Bis-Quinolinium Cyclophane Blockers of SK Potassium Channels Are Antagonists of M3 Muscarinic Acetylcholine Receptors.

Vladislav Bugay,Kelly Ann Berg,Derek J Wallace,Irving Salinas,Edward G Brooks,Hudson Ryan Smith,Peter Herbert Dube,Robert Brenner,Bin Wang,Adriana Paola Chapparo

doi:10.3389/fphar.2020.552211

Abstract

Dequalinium is used as an antimicrobial compound for oral health and other microbial infections. Derivatives of dequalinium, the bis-quinolinium cyclophanes UCL 1684 and UCL 1848, are high affinity SK potassium channel antagonists. Here we investigated these compounds as M3 muscarinic receptor (mACHR) antagonists. We used the R-CEPIAer endoplasmic reticulum calcium reporter to functionally assay for Gq-coupled receptor signaling, and investigated the bis-quinolinium cyclophanes as antagonists of M3 mACHR activation in transfected CHO cells. Given mACHR roles in airway smooth muscle (ASM) contractility, we also tested the ability of UCL 1684 to relax ASM. We find that these compounds antagonized M3 mACHRs with an IC50 of 0.27 μM for dequalinium chloride, 1.5 μM for UCL 1684 and 1.0 μM for UCL 1848. UCL 1684 also antagonized M1 (IC50 0.12 μM) and M5 (IC50 0.52 μM) mACHR responses. UCL 1684 was determined to be a competitive antagonist at M3 receptors as it increased the EC50 for carbachol without a reduction in the maximum response. The Ki for UCL1684 determined from competition binding experiments was 909 nM. UCL 1684 reduced carbachol-evoked ASM contractions (>90%, IC50 0.43 μM), and calcium mobilization in rodent and human lung ASM cells. We conclude that dequalinium and bis-quinolinium cyclophanes antagonized M3 mACHR activation at sub- to low micromolar concentrations, with UCL 1684 acting as an ASM relaxant. Caution should be taken when using these compounds to block SK potassium channels, as inhibition of mACHRs may be a side-effect if excessive concentrations are used.

Highlights

Dequalinium salts were first described as antibacterial agents (Babbs et al, 1956), that are currently in use in oral disinfectant mouthwashes (i.e. Dequadin and others) (Kaufman, 1981; Griffiths and Stock, 1986; Liberman et al, 1989), and for treatment of vaginal bacterial infections (Petersen et al, 2002; Mendling et al, 2016)
The antagonist effect was specific to the M3 muscarinic receptor, and not downstream Gq signaling since UCL 1684 did not block the effect of other Gq-coupled receptors such as purinergic receptor P2Y1 (Figure 1C, fractional inhibition 0.067 ± 0.06) or histamine receptor H1
A half maximal concentration of 1.5 mM UCL 1684 blocked the response of M3 muscarinic receptors to 0.5 mM CCH (Figure 1D)

Summary

Introduction

Dequalinium salts were first described as antibacterial agents (Babbs et al, 1956), that are currently in use in oral disinfectant mouthwashes (i.e. Dequadin and others) (Kaufman, 1981; Griffiths and Stock, 1986; Liberman et al, 1989), and for treatment of vaginal bacterial infections (Petersen et al, 2002; Mendling et al, 2016). Given that dequalinium was the first synthetic compound that could block SK potassium channels at micromolar concentrations, it was used as a template for modifications that would increase affinity for SK channels (Galanakis et al, 1995; Dunn et al, 1996; Galanakis et al, 1996). These efforts led to the synthesis of compounds that could block SK channels at nanomolar concentrations, and included the dequalinium-cyclophanes UCL 1684 (IC50 3 nM) (Campos Rosa et al, 1996) and UCL 1848 (IC50 2.7 nM) (Chen et al, 2000). It has been found that dequalinium and UCL 1684 inhibit TrpM3 and TrpM7 channels (~30%–50% reduction) albeit at much lower affinity (30 mM concentration) (Chubanov et al, 2012)

Methods

Results

Conclusion