Study Of Targeted Inhibitors Intervening SPOP Pathway Against Renal Carcinoma

ObjectiveApproximately 90%of patients with Renal Carcinoma have detected VHL mutations,which inactivate VHL protein.The inactivation of VHL protein lost the ability to regulate HIF gene,which leads to the over-activation of HIF gene.HIF gene can regulate the expression of SPOP gene at the transcriptional levels,and the over-expressed SPOP protein was mislocated in the cytoplasm and accumulated in a large amount in the cytoplasm.The overexpression of SPOP leads to the excessive degradation of downstream protein substrate,resulting in the activation of cancer-promoting gene-related pathways and accelerating cell proliferation,thereby promoting the occurrence of Renal Carcinoma.Studies have shown that SPOP protein binds to substrate proteins through the MATH domain,and binds to Cullin3 through the BTB domain.The purpose of this study is to find inhibitors that disrupt the interaction between SPOP and substrate proteins,inhibit the proliferation of Renal Carcinoma,thereby inhibiting the development of Renal Carcinoma.MethodStudy on chemical synthesis of small molecule inhibitors:First,the structure of the lead compound 6b was optimized,and the 65 compounds obtained.Then using nanoDSF method to screen the compounds to determine whether the compounds can bind to the SPOP protein;next using MTT method to screen 65 compounds to determine the effect of the compounds on the anti-proliferation effect of ccRCC cells.Combined with nanoDSF and MTT,it is determined that compound 61c can bind to SPOP protein in vitro and has strong anti-ccRCC cell proliferation activity.Then using nanoDSF,conventional DSF,NMR and SPR methods to confirm whether 61c can bind to SPOP protein in vitro;using DARTS experiment to verify whether 61c can improve the stability of the SPOP protein in cell;confirming whether 61c can destroy the interaction between SPOP and PTEN in cell through CO-IP experiment and UB experiment;using Western bolt method to verify the effect of 61c on SPOP-related pathway proteins.Next,in order to find new structural compounds for SPOP inhibitors,I screened the MCE Chinese medicine monomer compound library.First,use the fluorescence polarization FP method to detect whether the compound can bind to SPOP.and high-throughput screening of 720 small molecules in the MCE Chinese medicine monomer compound library was preformed;then use the conventional DSF to verify the 66 active ingredients of traditional Chinese medicine.The GST-Pull down experiment was used to determine whether small molecules can disrupt the interaction between SPOP and PTEN in vitro,and found that the small molecule of Chinese medicine Anacardic Acid.Then,colony formation assay and wound healing experiment were performed to verify the anti-cell proliferation and anti-cell migration effects of Anacardic Acid on ccRCC cells.Then use CETSA to verify whether Anacardic Acid can improve the thermal stability of SPOP protein in cells;through CO-IP experiment and UB experiment to confirm whether Anacardic Acid can destroy the interaction between SPOP and PTEN in cells.Finally,the Western bolt was used to verify the effect of Anacardic Acid on SPOP-related pathway.Researchers have found that most of the substrates that MATH binds contain SBC motifs.Based on this theoretical basis,this project wants to study SPOP peptide inhibitors.First,the method of computer virtual screening is adopted,the virtual screened peptide molecular fragments were scored and judged,and 101 peptides that might bind to the SPOP protein were determined.Fluorescence polarization FP method was used to verify whether the selected 101 synthetic peptides could compete with the substrate to bind to the SPOP protein,the FP experiment obtained the peptide Pep38.In order to determine why the peptide Pep38 can compete with the natural substrate to bind to the SPOP protein,the complex crystal structure of Pep3 8 and SPOPMATH was analyzed;then verify the membrane permeability of Pep38 and the peptide TAT38 with penetrating peptide added to the N-terminus the intracellular using laser confocal experiment.In vitro GST Pull-down experiment was used to verify the ability of Pep38 and TAT38 to inhibit the interaction of SPOP-substrate protein;Co-IP experiment was used to to verify the effect of Pep38 or TAT38 on the interaction between SPOP and PTEN protein in the cell lysate;Co-IP and UB experiments were used to verify the effect of TAT38 on the interaction between SPOP and PTEN proteins and the level of substrate protein ubiquitination in whole cells;Western bolt method to verify the effect of TAT38 on SPOP-related pathway proteins.Then the 11 peptides added with different types of penetrating peptides were verified to obtain peptides with better activity.The MTT experiment determined the effect of 11 peptides on the anti-proliferative activity of ccRCC cells;the GST Pull-down and Western bolt experiments were used to verify the effect of Pep38-3 and Pep38-9 on the interaction of SPOP-substrate protein.ResultsOptimize the structure of 6b from 5 ways,and 65 compounds were obtained.Combined with nanoDSF and MTT experiments,it is determined that compound 61c has strong binding ability to SPOP protein,and 61c has strong anti-proliferative activity on two kinds of ccRCC cells,with IC50 of 2.1?M and 3.5 ?M,respectively.In addition,compound 61c significantly inhibited the colony formation of A498 and OS-RC-2 cell lines,which was better than the previously reported 6b and other structural analogs.DSF,NMR titration and SPR experiments all confirmed that 61c can bind to SPOP protein in vitro.DARTS experiment verified that 61c can improve the stability of SPOP protein in cells,CO-IP and UB experiments proved that 61c destroyed The interaction between SPOP and substrate protein in two ccRCC cell lines,leading to the stabilization and accumulation of tumor suppressor factors PTEN and DUSP7,and reducing the abundance of downstream p-AKT and p-ERK protein abundance.High-throughput screening of traditional Chinese medicine using fluorescence polarization FP method,the results showed that 66 of the 720 active ingredients of traditional Chinese medicine were bound to the SPOPMATH protein domain.DSF method confirmed that 17 small Chinese medicine molecules have specific binding to SPOPMATH.In vitro GST-Pull down experiment verified that Anacardic Acid can destroy the interaction between SPOP and PTEN.Intracellular CETSA experiment verified that Anacardic Acid can stabilize intracellular SPOP protein,intracellular CO-IP and ubiquitination experiments verified that Anacardic Acid can disrupt the interaction between SPOP and PTEN,and inhibited the ubiquitination level of substrate proteins,increased the level of tumor suppressor gene substrate protein in SPOP-related pathways and decreased the level of phosphorylated protein related to oncogenes,thereby inhibiting oncogene pathways.The results of clone formation experiment and cell scratch experiment showed that anacardic acid can inhibit the proliferation and migration of renal hyaluronic cell carcinoma cells A498 and OS-RC-2.Based on computer virtual screening method,a total of 101 peptides were obtained.The peptide Pep38 was finally obtained after verification by fluorescence polarization FP experiment.After analyzing the crystal structure complex,it was found that Pep38 can be specifically recognized by the MATH domain.The extra amino acids in the peptide inhibitor Pep3 8 form a stable ? chain,thereby enhancing the binding of the inhibitor to the MATH domain.The addition of the TAT penetrating peptide sequence improves the permeability of the polypeptide,which allows TAT38 to retain the ability to disrupt the interaction between SPOP and PTEN proteins in vitro and in ccRCC cells.TAT38 inhibits the ubiquitination level of the substrate protein in the cell,inhibits the degradation of tumor suppressor protein in cancer cells,thereby inhibiting the proliferation of clear cell Renal Cell Carcinoma cell lines A498 and OS-RC-2.Through further optimization and adding different penetrating peptides,peptides with better activity were found,Pep38-3 and Pep38-9,but they could not inhibit the SPOP signaling pathway at the cellular level.ConclusionThrough structural modification,a series of 6b structural analogs were synthesized to clarify the structure-activity relationship of the compound,and verify how the small chemical molecule 61c interferes with the SPOP pathway to against kidney cancer.Screening of SPOP Chinese medicine inhibitor from MCE Chinese medicine monomer compound library,hoping to screen out small molecules with new structures for subsequent optimization to obtain SPOP inhibitors with better activity,from which the candidate compound anacardic acid-AA was screened and verified what is the effect of AA against kidney cancer.The screening of peptide inhibitors,the peptide Pep38,the TAT38 which can pass through the cell membrane,answered the impact of chemical intervention in SPOP against renal cancer from different aspects,and also suggested the possibility of developing SPOP peptide inhibitors.