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Modulation of P2X3 and P2X2/3 Receptors by Monoclonal Antibodies.
J Biol Chem 2016; 291(23):12254-70JB

Abstract

Purinergic homomeric P2X3 and heteromeric P2X2/3 receptors are ligand-gated cation channels activated by ATP. Both receptors are predominantly expressed in nociceptive sensory neurons, and an increase in extracellular ATP concentration under pathological conditions, such as tissue damage or visceral distension, induces channel opening, membrane depolarization, and initiation of pain signaling. Hence, these receptors are considered important therapeutic targets for pain management, and development of selective antagonists is currently progressing. To advance the search for novel analgesics, we have generated a panel of monoclonal antibodies directed against human P2X3 (hP2X3). We have found that these antibodies produce distinct functional effects, depending on the homomeric or heteromeric composition of the target, its kinetic state, and the duration of antibody exposure. The most potent antibody, 12D4, showed an estimated IC50 of 16 nm on hP2X3 after short term exposure (up to 18 min), binding to the inactivated state of the channel to inhibit activity. By contrast, with the same short term application, 12D4 potentiated the slow inactivating current mediated by the heteromeric hP2X2/3 channel. Extending the duration of exposure to ∼20 h resulted in a profound inhibition of both homomeric hP2X3 and heteromeric hP2X2/3 receptors, an effect mediated by efficient antibody-induced internalization of the channel from the plasma membrane. The therapeutic potential of mAb12D4 was assessed in the formalin, complete Freund's adjuvant, and visceral pain models. The efficacy of 12D4 in the visceral hypersensitivity model indicates that antibodies against P2X3 may have therapeutic potential in visceral pain indications.

Authors+Show Affiliations

From the Rinat Laboratories, Pfizer Inc., South San Francisco, California 94080, anatoly.shcherbatko@pfizer.com.From the Rinat Laboratories, Pfizer Inc., South San Francisco, California 94080.From the Rinat Laboratories, Pfizer Inc., South San Francisco, California 94080.From the Rinat Laboratories, Pfizer Inc., South San Francisco, California 94080.From the Rinat Laboratories, Pfizer Inc., South San Francisco, California 94080.From the Rinat Laboratories, Pfizer Inc., South San Francisco, California 94080.From the Rinat Laboratories, Pfizer Inc., South San Francisco, California 94080.From the Rinat Laboratories, Pfizer Inc., South San Francisco, California 94080.From the Rinat Laboratories, Pfizer Inc., South San Francisco, California 94080.From the Rinat Laboratories, Pfizer Inc., South San Francisco, California 94080.From the Rinat Laboratories, Pfizer Inc., South San Francisco, California 94080.From the Rinat Laboratories, Pfizer Inc., South San Francisco, California 94080.the Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455, and.the Department of Biochemistry and Biophysics, University of California, San Francisco, California 94158.From the Rinat Laboratories, Pfizer Inc., South San Francisco, California 94080.From the Rinat Laboratories, Pfizer Inc., South San Francisco, California 94080.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

27129281

Citation

Shcherbatko, Anatoly, et al. "Modulation of P2X3 and P2X2/3 Receptors By Monoclonal Antibodies." The Journal of Biological Chemistry, vol. 291, no. 23, 2016, pp. 12254-70.
Shcherbatko A, Foletti D, Poulsen K, et al. Modulation of P2X3 and P2X2/3 Receptors by Monoclonal Antibodies. J Biol Chem. 2016;291(23):12254-70.
Shcherbatko, A., Foletti, D., Poulsen, K., Strop, P., Zhu, G., Hasa-Moreno, A., ... Shelton, D. (2016). Modulation of P2X3 and P2X2/3 Receptors by Monoclonal Antibodies. The Journal of Biological Chemistry, 291(23), pp. 12254-70. doi:10.1074/jbc.M116.722330.
Shcherbatko A, et al. Modulation of P2X3 and P2X2/3 Receptors By Monoclonal Antibodies. J Biol Chem. 2016 Jun 3;291(23):12254-70. PubMed PMID: 27129281.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Modulation of P2X3 and P2X2/3 Receptors by Monoclonal Antibodies. AU - Shcherbatko,Anatoly, AU - Foletti,Davide, AU - Poulsen,Kris, AU - Strop,Pavel, AU - Zhu,Guoyun, AU - Hasa-Moreno,Adela, AU - Melton Witt,Jody, AU - Loo,Carole, AU - Krimm,Stellanie, AU - Pios,Ariel, AU - Yu,Jessica, AU - Brown,Colleen, AU - Lee,John K, AU - Stroud,Robert, AU - Rajpal,Arvind, AU - Shelton,David, Y1 - 2016/04/20/ PY - 2016/02/16/received PY - 2016/4/30/entrez PY - 2016/4/30/pubmed PY - 2016/12/15/medline KW - P2X3 KW - cell surface receptor KW - monoclonal antibody KW - pain KW - patch clamp KW - purinergic receptor KW - receptor internalization SP - 12254 EP - 70 JF - The Journal of biological chemistry JO - J. Biol. Chem. VL - 291 IS - 23 N2 - Purinergic homomeric P2X3 and heteromeric P2X2/3 receptors are ligand-gated cation channels activated by ATP. Both receptors are predominantly expressed in nociceptive sensory neurons, and an increase in extracellular ATP concentration under pathological conditions, such as tissue damage or visceral distension, induces channel opening, membrane depolarization, and initiation of pain signaling. Hence, these receptors are considered important therapeutic targets for pain management, and development of selective antagonists is currently progressing. To advance the search for novel analgesics, we have generated a panel of monoclonal antibodies directed against human P2X3 (hP2X3). We have found that these antibodies produce distinct functional effects, depending on the homomeric or heteromeric composition of the target, its kinetic state, and the duration of antibody exposure. The most potent antibody, 12D4, showed an estimated IC50 of 16 nm on hP2X3 after short term exposure (up to 18 min), binding to the inactivated state of the channel to inhibit activity. By contrast, with the same short term application, 12D4 potentiated the slow inactivating current mediated by the heteromeric hP2X2/3 channel. Extending the duration of exposure to ∼20 h resulted in a profound inhibition of both homomeric hP2X3 and heteromeric hP2X2/3 receptors, an effect mediated by efficient antibody-induced internalization of the channel from the plasma membrane. The therapeutic potential of mAb12D4 was assessed in the formalin, complete Freund's adjuvant, and visceral pain models. The efficacy of 12D4 in the visceral hypersensitivity model indicates that antibodies against P2X3 may have therapeutic potential in visceral pain indications. SN - 1083-351X UR - https://www.unboundmedicine.com/medline/citation/27129281/Modulation_of_P2X3_and_P2X2/3_Receptors_by_Monoclonal_Antibodies_ L2 - http://www.jbc.org/cgi/pmidlookup?view=long&pmid=27129281 DB - PRIME DP - Unbound Medicine ER -