Synthesis, inverse docking-assisted identification and in vitro biological characterization of Flavonol-based analogs of fisetin as c-Kit, CDK2 and mTOR inhibitors against melanoma and non-melanoma skin cancers

Abstract

Due to hurdles, including resistance, adverse effects, and poor bioavailability, among others linked with existing therapies, there is an urgent unmet need to devise new, safe, and more effective treatment modalities for skin cancers. Herein, a series of flavonol-based derivatives of fisetin, a plant-based flavonoid identified as an anti-tumorigenic agent targeting the mammalian targets of rapamycin (mTOR)-regulated pathways, were synthesized and fully characterized. New potential inhibitors of receptor tyrosine kinases (c-KITs), cyclin-dependent kinase-2 (CDK2), and mTOR, representing attractive therapeutic targets for melanoma and non-melanoma skin cancers (NMSCs) treatment, were identified using inverse-docking, in vitro kinase activity and various cell-based anticancer screening assays. Eleven compounds exhibited significant inhibitory activities greater than the parent molecule against four human skin cancer cell lines, including melanoma (A375 and SK-Mel-28) and NMSCs (A431 and UWBCC1), with IC₅₀ values ranging from 0.12 to < 15 μM. Seven compounds were identified as potentially potent single, dual or multi-kinase c-KITs, CDK2, and mTOR kinase inhibitors after inverse-docking and screening against twelve known cancer targets, followed by kinase activity profiling. Moreover, the potent compound F20, and the multi-kinase F9 and F17 targeted compounds, markedly decreased scratch wound closure, colony formation, and heightened expression levels of key cancer-promoting pathway molecular targets c-Kit, CDK2, and mTOR. In addition, these compounds downregulated Bcl-2 levels and upregulated Bax and cleaved caspase-3/7/8 and PARP levels, thus inducing apoptosis of A375 and A431 cells in a dose-dependent manner. Overall, compounds F20, F9 and F17, were identified as promising c-Kit, CDK2 and mTOR inhibitors, worthy of further investigation as therapeutics, or as adjuvants to standard therapies for the control of melanoma and NMSCs.

Keywords: Anticancer activity; Apoptosis; Fisetin-analogs; Flavonols; Inverse docking; Kinase activity; Melanoma; Non-melanoma skin cancer.

Figure 1.

List of fisetin analogs designed and synthesized (*% yields from the one-pot reactions).

Figure 2.

3-D interaction representations for the flavonol analogs superimposed over fisetin within the binding cavity of target enzymes a) EGFR b) cKIT c) Akt d) MET e) MEK1 f) VEGFR g) MAPK h) mTOR i) PI3K j) PIP5K1α k) CDK2 l) FGFR.

Figure 2.

3-D interaction representations for the flavonol analogs superimposed over fisetin within the binding cavity of target enzymes a) EGFR b) cKIT c) Akt d) MET e) MEK1 f) VEGFR g) MAPK h) mTOR i) PI3K j) PIP5K1α k) CDK2 l) FGFR.

Figure 3.. Potent flavonol F20 induces apoptosis through activation of the extrinsic and intrinsic apoptotic pathway caspases in melanoma and non-melanoma cancer cells.

The blots show the effect of F20 at IC₅₀ and 2×IC₅₀ concentrations on the protein expression levels of markers of apoptosis, including pro- and-cleaved caspase−3, −7 and −8, components of the intrinsic apoptosis pathway, after 48-h treatment, comparing with untreated controls. A375 and A431 cells were incubated in the absence or presence of F20 (0, IC₅₀, 2×IC₅₀; 48 h), and whole-cell lysates were subjected to SDS-polyacrylamide gel electrophoresis. The Western blot data shown are representative immunoblots from three independent experiments with similar results. Equal protein loading was confirmed by re-probing for β-actin or GAPDH as loading control, and the actual protein levels were normalized to the loading control to obtain % Expression values shown on the bar graphs (mean ± SD of relative quantitative density values are plotted). The data are expressed as the percentage to which derivatives F20 suppressed or induced the protein expression levels of pro- and-cleaved caspases in A375 and A431 cells as compared with controls. Statistical significance was assessed using one-way ANOVA and Dunn’s multiple comparison tests, and p < 0.05 (*) was considered significant.

Figure 4.. Potent flavonol F20 induces apoptosis through activation of mitochondrial pro-apoptotic pathways involving Bcl-2 family proteins and of PARP cleavage in melanoma and non-melanoma cancer cells.

The Western blot data show the effects of F20 at IC₅₀ and 2×IC₅₀ concentrations for 48 h on the protein expression levels of intact and cleaved PARP (85 kDa), as well as Bcl-2 family of proteins (Bax and Bcl-2), components of the intrinsic apoptosis pathway, in A375 and A431 cells. A375 and A431 cells were incubated in the absence or presence of F20 (0, IC₅₀, 2×IC₅₀; 48 h), and whole-cell lysates were subjected to SDS-polyacrylamide gel electrophoresis. The blots shown are representative of immunoblots from three independent experiments with similar results. The data are expressed as the percentage suppression of the protein expression levels of Bcl-2 and cleaved PARP (116 kDa), and percentage increase in levels of cleaved PARP ((85 kDa)) and of Bax, in treated A375 and A431 cells as compared with controls. Equal protein loading was confirmed by re-probing for β-actin as loading control, and the actual protein levels were normalized to the loading control to obtain % Expression values shown on the bar graphs (mean ± SD of relative quantitative density values). Statistical significance was assessed using one-way ANOVA and Dunn’s multiple comparison tests, and p < 0.05 (*) was considered significant.

Figure 5:. Flavonol analogs inhibit the protein expression levels of cyclin A/E, CDK2 and phosphorylated c-Kit and Stat3 in A375 melanoma and A431 non-melanoma cancer cells.

(A) Western blot data showing the effect of different concentrations of F20 on protein expression levels of cyclin A2, cyclin E2, CDK2, c-Kit, and phosphorylated c-Kit and Stat3 in 48 h-treated A375 (left panel) and A431 (right panel) cells. A375 and A431 cells were incubated with/without F20; 0, IC₅₀, 2×IC₅₀; μM, 48h), and western blotting performed as described in the method section. Equal protein loading was confirmed by reprobing for GAPDH as loading control, and protein levels were normalized to the loading control and expressed as percentage. The blot data shown are representative of immunoblots of three independent experiments with similar results. (B) The data expressed in the bar graphs represent mean ± SD of relative quantitative normalized density values in percentage with an internal loading control from three independent experiments. Analog F20 significantly suppressed the protein expression levels of these in A375 and A431 as compared with untreated control cells. Bar graphs, the statistical significance was determined using one-way ANOVA and Dunn’s multiple comparison test, and p < 0.05 (*) was considered significant.

Figure 6:. Potent flavonol analogs inhibit the protein expressions of activated c-Kit and downstream effectors levels, including phosphorylated mTOR/ ribosomal protein S6/ MAPK (ERK1/2) and p90RSK in A375 melanoma and A431 non-melanoma cells.

(A) Western blots displays are representatives of c-Kit downstream effectors the protein expression levels of phosphorylated mTOR, ribosomal protein S6, MAPK p44/42(ERK1/2) and p90RSK, in 48h treated A375 (left panel) and A431 (right panel) cells. A375/A431 cells were incubated with/without test compounds (F20; 0, IC₅₀, 2×IC₅₀; μM, 48h), and western blotting was carried out as described in the Methods section. Equal protein loading was confirmed by reprobing for Rab11 as loading control, and protein levels were normalized to the loading control and expressed as percentage. The data shown are representative of immunoblot of three independent experiments with similar results. (B) The data expressed in the bar graphs represent mean ± SD of relative quantitative normalized density values in percentage with an internal loading control. The analogs F20 significantly suppressed the protein expression levels of the phorphorylated form of the targets in A375 and A431 as compared with untreated control cells. Statistical significance was determined using one-way ANOVA and Dunn’s multiple comparison test, and p < 0.05 (*) was considered significant.

Figure 7:. The most-potent analog, F20, significantly inhibited Scratch wound healing in 2D cultures of A375 and A431 in a dose-dependent manner.

Effect of the flavonol analog F20 on the closure/migration into the initial cell-free scratch-wound areas compared to the percentage of control cell-free areas (A) (Upper panel) A431 and (lower panel) A375. (B) The data presented indicate significant dose-dependent decrease in cultured cells repopulating the scratch wound healing area in the presence of F20, as compared to untreated control cells, after 48 h of incubation. The data in the bar graphs represent mean ± SD of scratched-wound area values, expressed as percentage, compared to 0 h control and 48 h controls from three independent experiments. F20 significantly decreased wound healing area A431 (left panel) and A375 (right panel) as compared with untreated controls. Statistical significance was assessed using one-way ANOVA and Dunn’s multiple comparison test, and p < 0.05 (*) was considered significant.

Figure 8:. Potent analog F20 significantly inhibits colony formation in 2D cultures of A375 and A431 cells in a dose-dependent manner.

(A-B) Long-term effect of the most potent flavonol, F20, on clonogenic potential in A375, and A431 cells. Treatment with derivative F20 (½IC₅₀, IC₅₀ and 2×IC₅₀) of the respective cell lines, significantly reduced/suppressed the percentage of colonies formed in a dose-dependent manner when compared to the respective control untreated cutaneous carcinoma cells. (B) The data expressed in the bar graphs represent mean ± SD of values in percentage control from three-independent experiments. Statistical significance was assessed using one-way ANOVA and Bonferoni’s multiple comparison test; p < 0.05 (*) was considered significant.

Scheme 1.

Microwave assisted synthesis of 2’-hydroxychalcones

Scheme 2.

Synthesis of substituted flavonols via oxidative cyclization of 2’-hydroxychalcones at room temperature.

Scheme 3.

Microwave-assisted one-pot synthesis of substituted flavonol derivatives.