Hydrochloride Cheong Hin Star To Inhibit Herg Potassium Channel Molecular Mechanisms

Human ether a-go-go related gene (HERG) is initially discovered in the hippocampus of human brain and it encodes the a subunit of the delayed rectifier potassium channel, which plays an important role in the third step of the process of repolarization of the cardiac action potential. It has been established that the mutations in HERG are associated with inherited long QT syndrome type 2 (LQT2). While the blockade of HERG potassium channel by all kinds of drugs is the main cause of the acquired LQTS. As a result of reduction in HERG channel activity, disrupting repolarization of the cardiac action potential manifested as a prolonged QT intervals on electrocardiogram (ECG) increases the risk of ventricular arrhythmias, or leads to a life-threatening arrhythimia Torsades de pointes (Tdp), characterized with cardiac syncope and sudden death. The structure of HERG potassium channel determines its ability to bind with drugs. The inner cavity of HERG channel, different from other potassium channels, is relative large, and two aromatic amino acid residues, Tyr652 and Phe656, located on each of the four subunits that line the central cavity of the channel pore, which are essential for high-affinity of most blockers. Up to date, variety of drugs have been reported to produce HERG current (IHERG) blockade, including cardiovascular and non-cardiovascular drugs, such as antimalarial, antihistamines, anti-tumor, muscle relaxant, antibiotics, and antibacterials etc. through binding of the amino acids of the pore region in HERG channel. Post-marketing reports of TdP and sudden death have led to the withdrawal of multiple therapeutic compounds. Consequently, an assessment of drug's HERG affinity and propensity to prolong the QT interval in humans become an important index in the development of new medicines. Furthermore, an accurate understanding of the molecular basis of drug binding to HERG has increasingly drawn attention from clinic and basic scientists.Fluoroquinolones are the third generation of the quinolone antibacterial agents, which have been widely prescripted in clinic because of its absorption easily and antibacterial activity powerfully. However, with their widespread use in clinic, undesired side effects resulted from fluoroquinolones-induced QT-prolongation associated with ventricular arrhythmias Tdp hava been successively evidenced. For instance, sparfloxacin (SPX) has been reported to produces a dose-dependent increase in QT interval at normal clinical doses in human and rabbits. Therapeutic doses of grepafloxacin (GPX) have also been shown to prolong QT interval. In particular, GPX, gatifloxacin (GTX) and temafloxacin (TMX) have been withdrawn from the market due to their higher incidences of arrhythmias. Chinfloxacin hydrochloride (CFX) is a novel fluoroquinolone compound developed by Zhejiang Medecine Co.Ltd. Xinchang Pharmaceutiacl Factory in China. However, its relative potency for inhibiting HERG channel is unknown. Therefor, this study was performed to examine whether CFX, like other fluoroquinolone antibacterial agents, is of high potency to inhibit HERG potassium channel, and to detemine the molecular mechanisms of the interaction between CFX and HERG potassium channel. Thus, following experiments were conducted concering the issues mentioned above.First of all, whole-cell patch clamp technique was used to study the effects of CFX on HERG potassium channel in HEK293 cells transiently transfected with HERG. And its precurosor moxifloxacin hydrochloride (MOX) was used as the positive control (MOX is recommended by regulatory authorities as a non-cardiac positive control drug in clinical and nonclinic studies evaluating the HERG inhibition of new drugs due to its recognised QT-prolonging effect). The results showed that CFX preferentially inhibited open-state channel in concentration-, voltage-and time-dependent manner. A value of IC50 for CFX was 162.1μM, two more folds than that of MOX (62.5μM), indicating that the potency of drug-induced HERG inhibition was significantly attenuated in response to substitution OMe with two fluorine atoms at C8 in CFX compared with MOX. It is well considerated that the reasons of the durg-induced LQTS are complex. The susceptibility for pateints to drug-induced HERG blockade would be enhanced while they subject to metabolic disease or electrolyte disorders. For this reason, effects of CFX on HERG inhibition upon changing in extracellular potassium were investigated. It was evident that no matter the concentration of extracellular K+ increased or decreased, the inhibitory effects of CFX became stronger compared with that in normal external solution. This demonstrated that potency of CFX-produced HERG blockade may be strengthened under the condition of changing in concentration of extracellular K+Secondly, in the present study, the molecular mechanism of the interaction between CFX and HERG potassium channel was defined using computer simulation and molecular docking. The data revealed that the binding sites of CFX were Tyr652, Ser624, Phe656, and Gly648 on HERG potassium channel. Among those sites, Ser624 in partilar had high affinity for CFX.Finally, based on the prediction of molecular docking, the amino acids with high affinity for CFX were substituted for Ala or Cys using site-directed mutangensis and thereafter the effects of CFX on these mutations were assessed. The results showed that no currents were generated from cells expressing G648A. While, the inhibitory fraction of 500μM CFX on Y652A, S624A and F656C mutants were reduced by 20%,50% and 40% respectively as compare to WT, addressing important role of these amino acids in binding with CFX. In summary, the interaction between fluoroquinolone antibacterial agents and these amino acids must be seriously taken into account for new drug development and research.