Towardin vivo-relevant hERG safety assessment and mitigation strategies based on relationships between non-equilibrium blocker binding, three-dimensional channel-blocker interactions, dynamic occupancy, dynamic exposure, and cellular arrhythmia

The human ether-a-go-go-related voltage-gated cardiac ion channel (commonly known as hERG) conducts the rapid outward repolarizing potassium current in cardiomyocytes (I_Kr). Inadvertent blockade of this channel by drug-like molecules represents a key challenge in pharmaceutical R&D due to frequent overlap between the structure-activity relationships of hERG and many primary targets. Building on our previous work, together with recent cryo-EM structures of hERG, we set about to better understand the energetic and structural basis of promiscuous blocker-hERG binding in the context of Biodynamics theory. We propose a two-step blocker binding process consisting of:

• Diffusion of a single fully solvated blocker copy into a large cavity lined by the intracellular cyclic nucleotide binding homology domain (the initial capture step). Occupation of this cavity is a necessary but insufficient condition for ion current disruption.

• Translocation of the captured blocker along the channel axis (the I_Krdisruption step), such that:

  • The head group, consisting of a quasi-linear moiety, projects into the open pore, accompanied by partial de-solvation of the binding interface.

  • One tail moiety packs along a kink between the S6 helix and proximal C-linker helix adjacent to the intra-cellular entrance of the pore, likewise accompanied by mutual de-solvation of the binding interface (noting that the association barrier is comprised largely of the total head + tail group de-solvation cost).

  • Blockers containing a highly planar moiety that projects into a putative constriction zone within the<underline>closed</underline>channel become trapped upon closing, as do blockers terminating prior to this region.

  • A single captured blocker molecule may associate and dissociate from the pore many times before exiting the CNBHD cavity.

Lastly, we highlight possible flaws in the current hERG safety index (SI) and propose an alternatein vivo-relevantstrategy factoring in:

• Benefit/risk.

• The predicted arrhythmogenic fractional hERG occupancy (based on action potential simulations of the<underline>undiseased</underline>human ventricular cardiomyocyte).

• Alteration of the safety threshold due to underlying disease.

• Risk of exposure escalation toward the predicted arrhythmic limit due to patient-to-patient pharmacokinetic variability, drug-drug interactions, overdose, and use for off-label indications in which the hERG safety parameters may differ from their on-label counterparts.