Identification of a small-molecule inhibitor of the PICK1 PDZ domain that inhibits hippocampal LTP and LTD

Abstract

Proteins containing PSD-95/Discs-large/ZO-1 homology (PDZ) domains play key roles in the assembly and regulation of cellular signaling pathways and represent putative targets for new pharmacotherapeutics. Here we describe the first small-molecule inhibitor (FSC231) of the PDZ domain in protein interacting with C kinase 1 (PICK1) identified by a screening of approximately 44,000 compounds in a fluorescent polarization assay. The inhibitor bound the PICK1 PDZ domain with an affinity similar to that observed for endogenous peptide ligands (K(i) approximately 10.1 microM). Mutational analysis, together with computational docking of the compound in simulations starting from the PDZ domain structure, identified the binding mode of FSC231. The specificity of FSC231 for the PICK1 PDZ domain was supported by the lack of binding to PDZ domains of postsynaptic density protein 95 (PSD-95) and glutamate receptor interacting protein 1 (GRIP1). Pretreatment of cultured hippocampal neurons with FSC231 inhibited coimmunopreciptation of the AMPA receptor GluR2 subunit with PICK1. In agreement with inhibiting the role of PICK1 in GluR2 trafficking, FSC231 accelerated recycling of pHluorin-tagged GluR2 in hippocampal neurons after internalization in response to NMDA receptor activation. FSC231 blocked the expression of both long-term depression and long-term potentiation in hippocampal CA1 neurons from acute slices, consistent with inhibition of the bidirectional function of PICK1 in synaptic plasticity. Given the proposed role of the PICK1/AMPA receptor interaction in neuropathic pain, excitotoxicity, and cocaine addiction, FSC231 might serve as a lead in the future development of new therapeutics against these conditions.

Fig. 1.

FSC231 dose-dependently inhibits peptide binding to the PICK1 PDZ domain. (A) Chemical structure of FSC231 and the analog FSC231_9 lacking the cyano group. (B) FP competition curves for FSC231 and FSC231_9 using a fixed concentration of Oregon Green-labeled DAT peptide (OG-DAT C13, ~30 nM) and incubation (15 min) together with purified PICK1 and indicated compounds before FP measurements. Data are shown as bound relative to maximum bound OG-DAT C13 (means of triplicates ± SE) and representative of n = 9 (FSC231) and n = 3 (FSC231_9). (C) Pull-down of PICK1 by a C-terminal DAT GST fusion protein (GST DAT C24) is dose-dependently blocked by FSC231 but not by FSC231_9. (Upper) Representative BlueGel-stained SDS gel. (Lower) Quantified and pooled data from three independent experiments (means ± SE, n = 3). Sixty micromolars of GLT1b C10 peptide and 50 μM of FSC231_9 were used as positive and negative controls, respectively. (D) FSC231 binds in the ligand-binding groove of the PICK1 PDZ domain. The PDZ structure is colored by electrostatic potential calculated using Delphi software. Regions of negative potential are in red (at the −4 kT level); regions of positive potential are in blue and displayed at the +4 kT level. The FSC231 compound in mode 1 is rendered in a shaded representation.

Fig. 2.

FSC231 blocks binding between GluR2 and PICK1 in cells. (A) FSC231 inhibits FRET between eCFP–GluR2 C29 and eYFP–PICK1. Normalized FRET efficiency (N_FRET) is given for cotransfected eCFP–GluR2 C29 and eYFP–PICK1 without treatment (control) in response to 50 μM FSC231_9 or in response to 50 μM FSC231. As controls, we used eCeYFP (a covalent fusion of eCFP and eYFP) and cotransfection of eCFP and eYFP. Data are from three experimental days (means ± SE, n = 3). (*P < 0.05, ANOVA, post-hoc Bonferroni's test for multiple comparisons). (B) Co-IP of GluR2 with PICK1 in hippocampal neurons is inhibited by FSC231. PICK1 was immunopreciptated (IP) with rabbit anti-PICK1 antibody from extracts of hippocampal neurons pretreated with 50 μM FSC231 or vehicle. (Top left) Representative SDS–PAGE followed by immunoblotting (IB) with mouse anti-GluR2 antibody (IB: GluR2) shows diminished co-IP of GluR2 by FSC231. (Right) Normalized GluR2 co-IP after quantification by densitometry, mean ± SE, n = 5, **P < 0.002, one-sample t test). IB with mouse anti-PICK1 antibody (IB: PICK1) showed no change in PICK1 IP (middle left) or PICK1 input (bottom left) in response to FSC231.

Fig. 3.

FSC231 accelerates pHluorin-GluR2 recycling in CA1 hippocampal neurons. The pH-sensitive green fluorescent protein variant, pHluorin, was tagged to the N terminus of GluR2 (pH-GluR2) and transfected into hippocampal neurons. Internalization of pH-GluR2 was induced with 20 μM of NMDA for 5 min and fluorescence was recorded during the subsequent recovery period by confocal microscopy. (A) Representative series of images from control neurons and neurons treated with 50 μM of FSC231. (Scale bars: 10 μm.) (B) Representative time course of the average pHluorin-GluR2 fluorescence intensity (F) relative to initial fluorescence (F₀) from neurons treated with FSC231 and vehicle-treated control. (C) Average recycling half time (t_1/2 is the period from maximum endocytosis to 50% recycling) after NMDA washout and (D) average fluorescence intensity amplitude following NMDA stimulation (both means ± SE, n = 12 from four different transfections, *P < 0.02, unpaired t test).

Fig. 4.

FSC231 attenuates LTD and LTP in CA1 hippocampal neurons. (A) LTD expression in CA1 hippocampal neurons was significantly reduced when FSC231 was included in the patch pipette (solid circles) as compared to saline control (open circles) tested in parallel. LTD was induced by pairing a train of 900 stimulations at a frequency of 1 Hz with a depolarization of the postsynaptic cell to −40 mV. (B) Average normalized excitatory postsynaptic currents (EPSCs) of the experiments presented in A and C (means ± SE of n = 7–11, *P < 0.05 unpaired t test). (C) The PICK-specific peptide (EVKI, solid circles) significantly reduced LTD compared to a nonbinding peptide (SVKE, open circles) tested in parallel. (D) Representative EPSCs with or without treatment with FSC231 before and 30 min after application of LTD-inducing stimuli. (E) LTP in CA1 hippocampal neurons was significantly reduced when FSC231 was included in the patch pipette (solid circles) as compared to saline control (open circles). LTP was induced by pairing a train of 50 stimulations at a frequency of 1Hz with a depolarization of the postsynaptic cell to −5 mV. (F) Average normalized EPSCs of the experiment presented in D (means ± SE of n = 4–5, *P < 0.05 unpaired t test).