Screening And Antitumor Activity Of USP28 Inhibitors

Protein ubiquitination is one of the post-translational modifications of proteins.The ubiquitination of proteins plays a key role in regulating protein stability and related cellular functions.The regulation of many important activities in cells are achieved by ubiquitination of target proteins.In addition,deubiquitination catalyzed by ubiquitinated enzymes(DUBs)plays a similarly important role in regulating many biochemical pathways.Ubiquitin specific peptidase 28(USP28)has the effect of reversing ubiquitination and directly affects the occurrence and development of tumor through various pathways.USP28 plays an important role in regulating DNA damage,expression of transcription factors.The study found that USP28 is highly expressed in various tumor tissues such as breast cancer,non-small cell lung cancer,bladder cancer and colorectal cancer,which directly promotes the development of tumors and has the potential for carcinogenesis.These physiological functions are through its deubiquitination.Therefore,the establishment of USP28 inhibitor screening platform to obtain high-efficiency,low-toxicity,high-selectivity USP28 inhibitor is a hot spot in the research of anti-tumor drugs.In this study,compound 14 was used as the target(IC50=12.26±0.27 ?M),and a series of new structur compounds were designed and synthesized.The inhibitory activity against USP28 was evaluated,and a highly effective and highly selective USP28 inhibitor was obtained.Furthermore,its inhibition of proliferation,colony formation and metastasis in tumor cells and the mechanism were studied at the cellular levels.The research results and main contents of this thesis include the following aspects:1.Establishment of USP28 inhibitor screening platform and screening of USP28 inhibitors.We constructed the prokaryotic expression vector of USP28 by molecular cloning methods,and used genetic engineering to express and isolate the USP28 recombinant protein.For selective screening,we isolated and purified USP28 subtype 3(M1-E583)with His The label and the full-length truncated body(M1-S700)were labeled with MBP,and experimentally verified,the USP28 full-length truncated body(M1-S700)was finally selected for subsequent experiments.Since USP28 declears the C-terminus of glycine at position 76 of the ubiquitin molecule linked to the substrate isopeptide bond during ubiquitination.This study used a fluorescent group 7-amino-4-methylcoumarin(Ubl-72-Leu73-Arg74-Gly75-Gly76-AMC,Ub-AMC)which is a fluorogenic peptide-modified ubiquitin-based glycine substrate.The enzymatic activity of the recombinant protein USP28 can be quantitatively analyzed by means of a microplate reader(PE Envision)using a dichroic mirror excitation light excitation wavelength of 355 nm by measuring the fluorescence intensity of the released AMC at an emission wavelength of 460 run.To determine the activity of USP28 and screen the inhibitory effect of small molecule compounds on its activity,a USP28 inhibitor screening platform was established.This method is also the most time-saving and sensitive method of detection.2.The inhibitory effect and binding force of USP28 inhibitors on the activity of USP28 enzyme at the recombinant protein expression level were studied.We used the fluorescence method to screen the in vitro enzyme activity,and screened more than 600 compounds in our group.After screening the active target compounds,we designed and synthesized a series of new compounds.Compound 29 has the strongest inhibitory effect on USP28,IC50 is 1.10±0.02 ?M,but has no inhibitory activity on recombinant protein LSD1,compound 24 as negative control with IC50>100 ?M.Through the Biolayer Interferometry Technology(BLI)and Isothermal Titration Calorimetry(ITC),the study found that compound 29 exhibits reversible binding to USP28 through non-covalent bond forms.The dose-dependent inhibition of USP28 activity,but the negative control compound 24 did not bind to USP28.3.Activity of USP28 Inhibitor at Cellular Level and Induction of LSD 1 and c-Myc Degradation and Its Mechanism.We found that in gastric cancer cells HGC-27,the small molecule compound 29 down-regulated the expression levels of LSD1 and c-Myc,but P53 is a mutant in gastric cancer cell HGC-27,not a substrate for USP28.Its expression level is up-regulated.The protein synthesis was inhibited by cycloheximide(CHX),and the proteasome inhibitor(MG-13 2)was simultaneously treated with the gastric cancer cells HGC-27,and then treated with a concentration gradient of the small molecule compound 29,and the treated cells were collected at different time points.After comparing the negative control group 24 with the solvent control group(DMSO),we found that after treatment with the small molecule compound 29,the degradation of the protein LSD1 and the total c-Myc was significantly accelerated,and the half-life was shortened.We concluded that compound 29 promoted the degradation of LSD 1 and total c-Myc by inhibiting the activity of USP28 and degraded LSD1 and c-Myc by the proteasome pathway.4.Evaluation of the effect of USP28 inhibitors on tumor cell proliferation and apoptosis at the cellular level.We found that in gastric cancer cells,compound 29 specifically inhibited the clonal formation of MGC-803,HGC-27,and MKN45,thereby inhibited cell proliferation.Whereas,the proliferation of HGC-27 cells were enhanced after siRNA-USP28 knockdown and the inhibitory effect of compound 29 on HGC-27 cell proliferation was weakened.In addition,compound 29 can cause changes in the expression levels of some apoptosis-related proteins,including increased expression of P53,P21,Cleaved PARP1,and Cleaved caspase-7,while the expression of anti-apoptotic protein BCL-2 decreases and the expression of pro-apoptotic protein BAX increases.Compound 29 induced P53-mediated apoptosis and arrested the cell cycle in S phase.Compound 29 specifically inhibited the formation of USP28 overexpressing gastric cancer cell lines MGC-803,HGC-27,and MKN45,thereby inhibiting cell proliferation.Conversely,when USP28 siRNA knocked down the expression of USP28 protein,HGC-27 cell proliferation ability was enhanced,and after adding compound 29 for 48 h,the inhibitory effect of compound 29 on HGC-27 cell proliferation was weakened.In addition,compound 29 can cause changes in the expression levels of some apoptosis-related proteins,including increased expression of P53,P21,Cleaved PARP1,and Cleaved caspase-7,while the expression of anti-apoptotic protein BCL-2 decreases and the expression of pro-apoptotic protein Bax increases.5.The inhibition of USP28 inhibitors on gastric cancer cell metastasis and EMT and its mechanism;Through scratch test and transwell experiment,compound 29 had potent inhibitory effects on the cell migration and EMT progression in vitro.We observed that E-Cadherin was upregulated and Vimentin was downregulated after compound 29 treatment,indicating the inhibitory influence of EMT progression by the inhibitor.The upregulated E-Cadherin level was probably the corresponding compound 29 mediated degradation of LSD 1,interestingly,also induced ZEB1 reduction which is one of the significant transcriptional repressor of E-Cadherin,thereby suppressing the EMT transition in the gastric cancer cell lines.Through the above research results,we synthesized a total of 33 new compounds with novel structures.Due to the fluorescence interference of 4 compounds,29 compounds of the USP28 enzyme activities were screened and evaluated,and the best compound 29 IC50 was 1.10±0.02 ?M.The mechanism of tumor action had been studied in depth.Compound 29 reversibly inhibited USP28 activity and selectively suppressed the proliferation and metastasis on MGC-803 and HGC-27,meanwhile halted the EMT progression of HGC-27.The results of this study reported for the first time a new class of structural compounds that induced the degradation of LSD1 and c-Myc by inhibiting USP28 and repressed the proliferation,colony formation and metastasis of gastric cancer cells at lower concentrations.The inhibition molecular mechanism of EMT in HGC-27 cells were also discussed.From the above studies,exploring a novel USP28 inhibitor with better enzymatic activity and low toxicity should be possible,it is important to develop a novel antitumor drug targeting the ubiquitin proteasome pathway.