Studies On The Anticancer Activity And Mechanism Of Simmiparib,a Novel Inhibitor Of Poly(ADP-ribose) Polymerase

With potent and broad-spectrum anticancer activities,conventional cytotoxic drugs occupy the main position in clinical cancer therapy.However,these drugs mostly have severe toxicity for their undifferentiated damage on both tumor cells and normal ones.It becomes a new trend to develop drugs with high selectivity and low toxicity.Poly(ADP-ribose)polymerase 1(PARP1)is an important target for cancer therapy,inhibition of PARP1 blocks the base excision repair pathway,resulting in synthetic lethality in homologous recombination repair(HR)-deficient cancer cells.PARP1 inhibitors have been widely confirmed as anticancer drugs against cancers with HR defects.Olaparib(AZD2281)became the first Food and Drug Administration(FDA)-approved PARP1/2 inhibitor for cancer therapy in 2014;and then,rucaparib(AG014699)and niraparib(MK4827)were approved in 2016 and in 2017,respectively.However,olaparib has relatively weak anticancer activity(400 mg twice a day),low bioavailability of 11.1%,and potential safety risk.Therefore,Professor Zhang Ao's group and our group conducted extensive activity-guided structural optimization and obtained a new PARP1 inhibitor,simmiparib,which is in phase I clinical trials.In this study,simmiparib was investigated for its PARP inhibition,enzymatic and cellular selectivity,antitumor activities in vitro/in vivo,antitumor mechanism and pharmacodynamic biomarkers in molecular,cellular and animal models.Simmiparib displayed more potent PARP1 inhibition than olaparib in ELISA assays(IC50: 1.75 nM versus 8.09 nM).In biotinylated NAD+-based assays,simmiparib also displayed high selectivity for PARP1 and PARP2;it inhibited PARP1/2 >90-fold more potently than the other tested PARPs(PARP3,TNKS1,TNKS2 and PARP6).In cytotoxicity assays,simmiparib selectively killed BRCA-deficient cells and displayed higher selectivity than olaparib.Simmiparib caused averagely 43.8-fold higher proliferative inhibition(the average IC50: 2.76 ?M)than that of olaparib in 11 HR-deficient cancer cell lines.Simmiparib showed different sensitization capacity to several conventional anticancer drugs in HR-proficient cancer cells from 5 types of tissues.The average potentiation indexes were calculated as 6.8,2.8,2.8,1.70 for TMZ,Taxol,SN38 and OXA,respectively.Accordingly,simmiparib killed HR-deficient cancer cells by synthetic lethality effect,and potentiated the proliferative inhibition of several anticancer drugs against HR-proficient cells.Simmiparib showed potent in vivo antitumor effect in CDX models,at doses which were 25-fold lower than that of olaparib.Simmiparib apparently inhibited the growth of V-C8(BRCA2-/-)xenografts at doses of 8 mg/kg,4 mg/kg and 2 mg/kg with the T/C ratios of 5.47%,47.12% and 39.40%,respectively,and the T/C ratios were documented as 25.47%,47.12% and 39.40% at all three doses in MDA-MB-436(BRCA1-/-)xenografts.The combination treatment of simmiparib with TMZ at all the three groups showed prominent tumor inhibition(Combination Ratio >1)in HR-proficient SW620 xenografts,and no apparent tumor inhibition was observed in the animals with monotherapy of simmiparib.The data from PDX models showed that simmiparib caused approximately 10-fold greater growth inhibition than olaparib did;the inhibition rate caused by simmiparib at 10 mg/kg(76.73%)was similar to that caused by olaparib at 100 mg/kg(67.19%).It was revealed that the antitumor mechanism of simmiparib is similar to that of other PARP inhibitors.As a NAD+ competitive inhibitor,simmiparib reduced PAR formation by inhibiting cellular PARP1/2 activities,leading to blocking of the BER pathway,which repairs SSBs;unrepaired SSBs were transferred to replicative DSBs,which resulted in accumulation of lethal DSBs in HR-deficient cells,then cell cycle checkpoints were activated,resulting in G2/M arrest;and finally,simmiparib exerted antitumor effect by inducing apoptosis in HR-deficient tumor cells.According to the pharmacokinetic data in MDA-MB-436 xenografts,the elimination rate of simmiparib was much higher in the plasma than in xenografts.Consistently,the exposure in the xenografts was 2.2-to 7.2-fold higher than that in the plasma.In the groups of 8 mg/kg,4 mg/kg and 2 mg/kg,even 24 h post the treatment,the concentrations of simmiparib in the xenografts were equivalent to 0.17,0.12 and 0.05 ?M,which were 250-to 850-fold higher than IC50 of its in vitro proliferative inhibition.According to the pharmacodynamic study in MDA-MB-436 xenografts,5 and 30 min post administration with simmiparib at 2 and 8 mg/kg,PAR levels decreased both in the PBMC and the xenografts,and ?H2AX levels increased in the xenografts in concentration-and time-dependent manners.The results indicate that simmiparib can inhibit PARP1/2 and can also induce DSBs accumulation in vivo.In summary,simmiparib has been proved to be a potent and highly selective PARP1/2 inhibitor with potent antitumor activities against HR-deficient tumors in vitro and in vivo;it can sensitize several anticancer drugs with different mechanisms in HR-proficient tumor cells and in the xenografts.Simmiparib was more potent PARP inhibition,PARP1/2 selectivity and antitumor effect than olaparib,the first-proved PARP inhibitor.Moreover,PAR levels and ?H2AX levels in PBMC and/or in the the xenografts could be used to monitor the therapeutic effects of simmiparib as therapeutic biomarkers.