophysical studies, and X-ray crystallography to identify key residues within the ligand-binding domain of the GR, T524 and S617, whose phosphorylation results in binding of the representative 14-3-3 protein 14-3-3ζ. A kinase screen identified misshapen-like kinase 1 (MINK1) as responsible for phosphorylating T524 and Rho-associated protein kinase 1 for phosphorylating S617; cell-based approaches confirmed the importance of both GR phosphosites and MINK1 but not Rho-associated protein kinase 1 alone in inducing GR-14-3-3 binding. Together our results provide molecular-level insight into 14-3-3-mediated regulation of the GR and highlight both MINK1 and the GR-14-3-3 axis as potential targets for future therapeutic intervention.

Effects of a selective glucocorticoid receptor modulator (AZD9567) versus prednisolone in healthy volunteers: two phase 1, single-blind, randomised controlled trials.

BACKGROUND: Glucocorticoids are highly effective and widely used anti-inflammatory drugs, but their use is limited by serious side-effects, including glucocorticoid-induced hyperglycaemia and diabetes. AZD9567 is a non-steroidal, selective glucocorticoid receptor modulator that aims to reduce side-effects. We aimed to assess the safety, tolerability, and pharmacokinetics of AZD9567 in healthy volunteers. METHODS: Two phase 1 clinical studies were done. First, a randomised, placebo-controlled, single-blind, single-ascending dose study was done in healthy men who received single oral doses of AZD9567 2 mg, 10 mg, 20 mg, 40 mg, 80 mg, 100 mg, 125 mg, or 155 mg, or prednisolone 60 mg (n=8 per dose group, randomly assigned [6:2] to receive active drug or placebo). Second, a randomised, active-controlled, single-blind, multiple-ascending dose study was done, in which men and women received oral AZD9567 or prednisolone once daily for 5 days. One cohort of volunteers with prediabetes received AZD9567 10 mg (n=7) or prednisolone 20 mg (n=2). All other cohorts comprised healthy volunteers, receiving AZD9567 20 mg, 40 mg, 80 mg, or 125 mg (n=7 per dose group), or prednisolone 5 mg (n=13), 20 mg (n=16), or 40 mg (n=13). Participants and study centre staff were masked to treatment assignment for each cohort, although data were unmasked for safety review between cohorts. The primary outcome of the single-ascending dose study was the safety, tolerability, and pharmacokinetics of single ascending doses of AZD9567; for the multiple-ascending dose study it was the safety and tolerability of AZD9567 following multiple ascending doses. As a secondary outcome, effects on glycaemic control were ascertained with oral glucose tolerance tests (OGTTs) done at baseline and on day 1 of the single-ascending dose study, and at baseline and on day 4 of the multiple-ascending dose study. These trials are registered at ClinicalTrials.gov, NCT02512575 and NCT02760316. FINDINGS: In the single-ascending dose study, between Nov 18, 2015, and Sept 26, 2016, 72 healthy white men were enrolled, and all completed the study. In the multiple-ascending dose study, between May 2, 2016, and Sept 13, 2017, 77 predominantly white male volunteers (including nine individuals with prediabetes and eight women) were enrolled and 75 completed the study. All doses of AZD9567 and prednisolone were well tolerated, with no serious adverse events or events suggesting adrenal insufficiency. In the single-ascending dose study, nine adverse events of mild intensity were reported (five with AZD9567 and four with placebo); no adverse event was reported by more than one person. In the multiple-ascending dose study, 44 adverse events of mild or moderate intensity were reported (18 with AZD9567 and 26 with prednisolone). The most common were headache and micturition. Apparent clearance, volume of distribution, and half-life of AZD9567 were consistent across doses and for single versus repeated dosing. In the multiple-ascending dose study, OGTTs showed no significant difference with AZD9567 doses up to 80 mg compared with prednisolone 5 mg in glucose area under the curve from 0 h to 4 h post-OGTT (AUC0-4h) from baseline to day 4; the increase in glucose AUC0-4h from baseline to day 4 was significantly lower with all AZD9567 doses versus prednisolone 20 mg (AZD9567 20 mg p<0·0001, 40 mg p=0·0001, 80 mg p=0·0001, and 125 mg p=0·0237). INTERPRETATION: AZD9567 appears to be safe and well tolerated in healthy, predominantly white male volunteers and shows promising initial evidence for improved post-prandial glucose control. Studies of longer duration, with a greater proportion of women and other ethnic groups, and in patients requiring anti-inflammatory treatment are needed to characterise the clinical efficacy and safety profile of AZD9567. FUNDING: AstraZeneca.

Identification of Mineralocorticoid Receptor Modulators with Low Impact on Electrolyte Homeostasis but Maintained Organ Protection.

The mechanism-based risk for hyperkalemia has limited the use of mineralocorticoid receptor antagonists (MRAs) like eplerenone in cardio-renal diseases. Here, we describe the structure and property-driven lead generation and optimization, which resulted in identification of MR modulators ( S)-1 and ( S)-33. Both compounds were partial MRAs but still demonstrated equally efficacious organ protection as eplerenone after 4 weeks of treatment in uni-nephrectomized rats on high-salt diet and aldosterone infusion. Importantly, and in sharp contrast to eplerenone, this was achieved without substantial changes to the urine Na+/K+ ratio after acute treatment in rat, which predicts a reduced risk for hyperkalemia. This work led to selection of ( S)-1 (AZD9977) as the clinical candidate for treating MR-mediated cardio-renal diseases, including chronic kidney disease and heart failure. On the basis of our findings, we propose an empirical model for prediction of compounds with low risk of affecting the urinary Na+/K+ ratio in vivo.

Backbone 1H, 13C, and 15N resonance assignments of the ligand binding domain of the human wildtype glucocorticoid receptor and the F602S mutant variant.

The glucocorticoid receptor (GR) is a nuclear hormone receptor that regulates key genes controlling development, metabolism, and the immune response. GR agonists are efficacious for treatment of inflammatory, allergic, and immunological disorders. Steroid hormone binding to the ligand-binding domain (LBD) of GR is known to change the structural and dynamical properties of the receptor, which in turn control its interactions with DNA and various co-regulators and drive the pharmacological response. Previous biophysical studies of the GR LBD have required the use of mutant forms to overcome issues with limited protein stability and high aggregation propensity. However, these mutant variants are known to also influence the functional response of the receptor. Here we report a successful protocol for protein expression, purification, and NMR characterization of the wildtype human GR LBD. We achieved chemical shift assignments for 90% of the LBD backbone resonances, with 216 out of 240 non-proline residues assigned in the 1H-15N TROSY spectrum. These advancements form the basis for future investigations of allosteric effects in GR signaling.

Preclinical pharmacology of AZD9977: A novel mineralocorticoid receptor modulator separating organ protection from effects on electrolyte excretion.

Excess mineralocorticoid receptor (MR) activation promotes target organ dysfunction, vascular injury and fibrosis. MR antagonists like eplerenone are used for treating heart failure, but their use is limited due to the compound class-inherent hyperkalemia risk. Here we present evidence that AZD9977, a first-in-class MR modulator shows cardio-renal protection despite a mechanism-based reduced liability to cause hyperkalemia. AZD9977 in vitro potency and binding mode to MR were characterized using reporter gene, binding, cofactor recruitment assays and X-ray crystallopgraphy. Organ protection was studied in uni-nephrectomised db/db mice and uni-nephrectomised rats administered aldosterone and high salt. Acute effects of single compound doses on urinary electrolyte excretion were tested in rats on a low salt diet. AZD9977 and eplerenone showed similar human MR in vitro potencies. Unlike eplerenone, AZD9977 is a partial MR antagonist due to its unique interaction pattern with MR, which results in a distinct recruitment of co-factor peptides when compared to eplerenone. AZD9977 dose dependently reduced albuminuria and improved kidney histopathology similar to eplerenone in db/db uni-nephrectomised mice and uni-nephrectomised rats. In acute testing, AZD9977 did not affect urinary Na+/K+ ratio, while eplerenone increased the Na+/K+ ratio dose dependently. AZD9977 is a selective MR modulator, retaining organ protection without acute effect on urinary electrolyte excretion. This predicts a reduced hyperkalemia risk and AZD9977 therefore has the potential to deliver a safe, efficacious treatment to patients prone to hyperkalemia.

Discovery of a Novel Oral Glucocorticoid Receptor Modulator (AZD9567) with Improved Side Effect Profile.

Synthetic glucocorticoids (GC) are essential for the treatment of a broad range of inflammatory diseases. However, their use is limited by target related adverse effects on, e.g., glucose homeostasis and bone metabolism. Starting from a nonsteroidal GR ligand (4) that is a full agonist in reporter gene assays, we exploited key functional triggers within the receptor, generating a range of structurally diverse partial agonists. Of these, only a narrow subset exhibited full anti-inflammatory efficacy and a significantly reduced impact on adverse effect markers in human cell assays compared to prednisolone. This led to the discovery of AZD9567 (15) with excellent in vivo efficacy when dosed orally in a rat model of joint inflammation. Compound 15 is currently being evaluated in clinical trials comparing the efficacy and side effect markers with those of prednisolone.

Selective Nonsteroidal Glucocorticoid Receptor Modulators for the Inhaled Treatment of Pulmonary Diseases.

A class of potent, nonsteroidal, selective indazole ether-based glucocorticoid receptor modulators (SGRMs) was developed for the inhaled treatment of respiratory diseases. Starting from an orally available compound with demonstrated anti-inflammatory activity in rat, a soft-drug strategy was implemented to ensure rapid elimination of drug candidates to minimize systemic GR activation. The first clinical candidate 1b (AZD5423) displayed a potent inhibition of lung edema in a rat model of allergic airway inflammation following dry powder inhalation combined with a moderate systemic GR-effect, assessed as thymic involution. Further optimization of inhaled drug properties provided a second, equally potent, candidate, 15m (AZD7594), that demonstrated an improved therapeutic ratio over the benchmark inhaled corticosteroid 3 (fluticasone propionate) and prolonged the inhibition of lung edema, indicating potential for once-daily treatment.

Structural insight into allosteric modulation of protease-activated receptor 2.

Protease-activated receptors (PARs) are a family of G-protein-coupled receptors (GPCRs) that are irreversibly activated by proteolytic cleavage of the N terminus, which unmasks a tethered peptide ligand that binds and activates the transmembrane receptor domain, eliciting a cellular cascade in response to inflammatory signals and other stimuli. PARs are implicated in a wide range of diseases, such as cancer and inflammation. PARs have been the subject of major pharmaceutical research efforts but the discovery of small-molecule antagonists that effectively bind them has proved challenging. The only marketed drug targeting a PAR is vorapaxar, a selective antagonist of PAR1 used to prevent thrombosis. The structure of PAR1 in complex with vorapaxar has been reported previously. Despite sequence homology across the PAR isoforms, discovery of PAR2 antagonists has been less successful, although GB88 has been described as a weak antagonist. Here we report crystal structures of PAR2 in complex with two distinct antagonists and a blocking antibody. The antagonist AZ8838 binds in a fully occluded pocket near the extracellular surface. Functional and binding studies reveal that AZ8838 exhibits slow binding kinetics, which is an attractive feature for a PAR2 antagonist competing against a tethered ligand. Antagonist AZ3451 binds to a remote allosteric site outside the helical bundle. We propose that antagonist binding prevents structural rearrangements required for receptor activation and signalling. We also show that a blocking antibody antigen-binding fragment binds to the extracellular surface of PAR2, preventing access of the tethered ligand to the peptide-binding site. These structures provide a basis for the development of selective PAR2 antagonists for a range of therapeutic uses.

Exploring Binding Mechanisms in Nuclear Hormone Receptors by Monte Carlo and X-ray-derived Motions.

In this study, we performed an extensive exploration of the ligand entry mechanism for members of the steroid nuclear hormone receptor family (androgen receptor, estrogen receptor α, glucocorticoid receptor, mineralocorticoid receptor, and progesterone receptor) and their endogenous ligands. The exploration revealed a shared entry path through the helix 3, 7, and 11 regions. Examination of the x-ray structures of the receptor-ligand complexes further showed two distinct folds of the helix 6-7 region, classified as "open" and "closed", which could potentially affect ligand binding. To improve sampling of the helix 6-7 loop, we incorporated motion modes based on principal component analysis of existing crystal structures of the receptors and applied them to the protein-ligand sampling. A detailed comparison with the anisotropic network model (an elastic network model) highlights the importance of flexibility in the entrance region. While the binding (interaction) energy of individual simulations can be used to score different ligands, extensive sampling further allows us to predict absolute binding free energies and analyze reaction kinetics using Markov state models and Perron-cluster cluster analysis, respectively. The predicted relative binding free energies for three ligands binding to the progesterone receptor are in very good agreement with experimental results and the Perron-cluster cluster analysis highlighted the importance of a peripheral binding site. Our analysis revealed that the flexibility of the helix 3, 7, and 11 regions represents the most important factor for ligand binding. Furthermore, the hydrophobicity of the ligand influences the transition between the peripheral and the active binding site.

Discovery of new selective glucocorticoid receptor agonist leads.

We report on the discovery of two new lead series for the development of glucocorticoid receptor agonists. Firstly, the discovery of tetrahydronaphthalenes led to metabolically stable and dissociated compounds. Their binding mode to the glucocorticoid receptor could be elucidated through an X-ray structure. Closer inspection into the reaction path and analyses of side products revealed a new amino alcohol series also addressing the glucocorticoid receptor and demonstrating strong anti-inflammatory activity in vitro.

Structure-Based Drug Design of Mineralocorticoid Receptor Antagonists to Explore Oxosteroid Receptor Selectivity.

The mineralocorticoid receptor (MR) is a nuclear hormone receptor involved in the regulation of body fluid and electrolyte homeostasis. In this study we explore selectivity triggers for a series of nonsteroidal MR antagonists to improve selectivity over other members of the oxosteroid receptor family. A biaryl sulfonamide compound was identified in a high-throughput screening (HTS) campaign. The compound bound to MR with pKi =6.6, but displayed poor selectivity over the glucocorticoid receptor (GR) and the progesterone receptor (PR). Following X-ray crystallography of MR in complex with the HTS hit, a compound library was designed that explored an induced-fit hypothesis that required movement of the Met852 side chain. An improvement in MR selectivity of 11- to 79-fold over PR and 23- to 234-fold over GR was obtained. Given the U-shaped binding conformation, macrocyclizations were explored, yielding a macrocycle that bound to MR with pKi =7.3. Two protein-ligand X-ray structures were determined, confirming the hypothesized binding mode for the designed compounds.

Discovery of indazole ethers as novel, potent, non-steroidal glucocorticoid receptor modulators.

A structure-based design approach led to the identification of a novel class of indazole ether based, non-steroidal glucocorticoid receptor (GR) modulators. Several examples were identified that displayed cell potency in the picomolar range, inhibiting LPS-induced TNF-α release by primary peripheral blood mononuclear cells (PBMCs). Additionally, an improved steroid hormone receptor binding selectivity profile, compared to classical steroidal GR agonists, was demonstrated. The indazole ether core tolerated a broad range of substituents allowing for modulation of the physiochemical parameters. A small sub-set of indazole ethers, with pharmacokinetic properties suitable for oral administration, was investigated in a rat antigen-induced joint inflammation model and demonstrated excellent anti-inflammatory efficacy.

Binding Mode and Induced Fit Predictions for Prospective Computational Drug Design.

Computer-aided drug design plays an important role in medicinal chemistry to obtain insights into molecular mechanisms and to prioritize design strategies. Although significant improvement has been made in structure based design, it still remains a key challenge to accurately model and predict induced fit mechanisms. Most of the current available techniques either do not provide sufficient protein conformational sampling or are too computationally demanding to fit an industrial setting. The current study presents a systematic and exhaustive investigation of predicting binding modes for a range of systems using PELE (Protein Energy Landscape Exploration), an efficient and fast protein-ligand sampling algorithm. The systems analyzed (cytochrome P, kinase, protease, and nuclear hormone receptor) exhibit different complexities of ligand induced fit mechanisms and protein dynamics. The results are compared with results from classical molecular dynamics simulations and (induced fit) docking. This study shows that ligand induced side chain rearrangements and smaller to medium backbone movements are captured well in PELE. Large secondary structure rearrangements, however, remain challenging for all employed techniques. Relevant binding modes (ligand heavy atom RMSD < 1.0 Å) can be obtained by the PELE method within a few hours of simulation, positioning PELE as a tool applicable for rapid drug design cycles.

Ligand Binding Mechanism in Steroid Receptors: From Conserved Plasticity to Differential Evolutionary Constraints.

Steroid receptor drugs have been available for more than half a century, but details of the ligand binding mechanism have remained elusive. We solved X-ray structures of the glucocorticoid and mineralocorticoid receptors to identify a conserved plasticity at the helix 6-7 region that extends the ligand binding pocket toward the receptor surface. Since none of the endogenous ligands exploit this region, we hypothesized that it constitutes an integral part of the binding event. Extensive all-atom unbiased ligand exit and entrance simulations corroborate a ligand binding pathway that gives the observed structural plasticity a key functional role. Kinetic measurements reveal that the receptor residence time correlates with structural rearrangements observed in both structures and simulations. Ultimately, our findings reveal why nature has conserved the capacity to open up this region, and highlight how differences in the details of the ligand entry process result in differential evolutionary constraints across the steroid receptors.