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1.
Front Mol Neurosci ; 13: 166, 2020.
Article in English | MEDLINE | ID: mdl-33071753

ABSTRACT

The P2X7 receptor (P2X7) is a cell surface ligand-gated ion channel, activated by its physiological nucleotide agonist ATP and a synthetic analog (BzATP). However, it has also been suggested that there may be structurally unrelated, non-nucleotide agonists such as the amyloidogenic ß peptide. Here we aimed to reassess the effect of amyloid ß peptides in various in vitro cell models, namely HEK293 overexpressing human P2X7, the microglial BV-2 cell line, and BV-2 cells lacking P2X7. We measured YO-PRO-1 dye uptake in response to full-length amyloid ß peptide (1-42) or the shorter amyloid ß peptide (25-35) and there was a concentration-dependent increase in YO-PRO-1 dye uptake in HEK-hP2X7 cells. However, these amyloid ß peptide-induced increases in YO-PRO-1 dye uptake were also identical in non-transfected HEK-293 cells. We could observe small transient increases in [Ca2+] i induced by amyloid ß peptides in BV-2 cells, however these were identical in BV-2 cells lacking P2X7. Furthermore, our metabolic viability and LDH release experiments suggest no significant change in viability or cell membrane damage in HEK-hP2X7 cells. In the BV-2 cells we found that high concentrations of amyloid ß peptides (1-42) and (25-35) could reduce cell viability by up to 35% but this was also seen in BV-2 cells lacking P2X7. We found no evidence of LDH release by amyloid ß peptides. In summary, we found no evidence that amyloid ß peptides act as agonists of P2X7 in our in vitro models. Our study raises the possibility that amyloid ß peptides simply mimic features of P2X7 activation.

4.
Front Pharmacol ; 11: 627, 2020.
Article in English | MEDLINE | ID: mdl-32477120

ABSTRACT

The family of ligand-gated ion channels known as P2X receptors were discovered several decades ago. Since the cloning of the seven P2X receptors (P2X1-P2X7), a huge research effort has elucidated their roles in regulating a range of physiological and pathophysiological processes. Transgenic animals have been influential in understanding which P2X receptors could be new therapeutic targets for disease. Furthermore, understanding how inherited mutations can increase susceptibility to disorders and diseases has advanced this knowledge base. There has been an emphasis on the discovery and development of pharmacological tools to help dissect the individual roles of P2X receptors and the pharmaceutical industry has been involved in pushing forward clinical development of several lead compounds. During the discovery phase, a number of positive allosteric modulators have been described for P2X receptors and these have been useful in assigning physiological roles to receptors. This review will consider the major physiological roles of P2X1-P2X7 and discuss whether enhancement of P2X receptor activity would offer any therapeutic benefit. We will review what is known about identified compounds acting as positive allosteric modulators and the recent identification of drug binding pockets for such modulators.

6.
J Nat Prod ; 82(9): 2559-2567, 2019 09 27.
Article in English | MEDLINE | ID: mdl-31532206

ABSTRACT

Animal venoms can play an important role in drug discovery, as they are a rich source of evolutionarily tuned compounds that target a variety of ion channels and receptors. To date, there are six FDA-approved drugs derived from animal venoms, with recent work using high-throughput platforms providing a variety of new therapeutic candidates. However, high-throughput methods for screening animal venoms against purinoceptors, one of the oldest signaling receptor families, have not been reported. Here, we describe a variety of quantitative fluorescent-based high-throughput screening (HTS) cell-based assays for screening animal venoms against ligand-gated P2X receptors. A diverse selection of 180 venoms from arachnids, centipedes, hymenopterans, and cone snails were screened, analyzed, and validated, both analytically and pharmacologically. Using this approach, we performed screens against human P2X3, P2X4, and P2X7 using three different fluorescent-based dyes on stable cell lines and isolated the active venom components. Our HTS assays are performed in 96-well format and allow simultaneous screening of multiple venoms on multiple targets, improving testing characteristics while minimizing costs, specimen material, and testing time. Moreover, utilizing our assays and applying them to the other natural product libraries, rather than venoms, might yield other novel natural products that modulate P2X activity.


Subject(s)
Drug Discovery , High-Throughput Screening Assays/methods , Purinergic P2X Receptor Antagonists/chemistry , Receptors, Purinergic P2X/drug effects , Spectrometry, Fluorescence/methods , Venoms/chemistry , Animals , Cell Line , Humans , Purinergic P2X Receptor Antagonists/pharmacology
7.
J Biol Chem ; 294(45): 16587-16603, 2019 11 08.
Article in English | MEDLINE | ID: mdl-31467080

ABSTRACT

Membrane proteins can associate into larger complexes. Examples include receptor tyrosine complexes, ion channels, transporters, and G protein-coupled receptors (GPCRs). For the latter, there is abundant evidence indicating that GPCRs assemble into complexes, through both homo- and heterodimerization. However, the tools for studying and disrupting these complexes, GPCR or otherwise, are limited. Here, we have developed stabilized interference peptides for this purpose. We have previously reported that tetrahydrocannabinol-mediated cognitive impairment arises from homo- or heterooligomerization between the GPCRs cannabinoid receptor type 1 (CB1R) and 5-hydroxytryptamine 2A (5-HT2AR) receptors. Here, to disrupt this interaction through targeting CB1-5-HT2A receptor heteromers in HEK293 cells and using an array of biochemical techniques, including calcium and cAMP measurements, bimolecular fluorescence complementation assays, and CD-based helicity assessments, we developed a NanoLuc binary technology (NanoBiT)-based reporter assay to screen a small library of aryl-carbon-stapled transmembrane-mimicking peptides produced by solid-phase peptide synthesis. We found that these stapling peptides have increased α-helicity and improved proteolytic resistance without any loss of disrupting activity in vitro, suggesting that this approach may also have utility in vivo In summary, our results provide proof of concept for using NanoBiT to study membrane protein complexes and for stabilizing disrupting peptides to target such membrane complexes through hydrocarbon-mediated stapling. We propose that these peptides could be developed to target previously undruggable GPCR heteromers.


Subject(s)
Peptides/metabolism , Receptors, G-Protein-Coupled/metabolism , Amino Acid Sequence , Calcium/metabolism , Cyclic AMP/metabolism , Dimerization , HEK293 Cells , Humans , Nanotechnology , Peptides/chemical synthesis , Peptides/chemistry , Protein Interaction Domains and Motifs , Receptor, Cannabinoid, CB1/chemistry , Receptor, Cannabinoid, CB1/metabolism , Receptor, Serotonin, 5-HT2A/chemistry , Receptor, Serotonin, 5-HT2A/metabolism , Receptors, G-Protein-Coupled/chemistry
8.
Proc Natl Acad Sci U S A ; 116(9): 3863-3872, 2019 02 26.
Article in English | MEDLINE | ID: mdl-30733293

ABSTRACT

Although human epidermal growth factor receptor 2 (HER2)-targeted therapies have dramatically improved the clinical outcome of HER2-positive breast cancer patients, innate and acquired resistance remains an important clinical challenge. New therapeutic approaches and diagnostic tools for identification, stratification, and treatment of patients at higher risk of resistance and recurrence are therefore warranted. Here, we unveil a mechanism controlling the oncogenic activity of HER2: heteromerization with the cannabinoid receptor CB2R. We show that HER2 physically interacts with CB2R in breast cancer cells, and that the expression of these heteromers correlates with poor patient prognosis. The cannabinoid Δ9-tetrahydrocannabinol (THC) disrupts HER2-CB2R complexes by selectively binding to CB2R, which leads to (i) the inactivation of HER2 through disruption of HER2-HER2 homodimers, and (ii) the subsequent degradation of HER2 by the proteasome via the E3 ligase c-CBL. This in turn triggers antitumor responses in vitro and in vivo. Selective targeting of CB2R transmembrane region 5 mimicked THC effects. Together, these findings define HER2-CB2R heteromers as new potential targets for antitumor therapies and biomarkers with prognostic value in HER2-positive breast cancer.


Subject(s)
Breast Neoplasms/cerebrospinal fluid , Molecular Targeted Therapy , Receptor, Cannabinoid, CB2/genetics , Receptor, ErbB-2/genetics , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Line, Tumor , Dronabinol/pharmacology , Drug Resistance, Neoplasm/genetics , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Neoplasm Recurrence, Local/drug therapy , Neoplasm Recurrence, Local/genetics , Neoplasm Recurrence, Local/pathology , Protein Multimerization/drug effects , Proto-Oncogene Proteins c-cbl/genetics , Receptor, Cannabinoid, CB2/chemistry , Receptor, ErbB-2/chemistry , Signal Transduction
9.
Mol Pharmacol ; 95(2): 210-221, 2019 02.
Article in English | MEDLINE | ID: mdl-30545933

ABSTRACT

We investigated the selectivity of protopanaxadiol ginsenosides from Panax ginseng acting as positive allosteric modulators on P2X receptors. ATP-induced responses were measured in stable cell lines overexpressing human P2X4 using a YOPRO-1 dye uptake assay, intracellular calcium measurements, and whole-cell patch-clamp recordings. Ginsenosides CK and Rd were demonstrated to enhance ATP responses at P2X4 by ∼twofold, similar to potentiation by the known positive modulator ivermectin. Investigations into the role of P2X4 in mediating a cytotoxic effect showed that only P2X7 expression in HEK-293 cells induces cell death in response to high concentrations of ATP, and that ginsenosides can enhance this process. Generation of a P2X7-deficient clone of BV-2 microglial cells using CRISPR/Cas9 gene editing enabled an investigation of endogenous P2X4 in a microglial cell line. Compared with parental BV-2 cells, P2X7-deficient BV-2 cells showed minor potentiation of ATP responses by ginsenosides, and insensitivity to ATP- or ATP+ ginsenoside-induced cell death, indicating a primary role for P2X7 receptors in both of these effects. Computational docking to a homology model of human P2X4, based on the open state of zfP2X4, yielded evidence of a putative ginsenoside binding site in P2X4 in the central vestibule region of the large ectodomain.


Subject(s)
Ginsenosides/pharmacology , Receptors, Purinergic P2X4/metabolism , Adenosine Triphosphate/metabolism , Animals , Benzoxazoles/metabolism , Calcium/metabolism , Cell Death/drug effects , Cell Line , HEK293 Cells , Humans , Ivermectin/pharmacology , Mice , Microglia/drug effects , Microglia/metabolism , Quinolinium Compounds/metabolism , Receptors, Purinergic P2X7/metabolism , Sapogenins/pharmacology
10.
Science ; 361(6407): 1158, 2018 Sep 14.
Article in English | MEDLINE | ID: mdl-30213917
11.
Neuropsychopharmacology ; 43(5): 964-977, 2018 04.
Article in English | MEDLINE | ID: mdl-28102227

ABSTRACT

The dorsal striatum is a key node for many neurobiological processes such as motor activity, cognitive functions, and affective processes. The proper functioning of striatal neurons relies critically on metabotropic receptors. Specifically, the main adenosine and endocannabinoid receptors present in the striatum, ie, adenosine A2A receptor (A2AR) and cannabinoid CB1 receptor (CB1R), are of pivotal importance in the control of neuronal excitability. Facilitatory and inhibitory functional interactions between striatal A2AR and CB1R have been reported, and evidence supports that this cross-talk may rely, at least in part, on the formation of A2AR-CB1R heteromeric complexes. However, the specific location and properties of these heteromers have remained largely unknown. Here, by using techniques that allowed a precise visualization of the heteromers in situ in combination with sophisticated genetically modified animal models, together with biochemical and pharmacological approaches, we provide a high-resolution expression map and a detailed functional characterization of A2AR-CB1R heteromers in the dorsal striatum. Specifically, our data unveil that the A2AR-CB1R heteromer (i) is essentially absent from corticostriatal projections and striatonigral neurons, and, instead, is largely present in striatopallidal neurons, (ii) displays a striking G protein-coupled signaling profile, where co-stimulation of both receptors leads to strongly reduced downstream signaling, and (iii) undergoes an unprecedented dysfunction in Huntington's disease, an archetypal disease that affects striatal neurons. Altogether, our findings may open a new conceptual framework to understand the role of coordinated adenosine-endocannabinoid signaling in the indirect striatal pathway, which may be relevant in motor function and neurodegenerative diseases.


Subject(s)
Corpus Striatum/metabolism , Protein Structure, Quaternary , Receptor, Adenosine A2A/metabolism , Receptor, Cannabinoid, CB1/metabolism , Signal Transduction , Animals , Humans , Huntington Disease/metabolism , Mice , Neural Pathways/metabolism , Protein Subunits/biosynthesis
12.
Front Pharmacol ; 8: 291, 2017.
Article in English | MEDLINE | ID: mdl-28588493

ABSTRACT

Adenosine 5'-triphosphate is a well-known extracellular signaling molecule and neurotransmitter known to activate purinergic P2X receptors. Information has been elucidated about the structure and gating of P2X channels following the determination of the crystal structure of P2X4 (zebrafish), however, there is still much to discover regarding the role of this receptor in the central nervous system (CNS). In this review we provide an overview of what is known about P2X4 expression in the CNS and discuss evidence for pathophysiological roles in neuroinflammation and neuropathic pain. Recent advances in the development of pharmacological tools including selective antagonists (5-BDBD, PSB-12062, BX430) and positive modulators (ivermectin, avermectins, divalent cations) of P2X4 will be discussed.

13.
J Med Chem ; 59(15): 7152-66, 2016 08 11.
Article in English | MEDLINE | ID: mdl-27420737

ABSTRACT

Dimeric/oligomeric states of G-protein coupled receptors have been difficult to target. We report here bivalent ligands consisting of two identical oxytocin-mimetics that induce a three order magnitude boost in G-protein signaling of oxytocin receptors (OTRs) in vitro and a 100- and 40-fold gain in potency in vivo in the social behavior of mice and zebrafish. Through receptor mutagenesis and interference experiments with synthetic peptides mimicking transmembrane helices (TMH), we show that such superpotent behavior follows from the binding of the bivalent ligands to dimeric receptors based on a TMH1-TMH2 interface. Moreover, in this arrangement, only the analogues with a well-defined spacer length (∼25 Å) precisely fit inside a channel-like passage between the two protomers of the dimer. The newly discovered oxytocin bivalent ligands represent a powerful tool for targeting dimeric OTR in neurodevelopmental and psychiatric disorders and, in general, provide a framework to untangle specific arrangements of G-protein coupled receptor dimers.


Subject(s)
Drug Design , Oxytocin/pharmacology , Receptors, Oxytocin/agonists , Animals , Dimerization , Dose-Response Relationship, Drug , HEK293 Cells , Humans , Ligands , Mice , Models, Molecular , Molecular Conformation , Oxytocin/chemical synthesis , Oxytocin/chemistry , Structure-Activity Relationship
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