5 research outputs found
Caged Naloxone: Synthesis, Characterization, and Stability of 3‑<i>O</i>‑(4,5-Dimethoxy-2-nitrophenyl)carboxymethyl Naloxone (CNV-NLX)
The
photolabile analogue of the broad-spectrum opioid antagonist
naloxone, 3-<i>O</i>-(4,5-dimethoxy-2-nitrophenyl)carboxymethyl
naloxone (also referred to as “caged naloxone”, 3-<i>O</i>-(α-carboxy-6-nitroveratryl)naloxone, CNV-NLX), has
been found to be a valuable biochemical probe. While the synthesis
of CNV-NLX is simple, its characterization is complicated by the fact
that it is produced as a mixture of α<i>R</i>,5<i>R</i>,9<i>R</i>,13<i>S</i>,14<i>S</i> and α<i>S</i>,5<i>R</i>,9<i>R</i>,13<i>S</i>,14<i>S</i> diastereomers. Using long-range
and heteronuclear NMR correlations, the <sup>1</sup>H NMR and <sup>13</sup>C NMR resonances of both diastereomers have been fully assigned,
confirming the structures. Monitoring of solutions of CNV-NLX in saline
buffer, in methanol, and in DMSO has shown CNV-NLX to be stable for
over a week under fluorescent laboratory lights at room temperature.
Exposure of such solutions to λ 365 nm from a hand-held UV lamp
led to the formation of naloxone and CNV-related breakdown products
The Importance of Hydrogen Bonding and Aromatic Stacking to the Affinity and Efficacy of Cannabinoid Receptor CB<sub>2</sub> Antagonist, 5‑(4-chloro-3-methylphenyl)-1-[(4-methylphenyl)methyl]‑<i>N</i>‑[(1<i>S</i>,2<i>S</i>,4<i>R</i>)‑1,3,3-trimethylbicyclo[2.2.1]hept-2-yl]-1H-pyrazole-3-carboxamide (SR144528)
Despite
the therapeutic promise of the subnanomolar affinity cannabinoid
CB<sub>2</sub> antagonist, 5-(4-chloro-3-methylphenyl)-1-[(4-methylphenyl)methyl]-<i>N</i>-[(1<i>S</i>,2<i>S</i>,4<i>R</i>)-1,3,3-trimethylbicyclo[2.2.1]hept-2-yl]-1<i>H</i>-pyrazole-3-carboxamide (SR144528, <b>1</b>), little is known about its binding site interactions and
no primary interaction site for <b>1</b> at CB2 has been identified.
We report here the results of Glide docking studies in our cannabinoid
CB<sub>2</sub> inactive state model that were then tested via compound
synthesis, binding, and functional assays. Our results show that the
amide functional group of <b>1</b> is critical to its CB2 affinity
and efficacy and that aromatic stacking interactions in the TMH5/6
aromatic cluster of CB2 are also important. Molecular modifications
that increased the positive electrostatic potential in the region
between the fenchyl and aromatic rings led to more efficacious compounds.
This result is consistent with the EC-3 loop negatively charged amino
acid, D275 (identified via Glide docking studies) acting as the primary
interaction site for <b>1</b> and its analogues
Peripherally Selective Cannabinoid 1 Receptor (CB1R) Agonists for the Treatment of Neuropathic Pain
Alleviation of neuropathic pain by
cannabinoids is limited by their
central nervous system (CNS) side effects. Indole and indene compounds
were engineered for high hCB1R affinity, peripheral selectivity, metabolic
stability, and in vivo efficacy. An epithelial cell line assay identified
candidates with <1% blood–brain barrier penetration for
testing in a rat neuropathy induced by unilateral sciatic nerve entrapment
(SNE). The SNE-induced mechanical allodynia was reversibly suppressed,
partially or completely, after intraperitoneal or oral administration
of several indenes. At doses that relieve neuropathy symptoms, the
indenes completely lacked, while the brain-permeant CB1R agonist HU-210
(<b>1</b>) exhibited strong CNS side effects, in catalepsy,
hypothermia, and motor incoordination assays. Pharmacokinetic findings
of ∼0.001 cerebrospinal fluid:plasma ratio further supported
limited CNS penetration. Pretreatment with selective CB1R or CB2R
blockers suggested mainly CB1R contribution to an indene’s
antiallodynic effects. Therefore, this class of CB1R agonists holds
promise as a viable treatment for neuropathic pain
Novel Synthesis and Pharmacological Characterization of NOP Receptor Agonist 8-[(1<i>S</i>,3a<i>S</i>)-2,3,3a,4,5,6-Hexahydro-1<i>H</i>-phenalen-1-yl]-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one (Ro 64-6198)
The
nociceptin/orphanin FQ opioid peptide (NOP) receptor is a widely
expressed GPCR involved in the modulation of pain, anxiety, and motor
behaviors. Dissecting the functional properties of this receptor is
limited by the lack of systemically active ligands that are brain
permeant. The small molecule NOP receptor-selective, full agonist
8-[(1<i>S</i>,3a<i>S</i>)-2,3,3a,4,5,6-hexahydro-1<i>H</i>-phenalen-1-yl]-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one
(Ro 64-6198) hydrochloride is an active, brain penetrant ligand, but its difficult
and cost-prohibitive synthesis limits its widespread use and availability
for animal studies. Here, we detail a more efficient and convenient
method of synthesis, and use both in vitro and in vivo pharmacological
assays to fully characterize this ligand. Specifically, we characterize
the pharmacodynamics of Ro 64-6198 in cAMP and G-protein coupling
in vitro and examine, for the first time, the effects of nociceptin/orphanin
FQ and Ro 64-6198 in arrestin recruitment assays. Further, we examine
the effects of Ro 64-6198 on analgesia, anxiety, and locomotor responses
in vivo. This new synthesis and pharmacological characterization provide
additional insights into the useful, systemically active, NOP receptor
agonist Ro 64-6198
Identification of the GPR55 Antagonist Binding Site Using a Novel Set of High-Potency GPR55 Selective Ligands
GPR55 is a class A G protein-coupled
receptor (GPCR) that has been
implicated in inflammatory pain, neuropathic pain, metabolic disorder,
bone development, and cancer. Initially deorphanized as a cannabinoid
receptor, GPR55 has been shown to be activated by non-cannabinoid
ligands such as l-α-lysophosphatidylinositol (LPI).
While there is a growing body of evidence of physiological and pathophysiological
roles for GPR55, the paucity of specific antagonists has limited its
study. In collaboration with the Molecular Libraries Probe Production
Centers Network initiative, we identified a series of GPR55 antagonists
using a β-arrestin, high-throughput, high-content screen of
∼300000 compounds. This screen yielded novel, GPR55 antagonist
chemotypes with IC<sub>50</sub> values in the range of 0.16–2.72
μM [Heynen-Genel, S., et al. (2010) Screening for Selective
Ligands for GPR55: Antagonists (ML191, ML192, ML193) (Bookshelf ID
NBK66153; PMID entry 22091481)]. Importantly, many of the GPR55 antagonists
were completely selective, with no agonism or antagonism against GPR35,
CB1, or CB2 up to 20 μM. Using a model of the GPR55 inactive
state, we studied the binding of an antagonist series that emerged
from this screen. These studies suggest that GPR55 antagonists possess
a head region that occupies a horizontal binding pocket extending
into the extracellular loop region, a central ligand portion that
fits vertically in the receptor binding pocket and terminates with
a pendant aromatic or heterocyclic ring that juts out. Both the region
that extends extracellularly and the pendant ring are features associated
with antagonism. Taken together, our results provide a set of design
rules for the development of second-generation GPR55 selective antagonists