Mouse Brain Membranes Research Articles

Design, synthesis, ADME and biological evaluation of benzylpiperidine and benzylpiperazine derivatives as novel reversible monoacylglycerol lipase (MAGL) inhibitors

The degradation of the endocannabinoid 2-arachidonoylglycerol is mediated by the enzyme monoacylglycerol lipase (MAGL), thus generating arachidonic acid, the precursor of prostaglandins and other pro-inflammatory mediators. MAGL also contributes to the hydrolysis of monoacylglycerols into glycerol and fatty acids in peripheral body districts, which may act as pro-tumorigenic signals. For this reason, MAGL inhibitors have been considered as interesting therapeutic agents for their anti-nociceptive, anti-inflammatory, antioxidant and anti-cancer properties. So far, only a limited series of reversible MAGL inhibitors, which are devoid of side effects shown by irreversible inhibitors in animal models, have been reported. Here we optimized a class of benzylpiperidine and benzylpiperazine-based compounds for a reversible MAGL inhibition. The best MAGL inhibitors of this class, compounds 28 and 29, showed a very good inhibition potency, both on the isolated enzyme and in U937 cells, as confirmed by molecular modeling studies that predicted their binding mode into the MAGL active site. Both compounds are characterized by a high selectivity for MAGL versus other serine hydrolases including enzymes of the endocannabinoid system, as confirmed by ABPP experiments in mouse brain membranes. Moreover, very good properties concerning ADME parameters and low in vivo toxicity have been observed for both compounds.

Effect of moderate prenatal ethanol exposure on the differential expression of two histamine H3 receptor isoforms in different brain regions of adult rat offspring.

We have reported that prenatal alcohol exposure (PAE) elevates histamine H3 receptor (H3R) agonist-mediated inhibition of glutamatergic neurotransmission in the dentate gyrus. Here, we hypothesized that PAE alters the expression of two prominent H3R isoforms namely, the rH3A and rH3C isoforms, which have differing intrinsic activities for H3R agonists, in a manner that may contribute to heightened H3R function in PAE rats. In contrast to our predictions, we found different effects of sex and PAE in various brain regions with significant interactions between sex and PAE in dentate gyrus and entorhinal cortex for both isoforms. Subsequently, to confirm the PAE-and sex-induced differences on H3R isoform mRNA expression, we developed a polyclonal antibody selective for the rH3A inform. Western blots of rH3A mRNA-transfected HEK-293 cells identified a ~ 48 kDa band of binding consistent with the molecular weight of rH3A, thus confirming antibody sensitivity for rH3A protein. In parallel, we also established a pan-H3R knockout mice line to confirm antibody specificity in rodent brain membranes. Both qRT-PCR and H3R agonist-stimulated [35S]-GTPγS binding confirmed the absence of mH3A mRNA and H3 receptor-effector coupling in H3R knockout (KO) mice. Subsequent western blotting studies in both rat and mouse brain membranes were unable to detect rH3A antibody binding at ~48 kDa. Rather, the H3RA antibody bound to a ~ 55 kDa band in both rat and mouse membranes, including H3R KO mice, suggesting H3RA binding was not specific for H3Rs in rodent membranes. Subsequent LC/MS analysis of the ~55 kDa band in frontal cortical membranes identified the highly abundant beta subunit of ATPase in both WT and KO mice. Finally, LC/MS analysis of the ~48 kDa band from rH3A mRNA-transfected HEK-293 cell membranes was able to detect rH3A protein, but its presence was below the limits of quantitative reliability. We conclude that PAE alters rH3A and rH3C mRNA expression in some of the same brain regions where we have previously reported PAE-induced alterations in H3R-effector coupling. However, interpreting the functional consequences of altered H3R isoform expression was limited given the technical challenges of measuring the relatively low abundance of rH3A protein in native membrane preparations.

Structure-activity relationships for 5F-MDMB-PICA and its 5F-pentylindole analogs to induce cannabinoid-like effects in mice.

Synthetic cannabinoid receptor agonists (SCRAs) are an evolving class of new psychoactive substances found on recreational drug markets worldwide. The indole-containing compound, 5F-MDMB-PICA, is a popular SCRA associated with serious medical consequences, including overdose and hospitalizations. In vitro studies reveal that 5F-MDMB-PICA is a potent agonist at cannabinoid type 1 receptors (CB1), but little information exists regarding in vivo pharmacology of the drug. To this end, we examined the in vitro and in vivo cannabinoid-like effects produced by 5F-MDMB-PICA and related 5F-pentylindole analogs with differing composition of the head group moiety (i.e., 5F-NNEI, 5F-SDB-006, 5F-CUMYL-PICA, 5F-MMB-PICA). In mouse brain membranes, 5F-MDMB-PICA and its analogs inhibited binding to [3H]rimonabant-labeled CB1 and displayed agonist actions in [35S]GTPγS functional assays. 5F-MDMB-PICA exhibited the highest CB1 affinity (Ki = 1.24 nM) and functional potency (EC50 = 1.46 nM), but head group composition markedly influenced activity in both assays. For example, the 3,3-dimethylbutanoate (5F-MDMB-PICA) and cumyl (5F-CUMYL-PICA) head groups engendered high CB1 affinity and potency, whereas a benzyl (5F-SDB-006) head group did not. In C57BL/6J mice, all 5F-pentylindole SCRAs produced dose- and time-dependent hypothermia, catalepsy, and analgesia that were reversed by rimonabant, indicating CB1 involvement. In vitro Ki and EC50 values were positively correlated with in vivo ED50 potency estimates. Our findings demonstrate that 5F-MDMB-PICA is a potent SCRA, and subtle alterations to head group composition can have profound influence on pharmacological effects at CB1. Importantly, measures of CB1 binding and efficacy in mouse brain tissue seem to accurately predict in vivo drug potency in this species.

Peptide Sequence Mapping around Bisecting GlcNAc-Bearing N-Glycans in Mouse Brain.

N-glycosylation is essential for many biological processes in mammals. A variety of N-glycan structures exist, of which, the formation of bisecting N-acetylglucosamine (GlcNAc) is catalyzed by N-acetylglucosaminyltransferase-III (GnT-III, encoded by the Mgat3 gene). We previously identified various bisecting GlcNAc-modified proteins involved in Alzheimer’s disease and cancer. However, the mechanisms by which GnT-III acts on the target proteins are unknown. Here, we performed comparative glycoproteomic analyses using brain membranes of wild type (WT) and Mgat3-deficient mice. Target glycoproteins of GnT-III were enriched with E4-phytohemagglutinin (PHA) lectin, which recognizes bisecting GlcNAc, and analyzed by liquid chromatograph-mass spectrometry. We identified 32 N-glycosylation sites (Asn-Xaa-Ser/Thr, Xaa ≠ Pro) that were modified with bisecting GlcNAc. Sequence alignment of identified N-glycosylation sites that displayed bisecting GlcNAc suggested that GnT-III does not recognize a specific primary amino acid sequence. The molecular modeling of GluA1 as one of the good cell surface substrates for GnT-III in the brain, indicated that GnT-III acts on N-glycosylation sites located in a highly flexible and mobile loop of GluA1. These results suggest that the action of GnT-III is partially affected by the tertiary structure of target proteins, which can accommodate bisecting GlcNAc that generates a bulky flipped-back conformation of the modified glycans.

Major Metabolites of the Synthetic Cannabinoid 5F‐ADB Retain High Affinity and Full Efficacy at CB1 Receptors; Potential Mechanism Contributing to Enhanced Toxicity?

Synthetic cannabinoids (SCs) are a large group of abused psychoactive compounds that elicit numerous toxic effects not observed with marijuana, including death. SCs are abused worldwide and metabolites of these drugs are often detected as a means of drug identification. SC metabolites resulting from Phase I metabolism have been shown to retain affinity and activity at cannabinoid receptors. However, whether these active compounds contribute to toxicity of SCs is unknown. 5-Fluoro-ADB (5F-ADB), also known as 5-Fluoro-MDMB-PINACA, is a commonly abused third-generation synthetic cannabinoid agonist based on an indazole-structure. Significantly, over 40 recent fatalities have been attributed to 5F-ADB overdose. A potential mechanism underlying 5F-ADB-induced mortality is drug-related dysrhythmia resulting in sudden death. Analysis of postmortem central and peripheral blood samples have revealed abnormally high concentrations of an ester-hydrolysis Phase I metabolite of 5F-ADB, identified as metabolite 7 (5F-ADB-M7). We propose that high concentrations of potent and efficacious active metabolites, such as 5F-ADB-M7 and others, accumulate with the parent drug 5F-ADB and produce additive and/or synergistic effects that possibly contribute to fatal intoxication following overdose. As a first step to test this hypothesis, in vitro studies were conducted to compare the affinity and activity of tetrahydrocannabinol (THC; the primary psychoactive component in marijuana), 5F-ADB, 5F-ADB-M7 and a second major metabolite, 5F-ADB-M2 at cannabinoid type-1 (CB1) receptors. Competition receptor binding studies, using mouse brain membranes, showed that all four cannabinoids bound with high affinity to CB1 receptors with a rank order of affinity (Ki values) of 5F-ADB > 5F-ADB-M2 > THC > 5F-ADB-M7. Furthermore, functional experiments examining modulation of G-protein activity demonstrated that 5F-ADB and both Phase I metabolites acted as full agonists at CB1 receptors, relative to partial agonist effects produced by THC. Subsequently, in vivo studies were conducted utilizing the cannabinoid tetrad in mice to compare the effects of THC with those of 5F-ADB-M2. Both 5F-ADB-M2 and THC produced CB1-mediated dose-dependent hypothermia, analgesia, and locomotor suppression, within 30 minutes of injection; however, catalepsy was not elicited by the metabolite. Consistent with higher CB1 affinity determined from in vitro studies, 5F-ADB-M2 produced tetrad effects with higher potency compared to THC. Future studies are planned to examine in vivo effects of 5F-ADB-M7 compared to THC. If 5F-ADB metabolites exhibit in vivo activity similar to that predicted from the in vitro studies presented here, this suggests that the enhanced, often fatal, effects of 5F-ADB may be due to an accumulation of the parent and active, high affinity metabolites acting at CB1 receptors.

Design, synthesis and biological evaluation of second-generation benzoylpiperidine derivatives as reversible monoacylglycerol lipase (MAGL) inhibitors.

An interesting enzyme of the endocannabinoid system is monoacylglycerol lipase (MAGL). This enzyme, which metabolizes the endocannabinoid 2-arachidonoylglycerol (2-AG), has attracted great interest due to its involvement in several physiological and pathological processes, such as cancer progression. Experimental evidences highlighted some drawbacks associated with the use of irreversible MAGL inhibitors invivo, therefore the research field concerning reversible inhibitors is rapidly growing. In the present manuscript, the class of benzoylpiperidine-based MAGL inhibitors was further expanded and optimized. Enzymatic assays identified some compounds in the low nanomolar range and steered molecular dynamics simulations predicted the dissociation itinerary of one of the best compounds from the enzyme, confirming the observed structure-activity relationship. Biological evaluation, including assays in intact U937cells and competitive activity-based protein profiling experiments in mouse brain membranes, confirmed the selectivity of the selected compounds for MAGL versus other components of the endocannabinoid system. An antiproliferative ability in a panel of cancer cell lines highlighted their potential as potential anticancer agents. Future studies on the potential use of these compounds in the clinical setting are also supported by the inhibition of cell growth observed both in cancer organoids derived from high grade serous ovarian cancer patients and in pancreatic ductal adenocarcinoma primary cells, which showed genetic and histological features very similar to the primary tumors.

Characterization of binding of Varicella zoster virus vaccine strains to preparations of mouse brain membrane receptors

Purpose: characterization of vFiraVax (the causative agent of chickenpox - VZV) and vZelVax (the causative agent of shingles - HZ) vaccine strains by their ability to bind to preparations of brain membrane receptors of SPF BALB/c mice.Materials and Methods. The study was performed on cold-adapted vFiraVax VZV and vZelVax HZ vaccine strains developed by the authors on the basis of the wild-type parental pFira VZV virus (chickenpox causative agent) and the latent parental lpZel HZ virus (shingles causative agent); vOka vaccine strains isolated from vaccines against VZV infection from two manufactures (United Kingdom and USA); the HEL-3 strain of diploid cells from human embryonic lung tissue, the MC 27 strain of diploid cells from human embryonic musculocutaneous tissue, primary and diploid cells from guinea pig fetal fibroblasts. The VZV infectivity was estimated by the limiting dilution method using MC 27 cell cultures or guinea pig fetal fibroblasts. The virus titer was measured by the hemadsorption test performed with suspensions of red blood cells from guinea pig or human type 0 positive blood. Negative staining and electron microscopy were used to study the virus preparation. The immunogenicity of vFiraVax VZV and vZelVax HZ virus strains was compared with the immunogenicity of vOka VZV virus strains from different manufacturers by using a cross-neutralization test with immune sera.Results. The Russian cold-adapted vFiraVax VZV and vZelVax HZ vaccine strains, the latent parental lpZel HZ virus and the vOka VZV vaccine strain (United Kingdom) did not bind to preparations of brain neuroreceptors of SPF BALB/c mice as distinct from the wild-type parental pFira VZV variant and vOka VZV vaccine strains (USA); the absent neurotropism of Russian vFiraVax VZV and vZelVax HZ vaccine strains is not connected with the decreased immunogenicity in relation to foreign counterparts; the electron microscope study of the vFiraVax VZV virus containing liquid concentrate detected VZV nucleocapsids.Conclusion. The differences in the VZV ability to bind to preparations of brain membrane receptors of SPF BALB/c mice can be explained by the differences in the technology of vaccine manufacturing, including attenuation techniques, obtaining of the vaccine strain, specific characteristics of the latent parental lpZel HZ virus. The absence of the binding with brain neuroreceptors of SPF mice has been proved for the Russian vFiraVax VZV and vZelVax HZ vaccine strains which was is not connected with a decrease in their immunogenicity. The method of assessment of the binding ability of VZV vaccine strains can be used as a preliminary characteristic of neurotropism for newly created vaccine strains and for vaccine products.

Allosteric modulation of sigma receptors by BH3 mimetics ABT-737, ABT-263 (Navitoclax) and ABT-199 (Venetoclax)

ABT-737, ABT-263 (Navitoclax) and ABT-199 (Venetoclax) are under intensive preclinical and clinical investigation as treatments for hematologic and other malignancies. These small molecules mimic pro-death B-cell lymphoma-2 (Bcl-2) Homology 3 (BH3) domain-only proteins. They also bear a structural resemblance to certain sigma (σ) receptor ligands. Moreover, the Bcl-2 and σ receptor protein families are both located primarily at the endoplasmic reticulum, mediate cell death and survival through protein–protein interactions, and physically associate. Accordingly, we examined the ability of the ABT series of BH3 mimetics to interact with σ receptors using radioligand-binding techniques. Negative allosteric modulation of [3H](+)-pentazocine, an agonist, binding to σ1 receptors in guinea pig brain membranes was observed for ABT-737, ABT-263 and ABT-199. Findings included reduction of specific binding to distinct plateaus in concentration-dependent fashion, significant slowing of radioligand dissociation kinetics, and decreases in radioligand affinity with no or modest changes in maximal receptor densities. Using a ternary complex model, dissociation constants (KX) for modulator binding to the σ1 receptor ranged from 1 to 2.5 μM, while negative cooperativity factors (α), representing the changes in affinity of ligand and modulator when bound as a ternary complex with the receptor, ranged from 0.15 to 0.42. These observations were extended and reinforced by studies using intact small cell (NCI-H69) and non-small cell (NCI-H23) lung cancer cells, and by using an antagonist σ1 receptor radioligand, E-N-1-(3′-[125I]iodoallyl)-N′-4-(3″,4″-dimethoxyphenethyl)piperazine, in mouse brain membranes. By contrast, exploratory studies indicate marked enhancement of the σ2 receptor binding of [3H]1,3-di-(o-tolyl)guanidine/(+)-pentazocine in NCI-H23 cells and guinea pig brain membranes. These findings raise intriguing questions regarding mechanism and potential functional outcomes.

Yüksek dizi kapsama alanı ile fare beyin zarlarından VDAC (voltaj bağımlı anyon kanalı) kütle spektrometrik haritalama.

VDACs (voltage dependent anion channels) are integral membrane proteins serving as nonspecific diffusion pores or as specific systems for the transport of substrates through mitochondrial membranes. The functional role of VDAC has been investigated in many studies, and different functions of VDAC have been shown. Posttranslational modifications of VDAC are significant for its regulation. The aim of our research was to develop approach for characterization of primary structure and posttranslational modifications of VDACs and other membrane proteins. Mouse brain membranes were isolated from mouse brains by differential centrifugation. Primary structure of mitochondrial isoform VDAC1 from mouse brain membranes has been identified almost completely (95 %, 258 of 283 amino acids) by combination of SDS-PAGE and LTQ-FTMS mapping of peptide mixtures after proteolytic degradation with trypsin. Sequence of each found peptide of VDAC has been analysed and confirmed according to accurate mass, isotopic distribution and MS/MS tandem analysis. Posttranslational modifications of VDAC’speptides have been shown. High sequence coverage of VDAC has been obtained, including 11 transmembranes domains. Extensive sequence coverage has been also detected for some other proteins at 30-34 kDa. A repressor of estrogen receptor activity has been identified with 76 % coverage, malate dehydrogenase with 55 % sequence coverage, syntaxin 1A and syntaxin 1B2 have been sequenced with 60 % and 65 % coverage, respectively. These results demonstrated that mass spectrometric mapping is reliable and sensitive approach for characterization of primary structure membrane proteins and identification of their posttranslational modifications.

Characterization of 125‐I‐Angiotensin (1–7) Binding to Mouse Forebrain and Liver and Rat Liver

Angiotensin II (Ang II) is a potent regulator of blood pressure and is pathogenetic for cardiovascular disease. Identification of the ACE homologue ACE2, which forms Ang 1–7 from Ang II, and the GPCR Mas as a functional Ang 1–7 receptor has provided biochemical and molecular tools to study the role of Ang 1–7 as a counter regulator of the pathogenesis of Ang II. Despite the physiological importance of Ang1–7, no one has demonstrated saturable 125I‐Ang 1–7 binding in mammalian tissue. To date, only single concentration, specific binding of 125I‐Ang 1–7 to mouse kidney has been reported. To assess the saturability of 125I‐Ang 1–7 binding to mammalian tissue, we prepared a low specific activity 125/127I‐Ang 1–7 by radioiodinating Ang 1–7 with 1 part 125I:19 parts 127I, using chloramine T at pH 7.5 to limit radioiodination to tyr4, and pH 8.5 to radioiodinate both tyr4 and his6, with subsequent purification of monoradioiodinated Ang 1–7 using reverse‐phase HPLC. Forebrains from adult male athymic mice (nu/+) were dissected and homogenized in water and the membranes were precipitated by centrifugation at 48 kxG. Membranes were resuspended in Tris:EDTA (50:5 mM) pH 7.3 and incubated with 12 concentrations of 125/127I‐Ang 1–7 ranging from 3–110 nM for 30 min at 22 C, after which bound 125/127I‐Ang 1–7 was collected on GF/B filters and counted. Both radio ligands showed specific (100 μM Ang 1–7 displaceable) saturable binding: 35±8.3 and 35±4.2 fmol/mg initial wet weight (mean±SEM) with KD = 42.6±19.2 and 47.9±13.9 nM, for pH 7.5 and pH 8.5 preparations, respectively. This suggests that the presence of iodine on tyr4 or tyr4 + his6 does not differentially affect binding of Ang 1–7 to mouse brain membranes. Preliminary studies indicate that Ang II, Ang IV, Ang 3–7, Ang 4–7, Sar1, Ile8 Ang II, losartan and PD123319 have low affinity for the 125/127I‐Ang 1–7 binding site in mouse brain membranes. 125/127I‐Ang 1–7 (prepared at pH 8.5) binding to mouse liver using 50 mM Tris: 5 mM MgCl2, pH 7.2, also was saturable 179±10 fmol/g with KD = 20.4±4 nM. 125/127I‐Ang 1–7 (prepared at pH 7.5) binding to rat liver membranes using 50 mM Tris: 5 mM MgCl2 pH 7.2 also was saturable 66.7±1.8 fmol/g with KD = 7.1±1.7 nM. No saturable binding was observed in rat heart. Future studies will assess whether this binding is to the mas protein using other rodent tissues, e.g., kidney, as well as mas knockout mice. If these studies document that 125/127I‐Ang 1–7 binds to mas, this radio ligand can be used to assess the binding functionality of mas as well as to measure differences in the binding functionality of mas in animal models of cardiovascular disease.Support or Funding InformationF.F. Conti: CAPES: 99999.008874/2014‐00. A. Linares: None. L. Couling: None. M. Morris: DOD grant W81XWH‐13‐2‐0085. K. De Angelis: None. R.C. Speth: Research Grant; Significant; NIH HL‐113905; Pilot Award from the Translational Technologies Component of the Georgetown, Howard Universities Center for Clinical and Translational Science [UL1TR000101]. Other Research Support; Significant; Cardiovascular Neuroscience Research Fund, Nova Southeastern University.

Identification of Palmitoylated Transitional Endoplasmic Reticulum ATPase by Proteomic Technique and Pan Antipalmitoylation Antibody

Protein palmitoylation plays a significant role in a wide range of biological processes such as cell signal transduction, metabolism, apoptosis, and carcinogenesis. For high-throughput analysis of protein palmitoylation, approaches based on the acyl-biotin exchange or metabolic labeling of azide/alkynyl-palmitate analogs are commonly used. No palmitoylation antibody has been reported. Here, the palmitoylated proteome of human colon cancer cell lines SW480 was analyzed via a TS-6B-based method. In total, 151 putative palmitoylated sites on 92 proteins, including 100 novel sites, were identified. Except for 3 known palmitoylated transmembrane proteins, ATP1A1, ZDHHC5, and PLP2, some important proteins including kinases, ion channels, receptors, and cytoskeletal proteins were also identified, such as CLIC1, PGK1, PPIA, FKBP4, exportin-2, etc. More importantly, the pan antipalmitoylation antibody was developed and verified for the first time. Our homemade pan antipalmitoylation antiserum could differentiate well protein palmitoylation from mouse brain membrane fraction and SW480 cells, which affords a new technique for analyzing protein palmitoylation by detecting the palmitic acid moiety directly. Furthermore, the candidate protein transitional endoplasmic reticulum ATPase (VCP) identified in SW480 cells was validated to be palmitoylated by Western blotting with anti-VCP antibody and the homemade pan antipalmitoylation antibody.

Investigation of receptor binding and functional characteristics of hemopressin(1–7)

The orally active, α-hemoglobin derived hemopressin (PVNFKFLSH, Hp(1–9)) and its truncated (PVNFKFL, Hp(1–7) and PVNFKF, Hp(1–6)) and extended ((R)VDPVNFKFLSH, VD-Hp(1–9) and RVD-Hp(1–9)) derivatives have been postulated to be the endogenous peptide ligands of the cannabinoid receptor type 1 (CB1). In an attempt to create a versatile peptidic research tool for the direct study of the CB1 receptor–peptide ligand interactions, Hp(1–7) was radiolabeled and in vitro characterized in rat and CB1 knockout mouse brain membrane homogenates. In saturation and competition radioligand binding studies, [3H]Hp(1–7) labeled membrane receptors with high densities and displayed specific binding to a receptor protein, but seemingly not to the cannabinoid type 1, in comparison the results with the prototypic JWH-018, AM251, rimonabant, Hp(1–9) and RVD-Hp(1–9) (pepcan 12) ligands in both rat brain and CB1 knockout mouse brain homogenates. Furthermore, functional [35S]GTP γS binding studies revealed that Hp(1–7) and Hp(1–9) only weakly activated G-proteins in both brain membrane homogenates. Based on our findings and the latest literature data, we assume that the Hp(1–7) peptide fragment may be an allosteric ligand or indirect regulator of the endocannabinoid system rather than an endogenous ligand of the CB1 receptor.

Characterization of methadone as a β-arrestin-biased μ-opioid receptor agonist.

BackgroundMethadone is a unique µ-opioid receptor agonist. Although several researchers have insisted that the pharmacological effects of methadone are mediated through the blockade of NMDA receptor, the underlying mechanism by which methadone exerts its distinct pharmacological effects compared to those of other µ-opioid receptor agonists is still controversial. In the present study, we further investigated the pharmacological profile of methadone compared to those of fentanyl and morphine as measured mainly by the discriminative stimulus effect and in vitro assays for NMDA receptor binding, µ-opioid receptor-internalization, and µ-opioid receptor-mediated β-arrestin recruitment.ResultsWe found that fentanyl substituted for the discriminative stimulus effects of methadone, whereas a relatively high dose of morphine was required to substitute for the discriminative stimulus effects of methadone in rats. Under these conditions, the non-competitive NMDA receptor antagonist MK-801 did not substitute for the discriminative stimulus effects of methadone. In association with its discriminative stimulus effect, methadone failed to displace the receptor binding of MK801 using mouse brain membrane. Methadone and fentanyl, but not morphine, induced potent µ-opioid receptor internalization accompanied by the strong recruitment of β-arrestin-2 in µ-opioid receptor-overexpressing cells.ConclusionsThese results suggest that methadone may, at least partly, produce its pharmacological effect as a β-arrestin-biased µ-opioid receptor agonist, similar to fentanyl, and NMDA receptor blockade is not the main contributor to the pharmacological profile of methadone.

Revisiting 1,3,4-Oxadiazol-2-ones: Utilization in the Development of ABHD6 Inhibitors

This article describes our systematic approach to exploring the utility of the 1,3,4-oxadiazol-2-one scaffold in the development of ABHD6 inhibitors. Compound 3-(3-aminobenzyl)-5-methoxy-1,3,4-oxadiazol-2(3H)-one (JZP-169, 52) was identified as a potent inhibitor of hABHD6, with an IC50 value of 216nM. This compound at 10μM concentration did not inhibit any other endocannabinoid hydrolases, such as FAAH, MAGL and ABHD12, or bind to the cannabinoid receptors (CB1 and CB2). Moreover, in competitive activity-based protein profiling (ABPP), compound 52 (JZP-169) at 10μM selectively targeted ABHD6 of the serine hydrolases of mouse brain membrane proteome. Reversibility studies indicated that compound 52 inhibited hABHD6 in an irreversible manner. Finally, homology modelling and molecular docking studies were used to gain insights into the binding of compound 52 to the active site of hABHD6.

A Cannabinoid CB1 Receptor-Positive Allosteric Modulator Reduces Neuropathic Pain in the Mouse with No Psychoactive Effects.

The CB1 receptor represents a promising target for the treatment of several disorders including pain-related disease states. However, therapeutic applications of Δ(9)-tetrahydrocannabinol and other CB1 orthosteric receptor agonists remain limited because of psychoactive side effects. Positive allosteric modulators (PAMs) offer an alternative approach to enhance CB1 receptor function for therapeutic gain with the promise of reduced side effects. Here we describe the development of the novel synthetic CB1 PAM, 6-methyl-3-(2-nitro-1-(thiophen-2-yl)ethyl)-2-phenyl-1H-indole (ZCZ011), which augments the in vitro and in vivo pharmacological actions of the CB1 orthosteric agonists CP55,940 and N-arachidonoylethanolamine (AEA). ZCZ011 potentiated binding of [(3)H]CP55,940 to the CB1 receptor as well as enhancing AEA-stimulated [(35)S]GTPγS binding in mouse brain membranes and β-arrestin recruitment and ERK phosphorylation in hCB1 cells. In the whole animal, ZCZ011 is brain penetrant, increased the potency of these orthosteric agonists in mouse behavioral assays indicative of cannabimimetic activity, including antinociception, hypothermia, catalepsy, locomotor activity, and in the drug discrimination paradigm. Administration of ZCZ011 alone was devoid of activity in these assays and did not produce a conditioned place preference or aversion, but elicited CB1 receptor-mediated antinociceptive effects in the chronic constriction nerve injury model of neuropathic pain and carrageenan model of inflammatory pain. These data suggest that ZCZ011 acts as a CB1 PAM and provide the first proof of principle that CB1 PAMs offer a promising strategy to treat neuropathic and inflammatory pain with minimal or no cannabimimetic side effects.

A novel opioid for photoaffinity labeling opioid receptor complexes in natively‐expressing tissues

A novel radioiodinated opioid photoaffinity label bearing an aryl azide moiety was synthesized, permitting covalent attachment of the drug to its target following irradiation with UV light. In receptor binding assays using membranes from stably transfected cell lines, the drug demonstrated extremely high affinity for mu and kappa opioid receptors with high selectivity over delta receptors. The drug was readily incorporated into mu receptors, and autoradiography following denaturing polyacrylamide gel electrophoresis (SDS-PAGE) revealed a specific band corresponding to the mature, glycosylated form of the receptor which was fully competed by levallorphan (1uM). Photoaffinity labeling of mouse brain and human neuroblastoma cell membranes followed by immunoprecipitation also revealed specific bands corresponding to the mature forms of the receptor in these tissues. The molecular weight of the receptor differed between mouse brain and cell line membranes, consistent with differing glycosylation state of the receptor. Photolabeling the receptor in natively-expressing tissue also facilitated the optimization of a purification scheme under native conditions to isolate opioid receptor signaling complexes which were resolved using Blue Native-PAGE and could be further separated by a second dimension of SDS-PAGE.

A natural substrate-based fluorescence assay for inhibitor screening on diacylglycerol lipase α

The endocannabinoid 2-arachidonoylglycerol (2-AG) is predominantly biosynthesized by sn-1-diacylglycerol lipase α (DAGL-α) in the CNS. Selective inhibitors of DAGL-α will provide valuable insights in the role of 2-AG in endocannabinoid signaling processes and are potential therapeutics for the treatment of obesity and neurodegenerative diseases. Here, we describe the development of a natural substrate-based fluorescence assay for DAGL-α, using a coupled enzyme approach. The continuous setup of our assay allows monitoring of DAGL-α activity in real-time and in a 96-well plate format. This constitutes a major improvement to the currently available radiometric and LC/MS-based methods, which can be executed only in low-throughput formats. In addition, our assay circumvents the use of radioactive material. We demonstrate that our assay can be used to screen inhibitors of DAGL-α activity, using 1-stearoyl-2-arachidonoyl-sn-glycerol as the physiologically relevant natural substrate of DAGL-α. Furthermore, our method can be employed to measure DAGL activity and inhibition in the mouse brain membrane proteome. Consequently, our assay should serve as a valuable tool for rapid hit validation and lead optimization of DAGL-α inhibitors.

Optimization of 1,2,5-thiadiazole carbamates as potent and selective ABHD6 inhibitors.

At present, inhibitors of α/β-hydrolase domain 6 (ABHD6) are viewed as a promising approach to treat inflammation and metabolic disorders. This article describes the development of 1,2,5-thiadiazole carbamates as ABHD6 inhibitors. Altogether, 34 compounds were synthesized, and their inhibitory activity was tested using lysates of HEK293 cells transiently expressing human ABHD6 (hABHD6). Among the compound series, 4-morpholino-1,2,5-thiadiazol-3-yl cyclooctyl(methyl)carbamate (JZP-430) potently and irreversibly inhibited hABHD6 (IC50 =44 nM) and showed ∼230-fold selectivity over fatty acid amide hydrolase (FAAH) and lysosomal acid lipase (LAL), the main off-targets of related compounds. Additionally, activity-based protein profiling indicated that JZP-430 displays good selectivity among the serine hydrolases of the mouse brain membrane proteome. JZP-430 has been identified as a highly selective, irreversible inhibitor of hABHD6, which may provide a novel approach in the treatment of obesity and type II diabetes.

Discovery of Triterpenoids as Reversible Inhibitors of α/β-hydrolase Domain Containing 12 (ABHD12)

Backgroundα/β-hydrolase domain containing (ABHD)12 is a recently discovered serine hydrolase that acts in vivo as a lysophospholipase for lysophosphatidylserine. Dysfunctional ABHD12 has been linked to the rare neurodegenerative disorder called PHARC (polyneuropathy, hearing loss, ataxia, retinosis pigmentosa, cataract). In vitro, ABHD12 has been implicated in the metabolism of the endocannabinoid 2-arachidonoylglycerol (2-AG). Further studies on ABHD12 function are hampered as no selective inhibitor have been identified to date. In contrast to the situation with the other endocannabinoid hydrolases, ABHD12 has remained a challenging target for inhibitor development as no crystal structures are available to facilitate drug design.Methodology/Principal FindingsHere we report the unexpected discovery that certain triterpene-based structures inhibit human ABHD12 hydrolase activity in a reversible manner, the best compounds showing submicromolar potency. Based on structure activity relationship (SAR) data collected for 68 natural and synthetic triterpenoid structures, a pharmacophore model has been constructed. A pentacyclic triterpene backbone with carboxyl group at position 17, small hydrophobic substituent at the position 4, hydrogen bond donor or acceptor at position 3 accompanied with four axial methyl substituents was found crucial for ABHD12 inhibitor activity. Although the triterpenoids typically may have multiple protein targets, we witnessed unprecedented selectivity for ABHD12 among the metabolic serine hydrolases, as activity-based protein profiling of mouse brain membrane proteome indicated that the representative ABHD12 inhibitors did not inhibit other serine hydrolases, nor did they target cannabinoid receptors.Conclusions/SignificanceWe have identified reversibly-acting triterpene-based inhibitors that show remarkable selectivity for ABHD12 over other metabolic serine hydrolases. Based on SAR data, we have constructed the first pharmacophore model of ABHD12 inhibitors. This model should pave the way for further discovery of novel lead structures for ABHD12 selective inhibitors.

Benzophenanthridine alkaloid, piperonyl butoxide and (S)-methoprene action at the cannabinoid-1 receptor (CB1-receptor) pathway of mouse brain: Interference with [3H]CP55940 and [3H]SR141716A binding and modification of WIN55212-2-dependent inhibition of synaptosomal l-glutamate release

Benzophenanthridine alkaloids (chelerythrine and sanguinarine) inhibited binding of [3H]SR141716A to mouse brain membranes (IC50s: <1µM). Piperonyl butoxide and (S)-methoprene were less potent (IC50s: 21 and 63µM respectively). Benzophenanthridines and piperonyl butoxide were more selective towards brain CB1 receptors versus spleen CB2 receptors. All compounds reduced Bmax of [3H]SR141716A binding to CB1 receptors, but only methoprene and piperonyl butoxide increased Kd (3–5-fold). Benzophenanthridines increased the Kd of [3H]CP55940 binding (6-fold), but did not alter Bmax. (S)-methoprene increased the Kd of [3H]CP55940 binding (by almost 4-fold) and reduced Bmax by 60%. Piperonyl butoxide lowered the Bmax of [3H]CP55940 binding by 50%, but did not influence Kd. All compounds reduced [3H]SR141716A and [3H]CP55940 association with CB1 receptors. Combined with a saturating concentration of SR141716A, only piperonyl butoxide and (S)-methoprene increased dissociation of [3H]SR141716A above that of SR141716A alone. Only piperonyl butoxide increased dissociation of [3H]CP55940 to a level greater than CP55940 alone. Binding results indicate predominantly allosteric components to the study compounds action. 4-Aminopyridine-(4-AP-) evoked release of l-glutamate from synaptosomes was partially inhibited by WIN55212-2, an effect completely neutralized by AM251, (S)-methoprene and piperonyl butoxide. With WIN55212-2 present, benzophenanthridines enhanced 4-AP-evoked l-glutamate release above 4-AP alone. Modulatory patterns of l-glutamate release (with WIN-55212-2 present) align with previous antagonist/inverse agonist profiling based on [35S]GTPγS binding. Although these compounds exhibit lower potencies compared to many classical CB1 receptor inhibitors, they may have potential to modify CB1-receptor-dependent behavioral/physiological outcomes in the whole animal.