Identification Of Intracellular Targets Of TAK-632 By Click Affinity Chromatography Combined With Limited Proteolysis

Objective: Recent studies have shown that necroptosis is a updated and regulatable way to lead to cell death.Hepatocellular carcinoma cell can perform necroptosis spontaneously by releasing dangerous signals known as dangerous-associated molecular patterns(DAMPs),promoting cancer cell proliferation and metastasis by influencing the tumor microenvironment.Therefore,further research on the mechanism of necroptosis and the developing new inhibitors has become the frontier of pharmaceutical biology.The result of high-throughput(HT)screening has showed that the anti-tumor drug TAK-632 is a powerful necroptosis inhibitor in both vivo and vitro.However,during the research of TAK-632’s structural optimization and structure-activity relationship,it suggests that even the smallest structure changing could significantly reduce its anti-necroptosis activity,but the activity of inhibitory RIPK1(Receptor-interprotein kinase 1)and RIPK3(Receptorinterprotein kinase 3)kinases remains.The face above reminds us that TAK-632 may also interact with other effect targets,or effect targets outside of RIPK kinase domain.Moreover,the effect targets within cells that can be effected by TAK-632 differentiate temporally and spatially.Therefore,the answer to what are the effect targets and binding sites of TAK-632 after cells performed necroptosis is critical to figure out and improve TAK-632’s structual mode,it can also promote the development of other necroptosis inhibitors and the ways they work.Methods: On the basis of current research on target identification,we first selected limited proteolysis‐coupled with mass spectrometry(Li P-MS)to analyze and verify the effect target and binding site of TAK-632,the optimal concentration of Proteinase K(PK)and small molecule drug TAK-632.Then,we developed a new method combining Click reaction with Li P-MS to characterize TAK-632’s effect on targets and binding sites during the necroptosis at living cells level.The pure target protein was selected for validation of a specific binding sites,which are compared with the previous Li P-MS results.Finally,by practising methods of molecular biology,surface plasmon resonance and computer molecular simulation docking we will study on the function of the new targets discovered in the process of necroptosis.Results: Firstly,we identified 10837 peptide in cell lysate by Li P-MS.With the cutoff of Fold change ≥ 1.5 times and P-value ≤ 0.05,2043 different peptides were found,corresponding to 770 differential proteins,among which there were 1964 conformotypic peptides up-regulated significantly in the TAK-632 group.A total of 50 proteins related to tumor and cell death were founded by literature review.We identified five conformotypic peptides targets: Parkinson disease protein 7(PARK7),Peroxiredoxin-1(Prdx1),Eukaryotictranslation initiation 5A-1(IF5A1),NAD(P)H dehydrogenase [quinone] 1(NQO1),Ephrintype-A receptor 2(EPHA2).Previous reseach showed that EPHA2 was a known target of TAK-632 and NQO1 is a quinone oxidoreductase,highly expressed in many tumor types,and closely related to the pathway of necroptosis.Secondly,Click affinity chromatography combined with Li P-MS was constructed to identify the target in living cells.A total of 10738 credible protein peptides were found.Through differential screening: Fold change ≥ 1.5 times and P-value ≤ 0.05.There were 711 differential peptides,432 corresponding differential proteins,of which 306 conformation-preserving peptides were significantly up-regulated in the TAK-632 group;Using Li P-MS and Click combined Li PMS methods,258 coincident proteins were identified by Wayne analysis.The specific functional information of 258 proteins was searched and 30 proteins related to tumor and cell death were found that the four protein targets of NQO1,Acidic inucleine-rich nuclearho32 family member A(ANP32A),Appoptosis-Inducing1,mitochondrikinal(AIFM1)and Cyclin-ase 1(CDK1)were screened.CDK1 was found to be a known target of TAK-632 by investigating the applications and prospects of 4 protein targets in cell death and tumor therapy.NQO1 was highly enriched in both target identification methods,and the conformotypic peptides are several times larger than that of others.Therefore,NQO1 protein was locked on.Subsequently,the NQO1 recombinant protein was expressed by Li P-MS assay.The conformotypic peptides and the previous results could verify the binding sites.Finally,we used surface plasmon resonance to detect the binding constant,NQO1 enzyme activity evaluation,CRISPR/Cas9 lentivirus NQO1 knockout and other molecular biology techniques.This study reveals that small molecule TAK-632 also acts on a new potential target,NQO1 during necroptosis stage,inhibiting cell death by inhibiting the activity of NQO1 and reducing the production of Reactive Oxygen Species(ROS).Through the latent binding pockets obtained from Li P-MS and the results of molecular docking simulations,we found that the NQO1 protein activity site is a flexible hydrophobic bag with three latent hydrogen bond residues(Tyr126,Tyr128 and His161),which can adapt to the continuous binding of NAD(P)H with ligands of different sizes and shapes,and has multiple binding modes and interactions.Both NEC-1S and TAK-632 interact with the hydroxyl groups of Tyr128 and Tyr126.This high plasticity may provide a significant advantage for the design of NQO1 targeted antitumor drugs.Conclusion: This study is the first time to combine the advantages of both labeled and unlabeled target identification methods to develop a new method for intracellular necroptosis inhibitor effect target and binding site analysis basing on Click reaction combined with Li PMS.The discovery of new TAK-632 target NQO1 at the stage of necroptosis was carried out at the living cell level for the first time.Meanwhile,this method has significant scientific meaning for understanding the mechanism of necroptosis and development of new inhibitors.This method can also be applied to other drugs and complex disease models,and provides a new idea and method for dynamic sensitive analysis of drug targets and binding sites at the living cell level.