Disruption of a key ligand-H-bond network drives dissociative properties in vamorolone for Duchenne muscular dystrophy treatment

Abstract

Duchenne muscular dystrophy is a genetic disorder that shows chronic and progressive damage to skeletal and cardiac muscle leading to premature death. Antiinflammatory corticosteroids targeting the glucocorticoid receptor (GR) are the current standard of care but drive adverse side effects such as deleterious bone loss. Through subtle modification to a steroidal backbone, a recently developed drug, vamorolone, appears to preserve beneficial efficacy but with significantly reduced side effects. We use combined structural, biophysical, and biochemical approaches to show that loss of a receptor-ligand hydrogen bond drives these remarkable therapeutic effects. Moreover, vamorolone uniformly weakens coactivator associations but not corepressor associations, implicating partial agonism as the main driver of its dissociative properties. Additionally, we identify a critical and evolutionarily conserved intramolecular network connecting the ligand to the coregulator binding surface. Interruption of this allosteric network by vamorolone selectively reduces GR-driven transactivation while leaving transrepression intact. Our results establish a mechanistic understanding of how vamorolone reduces side effects, guiding the future design of partial agonists as selective GR modulators with an improved therapeutic index.

Keywords: allostery; dissociative agonist; glucocorticoid receptor; molecular dynamics; nuclear receptor.

Conflict of interest statement

Competing interest statement: The authors declare competing financial interests. J.M.D., K.N., and E.P.H. are employees of ReveraGen BioPharma, and hold stock. The other authors declare no competing interests.

Figures

Fig. 1.

Corticosteroids for DMD treatment have tight binding to AncGR2 LBD and induce similar GR dimerization effects. (A–C) Chemical structures of prednisolone (PRED), C21-desacetyl deflazacort (dDFZ), and vamorolone (VAM) used for DMD treatment. Key carbon and oxygen atom positions are highlighted. (D) All compounds bind to AncGR2 LBD directly with a nanomolar (nM) Ki, as measured by FP competition against fluorescein labeled dexamethasone (FAM-DEX). Error bars in D indicate SD from three replicates and from three independent experiments. (E and F) Effects of VAM on GR dimerization probed by BRETn and PCA assays with GR WT and Mon mutant upon treatment with 1,000 nM concentration of PRED and VAM. Bars indicate mean, and error bars represent SEM. Two-way ANOVA followed by Tukey’s post hoc test was performed. n.s., not significant.

Fig. 2.

PRED, dDFZ, and VAM adopt similar orientations within the AncGR2 LBD but engage different H-bonding networks. (A) Overall structure of AncGR2 LBD with PRED (green) bound to PGC1α (light purple), with α-helices shown in light blue, β-strands in yellow, and loops in gray. (B) 2Fo-Fc omit electron density map (contoured to 2.0 σ) surrounding PRED (green), dDFZ (orange), and VAM (purple) in the ligand binding pocket. (C) Extensive hydrogen bonds (dark blue residues) and hydrophobic interactions (light blue residues) are formed between AncGR2 LBD-PRED. (D) Select hydrophobic interactions between AncGR2 LBD and different ligands. (E) Different hydrogen bond networks are formed between AncGR2 LBD and ligands with bond distances labeled.

Fig. 3.

GR LBD-VAM complex has a distinct coregulator binding pattern. (A) Comparison of drug-induced coregulator peptide recruitment to hGR LBD by MARCoNI. (B) Hierarchical clustering of log-fold change (LFC) by comparing binding data of individual drug-bound and apo hGR LBD (dimethyl sulfoxide [DMSO] control) and colored continuously from blue to red. Statistically significant changes relative to DMSO control were identified by Student's t test, post hoc false discovery rate (FDR), *P < 0.05, **P < 0.01, or ***P < 0.001. (C) Box-and-whisker plot of LFC against DMSO control for hGR LBD in complex with PRED, dDFZ, VAM, and hydrocortisone (HCY). The line in the box is plotted at the median of all of the coregulator binding, whereas the whiskers are the 2.5 and 97.5 percentiles. (D) Summary of binding affinities for various coregulator peptides bound to AncGR2 LBD with different ligands are expressed as Kd (μM) with 95% confidence interval. (E) Fold change of binding to different coregulators compared to AncGR2 LBD with HCY as expressed by [Kd(cmpd)/Kd(HCY)] for each coregulator.

Fig. 4.

AncGR2 LBD with VAM has reduced stability and enhanced protein dynamics. (A) Thermal unfolding curves of AncGR2 LBD bound to PRED, dDFZ, and VAM. (B) Difference in thermostability (∆Tm) of GR2 LBD with different ligands in the presence of coregulators compared to GR2 LBD with HCY as expressed by ∆Tm = [Tm(ligand) − Tm(HCY)] for each coregulator. (C). Heat maps of deuterium uptake monitored by HDX-MS for AncGR2 LBD bound to PRED and VAM. Different time points of LBD incubation in D2O before measuring deuterium uptake are indicated on the Left. (D) Three representative HDX plots of peptic fragments from PRED- and VAM-bound AncGR2 LBD. (E) Differential deuterium uptakes are mapped on the structure of AncGR2 LBD in complex with VAM. Residues are colored in a continuous gradient from blue to red, with their intensity scaling to the difference in percentage of deuterium exchange [(VAM-bound) − (PRED-bound)]. Residues not covered by any peptides are shown in black.

Fig. 5.

VAM binding reduces allosteric communication between AncGR2 ligand and AFH. (A) Suboptimal paths connecting nodes Glu224 (shown in blue) and ligand (shown in cyan) with edges (shown in green) displayed. The other nodes in the protein are shown in gray. (B) Top nodes used in the top 1,000 suboptimal pathway analyses in five different AncGR2-ligand complexes. (C) Histograms of top 1,000 suboptimal paths in AncGR2 with three ligands. (D) Optimal path length and nodes used in these paths in the AncGR2 with three ligands.

Fig. 6.

Residue N33 (in AncGR2) or N564 (in hGR) is crucial for ligand binding and gene transcription. (A) Sequence alignment between hGR and AncGR2 centered on N33 (AncGR2 numbering). (B) Hydrogen bonds formed between N33 and ligands (overlaid structures of PRED and VAM) with the N33A mutation modeled and shown in red. (C) Binding affinities of PRED and VAM interacting with AncGR2WT and N33A mutant that expressed as Kd (M) with 95% confidence interval. (D and E) Effects of N564A mutation on transactivation and transrepression using luciferase reporters containing SGK1 promoter (D) and κBRE promoter (E) upon treatment with PRED and VAM. Error bars in D and E represent SEM.