Potent hERG channel inhibition by sarizotan, an investigative treatment for Rett Syndrome

Abstract

Rett Syndrome (RTT) is an X-linked neurodevelopmental disorder associated with respiratory abnormalities and, in up to~40% of patients, with prolongation of the cardiac QT c interval. QT c prolongation calls for cautious use of drugs with a propensity to inhibit hERG channels. The STARS trial has been undertaken to investigate the efficacy of sarizotan, a 5-HT 1A receptor agonist, at correcting RTT respiratory abnormalities. The present study investigated whether sarizotan inhibits hERG potassium channels and prolongs ventricular repolarization. Whole-cell patch-clamp measurements were made at 37°C from hERG-expressing HEK293 cells. Docking analysis was conducted using a recent cryo-EM structure of hERG. Sarizotan was a potent inhibitor of hERG current (I hERG ; IC 50 of 183 nM) and of native ventricular I Kr from guinea-pig ventricular myocytes. 100 nM and 1 μM sarizotan prolonged ventricular action potential (AP) duration (APD 90) by 14.1 ± 3.3% (n = 6) and 29.8 ± 3.1% (n = 5) respectively and promoted AP triangulation. High affinity I hERG inhibition by sarizotan was contingent upon channel gating and intact inactivation. Mutagenesis experiments and docking analysis implicated F557, S624 and Y652 residues in sarizotan binding, with weaker contribution from F656. In conclusion, sarizotan inhibits I Kr /I hERG , accessing key binding residues on channel gating. This action and consequent ventricular AP prolongation occur at concentrations relevant to those proposed to treat breathing dysrhythmia in RTT. Sarizotan should only be used in RTT patients with careful evaluation of risk factors for QT c prolongation.

An Update on the Structure of hERG

“…Support for binding of blockers within one of the hydrophobic pockets below the pore helix comes from observations that mutation of F557 on the outer pore helix (S5) to F557L (hERG F557A is poorly-expressible (Ju et al, 2009)) attenuates the effects of some hERG blockers (Saxena et al, 2016). A direct interaction between blocker and F557 would seem to require that the blocker lies deep within a hydrophobic pocket (see Figure 4C), and recently the serotonin receptor (5HT 1A ) agonist and dopamine (D2) receptor antagonist, sarizotan, was docked within a hydrophobic pocket in a configuration consistent with available mutagenesis data (Cheng et al, 2019). However evidence remains equivocal about direct interactions between drugs and F557; mutation of other residues that line the hydrophobic pockets (F619 and L622; see Figure 4C) have negligible (cisapride, haloperidol) or limited (dofetilide) effects on block by these drugs (Saxena et al, 2016), and this is surprising since computational docking indicates that drugs that bind deep within a hERG pore hydrophobic pocket are constrained within a compact binding site; as discussed below, mutations of F619 and L622 strongly attenuate the effects of a hERG activator, indicating that drug binding in this part of the channel is expected to be susceptible to mutation of these side chains.…”

“…However evidence remains equivocal about direct interactions between drugs and F557; mutation of other residues that line the hydrophobic pockets (F619 and L622; see Figure 4C) have negligible (cisapride, haloperidol) or limited (dofetilide) effects on block by these drugs (Saxena et al, 2016), and this is surprising since computational docking indicates that drugs that bind deep within a hERG pore hydrophobic pocket are constrained within a compact binding site; as discussed below, mutations of F619 and L622 strongly attenuate the effects of a hERG activator, indicating that drug binding in this part of the channel is expected to be susceptible to mutation of these side chains. Likewise, for all drugs so far tested except cisapride, the attenuation of block in F557L is similar to the effect of the hERG Y652A mutation (Saxena et al, 2016;Helliwell et al, 2018;Cheng et al, 2019;Perissinotti et al, 2019) suggesting that the contributions of these side chains to hERG block may be linked. This interpretation is reinforced by the finding that the voltage-dependence of hERG block by Cavalli-2 is lost in hERG F557L (Helliwell et al, 2018), similar to the loss of voltagedependence of block by this and other high-affinity blockers in hERG Y652A (Sanchez-Chapula et al, 2003).…”

Arrhythmogenic late Ca 2+ sparks in failing heart cells and their control by action potential configuration

“…Enzymatic dissociation was carried out using 1 mg/mL collagenase I (Worthington), 0.05 mg/mL protease (type XIV Sigma), and 0.1 mmol/L Ca 2+ , as described previously (22). Guinea pig, rat, mouse, and zebrafish myocytes were isolated using similar methods to those described previously (22,43,44), and methods for zebrafish myocyte isolation are given in SI Appendix.…”

In silico Exploration of Interactions Between Potential COVID-19 Antiviral Treatments and the Pore of the hERG Potassium Channel—A Drug Antitarget

“…For example, of the larger antiviral molecules studied only remdesivir showed a propensity to interact with the lateral binding pockets surrounding the central cavity and there was a marked difference between interactions with residues in this region observed using the MD based Dickson model ( 42 ) and the original cryo-EM structure ( 40 ). For ritonavir, the use of the Dickson model allowed the drug to be in close proximity to F557 [a residue implicated in binding of a number of drugs ( 47 , 62 – 64 )]. The future experimental investigation of pore cavity and lateral binding pocket residue mutants should be able to identify which of the different binding modes predicted here most accurately describes drug-channel interactions and whether or not any particular channel structure is used for the docking here outperforms the others in matching experimental observations.…”