Transmembrane domain 3 (TM3) plays a crucial role mediating muscarinic acetylcholine receptor activation by acetylcholine, carbachol, and other muscarinic agonists. We compared the effects of point mutations throughout TM3 on the interactions of carbachol, 4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl] piperidine hydrogen chloride (AC-42), a potent structural analog of AC-42 called 4-[3-(4-butylpiperidin-1-yl)-propyl]-7-fluoro-4H-benzo[1,4]oxazin-3-one (AC-260584), N-desmethylclozapine, and clozapine with the M 1 muscarinic receptor. The binding and activation profiles of these ligands fell into three distinct patterns; one exemplified by orthosteric compounds like carbachol, another by structural analogs of AC-42, and a third by structural analogs of N-desmethylclozapine. All mutations tested severely reduced carbachol binding and activation of M 1 . In contrast, the agonist actions of AC-42 and AC-260584 were greatly potentiated by the W101A mutation, slightly reduced by Y106A, and slightly increased by S109A. Clozapine and N-desmethylclozapine displayed substantially increased maximum responses at the Y106A and W101A mutants, slightly lower activity at S109A, but no substantial changes in potency. At L102A and N110A, agonist responses to AC-42, AC-260584, clozapine, and N-desmethylclozapine were all substantially reduced, but usually less than carbachol. D105A showed no functional responses to all ligands. Displacement and dissociation rate experiments demonstrated clear allosteric properties of AC-42 and AC-260584 but not for N-desmethylclozapine and clozapine, indicating that they may contact different residues than carbachol to activate M 1 but occupy substantially overlapping spaces, in contrast to AC-42 and AC-260584, which occupy separable spaces. These results show that M 1 receptors can be activated in at least three distinct ways and that there is no requirement for potent muscarinic agonists to mimic acetylcholine interactions with TM3.
The role of neuropeptide FF (NPFF) and its analogs in pain modulation is ambiguous. Although NPFF was first characterized as an antiopioid peptide, both antinociceptive and pronociceptive effects have been reported, depending on the route of administration. Currently, two NPFF receptors, termed FF1 and FF2, have been identified and cloned, but their roles in pain modulation remain elusive because of the lack of availability of selective compounds suitable for systemic administration in in vivo models. Ligand-binding studies confirm ubiquitous expression of both subtypes in brain, whereas only FF2 receptors are expressed spinally. This disparity in localization has served as the foundation of the hypothesis that FF1 receptors mediate the pronociceptive actions of NPFF. We have identified novel small molecule NPFF receptor agonists and antagonists with varying degrees of FF2/FF1 functional selectivity. Using these pharmacological tools in vivo has allowed us to define the roles of NPFF receptor subtypes as pertains to the modulation of nociception. We demonstrate that selective FF2 agonism does not modulate acute pain but instead ameliorates inflammatory and neuropathic pains. Treatment with a nonselective FF1/FF2 agonist potentiates allodynia in neuropathic rats and increases sensitivity to noxious thermal and to non-noxious mechanical stimuli in normal rats in an FF1 antagonist-reversible manner. Treatment with FF1 antagonists reversed established mechanical allodynia, indicating the possibility of increased NPFF tone through FF1 receptors. In conclusion, we provide evidence for the opposing roles of NPFF receptors and highlight selective FF2 agonism and/or selective FF1 antagonism as potential targets warranting further investigation.
Hybanthus enneaspermus is an Indian
folk medicinal herb that has been widely used as a libido enhancer.
This plant belongs to the Violaceae plant family, which ubiquitously
contains disulfide-rich cyclic peptides named cyclotides. Cyclotides
are an expanding plant-derived peptide family with numerous interesting
bioactivities, and their unusual stability against proteolysis has
attracted much attention in drug design applications. Recently, H. enneaspermus has been reported to be a rich source
of cyclotides, and hence, it was of interest to investigate whether
cyclotides contribute to its aphrodisiac activity. In this study,
we evaluated the in vivo aphrodisiac activity of
the herbal powder, extract, and the most abundant cyclotide, hyen
D, extracted from H. enneaspermus on
rats in a single dose regimen. After dosing, the sexual behaviors
of male rats were observed, recorded, analyzed, and compared with
those of the vehicle group. The results show that the extract and
hyen D significantly decreased the intromission latency of sexually
naïve male rats and the extract improved a range of other measured
sexual parameters. The results suggest that the extract could enhance
libido as well as facilitate erectile function in male rats and that
the cyclotide hyen D could contribute to the libido-enhancing activity
of this ethnomedicinal herb.
Cyclotides are a unique family of stable and cyclic mini-proteins
found in plants that have nematicidal and anthelmintic activities.
They are distributed across the Rubiaceae, Violaceae, Fabaceae, Cucurbitaceae,
and Solanaceae plant families, where they are posited to act as protective
agents against pests. In this study, we tested the nematicidal properties
of extracts from four major cyclotide-producing plants, Oldenlandia
affinis, Clitoria ternatea, Viola
odorata, and Hybanthus enneaspermus, against
the free-living model nematode Caenorhabditis elegans. We evaluated the nematicidal activity of the cyclotides kalata
B1, cycloviolacin O2, and hyen D present in these extracts and found
them to be active against the larvae of C. elegans. Both the plant extracts and isolated cyclotides exerted dose-dependent
toxicity on the first-stage larvae of C. elegans.
Isolated cyclotides caused death or damage upon interacting with the
worms’ mouth, pharynx, and midgut or membrane. Cycloviolacin
O2 and hyen D produced bubble-like structures around the C.
elegans membrane, termed blebs, implicating membrane disruption
causing toxicity and death. All tested cyclotides lost their toxicity
when the hydrophobic patches present on them were disrupted via a
single-point mutation. The present results provide a facile assay
design to measure and explore the nematicidal activities of plant
extracts and purified cyclotides on C. elegans.
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