title: Efficient and specific cardiac IK₁ inhibition by a new pentamidine analogue.
abstract: OBJECTIVE
  In excitable cells, KIR2.x ion-channel-carried inward rectifier current (IK₁) is thought to set the negative and stable resting membrane potential, and contributes to action potential repolarization. Loss- or gain-of-function mutations correlate with cardiac arrhythmias and pathological remodelling affects normal KIR2.x protein levels. No specific IK1 inhibitor is currently available for in vivo use, which severely hampers studies on the precise role of IK1 in normal cardiac physiology and pathophysiology. The diamine antiprotozoal drug pentamidine (P) acutely inhibits IK₁ by plugging the cytoplasmic pore region of the channel. We aim to develop more efficient and specific IK₁ inhibitors based on the P structure.
  
  
  RESULTS
  We analysed seven pentamidine analogues (PA-1 to PA-7) for IK₁ blocking potency at 200 nM using inside-out patches from KIR2.1 expressing HEK-293 cells. PA-6 showed the highest potency and was tested further. PA-6 blocked KIR2.x currents of human and mouse with low IC₅₀ values (12-15 nM). Modelling indicated that PA-6 had less electrostatic but more lipophilic interactions with the cytoplasmic channel pore than P, resulting in a higher channel affinity for PA-6 (ΔG -44.1 kJ/Mol) than for P (ΔG -31.7 kJ/Mol). The involvement of acidic amino acid residues E224 and E299 in drug-channel interaction was confirmed experimentally. PA-6 did not affect INav1.5, ICa-L, IKv4.3, IKv11.1, and IKv7.1/minK currents at 200 nM. PA-6 inhibited the inward (50 nM 40%; 100 nM 59%; 200 nM 77%) and outward (50 nM 40%; 100 nM 76%; 200 nM 100%) components of IK₁ in isolated canine adult-ventricular cardiomyocytes (CMs). PA-6 prolonged action potential duration of CMs by 8 (n = 9), 26 (n = 5), and 34% (n = 11) at 50, 100, and 200 nM, respectively. Unlike P, PA-6 had no effect on KIR2.1 channel expression at concentrations from 0.1 to 3 μM. However, PA-6 at 10 μM increased KIR2.1 expression levels. Also, PA-6 did not affect the maturation of hERG, except when applied at 10 μM.
  
  
  CONCLUSIONS
  PA-6 has higher efficiency and specificity to KIR2.x-mediated current than P, lengthens action potential duration, and does not affect channel trafficking at concentrations relevant for complete IK₁ block.
authors.affiliation: Department of Medical Physiology, Division of Heart & Lungs, University Medical Center Utrecht, Yalelaan 50, 3584 CM Utrecht, The Netherlands.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Takanari
authors.forename: Lukas
authors.initials: L
authors.lastname: Nalos
authors.forename: Anna
authors.initials: A
authors.lastname: Stary-Weinzinger
authors.forename: Kathy C G
authors.initials: KC
authors.lastname: de Git
authors.forename: Rosanne
authors.initials: R
authors.lastname: Varkevisser
authors.forename: Tobias
authors.initials: T
authors.lastname: Linder
authors.forename: Marien J C
authors.initials: MJ
authors.lastname: Houtman
authors.forename: Maaike
authors.initials: M
authors.lastname: Peschar
authors.forename: Teun P
authors.initials: TP
authors.lastname: de Boer
authors.forename: Richard R
authors.initials: RR
authors.lastname: Tidwell
authors.forename: Martin B
authors.initials: MB
authors.lastname: Rook
authors.forename: Marc A
authors.initials: MA
authors.lastname: Vos
authors.forename: Marcel A G
authors.initials: MA
authors.lastname: van der Heyden
mesh_terms: D000200:Action Potentials; D000818:Animals; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D051379:Mice; D062105:Molecular Docking Simulation; D015394:Molecular Structure; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000378:metabolism; D010419:Pentamidine / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology*; D011188:Potassium / Q000378:metabolism; D026902:Potassium Channel Blockers / Q000737:chemistry / Q000494:pharmacology*; D024661:Potassium Channels, Inwardly Rectifying / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport; D013329:Structure-Activity Relationship; D014162:Transfection
chemical_list: C489457:KCNJ2 protein, human; C489461:Kir2.1 channel; D026902:Potassium Channel Blockers; D024661:Potassium Channels, Inwardly Rectifying; D010419:Pentamidine; D011188:Potassium
keywords: Cardiomyocytes; Inward rectifier current; KIR2.1; KIR2.2; KIR2.3; Molecular modelling; Pentamidine; Structure activity relation; Trafficking