Deacylcortivazol-like pyrazole regioisomers reveal a more accommodating expanded binding pocket for the glucocorticoid receptor

Abstract

Glucocorticoids (GCs) are widely used, potent anti-inflammatory and chemotherapeutic drugs. They work by binding to the glucocorticoid receptor (GR), a ligand-activated transcription factor, inducing translocation to the nucleus and regulation of genes that influence a variety of cellular activities. Despite being effective for a broad number of conditions, GC use is limited by severe side effects. To identify ligands that are more selective, we synthesized pairs of regioisomers in the pyrazole ring that probe the expanded binding pocket of GR opened by deacylcortivazol (DAC). Using an Ullmann-type reaction, a deacylcortivazol-like (DAC-like) backbone was modified with five pendant groups at the 1'- and 2'-positions of the pyrazole ring, yielding 9 ligands. Most of the compounds were cytotoxic to leukemia cells, and all required GR expression. Both aliphatic and other aromatic groups substituted at the 2'-position produced ligands with GC activity, with phenyl and 4-fluorophenyl substitutions exhibiting high cellular affinity for the receptor and >5× greater potency than dexamethasone, a commonly used strong GC. Surprisingly, phenyl substitution at the 1'-position produced a high-affinity ligand with ∼10× greater potency than dexamethasone, despite little apparent room in the expanded binding pocket to accommodate 1'-modifications. Other 1'-modifications, however, were markedly less potent. The potency of the 2'-substituted and 1'-substituted DAC-like compounds tracked linearly with cellular affinity but had different slopes, suggesting a different mode of interaction with GR. These data provide evidence that the expanded binding pocket opened by deacylcortivazol is more accommodating that expected, allowing development of new, and possibly selective, GCs by substitution within the pyrazole ring.

Scheme 1. Traditional synthesis of ligands. Conditions for a: ref. .

Fig. 1. Cortivazol backbone with steroid numbering.

Scheme 2. Revised synthetic pathway for ligands. a) H₂NNH₂, HOAc; b) R–X, CuI, base, ligand (ref. 10); c) HF, THF.

Fig. 2. Synthesized ligands. Spectra confirming the identity and purity of these ligands are found in SpectraV2.pdf.

Fig. 3. Biological specificity and potency of each ligand measured by REH cell viability. (A) Western blot of GR from REH cells following infection with pMK1115-GFP-T2A-GR. GR levels correlate with GFP intensity (middle 50% GFP = med-GR-REH, top 10% GFP = high-GR-REH). The differences in normalized expression levels of GR are statistically significant (p < 0.05). (B) Reintroduction of GR by pMK1115-GFP-T2A-GR restores REH cell dex sensitivity. Control infected REH cells (black) are compared to high-GR-REH (pink), and med-GR-REH (teal). NALM6 cells are shown in purple for comparison. (C) EC50 for each ligand in high-GR-REH. *Ligands significantly different (p value < 0.05) from dex calculated by one-way ANOVA (Brown–Forsythe and Welch) with Dunnett T3 multiple comparisons (p values are in Table S2†). Red indicates ligands with pendant groups in the 2′ position, and blue indicates ligands with pendant groups in the 1′ position. The EC50 for D1′H could not be determined. (D) Relative potency of each DAC-like ligand compared to dex in high-GR-REH cells. Dotted line indicates potency equivalent to dex. EC50 assays were performed with at least 6 replicates per ligand.

Fig. 4. The effective affinity of 2′-substituted DAC-like compounds for GR are higher than 1′-substituted. (A) Representative images of HEK-293 T infected with GFP-GR and mCherry-H2B to mark nuclei. Cells treated with 10 μM dex (top panel) or DMSO (bottom panel) at time point 0 (T0) and ∼11 minutes later (T5) show translocation of GFP-GR to the nucleus with dexamethasone treatment but not with DMSO. (B) t_1/2 is the time at which half of the GFP-GR is translocated to the nucleus (see Materials and methods) after addition of ligand. *Ligands significantly different (p value < 0.05) than dexamethasone by one-way ANOVA (Brown–Forsythe and Welch) with Dunnett T3 multiple comparisons (p values are in Table S3†). (C) Determination of effective K_D (K_Deff) for dexamethasone using non-linear regression of normalized nuclear contrast (NNC) vs. concentration (see Materials and methods). Dashed lines indicate concentration at half translocation, defined as K_Deff. (D) K_Deff for dexamethasone, prednisone, and each of the DAC-like ligands. *Ligands significantly different (p value < 0.05) than dexamethasone by one-way ANOVA (p values are in Table S4†). (E) Pearson correlation plot of docking energy versus the K_Deff for each ligand (at least three biological replicates were performed for each experiment. Blue, 1′-modified compounds; red, 2′ modified compounds; black/grey, dexamethasone/prednisone).

Fig. 5. The affinity/potency correlation between 1′- and 2′-substituted DAC-like compounds are different. (A) Comparison of EC50 (x axis) vs. K_Deff (y axis) demonstrates that potency generally tracks with affinity for each of the DAC-like ligands for which both EC50 and K_Deff that could be determined (linear regression for all compounds – dotted line). The regression lines for effective affinity versus potency for 1′-(blue) and 2′-(red) substituted DAC-like ligands are significantly different (also included are points for dex (black) and pred (grey)). (B) Average EC50s and K_Deff for each of the ligands shown in panel A.