UR-DEBa242: A Py-5-Labeled Fluorescent Multipurpose Probe for Investigations on the Histamine H₃ and H₄ Receptors

Abstract

Oral presentationsBartole, E.; Littmann, T.; Bernhardt, G.; Buschauer, A. "2,4-Diaminopyrimidines: towards molecular tools for investigations on human and rodent histamine H 4 receptors." Mid-term evaluation event of the GRK 1910 by the "Deutsche Forschungsgemeinschaft" (Regensburg, 2017) Bartole, E.; Littmann, T.; Bernhardt, G.; Buschauer, A. "2,4-Diaminopyrimidines: towards molecular tools for investigations on human and rodent histamine H 4 receptors." 1 st Joint meeting of the European and Japanese Histamine Research Societies (Amsterdam, 2017) Bartole, E.; Bernhardt, G.; Buschauer, A. "Molecular tools for the investigation of GPCR orthologues: the histamine H 4 receptor as an example." Christmas Colloquium 2016 of the

Label-Free Investigations on the G Protein Dependent Signaling Pathways of Histamine Receptors

“…The HEK hH 1 R, HEK hH 2 R, HEK hH 3 R and HEK hH 4 R cells are abbreviated designations of the previously described stable single clone transfectants: HEK293T-SP-FLAG-hH 1 R K12 [ 111 ], HEK293T-SP-FLAG-hH 2 R K46 [ 111 ], HEK293T-SP-FLAG-hH 3 R K16 [ 89 ] and HEK293T-SP-FLAG-hH 4 R K3 cells [ 89 ], respectively. The procedures for molecular cloning of the receptors and the generation of the stable cell lines are described elsewhere [ 89 , 111 ]. The cells were cultured in DMEM supplemented with 10% FBS + P/S and 600 µg/mL G418.…”

“…For the generation of ΔGα x HEK hH 1–4 R cells CRISPR/Cas9 modified HEK293A cells lacking the Gα proteins Gα s + Gα olf (ΔGα s/l HEK) [ 63 ], Gα q + Gα 11 (ΔGα q/11 HEK) [ 44 ], Gα 12 + Gα 13 (ΔGα 12/13 HEK) [ 64 ] or Gα q + Gα olf + Gα 11 + Gα s + Gα 12 + Gα 13 (ΔGα six HEK) [ 41 ] were transfected with the pIRESneo3-SP-FLAG vector encoding the hH 1–4 Rs according to the procedure described for HEK hH 4 R cells [ 89 ] except that no single clone selection was performed. The cells were cultured in DMEM supplemented with 10% FBS + P/S and 600 µg/mL G418.…”

A Versatile Sub-Nanomolar Fluorescent Ligand Enables NanoBRET Binding Studies and Single-Molecule Microscopy at the Histamine H3 Receptor

“…BRET-Based Saturation/Real-Time Kinetics/Competition Binding at the NLuc-H 3 R. BRET-based saturation/real-time kinetics/competition binding at the NLuc-H 3 R were performed as previously described by Bartole et al with minor modifications. 23 For the determination of nonspecific binding, clobenpropit (500-fold excess) was used instead of thioperamide. Dissociation was initiated by addition of 250 nM clobenpropit (500-fold excess) instead of thioperamide.…”

“…48 All experiments were carried out on whole HEK cells instead of Sf9 membranes. Generation of the stable HEK293-SP-FLAG-hH 1 R and HEK293-SP-FLAG-hH 2 R cell lines was conducted as described for the HEK293-SP-FLAG-hH 3 R and HEK293-SP-FLAG-hH 4 R. 23 Ligand dilutions of 12 were prepared 10-fold concentrated in L-15 with 1% BSA, and 10 μL/ well was transferred to a flat-bottom polypropylene 96-well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), as well as 10 μL/ well of the respective radioligand. The cells were adjusted to a density of 1.25 × 10 6 cells/mL, and 80 μL of the cell suspension was added to each well (total volume of 100 μL).…”

“…We therefore aimed to develop a tool suitable for single-molecule imaging and as a tracer probe for binding studies of new putative drug candidates for the H 3 R in a recently developed NanoBRET binding assay that can be employed in high throughput analyses. 23 ■ RESULTS AND DISCUSSION Design Rationale. Fluorescent receptor ligands can be considered as a combination of three distinct parts: the parent ligand, often referred to as a pharmacophore, a linker region, and a fluorescent dye.…”