Journal: 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: Figure 1. 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 (pY542 and pY580) 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. (D) 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: For recombinant expression of the full-length SHP2 (1-594), a glutathione S-transferase (GST) fusion construct in pGEX-4T1 was used to produce a thrombin-cleavable construct (Addgene plasmid #8322).

Techniques: Control, Drug discovery, Cell Stimulation, Phospho-proteomics, Binding Assay, Activity Assay, Over Expression, Scaffolding, Activation Assay, Inhibition

Journal: Chemical reviews

Article Title: Regulatory Mechanisms and Novel Therapeutic Targeting Strategies for Protein Tyrosine Phosphatases

doi: 10.1021/acs.chemrev.7b00105

Figure Lengend Snippet: Allosteric regulation of SHP2. (A) The schematic representation of SHP2 structure and allosteric regulation. (B) The comparison of N-SH2 domain conformation at I (gray) and A (cyan) state. (C) The pY peptide binding surface in N-SH2 domain at I and A state. BG- and EF-loop are depicted in purple and yellow, respectively. (D) The N-SH2/PTP interaction surface in the N-SH2 domain at the I and A state.

Article Snippet: 262 – 263 Utilizing the full-length SHP2 construct and screening for compounds that can block SHP2 activation by an N-SH2 domain binding pTyr peptide, the Novartis team identified several small molecule compounds that stabilize the auto-inhibited SHP2 conformation.

Techniques: Binding Assay

Journal: Chemical reviews

Article Title: Regulatory Mechanisms and Novel Therapeutic Targeting Strategies for Protein Tyrosine Phosphatases

doi: 10.1021/acs.chemrev.7b00105

Figure Lengend Snippet: Disease associated SHP2 mutations. (A) NS/cancer-associated SHP2 mutations mainly reside at the interface of N-SH2 and PTP domains. (B) LS-associated SHP2 mutations only appear within the PTP domain.

Article Snippet: 262 – 263 Utilizing the full-length SHP2 construct and screening for compounds that can block SHP2 activation by an N-SH2 domain binding pTyr peptide, the Novartis team identified several small molecule compounds that stabilize the auto-inhibited SHP2 conformation.

Techniques:

Journal: Chemical reviews

Article Title: Regulatory Mechanisms and Novel Therapeutic Targeting Strategies for Protein Tyrosine Phosphatases

doi: 10.1021/acs.chemrev.7b00105

Figure Lengend Snippet: LS SHP2 mutations reduce SHP2 phosphatase activity by disturbing different step(s) in the catalytic process. In this figure, SHP2 wild-type (gray) and mutant (green) were superimposed onto PTP1B (cyan) structure representing transition state 1 or 2 to show mutation-induced disturbance at each specific step. Residue numbers are shown in blue for PTP1B and black for SHP2. Red dash lines represent mutation induced steric conflicts.

Article Snippet: 262 – 263 Utilizing the full-length SHP2 construct and screening for compounds that can block SHP2 activation by an N-SH2 domain binding pTyr peptide, the Novartis team identified several small molecule compounds that stabilize the auto-inhibited SHP2 conformation.

Techniques: Activity Assay, Mutagenesis

Journal: Chemical reviews

Article Title: Regulatory Mechanisms and Novel Therapeutic Targeting Strategies for Protein Tyrosine Phosphatases

doi: 10.1021/acs.chemrev.7b00105

Figure Lengend Snippet: The binding modes for two allosteric PTP inhibitors. (A) SHP099 binds at the inter-domain interfaces of SHP2 to stabilize the autoinhibited conformation. (B) Analog 3 binds at the trimer interfaces of PRL1 to prevent trimer formation.

Article Snippet: 262 – 263 Utilizing the full-length SHP2 construct and screening for compounds that can block SHP2 activation by an N-SH2 domain binding pTyr peptide, the Novartis team identified several small molecule compounds that stabilize the auto-inhibited SHP2 conformation.

Techniques: Binding Assay

Journal: bioRxiv

Article Title: The pathogenic T42A mutation in SHP2 rewires the interaction specificity of its N-terminal regulatory domain

doi: 10.1101/2023.07.10.548257

Figure Lengend Snippet: ( A ) Domain architecture diagram of SHP2. Relevant mutations and the catalytic cysteine (Cys 459 ) are indicated. ( B ) SHP2 is kept in its auto-inhibited state by interactions between the N-SH2 and PTP domain (PDB: 4DGP). In its active state, the catalytic cysteine is accessible. The structure of SHP2 E76K (PDB: 6CRF) is used to represent the active state. ( C ) The SH2 domains of SHP2 bind to upstream phosphoproteins, such as transmembrane receptors, inducing a conformational change that activates SHP2. ( D ) Disease-associated mutations cluster largely, but not exclusively, on the interdomain interface between the N-SH2 and the PTP domain (PDB: 4DGP). The E76K mutation is a canonical N-SH2/PTP interface mutation. ( E ) Mutations in or near the N-SH2 binding pocket (PDB: 6ROY). ( F ) Mutations in or near the C-SH2 binding pocket (PDB: 6R5G). The well-established specificity-determining regions of the SH2 domains, which dictate +1 to +5 residue preferences, are marked with black dashed lines.

Article Snippet: The SHP2 full-length, wild-type gene used as the template for all SHP2 constructs in this study was cloned from the pGEX-4TI SHP2 WT plasmid, which was a generous gift from Ben Neel (Addgene plasmid #8322) ( ).

Techniques: Mutagenesis, Binding Assay, Residue

Journal: bioRxiv

Article Title: The pathogenic T42A mutation in SHP2 rewires the interaction specificity of its N-terminal regulatory domain

doi: 10.1101/2023.07.10.548257

Figure Lengend Snippet: ( A ) Measured binding affinities of N-SH2 WT against peptides derived from various known SHP2 interactors ( B ) Fold-change in K D for N-SH2 T42A compared to N-SH2 WT , for each of the peptides shown in panel (A). ( C ) Same as (B), but for N-SH2 L43F . ( D ) Same as (B), but for N-SH2 T52S . For ( A )-( D ), N = 3–4 independent protein, peptide, and fluorescent peptide titrations. Source data can be found in .

Article Snippet: The SHP2 full-length, wild-type gene used as the template for all SHP2 constructs in this study was cloned from the pGEX-4TI SHP2 WT plasmid, which was a generous gift from Ben Neel (Addgene plasmid #8322) ( ).

Techniques: Binding Assay, Derivative Assay

Journal: bioRxiv

Article Title: The pathogenic T42A mutation in SHP2 rewires the interaction specificity of its N-terminal regulatory domain

doi: 10.1101/2023.07.10.548257

Figure Lengend Snippet: ( A ) Hydrogen bonding of Thr 42 in SHP2 N-SH2 WT to the phosphoryl group of phosphopeptide ligands in several crystal structures (PDB: 6ROY, 1AYA, 1AYB, 3TL0, 5DF6, 5X94, and 5X7B). ( B ) Representative structure of N-SH2 WT bound to the PD-1 pTyr 223 (ITIM) peptide at the end of one MD simulation. N = 3 simulations of 1 μs each. ( C ) Representative structure of N-SH2 T42A bound to the PD-1 pTyr 223 (ITIM) peptide at the end of one MD simulation. N = 3 simulations of 1 μs each. ( D ) Overlay of the representative states shown in panels B and C. The N-SH2 WT state is in yellow with a dark-gray ligand. The N-SH2 T42A state is in light gray, with a light gray ligand. ( E ) Distribution of distances between the Lys 55 Nζ atom and the phosphotyrosine phosphorus atοm in simulations of the PD-1 pTyr 223 peptide bound to N-SH2 WT (black) or N-SH2 T42A (red). ( F ) Distribution of distances between the Lys 55 Nζ atom and the +2 Glu Cδ atom in simulations of the PD-1 pTyr 223 peptide bound to N-SH2 WT (black) or N-SH2 T42A (red). ( G ) An ion pair between Lys 55 and the +2 Glu residue (Glu 225) in the PD-1 pTyr 223 (ITIM) peptide, frequently observed in N-SH2 T42A simulations. ( H ) Peptide-specific effects of the T42A mutation in the presence and absence of the K55R mutation. N = 2–5 independent titrations.

Article Snippet: The SHP2 full-length, wild-type gene used as the template for all SHP2 constructs in this study was cloned from the pGEX-4TI SHP2 WT plasmid, which was a generous gift from Ben Neel (Addgene plasmid #8322) ( ).

Techniques: Phospho-proteomics, Residue, Mutagenesis

Journal: bioRxiv

Article Title: The pathogenic T42A mutation in SHP2 rewires the interaction specificity of its N-terminal regulatory domain

doi: 10.1101/2023.07.10.548257

Figure Lengend Snippet: ( A ) SHP2 activation is measured by incubation with phosphopeptide ligands, followed by monitoring dephosphorylation of the small-molecule substrate DiFMUP to generate fluorescent DiFMU. ( B ) Representative activation curves for SHP2 WT . N = 3–17 independent titrations of protein, peptide, and DiFMUP. ( C ) Correlation between the EC 50 of SHP2 WT activation by phosphopeptides and the K D of those phosphopeptides for the N-SH2 WT domain. For EC 50 values in ( B )-( C ), N = 3 independent titrations of protein, peptide, and DiFMUP.( D ) Correlation between activation EC 50 values for SHP2 WT and SHP2 R138Q . For SHP2 R138Q EC 50 values, N = 3–5 independent titrations of protein, peptide, and DiFMUP. ( E ) Comparison of SHP2 WT and SHP2 T42A activation curves for the PD-1 pTyr248 peptide. N = 3–4 independent titrations of protein, peptide, and DiFMUP. ( F ) Comparison of SHP2 WT and SHP2 T42A activation curves for the Imhof-9 peptide. N = 6–17 independent titrations of protein, peptide, and DiFMUP. ( G ) Bubble plot juxtaposing the EC 50 values for activation of SHP2 WT and SHP2 T42A by nine peptides, alongside the fold-change in K D for binding of those peptides to N-SH2 WT vs N-SH2 T42A . The dotted line indicates where EC 50 for SHP2 WT equals EC 50 for SHP2 T42A . Peptides with a large fold-change in binding affinity (larger bubble) have a large fold-change in EC 50 values for SHP2 T42A over SHP2 WT (distance from dotted line). All EC 50 values can be found in .

Article Snippet: The SHP2 full-length, wild-type gene used as the template for all SHP2 constructs in this study was cloned from the pGEX-4TI SHP2 WT plasmid, which was a generous gift from Ben Neel (Addgene plasmid #8322) ( ).

Techniques: Activation Assay, Incubation, Phospho-proteomics, De-Phosphorylation Assay, Comparison, Binding Assay

Journal: bioRxiv

Article Title: The pathogenic T42A mutation in SHP2 rewires the interaction specificity of its N-terminal regulatory domain

doi: 10.1101/2023.07.10.548257

Figure Lengend Snippet: ( A ) Scheme depicting the co-immunoprecipitation experiments with SHP2 and either Gab1, Gab2, or PD-1 in HEK293 cells. SHP2 co-immunoprecipitation results with ( B ) Gab1, ( C ) Gab2, and ( D ) PD-1. For ( B ), ( C ), ( D ), N = 2, 3, and 4 independent cell transfections, respectively. Co-immunoprecipitation of Gab1/Gab2 was detected using an α-FLAG antibody and PD-1 was detected using a PD-1-specific antibody. Co-immunoprecipitation levels of each protein in T42A samples relative to wild-type are normalized for expression level and shown as bar graphs. ( E ) Schematic depiction of EGF stimulation and phospho-Erk signaling experiments in the presence of co-expressed SHP2 and either Gab1 or Gab2. ( F ) Comparison of phospho-Erk levels in response to EGF stimulation in cells expressing Gab1 and either SHP2 WT or SHP2 T42A . N = 4 independent cell transfections and separate stimulations. A paired, one-tailed t-test was used to test for significance. ( G ) Comparison of phospho-Erk levels in response to EGF stimulation in cells expressing Gab2 and either SHP2 WT or SHP2 T42A . N = 3 independent cell transfections and separate stimulations. A paired, one-tailed t-test was used to test for significance. For panels (F) and (G), the bar graphs below the blots indicate phospho-Erk levels, normalized to total Erk levels, relative to the highest p-Erk signal in SHP2 WT time course (2 minutes).

Article Snippet: The SHP2 full-length, wild-type gene used as the template for all SHP2 constructs in this study was cloned from the pGEX-4TI SHP2 WT plasmid, which was a generous gift from Ben Neel (Addgene plasmid #8322) ( ).

Techniques: Immunoprecipitation, Transfection, Expressing, Comparison, One-tailed Test