The manifestations of Parkinson’s disease are caused by reduced dopaminergic innervation of the striatum. Loss-of-function mutations in the
DJ-1 gene cause early-onset familial parkinsonism. To ...investigate a possible role for DJ-1 in the dopaminergic system, we generated a mouse model bearing a germline disruption of
DJ-1. Although
DJ-1
−/−
mice had normal numbers of dopaminergic neurons in the substantia nigra, evoked dopamine overflow in the striatum was markedly reduced, primarily as a result of increased reuptake. Nigral neurons lacking DJ-1 were less sensitive to the inhibitory effects of D2 autoreceptor stimulation. Corticostriatal long-term potentiation was normal in medium spiny neurons of
DJ-1
−/−
mice, but long-term depression (LTD) was absent. The LTD deficit was reversed by treatment with D2 but not D1 receptor agonists. Furthermore,
DJ-1
−/−
mice displayed hypoactivity in the open field. Collectively, our findings suggest an essential role for DJ-1 in dopaminergic physiology and D2 receptor-mediated functions.
Blockade of voltage-gated Ca
2+ channels on sensory nerves attenuates neurotransmitter release and membrane hyperexcitability associated with chronic pain states. Identification of small molecule Ca
...2+ channel blockers that produce significant antinociception in the absence of deleterious hemodynamic effects has been challenging. In this report, two novel structurally related compounds, A-686085 and A-1048400, were identified that potently block N-type (IC
50
=
0.8
μM and 1.4
μM, respectively) and T-type (IC
50
=
4.6
μM and 1.2
μM, respectively) Ca
2+ channels in FLIPR based Ca
2+ flux assays. A-686085 also potently blocked L-type Ca
2+ channels (EC
50
=
0.6
μM), however, A-1048400 was much less active in blocking this channel (EC
50
=
28
μM). Both compounds dose-dependently reversed tactile allodynia in a model of capsaicin-induced secondary hypersensitivity with similar potencies (EC
50
=
300–365
ng/ml). However, A-686085 produced dose-related decreases in mean arterial pressure at antinociceptive plasma concentrations in the rat, while A-1048400 did not significantly alter hemodynamic function at supra-efficacious plasma concentrations. Electrophysiological studies demonstrated that A-1048400 blocks native N- and T-type Ca
2+ currents in rat dorsal root ganglion neurons (IC
50
=
3.0
μM and 1.6
μM, respectively) in a voltage-dependent fashion. In other experimental pain models, A-1048400 dose-dependently attenuated nociceptive, neuropathic and inflammatory pain at doses that did not alter psychomotor or hemodynamic function. The identification of A-1048400 provides further evidence that voltage-dependent inhibition of neuronal Ca
2+ channels coupled with pharmacological selectivity vs. L-type Ca
2+ channels can provide robust antinociception in the absence of deleterious effects on hemodynamic or psychomotor function.
Salvinorin A is a naturally occurring hallucinogenic diterpenoid from the plant Salvia divinorum that selectively and potently activates κ-opioid receptors (KORs). Salvinorin A is unique in that it ...is the only known lipid-like molecule that selectively and potently activates a G-protein coupled receptor (GPCR), which has as its endogenous agonist a peptide; salvinorin A is also the only known non-nitrogenous opioid receptor agonist. In this paper, we identify key residues in KORs responsible for the high binding affinity and agonist efficacy of salvinorin A. Surprisingly, we discovered that salvinorin A was stabilized in the binding pocket by interactions with tyrosine residues in helix 7 (Tyr313 and Tyr320) and helix 2 (Tyr119). Intriguingly, activation of KORs by salvinorin A required interactions with the helix 7 tyrosines Tyr312, Tyr313, and Tyr320 and with Tyr139 in helix 3. In contrast, the prototypical nitrogenous KOR agonist U69593 and the endogenous peptidergic agonist dynorphin A (1−13) showed differential requirements for these three residues for binding and activation. We also employed a novel approach, whereby we examined the effects of cysteine-substitution mutagenesis on the binding of salvinorin A and an analogue with a free sulfhydryl group, 2-thiosalvinorin B. We discovered that residues predicted to be in close proximity, especially Tyr313, to the free thiol of 2-thiosalvinorin B when mutated to Cys showed enhanced affinity for 2-thiosalvinorin B. When these findings are taken together, they imply that the diterpenoid salvinorin A utilizes unique residues within a commonly shared binding pocket to selectively activate KORs.
Background and purpose:
The histamine H
4
receptor is widely expressed in cells of immune origin and has been shown to play a role in a variety of inflammatory processes mediated by histamine. In ...this report, we describe the
in vitro
and
in vivo
anti‐inflammatory properties of a potent histamine H
4
receptor antagonist, A‐940894 (4‐piperazin‐1‐yl‐6,7‐dihydro‐5H‐benzo6,7cyclohepta1,2‐dpyrimidin‐2‐ylamine).
Experimental approach:
We have analysed the pharmacological profile of A‐940894 at mouse native, rat recombinant and human recombinant and native, histamine H
4
receptors by radioligand binding, calcium mobilization, mast cell shape change, eosinophil chemotaxis assays and in the mouse model of zymosan‐induced peritonitis.
Key results:
A‐940894 potently binds to both human and rat histamine H
4
receptors and exhibits considerably lower affinity for the human histamine H
1
, H
2
or H
3
receptors. It potently blocked histamine‐evoked calcium mobilization in the fluorometric imaging plate reader assays and inhibited histamine‐induced shape change of mouse bone marrow‐derived mast cells and chemotaxis of human eosinophils
in vitro
. In a mouse mast cell‐dependent model of zymosan‐induced peritonitis, A‐940894 significantly blocked neutrophil influx and reduced intraperitoneal prostaglandin D
2
levels. Finally, A‐940894 has good pharmacokinetic properties, including half‐life and oral bioavailability in rats and mice.
Conclusions and Implications:
These data suggest that A‐940894 is a potent and selective histamine H
4
receptor antagonist with pharmacokinetic properties suitable for long‐term
in vivo
testing and could serve as a useful tool for the further characterization of histamine H
4
receptor pharmacology.
Voltage‐gated Ca2+ channels modulate neurotransmitter release and cellular excitability in neurons. While the N‐Type selective Ca2+ channel pore blocker Prialt® attenuates chronic pain in patients, ...its clinical utility is limited by intrathecal delivery. Identification of state‐dependent small molecule Ca2+ channels blockers has the potential to have similar analgesic efficacy to Prialt® with a superior cardiovascular safety profile. We have identified two novel compounds, A‐686085 and A‐1048400, that potently block N‐ (IC50 = 0.84 and 0.8 μM, respectively) and T‐Type (IC50 = 4.6 and 0.3 μM, respectively) Ca2+ channels. Functional selectivity versus L‐Type Ca2+ channels was determined by measuring relaxation of rat aorta rings (EC50 of 0.37μM for A‐686085 and 14.4 μM for A‐1048400). Both blockers reversed mechanical nociception in the capsaicin model of secondary hyperalgesia with similar potency (EC50 = 0.5 – 0.8 μg/ml). In an anesthetized rat cardiovascular assay, A‐686085 produced large dose‐dependent decreases in mean arterial pressure (13%, 37%, 42%) with no effect on heart rate at plasma levels of 0.1, 0.3 and 0.7 μg/ml, respectively. In contrast, A‐1048400 produced only modest effects on mean arterial pressure (4%) and no effects on heart rate up to 4.9 μg/ml. These data show that A‐1048400 has a superior therapeutic index relative to A‐686085 and demonstrates that analgesic calcium channel blockers can be identified that have reduced hemodynamic liabilities. Research supported by Abbott Laboratories.
Ca(V)2.2 (N-type) calcium channels are key regulators of neurotransmission. Evidence from knockout animals and localization studies suggest that Ca(V)2.2 channels play a critical role in nociceptive ...transmission. Additionally, ziconotide, a selective peptide inhibitor of Ca(V)2.2 channels, is clinically used to treat refractory pain. However, the use of ziconotide is limited by its low therapeutic index, which is believed, at least in part, to be a consequence of ziconotide inhibiting Ca(V)2.2 channels regardless of the channel state. Subsequent efforts have focused on the discovery of state-dependent inhibitors that preferentially bind to the inactivated state of Ca(V)2.2 channels in order to achieve an improved safety profile relative to ziconotide. Much less attention has been paid to understanding the binding kinetics of these state-dependent inhibitors. Here, we describe a novel electrophysiology-based assay on an automated patch platform designed to differentiate Ca(V)2.2 inhibitors based on their combined state dependence and kinetics. More specifically, this assay assesses inactivated state block, closed state block, and monitors the kinetics of recovery from block when channels move between states. Additionally, a use-dependent assay is described that uses a train of depolarizing pulses to drive channels to a similar level of inactivation for comparison. This use-dependent protocol also provides information on the kinetics of block development. Data are provided to show how these assays can be utilized to screen for kinetic diversity within and across chemical classes.
Treatment of salvinorin A (1a) with KOH in MeOH gave the enedione 3, for which the dienone structure 7 was recently proposed. Also isolated, after methylation, were the secotriesters 4a−c. A ...mechanism for this unusual series of autoxidations is proposed. Surprisingly, 4a showed weak affinity at the κ-opioid receptor. Divinatorins A−C (2a−c) showed no affinity at opioid receptors. Attempted reduction of 3 to a novel salvinorin diol (9d) was unsuccessful, but careful deacetylation of salvinorin C (9a) provided a viable route to this compound. A general method for identifying salvinorin 8-epimers by TLC is also presented.
Background and purpose: The histamine H4 receptor is widely expressed in cells of immune origin and has been shown to play a role in a variety of inflammatory processes mediated by histamine. In ...this report, we describe the in vitro and in vivo anti‐inflammatory properties of a potent histamine H4 receptor antagonist, A‐940894 (4‐piperazin‐1‐yl‐6,7‐dihydro‐5H‐benzo6,7cyclohepta1,2‐dpyrimidin‐2‐ylamine).
Experimental approach: We have analysed the pharmacological profile of A‐940894 at mouse native, rat recombinant and human recombinant and native, histamine H4 receptors by radioligand binding, calcium mobilization, mast cell shape change, eosinophil chemotaxis assays and in the mouse model of zymosan‐induced peritonitis.
Key results: A‐940894 potently binds to both human and rat histamine H4 receptors and exhibits considerably lower affinity for the human histamine H1, H2 or H3 receptors. It potently blocked histamine‐evoked calcium mobilization in the fluorometric imaging plate reader assays and inhibited histamine‐induced shape change of mouse bone marrow‐derived mast cells and chemotaxis of human eosinophils in vitro. In a mouse mast cell‐dependent model of zymosan‐induced peritonitis, A‐940894 significantly blocked neutrophil influx and reduced intraperitoneal prostaglandin D2 levels. Finally, A‐940894 has good pharmacokinetic properties, including half‐life and oral bioavailability in rats and mice.
Conclusions and Implications: These data suggest that A‐940894 is a potent and selective histamine H4 receptor antagonist with pharmacokinetic properties suitable for long‐term in vivo testing and could serve as a useful tool for the further characterization of histamine H4 receptor pharmacology.
An estimated 50% of currently marketed drugs target G protein-coupled receptors (GPCRs) for a wide variety of indications, including central nervous system (CNS) disorders. Although drug discovery ...efforts have focused on GPCRs, less than 10% of GPCRs are currently used as drug targets. Thus, GPCRs continue to represent a significant opportunity for future CNS drug development. Identifying the molecular targets of psychoactive compounds may result in the elucidation of novel targets for CNS drug discovery. This commentary will describe discovery-based approaches and provide several recent examples of novel ligand-receptor interactions discovered through systematic screening of the 'receptorome'.
Adenylate cyclase activity in NS20Y cells expressing D
2L dopamine receptors was examined following chronic treatment with norepinephrine and epinephrine. Initial acute experiments revealed that both ...norepinephrine and epinephrine inhibited forskolin-stimulated cyclic AMP accumulation via D
2 receptors. Furthermore, chronic (18 h) activation of D
2 dopamine receptors by norepinephrine or epinephrine induced a marked increase (>10-fold) in subsequent forskolin-stimulated cyclic AMP accumulation. This heterologous sensitization of adenylate cyclase activity was blocked by D
2 dopamine receptor antagonists and by pertussis toxin pretreatment. In contrast, concurrent activation of Gα
s or adenylate cyclase did not appear to alter noradrenergic agonist-induced sensitization.
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