Bumetanide Derivatives AqB007 and AqB011 Selectively Block the Aquaporin-1 Ion Channel Conductance and Slow Cancer Cell Migration

Abstract

Aquaporins (AQPs) in the major intrinsic family of proteins mediate fluxes of water and other small solutes across cell membranes. AQP1 is a water channel, and under permissive conditions, a nonselective cation channel gated by cGMP. In addition to mediating fluid transport, AQP1 expression facilitates rapid cell migration in cell types including colon cancers and glioblastoma. Work here defines new pharmacological derivatives of bumetanide that selectively inhibit the ion channel, but not the water channel, activity of AQP1. Human AQP1 was analyzed in the Xenopus laevis oocyte expression system by two-electrode voltage clamp and optical osmotic swelling assays. The aquaporin ligand bumetanide derivative AqB011 was the most potent blocker of the AQP1 ion conductance (IC 50 of 14 mM), with no effect on water channel activity (at up to 200 mM). The order of potency for inhibition of the ionic conductance was AqB011 . AqB007 .. AqB006 $ AqB001. Migration of human colon cancer (HT29) cells was assessed with a wound-closure assay in the presence of a mitotic inhibitor. AqB011 and AqB007 significantly reduced migration rates of AQP1-positive HT29 cells without affecting viability. The order of potency for AQP1 ion channel block matched the order for inhibition of cell migration, as well as in silico modeling of the predicted order of energetically favored binding. Docking models suggest that AqB011 and AqB007 interact with the intracellular loop D domain, a region involved in AQP channel gating. Inhibition of AQP1 ionic conductance could be a useful adjunct therapeutic approach for reducing metastasis in cancers that upregulate AQP1 expression.

Fundamental structural and functional properties of Aquaporin ion channels found across the kingdoms of life

“…AQP1 ionic current activation is less efficient when two key residues aspartate (D237) and lysine (K243) in the carboxyl terminal domain are mutated, further indicating the C‐terminal domain influences the efficacy of cGMP‐mediated activation. Site‐directed mutagenesis of a conserved pair of arginine residues in loop D decreases the efficacy of inhibition by the AQP1 ion channel blocker AqB011 (Kourghi et al., 2017), confirming an earlier proposal that the compound interacts at the loop D gating region . AQP1‐expressing HT29 colon cancer cells treated with AqB011 show impaired cell migration at doses that match the dose‐dependent block of the ion channel conductance, recorded from cloned human AQP1 channels expressed in Xenopus oocytes .…”

“…The development of selective blockers has been a long awaited milestone, and is now allowing the characterization of aquaporin functions in living cells. Block by AqB011 of the AQP1 ionic conductance demonstrated that the cation channel activity is a key component for HT29 cancer cell migration Figure . The continuing evaluation of traditional medicinal plants as sources of blockers of AQP1 has produced agents that selectively inhibit water permeability (such as bacopaside II), or that block both the water and ion channel pores (such as bacopaside I); these also are promising tools for controlling migration in the subset of cancers that rely on AQP1 expression .…”

“…AqB011 is a specific blocker of AQP 1 ion channel and impairs cancer cell migration in HT 29 cancer cells . AqB011 is predicted to interact with the loop D gating domain of the channel.…”

“…The double arginine site is proposed to be involved in binding of the agonist cGMP and antagonists AqB011 and bacopaside I. 15,38,109 Cysteine at position 189 (cyan) is the mercury binding site. 88 Tyrosine 186 (magenta) influences the binding of tetraethylammonium as a water pore blocker.…”

“…Highlighted in blue is the loop D domain, in red are the double arginine residues at positions 159 and 160 (R159 + R160) located in the loop D domain. The double arginine site is proposed to be involved in binding of the agonist cGMP and antagonists AqB011 and bacopaside I . Cysteine at position 189 (cyan) is the mercury binding site .…”