Manipulating JNK Signaling with (−)-Zuonin A

Recently, in a virtual screening strategy to identify new compounds targeting the D-recruitment site (DRS) of the c-Jun N-terminal kinases (JNKs), we identified the natural product (−)-zuonin A. Here we report the asymmetric synthesis of (−)-zuonin A and its enantiomer (+)-zuonin A. A kinetic analysis for the inhibition of c-Jun phosphorylation by (−)-zuonin A revealed a mechanism of partial competitive inhibition. Its binding is proposed to weaken the interaction of c-Jun to JNK by approximately 5-fold, without affecting the efficiency of phosphorylation within the complex. (−)-Zuonin A inhibits the ability of both MKK4 and MKK7 to phosphorylate and activate JNK. The binding site of (−)-zuonin A is predicted by docking and molecular dynamics simulation to be located in the DRS of JNK. (+)-Zuonin A also binds JNK but barely impedes the binding of c-Jun. (−)-Zuonin A inhibits the activation of JNK, as well as the phosphorylation of c-Jun in anisomycin-treated HEK293 cells, with the inhibition of JNK activation being more pronounced. (−)-Zuonin A also inhibits events associated with constitutive JNK2 activity, including c-Jun phosphorylation, basal Akt activation, and MDA-MB-231 cell migration. Mutations in the predicted binding site for (−)-zuonin A can render it significantly more or less sensitive to inhibition than wild type JNK2, allowing for the design of potential chemical genetic experiments. These studies suggest that the biological activity reported for other lignans, such as saucerneol F and zuonin B, may be the result of their ability to impede protein–protein interactions within MAPK cascades

Manipulating JNK Signaling with (−)-Zuonin A

Recently, in a virtual screening strategy to identify new compounds targeting the D-recruitment site (DRS) of the c-Jun N-terminal kinases (JNKs), we identified the natural product (−)-zuonin A. Here we report the asymmetric synthesis of (−)-zuonin A and its enantiomer (+)-zuonin A. A kinetic analysis for the inhibition of c-Jun phosphorylation by (−)-zuonin A revealed a mechanism of partial competitive inhibition. Its binding is proposed to weaken the interaction of c-Jun to JNK by approximately 5-fold, without affecting the efficiency of phosphorylation within the complex. (−)-Zuonin A inhibits the ability of both MKK4 and MKK7 to phosphorylate and activate JNK. The binding site of (−)-zuonin A is predicted by docking and molecular dynamics simulation to be located in the DRS of JNK. (+)-Zuonin A also binds JNK but barely impedes the binding of c-Jun. (−)-Zuonin A inhibits the activation of JNK, as well as the phosphorylation of c-Jun in anisomycin-treated HEK293 cells, with the inhibition of JNK activation being more pronounced. (−)-Zuonin A also inhibits events associated with constitutive JNK2 activity, including c-Jun phosphorylation, basal Akt activation, and MDA-MB-231 cell migration. Mutations in the predicted binding site for (−)-zuonin A can render it significantly more or less sensitive to inhibition than wild type JNK2, allowing for the design of potential chemical genetic experiments. These studies suggest that the biological activity reported for other lignans, such as saucerneol F and zuonin B, may be the result of their ability to impede protein–protein interactions within MAPK cascades

Additional file 1 of Involvement of the tumour necrosis factor receptor system in glioblastoma cell death induced by palbociclib-heptamethine cyanine dye conjugate

Additional file 1: Supplementary Figure 1. Cyclin-dependent kinase inhibitor-MHI-148 conjugates, 1 are not synergistic with TMZ. Patient-derived glioblastoma cells were treated with up to 100 μM of palbociclib, and 1 with and without 100 μM of TMZ for 96 h. Concentration-dependent effects of each compound on the toxicity of glioblastoma cells was measured by the percentage of Hoechst-positive cells after 96 h (EC50). A non-linear curve was fitted using Graphpad Prism using the concentration of each compound versus the percentage of hoechst-positive cells (A). Combination index (CI) was calculated from the CI equation algorithms using CompuSyn software. CI=1, 1 indicates additive effect, synergism, and antagonism, respectively (B). The pEC50 of each compound with and without TMZ (100 μM) on each glioblastoma cell line is summarised in B and D. The CI for palbociclib (A) and 1 (C), with TMZ across a range of effect sizes is presented per case. Data represents mean ±SEM for at least six independent glioblastoma cases, ns = p > 0.05. Supplementary Figure 2. Trafficking and expression of TNFR1 in response to 1 in patient-derived glioblastoma cells in the presence of vesicle trafficking inhibitor, BFA and protein translation inhibitor, CHX. Residual starting surface expression TNFR1 (Method A) (A). To investigate the net surface expression of TNFR1 (Method B) (B). C summarises method A and B detection of TNFR1. Total TNFR1 expression in response to 1 in the presence of absence of BFA or CHX (D) Summary data of the pEC50 (E) and Emax (F) of 1 on the total TNFR1 expression in the presence or absence of BFA or CHX. Basal total TNFR1 expression in G. Data represents mean ± SEM from three independent glioblastoma cases. ns = p > 0.05, * p < 0.05, ** p< 0.01, *** p < 0.001, One-way ANOVA relative to 1 plus vehicle inhibitor

From in Silico Discovery to Intracellular Activity: Targeting JNK–Protein Interactions with Small Molecules

The JNK–JIP1 interaction represents an attractive target for the selective inhibition of JNK-mediated signaling. We report a virtual screening (VS) workflow, based on a combination of three-dimensional shape and electrostatic similarity, to discover novel scaffolds for the development of non-ATP competitive inhibitors of JNK targeting the JNK–JIP interaction. Of 352 (0.13%) compounds selected from the NCI Diversity Set, more than 22% registered as hits in a biochemical kinase assay. Several compounds discovered to inhibit JNK activity under standard kinase assay conditions also impeded JNK activity in HEK293 cells. These studies led to the discovery that the lignan (−)-zuonin A inhibits JNK–protein interactions with a selectivity of 100-fold over ERK2 and p38 MAPKα. These results demonstrate the utility of a virtual screening protocol to identify novel scaffolds for highly selective, cell-permeable inhibitors of JNK–protein interactions