Most estrogen receptor α (ER)-positive breast cancers initially respond to antiestrogens, but many eventually become estrogen-independent and recur. We identified an estrogen-independent role for ER ...and the CDK4/Rb/E2F transcriptional axis in the hormone-independent growth of breast cancer cells. ER downregulation with fulvestrant or small interfering RNA (siRNA) inhibited estrogen-independent growth. Chromatin immunoprecipitation identified ER genomic binding activity in estrogen-deprived cells and primary breast tumors treated with aromatase inhibitors. Gene expression profiling revealed an estrogen-independent, ER/E2F-directed transcriptional program. An E2F activation gene signature correlated with a lesser response to aromatase inhibitors in patients' tumors. siRNA screening showed that CDK4, an activator of E2F, is required for estrogen-independent cell growth. Long-term estrogen-deprived cells hyperactivate phosphatidylinositol 3-kinase (PI3K) independently of ER/E2F. Fulvestrant combined with the pan-PI3K inhibitor BKM120 induced regression of ER(+) xenografts. These data support further development of ER downregulators and CDK4 inhibitors, and their combination with PI3K inhibitors for treatment of antiestrogen-resistant breast cancers.
Kappa opioid receptors and their endogenous neuropeptide ligand, dynorphin A, are densely localized in limbic and cortical areas comprising the brain reward system, and appear to play a key role in ...modulating stress and mood. Growing literature indicates that kappa receptor antagonists may be beneficial in the treatment of mood and addictive disorders. However, existing literature on kappa receptor antagonists has used extensively JDTic and nor-BNI which exhibit long-lasting pharmacokinetic properties that complicate experimental design and interpretation of results. Herein, we report for the first time the in vitro and in vivo pharmacological profile of a novel, potent kappa opioid receptor antagonist with excellent selectivity over other receptors and markedly improved drug-like properties over existing research tools. LY2456302 exhibits canonical pharmacokinetic properties that are favorable for clinical development, with rapid absorption (tmax: 1–2 h) and good oral bioavailability (F = 25%). Oral LY2456302 administration selectively and potently occupied central kappa opioid receptors in vivo (ED50 = 0.33 mg/kg), without evidence of mu or delta receptor occupancy at doses up to 30 mg/kg. LY2456302 potently blocked kappa-agonist-mediated analgesia and disruption of prepulse inhibition, without affecting mu-agonist-mediated effects at doses >30-fold higher. Importantly, LY2456302 did not block kappa-agonist-induced analgesia one week after administration, indicating lack of long-lasting pharmacodynamic effects. In contrast to the nonselective opioid antagonist naltrexone, LY2456302 produced antidepressant-like effects in the mouse forced swim test and enhanced the effects of imipramine and citalopram. LY2456302 reduced ethanol self-administration in alcohol-preferring (P) rats and, unlike naltrexone, did not exhibit significant tolerance upon 4 days of repeated dosing. LY2456302 is a centrally-penetrant, potent, kappa-selective antagonist with pharmacokinetic properties favorable for clinical development and activity in animal models predictive of efficacy in mood and addictive disorders.
•Noncanonical pharmacokinetic properties have hampered kappa antagonist development.•Chemistry improvements identified novel compounds with kappa antagonist activity.•LY2456302 exhibits canonical pharmacokinetic properties and kappa selectivity in vivo.•LY2456302 has antidepressant-like properties and reduces ethanol self-administration.•LY2456302 is a novel research tool with favorable properties for clinical development.
The κ-opioid receptor is a widely expressed G-protein-coupled receptor that has been implicated in biological responses to pain, stress, anxiety, and depression, and its potential as a therapeutic ...target in these syndromes is becoming increasingly apparent. However, the prototypical selective κ-opioid antagonists have very long durations of action that have been attributed to c-Jun N-terminal kinase (JNK) 1 activation in vivo. To test generality of this proposed noncompetitive mechanism, we used C57BL/6 wild type mice to determine the durations of antagonist action of novel κ-opioid receptor ligands and examined their efficacies for JNK1 activation compared with conventional competitive antagonists. Of the 12 compounds tested, 5 had long durations of action that positively correlated with JNK activation: RTI-5989-97 (3S)-7-hydroxy-N-(1S)-1-(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethyl-1-piperidinylmethyl}-(2-methylpropyl-2-methyl-1,2,3,4-tetrahydro-3-isoquinolinecarboxamide, RTI-5989-194 (3R)-7-hydroxy-N-(1S)-1-(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethyl-1-piperidinylmethyl}-(2-methylbutyl-1,2,3,4-tetrahydro-3-isoquinolinecarboxamide, RTI-5989-241 (3R)-7-hydroxy-N-(1S)-1-{(3R,4R)-4-(3-methoxyphenyl)-3,4-dimethyl-1-piperidinylmethyl}-2-methylpropyl-1,2,3,4-tetrahydroisoquinoline-3-carboxamide), nor-binaltorphimine (nor-BNI); and (3R)-7-hydroxy-N-((1S)-1-{(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethyl-1-piperidinylmethyl}-2-methylpropyl)-1,2,3,4-tetrahydro-3-isoquinolinecarboxamide (JDTic). Seven had short durations of action and did not increase phospho-JNK-ir: RTI-5989-212(3R)-N-(1S)-1-(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethyl-1-piperidinylmethyl}-(2-methylpropyl-7-methoxy-1,2,3,4-tetrahydroisoquinoline-3-carboxamide, RTI-5989-240 (3R)-7-hydroxy-N-(1S)-1-(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethylpiperidin-1-ylmethyl}-(2-methylpropyl-3-methyl-1,2,3,4-tetrahydroisoquinoline-3-carboxamide, JSPA0658 (S)-3-fluoro-4-(4-((2-(3,5-dimethylphenyl)pyrrolidin-1-yl)methyl)phenoxy)benzamide, JSPA071B (S)-3-fluoro-4-(4-((2-(3,5-bis(trifluoromethyl)phenyl)pyrrolidin-1-yl)methyl)phenoxy)benzamide. PF-4455242 2-methyl-N-((2'-(pyrrolidin-1-ylsulfonyl)biphenyl-4-yl)methyl)propan-1-amine, PF-4455242 2-methyl-N-((2'-(pyrrolidin-1-ylsulfonyl)biphenyl-4-yl)methyl)propan-1-amine, FP3FBZ (S)-3-fluoro-4-(4-((2-(3-fluorophenyl)pyrrolidin-1-yl)methyl)phenoxy)benzamide, and naloxone. After long-acting antagonist treatment, pJNK-ir did not increase in mice lacking the κ-opioid receptor; increased pJNK-ir returned to baseline by 48 h after treatment; and a second challenge with nor-BNI 72 h after the first did not increase pJNK-ir. Long-lasting antagonism and increased phospho-JNK-ir were not seen in animals lacking the JNK1 isoform. These results support the hypothesis that the duration of action of small molecule κ-opioid receptor antagonists in vivo is determined by their efficacy in activating JNK1 and that persistent inactivation of the κ-receptor does not require sustained JNK activation.
Metabotropic glutamate 2/3 (mGlu2/3) receptors are of considerable interest owing to their role in modulating glutamate transmission via presynaptic, postsynaptic and glial mechanisms. As part of our ...ongoing efforts to identify novel ligands for these receptors, we have discovered (1S,2R,3S,4S,5R,6R)-2-amino-3-(3,4-difluorophenyl)sulfanylmethyl-4-hydroxy-bicyclo3.1.0hexane-2,6-dicarboxylic acid; (LY3020371), a potent and selective orthosteric mGlu2/3 receptor antagonist. In this account, we characterize the effects of LY3020371 in membranes and cells expressing human recombinant mGlu receptor subtypes as well as in native rodent and human brain tissue preparations, providing important translational information for this molecule. In membranes from cells expressing recombinant human mGlu2 and mGlu3 receptor subtypes, LY3020371.HCl competitively displaced binding of the mGlu2/3 agonist ligand 3H-459477 with high affinity (hmGlu2 Ki = 5.26 nM; hmGlu3 Ki = 2.50 nM). In cells expressing hmGlu2 receptors, LY3020371.HCl potently blocked mGlu2/3 agonist (DCG-IV)-inhibited, forskolin-stimulated cAMP formation (IC50 = 16.2 nM), an effect that was similarly observed in hmGlu3-expressing cells (IC50 = 6.21 nM). Evaluation of LY3020371 in cells expressing the other human mGlu receptor subtypes revealed high mGlu2/3 receptor selectivity. In rat native tissue assays, LY3020371 demonstrated effective displacement of 3H-459477 from frontal cortical membranes (Ki = 33 nM), and functional antagonist activity in cortical synaptosomes measuring both the reversal of agonist-suppressed second messenger production (IC50 = 29 nM) and agonist-inhibited, K+-evoked glutamate release (IC50 = 86 nM). Antagonism was fully recapitulated in both primary cultured cortical neurons where LY3020371 blocked agonist-suppressed spontaneous Ca2+ oscillations (IC50 = 34 nM) and in an intact hippocampal slice preparation (IC50 = 46 nM). Functional antagonist activity was similarly demonstrated in synaptosomes prepared from epileptic human cortical or hippocampal tissues, suggesting a translation of the mGlu2/3 antagonist pharmacology from rat to human. Intravenous dosing of LY3020371 in rats led to cerebrospinal fluid drug levels that are expected to effectively block mGlu2/3 receptors in vivo. Taken together, these results establish LY3020371 as an important new pharmacological tool for studying mGlu2/3 receptors in vitro and in vivo.
This article is part of the Special Issue entitled ‘Metabotropic Glutamate Receptors, 5 years on’.
•LY3020371 is a highly potent and selective antagonist of human mGlu2/3 receptors.•Highly consistent antagonist potencies exhibited in rat brain tissue assays.•Dose-proportionate plasma and csf pharmacokinetics after i.v. dosing.•Pharmacologically relevant csf concentrations for i.v. doses between 0.3 and 10 mg/kg.•LY3020371 is a new tool for studying mGlu2/3 receptors in vitro and in vivo.
Arylphenylpyrrolidinylmethylphenoxybenzamides were found to have high affinity and selectivity for κ opioid receptors. On the basis of receptor binding assays in Chinese hamster ovary (CHO) cells ...expressing cloned human opioid receptors, (S)-3-fluoro-4-(4-((2-(3-fluorophenyl)pyrrolidin-1-yl)methyl)phenoxy)benzamide (25) had a K i = 0.565 nM for κ opioid receptor binding while having a K i = 35.8 nM for μ opioid receptors and a K i = 211 nM for δ opioid receptor binding. Compound 25 was also a potent antagonist of κ opioid receptors when tested in vitro using a 35S-guanosine 5′O-3-thiotriphosphate (35SGTP-γ-S) functional assay in CHO cells expressing cloned human opioid receptors. Compounds were also evaluated for potential use as receptor occupancy tracers. Tracer evaluation was done in vivo, using liquid chromatography-tandem mass spectrometry (LC/MS/MS) methods, precluding the need for radiolabeling. (S)-3-Chloro-4-(4-((2-(pyridine-3-yl)pyrrolidin-1-yl)methyl)phenoxy)benzamide (18) was found to have favorable properties for a tracer for receptor occupancy, including good specific versus nonspecific binding and good brain uptake.
As part of our ongoing efforts to identify novel ligands for the metabotropic glutamate 2 and 3 (mGlu2/3) receptors, we have incorporated substitution at the C3 and C4 positions of the ...(1S,2R,5R,6R)-2-amino-bicyclo3.1.0hexane-2,6-dicarboxylic acid scaffold to generate mGlu2/3 antagonists. Exploration of this structure–activity relationship (SAR) led to the identification of (1S,2R,3S,4S,5R,6R)-2-amino-3-(3,4-difluorophenyl)sulfanylmethyl-4-hydroxy-bicyclo3.1.0hexane-2,6-dicarboxylic acid hydrochloride (LY3020371·HCl, 19f), a potent, selective, and maximally efficacious mGlu2/3 antagonist. Further characterization of compound 19f binding to the human metabotropic 2 glutamate (hmGlu2) site was established by cocrystallization of this molecule with the amino terminal domain (ATD) of the hmGlu2 receptor protein. The resulting cocrystal structure revealed the specific ligand–protein interactions, which likely explain the high affinity of 19f for this site and support its functional mGlu2 antagonist pharmacology. Further characterization of 19f in vivo demonstrated an antidepressant-like signature in the mouse forced-swim test (mFST) assay when brain levels of this compound exceeded the cellular mGlu2 IC50 value.
Metabotropic glutamate
(mGlu
) receptors are of considerable interest owing to their role in modulating glutamate transmission via presynaptic, postsynaptic and glial mechanisms. As part of our ...ongoing efforts to identify novel ligands for these receptors, we have discovered (1S,2R,3S,4S,5R,6R)-2-amino-3-(3,4-difluorophenyl)sulfanylmethyl-4-hydroxy-bicyclo3.1.0hexane-2,6-dicarboxylic acid; (LY3020371), a potent and selective orthosteric mGlu
receptor antagonist. In this account, we characterize the effects of LY3020371 in membranes and cells expressing human recombinant mGlu receptor subtypes as well as in native rodent and human brain tissue preparations, providing important translational information for this molecule. In membranes from cells expressing recombinant human mGlu
and mGlu
receptor subtypes, LY3020371.HCl competitively displaced binding of the mGlu
agonist ligand
H-459477 with high affinity (hmGlu
K
= 5.26 nM; hmGlu
Ki = 2.50 nM). In cells expressing hmGlu
receptors, LY3020371.HCl potently blocked mGlu
agonist (DCG-IV)-inhibited, forskolin-stimulated cAMP formation (IC
= 16.2 nM), an effect that was similarly observed in hmGlu
-expressing cells (IC
= 6.21 nM). Evaluation of LY3020371 in cells expressing the other human mGlu receptor subtypes revealed high mGlu
receptor selectivity. In rat native tissue assays, LY3020371 demonstrated effective displacement of
H-459477 from frontal cortical membranes (K
= 33 nM), and functional antagonist activity in cortical synaptosomes measuring both the reversal of agonist-suppressed second messenger production (IC
= 29 nM) and agonist-inhibited, K
-evoked glutamate release (IC
= 86 nM). Antagonism was fully recapitulated in both primary cultured cortical neurons where LY3020371 blocked agonist-suppressed spontaneous Ca
oscillations (IC
= 34 nM) and in an intact hippocampal slice preparation (IC
= 46 nM). Functional antagonist activity was similarly demonstrated in synaptosomes prepared from epileptic human cortical or hippocampal tissues, suggesting a translation of the mGlu
antagonist pharmacology from rat to human. Intravenous dosing of LY3020371 in rats led to cerebrospinal fluid drug levels that are expected to effectively block mGlu
receptors in vivo. Taken together, these results establish LY3020371 as an important new pharmacological tool for studying mGlu
receptors in vitro and in vivo. This article is part of the Special Issue entitled 'Metabotropic Glutamate Receptors, 5 years on'.
Selective kappa opioid receptor antagonism is a promising experimental strategy for the treatment of depression. The kappa opioid receptor antagonist, LY2456302, exhibits ~30-fold higher affinity for ...kappa opioid receptors over mu opioid receptors, which is the next closest identified pharmacology.
Here, we determined kappa opioid receptor pharmacological selectivity of LY2456302 by assessing mu opioid receptor antagonism using translational pupillometry in rats and humans.
In rats, morphine-induced mydriasis was completely blocked by the nonselective opioid receptor antagonist naloxone (3mg/kg, which produced 90% mu opioid receptor occupancy), while 100 and 300 mg/kg LY2456302 (which produced 56% and 87% mu opioid receptor occupancy, respectively) only partially blocked morphine-induced mydriasis. In humans, fentanyl-induced miosis was completely blocked by 50mg naltrexone, and LY2456302 dose-dependently blocked miosis at 25 and 60 mg (minimal-to-no blockade at 4-10mg).
We demonstrate, for the first time, the use of translational pupillometry in the context of receptor occupancy to identify a clinical dose of LY2456302 achieving maximal kappa opioid receptor occupancy without evidence of significant mu receptor antagonism.
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Negative modulators of metabotropic glutamate 2 & 3 receptors demonstrate antidepressant-like activity in animal models and hold promise as novel therapeutic agents for the treatment ...of major depressive disorder. Herein we describe our efforts to prepare and optimize a series of conformationally constrained 3,4-disubstituted bicyclo3.1.0hexane glutamic acid analogs as orthosteric (glutamate site) mGlu2/3 receptor antagonists. This work led to the discovery of a highly potent and efficacious tool compound 18 (hmGlu2 IC50 46±14.2nM, hmGlu3 IC50=46.1±36.2nM). Compound 18 showed activity in the mouse forced swim test with a minimal effective dose (MED) of 1mg/kg ip. While in rat EEG studies it exhibited wake promoting effects at 3 and 10mg/kg ip without any significant effects on locomotor activity. Compound 18 thus represents a novel tool molecule for studying the impact of blocking mGlu2/3 receptors both in vitro and in vivo.
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![Novel bicyclo[3.1.0]hexane ...](http://api.summon.serialssolutions.com/2.0.0/image/issn/XR4PS5YY4K/0960-894X/small "Novel bicyclo[3.1.0]hexane analogs as antagonists of metabotropic glutamate 2/3 receptors for the treatment of depression")