Inhibition of the Aquaporin-1 Cation Conductance by Selected Furan Compounds Reduces Red Blood Cell Sickling

Abstract

In sickle cell disease (SCD), the pathological shift of red blood cells (RBCs) into distorted morphologies under hypoxic conditions follows activation of a cationic leak current (Psickle) and cell dehydration. Prior work showed sickling was reduced by 5-hydroxylmethyl-2-furfural (5-HMF), which stabilized mutant hemoglobin and also blocked the Psickle current in RBCs, though the molecular basis of this 5-HMF-sensitive cation current remained a mystery. Work here is the first to test the hypothesis that Aquaporin-1 (AQP1) cation channels contribute to the monovalent component of Psickle. Human AQP1 channels expressed in Xenopus oocytes were evaluated for sensitivity to 5-HMF and four derivatives known to have differential efficacies in preventing RBC sickling. Ion conductances were measured by two-electrode voltage clamp, and osmotic water permeability by optical swelling assays. Compounds tested were: 5-HMF; 5-PMFC (5-(phenoxymethyl)furan-2-carbaldehyde); 5-CMFC (5-(4-chlorophenoxymethyl)furan-2-carbaldehyde); 5-NMFC (5-(2-nitrophenoxymethyl)-furan-2-carbaldehyde); and VZHE006 (tert-butyl (5-formylfuran-2-yl)methyl carbonate). The most effective anti-sickling agent, 5-PMFC, was the most potent inhibitor of the AQP1 ion conductance (98% block at 100 µM). The order of sensitivity of the AQP1 conductance to inhibition was 5-PMFC > VZHE006 > 5-CMFC ≥ 5-NMFC, which corresponded with effectiveness in protecting RBCs from sickling. None of the compounds altered AQP1 water channel activity. Combined application of a selective AQP1 ion channel blocker AqB011 (80 µM) with a selective hemoglobin modifying agent 5-NMFC (2.5 mM) increased anti-sickling effectiveness in red blood cells from human SCD patients. Another non-selective cation channel known to be expressed in RBCs, Piezo1, was unaffected by 2 mM 5-HMF. Results suggest that inhibition of AQP1 ion channels and capacity to modify hemoglobin are combined features of the most effective anti-sickling agents. Future therapeutics aimed at both targets could hold promise for improved treatments for SCD.

Reducing the Invasiveness of Low- and High-Grade Endometrial Cancers in Both Primary Human Cancer Biopsies and Cell Lines by the Inhibition of Aquaporin-1 Channels

“…In both Ishikawa ( Figure 4 A) and MFE-280 ( Figure 4 B) cell lines, the most potent blockers of transwell invasion were 5-PMFC, curcumin, and resveratrol. 5-PMFC is known to block the AQP1 cation conductance through the central pore, but does not impair osmotic water flux through the parallel intrasubunit pores [ 29 ]. Transwell invasion of Ishikawa cells was blocked by AQP1 ion channel inhibitors, being reduced ~41% with 80 µM AqB011 and ~55% with 0.5 mM 5-PMFC.…”

“…AqB011 is specific for inhibiting the ion channel activity of AQP1 with no effect on parallel water channel permeation [ 11 , 28 ]. Concentrations of these compounds that blocked EC cell invasion aligned with the concentrations shown to block the human AQP1 ion conductance in electrophysiology assays [ 11 , 29 ]. The more subtle effect of AqB011 on MFE-280 invasiveness (~20% block at 60 µM) as compared to Ishikawa might reflect the lower dose used, since 80 µM, unusually, was toxic for MFE-280.…”

“…The important outcome of this work was the demonstration that pharmacological blockade of AQP1 ion channels substantially inhibited invasion both in EC cell lines and in primary EC cells derived from patient low- and high-grade tumors. The AQP1 ion channel blockers used in this study were AqB011 [ 11 , 20 , 28 , 64 ], previously shown to inhibit migration and invasion in AQP1-expressing colon cancers [ 11 , 12 ], and the furan compounds 5-HMF and 5-PMFC, shown to block the AQP1 cation conductance in red blood cells at 0.5 to 1 mM, as well as to reduce colon cancer motility at similar concentrations [ 29 , 30 ]. AqB011 significantly restrained the invasion of primary EC cells in biopsy samples of low- and high-grade ECs.…”

“…AqB011, a synthetic derivative of the diuretic agent bumetanide, has been shown to interact with the gating domain of AQP1 and block the ion conductance without impairing water channel activity [ 11 , 28 ]. Also shown to act as selective AQP1 ion channel blockers are the furan compounds, 5-hydroxymethyl-2-furfural (5-HMF) and 5 phenoxymethylfuran-2-carbaldehyde (5-PMFC), which, similarly, do not alter water flux through the parallel intrasubunit pores of the AQP1 tetramer [ 29 , 30 ]. IMD-0354 (N-(3, 5-bis-trifluoromethyl-phenyl)-5-chloro-2-hydroxy-benzamide) is suggested to interfere with the water channel activity of AQP4 by direct blocking or via downregulation of expression [ 13 ].…”

Proteoliposomes reconstituted with human aquaporin-1 reveal novel single-ion-channel properties

“…Combined inhibition of hAQP1 ion and water channel pores was shown to slow colon cancer cell migration in vitro more effectively than blocking single AQP1 permeation pathways alone ( 68 ). The cation conductance of hAQP1 was proposed to account for a hypoxia-induced ionic leak current in sickled erythrocytes that was blocked by the anti-sickling compound 5-hydroxylmethyl-2-furfural and structurally related furan compounds ( 69 ). The discovery of new medicinal compounds that regulate AQP ion channels has clear potential to expand therapeutic options, a goal that would benefit from establishment of a rapid screening technique free from background interference.…”