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

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

<div><p>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 <i>in vitro</i> 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 <i>in vitro</i> 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.</p></div

AZD9977 and eplerenone activities in reporter gene assays.

<p>Concentration response curves of AZD9977 and eplerenone tested in a reporter gene assay in (a) presence or (b) absence of 0.1 nM aldosterone. <i>n</i> = 4, average ± SD.</p

AZD9977 does not change urinary Na⁺/K⁺ ratio in rat urine electrolyte secretion model.

<p>a) Salt deprived rats were treated with increasing doses of eplerenone (a) or AZD9977 (b), urine collection for 8 hours after dose. Average +/- SEM; <i>n</i> = 8; *p<0.05 compared to vehicle. c) Salt deprived rats were treated with 10 or 30 mg kg^-1 eplerenone in absence or presence of 100 mg kg^-1 AZD9977, urine collection for 8 hours after dose. Average +/- SEM; <i>n</i> = 8, *p<0.05 vs vehicle. ^#p<0.05 vs eplerenone treatment in absence of AZD9977.</p

AZD9977 and eplerenone protect against aldosterone and high salt induced renal injury.

<p>Uni-nephrectomised rats with an aldosterone mini-pump and on a high salt diet were treated for 4 weeks with AZD9977 or eplerenone as food admixtures. Doses in mg kg^-1 d^-1 are indicated under the bars. (a) UACR was measured in urine collected for 24h at day 28. (b) UACR vs free steady average plasma drug exposure normalized to rat <i>in vitro</i> IC₅₀ (C_uss-average/<i>in vitro</i> IC₅₀). Drug exposure levels correspond to the exposure levels achieved at the studied doses (a). c) Renal pathology scores (renal fibrosis and glomerular nephritis). d) Histological sections stained with PAS.Representative photomicrographs of glomeruli/tubular sections from rats treated with vehicle (V), AZD9977 at 10 (AZ 10), 30 (AZ 30) or 100 mg kg^-1 d^-1 (AZ 100) or eplerenone at 10 (EP 10) or 30 mg kg^-1 d^-1 (EP 30). The scale bar corresponds to 200 μm original size. Average +/- SEM; <i>n</i> = 7–8; *p<0.05 compared to vehicle.</p

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

AZD9977 and eplerenone activities in reporter gene assays for human, mouse and rat MR.

<p>AZD9977 and eplerenone activities in reporter gene assays for human, mouse and rat MR.</p

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

AZD9977 and eplerenone activities on MR, GR, PR and AR in binding assays.

<p>AZD9977 and eplerenone activities on MR, GR, PR and AR in binding assays.</p

Structures of AZD9977 and eplerenone.

<p>Structures of AZD9977 ((S)-2-(7-Fluoro-4-(3-oxo-3,4-dihydro-2H-benzo[b][<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193380#pone.0193380.ref001" target="_blank">1</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193380#pone.0193380.ref004" target="_blank">4</a>]oxazine-6-carbonyl)-3,4-dihydro-2H-benzo[b][<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193380#pone.0193380.ref001" target="_blank">1</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193380#pone.0193380.ref004" target="_blank">4</a>]oxazin-3-yl)-N-methylacetamide) and eplerenone.</p