Bodilisant—A Novel Fluorescent, Highly Affine Histamine H₃ Receptor Ligand

Abstract

A piperidine-based lead structure for the human histamine H 3 receptor (hH 3 R) was coupled with the BODIPY fluorophore and resulted in a strong green fluorescent (quantum yield, 0.92) hH 3 R ligand with affinity in the nanomolar concentration range (K i hH 3 R = 6.51 ± 3.31 nM), named Bodilisant. Screening for affinities at histamine and dopamine receptor subtypes showed high hH 3 R preference. Bodilisant was used for visualization of hH 3 R in hH 3 R overexpressing HEK-293 cells with fluorescence confocal laser scanning microscopy. In addition, in native human brain tissues, Bodilisant showed clear and displaceable images of labeled hH 3 R.KEYWORDS: histamine, H 3 receptor, nonimidazole derivative, GPCR, BODIPY, HEK-293 cells, tissue labeling, fluorescence confocal laser scanning microscopy, displacement, pharmacological tool T he human histamine H 3 receptor is one of the four human histamine receptor subtypes (hH 1−4 R). It is a membranebound class A family G-protein-coupled receptor (GPCR) (G i/o coupled) mainly expressed in the central nervous system (CNS) acting as an auto-as well as a heteroreceptor.1 The human histamine H 3 receptor (hH 3 R) modulates the release of several neuronal neurotransmitters.2,3 Because of different central effects of several hH 3 R antagonists in preclinical and clinical trials, we have seen a need for labeled hH 3 R ligands to be taken as diagnostic tools to further investigate neurological disorders in the CNS based on receptor distribution, occupation, and regulation. In histochemistry, fluorescentlabeled antibodies are of common use. 4 For the manufacturing process of antibodies, mostly animal experiments are required. 5,6 These antibodies have to be labeled in a followup work step with a second labeled antibody for immunofluorescence; merely a few primary antibodies are directly labeled.7 Most antibodies are sensitive to temperature and have to be stored in freezers. The main disadvantage as compared to small fluorescent GPCR ligands is their undisplacement properties without target destruction. Small fluorescent GPCR ligands can be displaced by other ligands and consequently enable the development of fluorescence displacement assays. There are many efforts to design fluorescent GPCR ligands on the strength of their prominence as the largest and most versatile group of cell surface receptors, therefore responsible for various pharmacological functions. 8,9 Recent research in our working group resulted in chalconebased fluorescent hH 3 R ligands able to label hH 3 R in cells and human tissue 10 based on earlier results (Mirisant-405, Mirisant-470, and Benz-Mirisant-405).11−13 Because these early ligands were not usable under all conditions, we wanted to design fluorescent hH 3 R ligands with more properties in common in one molecule, that is, higher hH 3 R affinity, versatile fluorescent wavelength, optimized fluorescence intensity, and thus a better signal-to-noise ratio. The decision has been made for 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) dye as a...

The Histamine H3 Receptor Antagonist E159 Reverses Memory Deficits Induced by Dizocilpine in Passive Avoidance and Novel Object Recognition Paradigm in Rats

“…a [ 125 I]Iodoproxyfan binding assay at human H3Rstably expressed in CHO-K1 cells, n = 3 ( Ligneau et al, 1994 ; Ligneau et al, 2000 ; Lazewska et al, 2006 ). b [ 3 H]Histamine binding assay performed with cell membrane preparation of Sf9 cells transiently expressing the human histamine H4Rand co-expressed with G αi2 and Gβ1γ2 subunits, n = 3 ( Meier et al, 2001 ; Amon et al, 2007 ; Isensee et al, 2009 ; Tomasch et al, 2013 ). c [ 3 H]Pyrilamine binding assay performed with cell membrane preparation of CHO-hH1Rcells stably expressing the human H1R, n = 3 ( Schibli and Schubiger, 2002 ; van Staveren and Metzler-Nolte, 2004 ; Schlotter et al, 2005 ).…”

Pharmacological Characterization of Human Histamine Receptors and Histamine Receptor Mutants in the Sf9 Cell Expression System

“…Fluorescent hH 3 R-selective ligands were developed by using the chalcone partial structure (Tomasch et al 2012). Moreover, a compound named "Bodilisant," which has been reported recently, is a BODIPY-labeled non-imidazole ligand with nanomolar hH 3 R affinity (Tomasch et al 2013). Some progress has also been made in the field of fluorescence-based G protein activation assays.…”

The evolving small‐molecule fluorescent‐conjugate toolbox for Class A GPCRs

“…From the same laboratory, Tomasch et al . (2012b) have used the same piperidine‐based pharmacophore but now with the boron‐dipyrromethene scaffold as the fluorophore ( 19 ). Synthesis of the fluorophore moiety was completed by reaction with boron trifluoroetherate as the final reaction in a stepwise synthesis, rather than the more common convergent approach of coupling a pre‐activated (and often commercially available as the NHS ester) fluorophore to a complementary pharmacophore/linker.…”

“…From the same research laboratory as fluorescent H 3 R ligands 16–18 (Tomasch et al ., 2012b; 2012c) was the report of a fluorescent prostanoid EP 3 receptor (EP 3 R) antagonist ( 26 ) (Tomasch et al ., 2012a). Based on an ortho‐substituted cinnamic acid antagonist, a series of fluorescent conjugates were synthesized containing different fluorophores, with pyrylium‐containing 26 showing the most promise as an imaging tool.…”