In the intricate world of pharmaceutical research and development, understanding the nuances of cardiac safety is paramount. Our blog, “HERG Assay,” focuses on one of the most critical aspects of this domain: the Human Ether-à-Go-Related Gene (HERG) assay and its pivotal role in evaluating the cardiac safety of new drugs in the UK and beyond.
The HERG potassium channel is crucial in cardiac electrophysiology, and its interaction with new pharmaceutical compounds can have significant implications for drug safety. This blog aims to demystify the science behind the HERG assay, exploring its importance in the drug development process, from early discovery to clinical trials.
We delve into the technicalities of conducting HERG assays, discussing both in vitro and in silico approaches and the latest advancements in this field. We have designed our content to be accessible to a wide audience, including seasoned researchers, pharmaceutical professionals, students, and those new to the field.
Understanding the regulatory landscape surrounding drug safety in the UK is also a key focus. We provide insights into the guidelines set by agencies like the Medicines and Healthcare Products Regulatory Agency (MHRA) and how they shape the implementation and interpretation of HERG assays in drug development.
Whether you’re a professional in the pharmaceutical industry, a researcher in cardiac electrophysiology, or simply someone with a keen interest in the science of drug safety, “HERG Assay” is your go-to resource.
Join us as we explore the critical role of HERG assays in ensuring the safety of new medications, contributing to the advancement of healthcare and patient safety. Let’s dive into the world of cardiac safety in drug development together!
What is the hERG Assay?
In the intricate landscape of drug development and safety screening, the hERG (Human Ether-à-go-go-Related Gene) assay emerges as a pivotal tool, especially in the context of evaluating cardiac safety of new pharmaceutical compounds. This assay is named after the hERG gene, which encodes a specific type of potassium ion channel in the human heart. These channels are critical for the electrical activity of the heart, playing a key role in the repolarization phase of the cardiac cycle.
The significance of the hERG assay in drug development cannot be overstated. Many drugs have the unintended side effect of blocking the hERG ion channel. Such blockage can lead to a potentially dangerous condition known as Long QT Syndrome, which increases the risk of irregular heartbeats and can lead to sudden cardiac death. Therefore, the hERG assay is crucial for early detection of such risks.
In the UK, as in many parts of the world, the hERG assay has become a standard component of the preclinical phase of drug development. It’s a requirement set forth by regulatory bodies such as the Medicines and Healthcare products Regulatory Agency (MHRA) and the European Medicines Agency (EMA) to ensure cardiac safety of new drugs. The assay typically involves using in vitro techniques where cell lines expressing the hERG channel are exposed to the drug. The interaction between the drug and the ion channels is then meticulously measured and analysed.
The assay can be conducted using various methods, including manual patch-clamp techniques, automated electrophysiology, and newer, high-throughput screening methods. The choice of method often depends on the stage of drug development and the specific requirements of the study.
In the context of the UK’s pharmaceutical industry, the hERG assay is not just a regulatory requirement but a part of the ethical commitment to patient safety. It represents a balance between scientific innovation and the responsibility to bring safe medications to the market. Moreover, the assay aligns with the UK’s ongoing efforts in ethical research practices, offering an alternative to certain animal testing methods.
As drug development continues to evolve, so too does the hERG assay. Ongoing research in the UK and globally aims to enhance the sensitivity and specificity of the assay, making it an even more powerful tool in predicting cardiac safety. This research is crucial in the development of newer, safer pharmaceuticals and in ensuring the health and well-being of patients worldwide.
The hERG Assay as a Viable Screening Method
In the realm of pharmaceutical development and safety screening, the Human Ether-à-go-Related Gene (hERG) assay stands as a critical tool, especially in the context of cardiac safety. Originating from a gene that encodes a potassium ion channel in the heart, the hERG assay has become a cornerstone in assessing the cardiac toxicity of new drugs. Our UK-based blog delves into the intricacies of this assay, discussing its reliability, methodology, and paramount role in drug development and safety. Understanding the hERG assay is essential for professionals in the pharmaceutical and biomedical fields, as well as for those interested in the latest advancements in drug safety screening. Join us as we explore the significance and application of the hERG assay in the UK and globally, highlighting its role in advancing safe pharmaceutical practices.
This table provides a structured overview of the hERG assay, its role in drug safety screening, and considerations specific to the UK context.
| Aspect | Description | Significance in Drug Safety | Considerations in the UK |
|---|---|---|---|
| Fundamental Role | The hERG assay checks how well a drug can block the hERG potassium ion channel, which is an important part of heart repolarization. | Reliable identification of drugs that may cause QT interval prolongation, a risk factor for cardiac arrhythmias | Alignment with UK and European Medicines Agency (EMA) regulations for drug safety |
| Methodology | In vitro techniques using cell lines expressing the hERG channel to measure drug interaction | provides a cost-effective, standardised method for early cardiac safety assessment. | Adaptation to UK laboratory standards and practices |
| Technological Advancements | The development of automated, high-throughput screening methods has significantly advanced scientific research. | Enhances efficiency and accuracy in drug screening processes. | Integration of advanced technologies in UK-based pharmaceutical research and development |
| Complementary Tests | Use it in conjunction with other assays for a comprehensive cardiac safety profile. | Enhancing the predictive value of cardiac safety assessment | Consideration of other relevant cardiac ion channels and assays, as recommended by UK regulatory bodies, is necessary. |
| Regulatory Compliance | An essential part of preclinical safety assessment is to meet regulatory requirements. | ensures drugs meet safety standards to minimise cardiac risks. | Ensuring compliance with drug approval guidelines set by the Medicines and Healthcare products Regulatory Agency (MHRA) and the European Medicines Agency (EMA). |
| Ethical Considerations | Utilisation of cell lines as an alternative to animal testing | Aligning with ethical considerations is crucial in drug development. | reflects the UK’s commitment to ethical research practices and the reduction of animal testing in drug development. |
| Limitations and Challenges | recognising the limitations in predicting all cardiotoxic effects. | There is a need for further research and the development of more comprehensive assays. | The acknowledgement and addressing of these limitations within the UK pharmaceutical industry and research communities need improvement. |
| Future Prospects | Researchers are actively conducting ongoing research to enhance the sensitivity and specificity of the assay. | aims to improve cardiac safety predictions in drug development. | The UK’s role in global advancements and collaborations in pharmaceutical research, particularly in cardiac safety assays |
hERG channel
In cardiac myocytes, the hERG channel serves as a key regulator of depolarisation-activation transitions. These processes take place voltage-dependently and their kinetics may be altered by certain drugs like calcium channel blockers. Researchers have not yet determined the exact mechanism that connects the voltage sensor with pore opening, and the sensitivity of this process to drug blocking may be due to the high energy required to close the pores.
Tan et al. (2012), Villalba-Galea et al. (2014), Goodchild et al. (2015), and Thouta et al. (2017) have shown that during prolonged depolarisations, hERG channels exhibit robust mode shift behaviour, with the sensor return voltage-dependence being left-shifted from that of pore gate closure. This behaviour may be explained by energetic separation between activation and deactivation gating, which results in an increase in the probability that pores remain open while decreasing the rate of depolarization (Fig. 2A).
hERG channels are multi-domain structures, and their ability to switch between voltage sensing and pore opening depends on a series of interactions between various domains. Ligands can change the structure of the channel, which controls how it interacts with other molecules and changes the balance between open and closed states. This is necessary to make the channel more permeable to different substances.
As well as changing the shape of a pore, PAS-cap can also bind to negative membrane surface charges and alter membrane potential as sensed by channels. This interaction takes place through the L10 residue of the pore linker. Changes to this residue can quickly turn off gating in hERG channels by opening or closing pore access channels. Mutations or truncations in this region often result in rapid deactivation.
Using overlap extension PCR, we made point mutants of hERG that don’t have the L10 residue or have an arginine substitution (Fig. 3). We then generated a chimeric channel by combining bEAG and hERG-bEAG S5 pores to determine the essential residues for linking voltage sensing to deactivation gating. Our research showed that lowering the number of L10 residues made the open state more stable while lowering the chances of pores closing. This effect is similar to how raising Mg2+ speeds up deactivation gating.
hERG binding
Researchers have long identified the hERG binding pocket as an attractive drug target due to its size, which is one of the largest among voltage-gated potassium channels. Many drugs can cause proarrhythmic effects by binding to this pocket and stopping the channel from opening. However, the exact mechanisms governing its binding are still not well understood. This means that different binding mechanisms can lead to different state-dependent proarrhythmic risk predictions, which is a lot of uncertainty that could affect clinical decisions about patient safety.
Previous studies have identified key hERG residues involved in binding and gating, yet we lack in-depth knowledge of their interactions. Recent structural analyses have uncovered that an exceptionally conserved tyrosine residue (Y652) within the S4 subsite of VSD plays an essential role in binding with drugs targeting this receptor, leading to a paradigm shift in our thinking regarding binding and gating relationships for this protein.
A study of the structures of detergent micelles reveals that VSD takes on a closed shape when membrane potentials are nominally neutral. This is different from when physiological depolarizations cause it to block its open channel. An assembly, comprising several domains such as long C-linker helixes, S2-S3 loops characteristic of KCNH channels, and N-terminal arginine residues that make up PAS-cap, is likely responsible for maintaining the hERG pore in its place. These elements play key roles in determining the state-dependent channel opening rate.
Several electrophysiological tests can find hERG liability, but the most accurate ways to measure drug-hERG interactions and check for torsadogenicity are the traditional square pulse and step-ramp protocols. They use short depolarization periods of one or two seconds as measures.
hERG-binding pockets are large, accommodating multiple binding molecules at once. Unfortunately, the size and shape of hERG-binding pockets limit the amount of each molecule that can enter a channel at one time. For example, larger ligands may require modification to fit in these pockets, potentially increasing the risk of arrhythmia due to faster gate gating and resulting arrhythmias.
hERG toxicity
hERG is an essential channel for controlling the QT interval. Anything that interferes with its activity could prolong it and increase the risk of sudden cardiac death; hence, early evaluation of hERG toxicity for new drugs is so essential. Toxicity assessment is also a vital element in the drug approval process. One such approach for evaluating potential drugs’ toxicity and reducing this risk is quantitative high-throughput screening (qHTS). These steps use the thallium fluorescence assay platform to test thousands of chemicals three times at 14 different concentrations to see which ones stop hERG activity in U2OS cells. Chemomodulation analysis and chemical structure-based clustering techniques can evaluate the results of an HTS experiment, providing insight into molecular features associated with inhibiting hERG activity.
The hERG Thallium Fluorescence Assay is an easy and reliable method for measuring the inhibition of hERG by compounds. This assay utilises thallium-labelled cell membranes and an ion flux assay to assess the inhibition of hERG channels by compounds. Performing this assay requires only standard microplate assay equipment, eliminating the need for complex equipment. The reading of the assay is straightforward, making it a fast and efficient way to evaluate inhibition.
An hERG-inhibitory compound can be defined as any substance that produces a change in peak tail current at the end of an action potential (AP) that exceeds zero but falls below 50% of the maximum change. After a thallium ion pump depolarizes the cell, make an AP by sending two conditioning pre-pulses for two seconds at +25 or +45 mV each, then three test pulses for three seconds at -30 mV.
Many mutations can cause LQTS, but they do not all affect ion channels equally. Early nonsense mutations may cause loss of function right away, while missense mutations in the PAS domain of HERG lead to shorter repolarization phases or even the inability to do anything at all, which causes LQTS symptoms to show up early.
hERG pharmacology
To figure out if hERGs can cause delayed ventricular repolarization (QT interval prolongation), we need to know how they work chemically. The hERG potassium channel controls normal cell repolarization by suppressing IKr current. This is the same mechanism responsible for torsades de pointes arrhythmias with abnormally long QT intervals, one of the leading causes among patients taking antiarrhythmic drugs.
The pharmacology of hERG is determined by how drugs interact with its inactivation gating process. This includes drugs that bind very strongly to hERG. These drugs might change its shape, which could either make channel opening more dependent on voltage or make channels open over a smaller range. Either way, this could stop hERG activity permanently.
Many factors affecting hERG pharmacology remain poorly understood. For example, some compounds exhibit differing potencies and toxicity values in various experiments; these discrepancies could be the result of test conditions, the sensitivity of the system, or changes to ligand concentration levels. Also, their effect could depend on specific residues at the entrance to an inactivation gate.
Researchers have developed methods to increase the reliability of hERG tests by accounting for variations in test conditions and reagents. For instance, using an automated electrophysiology platform like QPatch HTX or SyncroPatch 384PE with HEK-293 cells can help minimise variations caused by differences in cell permeability or patch-clamp configuration, temperature variations, or free Ca2+. These techniques may also be beneficial in eliminating the effects of temperature, voltage variations, and free Ca2+.
To get accurate results and avoid false positives, every hERG assay should be done at 37 oC with 0.1 mM Mg2+ to keep the cells in the best possible condition. It is also advised that these assays be conducted on HEK-293 cells to minimise variations in cell permeability settings and patch clamp settings, as well as to maximise the signal-to-noise ratio to ensure maximum sensitivity while minimising variability due to assay conditions or reagents.