Streamlined Methods for Profiling Targeted Michael Acceptor Molecules Reveal Natural PTP1B Covalent Inhibitors

In natural products, Michael acceptor molecules (MAMs) contain electrophilic groups capable of forming covalent bonds with protein targets, making them promising candidates for covalent drug development. Leveraging the reactivity of MAMs, we developed a streamlined workflow to profile MAMs in natural products and investigate potential MAM-protein covalent interactions. Using the methoxypolyethylene glycol maleimide (mPEG) tag-shift technique, we have demonstrated that <i>Andrographis paniculata</i> extract exhibits covalent inhibition of protein tyrosine phosphatase 1B (PTP1B). Subsequent thiocholine iodide (TCI) labeling and mass spectrometry analysis revealed that compounds found in <i>A. paniculata</i> extract possess thiol-reactive properties and covalently inhibit PTP1B. Among these compounds, oridonin is the key agent modifying PTP1B, inhibiting PTPase activity, and inducing the accumulation of tyrosine-phosphorylated proteins in cells. Further studies have revealed that oridonin covalently modifies both Cys92 and the active-site residue Cys215 of PTP1B. Additionally, oridonin enhances the phosphorylation of IRβ, Akt, and ERK, while also upregulating Glut4 expression and promoting glucose uptake in cultured cells. These findings provide new insights into the role of oridonin in PTP1B inhibition, insulin signaling, and glucose metabolism. Furthermore, the successful application of TCI labeling for MAM discovery, alongside the mPEG tag-shift technique for target identification, highlights their potential utility in covalent drug development.