Structure-activity relationships of pentamidine-affected ion channel trafficking and dofetilide mediated rescue

Medicinal Chemistr

PA-6 inhibits inward rectifier currents carried by V93I and D172N gain-of-function KIR2.1 channels, but increases channel protein expression

Background: The inward rectifier potassium current IK1 contributes to a stable resting membrane potential and phase 3 repolarization of the cardiac action potential. KCNJ2 gain-of function mutations V93I and D172N associate with increased IK1, short QT syndrome type 3 and congenital atrial fibrillation. Pentamidine-Analogue 6 (PA-6) is an efficient (IC50 = 14 nM with inside-out patch clamp methodology) and specific IK1 inhibitor that interacts with the cytoplasmic pore region of the KIR2.1 ion channel, encoded by KCNJ2. At 10 μM, PA-6 increases wild-type (WT) KIR2. 1 expression in HEK293T cells upon chronic treatment. We hypothesized that PA-6 will interact with and inhibit V93I and D172N KIR2.1 channels, whereas impact on channel expression at the plasma membrane requires higher concentrations. Methods: Molecular modelling was performed with the human KIR2.1 closed state homology model using FlexX. WT and mutant KIR2.1 channels were expressed in HEK293 cells. Patch clamp single cell electrophysiology measurements were performed in the whole cell and inside-out mode of the patch clamp method. KIR2.1 expression level and localization were determined by western blot analysis and immunofluorescence microscopy, respectively. Results: PA-6 docking in the V93I/D172N double mutant homology model of KIR2.1 demonstrated that mutations and drug-binding site are >30 Å apart. PA-6 inhibited WT and V93I outward currents with similar potency (IC50 = 35.5 and 43.6 nM at +50 mV for WT and V93I), whereas D172N currents were less sensitive (IC50 = 128.9 nM at +50 mV) using inside-out patch-clamp electrophysiology. In whole cell mode, 1 μM of PA-6 inhibited outward IK1 at −50 mV by 28 ± 36%, 18 ± 20% and 10 ± 6%, for WT, V93I and D172N channels respectively. Western blot analysis demonstrated that PA-6 (5 μM, 24 h) increased KIR2.1 expression levels of WT (6.3 ± 1.5 fold), and V93I (3.9 ± 0.9) and D172N (4.8 ± 2.0) mutants. Immunofluorescent microscopy demonstrated dose-dependent intracellular KIR2.1 accumulation following chronic PA-6 application (24 h, 1 and 5 μM). Conclusions: 1) KCNJ2 gain-of-function mutations V93I and D172N in the KIR2.1 ion channel do not impair PA-6 mediated inhibition of IK1, 2) PA-6 elevates KIR2.1 protein expression and induces intracellular KIR2.1 accumulation, 3) PA-6 is a strong candidate for further preclinical evaluation in treatment of congenital SQT3 and AF

Structure-activity relationships of pentamidine-affected ion channel trafficking and dofetilide mediated rescue: Ion channel trafficking

Drug interference with normal hERG protein trafficking substantially reduces the channel density in the plasma membrane and thereby poses an arrhythmic threat. The chemical substructures important for hERG trafficking inhibition were investigated using pentamidine as a model drug. Furthermore, the relationship between acute ion channel block and correction of trafficking by dofetilide was studied

Computational Identification of Novel Kir6 Channel Inhibitors

KATP channels consist of four Kir6.x pore–forming subunits and four regulatory sulfonylurea receptor (SUR) subunits. These channels couple the metabolic state of the cell to membrane excitability and play a key role in physiological processes such as insulin secretion in the pancreas, protection of cardiac muscle during ischemia and hypoxic vasodilation of arterial smooth muscle cells. Abnormal channel function resulting from inherited gain or loss-of-function mutations in either the Kir6.x and/or SUR subunits are associated with severe diseases such as neonatal diabetes, congenital hyperinsulinism, or Cantú syndrome (CS). CS is an ultra-rare genetic autosomal dominant disorder, caused by dominant gain-of-function mutations in SUR2A or Kir6.1 subunits. No specific pharmacotherapeutic treatment options are currently available for CS. Kir6 specific inhibitors could be beneficial for the development of novel drug therapies for CS, particular for mutations, which lack high affinity for sulfonylurea inhibitor glibenclamide. By applying a combination of computational methods including atomistic MD simulations, free energy calculations and pharmacophore modeling, we identified several novel Kir6.1 inhibitors, which might be possible candidates for drug repurposing. The in silico predictions were confirmed using inside/out patch-clamp analysis. Importantly, Cantú mutation C166S in Kir6.2 (equivalent to C176S in Kir6.1) and S1020P in SUR2A, retained high affinity toward the novel inhibitors. Summarizing, the inhibitors identified in this study might provide a starting point toward developing novel therapies for Cantú disease

LUF7244 plus Dofetilide Rescues Aberrant Kv11.1 Trafficking and Produces Functional IKv11.1

Kv11.1 (hERG) channels play a critical role in repolarization of cardiomyocytes during the cardiac action potential (AP). Drug mediated Kv11.1 blockade results in AP prolongation, which poses an increased risk of sudden cardiac death. Many drugs, like pentamidine, interfere with normal Kv11.1 forward trafficking and thus reduce functional Kv11.1 channel densities. Although class III antiarrhythmics, e.g. dofetilide, rescue congenital and acquired forward trafficking defects, this is of little use due to their simultaneous acute channel blocking effect. We aimed to test the ability of a combination of dofetilide plus LUF7244, a Kv11.1 allosteric modulator/activator, to rescue Kv11.1 trafficking and produce functional Kv11.1 current. LUF7244 treatment by itself did not disturb or rescue WT or G601S Kv11.1 trafficking as shown by western blot and immunofluorescence microcopy analysis. Pentamidine-decreased maturation of WT Kv11.1 levels was rescued by 10 μM dofetilide or 10 μM dofetilide + 5 μM LUF7244. In trafficking defective G601S Kv11.1 cells, dofetilide (10 μM) or dofetilide+LUF7244 (10+5 μM) restored Kv11.1 trafficking also, as demonstrated by western blot and immunofluorescence microscopy. LUF7244 (10 μM) increased IKv11.1 despite the presence of dofetilide (1 μM) in WT Kv11.1 cells. In G601S expressing cells, long-term treatment (24-48 h) with LUF7244 (10 μM) and dofetilide (1 μM) increased IKv11.1 compared to non-treated, or acutely treated cells. We conclude that dofetilide plus LUF7244 rescues Kv11.1 trafficking and produces functional IKv11.1. Thus, combined administration of LUF7244 and an IKV11.1 trafficking corrector could serve as a new pharmacological therapy of both congenital and drug-induced Kv11.1 trafficking defects.Toxicolog

Physicochemical properties of pore residues predict activation gating of CaV1.2: A correlation mutation analysis

Single point mutations in pore-forming S6 segments of calcium channels may transform a high-voltage-activated into a low-voltage-activated channel, and resulting disturbances in calcium entry may cause channelopathies (Hemara-Wahanui et al., Proc Natl Acad Sci U S A 102(21):7553–7558, 16). Here we ask the question how physicochemical properties of amino acid residues in gating-sensitive positions on S6 segments determine the threshold of channel activation of CaV1.2. Leucine in segment IS6 (L434) and a newly identified activation determinant in segment IIIS6 (G1193) were mutated to a variety of amino acids. The induced leftward shifts of the activation curves and decelerated current activation and deactivation suggest a destabilization of the closed and a stabilisation of the open channel state by most mutations. A selection of 17 physicochemical parameters (descriptors) was calculated for these residues and examined for correlation with the shifts of the midpoints of the activation curve (ΔVact). ΔVact correlated with local side-chain flexibility in position L434 (IS6), with the polar accessible surface area of the side chain in position G1193 (IIIS6) and with hydrophobicity in position I781 (IIS6). Combined descriptor analysis for positions I781 and G1193 revealed that additional amino acid properties may contribute to conformational changes during the gating process. The identified physicochemical properties in the analysed gating-sensitive positions (accessible surface area, side-chain flexibility, and hydrophobicity) predict the shifts of the activation curves of CaV1.2

Disease Associated Mutations in KIR Proteins Linked to Aberrant Inward Rectifier Channel Trafficking

The ubiquitously expressed family of inward rectifier potassium (KIR) channels, encoded by KCNJ genes, is primarily involved in cell excitability and potassium homeostasis. Channel mutations associate with a variety of severe human diseases and syndromes, affecting many organ systems including the central and peripheral neural system, heart, kidney, pancreas, and skeletal muscle. A number of mutations associate with altered ion channel expression at the plasma membrane, which might result from defective channel trafficking. Trafficking involves cellular processes that transport ion channels to and from their place of function. By alignment of all KIR channels, and depicting the trafficking associated mutations, three mutational hotspots were identified. One localized in the transmembrane-domain 1 and immediately adjacent sequences, one was found in the G-loop and Golgi-export domain, and the third one was detected at the immunoglobulin-like domain. Surprisingly, only few mutations were observed in experimentally determined Endoplasmic Reticulum (ER)exit-, export-, or ER-retention motifs. Structural mapping of the trafficking defect causing mutations provided a 3D framework, which indicates that trafficking deficient mutations form clusters. These “mutation clusters” affect trafficking by different mechanisms, including protein stability