Characterization of the Aquaporin-9 Inhibitor RG100204 In Vitro and in <i>db/db</i> Mice
Marilina Florio,
Angelica Engfors,
Patrizia Gena,
Jessica Larsson,
Alessandro Massaro,
Stella Timpka,
Martina Kvist Reimer,
Per Kjellbom,
Eric Beitz,
Urban Johanson,
Michael Rützler,
Giuseppe Calamita
Affiliations
Marilina Florio
Department of Biosciences, Biotechnologies and Environment, University of Bari Aldo Moro, 70125 Bari, Italy
Angelica Engfors
Division of Biochemistry and Structural Biology, Department of Chemistry, Lund University, 22100 Lund, Sweden
Patrizia Gena
Department of Biosciences, Biotechnologies and Environment, University of Bari Aldo Moro, 70125 Bari, Italy
Jessica Larsson
Red Glead Discovery AB, Medicon Village, 22381 Lund, Sweden
Alessandro Massaro
Department of Management, Finance and Technology, Libera Università Mediterranea (LUM) “Giuseppe Degennaro” LUM University, 70010 Casamassima, Italy
Stella Timpka
Red Glead Discovery AB, Medicon Village, 22381 Lund, Sweden
Martina Kvist Reimer
Red Glead Discovery AB, Medicon Village, 22381 Lund, Sweden
Per Kjellbom
Division of Biochemistry and Structural Biology, Department of Chemistry, Lund University, 22100 Lund, Sweden
Eric Beitz
Department of Pharmaceutical and Medicinal Chemistry, Pharmaceutical Institute, Christian-Albrechts-University of Kiel, Gutenbergstr. 76, 24118 Kiel, Germany
Urban Johanson
Division of Biochemistry and Structural Biology, Department of Chemistry, Lund University, 22100 Lund, Sweden
Michael Rützler
Division of Biochemistry and Structural Biology, Department of Chemistry, Lund University, 22100 Lund, Sweden
Giuseppe Calamita
Department of Biosciences, Biotechnologies and Environment, University of Bari Aldo Moro, 70125 Bari, Italy
Aquaporin-9 (AQP9) is a facilitator of glycerol and other small neutral solute transmembrane diffusion. Identification of specific inhibitors for aquaporin family proteins has been difficult, due to high sequence similarity between the 13 human isoforms, and due to the limited channel surface areas that permit inhibitor binding. The few AQP9 inhibitor molecules described to date were not suitable for in vivo experiments. We now describe the characterization of a new small molecule AQP9 inhibitor, RG100204 in cell-based calcein-quenching assays, and by stopped-flow light-scattering recordings of AQP9 permeability in proteoliposomes. Moreover, we investigated the effects of RG100204 on glycerol metabolism in mice. In cell-based assays, RG100204 blocked AQP9 water permeability and glycerol permeability with similar, high potency (~5 × 10−8 M). AQP9 channel blocking by RG100204 was confirmed in proteoliposomes. After oral gavage of db/db mice with RG100204, a dose-dependent elevation of plasma glycerol was observed. A blood glucose-lowering effect was not statistically significant. These experiments establish RG100204 as a direct blocker of the AQP9 channel, and suggest its use as an experimental tool for in vivo experiments on AQP9 function.
