Modulation of P2X3 and P2X2/3 Receptors by Monoclonal Antibodies

Abstract

The ability to exogenously impose targeted epigenetic changes in the genome represents an attractive route for the simulation of genomic de novo epigenetic events characteristic of some diseases and for the study of their downstream effects and also provides a potential therapeutic approach for the heritable repression of selected genes. Here we demonstrate for the first time the ability of zinc finger peptides to deliver DNA cytosine methylation in vivo to a genomic integrated target promoter when expressed as fusions with a mutant prokaryotic DNA cytosine methyltransferase enzyme, thus mimicking cellular genomic de novo methylation events and allowing a direct analysis of the mechanics of de novo DNA methylation-mediated gene silencing at a genomic locus. We show that targeted methylation leads to gene silencing via the initiation of a repressive chromatin signature at the targeted genomic locus. This repression is maintained after the loss of targeted methyltransferase enzyme from the cell, confirming epigenetic maintenance purely through the action of cellular enzymes. The inherited DNA methylation pattern is restricted only to targeted sites, suggesting that the establishment of repressive chromatin structure does not drive further de novo DNA methylation in this system. As well as demonstrating the potential of these enzymes as tools for the exogenous, heritable control of cellular gene expression, this work also provides the most definitive confirmation to date for a transcriptionally repressive role for de novo DNA methylation in the cell and lends some weight to the hypothesis that the aberrant methylation associated with certain diseases may well be a cause rather than a consequence of transcriptional gene repression.

Purinergic drug targets for gastrointestinal disorders

“…P2X3R antagonists and anti-P2X3R antibodies are being explored as therapeutic agents against colic and colitis pain (Deiteren et al, 2015; Shcherbatko et al, 2016). …”

Ocular Purine Receptors as Drug Targets in the Eye

“…However, a range of other G protein-dependent and independent signaling mechanisms are associated with activation of ARs. 1 Nucleoside derivatives that activate ARs (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) are shown in Fig. 1, and AR antagonists (both nucleosides and nonnucleosides, 25-49) are shown in Fig.…”

Screening Strategies for the Discovery of Ion Channel Monoclonal Antibodies

“…While several of these approaches have successfully identified antibodies that modulate ion channel function, the number of reported examples of functional ion channel antibodies remains limited. Antibodies which modulate ion channel function have been identified for ligand‐gated ion channels (P2X 3 and P2X 7 ), members of the transient receptor potential family (TrpA1, TrpM8) and voltage‐gated ion channels, Na v 1.7, Eag1, K v 1.2, K v 1.3 (Danquah et al., ; Gomez‐Varela et al., ; Lee et al., , b; Mankouri et al., ; Miller et al., ; Shcherbatko et al., ). Therefore, of the 300 or so ion channel encoding genes, only limited progress has been made in exploring the potential of antibodies to modulate ion channel function.…”

“…Fluorescent dyes have been used as calcium indicators in secondary flux assays for the identification and characterization of ion channel antibodies (Cox et al., ; Danquah et al., ; Naylor et al., ; Shcherbatko et al., ). Danquah et al.…”

“…Parental cell lines for heterologous expression of the target ion channel are selected based on the lack of endogenous expression of the target, the ease of sealing the cell during whole cell patch clamp, and a lack of other confounding endogenous channels. For example, 1321N1 cells were selected for expression of P2X 3 or P2X 2/3 channels due to their lack of endogenous expression of other P2X channels or P2Y receptors (Herold, Li, Schachter, Harden, & Nicholas, ; Shcherbatko et al., ). The use of HEK293 cells for studying voltage‐gated sodium (Na v ) or potassium (K v ) channels requires caution as this cell line endogenously expresses small TTX‐sensitive Na v currents (He & Soderlund, ), and outwardly rectifying K v currents (Varghese, Tenbroek, Coles, & Sigg, ).…”