Manipulating JNK Signaling with (−)-Zuonin A

Abstract

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.

Elucidating binding modes of zuonin A enantiomers to JNK1 via in silico methods

“…The pose appears to be further stabilized by the stacking of one of its 1,3-benzodioxole rings between the groove formed by Arg127, Cys163, and Asp162 (Supplementary Figure 1A). This pose was quite similar to a (−)-zuonin A binding mode proposed by Kaoud et al[7] The highest scoring pose for (−)-zuonin A was pose 10 as shown in Table 1. This pose also interacted with the S 2 site and had a chemscore of 25.7.…”

“…Lastly, it is important to address a previous (−)-zuonin A/JNK interaction study performed by Kaoud et al[7] which proposed a model of (−)-zuonin binding where the inhibitor was bound to the S 2 region of JNK. Residues that were considered important for (−)-zuonin A binding to the S 2 site were mutated to alanines and kinetic/inhibition assays were performed on the mutants.…”

“…The perturbed binding of c-Jun by the R127A mutant makes it difficult to derive any conclusions regarding inhibitor binding, however the IC 50 values for mutants C163A and T164A both increased by over 1100% and 360% respectively while their maximal inhibition was nearly halved clearly showing that these mutations have some effect on inhibitor binding, which would be expected in either model. [7]…”

Small-molecule inhibitors of ERK-mediated immediate early gene expression and proliferation of melanoma cells expressing mutated BRaf

“…However, others suggested the differential effects of this compound on MK2 and ATF2 phosphorylation were a function of substrate:kinase stoichiometry and not due to the substrate selectivity of the compound [72]. A previous study indicated that the natural product zuonin A can target the DRS on JNK MAPK and inhibit phosphorylation of c-Jun [73]. Similarly, BI-78D3 was a small molecule identified to compete with a D-domain-containing peptide found on the JNK-interacting protein-1 (JIP1) and inhibits interactions with JNK1 [74].…”

A Fluorescence‐Based Assay for p38α Recruitment Site Binders: Identification of Rooperol as a Novel p38α Kinase Inhibitor

“…[10] Recently, inhibitors targeting the D-recruitment site (DRS) of JNK and ERK have been reported. [11][12][13][14][15] Inhibitors that bind to an allosteric site in the MAPK insert region of p38a, [16,17] and a substrate-selective inhibitor that binds in regions adjacent to the ATP-binding site [18,19] have been described, but no DRS-targeting p38a inhibitors have been reported. ATP-competitive and "DFG-out"-type p38a inhibitors have been evaluated in the clinic for treatment of inflammatory diseases [20][21][22][23] and p38a may also be a target for cancer treatment.…”