Discover the most talked about and latest scientific content & concepts.

Journal: Molecular pharmacology


This issue of Molecular Pharmacology is dedicated to Dr. Avram Goldstein, the journal’s founding Editor and one of the leaders in the development of modern pharmacology. This chapter focuses on his contributions to the discovery of the dynorphins and evidence that members of this family of opioid peptides are endogenous agonists for the kappa opioid receptor. In his original publication describing the purification and sequencing of dynorphin A, Avram described this peptide as ‘extraordinarily potent’ (‘dyn’ from the Greek, dynamis = power and ‘-orphin’ for endogenous morphine peptide). The name originally referred to its high affinity and great potency in the bioassay that was used to follow its activity during purification, but the name has come to have a second meaning: Studies of its physiological function in brain continue to provide powerful insights to the molecular mechanisms controlling the mood disorders and drug addiction. In the 30 years since its discovery, we have learned that the dynorphin peptides are released in brain during stress exposure. Once released, they activate kappa opioid receptors distributed throughout the brain and spinal cord where they trigger cellular responses resulting in different stress responses: analgesia, dysphoria-like behaviors, anxiety-like responses, and increased addiction behaviors in experimental animals. Avram predicted that a detailed molecular analysis of opiate drug actions would someday lead to better treatments for drug addiction, and he would be gratified to know that subsequent studies enabled by his discovery of the dynorphins resulted in insights that hold great promise for new treatments for addiction and depressive disorders.

Concepts: Opioid, Morphine, Opioid receptor, Heroin, Buprenorphine, Endorphin, Codeine, Kappa Opioid receptor


In this work we investigated the formation, reactivity and anti-platelet activity of various mixed disulfide conjugates of clopidogrel. Our results showed that the production of the active metabolite (AM) from 2-oxoclopidogrel by human liver microsomes (HLMs) is greatly affected by the thiol reductants used. Among the ten thiol compounds tested, glutathione (GSH) is most efficient in producing the AM at a rate of 167 pmoles AM/min/mg HLM. Interestingly, no AM but only the mixed disulfide conjugates were formed in the presence of 6-chloropyridazine-3-thiol (CPT), 2,5-dimethylfuran-3-thiol (DFT), and 3-nitropyridine-2-thiol (NPT). The MS and MS2 spectra of the conjugates of these thiol compounds confirmed the presence of a mixed disulfide bond linkage between the AM and the thiol reductants. Kinetic studies revealed that the mixed disulfide conjugates were capable of exchanging thiols with GSH to release the AM with second order rate constants ranging from 1.2 to 28 M(-1)s(-1). The mixed disulfide conjugates of CPT and NPT showed potent inhibition of platelet aggregation after pre-treatment with 1 mM GSH, confirming that the AM is responsible for the anti-platelet activity of clopidogrel. Collectively, our results provide strong support for a P450-mediated bioactivation mechanism involving the initial formation of a glutathionyl conjugate followed by thiol-disulfide exchange with another GSH molecule to release the AM. Furthermore, the stable mixed disulfide conjugates identified in this study provide a platform to quantitatively generate the therapeutic AM without the need for P450-mediated bioactivation. This property can be further explored in order to overcome the inter-individual variability in clopidogrel therapy.

Concepts: Platelet, Disulfide bond, Glutathione, Sulfur, Cysteine, Thiol, Dithiothreitol, Thiol-disulfide exchange


Purinergic signaling contributes significantly to pain mechanisms, and the nociceptor-specific P2X3 ATP receptor channel is considered a target in pain therapeutics. Recent evidence for co-expression of metabotropic P2Y receptors with P2X3 suggests that ATP release triggers the activation of both ionotropic and metabotropic purinoceptors, with strong potential for functional interaction. Modulation of native P2X3 function by P2Y receptor activation was investigated in rat dorsal root ganglia (DRG) neurons using whole-cell patch-clamp recordings. Application of the selective P2Y receptor agonist UTP decreased peak amplitudes of α,β-meATP-evoked homomeric P2X3-mediated currents, but had no effect on heteromeric P2X2/3-mediated currents. Treatment with phospholipase C (PLC) inhibitor U73122 significantly reversed P2X3 current inhibition induced by UTP-sensitive P2Y receptor activation. We previously reported the modulation of P2X receptors by phospholipids in DRG neurons and injection of exogenous phosphatidylinositol-4,5-bisphosphate (PIP2) fully reverses UTP-mediated regulation of P2X3 channel activity. Pharmacological as well as functional screening of P2Y receptor subtypes indicates the predominant involvement of P2Y2 receptor in P2X3 inhibition and immunolocalization confirms a significant cellular co-expression of P2X3 and P2Y2 in rat DRG neurons. In summary, the function of P2X3 ATP receptor can be inhibited by P2Y2-mediated depletion of PIP2. We propose that expression of P2Y2 purinoceptor in nociceptive sensory neurons provides an homeostatic mechanism to prevent excessive ATP signaling through P2X3 receptor channels.

Concepts: Nervous system, Signal transduction, Receptor, Action potential, Sensory system, Receptor antagonist, Serotonin, Inverse agonist


Lubeluzole, which acts on various targets in vitro, including voltage-gated sodium channels (NaChs), was initially proposed as neuroprotectant. Lubeluzole structure contains a benzothiazole moiety (R-like) related to riluzole and a phenoxy-propranol-amine moiety (A-core) recalling propranolol. Both riluzole and propranolol are efficient NaCh blockers. We studied in detail the effects of lubeluzole (racemic mixture and single isomers), aforementioned lubeluzole moieties, and riluzole on NaChs to increase our knowledge about drug-channel molecular interactions. Compounds were tested on hNav1.4 NaChs, and F1586C or Y1593C mutants functionally expressed in HEK293 cells, using patch-clamp. Lubeluzole blocked NaChs with a remarkable effectiveness. No stereoselectivity was found. Compared to mexiletine, dissociation constant for inactivated channels was ≈600 times lower (≈11 nM), conferring to lubeluzole a huge use-dependence of great therapeutic value. The F1586C mutation impaired use-dependent block only partially, suggesting that additional amino acids are critically involved in high-affinity binding. Lubeluzole moieties were modest NaCh blockers. Riluzole blocked NaChs efficiently but lacked use-dependence, similarly to R-like. F1586C fully abolished A-core use-dependence, suggesting that A-core binds to the local anesthetic receptor. Thus lubeluzole likely binds to the local anesthetic receptor through its phenoxy-propranol-amine moiety, with consequent use-dependent behavior. Nevertheless, compared to other known NaCh blockers, lubeluzole adds a third pharmacophoric point through its benzothiazole moiety, that greatly enhances high-affinity binding and use-dependent block. If sufficient isoform specificity can be attained, the huge use-dependent block may help in the development of new NaCh inhibitors to provide pharmacotherapy for membrane excitability disorders, such as myotonia, epilepsy, or chronic pain.

Concepts: Protein, Acid, Amine, Functional group, Ion channel, Local anesthetic, Racemic mixture, Adderall


We have previously described the development of genetic models to study the in vivo functions of the hepatic cytochrome P450 system, through the hepatic deletion of either cytochrome P450 oxidoreductase (POR; HRN line) or cytochrome b5 (Cyb5; HBN line). However, HRN mice still exhibit low levels of mono-oxygenase activity, in spite of the absence of detectable reductase protein. To investigate whether this is because cytochrome b5 and cytochrome b5 reductase can act as sole electron donors to the P450 system, we have crossed HRN with HBN mice to generate a line lacking hepatic expression of both electron donors (HBRN). HBRN mice exhibited exacerbation of the phenotypic characteristics of the HRN line - liver enlargement, hepatosteatosis and increased expression of certain cytochrome P450s. Also, drug metabolising activities in vitro were further reduced relative to the HRN model, in some cases to undetectable levels. Pharmacokinetic studies in vivo demonstrated that midazolam half-life, Cmax and area under the concentration-time curve (AUC) were increased, and clearance was decreased, to a greater extent in the HBRN line than in either the HBN or HRN model. Microsomal incubations using NADPH concentrations below the apparent Km of cytochrome b5 reductase, but well above that for POR, led to the virtual elimination of 7-benzyloxyquinoline turnover in HRN samples. These data provide strong evidence that cytochrome b5/cytochrome b5 reductase can act as a sole electron donors to the cytochrome P450 system in vitro and in vivo.

Concepts: Cytochrome P450, Paracetamol, CYP3A4, P450-containing systems, Cytochrome P450 reductase


Smoking is a common addiction and a leading cause of disease. Chronic nicotine exposure is known to activate nicotinic acetylcholine receptors (nAChRs) in immune cells. We demonstrate a novel role for α4 nAChRs in the effect of nicotine on T-cell proliferation and immunity. Using cell based sorting and proteomic analysis we define an α4 nAChR expressing helper T-cell population (α4+CD3+CD4+) and show that this group of cells is responsive to sustained nicotine exposure. In circulation, spleen, and thymus we find that nicotine promotes an increase in CD3+CD4+ cells via its activation of the α4 nAChR and regulation of Gαo, Gprin1, and CDC42 signaling within T-cells. In particular, nicotine is found to promote a Th2, adaptive, immunological response within T-cells, which was absent in α4-/- mice. We thus present a new mechanism of α4 nAChR signaling and immune regulation in T-cells, possibly accounting for the effect of smoking on the immune system.

Concepts: Immune system, White blood cell, Immunology, T helper cell, Nicotine, Cell signaling, Acetylcholine, Nicotinic acetylcholine receptor


The CB2 cannabinoid agonist LY2828360 lacked both toxicity and efficacy in a clinical trial for osteoarthritis. Whether LY2828360 suppresses neuropathic pain has not been reported and its signaling profile is unknown. In vitro, LY2828360 was a slowly acting but efficacious G protein-biased CB2 agonist, inhibiting cAMP accumulation and activating ERK1/2 signaling while failing to recruit arrestin, activate inositol phosphate signaling or internalize CB2 receptors. In wildtype (WT) mice, LY2828360 (3 mg/kg/day i.p. x 12 days) suppressed chemotherapy-induced neuropathic pain produced by paclitaxel without producing tolerance. Anti-allodynic efficacy of LY2828360 was absent in CB2KO mice. Morphine (10 mg/kg/day i.p. x 12 days) tolerance developed in CB2KO mice but not in WT mice with a history of LY2828360 treatment (3 mg/kg/day i.p. x 12 days). LY2828360-induced anti-allodynic efficacy was preserved in WT mice previously rendered tolerant to morphine (10 mg/kg/day i.p. x 12 days) but absent in morphine-tolerant CB2KO mice. Coadministration of LY2828360 (0.1 mg/kg/day i.p. x 12 days) with morphine (10 mg/kg/day x 12 days) blocked morphine tolerance in WT but not CB2KO mice. WT mice that received LY2828360 coadministered with morphine exhibited a trend (p=0.055) towards fewer naloxone-precipitated jumps compared to CB2KO mice. In conclusion, LY2828360 is a slowly signaling, G protein-biased CB2 agonist that attenuates chemotherapy-induced neuropathic pain without producing tolerance, and may prolong effective opioid analgesia while reducing opioid dependence. LY2828360 may be useful as a first line treatment in chemotherapy-induced neuropathic pain and may be highly efficacious in neuropathic pain states that are refractive to opioid analgesics.

Concepts: Opioid, Pain, Morphine, Cannabinoid receptor, Analgesic, Cannabis, Buprenorphine, Cannabinoids


Valproic acid (VPA) is a widely prescribed anti-convulsant for the treatment of epilepsy. Here we demonstrate that VPA is a novel activator of AMP-activated protein kinase (AMPK), a key regulator of cellular metabolism, using primary mouse and human hepatocytes. Incubation of primary mouse hepatocytes with VPA resulted in increased levels of phosphorylated AMPK and acetyl-CoA carboxylase (ACC). This finding was recapitulated using primary human hepatocytes. Pre-treatment of mouse hepatocytes with small molecule inhibitor of AMPK, Compound C, abrogated the phosphorylation of ACC following treatment with VPA. The cytochrome P450 inhibitor 1-aminobenzotriazole blocked the VPA-stimulated phosphorylation of AMPK, suggesting a requirement for biotransformation of VPA. In line with this, treatment of hepatocytes with metabolites of VPA resulted in increased phosphorylation of AMPK/ACC as compared to VPA. Treatment of ob/ob mice with VPA for 14 days resulted in decreased liver masses, hepatic fat accumulation, and serum glucose. These results paralleled those observed in mice treated with metformin. In addition, a targeted mass spectrometry-based metabolomics assay revealed several small molecules that were differentially abundant in the serum of ob/ob mice treated with VPA as compared to vehicle-treated mice. These studies are the first to establish VPA and its metabolites as in vitro activators of AMPK.

Concepts: Protein, Metabolism, Nutrition, Adenosine triphosphate, Enzyme, Glycogen, Enzyme inhibitor, Proteomics


Previous studies have demonstrated that the knockdown or knockout of the three Na(+)/Ca(2+) exchanger (NCX) isoforms, NCX1, NCX2, and NCX3, worsens ischemic brain damage. This suggests that the activation of these antiporters exerts a neuroprotective action against stroke damage. However, drugs able to increase the activity of NCXs are not yet available. We have here succeeded in synthesizing a new compound, named neurounina-1 (7-nitro-5-phenyl-1-(pyrrolidin-1-ylmethyl)-1H-benzo[e][1,4]diazepin-2(3H)-one), provided with an high lipophilicity index and able to increase NCX activity. Ca(2+) radiotracer, Fura-2 microfluorimetry, and patch-clamp techniques revealed that neurounina-1 stimulated NCX1 and NCX2 activities with an EC(50) in the picomolar to low nanomolar range, whereas it did not affect NCX3 activity. Furthermore, by using chimera strategy and site-directed mutagenesis, three specific molecular determinants of NCX1 responsible for neurounina-1 activity were identified in the α-repeats. Interestingly, NCX3 became responsive to neurounina-1 when both α-repeats were replaced with the corresponding regions of NCX1. In vitro studies showed that 10 nM neurounina-1 reduced cell death of primary cortical neurons exposed to oxygen-glucose deprivation followed by reoxygenation. Moreover, in vitro, neurounina-1 also reduced γ-aminobutyric acid (GABA) release, enhanced GABA(A) currents, and inhibited both glutamate release and N-methyl-d-aspartate receptors. More important, neurounina-1 proved to have a wide therapeutic window in vivo. Indeed, when administered at doses of 0.003 to 30 μg/kg i.p., it was able to reduce the infarct volume of mice subjected to transient middle cerebral artery occlusion even up to 3 to 5 hours after stroke onset. Collectively, the present study shows that neurounina-1 exerts a remarkable neuroprotective effect during stroke and increases NCX1 and NCX2 activities.

Concepts: Stroke, Traumatic brain injury, In vivo, Cerebral cortex, Middle cerebral artery, Anterior cerebral artery, Ischemia, In vitro


Estimates vary regarding the number of G protein-coupled receptors, GPCRs, the largest family of membrane receptors that are targeted by approved drugs and the number of such drugs that target GPCRs. We review current knowledge regarding GPCRs as drug targets by integrating data from public databases (CHEMBL, IUPHAR and DRUGBANK) and from the Broad drug repurposing initiative. To account for discrepancies among these sources, we curated a list of GPCRs currently targeted by approved drugs. As of November, 2017, 134 GPCRs are targets for drugs approved in the United States or European Union; 128 GPCRs are targets for drugs listed in the FDA orange book. We estimate that ~700 approved drugs target GPCRs, implying that approximately 35% of approved drugs target GPCRs. GPCRs and GPCR-related proteins, i.e. those upstream or downstream of GPCRs, represent ~17% of all protein targets for approved drugs, with GPCRs themselves accounting for ~12%. As such, GPCRs constitute the largest family of proteins targeted by approved drugs. Drugs that currently target GPCRs and GPCR related proteins are primarily small molecules and peptides. Since ~100 of the ~360 human endoGPCRs (other than olfactory, taste and visual GPCRs) are orphan receptors (lacking known physiologic agonists), the number of GPCR targets, the number of GPCR-targeted drugs and perhaps the types of drugs will likely increase, thus further expanding this GPCR repertoire and the many roles of GPCR-drugs in therapeutics.

Concepts: Protein, Pharmacology, Signal transduction, Cell membrane, G protein-coupled receptor, Metabotropic glutamate receptor, G protein, Orphan receptor