hERG is one of the key cardiac safety targets, and an unexpected blockade may result in arrhythmic side effects. Therefore, screening against hERG should be an essential component of all drug development programmes. The hERG assay plays a crucial role in the realm of drug development and safety pharmacology, serving as a key screening tool against potential cardiac toxicity. This assay, named after the human Ether-à-go-go-Related Gene, is instrumental in evaluating the risk of compounds prolonging the QT interval on the electrocardiogram, a predictor of serious cardiac arrhythmias like Torsades de Pointes.

Screening compounds against the hERG assay is crucial as it aids in early identification and mitigation of cardiotoxic risks in the drug development process. By doing so, it not only ensures the safety of future pharmaceutical products but also significantly reduces the cost and time associated with late-stage drug development failures. The hERG assay has become a standard in the industry and is pivotal in guiding researchers towards the development of safer therapeutic agents.

hERG ion channel

hERG is a voltage-gated potassium channel that regulates cardiac action potentials. It features an unusual gating behaviour characterised by slow activation and deactivation followed by rapid inactivation. Gating of hERG is controlled through the voltage-dependent opening and closing of an intracellular gate and by the closing of its extracellular counterpart; channels are activated by K+ ions while extracellular cations or tetraethyl ammonium (TEA) block them.

While hERG exhibits similar gating behaviour to other voltage-dependent potassium channels, it stands out for several unique traits. Notably, it is highly sensitive to extracellular cations such as tetraethylammonium; additionally, its inactivation occurs much quicker than its activation. These features make hERG an attractive target for drug discovery.

The human ERG protein is produced as an immature 135-kDa core glycosylated 135-kDa protein in the endoplasmic reticulum and then undergoes complex glycosylation at the Golgi apparatus before being secreted into the cell membrane as a mature 155-kDa protein.

One thing that makes hERG different from other voltage-gated ion channels is that it doesn’t have a proline-X-proline (PXP) domain, which is usually found near the S6 helices of other Kv channels and is thought to bend the helixes, which reduces the volume of the pore cavity. It may account for its promiscuous interactions with drugs; additionally, proteases like Calpain-1 and PK can cleave it apart.

hERG mutations

Human voltage-sensitive K+ channel hERG plays an essential role in cardiac action potential repolarization and QT interval control, with gain-of-function mutations attenuating inactivation to cause dangerous long QT syndrome (LQTS), while drug-induced block of hERG can also lead to arrhythmias. A recent cryo-EM structure of hERG offers us an opportunity to better understand its mechanisms as well as channelopathies or drug-induced LQTS caused by drugs.

Early studies demonstrated that hERG mutants with natural mutations that interfere with inactivation display a more severe phenotype than other mutants. For example, the I636T mutation on the S5 helix interacts closely with the selectivity filter to cause current to decrease significantly upon membrane repolarization, an essential factor for establishing genotype-phenotype correlations.

These observations indicate that the helix in question isn’t essential to high-affinity block by PD-118057; rather, helix D540C forms a disulfide bond with S666C in the tetrameric pore, and this interaction appears sufficient to achieve high-affinity block. Thus, its binding pocket appears to be primarily driven by interactions with S4 and S5 helices.

Studying human hERG mutants will enable researchers to establish more accurate correlations between genotype-phenotype correlations and rescue strategies and test if an individual hERG mutant is resistant to specific drugs, providing researchers with an opportunity to identify drug-specific phenotypes.

hERG inhibitors

hERG is an ion channel that can be blocked by several drugs to treat arrhythmias, but inhibition can cause QT interval prolongation if left unchecked during preclinical development. Evaluating its activity is crucial, as inhibiting it may increase QT interval lengthening. To assess proarrhythmic potential accurately, it’s also essential to evaluate other properties, such as its ALogP, molecular weight ratio between water and oil (octanol-water molecular weight ratio), rotatable bond number, hydrogen bond acceptor number, and topological polar surface area.

ALogP values and octanol-water binding energy are two useful physicochemical parameters for evaluating the potential proarrhythmic effects of compounds on hERG. A plot comparing ALogP versus inhibition percentage shows that acids have less affinity for the open state of hERG, while bases and neutrals show a stronger attraction to this target site.

Proarrhythmic properties of compounds depend heavily on their concentration, with an elevated IC50 value being an indicator of inhibitory potency; such a high value indicates an increased risk for arrhythmia.

Creative Bioarray employs an expert team with decades of experience in the development and application of ion channel safety assays. Additionally, Creative Bioarray offers manual patch clamp electrophysiology services as well as automated hERG assays designed specifically to evaluate library screening for cardiotoxicity evaluation and library screening for potential library screening or cardiotoxicity screening targets. Their automated systems can save researchers both time and improve results significantly.

Radioligand-binding assays

The hERG channel is vulnerable to inhibition by various compounds and its blockage can result in QT interval prolongation and cardiac arrhythmias, increasing the risk of potentially dangerous side effects. To minimise these potential side effects, it is vital that early assessment of the compound’s hERG liability take place as early as possible in drug discovery processes; the hERG binding and rubidium efflux assays provide accurate predictions of patch-clamp data and allow high-throughput screening methods.

Radioligand-binding assays can be used to measure the concentration of radiolabelled ligand bound to receptors and determine Bmax and Kd values, but results may be impacted by numerous variables, including buffer composition, incubation time, and temperature; each factor can impede binding equilibrium or disrupt it altogether. Furthermore, membrane preparation stability over an incubation period must also be confirmed; proteolysis should be avoided by adding proteolytic inhibitors when necessary.

The hERG binding assay is highly sensitive, capable of detecting potency inhibitors with IC50s below 0.1 M, less susceptible to false-negative results than the patch clamp method, easier to perform, and requires fewer cells. However, due to the time and labour-intensive nature of the experiment, it’s essential that optimal conditions be set up so as to maximise experiment efficiency.