Fig. 6: Characterization of compounds with extended cyclohexenone scaffolds in cell-based tests.

a Results with the NanoBiT luciferase fragment-complementation assay. The panel on the left shows the scheme of the dynamic, luciferase complementation-based protein-protein interaction (PPI) assay (NanoBiT)²⁹. The assay was done in live HEK293T cells using 10 μM inhibitor concentration and compounds were added 1.5-hours before the measurements (“-”: control with 0.05 % DMSO). Top panels show the effect of 28S, 32S, 6S,S and 6R,R on MAP2K-MAPK binding. MKK7, MKK1, and MKK6 are the cognate upstream activator kinases (MAP2Ks) of JNK1, ERK2, and p38α, respectively. Note that 32S is an extended version of 28S with the same configuration at C4, while 6S,S and 6R,R are stereoisomers containing two stereogenic centers (at C4 and C6; shown with arrows) with opposite configurations. MKK7_D0, MKK1_D0 and MKK6_D0 are MAP2K constructs that do not contain their D-groove binding docking motifs – located in their disordered N-terminal region – and were used to show that binding is indeed D-motif/docking dependent. Lower panels show the effect of 32S and 32R, which are stereoisomers with the same extension at C4, on MKK1-ERK2, ERK2-RSK1, and ERK2-MKP3 binary binding. Data are presented as mean values and error bars show SD (n = 3, upper panels, or n = 4, lower panels; luminescence for the latter is shown in arbitrary units; parallel independent measurements). (*p < 0.05, **p < 0.01, ***p < 0.001; two-sided, unpaired t-test). b Effect of inhibitors on AP-1 transcription factor promoter activity. The scheme shows that PMA treatment activates protein kinase C (PKC) which ultimately leads to the phosphorylation (P) of AP-1 transcription factor dimers promoting AP-1 mediated gene expression. The process, in addition to c-Jun phosphorylation by JNK, is also positively affected by phosphorylation of c-Jun dimerization partners (ATF and c-Fos). This latter is promoted by p38 and/or ERK1/2. In the Reporter AP-1 – HEK293 Recombinant Cell Line PMA stimulation increases the transcription of the reporter luciferase gene (Luc). AP-1 promoter driven transcription of the luciferase reporter gene was monitored by measuring luminescence after 6 h in unstimulated cells (-) or cells stimulated with phorbol 12-myristate 13-acetate ( + PMA). Inhibitors were co-administered with PMA. The first panel shows that p38i and ERKi – two ATP competitive inhibitors, SB202190 and SCH772984, respectively, used in 1 μM concentration – indeed attenuate AP-1 mediated transcription as expected. D-groove binding inhibitors (6 R,R, 32S or 32 R) were then used in 1, 3, or 10 μM concentrations in another experiment. 6R,R shows significant inhibition at 3 μM concentration and above, while 32S and 32R, which are enantiomeric pairs with extended moieties at C4, appear to be stronger inhibitors in this MAPK dependent cell-based test. 32S performs significantly better than 32R at all three concentrations tested. The next panel at the bottom shows the results of another experiment with 34S and 35S, containing N,N-dimethylaniline or pyridine moiety, respectively (but C4 was extended by the same chemical solution, i.e. Sonogoshira coupling). 28S, 34S, and 35S are all S stereoisomers at C4 but they contain different moieties (propargyl ester, N,N-dimethylaniline or pyridine) or different linkers for C4 extension (1,4-substituated 1,2,3-triazole: nonlinear or arylalkyne: linear). The last panel shows that extending 28S with an appropriate extra moiety such as in 35S greatly increases the potency of the compound in this MAPK-dependent assay. 35S exerts a greater inhibitory effect at each concentration tested below 10 μM. Data are presented as mean values and error bars show SD (n = 3, parallel independent measurements). (*p < 0.05, **p < 0.01, ***p < 0.001; two-sided, unpaired t-test). Source data are provided as a Source Data file.