Novel dual inhibitor targeting CDC25 and HDAC for treating triple-negative breast cancer

Abstract

Triple-negative breast cancer (TNBC) presents a significant challenge for treatment due to its aggressive nature and the lack of effective therapies. This study developed dual inhibitors against cell division cycle 25 (CDC25) and histone deacetylases (HDACs) for TNBC treatment. CDC25 phosphatases are crucial for activating cyclin-dependent kinases (CDKs), the master regulators of cell cycle progression. HDACs regulate various biological processes by deacetylating histone and non-histone proteins, affecting gene expression, chromatin structure, cell differentiation, and proliferation. Dysregulations of HDAC and CDC25 are associated with several human malignancies. We generated a group of dual inhibitors for CDC25 and HDAC by combining the molecular structures of CDC25 (quinoline-5,8-dione) and HDAC (hydroxamic acid or benzamide) pharmacophores. The newly developed compounds were evaluated against various solid-tumor, leukemia, and non-malignant breast epithelial cells. Among the synthesized compounds, 18A emerged as a potent inhibitor, demonstrating significant cytotoxicity against TNBC cells, superior to its effects on other cancer types while sparing non-malignant cells. 18A possessed similar HDAC inhibitory activity as MS-275 and potently suppressed CDC25 activity in vitro and the CDK1 dephosphorylation in cells. Additionally, 18A hindered the progression of S and G₂/M phases, triggered DNA damage, and induced apoptosis. These findings underscore the potential of 18A as a targeted therapy for TNBC and warrants further preclinical development.

Keywords: Apoptosis; CDC25 inhibitor; Dual-target; Genome instability; HDAC inhibitor; Triple-negative breast cancer.

Fig. 1

HDAC and CDC25 inhibitors. A Representative HDAC inhibitors, including long-chain hydroxamic acids (SAHA; 1), a cinnamoyl hydroxamic acid (Belinostat; 2), a natural cyclic depsipeptide (Romidepsin; 3), a phenyl hydroxamic acid (4), and an ortho-benzamide (Entinostat; 5), for the treatment of various cancers in clinical. B Chemical structures of quinone- and non-quinone-based CDC25 inhibitors (6–12)

Fig. 2

Design rationale of dual CDC25-HDAC inhibitors based on the chemical structure of quinoline-5, 8-dione as CDC25 inhibitor (NSC663284/6a), and hydroxamic acid/benzamide pharmacophores as HDAC inhibitors

Scheme 1

Synthesis of 13A, 13B, 14A, and 14B (Reagents and conditions): (a) Phenylboronic acid, Pd(PPh₃)₄, K₂CO₃, DMF, 110 ^oC, Inert (Ar), 16 h, (b) NBS, AICN, CCl₄, 90 ^oC, Inert (Ar), 5 h, (c) p-Phenylenediamine, DMF, 140 ^oC Reflux, o.n., (d) methyl 4-(bromomethyl)benzoate or 20, K₂CO₃, DMF, rt, 1–2 days, (e) LiOH (1 M), MeOH, 55 ^oC, o.n., (f) NH₂-OTHP, EDC.HCl, HOBt, NMM, DMF, rt, 3 h, (g) Hydrazine monohydrate (64%), Ethanol, Ar-gas, 60–65 ^oC, 3–4 h, (h) 6,7-dichloroquinoline-5,8-dione, THF, DIPEA, rt, 1 h, (i) 10% HCl in MeOH, 0 ^oC - rt, 3 h

Scheme 2

Synthesis of 15A, 15B, 16A, 16B, 17A, and 17B (Reagents and conditions): (a) NH₂-OTHP, EDC.HCl, HOBt, NMM, DMF, rt, o.n. (b) LiOH (1 M), MeOH, rt, o.n., (c) 38, or 39, or 40 respectively, EDC.HCl, HOBt, NMM, DMF, rt, 5–6 h (d) Hydrazine monohydrate (64%), Ethanol, Ar-gas, 60–65 ^oC, 3–4 h, (e) 6,7-dichloroquinoline-5,8-dione, DCM, DIPEA, rt, 1 h, (f) 10% HCl in MeOH, 0 ^oC - rt, 3 h

Scheme 3

Synthesis of 18A and 18B (Reagents and conditions): (a) Methyl 4-(bromomethyl) benzoate, K₂CO₃, DMF, rt, 5 h (b) LiOH 1.0 M in MeOH, 60 ^oC, 3 h, (c) o-Phenylenediamine, EDC.HCl, HOBt, NMM, DMF, rt, o.n., (d) 20% TFA in DCM, 0 ^oC-rt, 2 h, (e) 6,7-dichloroquinoline-5,8-dione, DIPEA, MeOH, rt, 7 h

Fig. 3

Cytotoxicity screening of synthesized compounds in triple-negative breast cancer and non-malignant mammary cells. A Cytotoxicity of synthesized compounds. MDA-MB-231 cells were treated with 8 µM of indicated compounds for 48 h, and the percentages of surviving cells were determined by the MTT assay. Results are shown as means with SDs from at least three independent experiments (n ≥ 9). B IC₅₀ values of compounds in MDA-MB-231 cells. Cells were treated with a series of concentrations of indicated compounds for 48 h. Surviving cells were analyzed by the MTT assay, and IC₅₀ values were calculated by the CompuSyn software and displayed with means and SDs from at least three biological replicates (n ≥ 9). C Cytotoxicity of 18A in TNBC and normal epithelium cells. MDA-MB-231, MDA-MB-436, MCF-7, and M10 cell lines were treated with 2 µM of indicated compounds for 48 h, and the percentages of surviving cells were determined by the MTT assay. Results are shown as means with SDs from at least three independent experiments (n ≥ 9). Relative surviving adherent cells were obtained by normalizing individual values to the mean of the corresponding untreated control group. Dot: Normalized values of technical repeats of each biological replicate labeled with the corresponding color. Triangle: Mean of each biological replicate. Line: Mean ± SDs of the means from biological replicates. Statistics: One sample t-test. *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001; n.s.: No significance

Fig. 4

HDAC-inhibitory activity of 18A in cells. Western blot analysis for acetylation levels of indicated proteins in MDA-MB-231 cells treated with various concentrations of indicated compounds for 6 h. The left side of the figure shows representative results from one of three biological repeats. The bar graphs on the right illustrate the quantification of intensities of the indicated protein acetylation. Relative intensities of acH3K9, acH4K5/8/12/16, and acSMC3K105/106 were obtained by normalizing with the corresponding total protein level of SMC3, while relative intensities of ac-α-Tubulin were obtained by normalizing with the total α-Tubulin level. The results are compared to the untreated group and are presented as the means ± SEMs of three independent experiments. There were no significant differences between MS-275- and 18A-treated groups for acH3K9 and acH4K5/8/12/16 at the same concentration (One-way ANOVA). No considerable changes were observed between the control and treated groups for acSMC3K105/106 and ac-α-Tubulin (One sample t-test)

Fig. 5

18A treatment prevents CDK1 dephosphorylation and mitotic signaling activation. A Western blot analysis for CDK1 phosphorylation and mitotic signaling in thymidine-synchronized MDA-MB-231 cells co-treated with 100 ng/ml of nocodazole and various concentrations of indicated compounds. Experimental design (left upper), representative cell cycle profiles (right), and western blot results (left lower) from one of three biological replicates are shown. Asyn: Asynchronous; Cpd: Compound; Noco: Nocodazole. B Quantification of intensities of the indicated proteins. Relative intensities of CDK1pY15, H3pS10, CyclinB1pS126, and MPM2 were obtained by normalizing with the corresponding total protein level of histone H3. The results are compared to the asynchronous sample and are presented as the means ± SEMs of three independent experiments. Statistics: One-way ANOVA. *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001; n.s.: No significance

Fig. 6

18A treatment impairs cell cycle progression. A Analysis of cell cycle progression of thymidine-synchronized MDA-MB-231 cells treated with various concentrations of indicated compounds. Experimental design (upper) and representative results from one of two biological replicates (lower) are shown. Asyn: Asynchronous. B Immunofluorescence analysis of DNA replication efficiency of MDA-MB-231 cells treated with 5 µM of indicated compounds for 6 h. Representative images from the high-content microscope (upper) and quantification results of three independent experiments (lower) are shown. Relative EdU intensities in replicating cells positive to PCNA were obtained by normalizing individual values to the median of the corresponding untreated control group. Dot: Normalized values of individual cells from each biological replicate labeled with corresponding colors. Triangle: Median of each biological replicate. Line: Mean ± SDs of the medians from biological replicates. Statistics: One sample t-test. **: P < 0.01; ***: P < 0.001; n.s.: No significance

Fig. 7

18A triggers DNA damage. A Immunofluorescence analysis of DNA damage response of MDA-MB-231 cells treated with 5 µM of indicated compounds for 6 h. Representative images from the high-content microscope (upper) and quantification results of three independent experiments (lower) are shown. Relative H2AX phosphorylation at S126 (γH2AX) was obtained by normalizing individual values to the median of the corresponding untreated control group. Dot: Normalized values of individual cells from each biological replicate labeled with corresponding colors. Triangle: Median of each biological replicate. Line: Mean ± SDs of the medians from biological replicates. Statistics: One sample t-test. *: P < 0.05; **: P < 0.01; ***: P < 0.001; n.s.: No significance. B Western blot analysis of DNA damage response in MDA-MB-231 cells treated with indicated concentrations of 18A, 6a, or MS-275 for 6 h. Representative blot and quantification results from one of two biological replicates are shown. Relative intensities were obtained by normalizing with the corresponding total protein level of histone H2A, and the results are compared to the untreated control

Fig. 8

18A treatment triggers apoptosis. A Analysis of cell cycle profiles of MDA-MB-231 cells treated with indicated concentrations of MS-275, 6a, and 18A for 48 h. Representative profiles and percentages of the sub-G₁ population from one of two biological replicates are shown. B Analysis of apoptotic proteins in MDA-MB-231 cells treated with 2 µM of 18A, 6a, and MS-275 or for 48 h. Representative blot and quantification results from one of two biological replicates are shown. Relative intensities were obtained by normalizing with the corresponding total protein level of actin, and the results are compared to the untreated control. Data were collected from different sets of gel electrophoresis with equal loading of the same samples. p-Casp: precursor caspase; a-Casp: active caspase. * indicates a non-specific band