14 research outputs found
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 M<sub>1</sub> Receptor
Aiming
to design partial agonists as well as allosteric modulators
for the M<sub>1</sub> muscarinic acetylcholine (M<sub>1</sub>AChR)
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)Âoxy)-N,N-dimethylbut-2-yn-1-amine
(base of iperoxo) <b>1</b> or the endogenous ligand 2-(dimethylamino)Âethyl
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 M<sub>1</sub>AChR. 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<sub>i1</sub>-sensor.
<p>(A) Representative image showing the plasma membrane localization of Gα<sub>i1</sub> fused to mTurquoise2-Δ9, expressed in HeLa cells. (B) Schematic overview of the plasmid containing pGβ-2A-YFP-Gγ<sub>2</sub>-IRES-Gα<sub>i1</sub>-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<sub>i1</sub> 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<sub>i1</sub>-mTurquoise2-Δ9 (<i>top row</i>) and cp173Venus-Gγ<sub>2</sub> (<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<sub>i1</sub>-sensor. (D) Quantitative co-expression analysis of the CFP and YFP channels of the cp173Venus-Gγ<sub>2</sub> and Gα<sub>i1</sub>-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 r<sup>2</sup> is the coefficient of determination. Width of the individual images in A and C is 143μm.</p
Performance of the Gα<sub>i1</sub>-sensor in kinetic measurements.
<p>(A) HEK293 cells transfected with the Gα<sub>i1</sub>-sensor and the α<sub>2</sub>AR 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<sub>i1</sub>-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<sub>i1</sub> 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<sub>i1</sub>-sensor and the α<sub>2</sub>AR stimulated with 100μM norepinephrine or pooled data (n = 14) from individual fits of the Gα<sub>i1</sub>-sensor and the Adenosine A1-receptor stimulated with 30μM adenosine, respectively. Error bars indicate 95% CI.</p
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