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NSC-640358 acts as RXRα ligand to promote TNFα-mediated apoptosis of cancer cell.

Chen F, Chen J, Lin J, Cheltsov AV, Xu L, Chen Y, Zeng Z, Chen L, Huang M, Hu M, Ye X, Zhou Y, Wang G, Su Y, Zhang L, Zhou F, Zhang XK, Zhou H - Protein Cell (2015)

Bottom Line: Retinoid X receptor α (RXRα) and its N-terminally truncated version tRXRα play important roles in tumorigenesis, while some RXRα ligands possess potent anti-cancer activities by targeting and modulating the tumorigenic effects of RXRα and tRXRα.Using mutational analysis and computational study, we determine that Arg316 in RXRα, essential for 9-cis-retinoic acid binding and activating RXRα transactivation, is not required for antagonist effects of N-6, whereas Trp305 and Phe313 are crucial for N-6 binding to RXRα by forming extra π-π stacking interactions with N-6, indicating a distinct RXRα binding mode of N-6.N-6 inhibits TR3-stimulated transactivation of Gal4-DBD-RXRα-LBD by binding to the ligand binding pocket of RXRα-LBD, suggesting a strategy to regulate TR3 activity indirectly by using small molecules to target its interacting partner RXRα.

View Article: PubMed Central - PubMed

Affiliation: School of Pharmaceutical Sciences, Xiamen University, Xiamen, 361102, China.

ABSTRACT
Retinoid X receptor α (RXRα) and its N-terminally truncated version tRXRα play important roles in tumorigenesis, while some RXRα ligands possess potent anti-cancer activities by targeting and modulating the tumorigenic effects of RXRα and tRXRα. Here we describe NSC-640358 (N-6), a thiazolyl-pyrazole derived compound, acts as a selective RXRα ligand to promote TNFα-mediated apoptosis of cancer cell. N-6 binds to RXRα and inhibits the transactivation of RXRα homodimer and RXRα/TR3 heterodimer. Using mutational analysis and computational study, we determine that Arg316 in RXRα, essential for 9-cis-retinoic acid binding and activating RXRα transactivation, is not required for antagonist effects of N-6, whereas Trp305 and Phe313 are crucial for N-6 binding to RXRα by forming extra π-π stacking interactions with N-6, indicating a distinct RXRα binding mode of N-6. N-6 inhibits TR3-stimulated transactivation of Gal4-DBD-RXRα-LBD by binding to the ligand binding pocket of RXRα-LBD, suggesting a strategy to regulate TR3 activity indirectly by using small molecules to target its interacting partner RXRα. For its physiological activities, we show that N-6 strongly inhibits tumor necrosis factor α (TNFα)-induced AKT activation and stimulates TNFα-mediated apoptosis in cancer cells in an RXRα/tRXRα dependent manner. The inhibition of TNFα-induced tRXRα/p85α complex formation by N-6 implies that N-6 targets tRXRα to inhibit TNFα-induced AKT activation and to induce cancer cell apoptosis. Together, our data illustrate a new RXRα ligand with a unique RXRα binding mode and the abilities to regulate TR3 activity indirectly and to induce TNFα-mediated cancer cell apoptosis by targeting RXRα/tRXRα.

No MeSH data available.


Related in: MedlinePlus

Arg316 is not required for N-6 binding to RXRα. (A) MCF-7 cells cotransfected with pG5-Gaussia-Dura reporter vector and pBIND-RXRα-LBD or pBIND-RXRα-LBD/R316E expression vectors were treated with or without N-6 (10 μmol/L) in the presence or absence of CD3254 (10−7 mol/L) for 18 h. Reporter activities were measured and normalized. Data shown are mean ± SD (*P < 0.05). (B) Comparison of the docked conformation of N-6 (gray) with the crystal structure of LG100754 (green). (C) N-6 was docked into the LBP of the co-crystal structure of LG100754 and RXRα-LBD (PDB 3A9E). Salt bridges are shown as dotted yellow lines, and residues interacting with N-6 are shown in magenta. (D–E) Gradient concentrations of N-6 were injected through flow cells immobilized with RXRα-LBD/Trp305Ala (D) and RXRα-LBD/Phe313Ala (E), respectively. The kinetic profiles are shown and the dissociation constants (Kd) of the N-6/RXRα-LBD complex were calculated to be 1.0 × 10−3 mol/L (D) and more than 1.0 × 10−2 mol/L (E). (F) RXRα-LBD proteins (2 mg/mL) was incubated with DMSO, 10 μmol/L 9-cis-RA, 25 μmol/L N-6 or 50 μmol/L N-6 for 3 h, and proteins were separated by 8% nondenaturing PAGE followed by Commassie Blue staining
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Fig4: Arg316 is not required for N-6 binding to RXRα. (A) MCF-7 cells cotransfected with pG5-Gaussia-Dura reporter vector and pBIND-RXRα-LBD or pBIND-RXRα-LBD/R316E expression vectors were treated with or without N-6 (10 μmol/L) in the presence or absence of CD3254 (10−7 mol/L) for 18 h. Reporter activities were measured and normalized. Data shown are mean ± SD (*P < 0.05). (B) Comparison of the docked conformation of N-6 (gray) with the crystal structure of LG100754 (green). (C) N-6 was docked into the LBP of the co-crystal structure of LG100754 and RXRα-LBD (PDB 3A9E). Salt bridges are shown as dotted yellow lines, and residues interacting with N-6 are shown in magenta. (D–E) Gradient concentrations of N-6 were injected through flow cells immobilized with RXRα-LBD/Trp305Ala (D) and RXRα-LBD/Phe313Ala (E), respectively. The kinetic profiles are shown and the dissociation constants (Kd) of the N-6/RXRα-LBD complex were calculated to be 1.0 × 10−3 mol/L (D) and more than 1.0 × 10−2 mol/L (E). (F) RXRα-LBD proteins (2 mg/mL) was incubated with DMSO, 10 μmol/L 9-cis-RA, 25 μmol/L N-6 or 50 μmol/L N-6 for 3 h, and proteins were separated by 8% nondenaturing PAGE followed by Commassie Blue staining

Mentions: Unlike many natural and synthetic RXRα ligands, N-6 lacks a carboxylate moiety known to form salt bridges with Arg316 in RXRα-LBP. To examine the requirement of Arg316 for N-6, it was replaced with Glutamic acid, and the resulting mutant Gal4-DBD-RXRαLBD/R316E was evaluated for its transactivation regulated by N-6. As shown in Fig. 4A, CD3254 was able to stimulate the transactivation of the mutant. Similar to RXRα-LBD, CD3254-induced transactivation of RXRα-LBD/R316E was potently inhibited by N-6 (Fig. 4A), implying that Arg316 was not required for N-6 binding to RXRα. We then used computer-aided and docking-based techniques to analyze the binding mode of N-6. In light of the antagonist activity of N-6 and a large degree of structural overlapping between N-6 and LG100754 (Fig. 4B), an antagonist of RARα/RXRα heterodimer (Sato et al., 2010), it was reasonable that the model of RXRα under its antagonist conformation should be used for the docking of N-6. In fact, our docking study showed that N-6 was well accommodated into the LBP of RXRα with an antagonist conformation (Fig. 4C). Unlike LG100754, N-6 did not form ionic bonds (2.3 Å and 1.5 Å for LG100754) with Arg316. However, N-6 possesses two aromatic rings that could establish extra π-π stacking interactions with Phe313 and Trp305 (Fig. 4C). The essential role of Phe313 and Trp305 in N-6 binding was confirmed by our SPR assays, showing that N-6 had much lower binding affinity to two RXRα-LBD point mutants with Ala substitution of Phe313 or Trp305 (Fig. 4D and 4E). Furthermore, our docking study showed that two hydrophobic residues Ile324 and Leu326 could produce additional hydrophobic interactions with N-6 (Fig. 4C). Ligand binding often leads to the conformational change of RXRα protein, which was investigated for N-6 by our native gel electrophoresis assay. Consistent with previous reports, 9-cis-RA strongly induced the homodimeric formation of RXRα-LBD protein. However, N-6 dose-dependently induced the conformational changes of RXRα-LBD dimer, indicated by the pattern changes of the dimer bands (Fig. 4F). Taken together, our data indicate that N-6 has a distinct binding model comparing with classic RXRα ligands.Figure 4


NSC-640358 acts as RXRα ligand to promote TNFα-mediated apoptosis of cancer cell.

Chen F, Chen J, Lin J, Cheltsov AV, Xu L, Chen Y, Zeng Z, Chen L, Huang M, Hu M, Ye X, Zhou Y, Wang G, Su Y, Zhang L, Zhou F, Zhang XK, Zhou H - Protein Cell (2015)

Arg316 is not required for N-6 binding to RXRα. (A) MCF-7 cells cotransfected with pG5-Gaussia-Dura reporter vector and pBIND-RXRα-LBD or pBIND-RXRα-LBD/R316E expression vectors were treated with or without N-6 (10 μmol/L) in the presence or absence of CD3254 (10−7 mol/L) for 18 h. Reporter activities were measured and normalized. Data shown are mean ± SD (*P < 0.05). (B) Comparison of the docked conformation of N-6 (gray) with the crystal structure of LG100754 (green). (C) N-6 was docked into the LBP of the co-crystal structure of LG100754 and RXRα-LBD (PDB 3A9E). Salt bridges are shown as dotted yellow lines, and residues interacting with N-6 are shown in magenta. (D–E) Gradient concentrations of N-6 were injected through flow cells immobilized with RXRα-LBD/Trp305Ala (D) and RXRα-LBD/Phe313Ala (E), respectively. The kinetic profiles are shown and the dissociation constants (Kd) of the N-6/RXRα-LBD complex were calculated to be 1.0 × 10−3 mol/L (D) and more than 1.0 × 10−2 mol/L (E). (F) RXRα-LBD proteins (2 mg/mL) was incubated with DMSO, 10 μmol/L 9-cis-RA, 25 μmol/L N-6 or 50 μmol/L N-6 for 3 h, and proteins were separated by 8% nondenaturing PAGE followed by Commassie Blue staining
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Related In: Results  -  Collection

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Fig4: Arg316 is not required for N-6 binding to RXRα. (A) MCF-7 cells cotransfected with pG5-Gaussia-Dura reporter vector and pBIND-RXRα-LBD or pBIND-RXRα-LBD/R316E expression vectors were treated with or without N-6 (10 μmol/L) in the presence or absence of CD3254 (10−7 mol/L) for 18 h. Reporter activities were measured and normalized. Data shown are mean ± SD (*P < 0.05). (B) Comparison of the docked conformation of N-6 (gray) with the crystal structure of LG100754 (green). (C) N-6 was docked into the LBP of the co-crystal structure of LG100754 and RXRα-LBD (PDB 3A9E). Salt bridges are shown as dotted yellow lines, and residues interacting with N-6 are shown in magenta. (D–E) Gradient concentrations of N-6 were injected through flow cells immobilized with RXRα-LBD/Trp305Ala (D) and RXRα-LBD/Phe313Ala (E), respectively. The kinetic profiles are shown and the dissociation constants (Kd) of the N-6/RXRα-LBD complex were calculated to be 1.0 × 10−3 mol/L (D) and more than 1.0 × 10−2 mol/L (E). (F) RXRα-LBD proteins (2 mg/mL) was incubated with DMSO, 10 μmol/L 9-cis-RA, 25 μmol/L N-6 or 50 μmol/L N-6 for 3 h, and proteins were separated by 8% nondenaturing PAGE followed by Commassie Blue staining
Mentions: Unlike many natural and synthetic RXRα ligands, N-6 lacks a carboxylate moiety known to form salt bridges with Arg316 in RXRα-LBP. To examine the requirement of Arg316 for N-6, it was replaced with Glutamic acid, and the resulting mutant Gal4-DBD-RXRαLBD/R316E was evaluated for its transactivation regulated by N-6. As shown in Fig. 4A, CD3254 was able to stimulate the transactivation of the mutant. Similar to RXRα-LBD, CD3254-induced transactivation of RXRα-LBD/R316E was potently inhibited by N-6 (Fig. 4A), implying that Arg316 was not required for N-6 binding to RXRα. We then used computer-aided and docking-based techniques to analyze the binding mode of N-6. In light of the antagonist activity of N-6 and a large degree of structural overlapping between N-6 and LG100754 (Fig. 4B), an antagonist of RARα/RXRα heterodimer (Sato et al., 2010), it was reasonable that the model of RXRα under its antagonist conformation should be used for the docking of N-6. In fact, our docking study showed that N-6 was well accommodated into the LBP of RXRα with an antagonist conformation (Fig. 4C). Unlike LG100754, N-6 did not form ionic bonds (2.3 Å and 1.5 Å for LG100754) with Arg316. However, N-6 possesses two aromatic rings that could establish extra π-π stacking interactions with Phe313 and Trp305 (Fig. 4C). The essential role of Phe313 and Trp305 in N-6 binding was confirmed by our SPR assays, showing that N-6 had much lower binding affinity to two RXRα-LBD point mutants with Ala substitution of Phe313 or Trp305 (Fig. 4D and 4E). Furthermore, our docking study showed that two hydrophobic residues Ile324 and Leu326 could produce additional hydrophobic interactions with N-6 (Fig. 4C). Ligand binding often leads to the conformational change of RXRα protein, which was investigated for N-6 by our native gel electrophoresis assay. Consistent with previous reports, 9-cis-RA strongly induced the homodimeric formation of RXRα-LBD protein. However, N-6 dose-dependently induced the conformational changes of RXRα-LBD dimer, indicated by the pattern changes of the dimer bands (Fig. 4F). Taken together, our data indicate that N-6 has a distinct binding model comparing with classic RXRα ligands.Figure 4

Bottom Line: Retinoid X receptor α (RXRα) and its N-terminally truncated version tRXRα play important roles in tumorigenesis, while some RXRα ligands possess potent anti-cancer activities by targeting and modulating the tumorigenic effects of RXRα and tRXRα.Using mutational analysis and computational study, we determine that Arg316 in RXRα, essential for 9-cis-retinoic acid binding and activating RXRα transactivation, is not required for antagonist effects of N-6, whereas Trp305 and Phe313 are crucial for N-6 binding to RXRα by forming extra π-π stacking interactions with N-6, indicating a distinct RXRα binding mode of N-6.N-6 inhibits TR3-stimulated transactivation of Gal4-DBD-RXRα-LBD by binding to the ligand binding pocket of RXRα-LBD, suggesting a strategy to regulate TR3 activity indirectly by using small molecules to target its interacting partner RXRα.

View Article: PubMed Central - PubMed

Affiliation: School of Pharmaceutical Sciences, Xiamen University, Xiamen, 361102, China.

ABSTRACT
Retinoid X receptor α (RXRα) and its N-terminally truncated version tRXRα play important roles in tumorigenesis, while some RXRα ligands possess potent anti-cancer activities by targeting and modulating the tumorigenic effects of RXRα and tRXRα. Here we describe NSC-640358 (N-6), a thiazolyl-pyrazole derived compound, acts as a selective RXRα ligand to promote TNFα-mediated apoptosis of cancer cell. N-6 binds to RXRα and inhibits the transactivation of RXRα homodimer and RXRα/TR3 heterodimer. Using mutational analysis and computational study, we determine that Arg316 in RXRα, essential for 9-cis-retinoic acid binding and activating RXRα transactivation, is not required for antagonist effects of N-6, whereas Trp305 and Phe313 are crucial for N-6 binding to RXRα by forming extra π-π stacking interactions with N-6, indicating a distinct RXRα binding mode of N-6. N-6 inhibits TR3-stimulated transactivation of Gal4-DBD-RXRα-LBD by binding to the ligand binding pocket of RXRα-LBD, suggesting a strategy to regulate TR3 activity indirectly by using small molecules to target its interacting partner RXRα. For its physiological activities, we show that N-6 strongly inhibits tumor necrosis factor α (TNFα)-induced AKT activation and stimulates TNFα-mediated apoptosis in cancer cells in an RXRα/tRXRα dependent manner. The inhibition of TNFα-induced tRXRα/p85α complex formation by N-6 implies that N-6 targets tRXRα to inhibit TNFα-induced AKT activation and to induce cancer cell apoptosis. Together, our data illustrate a new RXRα ligand with a unique RXRα binding mode and the abilities to regulate TR3 activity indirectly and to induce TNFα-mediated cancer cell apoptosis by targeting RXRα/tRXRα.

No MeSH data available.


Related in: MedlinePlus