Discovery Of SHP2 Small Molecule Degraders And Its Anti-tumor Effect Exploration

PTPN11 is the first confirmed proto-oncogene in the tyrosine phosphatase family,and SHP2 is the non-receptor tyrosine phosphatase encoded by PTPN11.It is involved in RAS/RAF/ERK、PI3K/AKT and JAK-STAT signaling pathways,and is abnormal in various malignant tumors including leukemia,non-small cell lung cancer,breast cancer and other malignant tumors,and is an important potential target of anti-tumor.Due to the high conservatism of the PTP family,the development of SHP2 small molecule inhibitors has been slow until 2016,when Novartis first discovered SHP099,a highly selective allosteric inhibitor of SHP2.Subsequently,several literatures reported the anti-tumor effect and mechanism of SHP099 in inhibiting SHP2 phosphatase activity.Currently,several SHP2 small molecule inhibitors have entered clinical trials,but the skeleton of small molecule inhibitors is relatively simple.In addition,SHP2 is mutated in a variety of lymphomas,and SHP099significantly reduced its inhibitory ability against most of the mutants.Therefore,it is urgent to develop more SHP2 targeting regulatory molecules with different structures and different discovery strategies to promote the research and development of SHP2 targeting drugs.Proteolytic targeting chimera（PROTAC）has become a hot spot and frontier in the field of small molecule drug development.Proteolytic targeting chimera（PROTAC）can induce the degradation of target proteins by using the existing protein degradation system in cells to exert biological effects.At the time of project approval,this technology had not been reported to be useful in the targeted degradation of PTP,so whether PROTAC technology can be used in the degradation of PTP phosphatase,especially the targeted degradation of SHP2.Also its corresponding biological activities are unknown.Therefore,this paper explores the feasibility of using PROTAC technology to target SHP2 degradation,and explores whether the degradation of SHP2 protein through the ubiquitin-proteasome system can achieve the purpose of tumor growth inhibition.We used SHP099 parent nucleus structure as the binding part of SHP2 to explore the influence of different E3 ubiquitin ligase conjugates and connection lengths on SHP2degradation ability.Firstly,the activity evaluation of SHP2 degrader using CRBN,VHL and MDM2 as E3 were attempted.The promising molecule ZB-S-29 was found in 6 compounds.After the modification of Linker,a total of 14 PROTAC compounds were designed and synthesized using CRBN as E3,and the molecular level showed that the compounds all maintained good inhibitory activity against SHP2.The best degradation compound11（ZB-S-29）was further evaluated for its biological activity and mechanism of action.Gradient-dependent experiments showed that compound 11 exhibited a dose-dependent and time-dependent degradation of SHP2 protein,and the DC₅₀ of SHP2 degradation within 12h was 6.02 n M.In addition,compound 11 was superior to the positive compound SHP099 in inhibiting cell proliferation,inducing cell apoptosis and causing cell cycle arrest.Then,CRBN knockout、linalidomide competition and proteasome inhibitor reversal experiments confirmed that the down-regulation of compound 11 on SHP2 protein was dependent on the protein degradation pathway of CRBN,indicating that it was indeed a PROTAC molecule targeting SHP2 degradation.In addition,we also made a preliminary exploration for the extension of the indication of PROTAC-SHP2 compound,and found that compound 11 was sensitive to multiple myeloma MM1S cells.Further study on the mechanism showed that 11 not only degraded SHP2,but also had a good degradation effect on IKZF1,the degradation substrate of degree amine molecules.At present,SHP2 small molecule modulators mostly use SHP099 as the parent nucleus structure,so in this paper,we designed and synthesized several small molecules binding to SHP2 protein with non-SHP099 as the parent nucleus structure.In addition,since the regulation of activated mutated SHP2 is still an unsolved problem at present,preliminary exploration work has been carried out on small molecule degraders related to targeted mutated SHP2,which can provide an important reference for the molecular design of targeted degradation of mutated SHP2 in the future.To sum up,this study focused on SHP2 as an important target,and synthesized several series of potential SHP2 degraders using protein targeted degradation technology.Through systematic biological evaluation,compound 11,which has a good degradation effect on SHP2protein,was found to have a better inhibition effect on cell proliferation,and its activity was superior to that of the reported inhibitor SHP099.In addition,relevant attempts were made in expanding indications and designing different parent nucleus structures to synthesize degradation molecules of targeted mutant SHP2,which will provide reference for further work in the future.