Discovery Of Inhibitors Of Kv10.1 From Natural Compounds And Validation Of Its Efficacy On Anti-hepatoma

Kv10.1,also known as Eag1/KCNH1,is a voltage-gated potassium channel.Dysfunction or aberrant expression of Kv10.1 channel contributes to a variety of tumorigenesis process,including proliferation,migration,invasion,metastasis,recurrence and prognosis.Increasing evidence by our previous work suggests that Kv10.1 channel may serve as a biomarker and anti-tumor drug target for hepatoma.Potent and selective inhibitors of Kv10.1 are therefore much sought after,both as pharmacological tools for studying the（patho）physiological functions of this enigmatic channel and as potential leads for development of anti-tumor drugs.Here,the authors set up a high-throughput Rb⁺efflux assay and identified a natural compound,Pr B1,as a novel Kv10.1 inhibitor.To verify the inhibition mechanism of Procyanidin B1,intracellular[Ca²⁺]assay was used to detect the effect of drugs on intracellular[Ca²⁺],on the other hand,rat Kv10.1 crystal structure was used as a template to obtain human-derived Kv10.1 by homology modeling,and docking to obtain the main pocket of procyanidin B1 binding to Kv10.1 and confirm by mutation experiments and electrophysiology experiments.Finally,the authors validated the regulation of Pr B1 on proliferation,migration and cell cycle of normal liver cells（L-O2）and liver cancer cells（Hep G2）,and the suppression of Pr B1 on xenograft liver tumors through inhibiting the function of Kv10.1.The main innovations are as follows.1.Pr B1,a natural compound extracted from the grape seed,was identified as a potent,specific inhibitor,which can inhibit the Kv10.1 channel in a concentration-dependent manner(IC₅₀=10.38±0.87μM),but has no effect on other voltage depended or independent potassium channels,including Kir2.1,HERG or KCNQ1.2.Pr B1 inhibits the currents of Kv10.1 through binding with the channel rather than elevates the intracellular[Ca²⁺].There is a binding pocket for inhibitors at the C-linker domain of the Kv10.1 channel which comprise of three residues I550,T552 and Q557 and are are critical for the binding of Procyanidin B1.3.By targeting the Kv10.1 channel,Pr B1 can inhibit the proliferation and migration of liver cancer cells in vitro,and can inhibit the growth of mouse xenograft liver tumors（Hep G2）significantly in vivo.Moreover,both in vitro and in vivo,Pr B1 did not show any side effects.