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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

N-6 binds to RXRα. (A) Structure of NSC-640358 (N-6). (B) RXRα-LBD protein was incubated with [3H]9-cis-RA in the presence or absence of unlabeled 9-cis-RA or N-6. Bound [3H]9-cis-RA was quantitated by liquid scintillation counting. (C) Gradient concentrations of N-6 were injected through flow cells immobilized with RXRα-LBD. The kinetic profiles are shown and the dissociation constant (Kd) of the N-6/RXRα-LBD complex was calculated to be 15.755 × 10−6 mol/L. (D) RXRα-LBD protein enhanced fluorescent intensity of N-6 (ex 278 nm, em 338 nm, cutoff 325 nm, delay 50 μs, integration 450 μs). One of three similar experiments is shown
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Fig1: N-6 binds to RXRα. (A) Structure of NSC-640358 (N-6). (B) RXRα-LBD protein was incubated with [3H]9-cis-RA in the presence or absence of unlabeled 9-cis-RA or N-6. Bound [3H]9-cis-RA was quantitated by liquid scintillation counting. (C) Gradient concentrations of N-6 were injected through flow cells immobilized with RXRα-LBD. The kinetic profiles are shown and the dissociation constant (Kd) of the N-6/RXRα-LBD complex was calculated to be 15.755 × 10−6 mol/L. (D) RXRα-LBD protein enhanced fluorescent intensity of N-6 (ex 278 nm, em 338 nm, cutoff 325 nm, delay 50 μs, integration 450 μs). One of three similar experiments is shown

Mentions: We used Q-MOL molecular modeling package to perform a virtual screening of a compound library for RXRα ligands, and found that NSC-640358 ((NZ)-N-[1-[2-(3,5-diphenylpyrazol-1-yl)-4-methyl-1,3-thiazol-5-yl]ethylidene]droxylamine, N-6), a thiazolyl-pyrazole derived compound, was a potential RXRα ligand (Fig. 1A). The interaction of N-6 and RXRα was first examined by ligand competition binding assay. As a positive control, unlabeled 9-cis-RA displaced [3H]-labeled 9-cis-RA for binding to RXRα-LBD protein in a dose-dependent manner (Fig. 1B). Similarly, N-6 dose-dependently competed with [3H]-labeled 9-cis-RA for binding to RXRα-LBD protein with an IC50 at 11.3 μmol/L (Fig. 1B), suggesting the direct binding of N-6 to RXRα. Then we performed surface plasmon resonance (SPR) technology-based experiment to confirm their physical binding. As shown in Fig. 1C, N-6 dose-dependently binds to RXRα-LBD with a Kd value of 15.7 μmol/L. Furthermore, our fluorescence ligand binding assay showed that RXRα-LBD protein potently enhanced the fluorescence intensity of N-6 (Fig. 1D). Together, these results demonstrate that N-6 is able to bind to RXRα directly.Figure 1


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)

N-6 binds to RXRα. (A) Structure of NSC-640358 (N-6). (B) RXRα-LBD protein was incubated with [3H]9-cis-RA in the presence or absence of unlabeled 9-cis-RA or N-6. Bound [3H]9-cis-RA was quantitated by liquid scintillation counting. (C) Gradient concentrations of N-6 were injected through flow cells immobilized with RXRα-LBD. The kinetic profiles are shown and the dissociation constant (Kd) of the N-6/RXRα-LBD complex was calculated to be 15.755 × 10−6 mol/L. (D) RXRα-LBD protein enhanced fluorescent intensity of N-6 (ex 278 nm, em 338 nm, cutoff 325 nm, delay 50 μs, integration 450 μs). One of three similar experiments is shown
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Related In: Results  -  Collection

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Fig1: N-6 binds to RXRα. (A) Structure of NSC-640358 (N-6). (B) RXRα-LBD protein was incubated with [3H]9-cis-RA in the presence or absence of unlabeled 9-cis-RA or N-6. Bound [3H]9-cis-RA was quantitated by liquid scintillation counting. (C) Gradient concentrations of N-6 were injected through flow cells immobilized with RXRα-LBD. The kinetic profiles are shown and the dissociation constant (Kd) of the N-6/RXRα-LBD complex was calculated to be 15.755 × 10−6 mol/L. (D) RXRα-LBD protein enhanced fluorescent intensity of N-6 (ex 278 nm, em 338 nm, cutoff 325 nm, delay 50 μs, integration 450 μs). One of three similar experiments is shown
Mentions: We used Q-MOL molecular modeling package to perform a virtual screening of a compound library for RXRα ligands, and found that NSC-640358 ((NZ)-N-[1-[2-(3,5-diphenylpyrazol-1-yl)-4-methyl-1,3-thiazol-5-yl]ethylidene]droxylamine, N-6), a thiazolyl-pyrazole derived compound, was a potential RXRα ligand (Fig. 1A). The interaction of N-6 and RXRα was first examined by ligand competition binding assay. As a positive control, unlabeled 9-cis-RA displaced [3H]-labeled 9-cis-RA for binding to RXRα-LBD protein in a dose-dependent manner (Fig. 1B). Similarly, N-6 dose-dependently competed with [3H]-labeled 9-cis-RA for binding to RXRα-LBD protein with an IC50 at 11.3 μmol/L (Fig. 1B), suggesting the direct binding of N-6 to RXRα. Then we performed surface plasmon resonance (SPR) technology-based experiment to confirm their physical binding. As shown in Fig. 1C, N-6 dose-dependently binds to RXRα-LBD with a Kd value of 15.7 μmol/L. Furthermore, our fluorescence ligand binding assay showed that RXRα-LBD protein potently enhanced the fluorescence intensity of N-6 (Fig. 1D). Together, these results demonstrate that N-6 is able to bind to RXRα directly.Figure 1

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