Alkylphenol inverse agonists of HCN1 gating: H-bond propensity, ring saturation and adduct geometry differentially determine efficacy and potency.

Background And Purpose: In models of neuropathic pain, inhibition of HCN1 is anti-hyperalgesic. 2,6-di-iso-propyl phenol (propofol) and its non-anesthetic congener, 2,6-di-tert-butyl phenol, inhibit HCN1 channels by stabilizing closed state(s).

Experimental Approach: Using in vitro electrophysiology and kinetic modeling, we systematically explore the contribution of ligand architecture to alkylphenol-channel coupling.

Key Results: When corrected for changes in hydrophobicity (and propensity for intra-membrane partitioning), the decrease in potency upon 1-position substitution (NCO∼OH >> SH >>> F) mirrors the ligands' H-bond acceptor (NCO > OH > SH >>> F) but not donor profile (OH > SH >>> NCO∼F). H-bond elimination (OH to F) corresponds to a ΔΔG of ∼4.5 kCal mol loss of potency with little or no disruption of efficacy. Substitution of compact alkyl groups (iso-propyl, tert-butyl) with shorter (ethyl, methyl) or more extended (sec-butyl) adducts disrupts both potency and efficacy. Ring saturation (with the obligate loss of both planarity and π electrons) primarily disrupts efficacy.

Conclusions And Implications: A hydrophobicity-independent decrement in potency at higher volumes suggests the alkylbenzene site has a volume of ≥800 Å. Within this, a relatively static (with respect to ligand) H-bond donor contributes to initial binding with little involvement in generation of coupling energy. The influence of π electrons/ring planarity and alkyl adducts on efficacy reveals these aspects of the ligand present towards a face of the channel that undergoes structural changes during opening. The site's characteristics suggest it is "druggable"; introduction of other adducts on the ring may generate higher potency inverse agonists.