HERG and its evaluation in drug development

Drug safety evaluation plays an extremely important role in drug development. As some non-cardiovascular drugs have been found to induce acquired prolonged QT syndrome (LQTS) and lead to severe arrhythmias (Torsade de Pointes, TDP), they have been withdrawn from the market.It is particularly important to evaluate the safety of drugs to the heart.QT interval of the heart refers to the period from the beginning of QRS wave group to the end of T wave, including the process of ventricular depolarization and repolarization. QT interval prolongation is receiving more and more attention, and is considered as one of the key indicators for the safety evaluation of new drugs.

In cardiomyocytes, the potassium channel encoded by hERG (human Ether-a-go-go Related Gene) mediates a delayed rectifier potassium current (IKr), and IKr inhibition is the most important mechanism by which drugs lead to prolongation of the QT interval. Because of its special molecular structure (Figure 1), the loss of its function or drug inhibition of hERG will affect the repolarization process of the cardiac action potential and cause the QT interval to be prolonged. At the same time, it may induce torsade de pointes ventricular tachycardia, leading to heart rhythm. Abnormal.

As the target of antiarrhythmic drug therapy, HERG potassium channels are increasingly reflected in the process of drug safety testing and drug development.Currently, testing the action of a compound on the HERG potassium channel is a key step in preclinical evaluation of the cardiac safety of a compound. It is also required by regulatory agencies such as the US Food and Drug Administration (FDA) and the European Agency for the Evaluation of Medicines (EMEA) for the approval of new drugs, in order to avoid the risk of cardiotoxicity.It has been estimated that a 10% increase in the predicted rate of arrhythmias caused by a drug could save $100 million per drug in development costs over the course of drug development.Herg potassium channels play such an important role in guiding drug development. Here, we will briefly introduce the knowledge of HERG inhibition.

hERG introduction

The hERG gene was originally isolated and identified from the human hippocampal cDNA library by Warmke et al in 1994, and it has homology with the Drosophila EAG gene. The hERG gene is located on human chromosome 7 (7q35～q36), about 55kb, has 16 exons, and encodes 1159 amino acid residues. It is expressed in human body tissues such as myocardium, brain, liver, spleen, etc., and is highest in myocardial tissue. Recent studies have shown that hERG genes are expressed in many tumor cell lines. The α subunit of hERG gene encoding Ikr and the β subunit minK-related protein encoded by minK together form Ikr. The hERG potassium ion channel is composed of 4 identical α subunits to form a tetramer, and the intermediate type forms an ion channel. Because the protein encoded by the hERG gene has the structure of a voltage-gated channel protein, which includes 6 transmembrane α-helix fragments (S1～S6), the P loop between S5 and S6, the carboxyl terminal (C terminal) and the amino terminal ( N end) (Figure 2). In 1995, Sanguinetti et al. first transfected the HERG gene into cells to express the HERG protein channel, and its biological characteristics were almost the same as that of encoding IKR.Herg gene plays an important role in physiological process. The potassium channel expressed by it has the property of introverted rectification. The introverted IKR is open at all stages of cardiac action potential, and its conductivity is maximum at the repolarization stage of action potential 3 and the resting potential stage.In order to maintain the stability of resting potential, it shows obvious inward current when it is hyperpolarized and outward current when it is slightly depolarized.The HERG potassium channel activates rapidly when the action potential is depolarized to about -30mV and continues to the end of the 3-phase action potential.Therefore, HHERG potassium channels can regulate the premature arrival of impulses from the myocardial sinoatrial node or heteroexcitatory impulses and effectively inhibit the spread of prephase contraction.

hERG inhibition

Herg gene is involved in encoding the α subunit of IKR. When the IKR current encoded by Herg gene is blocked, the potassium ion outflow during the action potential repolarization of cardiomyocytes is reduced, and the action potential duration is prolonged. On electrocardiogram, the QT interval is prolonged, which refers to the time of ventricular depolarization and subsequent repolarization.On an electrocardiogram, the process from the beginning of QRS wave to the end of T wave is represented.There are two types of HERG inhibition: hereditary (primary) and non-hereditary (secondary).Hereditary prolongation of QT mainly includes gene mutation that can produce ion channel dysfunction and congenital prolongation of QT syndrome.Non-hereditary prolonged QT interval can be caused by metabolic abnormalities, diseases, and medications.

In the past few decades, many drugs have been withdrawn from the market or restricted from use due to cardiotoxicity due to hERG inhibition. As people’s understanding of drugs inhibiting hERG (the drug binding site of hERG is shown in Figure 3) continues to deepen, many antibiotics, antiarrhythmic, antipsychotic, antifungal, and antimalarial drugs have hERG inhibition. For example, the antihistamine terfenadine was marketed in the 1980s. Since pharmacologists had little knowledge of hERG inhibition at that time, they did not consider the need to test terfenadine for cardiotoxicity, and it was impossible to find the specific drug. The phenadine molecule itself has a strong inhibitory effect on potassium ion channels (i.e. hERG inhibition), so when a large number of patients had heart rhythm side effects, in 1990, the FDA had to add a warning about the heart rhythm side effects to terfenadine. Two years later, it was upgraded to a serious “black box warning.” In 1997, not long after it had just been approved as an over-the-counter drug, the FDA finally ordered terfenadine to withdraw from the market, ending the commercial journey of this original drug, thus creating another blockbuster antihistamine loratadine without hERG inhibition. Market dominance.

Evaluation technology for hERG inhibition

With the deepening of the research, the safety evaluation technology of HERG inhibition is becoming more and more mature. At present, there are a lot of mature evaluation methods on the market, such as Automated patch-clamp technology.Conventional patch-clamp technology and Fluxortm Thallium Assay.

Fully automatic patch clamp technology (hERG Qpatch assay): The traditional patch clamp technology is adsorbed on the cell surface through a special glass tube to form a high-impedance gigaohm seal, which can accurately record the changes in the current mediated by the ion channel. It is the “gold standard” for the study of ion channels, but it has high technical requirements for operators and low throughput, which cannot meet the current large demand for hERG toxicity evaluation in drug research and development.

Traditional patch-clamp technique (hERG manual patch-clamp asssay): The patch-clamp technique (Conventional Patch-Clamp) is the most important technical means for studying ion channels, and is recognized as the “gold standard” for ion channel research, and is the most accurate The experimental method of measuring ion channels is suitable for studying the mechanism of interaction between compounds and ion channels, and can also be used for the toxicity evaluation of candidate drugs and the structure optimization of lead compounds in the process of new drug application.

Fluxortm Thallium Assay: The Fluxortm Thallium Assay assays the effect of compounds on the HERG potassium channel using the Fluxortm fluorescent dye luminescence method.The fluorescent dye sensitive to thallium was loaded into the cell membrane, and the extracellular thallium ions entered the cell through the open HERG potassium ion channel along the concentration gradient, and then combined with the fluorescent dye to produce fluorescence.Fluxortm Thallium Assay has been widely used in the international pharmaceutical companies and scientific research institutions for HERG activity detection, because it can be tested in 96 well or 384-well plate, to achieve high throughput requirements, suitable for compound preliminary screening and lead compound optimization.

With advanced drug cardiotoxicity evaluation equipment and strong experimental data for endorsement, not only the risk of drug research and development is reduced, but the process of drug research and development is also greatly promoted. Of course, it also provides a certain guarantee for patients to use safer and more effective drugs.

Conclusion

At present, in order to meet the needs of patients, the world has invested a lot of financial and material resources for new drug research and development. New drug research and development is a costly and long period of systematic engineering, safety and effectiveness are the key factors to determine the success of new drug research and development.The U.S. Food and Drug Administration estimates that about 30 percent of new drugs fail to develop because they fail to meet safety standards.In the last century, the toxicological study of drugs in the process of drug development only carried out toxicity evaluation in the middle and late stage of new drug development, instead of actively testing and evaluating in the early stage of development, which led to many drugs with good development prospects died midway due to toxicity or other safety factors.  Therefore, more and more pharmaceutical companies are now more cautious, and the basic research on related projects is more comprehensive and detailed to minimize the risk of death due to hERG inhibition. For the entire new drug development cycle and investment, this aspect of research The time is not long and the investment is not much. The later you start related research, the more serious the loss caused by this risk.

References

[1] Warmke JW, GanetzkyB. A family of potassium channel genes related to eag in Drosophila and mammals Proc Natl Acad Sci USA 1994,91 (8):3438-3442.

[2] https://en.wikipedia.org/wiki/HERG

[3] Sanguinetti MC, Jiang C, Curran ME, et al. A mechanistic link between an inherited and an acquired cardiac arrhythmia: HERG encodes the Ikr potassium channel. Cell, 1995,127:243-251.

[4] Tsujimae K, Suzuki S, Yamada M, et al. Comparison of kinetic properties of quinidine and dofetilide block of HERG channels. Eur J Pharmacol, 2004, 493(1-3):29-40 [5] Brown AM. Drugs, hERG and sudden death. Cell Calcium, 2005, 35(6):543-547.

[5] StephenB. Long et al. Crystal Structure of a Mammalian Voltage-Dependent yShaker family K+channel. Science 309,897(2005).

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