MMB triazole analogs are potent NF-κB inhibitors and anti-cancer agents against both hematological and solid tumor cells

Abstract

Triazole derivatives of melampomagnolide B (MMB) have been synthesized via click chemistry methodologies and screened against a panel of 60 human cancer cell lines. Several derivatives showed promising anti-cancer activity, affording growth inhibition (GI₅₀) values in the nanomolar range (GI₅₀ = 0.02-0.99 μM). Lead compound 7h exhibited EC₅₀ values of 400 nM and 700 nM, respectively, against two AML clinical specimens. Compound 7h was significantly more potent than parthenolide as an inhibitor of p65 phosphorylation in both hematological and solid tumor cell lines, indicating its ability to inhibit the NF-κB pathway. In TMD-231 breast cancer cells, treatment with 7h reduced DNA binding activity of NF-κB through inhibition of IKK-β mediated p65 phosphorylation and caused elevation of basal IκBα levels through inhibition of constitutive IκBα turnover and NF-κB activation. Molecular docking and dynamic modeling studies indicated that 7h interacts with the kinase domain of the monomeric IKKβ subunit, leading to inhibition of IKKβ activation, and compromising phosphorylation of downstream targets of the NF-κB pathway; dynamic modeling studies show that this interaction also causes unwinding of the α-helix of the NEMO binding site on IKKβ. Molecular docking studies with 10, a water-soluble analog of 7h, demonstrate that this analog interacts with the dimerization/oligomerization domain of monomeric IKKβ and may inhibit oligomer formation and subsequent autophosphorylation. Sesquiterpene lactones 7h and 10 are considered ideal candidates for potential clinical development.

Keywords: Click chemistry; Leukemia cell lines; Melampomagnolide B; NF-κB pathway; Triazole derivatives.

Figure 1:

The anti-cancer sesquiterpene lactones parthenolide (PTL), DMAPT, melampomagnolide B (MMB), and MMB carbonate and carbamate dimers 1 and 2, respectively.

Figure 2:

Proposed mechanism of action of the sesquiterpene lactones parthenolide (PTL), DMAPT and melampomagnolide B (MMB): A primary mechanism involves inhibition of IKKβ activation, causing decreased phosphorylation of IkB and/or p65, resulting in down-regulation of anti-apoptotic gene transcription and sensitization of cancer cells to apoptotic signals. A secondary synergistic mechanism involves inhibition of the mitochondrial glutathione system, causing an increase in reactive oxygen species (ROS) and oxidative stress leading to mitochondrial dysfunction and activation of intrinsic apoptosis in cancer cells.

Figure 3:

Comparative anti-leukemic activity of PTL, DMAPT, MMB, and compounds 7a-7s, 9a-9c and 10 against M9 ENL-1 cells

Figure 4:

Comparative anti-leukemic activity of PTL, DMAPT, MMB, and compounds 7d-7h, 7j, 7l and 10, against primary leukemia cells from two AML patients.

Fig. 5.

Inhibition of p65 phosphorylation by PTL and 7h in primary leukemia cells (AML#1).

Figure 6:

Effect of PTL (2.5, 5 and 10 μM), 7h (0.625, 1.25, 2.5 and 5 μM), and 10 (0.625, 1.25, 2.5 and 5 μM) on GSH and GSSG levels in primary leukemia cells (AML#2).

Figure 7:

Anti-cancer activity if MMB triazole analog 7h against HSR-GBM1 stem-like gliobastoma cells. (One-Way ANOVA: ****p<0.001; ns = not significant).

Figure 8:

Compound 7h inhibits cell proliferation and NF-κB. A) The effect of 7h on proliferation of MDA-MB-231, MCF-7, and MDA-MB-436 breast cancer cell lines. Cells were treated with the indicated concentrations of 7h and cell proliferation was measured by bromodeoxyuridine ELISA assay 4–5 days after treatment. Results are average values from four experiments, each experiment with six technical replicates. B) Compound 7h inhibits NF-κB DNA binding activity in TMD-231 cells. Cells were incubated with different concentrations of 7h for three hours and EMSA was performed to measure NF-κB and Oct-1 DNA binding activity. Antibody supershift assay was used to identify NF-κB subunits in DNA-protein complex (last two lanes). C) Compound 7h prevents IKBα degradation. TMD-231 cells were incubated with vehicle or 2.5 μM 7h for one hour prior to treatment with TNFα (4 ng/mL). Cells were harvested at the indicated time after TNFα treatment and subjected to Western blotting.

Figure 9:

(A) Full length structure of IKKβ containing the Kinase Domain (KD), the ubiquitin-like domain (ULD), the Scaffold Dimerization Domain (SDD) and predicted NEMO binding domain (NBD). (B & C) Predicted region of interaction of compounds 7h and 10, (B) compound 10 binding to the SDD of IKKβ, (C) compound 7h binding to the KD of IKKβ.

Figure 10:

(A) Sites of interaction of compounds screened against IKKβ; (B and C) Predicted site of interaction of MMB, PTL, DMAPT and the MMB-triazole derivatives 7h and 10, with full length IKKβ: compounds 7h, MMB, and PTL are predicted to interact with the KD (activation site) of IKKβ, whereas DMAPT and compound 10 are predicted to interact at the interface of the SDD and the KD of IKKβ (C). (D) Binding free energies of each compound were calculated from Raccoon/AutoDock-Vina [66].

Figure 11:

(A & B) Detailed structure of IKKβ alone (A) or complexed with 7h (B) after 50ns simulation. Structures where viewed in Discovery Studio visualizer. (C) Secondary structure assignment over 50ns simulation with IKKβ alone (top panel) or when complexed with 7h (bottom panel), where red indicates beta-sheet, white indicates loops and turns, and blue indicates alpha-helix. Desmond trajectories were analyzed using the simulation interaction diagram module from Maestro (Schrodinger, Inc.).

Figure 12:

Interaction of compounds 7 and 10 with IKKβ and the subsequent events in the IKKβ oligomerization activation model. Compound 7h interacts with IKKβ at both the NEMO binding domain (NBD) and the kinase domain (KD), blocking transient oligomerization of IKKβ and subsequent trans autophosphorylation, while inhibition of the scaffold dimerization domain (SDD) by compound 10 results in inhibition of IKKβ dimerization and subsequent curtailment of oligomerization. Abbreviations: NBD: NEMO binding domain; SDD: scaffold dimerization domain; ULD: ubiquitin-like domain; KD: kinase domain; APD: auto phosphorylation domain

Scheme 1:

Synthesis of triazole derivatives of MMB

Scheme 2.

Synthesis of triazole dimers of MMB

Scheme 3.

Synthesis of compound 10, the dimethylamino Michael adduct of 7h