Paraspeckles are ribonucleoprotein granules assembled by NEAT1_2 lncRNA, an isoform of Nuclear Paraspeckle Assembly Transcript 1 (NEAT1). Dysregulation of NEAT1_2/paraspeckles has been linked to ...multiple human diseases making them an attractive drug target. However currently NEAT1_2/paraspeckle-focused translational research and drug discovery are hindered by a limited toolkit. To fill this gap, we developed and validated a set of tools for the identification of NEAT1_2 binders and modulators comprised of biochemical and cell-based assays. The NEAT1_2 triple helix stability element was utilized as the target in the biochemical assays, and the cellular assay ('ParaQuant') was based on high-content imaging of NEAT1_2 in fixed cells. As a proof of principle, these assays were used to screen a 1,200-compound FDA-approved drug library and a 170-compound kinase inhibitor library and to confirm the screening hits. The assays are simple to establish, use only commercially-available reagents and are scalable for higher throughput. In particular, ParaQuant is a cost-efficient assay suitable for any cells growing in adherent culture and amenable to multiplexing. Using ParaQuant, we identified dual PI3K/mTOR inhibitors as potent negative modulators of paraspeckles. The tools we describe herein should boost paraspeckle studies and help guide the search, validation and optimization of NEAT1_2/paraspeckle-targeted small molecules.
Lithium, which is still the gold standard in the treatment of bipolar disorder, has been proposed to inhibit inositol monophosphatase (IMPase) and is hypothesized to exert its therapeutic effects by ...attenuating phosphatidylinositol (PI) cell signalling. Drug‐discovery efforts have focused on small‐molecule lithium mimetics that would specifically inhibit IMPase without exhibiting the undesired side effects of lithium. L‐690,330 is a potent bisphosphonate substrate‐based inhibitor developed by Merck Sharp & Dohme. To aid future structure‐based inhibitor design, determination of the exact binding mechanism of L‐690,330 to IMPase was of interest. Here, the high‐resolution X‐ray structure of human IMPase in complex with L690,330 and manganese ions determined at 1.39 Å resolution is reported.
The crystal structure of human inositol monophosphatase in complex with the substrate‐based inhibitor L‐690,330 is reported.
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Benzodiazepines exert their effects by binding to multiple subtypes of the GABA A receptor, the predominant subtypes in the brain being those that contain α 1 -, α 2 -, α 3 -, and α 5 -subunits. To ...understand the potentially different roles of these subtypes in the therapeutic and side effects of benzodiazepines, we evaluated GABA A receptor subtype-preferring compounds in nonhuman primate models predictive of anxiolytic, sedative, motor, subjective, and reinforcing effects of benzodiazepine-type drugs. These compounds included zolpidem, which shows preferential binding to GABA A receptors containing α 1 -subunits (α 1 GABA A receptors); L-838,417, which shows functional selectivity for α 2 GABA A , α 3 GABA A , and α 5 GABA A receptors; and nonselective conventional benzodiazepines. The results provide evidence in nonhuman primates that α 1 GABA A receptors do not play a key role in the anxiolytic and muscle-relaxant properties of benzodiazepine-type drugs; instead, these effects involve α 2 GABA A , α 3 GABA A , and/or α 5 GABA A subtypes. Our results also suggest that the α 1 GABA A receptor subtype might be critically involved in the subjective, sedative, and motor effects of benzodiazepine-type drugs. In contrast, stimulation of α 1 GABA A receptors is sufficient, but not necessary, for mediation of the abuse potential of these drugs. addiction anxiety
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LIM domain kinases 1 and 2 (LIMK1 and LIMK2) regulate actin dynamics and subsequently key cellular functions such as proliferation and migration. LIMK1 and LIMK2 phosphorylate and inactivate cofilin ...leading to increased actin polymerization. As a result, LIMK inhibitors are emerging as a promising treatment strategy for certain cancers and neurological disorders. High-quality chemical probes are required if the role of these kinases in health and disease is to be understood. To that end, we report the results of a comparative assessment of 17 reported LIMK1/2 inhibitors in a variety of in vitro enzymatic and cellular assays. Our evaluation has identified three compounds (TH-257, LIJTF500025, and LIMKi3) as potent and selective inhibitors suitable for use as in vitro and in vivo pharmacological tools for the study of LIMK function in cell biology.
Calbindin‐D28K is a widely expressed calcium‐buffering cytoplasmic protein that is involved in many physiological processes. It has been shown to interact with other proteins, suggesting a role as a ...calcium sensor. Many of the targets of calbindin‐D28K are of therapeutic interest: for example, inositol monophosphatase, the putative target of lithium therapy in bipolar disorder. Presented here is the first crystal structure of human calbindin‐D28K. There are significant deviations in the tertiary structure when compared with the NMR structure of rat calbindin‐D28K (PDB entry 2g9b), despite 98% sequence identity. Small‐angle X‐ray scattering (SAXS) indicates that the crystal structure better predicts the properties of calbindin‐D28K in solution compared with the NMR structure. Here, the first direct visualization of the calcium‐binding properties of calbindin‐D28K is presented. Four of the six EF‐hands that make up the secondary structure of the protein contain a calcium‐binding site. Two distinct conformations of the N‐terminal EF‐hand calcium‐binding site were identified using long‐wavelength calcium single‐wavelength anomalous dispersion (SAD). This flexible region has previously been recognized as a protein–protein interaction interface. SAXS data collected in both the presence and absence of calcium indicate that there are no large structural differences in the globular structure of calbindin‐D28K between the calcium‐loaded and unloaded proteins.
The X‐ray structure of human calbindin‐D28K, a calcium‐buffering protein that is highly expressed in the central nervous system, is reported.
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Modulation of the metabotropic glutamate type 2 (mGlu2) receptor is considered a promising target for the treatment of central nervous system diseases such as schizophrenia. Here, we describe the ...pharmacological properties of the novel mGlu2 receptor positive allosteric modulator (PAM) 3-cyano-1-cyclopropylmethyl-4-(4-phenyl-piperidin-1-yl)-pyridine-2(1H)-one (JNJ-40068782) and its radioligand (3)HJNJ-40068782. In guanosine 5'-O-(3-(35)Sthio)triphosphate binding, JNJ-40068782 produced a leftward and upward shift in the glutamate concentration-effect curve at human recombinant mGlu2 receptors. The EC50 of JNJ-40068782 for potentiation of an EC20-equivalent concentration of glutamate was 143 nM. Although JNJ-40068782 did not affect binding of the orthosteric antagonist (3)H2S-2-amino-2-(1S,2S-2-carboxycyclopropyl-1-yl)-3-(xanth-9-yl)propanoic acid (LY-341495), it did potentiate the binding of the agonist (3)H(2S,2'R,3'R)-2-(2',3'-dicarboxylcyclopropyl)glycine (DCG-IV), demonstrating that it can allosterically affect binding at the agonist recognition site. The binding of (3)HJNJ-40068782 to human recombinant mGlu2 receptors in Chinese hamster ovary cells and rat brain receptors was saturable with a KD of ∼10 nM. In rat brain, the anatomic distribution of (3)HJNJ-40068782 was consistent with mGlu2 expression previously described and was most abundant in cortex and hippocampus. The ability of structurally unrelated PAMs to displace (3)HJNJ-40068782 suggests that PAMs may bind to common determinants within the same site. It is noteworthy that agonists also increased the binding affinity of (3)HJNJ-40068782. JNJ-40068782 influenced rat sleep-wake organization by decreasing rapid eye movement sleep with a lowest active dose of 3 mg/kg PO. In mice, JNJ-40068782 reversed phencyclidine-induced hyperlocomotion with an ED50 of 5.7 mg/kg s.c. Collectively, the present data demonstrate that JNJ-40068782 has utility in investigating the potential of mGlu2 modulation for the treatment of diseases characterized by disturbed glutamatergic signaling and highlight the value of (3)HJNJ-40068782 in exploring allosteric binding.
The recent approval of formulations of the endogenous neurosteroid allopregnanolone (brexanolone) and the synthetic neuroactive steroid SAGE-217 (zuranolone) to treat postpartum depression (PPD) has ...encouraged further research to elucidate why these potent enhancers of GABAAR function are clinically effective in this condition. Dopaminergic projections from the ventral tegmental area (VTA) to the nucleus accumbens are associated with reward/motivation and brain imaging studies report that individuals with PPD show reduced activity of this pathway in response to reward and infant engagement. However, the influence of neurosteroids on GABA-ergic transmission in the nucleus accumbens has received limited attention. Here, we investigate, in the medium spiny neurons (MSNs) of the mouse nucleus accumbens core, the effect of allopregnanolone, SAGE-217 and other endogenous and synthetic steroids of interest on fast phasic and tonic inhibition mediated by synaptic (α1/2βγ2) and extrasynaptic (α4βδ) GABAARs, respectively. We present evidence suggesting the resident tonic current results from the spontaneous opening of δ-GABAARs, where the steroid-enhanced tonic current is GABA-dependent. Furthermore, we demonstrate local neurosteroid synthesis in the accumbal slice preparation and reveal that GABA-ergic neurotransmission of MSNs is influenced by an endogenous neurosteroid tone. Given the dramatic fluctuations in allopregnanolone levels during pregnancy and postpartum, this neurosteroid-mediated local fine-tuning of GABAergic transmission in the MSNs will probably be perturbed.
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Multiple cognitive and psychiatric disorders are associated with an increased tonic inhibitory conductance that is generated by α5 subunit-containing γ-aminobutyric acid type A (α5 GABAA) receptors. ...Negative allosteric modulators that inhibit α5 GABAA receptors (α5-NAMs) are being developed as treatments for these disorders. The effects of α5-NAMs have been studied on recombinant GABAA receptors expressed in non-neuronal cells; however, no study has compared drug effects on the tonic conductance generated by native GABAA receptors in neurones, which was the goal of this study.
The effects of five α5-NAMs (basmisanil, Ono-160, L-655,708, α5IA, and MRK-016) on tonic current evoked by a low concentration of GABA were studied using whole-cell recordings in cultured mouse hippocampal neurones. Drug effects on current evoked by a saturating concentration of GABA and on miniature inhibitory postsynaptic currents (mIPSCs) were also examined.
The α5-NAMs caused a concentration-dependent decrease in tonic current. The potencies varied as the inhibitory concentration for 50% inhibition (IC50) of basmisanil (127 nM) was significantly higher than those of the other compounds (0.4–0.8 nM). In contrast, the maximal efficacies of the drugs were similar (35.5–51.3% inhibition). The α5-NAMs did not modify current evoked by a saturating GABA concentration or mIPSCs.
Basmisanil was markedly less potent than the other α5-NAMs, an unexpected result based on studies of recombinant α5 GABAA receptors. Studying the effects of α5 GABAA receptor-selective drugs on the tonic inhibitory current in neurones could inform the selection of compounds for future clinical trials.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
In nonhuman primates we tested a new set of behavioral categories for observable sedative effects using pediatric anesthesiology classifications as a basis. Using quantitative behavioral observation ...techniques in rhesus monkeys, we examined the effects of alprazolam and diazepam (nonselective benzodiazepines), zolpidem (preferential binding to
1 subunit-containing GABA
receptors), HZ-166 (8-ethynyl-6-(2'-pyridine)-4
-2,5,10b-triaza-benzo
azulene-3-carboxylic acid ethyl ester; functionally selective with relatively high intrinsic efficacy for
2 and
3 subunit-containing GABA
receptors), MRK-696 7-cyclobutyl-6-(2-methyl-2
-1,2,4-triazol-2-ylmethoxy)-3-(2-flurophenyl)-1,2,4-triazolo(4,3-
)pyridazine; no selectivity but partial intrinsic activity, and TPA023B 6,2'-diflouro-5'-3-(1-hydroxy-1-methylethyl)imidazo1,2-
1,2,4triazin-7-ylbiphenyl-2-carbonitrile; partial intrinsic efficacy and selectivity for
2,
3,
5 subunit-containing GABA
receptors. We further examined the role of
1 subunit-containing GABA
receptors in benzodiazepine-induced sedative effects by pretreating animals with the
1 subunit-preferring antagonist
-carboline-3-carboxylate-
-butyl ester (
CCT). Increasing doses of alprazolam and diazepam resulted in the emergence of observable ataxia, rest/sleep posture, and moderate and deep sedation. In contrast, zolpidem engendered dose-dependent observable ataxia and deep sedation but not rest/sleep posture or moderate sedation, and HZ-166 and TPA023 induced primarily rest/sleep posture. MRK-696 induced rest/sleep posture and observable ataxia. Zolpidem, but no other compounds, significantly increased tactile/oral exploration. The sedative effects engendered by alprazolam, diazepam, and zolpidem generally were attenuated by
CCT pretreatments, whereas rest/sleep posture and suppression of tactile/oral exploration were insensitive to
CCT administration. These data suggest that
2/3-containing GABA
receptor subtypes unexpectedly may mediate a mild form of sedation (rest/sleep posture), whereas
1-containing GABA
receptors may play a role in moderate/deep sedation.
Alzheimer's disease (AD) is the most common type of dementia and, after age, the greatest risk factor for developing AD is the allelic variation of apolipoprotein E (ApoE), with homozygote carriers ...of the ApoE4 allele having an up to 12-fold greater risk of developing AD than noncarriers. Apolipoprotein E exists as three isoforms that differ in only two amino acid sites, ApoE2 (Cys112/Cys158), ApoE3 (Cys112/Arg158), and ApoE4 (Arg112/Arg158). These amino acid substitutions are assumed to alter ApoE structure and function, and be responsible for the detrimental effects of ApoE4 via a mechanism that remains unclear. The hypothesis that a structural difference between ApoE4 and ApoE3 (and ApoE2) is the cause of the ApoE4-associated increased risk for AD forms the basis of a therapeutic approach to modulate ApoE4 structure, and we were therefore interested in screening to identify new chemical probes for ApoE4. In this regard, a high-yield protocol was developed for the expression and purification of recombinant full-length ApoE, and three diverse biophysical screening assays were established and characterized; an optical label-free assay (Corning Epic) for hit identification and microscale thermophoresis (MST) and isothermal titration calorimetry (ITC) as orthogonal assays for hit confirmation. The 707 compounds in the National Institute of Health clinical collection were screened for binding to ApoE4, from which six confirmed hits, as well as one analogue, were identified. Although the compounds did not differentiate between ApoE isoforms, these data nevertheless demonstrate the feasibility of using a biophysical approach to identifying compounds that bind to ApoE and that, with further optimization, might differentiate between isoforms to produce a molecule that selectively alters the function of ApoE4.
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