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Addgene inc full length shp2

Allosteric control mechanisms for <t>SHP2</t> 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.

Full Length Shp2, supplied by Addgene inc, used in various techniques. Bioz Stars score: 91/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "A cellular target engagement assay for the characterization of SHP2 (PTPN11) phosphatase inhibitors"

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

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.RA119.010838

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.

Figure Legend 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.

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

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

Techniques Used: Inhibition

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.

Figure Legend 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.

Techniques Used: Binding Assay, Mutagenesis, Inhibition

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.

Figure Legend 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.

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

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.

Figure Legend 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.

Techniques Used: Generated

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).

Figure Legend 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).

Techniques Used: Concentration Assay, Positive Control, Recombinant

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Phosphorylated Tg WIP binds to host cell tyrosine phosphatases Shp1 and Shp2. A Schematic showing the amino acid sequence of Tg WIP. Magenta color indicates the position of the three tyrosines in Tg WIP. Orange region represents the WRC interacting receptor sequence (WIRS) domain that could mediate Tg WIP’s interaction with the WAVE complex. Blue regions represent the proline rich regions (PRRs) that could mediate Tg WIP’s interactions with Nck and Grb2. B DCs were infected with parasites expressing wild-type Tg WIP ( Tg WIP WT ), or Tg WIP in which its SH2-binding motifs Y150 ( Tg WIP Y150A ), Y199 ( Tg WIP Y199A ), or both ( Tg WIP Y150A/Y199A ) are mutated to an alanine. Shown are the Western blots using phosphorylated tyrosine (pY) (1st membrane-probing antibody), HA (2nd membrane-probing antibody after stripping membrane), and Shp2 (3rd membrane-probing antibody after stripping membrane twice) antibodies on total lysate or immunoprecipitated Tg WIP. C The murine DC (DC2.4) and human foreskin fibroblast (HFF) cell lines were infected with type II ME49 Δtgwip complemented with HA-tagged wild-type Tg WIP ( Tg WIP WT ) for 4 h (intracellular) or until parasite egress (extracellular). Lysates were generated and Tg WIP immunoprecipitated. Shown are the Western blots using phosphorylated tyrosine (pY), HA, Shp1, and Shp2 antibodies, (probed in the same order as in C). D DC2.4 were treated with the Src kinase inhibitor Dasatinib at the indicated concentrations for 1 h and throughout infection with WT Toxoplasma . DMSO is the solvent control. Western blot analysis using antibodies against pY and HA. DCs were infected with Tg WIP WT or with either E Tg WIP Y150A or with F Tg WIP Y150A/Y199A Toxoplasma. Shown are the Western blots using antibodies against Shp2 in E and Shp1 in F . G-H In vitro phosphatase assay of Shp2 in G Shp1 in H incubated with recombinant Tg WIP, with or without prior phosphorylation by recombinant Src kinase. Reaction rates, represented by linear fluorescence changes over time, were normalized against the rate of Shp2 or Shp1 without Tg WIP. Data from 3 biological repeats are indicated by different colors, with each data point showing the average from three technical repeats. Bar graph represents the mean and standard error of the 3 biological repeats. ANOVA with Tukey’s post hoc test was used. n = 3; *** for p < 0.0005 against the first column). n.s.: not significant. I-J Coomassie blue-stained SDS-PAGE gel showing GST-tagged, Src-phosphorylated Tg WIP (GST-TgWIP P ) pulling down MBP-tagged full-length (FL) or individual SH2 domains of Shp2 in I or Shp1 in J . Loading controls contain 6 pmol of indicated prey proteins

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: The Toxoplasma secreted effector Tg WIP modulates dendritic cell motility by activating host tyrosine phosphatases Shp1 and Shp2

doi: 10.1007/s00018-024-05283-3

Figure Lengend Snippet: Phosphorylated Tg WIP binds to host cell tyrosine phosphatases Shp1 and Shp2. A Schematic showing the amino acid sequence of Tg WIP. Magenta color indicates the position of the three tyrosines in Tg WIP. Orange region represents the WRC interacting receptor sequence (WIRS) domain that could mediate Tg WIP’s interaction with the WAVE complex. Blue regions represent the proline rich regions (PRRs) that could mediate Tg WIP’s interactions with Nck and Grb2. B DCs were infected with parasites expressing wild-type Tg WIP ( Tg WIP WT ), or Tg WIP in which its SH2-binding motifs Y150 ( Tg WIP Y150A ), Y199 ( Tg WIP Y199A ), or both ( Tg WIP Y150A/Y199A ) are mutated to an alanine. Shown are the Western blots using phosphorylated tyrosine (pY) (1st membrane-probing antibody), HA (2nd membrane-probing antibody after stripping membrane), and Shp2 (3rd membrane-probing antibody after stripping membrane twice) antibodies on total lysate or immunoprecipitated Tg WIP. C The murine DC (DC2.4) and human foreskin fibroblast (HFF) cell lines were infected with type II ME49 Δtgwip complemented with HA-tagged wild-type Tg WIP ( Tg WIP WT ) for 4 h (intracellular) or until parasite egress (extracellular). Lysates were generated and Tg WIP immunoprecipitated. Shown are the Western blots using phosphorylated tyrosine (pY), HA, Shp1, and Shp2 antibodies, (probed in the same order as in C). D DC2.4 were treated with the Src kinase inhibitor Dasatinib at the indicated concentrations for 1 h and throughout infection with WT Toxoplasma . DMSO is the solvent control. Western blot analysis using antibodies against pY and HA. DCs were infected with Tg WIP WT or with either E Tg WIP Y150A or with F Tg WIP Y150A/Y199A Toxoplasma. Shown are the Western blots using antibodies against Shp2 in E and Shp1 in F . G-H In vitro phosphatase assay of Shp2 in G Shp1 in H incubated with recombinant Tg WIP, with or without prior phosphorylation by recombinant Src kinase. Reaction rates, represented by linear fluorescence changes over time, were normalized against the rate of Shp2 or Shp1 without Tg WIP. Data from 3 biological repeats are indicated by different colors, with each data point showing the average from three technical repeats. Bar graph represents the mean and standard error of the 3 biological repeats. ANOVA with Tukey’s post hoc test was used. n = 3; *** for p < 0.0005 against the first column). n.s.: not significant. I-J Coomassie blue-stained SDS-PAGE gel showing GST-tagged, Src-phosphorylated Tg WIP (GST-TgWIP P ) pulling down MBP-tagged full-length (FL) or individual SH2 domains of Shp2 in I or Shp1 in J . Loading controls contain 6 pmol of indicated prey proteins

Article Snippet: The impact of phosphorylated Tg WIP on both Shp1 and Shp2 activity was measured using a Shp2 activity assay kit (BPS bioscience, Cat# 79,330 [ ]), following the manufacturer’s instructions.

Techniques: Sequencing, Infection, Expressing, Binding Assay, Western Blot, Membrane, Stripping Membranes, Immunoprecipitation, Generated, Solvent, Control, In Vitro, Phosphatase Assay, Incubation, Recombinant, Fluorescence, Staining, SDS Page

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

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.

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.

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.

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).

Techniques: Concentration Assay, Positive Control, Recombinant

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