frax597 tgm6014  (TargetMol)

Journal: bioRxiv

Article Title: PERK inhibition rewires translational and CMGC protein kinase networks into an antiviral state

doi: 10.1101/2025.10.08.681090

Figure Lengend Snippet: (A) P-peptides from the same samples described in were extracted by IMAC and analyzed by LC-MS/MS resulting in 12585 peptide IDs. The heatmap demonstrates mean changes of P-peptides, their sequences and the identified P-sites (in red) corresponding to the N or S proteins (green or yellow colors) and to host proteins c-Jun and p38 MAPK (MAPK14) for each of the six conditions analyzed. Data are from four biologically independent experiments and two technical replicates. (B) Volcano plots show mean Log 2 -fold changes of all P-peptides and their significance from four biologically independent experiments and two technical replicates. Numbers in brackets and red or blue colors indicate up- or down-regulated P-peptides in response to CoV or PERKi treatment (-Log 10 p value ≥ 1.3, student’s t-test). P-peptides corresponding to N or S protein and individual P-sites are highlighed in green or yellow, respectively. Red text indicates sets of P-peptides suppressed by PERKi treatment in infected cells. (C) Venn diagrams indicating the overlap of P-peptides differentially phosphorylated in response to CoV or PERKi based on (-Log 10 p value ≥ 1.3, student’s t-test). Numbers in brackets indicate the overlap of gene IDs corresponding to the identified P-peptides. (D) Overrepresentation analysis was performed side by side based on gene IDs corresponding to P- peptides for factors that were up- or down-regulated in response to PERKi after 12 h or 24 hpi. The Venn diagram shows the overlap of the top100 enriched pathway terms across all conditions. (E) Heatmap showing the top5 most strongly enriched pathway terms representing exclusively suppressed P-protein sets (upper table) or those with a mixed pattern of regulation in response to PERKi (lower table). Yellow color highlights the Rho GTPase pathway. (F) Scatter plots showing mean abundancies of the P-peptides that are suppressed by PERKi 24 hpi, segregated according to the pathways identified in (E), along with their values across all conditions. Data are from four biological replicates and two technical replicates, black lines indicate medians. Red colors mark P-peptides belonging to protein kinases. Blue colors highlight P-peptides of PAK2. (G-J) Huh7 cells were infected for 24 h with HCoV-229E (H, MOI=l) and treated with group I PAK inhibitors FRAX469 (G, I) or FRAX597 (H-J) at the indicated concentrations, thapsigargin (l µM), solvent (DMSO) or were left untreated. (G-H) Whole cell extracts were used to analyze phosphorylation and expression of proteins by Western blotting. One representative out of four biologically independent experiments is shown. (I-J) Quantification of the indicated (P)-proteins from four replicates. Shown are mean values ± s.d. including non-linear regression lines used to estimate IC 50 values. (K) Experiments were performed as described in (G-J), including two additional concentrations of PAKi (10 µM, 25 µM). Supernatants of cells were used to analyze viral titers by plaque assays and parallel cell cultures were used to assess cell viability. Left graphs show mean titers ± s.d. from four biologically independent experiments. The same data were used to estimate EC 50 values by non-linear regression (middle graphs). Right graphs show mean normalized cell viability values ± s.d. including non-linear regression lines to estimate CC 50 values as indicated, obtained from three biologically independent experiments. Asterisks indicate p values (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001) derived from Kruskal Wallis tests. (C-F) Amber rectangles denote the sets of P-peptides and their corresponding gene IDs suppressed significantly by PERKi at 24 hpi (-Log 10 p value ≥ 1.3, student’s t-test).

Article Snippet: FRAX486 (TGM6840) and FRAX597 (TGM6014) were obtained from TargetMol and KIRA6 (532281) was purchased from Calbiochem through Merck-Millipore.

Techniques: Liquid Chromatography with Mass Spectroscopy, Infection, Solvent, Phospho-proteomics, Expressing, Western Blot, Derivative Assay