Journal: EMBO Molecular Medicine

Article Title: SFX-01 is therapeutic against myeloproliferative disorders caused by activating mutations in Shp2

doi: 10.1038/s44321-025-00267-7

Figure Lengend Snippet: ( A ) Recombinant human Shp2 activity after 30-min incubation with SFX-01. Data represent mean activity ( ± SEM; n = 3 biological replicates) and were fitted to a one-phase exponential decay curve (gray line; r 2 = 0.987). ( B ) Shp2 activity after incubation with SFX-01 for the indicated times and concentrations. Data shown are mean activity ( ± SEM; n = 3 biological replicates). ( C ) Shp2 activity after 30-minute incubation with or without bisphosphorylated IRS1 and SFX-01. Bar represents mean activity (± SEM; n = 4 biological replicates) and P values calculated by two-way ANOVA with Sîdak post hoc test. ( D ) Representative immunoblots showing SFN-modification of recombinant Shp2 (1.6 nM) following incubation with 1.75 or 0.109 µM SFX-01 for 30 min. ( E ) Precursor isotopic envelop spectrum of 0.1 µM recombinant human Shp2 protein incubated with equimolar SFN for 6 h at 37 °C corresponding to a dithiolethione modification adducted between Cys 333 and Cys 367 . ( F ) Schematic representing the proposed mechanism of Shp2-dithiolethione formation by SFN. The isothiocyanate group reacts with a cysteine residue to form a dithiocarbamate intermediate, which further reacts with a second cysteine residue to yield the dithiolethione modification. ( G ) Immunoblot of immunoprecipitated WT or active site mutant Shp2 and SFN-modification from HEK cells treated with SFX-01. “E” represents non-transfected cells, and 0 h represents untreated cells. The graph represents densitometric analysis of Shp2-SFN adduct formation in WT or mutant Shp2 exposed to SFX-01 for 2 or 4 h. Bars represent mean values (± SEM; n = 3 biological replicates) and P values calculated by two-way ANOVA with Sîdak post hoc test. .

Article Snippet: bisphosphorylated IRS1 peptide , BPS Bioscience , N/A.

Techniques: Recombinant, Activity Assay, Incubation, Western Blot, Modification, Residue, Immunoprecipitation, Mutagenesis, Transfection

Journal: EMBO Molecular Medicine

Article Title: SFX-01 is therapeutic against myeloproliferative disorders caused by activating mutations in Shp2

doi: 10.1038/s44321-025-00267-7

Figure Lengend Snippet: ( A ) Recombinant human Shp2 activity after 30-min incubation with SFX-01. Data represent mean activity ( ± SEM; n = 3 biological replicates) and were fitted to a one-phase exponential decay curve (gray line; r 2 = 0.987). ( B ) Shp2 activity after incubation with SFX-01 for the indicated times and concentrations. Data shown are mean activity ( ± SEM; n = 3 biological replicates). ( C ) Shp2 activity after 30-minute incubation with or without bisphosphorylated IRS1 and SFX-01. Bar represents mean activity (± SEM; n = 4 biological replicates) and P values calculated by two-way ANOVA with Sîdak post hoc test. ( D ) Representative immunoblots showing SFN-modification of recombinant Shp2 (1.6 nM) following incubation with 1.75 or 0.109 µM SFX-01 for 30 min. ( E ) Precursor isotopic envelop spectrum of 0.1 µM recombinant human Shp2 protein incubated with equimolar SFN for 6 h at 37 °C corresponding to a dithiolethione modification adducted between Cys 333 and Cys 367 . ( F ) Schematic representing the proposed mechanism of Shp2-dithiolethione formation by SFN. The isothiocyanate group reacts with a cysteine residue to form a dithiocarbamate intermediate, which further reacts with a second cysteine residue to yield the dithiolethione modification. ( G ) Immunoblot of immunoprecipitated WT or active site mutant Shp2 and SFN-modification from HEK cells treated with SFX-01. “E” represents non-transfected cells, and 0 h represents untreated cells. The graph represents densitometric analysis of Shp2-SFN adduct formation in WT or mutant Shp2 exposed to SFX-01 for 2 or 4 h. Bars represent mean values (± SEM; n = 3 biological replicates) and P values calculated by two-way ANOVA with Sîdak post hoc test. .

Article Snippet: In all, 25 μl of assay buffer (25 mM Tris-HCl, 75 mM NaCl, 2 mM EDTA, 0.16 mM DTT pH 7.2) was added to 25 μl of Shp2-containing solution of either: 0.011 ng/μl recombinant human Shp2 protein (Abcam) (desalted into an experimental buffer (200 μM DiFMUP, 25 mM Tris-HCl, 75 mM NaCl, 0.05% Tween-20, 2 mM EDTA, 0.16 mM DTT, pH 7.2)) which had been treated for 0.5, −6 h −/+ 0.007–1.75 μM SFX-01 and/or 0.5 μM of bisphosphorylated IRS1 peptide (sequence: H2N-LN(pY)IDLDLV(dPEG8)LST(pY)-ASINFQK-amide (BPS Bioscience), or Shp2 immunoprecipitated from cardiac or spleen tissue of WT or Ptpn11 D61G (−/+) mice treated or untreated with SFX-01 for 4 days to 10 weeks, +/− subsequent 30 min treatment with 5 mM DTT.

Techniques: Recombinant, Activity Assay, Incubation, Western Blot, Modification, Residue, Immunoprecipitation, Mutagenesis, Transfection