61 - Sulforaphane-mediated inhibition of SHP2 as a potential pharmacotherapy for Noonan syndrome

Abstract

Sulforaphane (SFN) is an electrophilic isothiocyanate capable of post-translationally modifying protein cysteine thiolates. Immunoprecipitation using a validated polyclonal antibody developed in-house to pan-specifically detect SFN adducted to cysteines, combined with quantitative proteomics confirmed the protein tyrosine phosphatase SHP2 as a target of SFN in cardiac tissue of wildtype (WT) mice orally administered the compound. A chemically-stabilised variant of SFN known as Sulforadex (SFX-01), which was used in subsequent studies, inhibited recombinant SHP2 in vitro, as well as that in cardiac tissue of mice administered the compound for 7-days. We speculated that SFX-01 may be therapeutic in diseases where SHP2 is hyperactive, such as Noonan syndrome. Indeed, using a Noonan syndrome mouse model, Ptpn11D61G/+, SFX-01 time-dependently inhibited cardiac SHP2 activity. Ptpn11D61G/+ mice, like many humans with Noonan syndrome, show myeloproliferative disease characterised by increased white blood cell count, spleen size, as well as increased myeloid cell number in blood, bone marrow and spleen. Sustained treatment of Ptpn11D61G/+ mice with SFX-01 normalised these myeloproliferation indices towards WT levels. SFX-01 also attenuated spleen SHP2 phosphatase activity in both WT and Ptpn11D61G/+ mice, consistent with this inhibitory mechanism mediating the therapeutic action of this electrophilic drug. Biochemical analyses showed SFN induced SHP2 oxidation in cardiac tissue of WT and Ptpn11D61G/+ mice despite the electrophile itself not remaining conjugated to the phosphatase. Using bovine serum albumin or SHP2 with conjugated SFN, we observed transfer (i.e. trans-thiolation) of the adducted SFN to other thiol-containing molecules such as haemoglobin or glutathione. SFN-labelling studies with SHP2 cysteine-to-serine mutants support the concept that the electrophile initially adducts the phosphatase, but a proximal cysteine thiol mediates its removal, which is associated with the formation of an inhibitory disulfide bond. In conclusion, SFX-01 shows promise in the treatment of Noonan syndrome by normalising the hyperactive SHP2 phosphatase activity that characterises these patients.