Design, synthesis, and biological evaluation of novel EF24 and EF31 analogs as potential IκB kinase β inhibitors for the treatment of pancreatic cancer

Figures & data

Figure 1 Chemical structures of some reported IKKβ inhibitors.

Abbreviation: IKKβ, inhibitory kappa B kinase β.

Figure 2 General synthesis route for the four series of compounds.

Figure 3 Inhibition of IKKβ kinase by the (A–D) series of analogs of EF24 and EF31 at a concentration of 20 μM was screened through a Caliper mobility shift assay.

Note: IR^a refers to the inhibitory ratio of the measured compounds at a concentration of 20 μM.
Abbreviations: IKKβ, inhibitory kappa B kinase β; IR, inhibitory ratio; Me, methyl; OMe, methoxy.

Figure 4 Direct-binding affinity between D6 and IKKβ demonstrated by SPR. Note: K_a, K_d, equilibrium dissociation constant.

Abbreviations: IKKβ, inhibitory kappa B kinase β; KD, kinase domain; RU, response units; SPR, surface plasmon resonance.

Figure 5 Molecular docking and MD simulation analysis of binding of D6 to the activity cavity of IKKβ.

Notes: (A) Backbone RMSDs are shown as a function of time for IKKβ/D6 in 60 ns MD simulation. (B) Per-residue value of the top ten contributions to the binding effective energy of D6. Per-residue contributions were calculated by the MM-GBSA decomposition method. (C) Last snapshot of IKKβ/D6 in 60 ns MD simulation.
Abbreviations: IKKβ, inhibitory kappa B kinase β; MD, molecular dynamics; MM-GBSA, molecular mechanics/generalized Born surface area; RMSD, root-mean-square deviation.

Table 1 Six compounds (B1, B2, B4, C6, D3, D6, and CUR) were selected for determining the IC₅₀ values of IKKβ kinase activity, and the active compounds B1, B2, B4, and D6 (IKKβ IC₅₀ <10 μM) were further measured for the antiproliferative effects in three PC cell lines (PANC-1, MiaPaCa-2, and BxPC-3) by the well-established MTT assay

Compound	IC50 (µM)
	IKKβ	PANC-1	MiaPaCa-2	BxPC-3
B1	8.9±1.1	12.8±6.9	11.8±1.8	21.5±13.3
B2	5.6±0.5	9.6±2.7	10.6±4.3	12.0±0.9
B4	3.4±1.4	8.2±0.9	6.3±1.3	5.2±1.1
C6	12.4±5.6	ND^a	ND^a	ND^a
D3	>20	ND^a	ND^a	ND^a
D6	0.8±0.3	0.7±1.1	1.3±0.4	1.9±1.3
EF31	1.92^b	1.7±1.8	5.0±0.5	2.1±0.9
EF24	1.9^b	2.8±3.0	4.2±1.5	5.9±5.9
CUR	20.5±1.5	21.5±17.3	24.8±7.4	17.9±2.3

Notes: The test results at both enzyme and cell levels are shown. The lead compounds EF31, EF24, and CUR are used for comparison. 1.92^a and 1.9^a were the IC₅₀ values of EF31 and EF24, respectively, against IKKβ.^31,32 All data are presented as the mean value of three to five independent tests. ND^a means not determined; 1.92^b and 1.9^b are the IC₅₀ values in the previous reported reference. The bold values represent the favorable inhibitory activity of two most active derivatives of EF24 and EF31.

Abbreviations: CUR, curcumin; IC₅₀, half-maximal inhibitory concentration; IKKβ, inhibitory kappa B kinase β; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ND, not determined.

Figure 6 Compound D6 suppressed TNF-α induced phosphorylation of IKKβ and degradation of the downstream signal molecule IκB in a dose-dependent manner.

Notes: (A) PANC-1 cells were incubated with the compounds D6 (1, 2.5, and 5 μM), EF31 (5 μM), or DMSO. After stimulation with TNF-α (1 ng/mL) for 30 min, the protein levels of pIKKβ and IκB were detected by Western blotting with the corresponding total IKKβ protein or GAPDH as loading control. (B and C) The columns show the normalized optical density as a percentage of the control. Bars represent the mean ± SEM of three to four independent experiments. **P<0.01; ***P<0.001; “ns” means no significance.
Abbreviations: DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IKKβ, inhibitory kappa B kinase β; pIKKβ, phosphorylated IKKβ; SEM, standard error of the mean; TNF, tumor necrosis factor.

Figure 7 Compound D6 induced PC cell apoptosis.

Notes: (A) Hoechst staining was performed in PANC-1 cells cultured with (a) DMSO, (b and c) D6 (2.5 and 5 μM) or (d) EF31 (5 μM) for 12 h (200×). (B) Effects of D6 on the activation of PARP and inhibition of Bcl-2 in PANC-1 and BxPC-3 cells. (C–F) The quantitative analysis of cleaved PARP and Bcl-2 band was performed using ImageJ image processing program. Data are presented as the mean ± SD of three independent experiments performed in triplicate: *P<0.05; **P<0.01; ***P<0.001; “ns” means no significance.
Abbreviations: DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; SD, standard deviation.

Scheme 1 The design of (A–D) series of derivatives of EF24 and EF31.

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