Hauwert NJ et al. (2019), A Photoswitchable Agonist for the Histamine H3 ...

XB-ART-55681

Angew Chem Int Ed Engl 2019 Mar 26;5814:4531-4535. doi: 10.1002/anie.201813110.

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A Photoswitchable Agonist for the Histamine H3 Receptor, a Prototypic Family A G-Protein-Coupled Receptor.

Hauwert NJ , Mocking TAM , Da Costa Pereira D , Lion K , Huppelschoten Y , Vischer HF , De Esch IJP , Wijtmans M , Leurs R .

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Spatiotemporal control over biochemical signaling processes involving G protein-coupled receptors (GPCRs) is highly desired for dissecting their complex intracellular signaling. We developed sixteen photoswitchable ligands for the human histamine H3 receptor (hH3 R). Upon illumination, key compound 65 decreases its affinity for the hH3 R by 8.5-fold and its potency in hH3 R-mediated Gi protein activation by over 20-fold, with the trans and cis isomer both acting as full agonist. In real-time two-electrode voltage clamp experiments in Xenopus oocytes, 65 shows rapid light-induced modulation of hH3 R activity. Ligand 65 shows good binding selectivity amongst the histamine receptor subfamily and has good photolytic stability. In all, 65 (VUF15000) is the first photoswitchable GPCR agonist confirmed to be modulated through its affinity and potency upon photoswitching while maintaining its intrinsic activity, rendering it a new chemical biology tool for spatiotemporal control of GPCR activation.

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Species referenced: Xenopus
Genes referenced: gprc6a kcnj3

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	Figure 1. General design and concept of photoswitchable H3R full agonists.
	Scheme 1. General synthetic scheme for photoswitchable H3R agonists. See the Supporting Information for detailed experimental procedures.
	Figure 2. A) Representative part of 1H NMR spectra of 10 mm 65 in [D6]DMSO illuminated at 360±20 nm displayed at various time points (seconds). The presented peak belongs to the hydrogen atoms explicitly drawn in the structure shown above the spectrum. Full spectra are available in Figure S4 in the Supporting Information. B) Representative part of LC‐MS chromatograms belonging to the illuminated NMR sample in Figure 2 A. Full chromatograms are available in Figure S5 in the Supporting Information. C) UV/Vis spectra of 25 μm of 65 in 50 mm Tris‐HCl pH 7.4 buffer + 1 % [D6]DMSO. PSS cis represents a sample which has been illuminated for 300 s using 360±20 nm light. PSS trans represents subsequent illumination for 300 s using 434±9 nm light. D) Repeated isomerization of 25 μm of 65 in 50 mm Tris‐HCl pH 7.4 buffer + 1 % [D6]DMSO analyzed at 320 nm. PSS cis was obtained by illuminating 65 for 40 s at 360±20 nm. PSS trans was obtained by illuminating 65 for 40 s at 434±9 nm. E) Absorbance at 320 nm of 25 μm of 65 in 50 mm Tris‐HCl pH 7.4 buffer + 1 % [D6]DMSO. UV/Vis spectra were obtained at 1 s intervals under alternating illumination at 360±20 nm and 434±9 nm perpendicular to the light source of the UV/Vis spectrometer.
	Figure 3. Representative curves of 65 (A) in competition binding with [3H]‐NAMH or (B) in Gi protein activation, as measured by [35S]‐GTPγS accumulation on HEK293T cell homogenates transiently expressing hH3R. Black lines refer to a sample containing more than 99 % trans 65, while magenta lines refer to a sample of 65 illuminated to PSS cis with 360±20 nm prior to the assay. C) Schematic drawing of the TEVC setup used for dynamic hH3R and GIRK current activation in Xenopus laevis oocytes expressing hH3R and GIRK. D) Representative part of a GIRK‐mediated current trace during continuous perfusion with 1 μm 65, while illuminating the oocyte with alternating 360±20 and 434±9 nm wavelength, as measured by TEVC. Error bars shown are mean±SD.

References [+] :

Ahmed, Controlling azobenzene photoswitching through combined ortho-fluorination and -amination. 2017, Pubmed