Conformationally Defined Rexinoids for the Prevention of Inflammation and Nonmelanoma Skin Cancers

Abstract

Compound 1 is a potent rexinoid that is highly effective in cancer chemoprevention but elevates serum triglycerides. In an effort to separate the lipid toxicity from the anticancer activity of 1, we synthesized four new analogs of rexinoid 1, of which three rexinoids did not elevate serum triglycerides. Rexinoids 3 and 4 are twice as potent as rexinoid 1 in binding to Retinoid X receptor (RXR). All-trans retinoic acid (ATRA) plays a key role in maintaining skin homeostasis, and rexinoids 3-6 are highly effective in upregulating the genes responsible for the biosynthesis of ATRA. Inflammation plays a key role in skin cancer, and rexinoids 3 and 4 are highly effective in diminishing LPS-induced inflammation. Rexinoids 3 and 4 are highly effective in preventing UVB-induced nonmelanoma skin cancer (NMSC) without displaying any overt toxicities. Biophysical studies of rexinoids 3 and 5 bound to hRXRα-ligand binding domain (LBD) reveal important conformational and dynamical differences in the ligand binding domain.

Figure 1.

Structures of bexarotene, UAB30 analogs, and rexinoids 1–6.

Figure 2.

Overlay of X-ray crystal structures of hRXRα–LBD–GRIP-1 bound to rexinoids 3 (green, PDB code 7UW4) and 5 (cyan, PDB code 7UW2).

Figure 3.

(A) General rexinoid structure and numbering. (B) Dihedral angles. (C) Overlay of rexinoids 1 (yellow, PDB code 4RME) and 5 (purple, PDB code 7UW2). (D) Overlay of rexinoids 1 (yellow, PDB code 4RME) and 3 (pink, PDB code 7UW4) (E) Overlay of rexinoids 2 (orange, PDB code 4RMD) and 3 (pink, PDB code 7UW4).

Figure 4.

(A) Electron density map for the ligand binding pocket (LBP) containing 1 (PDB code 4RME). (B) Electron density map for the LBP containing 3 (PDB code 7UW4). (C) Electron density map for the LBP containing 5 (PDB code 7UW2).

Figure 5.

HDX MS results mapped on X-ray crystal structures. Representation of HDX MS dynamics analysis of RXRα–LBD bound to (A) rexinoid 3 (cyan) and (B) rexinoid 5 (orange). Significant results are reported as a decrease (shades of blue) or increase (shades of red) in deuterium incorporation corresponding to reduced or increased regions of protein dynamics. RXR in complex with rexinoid 3 showed the greatest reduction of dynamics in helices 3 and 5. When rexinoid 5 is bound, there is an increase in deuterium incorporation in helices 8 and 9 and the loop between them, indicating an increase in solvent accessibility and dynamics in this region. This implies that there is an outward push from the ligand binding pocket toward the dimer interface. Regions shaded in gray showed no significant difference in deuterium incorporation when compared to the apo-RXRα–LBD control.

Figure 6.

Changes in epithelial gene expression upon treatment with rexinoids. Human organotypic epithelial cultures were treated with bexarotene and rexinoids 3 or 6 each at 0.2 μM, as described in the Experimental Section. The relative expression was normalized to that observed in vehicle-treated cultures; *, p < 0.05, **, p < 0.01, ***, p < 0.001.

Figure 7.

Rexinoids 2, 3, and 5 downregulate inflammatory markers in LPS-induced Raw264.7 macrophage cells.

Figure 8.

Ptch+/−/SKH-1 mice in the positive control group (group II, red) received UVB treatment and vehicle; group III (green) received rexinoid 3 (24 mg/kg body wt.); and group IV (dark green) received rexinoid 4 (25 mg/kg body wt.). Rexinoids 3 and 4 are formulated in 0.5% of methyl cellulose and administered by gavage thrice a week (M, W, F) for the duration of the experiment.

Figure 9.

Overlaid crystal structures of bexarotene (green), 7-Me-UAB30 (aqua), and 4-Me-UAB30 (yellow) show that the methyl groups are oriented toward Phe346 of helix 7.

Scheme 1.. Synthesis of Rexinoids 3–6^a

^a(a) RLi, ether, 0 °C for 1 h, rt for 48 h, 70–75% yield; (b) ethyl 4-bromo-3-methylbut-2-enoate, Zn, dioxane, 100 °C, 4 h, 60–70%; (c) LAH, ether, 0 °C for 2 h, –78 °C for 1 h, 100% crude yield; (d) IBX, acetone; 55 °C for 1.5 h, 60–75% yield; (e) triethyl phosphonosenecioate, n-butyllithium, tetrahydrofuran (THF), 0 °C for 0.5 h, rt for 1.5 h, 80–90% yield (85:15 mixture of (9Z): (9Z, 13Z)); and (f) KOH, methanol/H₂O, reflux for 1.5 h, 58–65% yield.