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

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