The Mechanism Underlying The Effects Of A New Oncology Candidate Compound FHND004 On The Cardiac HERG Potassium Channels

The HERG(Human ether-?-go-go-related gene)encodes the pore-forming subunit(?subunit)of the rapid delayed rectifier K+channels,Kv11.1,which conduct the rapiddelayed rectifier K+current(also referred as IHERG)and play an indispensable role inthe repolarization phase of cardiac action potential.This gene is located in humanchromosome 7 and mutations in HERG are associated with inherited long QTsyndrome type 2(LQT2),an inherited disorder which predisposes affected individualsto ventricular arrhythmias,such as torsade de pointes(TdP).However,drug-inducedLQTS can be more common than the inherited form and is a side effect of both cardiac and noncardiac medications.It has been reported that a huge variety of drugs can trigger ventricular arrhythmia or sudden death through block of HERG channels,such as Class ? antiarrhythmics,antibiotics,antihistamines,antipsychotic drugs or anti cancer agents.Consequently,in vitro HERG assays therefore have becomeroutine preclinical practice for most promising drug candidates.Epidermal growth factor receptor(EGFR)belongs to the receptor tyrosine kinase(RTK)super-family,which has a close relationship with the development of many human cancers,thus making it an important anticancer target.For example,EGFR tyrosine kinase inhibitors(EGFR-TKIs)Gefitinib,Erlotinib,and Afatinib have been already used in clinic for the treatment of different cancers,offering new therapeutic approaches and an effective long-term cancer therapy.Unfortunately,acquired resistance to conventional EGFR-TKIs develops within a median period,which has become a difficult problem in anti-cancer agent development.AZD9291 was newly approved by FDA in November 2015 for treating non-small cell lung cancer(NSCLC).Preclinical evidence confirmed that patients treated with AZD9291 didn't show obvious resistance,however,a few common adverse events such as diarrhea,rash,decreased appetite and cardiotoxicities had been reported in clinical trials.Therefore,how to develop more efficient but less cardiotoxic TKI has become a heated research area.FHND004 is a newly synthesized EGFR-TKI based on thestructure of AZD9291 for treating NSCLC which was already in clinical application.Earlier animal toxicity studies demonstrated that FHND004 is much safer than.AZD9291.While as the golden standard in evaluating cardiotoxicities of a particular drug,the aim of our study is to investigate whether FHND004 can block HERG potassium channels and its molecular mechanism.In this present study,whole-cell patch clamp technique was used to explore the effects of FHND004 on IHERG in HEK293 cells transiently transfected with HERG potassium channels and on native HL-1 cells.Also its precurosor AZD9291 was used as the positive control to compare the variation with FHND004.Experiment results demonstrated that FHND004 preferentially inhibited open-state HERG channels in a concentration-,voltage-and time-dependent manner(including step current and tail current)without changing their steady-state activation,inactivation,recovery from inactivation or deactivation.However,the inhibitory effect of FHND004 was significantly less potent than AZD9291.In heterologous expressing system,the value of IC50 for FHND004 blocking HERG tailcurrent was 8.46?M,about 15-fold larger than that of AZD9291(The IC50 of FHND004 was 0.57?M),indicating that the potency of drug-induced IHERG inhibition was dramatically attenuated.And the results we gained on HL-1 cells was similar(IC50=7.21?M).Multiple factors have been implicated in LQTS,including electrolyte abnormalities and LQT2-assocaited KCNH2 mutations.Here,we confirmed that for the electrolyte abnormal simulation and LQTS associated heterozygous mutations WT/A422T and WT/H562P-HERG simulations,FHND004 exhibited more potent inhibition.It has been reported that Y652,F656,S624 and F557 are important amino acids in mediating interaction of HERG channels with different drugs,after mutations in these sites,the IC50 values of FHND004 for inhibiting these mutant HERG tail current were increased to 66.47?M,46.12?M,47.60?M and 19.66?M,respectively,suggesting weaker binding abilities to HERG mutant channels and this conclusion was also supported by computational simulation.And western blot and immunofluorescence results elucidated that the membrane expression of HERG was not affected by:FHND004.This present study elucidated the molecular mechanism of FHND004 in inhibiting HERG channels and demonstrated that the inhibitory effects were significantly smaller than its precurosor AZD9291,providing experimental evidence for the clinical application of FHND004.