Fig 2: Chemoproteomic kinase profiling of 51-106. (A) Chemical structure of desthiobiotinylated acylphosphate-nucleotide probe and schematic of the probe in ATP binding site of a kinase targeting a conserved lysine residue. (B) Schematic of the KiNativ method. Protein lysates are treated with inhibitor (51-106) followed by the addition of the acylphosphate probe. Proteins are digested by trypsin, and the biotinylated peptides are enriched by streptavidin. The probe-labeled peptides are quantified using a targeted LC-MS2 strategy. (C) Kinome profiling (KiNativ) of 51-106 in a pancreatic cancer cell line (PANC-1). Each colored dot represents a quantified kinase-associated peptide. This kinome dataset is the result of an analysis of duplicate treated and control samples. The % changes in MS signals being reported are statistically significant (p-value < 0.04, Student’s t-test). (D) There are no available structures of MAP3K1. We used the available AlphaFold MAP3K1 structure (AF-Q13233-F1-model.pdb) and docked 51-106 into the ATP binding site using Schrödinger GLIDE.

Fig 3: Phosphoproteomic analysis following MAP3K1 inhibition with 51-106 treatment. PANC-1 cells were treated with either DMSO or 51-106 (10 µM, 5 µM, 2.5 µM, 1.25 µM, 0.625 µM) for 2 h. The were processed and analyzed for phosphoproteomic and total proteomic analysis using LC-MS/MS analysis. (A) Heatmap of the significant sites identified with RokaiXplorer analysis, which decreased (blue) or increased (red) in a 51-106 dose-dependent manner (log2 FC > ±0.32, FDR < 0.05) across the 10 µM, 5 µM, 2.5 µM, and 1.25 µM doses. Proteins known to interact with MAP3K1 from the Pathway Commons Database are highlighted in blue. (B) Top 10 significant GO Biological Processes enriched using Enrichr program, containing Ser/Thr-Pro phosphosites that decrease in a dose-response fashion with 51-106 treatment. (C) STRING network of medium-confidence interactions (minimum confidence score of 0.400) among proteins containing Ser/Thr-Pro phosphosites that decrease in a dose-dependent manner with 51-106 treatment. Disconnected nodes are not shown. Color of the nodes is arbitrary. (D) Western blot validation of phosphoproteomics results of NPM1 T199 phosphorylation inhibition. PANC-1 cells were treated with DMSO, 51-106, or Staurosporine, a multi-kinase inhibitor, for 2 h and membrane probed for phosphorylation NPM1 at T199 and α-Tubulin loading control. (E) The top 30 predicted inhibited kinases based on substrate phosphorylation changes by RokaiXplorer (FDR < 0.05) sorted by Z-score. (F) Results from the KiNativ kinome screen of the top 30 predicted kinases. NB = no binding, ND = not detected.