Journal: Nature Communications

Article Title: Modulating multi-functional ERK complexes by covalent targeting of a recruitment site in vivo

doi: 10.1038/s41467-019-12996-8

Figure Lengend Snippet: BI-78D3 labels C159 of ERK2 . a Chemical structure of BI-78D3. b BI-78D3 inhibits ERK1/2 phosphorylation by constitutively active MKK1 (MKK1G7B) and inhibits the phosphorylation of Ets-1 by activated ERK1/2. Different concentrations of BI-78D3 were incubated with either unphosphorylated or activated ERK1/2 for 30 min before the addition of [γ- 32 P] ATP and MKK1G7B or Ets-1, respectively (data are from three independent experiments for Ets-1 assays and two for MKK1G7B assays, and bars represent mean ± SD (standard deviation)). c The top panel shows an expansion of the region corresponding to C159 in 15 N, 1 H TROSY (600 MHz) spectra of inactive ERK2 (200 μM) in the presence of increasing amounts of BI-78D3. The bottom panel shows the change in the relative intensity of the C159 resonance in these spectra with increasing concentrations of BI-78D3. d Residues that show significant spectral perturbations calculated from 15 N, 1 H TROSY (800 MHz) or 13 C, 1 H HMQC (800 MHz) spectra of ERK2 in the presence of approximately equimolar amounts of BI-78D3 for backbone amide (left panel) or Ile (δ1), Val and Leu methyls (right panel) resonances are indicated on the structure of ERK2. Residues for which the amide or methyl chemical shift perturbations exceed the corresponding average plus twice the standard deviation are colored dark green and cyan, respectively. Residues for which amide resonances that are broadened to below the noise are colored light green. C159 is shown in yellow (and labeled in blue) and the activation loop T183 and Y185 are shown in red stick representation. All of the spectral perturbations in both cases are centered in and around the DRS of ERK2. e Fluorescence anisotropy was employed to assess the ability of BI-78D3 to competitively displace a fluorescent D-site-containing peptide from the DRS of activated ERK2 and ERK2 C159S (represents one experiment out of two repetitions). f Specificity of BI-78D3 towards C159 and C164. ERK proteins (5 µM) were incubated with 100 µM BI-78D3 for 60 min. Free thiol was titrated using Ellman’s reagent (data are from three independent experiments, and bars represent mean ± SD)

Article Snippet: FFPE sections were stained with an antibody against pp-ERK1/2 (Thr202/Tyr204—clone 20G11/Cell Signaling) by the MD Anderson Cancer Center Research Histology Core Lab (NCI CA#16672).

Techniques: Incubation, Standard Deviation, Labeling, Activation Assay, Fluorescence

Journal: Nature Communications

Article Title: Modulating multi-functional ERK complexes by covalent targeting of a recruitment site in vivo

doi: 10.1038/s41467-019-12996-8

Figure Lengend Snippet: BI-78D3 reaction with free thiols afford a stable tetrahedral intermediate. a , b Observed change in the UV-visible spectrum for the reaction of a 2-methoxyethanethiol (100 µM) or b ERK2 (50 µM) with BI-78D3 (10 µM) in 50 mM phosphate buffer, pH 7.5 and 2% dioxane (spectra recorded every 10 s for 1000 s). c Deconvoluted mass spectra of ERK2 showing the addition of approximately 386 Da after incubation of ERK2 (5 µM) with BI-78D3 (100 µM) for 15–20 min, followed by buffer exchange using a PD-10 column (calculated molecular weights of BI-78D3 and ERK2 are 380 and 42,329 Da respectively). d Proposed mechanism of the reaction between BI-78D3 and ERK1/2

Article Snippet: FFPE sections were stained with an antibody against pp-ERK1/2 (Thr202/Tyr204—clone 20G11/Cell Signaling) by the MD Anderson Cancer Center Research Histology Core Lab (NCI CA#16672).

Techniques: Incubation, Buffer Exchange

Journal: Nature Communications

Article Title: Modulating multi-functional ERK complexes by covalent targeting of a recruitment site in vivo

doi: 10.1038/s41467-019-12996-8

Figure Lengend Snippet: BI-78D3 exhibits a unique selectivity towards ERK1/2. a Kinetic behavior of ERK2 inactivation by BI-78D3. Data were fit to Eq. as described in the methods section and according to the two-step mechanism of activation shown in Scheme 1 (data represents one experiment out of two repetitions; both produced consistent kinetic parameters, values of K i and k inact ± standard error). b The final frame of a 100 ns molecular dynamics trajectory for BI-78D3 binding to ERK2 (PDB 4ERK). The figure was generated using UCSF Chimera software ( https://www.cgl.ucsf.edu/chimera/ ) . c Sequence alignment of Human MAPKs encompassing the D-recruitment site. The cysteines corresponding to C159 in ERK2 (targeted by BI-78D3) are indicated by the black rectangle; the numbering corresponds to human ERK1 sequence. C178 in Homo sapiens ERK1 corresponds to C161 in Homo sapiens ERK2 and C159 in Rattus norvegicus ERK2. d Reversibility of JNK1, but not ERK2 inhibition by BI-78D3. Each enzyme (5 µM) was treated with BI-78D3 (100 µM) or DMSO (control) for 1 h. The activity of each enzyme was estimated before and after excessive dialysis (data are from three independent experiments, and bars represent mean ± SD)

Article Snippet: FFPE sections were stained with an antibody against pp-ERK1/2 (Thr202/Tyr204—clone 20G11/Cell Signaling) by the MD Anderson Cancer Center Research Histology Core Lab (NCI CA#16672).

Techniques: Activation Assay, Produced, Binding Assay, Generated, Software, Sequencing, Inhibition, Activity Assay

Journal: Nature Communications

Article Title: Modulating multi-functional ERK complexes by covalent targeting of a recruitment site in vivo

doi: 10.1038/s41467-019-12996-8

Figure Lengend Snippet: BI-78D3 inhibits ERK in human BRAF- mutant A375 xenograft model. a Female athymic nude mice were injected subcutaneously with A375 cells. Once the tumor volume reached 150 mm 3 , mice were randomly divided into two groups ( n = 10) and dosed daily with vehicle or BI-78D3 (15 mg kg −1 by intraperitoneal administration) for 10 days. Tumor volumes were measured daily by caliper, and the values plotted as mean ± SEM; *** P < 0.001; **** P < 0.0001; data were analyzed following a two-way repeated measures ANOVA using the Bonferroni post-test method (GraphPad Prism 7 software). b To confirm target engagement of BI-78D3, tumor lysates from five mice of each group were subjected to western blotting and analyzed for pp-ERK1/2, ERK, pp-JNK , and β-actin (loading control). Images are cropped for presentation; uncropped images are shown in Supplementary Fig. . c Formalin-fixed paraffin-embedded tumor sections were subjected to immunohistochemistry. Representative images of the tumor sections stained with pp-ERK are shown. Comparisons of stained cells normalized to mm 2 of tumor area revealed significant suppression of pp-ERK in treated mice (the values were plotted as mean ± S.D., n = 5 for each group). d Estimated amount of active ERK in tumor lysates from five mice of each group, obtained using peptide-based biosensors and chemometric modeling using calibration regression model as described in Zamora-Olivares et al. (the values were plotted as mean ± SEM, n = 4 for each lysate)

Article Snippet: FFPE sections were stained with an antibody against pp-ERK1/2 (Thr202/Tyr204—clone 20G11/Cell Signaling) by the MD Anderson Cancer Center Research Histology Core Lab (NCI CA#16672).

Techniques: Mutagenesis, Injection, Software, Western Blot, Formalin-fixed Paraffin-Embedded, Immunohistochemistry, Staining