Identification of novel inhibitors of the transcriptional coactivator MRTF-A for HCC therapy

Abstract

Myocardin-related transcription factor A (MRTF-A) is a coactivator of serum response factor (SRF), which regulates the expression of genes involved in cell proliferation, migration, and differentiation and has been implicated in hepatocellular carcinoma (HCC) progression. We recently established inhibition of the transcriptional activity of MRTF-A by NS8593 as a novel therapeutic approach for HCC therapy. NS8593 is a negative gating modulator of the transient receptor potential cation channel TRPM7. In this report, we identify an aminobenzimidazole that is highly potent in inhibiting TRPM7 and its interaction with RhoA, leading to decreased SRF transcriptional activity and enhanced nuclear export of MRTF-A, as determined by fluorescence loss in photobleaching (FLIP). This resulted in reduced expression of the MRTF/SRF target genes transforming growth factor β1 (TGF-β1) and tetraspanin 5 (TSPAN5), senescence induction, and growth arrest in HCC cells. Replacement of the tetraline core by a 3-aminophenyl substructure yielded inhibitor 10 with higher potency than inhibitor 5, and further structural modifications yielded highly potent inhibitors of SRF activity, 14 and 16. Both compounds were capable of inhibiting cell proliferation and inducing senescence in HCC cells with improved efficacy compared to NS8593. These inhibitors represent valuable tools for understanding the molecular basis of drug development targeting TRPM7 and MRTFs.

Keywords: HCC; MKL1; MRTF; MT: Regular Issue; NS8593; RhoA; SK; SRF; TGF-ß1; TRPM7; TSPAN5.

Graphical abstract

Figure 1

Synthesis of target compounds 1–8 and determination of concentration dependencies for inhibition of TRPM7 and cell proliferation (A) Synthesis of target compounds 1–8. Reagents and conditions: (i) titanium(IV) isopropoxide, NaB(O₂CCH₃)₃, THF, rt, N₂, 38%–58%; (ii) 6,7-dihydroisoquinolin-8(5H)-one, titanium(IV) isopropoxide, NaB(O₂CCH₃)₃, THF, rt, N₂, 34%; (iii) 4-keto-4,5,6,7-tetrahydrothianaphthene, titanium(IV) isopropoxide, NaB(O₂CCH₃)₃, THF, rt, N₂, 41%. (B) Concentration dependencies for inhibition of TRPM7 by NS8593 (NS), rNS (1) and 2–8 generated by the Ca²⁺ influx assay. Curves through the points (mean ± SEM) are logistic equation fits. The values of IC₅₀ and hill slopes are provided in Table S1. (C) TRPM7 and SK3 channel affinities (taken from Sørensen et al.²²) of NS derivatives compared to NS. (D and E) Proliferation rates in HuH7 cells treated with (D) 30 μM NS, rNS, 2, 3, 7, 8, and DMSO as a control and (E) 10 μM NS, 4, 5, 6, and DMSO. Data are means ± SD (n = 3); ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (F) Concentration dependencies for inhibition of HuH7 cell proliferation upon administration of 2.5, 5, 7.5, and 10 μM compound 5. Data are means ± SD (n = 3); ∗p < 0.05, ∗∗p < 0.01.

Figure 2

Inhibition of TRPM7 by NS and analogs induces cellular senescence (A–C) Quantification of SA-β-Gal-positive HuH7 cells treated with compounds 5 (A), 8 (B), and rNS (C) as indicated and DMSO as a control (left). β-Gal-positive cells were counted in 100 cells per condition. All data are means ± SD (n = 3); ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, n.s. not significant. Shown are representative pictures of SA-β-gal staining with 5 (5 μM), rNS, and 8 (30 μM) (right). (D) Immunofluorescence staining with anti-PML antibody and DAPI for nuclear counterstaining in HuH7 cells treated with inhibitor of 5 (5 μM), 8, and rNS (30 μM) and DMSO as a control. Shown is quantification of PML nuclear body accumulation in 100 cells per condition. Scale bar, 10 μm. Data are means ± SD (n = 3); ∗∗p < 0.01. (E) HuH7 cells were treated as described in (D) and subjected to Matrigel invasion assay chambers, and after 20 h, invaded cells were stained by crystal violet and counted. All data are means ± SD (n = 3); ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 3

Impairment of TRPM7/RhoA interaction leads to enhanced MRTF-A nuclear export (A) Immunofluorescence analysis and quantification of proximity ligation assay (PLA) for endogenous TRPM7 and RhoA in HuH7 cells treated for 6 h with compounds 5 (5 μM) and 8 and rNS (30 μM). Scale bar, 10 μm. PLA signals were counted in 15 cells per condition. Data are means ± SD (n = 3); ∗p < 0.05, ∗∗∗p < 0.001. (B) Immunofluorescence staining with anti-MRTF-A antibody and DAPI for nuclear counterstaining in HuH7 cells treated as above. Scale bar, 10 μm. Data are means ± SD (n = 3); ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (C) Fluorescence loss curves from the FLIP assay, representing nuclear export of HuH7 cells treated with compound 5 (5 μM) and NS (30 μM). Data are mean (N ≥ 8 cells per condition), normalized to pre-bleach ±SD. (D) Fluorescence remaining in the nucleus after 80 s in the FLIP assay. Data were evaluated with a Mann-Whitney test; ∗∗∗p < 0.001.

Figure 4

Novel inhibitor 5 reduces SRF activity and MRTF/SRF target gene expression (A) HuH7 cells expressing an SRE-dependent luciferase reporter gene (5×SRE) and a Renilla luciferase internal control (pRL-SV40P) were treated with 30 μM 5, 8, rNS, or DMSO as a control, and 24 h later, luciferase assays were performed for firefly luciferase and normalized to Renilla luciferase. Data are means ± SD (n = 3); ∗∗∗p < 0.001. (B) HuH7 cells were treated as above. A concentration of 5 μM was used. Data are means ± SD (n = 3); ∗∗p < 0.01, ∗∗∗p < 0.001, n.s. not significant. (C) Immunoblotting (left) for TSPAN5, TGF-β1, and HSP90 as a loading control and quantification (right) of HuH7 cells treated with compound 5 as indicated. Data are means ± SD (n = 3); ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, n.s. not significant. (D) Immunoblotting (left) for TSPAN5 and HSP90 as a loading control and quantification (right) of lysates of HuH7 cells treated with 8 and DMSO as a control group with anti-TSPAN5 and anti-HSP90 antibody. Data are means ± SD (n = 3); ∗p < 0.05, ∗∗p < 0.01, n.s. not significant.

Figure 5

Constructing novel inhibitors of MRTF/SRF activity based on compound 5 (A) Docking of NS using the cryo-EM structure of TRPM7 (PDB: 8SIA) and AutoDockVina. (B) Structure of NS and orientations A and B for substitution on the tetraline core. (C) Aminobiphenyl derivatives incorporating a 2- or 3-aminobiphenyl substructure (attachment of rings D or C). (D) The biphenyl inhibitors 9–21 were synthesized from the corresponding 2- or 3-aminobiphenyls, which were prepared previously by either a radical Gomberg-Bachmann reaction or a Suzuki cross-coupling reaction.^, The inhibitors 9–21 were then obtained through a nucleophilic aromatic substitution of 2-chlorobenzimidazole with the respective aminobiphenyl applying microwave irradiation. The desired target compounds 9–21 were isolated in yields ranging from 49%–87%. (E) HuH7 cells expressing 5xSRE and pRL-SV40P were treated with 10 μM 9–13 or DMSO as a control, and 24 h later, luciferase assays were performed for firefly luciferase and normalized to Renilla luciferase. Data are means ± SD (n = 3); ∗∗p < 0.01, ∗∗∗p < 0.001, n.s. not significant. (F) Immunofluorescence staining with anti-MRTF-A antibody and DAPI for nuclear counterstaining in HuH7 cells treated for 18 h with 5 μM 10 and DMSO as a control group. Scale bar, 10 μm. Data are means ± SD (n = 3); ∗∗∗p < 0.001.

Figure 6

Constructing novel inhibitors of MRTF/SRF activity based on compound 10 (A) Structures of compounds 14–21. The synthesis was performed according to Figure 5D. (B) Proliferation rates in HuH7 cells treated for 5 days with 10 μM 10 and 14–21 and DMSO as a control group. Data are means ± SD (n = 3); ∗∗p < 0.01, ∗∗∗p < 0.001. (C) Proliferation rates in HuH7 cells treated with 10 μM 14 and 16 and DMSO as a control group. Data are means ± SD (n = 3); ∗∗∗p < 0.001. (D) Concentration dependencies for inhibition of HuH7 cell proliferation upon administration of 2.5, 5, 7.5, and 10 μM compounds 14 and 16. Data are means ± SD (n = 3); ∗∗p < 0.01, ∗∗∗p < 0.001. (E) Quantification of SA-β-Gal-positive HuH7 cells treated with compounds 14 and 16 as indicated and DMSO as a control (left). β-Gal-positive cells were counted in 100 cells per condition. All data are means ± SD (n = 3); ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, n.s. not significant. Representative pictures of SA-β-gal staining with 14 and 16 (5 μM) (right). (F) Immunofluorescence staining with anti-PML antibody and DAPI for nuclear counterstaining in HuH7 cells treated with 14 and 16 and DMSO as a control. Quantification of PML nuclear body accumulation in 100 cells per condition. Scale bar, 10 μm. Data are means ± SD (n = 3); ∗p < 0.05, n.s. not significant.

Figure 7

Compounds 14 and 16 inhibit MRTF/SRF activity (A) HuH7 cells expressing 5×SRE and pRL-SV40P were treated with compounds 14 and 16 or DMSO as a control, and 24 h later, luciferase assays were performed for firefly luciferase and normalized to Renilla luciferase. Data are means ± SD (n = 3); ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (B) HuH7 cells were treated as above. A concentration of 2.5 μM of 5, 14, and 16 was used. Data are means ± SD (n = 3); ∗p < 0.05, ∗∗p < 0.01, n.s. not significant. (C) Immunofluorescence staining with anti-MRTF-A antibody and DAPI for nuclear counterstaining in HuH7 cells treated for 18 h with 5 μM 14 and 16 and DMSO as a control group. Scale bar, 10 μm. Data are means ± SD (n = 3); ∗∗∗p < 0.001. (D) The model for MRTF/SRF inhibition by NS and derivatives. TRPM7 blockade inhibits RhoA activation, which leads to disassembly of actin stress fibers and an increase in G-actin levels. Export of G-actin-bound MRTF-A reduces the amount of MRTF-A available to bind SRF and activate transcription of target genes such as TSPAN5 and TGF-β1, thereby reducing proliferation and inducing senescence.