Screening The Inhibitors Of Protein Tyrosine Phosphatase Shp2 From Natural Product

In recent years,cancer has become an important disease that threatens our health,due to ageing population,poor living habits,environmental pollution,and low cure rate of traditional methods.However,many cancers lack effective medications.With the development of molecular cell biology,molecular targeted therapy has received more and more attention from researchers and clinicians.It is an effective cancer treatment method that has been widely studied.In the clinic,successful drugs have been used for the treatment of individual cancers,effectively prolonging the survival of patients.In the occurrence and development of cancer,kinases and phosphatases play an important role.Phosphorylation and dephosphorylation mediated by kinase and phosphatase is a basic cellular signaling regulation.Many cell functions are regulated by reversible phosphorylation,abnormalities of reversible phosphorylation can induce the production of a variety of diseases,such as inflammation,tumors.Shp2,an intracellular non-receptor protein tyrosine phosphatase encoded by the PTPN11 gene,is a member of the PTPs family.It is widely expressed in embryos and various adult tissues and participates in the regulation of various signaling pathways,such as Ras/Erk,PI3K/Akt,and Jak/Stat,thus regulating cell proliferation,differentiation,migration,and apoptosis.Previous studies have shown that Shp2 is a proto-oncogene that can induce the occurrence of cancers and has a promoting effect in leukemia,breast cancer,gastr:ic cancer,non-small cell lung cancer and other diseases,gain-of-function or loss-of-function mutations of Shp2 will cause Noonan syndrome,LEOPARD syndrome and JMML,animal experiments show that knocking out the Shp2 gene or inhibiting the high activity of Shp2 can block or delay the development of tumors.Therefore,Shp2 is a excellent target for drug development,developing the specific inhibitors of Shp2 is of great practical significance.In order to discover inhibitors of protein tyrosine phosphatase Shp2,we used the Escherichia coli expression system to express the catalytic domain of Shp2(PTP-Shp2 protein)and Shpl protein,VHR protein,HePTP protein which is belong to the protein tyrosine phosphatase family,these protein are used as a test to determine whether the inhibitors we screened specifically inhibit PTP-Shp2.Next,we established an in vitro high-throughput screening model of enzyme and substrate reactions based on pNPP(a universal substrate for protein tyrosine phosphatases).First,using this model,we screened the total of 223 kinds of endophytic active ingredients of 4 kinds of Chinese herbs,including Radix pseudostellariae,Carpet Bugle,Anoectochilus roxburghii,Russulaceae and found that an active fraction of the endophytic bacteria of Carpet Bugle(named g28)is a specific inhibitor of PTP-Shp2;from the crude fraction of 30 kinds of traditional Chinese medicine,we found the crude fractions of Houttuynia cordata,Dandelion,Sarcandra glabra,and Prunella vulgaris specifically inhibit PTP?Shp2;Next,screening from 329 kinds of natural monomers of Chinese herbal medicine,H320(isoferulic acid)and H335(cinnamic acid)were found to have a strong inhibitory effect on PTP-Shp2.As for cellular experiments,the results indicated that H320 can inhibit the activation of Shp2-dependent MAPK induced by EGF in 293T cells.Previous studies found that MDA-MB-468 cells depend on Shp2 for proliferation and migration,and we used H335 to treat MDA-MB-468 cell line and the MDA-MB-468 cell line stably expressed Shp2 Q79P,We found that H335 can inhibit the activation of Shpt-dependent MAPK induced by EGF and can inhibit the migration of MDA-MB-468 cells.Therefore,we identified two specific inhibitors of Shp2,H320(isoferulic acid)and H335(cinnamic acid),from the natural monomers of traditional Chinese medicines for the first time,and through related cell function studies and structural transformation,H320 and H335 are hopeful to be developed as drugs for the treatment of Shp2 related cancers.