Journal: Communications Chemistry

Article Title: Design of a covalent protein-protein interaction inhibitor of SRPKs to suppress angiogenesis and invasion of cancer cells

doi: 10.1038/s42004-024-01230-2

Figure Lengend Snippet: a List of peptides designed with lysine-reactive modifications. The peptides comprise 20 amino acids with reactive chemical warheads installed. The chosen reactive groups were aryl-sulfonyl fluoride (X1), 4-nitrophenyl (X2), 1-fluoro-2,4-dinitrobenzene (FDNB) (X3), 4-fluorophenyl (X4), and phenyl (X5) groups. b–d Adduct formation assays (left panel) and in vitro kinase activity assays (right panel) of the modified peptides. The adduction assays were performed in a concentration of 1:4 (protein: peptide) for the indicated time periods. The samples were resolved by SDS-PAGE and visualized by Coomassie Blue. Kinase activity assays were performed using [ 32 P]ATP in the presence of the modified peptides for 2 min. The reaction samples were resolved by SDS-PAGE and visualized by autoradiography. DBS1-1p (hereafter named as C-DBS) showed the best conjugation and inhibition of SRPK1. e C-DBS inhibited SRPK1-mediated SRSF1 phosphorylation with an IC 50 value of 142 nM. Data represent means ± SEM from three independent experiments. f C-DBS disrupted the interaction between SRPK1 and SRSF1 dose-dependently. SRPK1 was preincubated with C-DBS at the indicated concentrations before the addition of GST-SRSF1. Samples were resolved by SDS-PAGE and visualized by Coomassie Blue.

Article Snippet: Recombinant GST-tagged SRSF1 protein in pGEX-4T-2 vector was purified using anion exchange (Q-Sepharose) and GST columns (GenScript) and dialyzed into a buffer containing 20 mM Tris-HCl pH 7.5, 300 mM KCl, 10% glycerol, and 1 mM DTT.

Techniques: In Vitro, Activity Assay, Modification, Concentration Assay, SDS Page, Autoradiography, Conjugation Assay, Inhibition, Phospho-proteomics

Journal: Communications Chemistry

Article Title: Design of a covalent protein-protein interaction inhibitor of SRPKs to suppress angiogenesis and invasion of cancer cells

doi: 10.1038/s42004-024-01230-2

Figure Lengend Snippet: a FAM-C-DBS conjugated with recombinant His-tagged SRPK1ΔNS3 in the absence or presence of HeLa cell lysate. The samples were resolved by SDS-PAGE and the adduct bands were visualized using Coomassie Blue (top panel), in-gel fluorescence (middle panel), and Western blotting (bottom panel). b C-DBS conjugated with endogenous SRPK1. MDA-MB-231 cell lysate samples before and after incubation with C-DBS were resolved by SDS-PAGE. SRPK1 and the adducts were detected using the anti-SRPK1 antibody. c C-DBS conjugated with SRPK2ΔS1. C-DBS was incubated with SRPK2ΔS1, CLK1 kinase domain, BSA, and GST for the indicated time periods. Only SRPK2 formed adducts. d C-DBS selectively inhibited SRPKs. Kinase activity assays were performed toward SRPK1ΔNS3, SRPK2ΔS1, CLK1, and Akt in the presence of the indicated concentrations of C-DBS. C-DBS inhibited both SRPK1 and SRPK2 but not CLK1 or Akt. The p-SRSF1 levels were quantified using ImageJ. Data represents means ± SEM from three independent experiments. Statistical analysis was performed using one-way ANOVA. *** p < 0.001.

Article Snippet: Recombinant GST-tagged SRSF1 protein in pGEX-4T-2 vector was purified using anion exchange (Q-Sepharose) and GST columns (GenScript) and dialyzed into a buffer containing 20 mM Tris-HCl pH 7.5, 300 mM KCl, 10% glycerol, and 1 mM DTT.

Techniques: Recombinant, SDS Page, Fluorescence, Western Blot, Incubation, Activity Assay