Thor S. Thorsen

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

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 ~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 (Ki~10.1 μM). 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.

Protein Interacting with C Kinase 1 (PICK1) Reduces Reinsertion Rates of Interaction Partners Sorted to Rab11-dependent Slow Recycling Pathway

Background: The role of PICK1 in regulating trafficking of its PDZ domain binding partners (e.g. AMPA receptors) remains unclear. Results: PICK1 clusters and reduces recycling only of PDZ binding partners sorted to Rab11-dependent recycling. Conclusion: Contrary to other PDZ domain proteins, which regulate postendocytic sorting, PICK1 determines the trafficking rate through an endocytic compartment. Significance: This function might explain the role of PICK1 in synaptic plasticity. The scaffolding protein PICK1 (protein interacting with C kinase 1) contains an N-terminal PSD-95/Discs large/ZO-1 (PDZ) domain and a central lipid-binding Bin/amphiphysin/Rvs (BAR) domain. PICK1 is thought to regulate trafficking of its PDZ binding partners but different and even opposing functions have been suggested. Here, we apply ELISA-based assays and confocal microscopy in HEK293 cells with inducible PICK1 expression to assess in an isolated system the ability of PICK1 to regulate trafficking of natural and engineered PDZ binding partners. The dopamine transporter (DAT), which primarily sorts to degradation upon internalization, did not form perinuclear clusters with PICK1, and PICK1 did not affect DAT internalization/recycling. However, transfer of the PICK1-binding DAT C terminus to the β2-adrenergic receptor, which sorts to recycling upon internalization, led to formation of PICK1 co-clusters in Rab11-positive compartments. Furthermore, PICK1 inhibited Rab11-mediated recycling of the receptor in a BAR and PDZ domain-dependent manner. In contrast, transfer of the DAT C terminus to the δ-opioid receptor, which sorts to degradation, did not result in PICK1 co-clusters or any change in internalization/recycling. Further support for a role of PICK1 determined by its PDZ cargo was obtained for the PICK1 interaction partner prolactin-releasing peptide receptor (GPR10). GPR10 co-localized with Rab11 and clustered with PICK1 upon constitutive internalization but co-localized with the late endosomal marker Rab7 and did not cluster with PICK1 upon agonist-induced internalization. Our data suggest a selective role of PICK1 in clustering and reducing the recycling rates of PDZ domain binding partners sorted to the Rab11-dependent recycling pathway.

Structure-activity relationships of a small-molecule inhibitor of the PDZ domain of PICK1.

Recently, we described the first small-molecule inhibitor, (E)-ethyl 2-cyano-3-(3,4-dichlorophenyl)acryloylcarbamate (1), of the PDZ domain of protein interacting with Calpha-kinase 1 (PICK1), a potential drug target against brain ischemia, pain and cocaine addiction. Herein, we explore structure-activity relationships of 1 by introducing subtle modifications of the acryloylcarbamate scaffold and variations of the substituents on this scaffold. The configuration around the double bond of 1 and analogues was settled by a combination of X-ray crystallography, NMR and density functional theory calculations. Thereby, docking studies were used to correlate biological affinities with structural considerations for ligand-protein interactions. The most potent analogue obtained in this study showed an improvement in affinity compared to 1 and is currently a lead in further studies of PICK1 inhibition.

Structure of Dimeric and Tetrameric Complexes of the BAR Domain Protein PICK1 Determined by Small-Angle X-Ray Scattering

PICK1 is a neuronal scaffolding protein containing a PDZ domain and an auto-inhibited BAR domain. BAR domains are membrane-sculpting protein modules generating membrane curvature and promoting membrane fission. Previous data suggest that BAR domains are organized in lattice-like arrangements when stabilizing membranes but little is known about structural organization of BAR domains in solution. Through a small-angle X-ray scattering (SAXS) analysis, we determine the structure of dimeric and tetrameric complexes of PICK1 in solution. SAXS and biochemical data reveal a strong propensity of PICK1 to form higher-order structures, and SAXS analysis suggests an offset, parallel mode of BAR-BAR oligomerization. Furthermore, unlike accessory domains in other BAR domain proteins, the positioning of the PDZ domains is flexible, enabling PICK1 to perform long-range, dynamic scaffolding of membrane-associated proteins. Together with functional data, these structural findings are compatible with a model in which oligomerization governs auto-inhibition of BAR domain function.

Serine 77 in the PDZ domain of PICK1 is a protein kinase Cα phosphorylation site regulated by lipid membrane binding.

PICK1 (protein interacting with C kinase 1) contains an N-terminal protein binding PDZ domain and a C-terminal lipid binding BAR domain. PICK1 plays a key role in several physiological processes, including synaptic plasticity. However, little is known about the cellular mechanisms governing the activity of PICK1 itself. Here we show that PICK1 is a substrate in vitro both for PKCα (protein kinase Cα), as previously shown, and for CaMKIIα (Ca(2+)-calmodulin-dependent protein kinase IIα). By mutation of predicted phosphorylation sites, we identify Ser77 in the PDZ domain as a major phosphorylation site for PKCα. Mutation of Ser77 reduced the level of PKCα-mediated phosphorylation ~50%, whereas no reduction was observed upon mutation of seven other predicted sites. Addition of lipid vesicles increased the level of phosphorylation of Ser77 10-fold, indicating that lipid binding is critical for optimal phosphorylation. Binding of PKCα to the PICK1 PDZ domain was not required for phosphorylation, but a PDZ domain peptide ligand reduced the overall level of phosphorylation ~30%. The phosphomimic S77D reduced the extent of cytosolic clustering of eYFP-PICK1 in COS7 cells and thereby conceivably its lipid binding and/or polymerization capacity. We propose that PICK1 is phosphorylated at Ser77 by PKCα preferentially when bound to membrane vesicles and that this phosphorylation in turn modulates its cellular distribution.

Invisible detergents for structure determination of membrane proteins by small-angle neutron scattering

A novel and generally applicable method for determining structures of membrane proteins in solution via small‐angle neutron scattering (SANS) is presented. Common detergents for solubilizing membrane proteins were synthesized in isotope‐substituted versions for utilizing the intrinsic neutron scattering length difference between hydrogen and deuterium. Individual hydrogen/deuterium levels of the detergent head and tail groups were achieved such that the formed micelles became effectively invisible in heavy water (D2O) when investigated by neutrons. This way, only the signal from the membrane protein remained in the SANS data. We demonstrate that the method is not only generally applicable on five very different membrane proteins but also reveals subtle structural details about the sarco/endoplasmatic reticulum Ca2+ ATPase (SERCA). In all, the synthesis of isotope‐substituted detergents makes solution structure determination of membrane proteins by SANS and subsequent data analysis available to nonspecialists.

Structure and Affinity of Two Bicyclic Glutamate Analogues at AMPA and Kainate Receptors

Ionotropic glutamate receptors (iGluRs) are involved in most of the fast excitatory synaptic transmission in the central nervous system. These receptors are important for learning and memory formation, but are also involved in the development of diseases such as Alzheimers disease, epilepsy and depression. To understand the function of different types of iGluRs, selective agonists are invaluable as pharmacological tool compounds. Here, we report binding affinities of two bicyclic, conformationally restricted analogues of glutamate (CIP-AS and LM-12b) at AMPA (GluA2 and GluA3) and kainate receptor subunits (GluK1-3 and GluK5). Both CIP-AS and LM-12b were found to be GluK3-preferring agonists, with Ki of 6 and 22 nM, respectively, at recombinant GluK3 receptors. The detailed binding mode of CIP-AS and LM-12b in the ligand-binding domains of the AMPA receptor subunit GluA2 (GluA2-LBD) and the kainate receptor subunits GluK1 (GluK1-LBD) and GluK3 (GluK3-LBD) was investigated by X-ray crystallography. CIP-AS stabilized all three receptor constructs in conformations similar to those with kainate. Remarkably, whereas LM-12b bound in a similar manner to CIP-AS in GluA2-LBD and GluK3-LBD, it introduced full closure of the ligand-binding domain in GluK1-LBD and formation of a D1-D2 interlobe hydrogen bond between Glu441 and Ser721, as also observed with glutamate. As the binding affinity of LM-12b at GluK1 is ∼8-fold better than that for CIP-AS (Ki of 85 and 656 nM, respectively), it shows that small changes in agonist structure can lead to prominent differences in structure and function.