Development of a Conformational Histamine H3 Receptor Biosensor for the Synchronous Screening of Agonists and Inverse Agonists.

Hannes Schihada,Henry F Vischer,Rob Leurs,Martin J Lohse,Steffen Pockes,Ulrike Zabel,Xiaoyuan Ma,Gunnar Schulte

doi:10.1021/acssensors.0c00397

Abstract

The histamine H3 receptor (H3R) represents a highly attractive drug target for the treatment of various central nervous system disorders, but the discovery of novel H3R targeting compounds relies on the assessment of highly amplified intracellular signaling events that do not only reflect H3R modulation and carry the risk of high false-positive and -negative screening rates. To address these limitations, we designed an intramolecular H3R biosensor based on the principle of bioluminescence resonance energy transfer (BRET) that reports the receptor’s real-time conformational dynamics and provides an advanced tool to screen for both H3R agonists and inverse agonists in a live cell screening-compatible assay format. This conformational G-protein-coupled receptor (GPCR) sensor allowed us to characterize the pharmacological properties of known and new H3 receptor ligands with unprecedented accuracy. Interestingly, we found that one newly developed H3 receptor ligand possesses even stronger inverse agonistic activity than reference H3R inverse agonists including the current gold standard pitolisant. Taken together, we describe here the design and validation of the first screening-compatible H3R conformational biosensor that will aid in the discovery of novel H3R ligands and can be employed to gain deeper insights into the (in-)activation mechanism of this highly attractive drug target.

Highlights

H3 receptor (H3R) biosensor based on the principle of bioluminescence resonance energy transfer (BRET) that reports the receptor’s real-time conformational dynamics and provides an advanced tool to screen for both H3R agonists and inverse agonists in a live cell screening-compatible assay format
The group of histamine receptors belongs to the superfamily of G-protein-coupled receptors (GPCRs) and comprises four distinct subtypes, named H1, H2, H3, and H4 receptor (H1−4R), that are of highest interest for modern drug discovery.[1]
We have previously shown that the combination of Nluc and HaloTag(618) yields the most sensitive conformational sensors for three model GPCRs19 and employed this BRET pair to create two distinct variants of conformational H3RNluc/Halo biosensors and monitor their conformational dynamics in a 96-well microtiter format (Scheme 1a,b)

Summary

Introduction

H3R biosensor based on the principle of bioluminescence resonance energy transfer (BRET) that reports the receptor’s real-time conformational dynamics and provides an advanced tool to screen for both H3R agonists and inverse agonists in a live cell screening-compatible assay format. H3R represents an attractive approach to treat various central nervous system diseases such as Parkinson’s, Huntington’s, and Alzheimer’s diseases, as well as tic disorders.[1,6−9] In addition, pitolisant, an H3R inverse agonist that reduces the basal signaling capacity of the receptor (known as receptor constitutive activity), has entered the market 4 years ago for the treatment of narcolepsia.[10] Despite increasing efforts to develop novel H3R-modulating compounds,[11−17] far, pitolisant represents the only H3R ligand that is approved by health authorities This is partially due to the limited power of our current screening technologies that are mainly based on receptor downstream signaling events. Since the receptor’s conformational change follows directly upon ligand binding without any signal amplification, this approach represents the most undistorted way to assess ligand efficacy and potency; as a consequence, these conformational readouts reveal differences among distinct test compounds where downstream-dependent assays report indiscernible activities.[19−21] singlecell studies employing FRET-based conformational biosensors of H1R and H3R have recently provided precious insights into the mechano-sensing mechanism and the activation kinetics of histamine receptors, respectively.[22,23] Recently, the suitability of such sensors for high-throughput screening has been demonstrated by employing a novel BRET system composed of the small engineered luciferase NanoLuciferase (Nluc)[24] and a red fluorescent HaloTag dye,[25] as demonstrated for two class A (α2A-adrenergic and β2-adrenergic receptor) and one class B GPCRs (parathyroid hormone receptor 1).[19]

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Results

Conclusion