Discovery of a Novel, Isothiazolonaphthoquinone-Based Small Molecule Activator of FOXO Nuclear-Cytoplasmic Shuttling

Abstract

FOXO factors are tumour suppressor proteins commonly inactivated in human tumours by posttranslational modifications. Furthermore, genetic variation within the FOXO3a gene is consistently associated with human longevity. Therefore, the pharmacological activation of FOXO proteins is considered as an attractive therapeutic approach to treat cancer and age-related diseases. In order to identify agents capable of activating FOXOs, we tested a collection of small chemical compounds using image-based high content screening technology. Here, we report the discovery of LOM612 (compound 1a), a newly synthesized isothiazolonaphthoquinone as a potent FOXO relocator. Compound 1a induces nuclear translocation of a FOXO3a reporter protein as well as endogenous FOXO3a and FOXO1 in U2OS cells in a dose-dependent manner. This activity does not affect the subcellular localization of other cellular proteins including NFkB or inhibit CRM1-mediated nuclear export. Furthermore, compound 1a shows a potent antiproliferative effect in human cancer cell lines.

Fig 1. Screening strategy to identify specific and potent FOXO translocator compounds.

As a primary filter a FOXO translocation assay has been used, followed by dose response analysis of the confirmed hit compounds. The effect of the hit compounds on endogenous FOXO proteins has been assessed by immunohistochemistry. Then inhibitors of the general nuclear export machinery were excluded using a nuclear export assay. Finally, the effect on other transcription factors has been determined by immunohistochemistry using a NFKB2 specific antibody.

Fig 2. Synthesis of compounds 1a-c (LOM612/621/604).

(a) chlorocarbonylsulphenyl chloride, N, N-dimethylurea or N,N-dibenzylurea, acetonitrile, 2h RT for 3a: 84%, 3b: 62%; chlorocarbonylsulphenyl chloride, benzamide, toluene, 3h, reflux 3c: 70%; (b) 1,4-naftochinone, 3a, xilene, 80°C, 3h, 23%; 1,4-naftochinone, 3b, xylene, 80°C, 3h, 63%; (C) 1,4-naftochinone, 3c, xilene, 8h, reflux, 3h, 38%; d) CAN, CH₃CN: H₂O 9:1, 1h, RT, 89%.

Fig 3. Primary screening identifies compounds capable of inducing FOXO translocation.

(A) U2fox RELOC cells were treated either with DMSO, 4nM LMB, 10μM of compound LOM 612 or compound LOM 621 for 30 min. representative images are shown. (B) Dose-response relationship of the nuclear–cytoplasmic shuttling of FOXO following LOM 612 treatment. LOM612 induces nuclear translocation in a dose dependent manner. Represented is the percentage of cells with more GFP fluorescence accumulation in nucleus than in cytoplasm. Results represent the mean of three independent experiments.

Fig 4. LOM612 specifically induces the nuclear translocation of endogenous FOXO proteins.

(A) Compound LOM612 induces the nuclear translocation of endogenous FOXO3a and FOXO1 protein detected by using a specific antibodies after 30 min of drug exposure. (B) LOM612 does not inhibit the nuclear export. U2OS cells stably expressing nuclear export signal (NES) (Rev-NES-EGFP) reporter were treated with DMSO, LMB, LOM612 and LOM621 for 30 min. (C) LOM612 does not induce nuclear translocation of endogenous nuclear factor (NF)–κB2 protein. Representative images of the compound-treated cells using a Leica SPE confocal imaging system. Cells were seeded automatically at appropriate density in 96-well black-wall clear-bottom tissue culture plates and allowed to attach overnight. Cells were then treated with compounds for 30 min before paraformaldehyde (Rev-NES-EGFP) or methanol (FOXO3a, NFKB2) fixation and DAPI staining.

Fig 5. LOM612 compromises the viability of human cancer cell lines.

(A) Breast cancer cell line MCF7, melanoma cell line A2058 and the neuroblastoma cell line SH-SY5 were seeded at a concentration of 1× 10⁴ cells/well in 200 μl and treated with compounds LOM612 and LOM621 for 72 hours with eight 2-fold serial dilutions of each compound spanning concentrations from 50μM to 0.39μM. Data is shown as mean ± SEM of three independent experiments. ****P<0.0001 by two-way ANOVA (ns, not significant). (B) Human liver cancer cell lines HepG2 and THLE-2 (cell line derived from primary normal liver epithelial cells) were seeded at a concentration of 1× 10⁴ cells/well in 200 μl and treated with 20 different concentrations of LOM612 from 50μM to 95pM. Data is shown as mean ± SEM of three independent experiments. ****P<0.0001 by two-way ANOVA.