NMR-Guided Repositioning of Non-Steroidal Anti-Inflammatory Drugs into Tight Junction Modulators.

Bioavailability is a major bottleneck in the clinical application of medium molecular weight therapeutics, including protein and peptide drugs. Paracellular transport of these molecules is hampered by intercellular tight junction (TJ) complexes. Therefore, safe chemical regulators for TJ loosening are desired. Here, we showed a potential application of select non-steroidal anti-inflammatory drugs (NSAIDs) as TJ modulators. Based on our previous observation that diclofenac and flufenamic acid directly bound various PDZ domains with a broad specificity, we applied solution nuclear magnetic resonance techniques to examine the interaction of other NSAIDs and the first PDZ domain (PDZ1) of zonula occludens (ZO)-1, ZO-1(PDZ1). Inhibition of ZO-1(PDZ1) is expected to provide loosening of the epithelial barrier function because the domain plays a crucial role in maintaining TJ integrity. Accordingly, diclofenac and indomethacin were found to decrease the subcellular localization of claudin (CLD)-2 but not occludin and ZO-1 at the apicolateral intercellular compartment of Madin-Darby canine kidney (MDCK) II cells. These NSAIDs exhibited 125-155% improved paracellular efflux of fluorescein isothiocyanate insulin for the Caco-2 cell monolayer. We propose that these NSAIDs can be repurposed as drug absorption enhancers for peptide drugs.

Peptide Targeting of PDZ-Dependent Interactions as Pharmacological Intervention in Immune-Related Diseases.

PDZ (postsynaptic density (PSD95), discs large (Dlg), and zonula occludens (ZO-1)-dependent interactions are widely distributed within different cell types and regulate a variety of cellular processes. To date, some of these interactions have been identified as targets of small molecules or peptides, mainly related to central nervous system disorders and cancer. Recently, the knowledge of PDZ proteins and their interactions has been extended to various cell types of the immune system, suggesting that their targeting by viral pathogens may constitute an immune evasion mechanism that favors viral replication and dissemination. Thus, the pharmacological modulation of these interactions, either with small molecules or peptides, could help in the control of some immune-related diseases. Deeper structural and functional knowledge of this kind of protein-protein interactions, especially in immune cells, will uncover novel pharmacological targets for a diversity of clinical conditions.

Validation of chemical compound library screening for transcriptional co-activator with PDZ-binding motif inhibitors using GFP-fused transcriptional co-activator with PDZ-binding motif.

Transcriptional co-activator with PDZ-binding motif (TAZ) plays versatile roles in cell proliferation and differentiation. It is phosphorylated by large tumor suppressor kinases, the core kinases of the tumor-suppressive Hippo pathway. Phosphorylation induces the cytoplasmic accumulation of TAZ and its degradation. In human cancers, the deregulation of the Hippo pathway and gene amplification enhance TAZ activity. TAZ interacts with TEA domain family members (TEAD), and upregulates genes implicated in epithelial-mesenchymal transition. It also confers stemness to cancer cells. Thus, TAZ activation provides cancer cells with malignant properties and worsens the clinical prognosis. Therefore, TAZ attracts attention as a therapeutic target in cancer therapy. We applied 18 606 small chemical compounds to human osteosarcoma U2OS cells expressing GFP-fused TAZ (GFP-TAZ), monitored the subcellular localization of GFP-TAZ, and selected 33 compounds that shifted GFP-TAZ to the cytoplasm. Unexpectedly, only a limited number of compounds suppressed TAZ-mediated enhancement of TEAD-responsive reporter activity. Moreover, the compounds that weakened TEAD reporter activity did not necessarily decrease the unphosphorylated TAZ. In this study, we focused on three compounds that decreased both TEAD reporter activity and unphosphorylated TAZ, and treated several human cancer cells with these compounds. One compound did not show a remarkable effect, whereas the other two compounds compromised the cell viability in certain cancer cells. In conclusion, the GFP-TAZ-based assay can be used as the first screening for compounds that inhibit TAZ and show anticancer properties. To develop anticancer drugs, we need additional assays to select the compounds.

Importance of a Conserved Lys/Arg Residue for Ligand/PDZ Domain Interactions as Examined by Protein Semisynthesis.

PDZ domains are ubiquitous small protein domains that are mediators of numerous protein-protein interactions, and play a pivotal role in protein trafficking, synaptic transmission, and the assembly of signaling-transduction complexes. In recent years, PDZ domains have emerged as novel and exciting drug targets for diseases (in the brain in particular), so understanding the molecular details of PDZ domain interactions is of fundamental importance. PDZ domains bind to a protein partner at either a C-terminal peptide or internal peptide motifs. Here, we examined the importance of a conserved Lys/Arg residue in the ligand-binding site of the second PDZ domain of PSD-95, by employing a semisynthetic approach. We generated six semisynthetic PDZ domains comprising different proteogenic and nonproteogenic amino acids representing subtle changes of the conserved Lys/Arg residue. These were tested with four peptide interaction partners, representing the two different binding modes. The results highlight the role of a positively charged amino acid in the ß1-ß2 loop of PDZ domains, and show subtle differences for canonical and noncanonical interaction partners, thus providing additional insight into the mechanism of PDZ/ligand interaction.

Targeted inhibition of disheveled PDZ domain via NSC668036 depresses fibrotic process.

In this study, we determined the effects of transforming growth factor-beta (TGF-ß) and Wnt/ß-catenin signaling on myofibroblast differentiation of NIH/3T3 fibroblasts in vitro and evaluated the therapeutic efficacy of NSC668036 in bleomycin-induced pulmonary fibrosis murine model. In vitro study, NSC668036, a small organic inhibitor of the PDZ domain in Dvl, suppressed ß-catenin-driven gene transcription and abolished TGF-ß1-induced migration, expression of collagen I and α-smooth muscle actin (α-SMA) in fibroblasts. In vivo study, we found that NSC668036 significantly suppressed accumulation of collagen I, α-SMA, and TGF-ß1 but increased the expression of CK19, Occludin and E-cadherin that can inhibit pulmonary fibrogenesis. Because fibrotic lung exhibit aberrant activation of Wnt/ß-catenin signaling, these data collectively suggest that inhibition of Wnt/ß-catenin signaling at the Dvl level may be an effective approach to the treatment of fibrotic lung diseases.

A high-affinity, dimeric inhibitor of PSD-95 bivalently interacts with PDZ1-2 and protects against ischemic brain damage.

Inhibition of the ternary protein complex of the synaptic scaffolding protein postsynaptic density protein-95 (PSD-95), neuronal nitric oxide synthase (nNOS), and the N-methyl-D-aspartate (NMDA) receptor is a potential strategy for treating ischemic brain damage, but high-affinity inhibitors are lacking. Here we report the design and synthesis of a novel dimeric inhibitor, Tat-NPEG4(IETDV)(2) (Tat-N-dimer), which binds the tandem PDZ1-2 domain of PSD-95 with an unprecedented high affinity of 4.6 nM, and displays extensive protease-resistance as evaluated in vitro by stability-measurements in human blood plasma. X-ray crystallography, NMR, and small-angle X-ray scattering (SAXS) deduced a true bivalent interaction between dimeric inhibitor and PDZ1-2, and also provided a dynamic model of the conformational changes of PDZ1-2 induced by the dimeric inhibitor. A single intravenous injection of Tat-N-dimer (3 nmol/g) to mice subjected to focal cerebral ischemia reduces infarct volume with 40% and restores motor functions. Thus, Tat-N-dimer is a highly efficacious neuroprotective agent with therapeutic potential in stroke.

Intracellular Delivery of Peptidyl Ligands by Reversible Cyclization: Discovery of a PDZ Domain Inhibitor that Rescues CFTR Activity.

A general strategy was developed for the intracellular delivery of linear peptidyl ligands through fusion to a cell-penetrating peptide and cyclization of the fusion peptides via a disulfide bond. The resulting cyclic peptides are cell permeable and have improved proteolytic stability. Once inside the cell, the disulfide bond is reduced to produce linear biologically active peptides. This strategy was applied to generate a cell-permeable peptide substrate for real-time detection of intracellular caspase activities during apoptosis and an inhibitor for the CFTR-associated ligand (CAL) PDZ domain as a potential treatment for cystic fibrosis.

Interactions outside the boundaries of the canonical binding groove of a PDZ domain influence ligand binding.

The postsynaptic density protein-95/discs large/zonula occludens-1 (PDZ) domain is a protein-protein interaction module with a shallow binding groove where protein ligands bind. However, interactions that are not part of this canonical binding groove are likely to modulate peptide binding. We have investigated such interactions beyond the binding groove for PDZ3 from PSD-95 and a peptide derived from the C-terminus of the natural ligand CRIPT. We found via nuclear magnetic resonance experiments that up to eight residues of the peptide ligand interact with the PDZ domain, showing that the interaction surface extends far outside of the binding groove as defined by the crystal structure. PDZ3 contains an extra structural element, a C-terminal helix (α3), which is known to affect affinity. Deletion of this helix resulted in the loss of several intermolecular nuclear Overhauser enhancements from peptide residues outside of the binding pocket, suggesting that α3 forms part of the extra binding surface in wild-type PDZ3. Site-directed mutagenesis, isothermal titration calorimetry, and fluorescence intensity experiments confirmed the importance of both α3 and the N-terminal part of the peptide for the affinity. Our data suggest a general mechanism in which different binding surfaces outside of the PDZ binding groove could provide sites for specific interactions.

Hybrid organic-inorganic inhibitors of a PDZ interaction that regulates the endocytic fate of CFTR.

Together strong: Cooperative binding of organic (see picture, red) and inorganic fragments provides a strategy for the potent inhibition of protein-protein interactions. By targeting specific Lewis basic side chains in peripheral regions of the binding site for coordination to a rhodium(II) center, the affinity of otherwise weak ligands is improved.

Coincidence detection and stress modulation of spike time-dependent long-term depression in the hippocampus.

Associative long-term depression (LTD) in the hippocampus is a form of spike time-dependent synaptic plasticity that is induced by the asynchronous pairing of postsynaptic action potentials and EPSPs. Although metabotropic glutamate receptors (mGluRs) and postsynaptic Ca(2+) signaling have been suggested to mediate associative LTD, mechanisms are unclear further downstream. Here we show that either mGluR1 or mGluR5 activation is necessary for LTD induction, which is therefore mediated by group I mGluRs. Inhibition of postsynaptic phospholipase C, inositol-1,4,5-trisphosphate, and PKC prevents associative LTD. Activation of PKC by a phorbol ester causes a presynaptic potentiation of synaptic responses and facilitates LTD induction by a postsynaptic mechanism. Lithium, an inhibitor of the PKC pathway, inhibits LTD and the presynaptic and postsynaptic effects of the phorbol ester. Furthermore, LTD is sensitive to the postsynaptic application of synthetic peptides that inhibit the interaction of AMPA receptors with PDZ domains, suggesting an involvement of protein interacting with C-kinase 1 (PICK1)-mediated receptor endocytosis. Finally, enhanced PKC phosphorylation, induced by behavioral stress, is associated with enhanced LTD. Both increased PKC phosphorylation and stress-induced LTD facilitation can be reversed by lithium, indicating that this clinically used mood stabilizer may act on synaptic depression via PKC modulation. These data suggest that PKC mediates the expression of associative LTD via the PICK1-dependent internalization of AMPA receptors. Moreover, modulation of the PKC activity adjusts the set point for LTD induction in a behavior-dependent manner.

Dopamine regulation of Na+-K+-ATPase requires the PDZ-2 domain of sodium hydrogen regulatory factor-1 (NHERF-1) in opossum kidney cells.

Na(+)-K(+)-ATPase activity in renal proximal tubule is regulated by several hormones including parathyroid hormone (PTH) and dopamine. The current experiments explore the role of Na(+)/H(+) exchanger regulatory factor 1 (NHERF-1) in dopamine-mediated regulation of Na(+)-K(+)-ATPase. We measured dopamine regulation of ouabain-sensitive (86)Rb uptake and Na(+)-K(+)-ATPase α1 subunit phosphorylation in wild-type opossum kidney (OK) (OK-WT) cells, OKH cells (NHERF-1-deficient), and OKH cells stably transfected with full-length human NHERF-1 (NF) or NHERF-1 constructs with mutated PDZ-1 (Z1) or PDZ-2 (Z2) domains. Treatment with 1 µM dopamine decreased ouabain-sensitive (86)Rb uptake, increased phosphorylation of Na(+)-K(+)-ATPase α1-subunit, and enhanced association of NHERF-1 with D1 receptor in OK-WT cells but not in OKH cells. Transfection with wild-type, full-length, or PDZ-1 domain-mutated NHERF-1 into OKH cells restored dopamine-mediated regulation of Na(+)-K(+)-ATPase and D1-like receptor association with NHERF-1. Dopamine did not regulate Na(+)-K(+)-ATPase or increase D1-like receptor association with NHERF-1 in OKH cells transfected with mutated PDZ-2 domain. Dopamine stimulated association of PKC-ζ with NHERF-1 in OK-WT and OKH cells transfected with full-length or PDZ-1 domain-mutated NHERF-1 but not in PDZ-2 domain-mutated NHERF-1-transfected OKH cells. These results suggest that NHERF-1 mediates Na(+)-K(+)-ATPase regulation by dopamine through its PDZ-2 domain.

Synthesis and evaluation of bidentate ligands designed to interact with PDZ domains.

We designed bidentate ligands to target PDZ domains through two binding sites: site S0, delimited by the GLGF loop, and site S1, a zone situated around loop ß(B)/ß(C). A molecular docking study allowed us to design a generic S0 binder, to which was attached a variable size linker, itself linked to an amino acid aimed to interact with the S1 site of PDZ domains. A series of 15 novel bidentate ligands was prepared in 6-11 steps in good overall yield (24-43%). Some of these ligands showed an inhibitory activity against serotonin 5-HT2A receptor/PSD-95 interaction. This was assessed by pull-down assay using a synthetic decapeptide corresponding to the C-terminal residues of the receptor as a bait.

Identification of Novel Fragments Binding to the PDZ1-2 Domain of PSD-95.

Inhibition of PSD-95 has emerged as a promising strategy for the treatment of ischemic stroke, as shown with peptide-based compounds that target the PDZ domains of PSD-95. In contrast, developing potent and drug-like small molecules against the PSD-95 PDZ domains has so far been unsuccessful. Here, we explore the druggability of the PSD-95 PDZ1-2 domain and use fragment screening to investigate if this protein is prone to binding small molecules. We screened 2500 fragments by fluorescence polarization (FP) and validated the hits by surface plasmon resonance (SPR), including an inhibition counter-test, and found four promising fragments. Three ligand efficient fragments were shown by 1 H,15 N HSQC NMR to bind in the small hydrophobic P0 pockets of PDZ1-2, and one of them underwent structure-activity relationship (SAR) studies. Overall, we demonstrate that fragment screening can successfully be applied to PDZ1-2 of PSD-95 and disclose novel fragments that can serve as starting points for optimization towards small-molecule PDZ domain inhibitors.

Regulation of stargazin synaptic trafficking by C-terminal PDZ ligand phosphorylation in bidirectional synaptic plasticity.

Stargazin is a transmembrane alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor regulatory protein that controls the surface and synaptic expression of AMPA-type glutamate receptors (AMPARs). Synaptic anchoring of AMPARs is influenced by the interaction between stargazin's C-terminal post-synaptic density-95 (PSD-95)/discs large/zona occludens-1 (PDZ) ligand and the synaptic scaffolding protein PSD-95. Phosphorylation of the stargazin PDZ ligand by protein kinase A (PKA) disrupts stargazin's interaction with PSD-95, but whether this phosphorylation plays a role in activity-dependent regulation of stargazin/AMPAR synaptic trafficking is unknown. Here, we show that stargazin is phosphorylated within the PDZ ligand at threonine residue 321 (T321) by mitogen-activated protein kinases (MAPKs) as well as PKA. By expressing constructs that selectively block T321 phosphorylation by either PKA or MAPKs, we show that stargazin T321 phosphorylation is required for activity-dependent changes in stargazin synaptic clustering in dissociated rat hippocampal neuron cultures. Specifically, we find that mutations that block stargazin T321 phosphorylation by PKA prevent activity-dependent increases in stargazin synaptic clustering, whereas a point mutant that blocks MAPK phosphorylation of T321 prevents activity-dependent decreases in stargazin synaptic clustering. Taken together, our studies implicate phosphorylation of stargazin T321 by PKA and MAPKs in bidirectional control of stargazin/AMPAR synaptic clustering during synaptic plasticity.

High dose of baicalin or baicalein can reduce tight junction integrity by partly targeting the first PDZ domain of zonula occludens-1 (ZO-1).

The tight junction (TJ) is the apical-most intercellular junction complex, serving as a biological barrier of intercellular spaces between epithelial cells. The TJ's integrity is maintained by a key protein-protein interaction between C-terminal motifs of claudins (CLDs) and the postsynaptic density 95 (PSD-95)/discs large/zonula occludens 1 (ZO-1; PDZ) domains of ZO-1. Weak but direct interaction of baicalin and its aglycon, baicalein-which are pharmacologically active components of Chinese skullcap (Radix scutellariae)-with ZO-1(PDZ1) have been observed in NMR experiments. Next, we observed TJ-mitigating activity of these flavonoids against Madin-Darby canine kidney (MDCK) II cells with the downregulation of subcellular localization of CLD-2 at TJs. Meanwhile, baicalein-but not baicalin-induced a slender morphological change of MDCK cells' shape from their normal cobblestone-like shapes. Since baicalin and baicalein did not induce a localization change of occludin (OCLN), a "partial" epithelial-mesenchymal transition (EMT) induced by these flavonoids was considered. SB431542, an ALK-5 inhibitor, reversed the CLD-2 downregulation of both baicalin and baicalein, while SB431542 did not reverse the slender morphology. In contrast, the MEK/ERK inhibitor U0126 reversed the slender shape change. Thus, in addition to inhibition of the ZO-1-CLD interaction, activation of both transforming growth factor-ß (TGF-ß) and MEK/ERK signaling pathways have been suggested to be involved in TJ reduction by these flavonoids. Finally, we demonstrated that baicalin enhanced the permeability of fluorescence-labeled insulin via the paracellular pathway of the Caco-2 cell layer. We propose that baicalin, baicalein, and Radix scutellariae extract are useful as drug absorption enhancers.

Administration of a novel high affinity PICK1 PDZ domain inhibitor attenuates cocaine seeking in rats.

Protein interacting with C kinase-1 (PICK1) regulates intra-cellular trafficking of GluA2-containing AMPA receptors, a process known to play a critical role in cocaine-seeking behavior. This suggests that PICK1 may represent a molecular target for developing novel pharmacotherapies to treat cocaine craving-induced relapse. Emerging evidence indicates that inhibition of PICK1 attenuates the reinstatement of cocaine-seeking behavior, an animal model of relapse. Here, we show that systemic administration of TAT-P4-(DATC5)2, a novel high-affinity peptide inhibitor of the PICK1 PDZ domain, dose-dependently attenuated the reinstatement of cocaine seeking in rats at doses that did not produce operant learning deficits or suppress locomotor activity. We also show that systemic TAT-P4-(DATC5)2 penetrated the brain where it was visualized in the nucleus accumbens shell. Consistent with these effects, infusions of TAT-P4-(DATC5)2 directly into the accumbens shell reduced cocaine, but not sucrose, seeking. The effects of TAT-P4-(DATC5)2 on cocaine seeking are likely due, in part, to inhibition of PICK1 in medium spiny neurons (MSNs) of the accumbens shell as TAT-P4-(DATC5)2 was shown to accumulate in striatal neurons and bind PICK1. Taken together, these findings highlight a novel role for PICK1 in the reinstatement of cocaine seeking and support future studies examining the efficacy of peptide inhibitors of PICK1 in animal and human models of cocaine relapse.

Emerging Themes in PDZ Domain Signaling: Structure, Function, and Inhibition.

Post-synaptic density-95, disks-large and zonula occludens-1 (PDZ) domains are small globular protein-protein interaction domains widely conserved from yeast to humans. They are composed of ∼90 amino acids and form a classical two α-helical/six ß-strand structure. The prototypical ligand is the C-terminus of partner proteins; however, they also bind internal peptide sequences. Recent findings indicate that PDZ domains also bind phosphatidylinositides and cholesterol. Through their ligand interactions, PDZ domain proteins are critical for cellular trafficking and the surface retention of various ion channels. In addition, PDZ proteins are essential for neuronal signaling, memory, and learning. PDZ proteins also contribute to cytoskeletal dynamics by mediating interactions critical for maintaining cell-cell junctions, cell polarity, and cell migration. Given their important biological roles, it is not surprising that their dysfunction can lead to multiple disease states. As such, PDZ domain-containing proteins have emerged as potential targets for the development of small molecular inhibitors as therapeutic agents. Recent data suggest that the critical binding function of PDZ domains in cell signaling is more than just glue, and their binding function can be regulated by phosphorylation or allosterically by other binding partners. These studies also provide a wealth of structural and biophysical data that are beginning to reveal the physical features that endow this small modular domain with a central role in cell signaling.

Rational design of the first small-molecule antagonists of NHERF1/EBP50 PDZ domains.

This report describes the first small-molecule antagonists that specifically target the ligand-binding pocket of PDZ domains of NHERF1 multi-functional adaptor protein. Comparison of the peptide sequence homology between the native ligand of NHERF1 PDZ domains and an indole-based non-peptide chemical scaffold allowed the design of a small-molecule antagonist of NHERF1 PDZ domains.

Indole-2-amide based biochemical antagonist of Dishevelled PDZ domain interaction down-regulates Dishevelled-driven Tcf transcriptional activity.

We designed and synthesized a series of indole-2-amide-based compounds that antagonize interaction between the Dishevelled (Dvl) PDZ domain and a peptide derived from the natural PDZ ligand Frizzled-7 (Fz7). These compounds inhibit Tcf-mediated transcription activated by exogenous Dvl via the biochemical antagonism. We confirmed tumor cell-selective activation of caspases by these compounds.

Denaturants Alter the Flux through Multiple Pathways in the Folding of PDZ Domain.

Although we understand many aspects of how small proteins (number of residues less than about hundred) fold, it is a major challenge to quantitatively describe how large proteins self-assemble. To partially overcome this challenge, we performed simulations using the self-organized polymer model with side chains (SOP-SC) in guanidinium chloride (GdmCl), using the molecular transfer model (MTM), to describe the folding of the 110-residue PDZ3 domain. The simulations reproduce the folding thermodynamics accurately including the melting temperature (Tm), the stability of the folded state with respect to the unfolded state. We show that the calculated dependence of ln kobs (kobs is the relaxation rate) has the characteristic chevron shape. The slopes of the chevron plots are in good agreement with experiments. We show that PDZ3 folds by four major pathways populating two metastable intermediates, in accord with the kinetic partitioning mechanism. The structure of one of the intermediates, populated after polypeptide chain collapse, is structurally similar to an equilibrium intermediate. Surprisingly, the connectivities between the intermediates and hence, the fluxes through the pathways depend on the concentration of GdmCl. The results are used to predict possible outcomes for unfolding of PDZ domain subject to mechanical forces. Our study demonstrates that, irrespective of the size or topology, simulations based on MTM and SOP-SC offer a theoretical framework for describing the folding of proteins, mimicking precisely the conditions used in experiments.