Tricyclic guanidine alkaloids from the marine sponge Acanthella cavernosa that stabilize the tumor suppressor PDCD4

A cell-based high-throughput screen that assessed the cellular stability of a tumor suppressor protein PDCD4 (Programmed cell death 4) was used to identify a new guanidine-containing marine alkaloid mirabilin K (3), as well as the known compounds mirabilin G (1) and netamine M (2). The structures of these tricyclic guanidine alkaloids were established from extensive spectroscopic analyses. Compounds 1 and 2 inhibited cellular degradation of PDCD4 with EC50 values of 1.8 μg/mL and 2.8 μg/mL, respectively. Mirabilin G (1) and netamine M (2) are the first marine natural products reported to stabilize PDCD4 under tumor promoting conditions

Cell cycle.

<p>HEK293 cells stably expressing Pdcd4_(39–91)luc were incubated for 24 h with DMSO (blue) or increasing concentrations of compounds <b>1</b>, <b>2</b>, <b>3</b>, <b>4</b>, <b>5</b>, and <b>6</b> (3 μM (violet), 10 μM (yellow), and 30 μM (red)). Cell cycle analysis was performed after permeabilization using 7-AAD. Data are presented as means ± SEM of at least 3 independent experiments (* p<0.05, ** p<0.01, *** p<0.01).</p

Conformational analysis of compounds 1, 2 and 4.

<p>(A) A systematic approach was chosen to explore the conformational space around the three rotable bonds. (B-D) Global minimum energy conformations (<i>left panel</i>) and energy plots (<i>right panel</i>) of compounds <b>1</b> (B), <b>2</b> (C), and <b>4</b> (D).</p

Pdcd4 stabilizing activity.

<p>HEK293 cells stably expressing the Pdcd4 stability reporter Pdcd4_(39–91)luc were incubated for 8 h with TPA [10 nM] in combination with increasing concentrations (0.1–30 μM) of compounds <b>1</b>, <b>2</b>, <b>3</b>, <b>4</b>, <b>5</b>, and <b>6</b>. Pdcd4 stabilizing activity was determined relative to Δ(RLU_DMSO–RLU_TPA). Data are presented as means ± SEM of at least 3 independent experiments (* p<0.05, ** p<0.01, *** p<0.01).</p

Chemical structures.

<p>Structures of compounds <b>1</b> (1,2-bis(4-chlorophenyl)disulfide), <b>2</b> (4-chloro-<i>N</i>-(4-chlorophenyl)benzamide), <b>3</b> ((<i>E</i>)-1,2-bis(4-chlorophenyl)diazene), <b>4</b> (1,2-bis(4-chlorophenyl)hydrazine), <b>5</b> (1,2-bis(4-methoxyphenyl)disulfide), and <b>6</b> (1,2-bis(4-nitrophenyl)disulfide).</p

Erioflorin inhibits AP-1- and NF-κB-trancriptional activities.

<p>(A) HEK293 cells were co-transfected with an AP-1 <i>firefly</i> reporter and a <i>renilla</i> luciferase plasmid one day prior to experiments. Transfected cells were treated for 16 h with TPA (10 nM) with or without erioflorin (2.5 and 5 µM). Relative AP-1 activity was normalized to <i>renilla</i> luciferase and presented relative to TPA-only treated cells. (B) HEK293 cells were co-transfected with a NF-κB <i>firefly</i> reporter and a <i>renilla</i> luciferase plasmid one day prior to experiments. Transfected cells were treated for 16 h with TNFα (20 mg mL^–1) with or without erioflorin (2.5 and 5 µM). Relative NF-κB activity was normalized to <i>renilla</i> luciferase and presented relative to TNFα-only treated cells. All data are given as means ± SEM (n≥3, **p<0.01).</p

Erioflorin specifically inhibits E3-ligase β-TrCP1 activity.

<p>(A) HEK293 cells were treated with TNFα (20 ng mL^–1) for the indicated times with or without erioflorin (10 µM). (B) HEK293 cells were maintained under full medium conditions (10% serum) or serum deprived for 24 h following treatment with erioflorin (5 µM) for 8 h. (C) HEK293 cells were treated for 8 h with erioflorin (1.25–10 µM) or the prolyl-hydroxylase inhibitor dimethyloxallylglycine (DMOG, 1 µM). (D) HeLa cells were serum deprived for 48 h prior to treatment with erioflorin (10 µM) or the proteasome inhibitor MG132 (10 µM) for 8 h. Whole cell extracts were subjected to western analysis and probed with the indicated antibodies. Blots are representative for at least three independent experiments.</p

Viability.

<p>HEK293 cells stably expressing Pdcd4_(39–91)luc were incubated for 24 h with DMSO (blue) or increasing concentrations of compounds <b>1</b>, <b>2</b>, <b>3</b>, <b>4</b>, <b>5</b>, and <b>6</b> (3 μM (violet), 10 μM (yellow), and 30 μM (red)). Cell death analysis was performed using Annexin V and 7-AAD co-staining. Data are presented as means ± SEM of at least 3 independent experiments (* p<0.05, ** p<0.01, *** p<0.01).</p