Identification of Michael acceptor-centric pharmacophores with substituents that yield strong thioredoxin reductase inhibitory character correlated to antiproliferative activity

Abstract

Aims: The role of thioredoxin reductase (TrxR) in tumorigenesis has made it an attractive anticancer target. A systematic approach for development of novel compounds as TrxR inhibitors is currently lacking. Structurally diversified TrxR inhibitors share in common electrophilic propensities for the sulfhydryl groups, among which include the Michael reaction acceptors containing an α,β-unsaturated carbonyl moiety. We aimed to identify features among structurally diversified Michael acceptor-based compounds that would yield a strong TrxR inhibitory character.

Results: Structurally dissimilar Michael acceptor-based natural compounds such as isobutylamides, zerumbone, and shogaols (SGs) were found to possess a poor TrxR inhibitory activity, indicating that a sole Michael acceptor moiety was insufficient to produce TrxR inhibition. The 1,7-diphenyl-hept-3-en-5-one pharmacophore in 3-phenyl-3-SG, a novel SG analog that possessed comparable TrxR inhibitory and antiproliferative potencies as 6-SG, was modified to yield 1,5-diphenyl-pent-1-en-3-one (DPPen) and 1,3-diphenyl-pro-1-en-3-one (DPPro, also known as chalcone) pharmacophores. These Michael acceptor-centric pharmacophores, when substituted with the hydroxyl and fluorine groups, gave rise to analogs displaying a TrxR inhibitory character positively correlated to their antiproliferative potencies. Lead analogs 2,2'-diOH-5,5'-diF-DPPen and 2-OH-5-F-DPPro yielded a half-maximal TrxR inhibitory concentration of 9.1 and 10.5 μM, respectively, after 1-h incubation with recombinant rat TrxR, with the C-terminal selenocysteine residue found to be targeted.

Innovation: Identification of Michael acceptor-centric pharmacophores among diversified compounds demonstrates that a systematic approach to discover and develop Michael acceptor-based TrxR inhibitors is feasible.

Conclusion: A strong TrxR inhibitory character correlated to the antiproliferative potency is attributed to structural features that include an α,β-unsaturated carbonyl moiety centered in a DPPen or DPPro pharmacophore bearing hydroxyl and fluorine substitutions.

FIG. 1.

In vitro inhibitory activity of α,β-unsaturated carbonyl (Michael acceptor) moiety-bearing compounds on mammalian thioredoxin reductase (TrxR). Dose-dependent (left panels) and time-dependent (right panels) TrxR inhibitory activities of (A) Michael acceptor-based natural compounds, (B) analogs bearing a 1,5-diphenyl-pent-1-en-3-one (DPPen) pharmacophore, and (C) analogs bearing a 1,3-diphenyl-pro-1-en-3-one (DPPro) (also known as chalcone) pharmacophore. Enzyme activities were evaluated by the 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) assay, upon 1-h incubation of the compounds at indicated concentrations with 110 nM recombinant rat TrxR (for dose-dependence assay conditions) or incubation of 25 μM of the compounds with 110 nM recombinant rat TrxR for different times (for time-dependence assay conditions). All data points represent means±SD of at least two independent experiments.

FIG. 2.

Correlation of the TrxR inhibitory and antiproliferative potencies of Michael acceptor moiety-bearing compounds. The in vitro 50% TrxR inhibition concentration (IC₅₀) values of 4-, 6-, and 3-Ph-3-SG, as well as the DPPen and DPPro analogs were plotted against their corresponding 50% growth inhibition concentration (GI₅₀) values obtained in HCT 116 cells. The DPPen and DPPro analogs, but not the shogaols (SGs), showed a positive correlation with the linear correlation coefficient r=0.81.

FIG. 3.

In vitro effects of the DPPen and DPPro lead analogs on glutathione reductase (GR) and glutathione peroxidase (GPx) activities. The activity of yeast GR (20 nM) incubated with 200 μM NADPH and indicated compounds at various concentrations (1 to 100 μM) for (A) 30 min and (B) 1 h was determined by following the absorbance at 340 nm after adding glutathione disulfide and NADPH. The activity of bovine GPx (100 nM) incubated with 20 nM yeast GR, 200 μM NADPH, 1 mM glutathione, and indicated compounds at various concentrations for (A) 30 min and (B) 1 h was determined by after the absorbance at 340 nm after adding H₂O₂. All data points are means±SD of two independent experiments.

FIG. 4.

Effects of the DPPen and DPPro lead analogs on enzyme activities in HCT 116 and MCF-7 cells. Lysates of HCT 116 and MCF-7 cells treated with indicated concentrations of SG, DPPen, and DPPro analogs for ∼12 h were collected for enzyme activity and protein expression determination. TrxR (A), Trx (B), GR (C), and GPx (D) activities were expressed as a percentage of dimethyl sulphoxide control. All data points are means±SE of two to four independent experiments. *Statistically significant difference (p<0.05) in enzyme activity compared to control. (E) The cell lysates with equal protein were analyzed by Western blotting with indicated antibodies for TrxR and Trx expression. Western blot images are representative of at least two independent experiments.

FIG. 5.

Effects of the DPPen and DPPro lead analogs on the Trx-apoptosis signal-regulating kinase 1 (ASK1) interaction and apoptosis induction in HCT 116 cells. (A) Dose-dependent induction of apoptosis, indicated by presence of cleaved caspase 3 (17/19 kDa) and poly (ADP-ribose) polymerase (PARP) (89 kDa), after 12-h treatment with 2,2′-diOH-5,5′-diF-DPPen or 2-OH-5-F-DPPro. (B) Time-dependent changes in the redox state of Trx after treatment with 2,2′-diOH-5,5′-diF-DPPen or 2-OH-5-F-DPPro at a lethal concentration of 40 μM at indicated timepoints. (C) The collected lysates of ASK1-transfected cells treated or untreated for 8 h with 40 μM 2,2′-diOH-5,5′-diF-DPPen or 2-OH-5-F-DPPro were subjected with immunoprecipitation using an anti-human Trx antibody. Immunoprecipitates and aliquots of the cell lysates were analyzed by Western blotting with the indicated antibodies. The images shown are representative Western blot results of three independent experiments.

FIG. 6.

Irreversibility of TrxR inhibition and selenocysteine (Sec) residue targeting by the DPPen and DPPro lead analogs. (A) Oxidized recombinant rat TrxR (110 nM) in the presence or absence of 200 μM NADPH was incubated with different concentrations of 2,2′-diOH-5,5′-diF-DPPen or 2-OH-5-F-DPPro for 20 min, and the TrxR activity was determined by the DTNB assay. All data points are means of two independent experiments. (B) Recombinant rat TrxR (0.9 μM) was incubated with 20 μM of 2,2′-diOH-5,5′-diF-DPPen or 2-OH-5-F-DPPro for 20 min in a 50 mM Tris-HCl, 1 mN EDTA, pH 7.5 buffer containing 200 μM NADPH. The excess drug was removed by passing the protein through a NAP-5-desalting column, followed by determination of the activity of the eluted protein at indicated times using the DTNB assay. (C) Recombinant rat TrxR (0.9 μM) was incubated with 200 μM NADPH and 20 μM of 2,2′-diOH-5,5′-diF-DPPen or 2-OH-5-F-DPPro. At different timepoints, an aliquot of enzyme mixture was withdrawn for TrxR activity measurement by the DTNB assay and N-(Biotinoyl)-N′-(Iodoacetyl) Ethylenediamine (BIAM) labeling of Sec at pH 6.5 and 8.5. Top panel: time course of TrxR enzyme activity; middle panel: horseradish peroxidase (HRP)-conjugated streptavidin detection of BIAM labeling of free selenol at pH 6.5 at various incubation times; bottom panel: HRP-conjugated streptavidin detection of BIAM labeling of free selenol and sulfhydryls at pH 8.5 at various incubation times. Results shown are representative of three independent experiments.

FIG. 7.

Peptide mass analysis of TrxR-2,2′-diOH-5,5′-diF-DPPen protein adduct by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. NADPH-reduced recombinant rat TrxR (3 μM) was incubated at 37°C with or without 50 μM 2,2′-diOH-5,5′-diF-DPPen for 30 min, desalted to remove excess drug, denatured in urea, and trypsin-digested. Finally, MALDI mass spectrometry was performed to obtain the peptide mass spectrum of the (A) untreated and (B) 2,2′-diOH-5,5′-diF-DPPen-treated protein sample. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 8.

Proposed mechanism of inhibition of mammalian TrxR by 2,2′-diOH-5,5′-diF-DPPen. The electrophilic β-carbon atom in 2,2′-diOH-5,5′-diF-DPPen undergoes a Michael addition reaction with the Sec residue in NADPH-reduced TrxR to form a Michael acceptor adduct.

FIG. 9.

Molecular simulation of the interaction between C-terminal active site in TrxR with SGs, DPPen, and DPPro compounds. Molecular docking exercises were conducted for (A) 6-SG, (B) 3-Ph-3-SG, (C) 2,2′-diOH-5,5′-diF-DPPen, and (D) 2-OH-5-F-DPPro, with the ligand interaction maps of the top five poses obtained for each ligand presented in descending order.