Journal: The Journal of Biological Chemistry

Article Title: A cellular target engagement assay for the characterization of SHP2 (PTPN11) phosphatase inhibitors

doi: 10.1074/jbc.RA119.010838

Figure Lengend Snippet: Allosteric control mechanisms for SHP2 are the basis for drug discovery. A, in normal cells under basal conditions, SHP2 adopts an autoinhibited closed conformation in which its N-terminal SH2 domain binds and blocks the PTP active site. Cell stimulation leads to Tyr phosphorylation of SHP2-binding proteins that then recruit SHP2 via its SH2 domains, causing SHP2 to open into its active conformation; tyrosine phosphorylation within the C-terminal tail (Tyr(P)-542 and Tyr(P)-580) further enhances SHP2 activity. B, in solid tumors, overexpression or aberrant phosphorylation of RTKs or scaffolding adapters result in hyperactivation of SHP2. C, in leukemias, somatic mutations located at the interface between the N-SH2 and PTP domains prevent SHP2 from closing, resulting in a constitutively active SHP2. D, crystal structure of the SHP2:SHP099 complex (PDB accession number 5EHR) with the N-SH2 (blue), C-SH2 (green), and phosphatase domain (orange) in the closed, autoinhibited conformation. The allosteric inhibitor SHP099 binds in a “tunnel” formed at an interface of the three domains and stabilizes SHP2 in its inactive conformation. E, allosteric SHP2 inhibitors such as SHP099 or RMC-4550 compete with SHP2 activation, as the allosteric binding site only exists in the closed conformation. The effect of SHP2 gain-of-function mutations is to destabilize the autoinhibited confirmation of SHP2. Therefore, many oncogenic SHP2 mutants are resistant to inhibition by the SHP099 class of compounds.

Article Snippet: Molecular cloning For recombinant expression of the full-length SHP2 (amino acid residues 1–594), a GST fusion construct in pGEX-4T1 was used to produce a thrombin-cleavable construct (Addgene plasmid 8322).

Techniques: Cell Stimulation, Binding Assay, Activity Assay, Over Expression, Scaffolding, Activation Assay, Inhibition

Journal: The Journal of Biological Chemistry

Article Title: A cellular target engagement assay for the characterization of SHP2 (PTPN11) phosphatase inhibitors

doi: 10.1074/jbc.RA119.010838

Figure Lengend Snippet: Characterization of SHP2 allosteric inhibitors in PTS and biochemical inhibition assays

Article Snippet: Molecular cloning For recombinant expression of the full-length SHP2 (amino acid residues 1–594), a GST fusion construct in pGEX-4T1 was used to produce a thrombin-cleavable construct (Addgene plasmid 8322).

Techniques: Inhibition

Journal: The Journal of Biological Chemistry

Article Title: A cellular target engagement assay for the characterization of SHP2 (PTPN11) phosphatase inhibitors

doi: 10.1074/jbc.RA119.010838

Figure Lengend Snippet: Differential scanning fluorimetry (protein thermal shift) results for SHP2-WT, SHP2-E76K, and SHP2cat. A, derivative plot of the thermal denaturation curves of SHP2-WT in the presence of SHP099 (blue) or vehicle (DMSO, red). The melting temperature (Tm) is defined at the peak maximum representing the inversion point. SHP099, at 50 μm, substantially stabilizes the SHP2-WT protein and shifts its Tm by 4.8 °C, indicating strong binding. B, the stabilization of SHP2-WT by SHP099 is dose-dependent. C, compared with SHP2-WT, the effect of SHP099 on the SHP2-E76K mutant protein is greatly reduced (ΔTm = 1.2 °C), indicating weaker binding of SHP099 to the mutant protein. D, SHP099 does not affect the Tm of the SHP2 catalytic domain alone, which is in agreement with the compound's binding mode as well as biochemical inhibition data.

Article Snippet: Molecular cloning For recombinant expression of the full-length SHP2 (amino acid residues 1–594), a GST fusion construct in pGEX-4T1 was used to produce a thrombin-cleavable construct (Addgene plasmid 8322).

Techniques: Binding Assay, Mutagenesis, Inhibition

Journal: The Journal of Biological Chemistry

Article Title: A cellular target engagement assay for the characterization of SHP2 (PTPN11) phosphatase inhibitors

doi: 10.1074/jbc.RA119.010838

Figure Lengend Snippet: Development of a cellular target engagement assay for WT and oncogenic mutant (E76K) SHP2 proteins. Transiently transfected HEK293T cells were used to investigate the utility of a cellular thermal shift assay based on the InCell Pulse technology. A, thermal profiles of the control protein MTH1 in the presence (blue) or absence (vehicle, red) of the MTH1 inhibitor TH588 (10 μm). B, thermal profiles of the SHP2 catalytic domain in the presence (blue) or absence (vehicle, red) of the SHP2 allosteric inhibitor SHP099 (10 μm). As expected, SHP099 does not engage with SHP2cat. C, thermal profiles of WT SHP2 (SHP2-WT) in the presence (blue) or absence (vehicle, red) of SHP099 (10 μm). SHP099 substantially stabilizes SHP2-WT, indicating target engagement in the cell. D, thermal profiles of oncogenic mutant SHP2-E76K in the presence (blue) or absence (vehicle, red) of SHP099 (10 μm). The E76K mutation in SHP2 ablates the response to the SHP099 allosteric inhibitor. E, thermal profiles of SHP2-WT in the absence (vehicle, red) or presence of the SHP099-like allosteric inhibitors RMC-4550 (10 μm, green), Ex-57 (10 μm, blue), or SHP836 (50 μm, violet). RMC-4550 and Ex-57 exhibit a greater stabilization of SHP2-WT than SHP099, in agreement with the greater potency of these compounds compared with SHP099 in both the in vitro PTS and biochemical inhibition assays. Similarly, the muted effect of SHP836 on SHP2-WT in cells corresponds with the lower potency of this compound in the in vitro assays. F, thermal profiles of SHP2-E76K in the absence (vehicle, red) or presence of SHP099-like allosteric inhibitors RMC-4550 (10 μm, green), Ex-57 (10 μm, blue), or SHP836 (50 μm, violet). All compounds exhibit an attenuated effect on the SHP2-E76K mutant in cells, which is also in agreement with the in vitro PTS binding and enzymatic inhibition data. The data points and error bars (±S.D.) represent duplicate measurements.

Article Snippet: Molecular cloning For recombinant expression of the full-length SHP2 (amino acid residues 1–594), a GST fusion construct in pGEX-4T1 was used to produce a thrombin-cleavable construct (Addgene plasmid 8322).

Techniques: Mutagenesis, Transfection, Thermal Shift Assay, In Vitro, Inhibition, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: A cellular target engagement assay for the characterization of SHP2 (PTPN11) phosphatase inhibitors

doi: 10.1074/jbc.RA119.010838

Figure Lengend Snippet: Cellular thermal shift isothermal dose-response assay for SHP2 WT. A, experiment to establish optimal isothermal conditions to evaluate the dose-dependent target engagement of SHP2 inhibitors. Applying a thermal gradient (50–65 °C) across the “short” axis of a 384-well plate (see also Fig. S2) allowed efficient optimization of cellular inhibitor dose response and temperature using SHP099 (3–50 μm). Five-point dose-response curves were generated for each temperature as indicated. B, full 10-point isothermal cellular dose-response for SHP099. The EC50 at an optimized temperature of 55.0 °C is indicated. The data points and error bars (±S.D.) represent quadruplicate measurements.

Article Snippet: Molecular cloning For recombinant expression of the full-length SHP2 (amino acid residues 1–594), a GST fusion construct in pGEX-4T1 was used to produce a thrombin-cleavable construct (Addgene plasmid 8322).

Techniques: Generated

Journal: The Journal of Biological Chemistry

Article Title: A cellular target engagement assay for the characterization of SHP2 (PTPN11) phosphatase inhibitors

doi: 10.1074/jbc.RA119.010838

Figure Lengend Snippet: Application of the SHP2 cellular target engagement assay. A, isothermal CETSA screening of biochemically active SHP2 inhibitor analogs from two distinct chemical scaffolds. Compounds were tested at 30 μm concentration against SHP2-WT (54 °C) and SHP2-E76K (50 °C). Luminescence measurements are indicated as a ratio to the DMSO vehicle control. SHP099 was included as a positive control. The data points and error bars (±S.E.) represent quadruplicate measurements. B and C, chemical structures and biochemical IC50 values against recombinant SHP2-WT and SHP2-E76K of representative compounds SBI-221 (B) and SBI-668 (C) are shown. D, CETSA thermal profiles for SHP2-WT in the presence (red) or absence (black) of 30 μm SBI-221. E, CETSA thermal profiles for SHP2-E76K in the presence (blue) or absence (black) of 30 μm SBI-668. F and G, SBI-221 (red) and SBI-668 (blue) dose-response isothermal CETSA experiments with SHP2-WT (55 °C; F) and SHP2-E76K (50 °C; G). The data points and error bars (±S.E.) represent quadruplicate measurements. The significance of the inhibitor effects was calculated using a multiple t test compared with the vehicle (DMSO) control with a false discovery rate approach by the two-stage step-up method of Benjamini, Krieger, and Yekutieli using GraphPad Prism, version 8. *, p < 0.001; **, p < 0.0001).

Article Snippet: Molecular cloning For recombinant expression of the full-length SHP2 (amino acid residues 1–594), a GST fusion construct in pGEX-4T1 was used to produce a thrombin-cleavable construct (Addgene plasmid 8322).

Techniques: Concentration Assay, Positive Control, Recombinant

Journal: Molecular cell

Article Title: Receptor tyrosine kinases regulate signal transduction through a liquid-liquid phase separated state.

doi: 10.1016/j.molcel.2022.02.005

Figure Lengend Snippet: Figure 1. Phosphorylated RTK-mediated condensation of protein complexes (A) (Above) Images of recombinant phosphorylated receptors from the EGFR, FGFR, and VEGFR families (Atto-488 labeled) droplet formation upon adding SHP2C459S (top panel) or SHC (middle panel); phosphorylated RTK proteins alone do not form droplets (third panel). Concentrations of each RTK-SHP2C459S or SHC pair were shown (x axis: RTK concentration; y axis: SHP2C459S or SHC concentration) and scale bars, 10 mm. (Below) Schematic diagram with residue numbers shows the defined boundaries of RTK intracellular regions of SHP2, SHC, and PLCg1 proteins and polypeptides used in this study. (B) Phase diagrams of phosphorylated EGFR, FGFR, and VEGFR family proteins (Atto-488 labeled) with concentrations shown in x axis and SHP2C459S (y axis) in 20 mM HEPES (pH7.5), 150 mM NaCl, and 1 mM TCEP. The sizes of the circles represent the average sizes of droplets (mm2), and the color scale bars represent the number of droplets in a 0.0256-mm2 area.

Article Snippet: The full length SHP2, EGFR, Her2, and Her4 plasmid templates were obtained from Addgene (SHP2: #8381, EGFR: #81926, Her2: #16257, Her4: #29527).

Techniques: Recombinant, Labeling, Concentration Assay, Residue

Journal: Molecular cell

Article Title: Receptor tyrosine kinases regulate signal transduction through a liquid-liquid phase separated state.

doi: 10.1016/j.molcel.2022.02.005

Figure Lengend Snippet: Figure 3. The formation of LLPS pFGFR2-SHP2C459S-pPLCg1 condensates on supported lipid bilayers and plasma membranes (A) pFGFR2Cyto-SHP2C459S-pPLCg1 condensates on supported lipid bilayers. (i) Confocal images of homogeneously distributed pFGFR2Cyto Atto-488 (20 mM, 6xHis tagged) on membrane bilayers, (ii) pFGFR2Cyto Atto-488 gradually clustered upon the addition of SHP2C459S Atto-594 (60 mM, untagged), and (iii) pPLCg1 Atto-647 (6 mM, untagged), followed by (iv) additional 36 mM of untagged pPLCg1 Atto-647. Scale bars, 10 mm. (B) FRAP analysis showing the dynamic nature of pFGFR2Cyto-SHP2C459S-pPLCg1 condensates on supported lipid bilayers as all pFGFR2Cyto, SHP2C459S, and pPLCg1 exchanged with their counterparts in the dilute phase. Data are presented as mean ± SD, n = 2 experiments. (C) Immunofluorescence staining images showing colocalized SHP2-Alexa 488 and PLCg1-Alexa 647 droplet formation on plasma membrane in FGF9-stim- ulated (10 ng/ml, 15 min) Caco-2 cells and Caco-2 FGFR2i cells. Inset image: magnification of regions shown to exemplify endogenous SHP2-PLCg1 clusters on membranes. Graph (right of image): statistical analysis of droplet formation in parental Caco-2 cells and Caco-2 FGFR2i cells. Only the SHP2-Alexa 488 and

Article Snippet: The full length SHP2, EGFR, Her2, and Her4 plasmid templates were obtained from Addgene (SHP2: #8381, EGFR: #81926, Her2: #16257, Her4: #29527).

Techniques: Clinical Proteomics, Membrane, Staining

Journal: Molecular cell

Article Title: Receptor tyrosine kinases regulate signal transduction through a liquid-liquid phase separated state.

doi: 10.1016/j.molcel.2022.02.005

Figure Lengend Snippet: Figure 4. Characterization of the interactions between FGFR2DVT-SHP2C459S droplets (A) (Left) Pull-down experiments using GST-SHP2C459S or GST-SHP22SH2 (see schematic in Figure 1A) show that the binding of SHP2 requires phosphorylation of FGFR2DVT. FGFR2DVT or FGFR2DVT-KD (double mutant Y656/657F) stably expressing HEK293T cells were unstimulated or FGF9-stimulated (10 ng/ml, 15 min). Arrows highlight GST fusion as part of the SHP2 constructs. The lower level of interaction without FGF9 stimulation due to protein recruitment by the basally activated FGFR2 as shown in the pFGFR2 blot (Input). (Right) Densitometry analysis of GST pull down, n = 3. Data are presented as mean ± SD. Replicate data are shown in Data S1A.

Article Snippet: The full length SHP2, EGFR, Her2, and Her4 plasmid templates were obtained from Addgene (SHP2: #8381, EGFR: #81926, Her2: #16257, Her4: #29527).

Techniques: Binding Assay, Phospho-proteomics, Mutagenesis, Stable Transfection, Expressing, Construct

Journal: Molecular cell

Article Title: Receptor tyrosine kinases regulate signal transduction through a liquid-liquid phase separated state.

doi: 10.1016/j.molcel.2022.02.005

Figure Lengend Snippet: Figure 5. Interactions between SHP2 and PLCg1 droplets and the formation of ternary complexes (A) Plot of the chemical shift changes (ppm) of the backbone amide peaks of 1H, 15N-labeled PLCg12SH2 (200 mM) upon the addition of 3 mol L1 equivalent of SHP22SH2. The residue numbers are indicated on the x axis. (B) CSP of residues mapped on to the crystal structure of the PLCg12SH2 (PDB code: 4FBN). The gradient indicates the strength of the perturbation. The pY binding pockets for NSH2 and CSH2 are shown in cyan (R562, R586, S588, E589, T590, and T596) and green (R675, R694, R696 and A703), respectively. Left hand image shows putative binding region (highlighted by increasing CSP). Right hand image shows the structure rotated into plane by 180 to show the comparatively negligible CSP on the ‘non-binding’ surface. (C) Plot of the chemical shift changes (ppm) of the backbone amide peaks of 1H, 15N-labeled SHP22SH2 (100 mM) upon the addition of 6 mol L1 equivalent of PLCg12SH2. The residue numbers are indicated on the x axis.

Article Snippet: The full length SHP2, EGFR, Her2, and Her4 plasmid templates were obtained from Addgene (SHP2: #8381, EGFR: #81926, Her2: #16257, Her4: #29527).

Techniques: Labeling, Residue, Binding Assay

Journal: Molecular cell

Article Title: Receptor tyrosine kinases regulate signal transduction through a liquid-liquid phase separated state.

doi: 10.1016/j.molcel.2022.02.005

Figure Lengend Snippet: Figure 6. Characterization of the ternary complex formation (A) (i) In vitro phase separation assay using Atto-labeled pFGFR2Cyto (10 mM) and truncated SHP22SH2 (30 mM). The addition of a pY769 peptide (ii) or a general pY peptide (ii) to compete SH2 domain binding reduces droplet formation. Scale bars, 10 mm. (B) R to A mutation of residues 32 or/and 138 in the pY binding sites show that both wild-type SH2 domains of SHP2 are required (30 mM of each mutant) for LLPS with pFGFR2Cyto (10 mM). (i) Wild-type SHP22SH2. (ii) SHP22SH2 R32A. (iii) SHP22SH2 R138A. (iv) SHP22SH2 R32/138A. Scale bars, 10 mm. (C) In vitro phase separation assay using Atto-labeled pFGFR2Cyto (10 mM), SHP22SH2 (30 mM), and pPLCg12SH2 (12 mM). (i) Individual proteins showed no evidence of droplet formation. Droplet formation was observed after 1 min: (ii) with all three proteins; (iii) with pFGFR2Cyto and SHP22SH2, not with pFGFR2Cyto with

Article Snippet: The full length SHP2, EGFR, Her2, and Her4 plasmid templates were obtained from Addgene (SHP2: #8381, EGFR: #81926, Her2: #16257, Her4: #29527).

Techniques: In Vitro, Labeling, Binding Assay, Mutagenesis

Journal: Molecular cell

Article Title: Receptor tyrosine kinases regulate signal transduction through a liquid-liquid phase separated state.

doi: 10.1016/j.molcel.2022.02.005

Figure Lengend Snippet: Figure 7. Phase transition of FGFR2-SHP2-PLCg1 upregulates downstream signaling (A) (Left) Depletion of SHP2 upregulates PLCg1 through phosphorylation of Y783 but downregulates its downstream effectors (shown by reduced phosphor- ylation of PKCbII-S660 and AKT-S473) in FGF9-stimulated (10 ng/ml) MCF7 cells and A431 cells. (Right) Densitometry analysis of SHP2 expression and the activation levels of various signaling proteins (dark green: parental cells; light green: SHP2 depletion cells). MCF7 cells: n = 3; A431 cells: n = 2. Data are presented as mean ± SD. Replicate data are shown in Data S3A.

Article Snippet: The full length SHP2, EGFR, Her2, and Her4 plasmid templates were obtained from Addgene (SHP2: #8381, EGFR: #81926, Her2: #16257, Her4: #29527).

Techniques: Sublimation, Phospho-proteomics, Expressing, Activation Assay