Figure 1.
Chemical synthesis of F-53 (1) and its CoA conjugate (2), and the structure of PTC124.
Conditions: a) pyridine, EtOH, reflux, 24 h, 62.3%; b) H2, 10% Pd-C, benzene, RT, 93.6%; c) HCHOaq., NaBH3CN, AcOH, THF, RT, 7.5 h, 88.4%; d) NaOH aq., EtOH, RT, 10 h, 77.6%; e) 3LiCoASH, PyBOP, K2CO3, THF, RT, 50%
Figure 2.
Inhibition of firefly luciferase (Luc) activity by F-53, as determined using a reporter assay.
The dose-response curve for F-53 was constructed by using the human retinoid orphan receptor α1 (hRORα1) reporter assay. Mammalian expression vectors for full-length hRORα1 (pcDNA3-RORα1), thymidine kinase promoter-based Luc reporter, (Rev-DR2×3-TK-Luc), and pCMVβ were transfected into HeLa cells. The cells were treated with F-53 (10−6–2×10−11 M) for 1 d. Luc activity was determined after the addition of PicaGene LT 2.0 and normalized relative to β-gal activity. The results (Y-axes) are expressed as the fold increase in Luc activity after F-53 treatment, relative to the increase after treatment with DMSO. Each value represents the mean±SEM of six transfection experiments.
Figure 3.
Effect of F-53 on reporter enzymes.
The effects of F-53 on firefly luciferase (A), Renilla luciferase (B), β-galactosidase (β-gal) (C), and chloramphenicol acetyl transferase (CAT) (D) are shown. A–C: All individual reporter plasmids, that is, pCMVluc+ (A), pRL-CMV (B), and pCMVβ (C), were transfected into HeLa cells. The relevant enzymatic activity in cell lysates was measured after treatment with DMSO or F-53 (1 and 10 µM), in the presence of the relevant enzymatic substrate. D: pSG5-hRARα1 DR5G-TK-CAT, and pCMVβ were transfected into COS-1 cells. After treatment with DMSO, F-53, and LE540 (RAR pan-antagonist) with or without Am80 (RARα,β-selective agonist), CAT activity in cell lysates was measured by a CAT ELISA, and the results were normalized relative to β-gal activity. The asterisks indicate statistically significant differences (** p<0.01) between F-53 and DMSO treatments, which were determined using post-hoc analysis with Fisher's LSD test. D: DMSO, CAT: chloramphenicol acetyl transferase, RLU: relative light unit, RFU: relative fluorescence unit. Each value represents the mean±SEM of three transfection experiments.
Figure 4.
Time course of changes in firefly luciferase (Luc) activities.
A: Protocol for treating cells and measuring Luc activity. HeLa cells were transiently transfected with the pCMVluc+ and the pCMVβ to normalize differences in transfection efficiency. The cells were then treated with F-53 (B: 1 µM, D: 0.1–10 µM,) or PTC124 (C: 0.1 µM) for the incubation periods indicated in the figure (B–C). B and C: Luc activity was determined by measurement-1 in (A). D: Luc activity after the addition of F-53 in culture medium was determined by measurement-1 (black bar) and measurement-2 (white bar) in (A). Luc activity after the addition of F-53 at the same time as lysis was determined by measurement-3 (black bar) and measurement-4 (white bar) in (A). Luc activity after the addition of F-53 after lysis was determined by measurement-5 in (A). Treatment with F-53 after cell lysis involved treatment with a solution of F-53 in lysis buffer solution. Luc activity was normalized relative to β-gal activity. The results (Y-axes) are expressed as the fold increase in Luc activity after F-53 or PTC124 treatment, relative to the increase after treatment with DMSO. Each value represents the mean±SEM of three transfection experiments.
Figure 5.
Effects of F-53 on levels of firefly luciferase (Luc) mRNA and protein in culture medium.
A: Luc mRNA expression was determined by RT-PCR. Total RNA was isolated from HeLa cells transiently transfected with pCMVluc+ and treated with F-53 or DMSO for 20–24 h. The expression of Luc was normalized relative to the abundance of the endogenous hGAPDH transcripts. B and C: Western blotting after SDS-PAGE (B) and native PAGE (C). Protein samples for SDS- or native PAGE and western blot analysis were obtained from HeLa cells that had been transiently transfected with pCMVluc+ and pCMVβ and treated with F-53, PTC124, or DMSO for 20–24 h before lysis. The protein level of Luc was normalized relative to that of β-gal. The asterisks indicate statistically significant differences (* p<0.05, ** p<0.01) between F-53 and DMSO treatments, determined using post-hoc analysis with Fisher's LSD test. D: DMSO, Luc: Luciferase. Each value represents the mean±SEM of three independent experiments.
Figure 6.
Effect of F-53 on recombinant luciferase (Luc) activity in vitro.
The inhibitory effects of F-53 on recombinant Luc enzymatic activity in lysis buffer (A) and HeLa cell lysate (B) are shown. The fold increase (Y axes) in Luc activity after treatment with either F-53 or PTC124 was normalized relative to the level obtained after DMSO treatment. The values shown above the bars represent the mean, and the error bars indicate SEM of three independent experiments. C: Western blotting after native PAGE by using the Luc protein sample used in (A). We added protein samples for native PAGE (12.5 ng protein), obtained from HeLa cells, to each Luc protein sample used in (A) because the band representing recombinant Luc protein alone was not clearly visible on western blotting of native PAGE gels, and then used western blotting to analyze these proteins after native PAGE. Cont represents samples with 12.5 ng of HeLa protein only; no Luc band was detected in this control.
Figure 7.
MALDI-TOF-MS and MS/MS analyses of firefly luciferase (Luc) protein modified by F-53 and synthetic peptides.
A: Total masses of Luc proteins after F-53 treatment compared with those after the control treatment (DMSO). B: Peptide mass spectra of Luc proteins after F-53 (upper) and DMSO (lower) treatments. C: MS/MS analysis of the 1,250 Da peak in (B). MS/MS analysis estimated that the peptide sequence of 1,250 Da peak is 525GLTGK*LDAR533 which was not digested at the lysine-529 position by trypsin. The molecular weight (1,250 Da) was 320 Da greater than that calculated from the actual peptide sequence. The difference was attributable to the presence or absence of F-53 at the amino group of lysine-529. D: MALDI-TOF-MS analysis of the synthetic peptides GLTGKLDAR (upper) and GLTGK(F-53)LDAR (lower), in which the amino group of the lysine and the carboxyl group of F-53 were dehydration-condensed. GLTGK(F-53)LDAR is the estimated sequence in (C). E and F: MS/MS analysis of the synthetic peptides GLTGKLDAR (E) and GLTGK(F-53)LDAR (F). The MS/MS data of (C) were the same data as those of (F).
Figure 8.
In vitro production of F-53-CoA.
The typical HPLC/UV chromatograms obtained from standard mixture (A), the assay samples without enzyme (negative control, B), by using recombinant luciferase (0.133 µM, C), and by using mouse liver microsomes (27.6 µg, D) with ATP, MgCl2, and CoA-SH are shown. Enzymatic in vitro assays were performed at 37°C for 15 min with F-53 (20 µM) as the substrate. The formation of F-53-CoA (product) was measured by HPLC/UV at 260 nm by using 10%–90% CH3CN in water with 10 mM AcONH4 (pH 7.0) as the mobile phase. Mass analysis was performed by HPLC/MS (positive-mode ESI) using the same mobile phase.
Figure 9.
Effect of a kinase inhibitor on firefly luciferase (Luc) modification by F-53.
A: The structure of PD98059. B: Effect of PD98059, a mitogen-activated protein kinase kinase (MEK) inhibitor for F-53 inhibition. PD98059 was added to HeLa cells transiently transfected with pCMVluc+ and pCMVβ to normalize differences in transfection efficiency at 3 h prior to the addition of F-53 (0.1 µM). Luc activity was determined after the addition of PicaGene LT 2.0 and normalized relative to β-gal activity. The results (Y-axes) are shown as the percentage of Luc activity relative to the DMSO control. C: Western blotting after native PAGE. PD98059 was added to HeLa cells transiently transfected with pCMVluc+ and pCMVβ at 1 h prior to F-53 or DMSO treatment for 3.5 h. HeLa cells lysates were subjected to native PAGE and western blotting. Luc: Luciferase. Each value represents the mean±SEM of three transfection experiments.
Figure 10.
Proposed mechanism for the reaction of F-53 with firefly luciferase (Luc).
The mechanism might involve F-53 activation to its CoA-derivative by Luc, which functions as an acyl-CoA synthetase in living cells. F-53-CoA is proposed to be transferred to lysine-529 of Luc by an unknown cellular acetyltransferase. Lysine-529 might be the most important lysine for the regulation of enzyme activity by acetylation and deacetylation. We also suggest that after F-53-CoA is activated by native acyl-CoA synthetase in mammalian liver microsomes, F-53-CoA may be transferred to the lysine residue, which induces the acetylation of the same enzyme.