Advances in Targeting GIRK Channels in Disease.

GIRK Channel Structure and Modulatory Sites.A) Left: Side view model of the GIRK2 channel and the G-protein β (cyan) and γ subunits (orange) (PDB: 4kfm). Two subunits are removed for clarity. Transmembrane (TM) domains are in contact with PIP2 (magenta spheres) near the intracellular interface. The cytoplasmic domain (CTD) interacts directly with the Gβγ subunits. Right: view of the top extracellular side of all four subunits. B) Model of GIRK2 tetramer with four subunits. A single subunit is highlighted (light blue) with its corresponding PIP2 molecule (magenta). Expansion of the regions containing key residues implicated in activation by ML297 (top) and GiGA1 (bottom) are shown on the right. Amino acids involved in ML297 activation of GIRK1/GIRK2 channels: GIRK1 F137 and D173 (top). Amino acids involved in GiGA1 interactions, GIRK1 R43 and L246 (not shown), and GIRK2 D346 and L257; in EtOH and GIRK2 I55, F254, P256, L342, L257 and Y349 (bottom). A key residue for EtOH/GiGA1 interaction, L257, is shown in orange. All residues numbers provided are for mouse GIRK1 and GIRK2 isoforms.

Discovery of New Small Molecule GIRK Modulators.A) ML297 was initially discovered through high-throughput screening flux assays with the molecular libraries small molecule repository (MLSMR) and followed by structure-activity relationship (SAR)-based optimization [25]. ML297 is an agonist for GIRK1-containing channels. The second generation of compounds were discovered by structure modification of ML297. The essential chemical groups of ML297 are highlighted. The flow chart shows both the urea and non-urea scaffold compounds [31, 34, 35, 38, 40]. Chemical structures and names of the compounds are indicated. B) The flow chart shows the discovery of GiGA1 through structure-based virtual screening method. A structural model of an alcohol pocket in GIRK2 was built based on the GIRK2 crystal structure [20, 21] and alcohol-bound IRK1 structure [10]. The screening was conducted by docking molecules from a ZINC15 library and National Center for Advancing Translational Sciences (NCATS) small-molecule library to the alcohol pocket in GIRK2. Top hits were further screened with whole-cell patch-clamp recordings on HEK cells expressing different GIRK channels. NCATS_2 was discovered to activate both GIRK2 and GIRK1/GIRK2 channels. Following screenings tested NCATS_2 analogs, and found that NCATS_2.3 (GiGA1) showed selectivity for activating GIRK1-containing channels [46]. Chemical structures and names of the compounds are indicated.

In vivo Biological Activities of Small Molecule GIRK Modulators.A) Antiepileptic effect of GIRK activators ML297 and GiGA1 in chemically and/or electroshock induced epilepsy mouse models. B) Sleep-inductive effect of ML297 in mice, reducing wake activity, and increasing the nonrapid eye movement (NREM) sleep. C) Analgesic effect of VU0466551 in the hot-plate nociception test in mice. D) Reduction of the swimming motility in zebrafish larvae by LOGO5 and CLOGO under 365 nm or 420 nm light irradiation (respectively). E) Anxiolytic effect of ML297 in mice in the elevated plus maze (EPM) test, increasing the time spent in the open arm. F) Anxiolytic effect of ML297 and VU0810464 measured as reduction of stress-induced hyperthermia in mice. G) Fear extinction enhancement (reducing freezing behavior) of GAT1508 in rats trained in a Pavlovian conditioned fear paradigm.