Hybridization into a Bitopic Ligand Increased Muscarinic Receptor Activation for Isopilocarpine but Not for Pilocarpine Derivatives

Pilocarpine (1), a secondary metabolite of several Pilocarpus species, is a therapeutically used partial agonist of muscarinic acetylcholine receptors (mAChRs). The available pharmacological data and structure–activity relationships do not provide comparable data for all five receptor subtypes. In this study, pilocarpine (1), its epimer isopilocarpine (2), racemic analogues pilosinine (3) and desmethyl pilosinine (4), and the respective hybrid ligands with a naphmethonium fragment (5-C6 to 8-C6) were synthesized and analyzed in mini-G nano-BRET assays at the five mAChRs. In line with earlier studies, pilocarpine was the most active compound among the orthosteric ligands 1–4. Computational docking of pilocarpine and isopilocarpine to the active M2 receptor suggests that the trans-configuration of isopilocarpine leads to a loss of the hydrogen bond from the lactone carbonyl to N6.52, explaining the lower activity of isopilocarpine. Hybrid formation of pilocarpine (1) and isopilocarpine (2) led to an inverted activity rank, with the trans-configured isopilocarpine hybrid (6-C6) being more active. The hydrogen bond of interest is formed by the isopilocarpine hybrid (6-C6) but not by the pilocarpine hybrid (5-C6). Hybridization thus leads to a modified binding mode of the orthosteric moiety, as the binding mode of the hybrid is dominated by the high-affinity allosteric moiety

FRET Studies of Quinolone-Based Bitopic Ligands and Their Structural Analogues at the Muscarinic M₁ Receptor

Aiming to design partial agonists as well as allosteric modulators for the M₁ muscarinic acetylcholine (M₁AChR) receptor, two different series of bipharmacophoric ligands and their structural analogues were designed and synthesized. The hybrids were composed of the benzyl quinolone carboxylic acid (BQCA)-derived subtype selective allosteric modulator <b>3</b> and the orthosteric building block 4-((4,5-dihydroisoxazol-3-yl)­oxy)-N,N-dimethylbut-2-yn-1-amine (base of iperoxo) <b>1</b> or the endogenous ligand 2-(dimethylamino)­ethyl acetate (base of acetylcholine) <b>2</b>, respectively. The two pharmacophores were linked <i>via</i> alkylene chains of different lengths (C4, C6, C8, and C10). Furthermore, the corresponding structural analogues of <b>1</b> and <b>2</b> and of modified BQCA <b>3</b> with varying alkyl chain length between C2 and C10 were investigated. Fluorescence resonance energy transfer (FRET) measurements in a living single cell system were investigated in order to understand how these compounds interact with a G protein-coupled receptor (GPCR) on a molecular level and how the single moieties contribute to ligand receptor interaction. The characterization of the modified orthosteric ligands indicated that a linker attached to an orthoster rapidly attenuates the receptor response. Linker length elongation increases the receptor response of bitopic ligands, until reaching a maximum, followed by a gradual decrease. The optimal linker length was found to be six methylene groups at the M₁AChR. A new conformational change is described that is not of inverse agonistic origin for long linker bitopic ligands and was further investigated by exceptional fragment-based screening approaches

Development and characterization of the new Gα_i1-sensor.

<p>(A) Representative image showing the plasma membrane localization of Gα_i1 fused to mTurquoise2-Δ9, expressed in HeLa cells. (B) Schematic overview of the plasmid containing pGβ-2A-YFP-Gγ₂-IRES-Gα_i1-CFP, driven by a CMV promoter. The inset shows the DNA sequence encoding the end of the IRES sequence and the start of the Gα_i1 sequence. The proposed protein translation is shown in the line below the DNA sequence (single letter abbreviations of the amino acids). (C) Confocal images of the localization of Gα_i1-mTurquoise2-Δ9 (<i>top row</i>) and cp173Venus-Gγ₂ (<i>bottom row</i>) in HeLa cells, for variant 1.0 (<i>left column</i>) and variant 2.0 (<i>right column</i>) of the Gα_i1-sensor. (D) Quantitative co-expression analysis of the CFP and YFP channels of the cp173Venus-Gγ₂ and Gα_i1-mTurquoise2-Δ9 transfections in HeLa cells. Single plasmid transfection (<i>left</i>) versus the transfection of the separate plasmids (<i>right</i>). The dots depict the CFP and YFP intensity, quantified from individual single cells. The r² is the coefficient of determination. Width of the individual images in A and C is 143μm.</p

Performance of the Gα_i1-sensor in kinetic measurements.

<p>(A) HEK293 cells transfected with the Gα_i1-sensor and the α₂AR were repeatedly stimulated with 20μM norepinephrine during intervals that are indicated by short horizontal lines. The presented data is representative for at least six different transfections performed on six experimental days. Top panel: YFP emission, center panel: CFP emission, bottom panel: corrected and normalized FRET ratio. (B) HEK293 cells transfected with the Gα_i1-sensor and the Adenosine A1-receptor were stimulated with 30μM adenosine, indicated by the short horizontal line. The presented data is representative for at least six different transfections performed on six experimental days. Top panel: YFP emission, center panel: CFP emission, bottom panel: corrected and normalized FRET ratio. (C) A close-up of the on-kinetics of Gα_i1 activation, showing the normalized FRET ratio during the first stimulation of the experiment in (A), fitted to a one component exponential decay function with tau = 1160ms and amplitude = 0.18 (R = 0.99). (D) Scatter plot showing the average exponential time constants (tau) of pooled data from (n = 10) individual fits of HEK293 cells transfected with the Gα_i1-sensor and the α₂AR stimulated with 100μM norepinephrine or pooled data (n = 14) from individual fits of the Gα_i1-sensor and the Adenosine A1-receptor stimulated with 30μM adenosine, respectively. Error bars indicate 95% CI.</p

Giese et al 2013_suppl_biomass

biomass data of the experimental sites from 2004 - 200

Giese et al 2013_suppl_image_dung heap

image of a dung heap used for cooking and heating during winte

Giese et al 2013_suppl_image_Inner Mongolia grassland Xilin River

panorama image of the Xilingole grassland near the experimental site

Giese et al 2013_suppl_temperature and precipitation IMGERS 1982-2003

Monthly mean values of precipitation and temperature 1982-200

Giese et al 2013_suppl_meteorological data IMGERS 2004-2006

daily meteorological data of the experimental years 2004 -200

Giese et al 2013_suppl_image_farm heavy grazing

image of the heavy grazing site with farm, sheep fold and grazing sheep her