Study On The Anti-multiple Myeloma Effect And Mechanism Of DCZ0415 Targeting TRIP13

Background:Multiple myeloma is a malignant plasma cell tumor with high incidence rate,poor prognosis,easy relapse and drug resistance.It is an incurable malignancy.Scientists have been trying to develop effective and safe drugs for many years.Studies have shown that the application of inhibitors targeting oncogenes can produce good results,which may become a breakthrough in MM therapy.Thyroid hormone receptor interacting molecule 13（TRIP13）is a key gene to induce the occurrence,development and drug resistance of MM.It is an excellent target for anti-MM therapy.Objective:The aim of this study is to design and synthesize small molecule inhibitors targeting TRIP13,and to study their anti-MM effects and related mechanisms.Methods:According to the three-dimensional crystal of TRIP13,using the structure-based computer-aided drug design method,we designed specific small molecule compounds,detected the activity of small molecule compounds,and screened the small molecule compounds with the best activity,namely DCZ0415.Cell counting kit-8（CCK-8）was used to detect cell survival.The combination of TRIP13and DCZ0415 was studied by nuclear magnetic resonance（NMR）,surface plasmon resonance（SPR）and pull down experiment.ATPase activity experiment was used to detect the effect of DCZ0415 on the ATPase activity of TRIP13.Soft agar colony formation assay were used to detect the effect of DCZ0415 on the proliferation of MM cells.The primary MM cells obtained by Ficoll Hypaque density gradient centrifugation,and the toxic effect of DCZ0415 on primary cells was detected.Annexin-V/PI were used to detect the effect of DCZ0415 on the cell apoptosis.The regulation of cell cycle by DCZ0415 was detected by PI staining.The effect of DCZ0415 onγH2AX was detected by immunofluorescence.The regulation of DCZ0415 on repair of DNA damage was detected by GFP based reporting system.The regulation of DCZ0415 on NF-κB signal pathway was studied by luciferase reporter gene experiment.Western blot was used to detect the regulation of DCZ0415on apoptosis,cell cycle,repair of DNA damage and NF-κB signal related proteins.CCK-8 method was used to detect the combined effect of DCZ0415 with melphalan and panobistat.The anti-MM activity of DCZ0415 was studied by tumorigenesis experiment in nude mice and the survival of animals was monitored.The transplanted tumor model of normal immune mice was established to study the anti-MM effect of DCZ0415 in vivo,and the effect of DCZ0415 on immune molecules was studied by immunohistochemistry to reflect the regulation of DCZ0415 on immune microenvironment in mice.The results:（1）DCZ0415 inhibited the activity of many MM cells,and the half inhibitory concentration(IC₅₀)of these cell lines was less than 10μM;（2）NMR,SPR and pull down experiments showed that DCZ0415 could closely bind to TRIP13,and ATPase activity experiment showed that DCZ0415 could inhibit the ATPase activity of TRIP13;（3）DCZ0415 inhibited the proliferation and clonal formation of MM cells,induced apoptosis and caused cell cycle arrest;（4）Mechanism studies showed that DCZ0415 inhibited NHEJ repair and NF-κB signal activity;（5）The drug combination experiment showed that DCZ0415 had combined lethal effect with traditional anti-MM drugs melphalan and panobistat,and enhanced the activity of traditional anti-MM drugs;（6）The results of tumor formation in nude mice showed that DCZ0415 could significantly inhibit the growth of multiple myeloma;animal survival experiments showed that DCZ0415 could significantly prolong the survival time of nude mice compared with the control group;（7）The results showed that DCZ0415could inhibit the growth of MM and significantly enhance the activity of immune molecules in the immune microenvironment.Conclusion:The results in vivo and in vitro showed that DCZ0415 has obvious anti-MM effect.The study on mechanism showed that DCZ0415 inhibited NHEJ repair and NF-κB signal by targeting TRIP13;More importantly,DCZ0415 can produce combined lethal effect with traditional anti-MM drugs melphalan and pabistat,and enhance the sensitivity of traditional anti-MM drugs.It can be seen that DCZ0415has the prospect of clinical transformation and is likely to become the lead compound of anti-MM drugs.