Irreversible Antagonist Research Articles

Gαs signalling of the CB1 receptor and the influence of receptor number.

CB1 receptor signalling is canonically mediated through inhibitory Gαi proteins, but occurs through other G proteins under some circumstances, Gαs being the most characterized secondary pathway. Determinants of this signalling switch identified to date include Gαi blockade, CB1 /D2 receptor co-stimulation, CB1 agonist class and cell background. Hence, we examined the effects of receptor number and different ligands on CB1 receptor signalling. CB1 receptors were expressed in HEK cells at different levels, and signalling characterized for cAMP by real-time BRET biosensor -CAMYEL - and for phospho-ERK by AlphaScreen. Homogenate and whole cell radioligand binding assays were performed to characterize AM6544, a novel irreversible CB1 receptor antagonist. In HEK cells expressing high levels of CB1 receptors, agonist treatment stimulated cAMP, a response not known to be mediated by receptor number. Δ9 -THC and BAY59-3074 increased cAMP only in high-expressing cells pretreated with pertussis toxin, and agonists demonstrated more diverse signalling profiles in the stimulatory pathway than the canonical inhibitory pathway. Pharmacological CB1 receptor knockdown and Gαi 1 supplementation restored canonical Gαi signalling to high-expressing cells. Constitutive signalling in both low- and high-expressing cells was Gαi -mediated. CB1 receptor coupling to opposing G proteins is determined by both receptor and G protein expression levels, which underpins a mechanism for non-canonical signalling in a fashion consistent with Gαs signalling. CB1 receptors mediate opposite consequences in endpoints such as tumour viability depending on expression levels; our results may help to explain such effects at the level of G protein coupling.

Extrasynaptic NMDA receptor dependent long-term potentiation of hippocampal CA1 pyramidal neurons

In the adult mouse hippocampus, NMDA receptors (NMDARs) of CA1 neurons play an important role in the synaptic plasticity. The location of NMDARs can determine their roles in the induction of long-term potentiation (LTP). However, the extrasynaptic NMDARs (ES-NMDARs) dependent LTP haven’t been reported. Here, through the use of a 5-Hz stimulation and MK-801 (an irreversible antagonist of NMDARs) in the CA1 neurons of adult mice hippocampal slices, synaptic NMDARs were selectively inhibited and NMDAR-mediated excitatory postsynaptic currents were not recovered. We found that a robust LTP was induced by 3-train 100-Hz stimulation when the synaptic NMDARs and extrasynaptic NR2B containing NMDARs were blocked, but not in the any of the following conditions: blocking of all NMDARs (synaptic and extrasynaptic), blocking of the synaptic NMDARs, and blocking of the synaptic NMDARs and extrasynaptic NR2A-containing NMDARs. The results indicate that this LTP is ES-NMDARs dependent, and NR2B-containing ES-NMDARs modulates the threshold of LTP induction.

Methodical Challenges and a Possible Resolution in the Assessment of Receptor Reserve for Adenosine, an Agonist with Short Half-Life.

The term receptor reserve, first introduced and used in the traditional receptor theory, is an integrative measure of response-inducing ability of the interaction between an agonist and a receptor system (consisting of a receptor and its downstream signaling). The underlying phenomenon, i.e., stimulation of a submaximal fraction of receptors can apparently elicit the maximal effect (in certain cases), provides an opportunity to assess the receptor reserve. However, determining receptor reserve is challenging for agonists with short half-lives, such as adenosine. Although adenosine metabolism can be inhibited several ways (in order to prevent the rapid elimination of adenosine administered to construct concentration–effect (E/c) curves for the determination), the consequent accumulation of endogenous adenosine biases the results. To address this problem, we previously proposed a method, by means of which this bias can be mathematically corrected (utilizing a traditional receptor theory-independent approach). In the present investigation, we have offered in silico validation of this method by simulating E/c curves with the use of the operational model of agonism and then by evaluating them using our method. We have found that our method is suitable to reliably assess the receptor reserve for adenosine in our recently published experimental setting, suggesting that it may be capable for a qualitative determination of receptor reserve for rapidly eliminating agonists in general. In addition, we have disclosed a possible interference between FSCPX (8-cyclopentyl-N3-[3-(4-(fluorosulfonyl)benzoyloxy)propyl]-N1-propylxanthine), an irreversible A1 adenosine receptor antagonist, and NBTI (S-(2-hydroxy-5-nitrobenzyl)-6-thioinosine), a nucleoside transport inhibitor, i.e., FSCPX may blunt the effect of NBTI.

Importance of D1 and D2 receptor stimulation for the induction and expression of cocaine-induced behavioral sensitization in preweanling rats

The behavioral manifestations of psychostimulant-induced sensitization vary markedly between young and adult rats, suggesting that the neural mechanisms mediating this phenomenon differ across ontogeny. In this project we examined the importance of D1 and D2 receptors for the induction and expression of cocaine-induced behavioral sensitization during the preweanling period. In the behavioral experiments, rats were injected with reversible D1 and/or D2 antagonists (SCH23390 and/or raclopride) or an irreversible receptor antagonist (EEDQ) either before cocaine administration on the pretreatment day (induction) or before cocaine challenge on the test day (expression). In the EEDQ experiments, receptor specificity was assessed by using selective dopamine antagonists to protect D1 and/or D2 receptors from inactivation. Receptor binding assays showed that EEDQ caused substantial reductions in dorsal striatal D1 and D2 binding sites, while SCH23390 and raclopride fully protected D1 and D2 receptors from EEDQ-induced alkylation. Behavioral results showed that neither D1 nor D2 receptor stimulation was necessary for the induction of cocaine sensitization in preweanling rats. EEDQ disrupted the sensitization process, suggesting that another receptor type sensitive to EEDQ alkylation was necessary for the induction process. Expression of the sensitized response was prevented by an acute injection of a D1 receptor antagonist. The pattern of DA antagonist-induced effects described for preweanling rats is, with few exceptions, similar to what is observed when the same drugs are administered to adult rats. Thus, it appears that maturational changes in D1 and D2 receptor systems are not responsible for ontogenetic differences in the behavioral manifestation of cocaine sensitization.

Modification of the Orthosteric PPARγ Covalent Antagonist Scaffold Yields an Improved Dual-Site Allosteric Inhibitor.

GW9662 and T0070907 are widely used commercially available irreversible antagonists of peroxisome proliferator-activated receptor gamma (PPARγ). These antagonists covalently modify Cys285 located in an orthosteric ligand-binding pocket embedded in the PPARγ ligand-binding domain and are used to block binding of other ligands. However, we recently identified an alternate/allosteric ligand-binding site in the PPARγ LBD to which ligand binding is not inhibited by these orthosteric covalent antagonists. Here, we developed a series of analogs based on the orthosteric covalent antagonist scaffold with the goal of inhibiting both orthosteric and allosteric cellular activation of PPARγ by MRL20, an orthosteric agonist that also binds to an allosteric site. Our efforts resulted in the identification of SR16832 (compound 22), which functions as a dual-site covalent inhibitor of PPARγ transcription by PPARγ-binding ligands. Molecular modeling, protein NMR spectroscopy structural analysis, and biochemical assays indicate the inhibition of allosteric activation occurs in part through expansion of the 2-chloro-5-nitrobenzamidyl orthosteric covalent antagonist toward the allosteric site, weakening of allosteric ligand binding affinity, and inducing conformational changes not competent for cellular PPARγ activation. Furthermore, SR16832 better inhibits binding of rosiglitazone, a thiazolidinedione (TZD) that weakly activates PPARγ when cotreated with orthosteric covalent antagonists, and may better inhibit binding of endogenous PPARγ ligands such as docosahexaenoic acid (DHA) compared to orthosteric covalent antagonists. Compounds such as SR16832 may be useful chemical tools to use as a dual-site bitopic orthosteric and allosteric covalent inhibitor of ligand binding to PPARγ.

Stimulation of glycerol kinase in grass carp preadipocytes by EPA.

This study was conducted to assess the effect of eicosapentaenoic acid (EPA) on grass carp preadipocyte glycerol kinase (GyK) expression, as well as to explore the mechanism. Here, we cloned partial sequence of grass carp GyK gene and analyzed its tissue distribution. The result showed that GyK gene expressed most in the liver, followed by adipose tissue and the kidney. Besides, 400μM oleic acid (18:1n-9, OA) was used to establish a hypertrophic preadipocyte model. GyK gene expression and enzyme activity were significantly enhanced after model cells were treated with 100μM eicosapentaenoic acid (20:5n-3, EPA) for 6, 12, and 24h. Meanwhile, peroxisome proliferative-activated receptor (PPAR)γ, adipose triglyceride lipase (ATGL), and the two isoforms of grass carp HSL gene were first identified by Sun et al (2016), and they defined the two isoforms as HSLa and HSLb. Therefore, maybe HSLa and HSLb are appropriate.. The content of triglyceride was dramatically increased by EPA treatment for 24h. Further, a competitive ATGL antagonist, HY-15859, attenuated the increase in GyK induced by EPA at 12h. Surprisingly, the enhanced lipolysis and PPARγ gene expression induced by serum deprivation were paralleled by an increase in GyK gene expression, whereas a stabilization in GyK enzyme activity. Other fatty acids, including docosahexaenoic acid, alpha-linolenic acid, linoleic acid, and OA also promoted GyK gene expression. Moreover, an irreversible PPARγ antagonist, GW9662, was used to investigate the role of PPARγ in GyK induction. Data showed that GW9662 abolished the induction of GyK by EPA at 12h. Together, these data suggested that EPA elevated grass carp preadipocytes GyK expression. ATGL and PPARγ contributed to the induction of GyK. PPARγ may be a key regulator in response to GyK expression induced by EPA.

Cardiovascular/Pulmonary Medications and Male Reproduction.

Cardiovascular and respiratory medications are used by men of reproductive age although use of the former is most prevalent in advanced age. Many of these drugs have been associated with sexual dysfunction, including erectile and ejaculatory dysfunction, but for most there is insufficient evidence to link their use with testicular dysfunction, reduced semen quality or infertility. Some exceptions are the irreversible α1-adrenergic antagonist phenoxybenzamine, which carries a high risk of retrograde ejaculation; the specific α1A-adrenergic antagonists silodosin and tamsulosin, used primarily to treat BPH/lower urinary tract symptoms, which can cause retrograde ejaculation; and the peripheral β1-adrenergic antagonist atenolol, used to treat hypertension, which may decrease testosterone/free-testosterone levels. In this chapter, we review the evidence available regarding adverse reactions on male reproduction of adrenergic receptor agonists/antagonists, calcium channel blockers, angiotensin converting enzyme (ACE) inhibitors, diuretics, digoxin, and hydralazine. For some of these medications, there is some evidence for male reproductive effects, along with some solid work in experimental and companion animal species suggesting negative effects. In contrast, and of special note, are calcium channel blockers, which have long been included on lists of medications with the potential to cause male infertility. This turns out to be a good example of a substance with profound effects on sperm function in vitro, but with limited evidence for in vivo effects on semen quality or fertility, even in experimental species. We hope that the evidence provided in this chapter will stimulate additional studies for these important classes of medications.

Spotlight on afatinib and its potential in the treatment of squamous cell lung cancer: the evidence so far

Compared to adenocarcinoma, fewer effective treatment options are available for advanced or metastatic squamous cell carcinoma (SCC) of the lung. Afatinib is an orally administered, irreversible EGFR antagonist. As a second-generation tyrosine kinase inhibitor, it has been applied in the treatment of patients with EGFR-mutant non-small-cell lung cancer. Recently, several clinical trials have shown that afatinib leads to a significant improvement in progression-free survival and overall survival of patients with SCC. Moving forward, afatinib should be one of the options among tyrosine kinase inhibitors, monoclonal antibodies, and cytotoxicity chemotherapy drugs for SCC.

The antinociceptive effects of ferulic acid on neuropathic pain: involvement of descending monoaminergic system and opioid receptors.

Neuropathic pain can be considered as a form of chronic stress that may share common neuropathological mechanism between pain and stress-related depression and respond to similar treatment. Ferulic acid (FA) is a major active component of angelica sinensis and has been reported to exert antidepressant-like effects; however, it remains unknown whether FA ameliorate chronic constriction injury (CCI)-induced neuropathic pain and the involvement of descending monoaminergic system and opioid receptors. Chronic treatment with FA (20, 40 and 80 mg/kg) ameliorated mechanical allodynia and thermal hyperalgesia in von Frey hair and hot plate tasks, accompanied by increasing spinal noradrenaline (NA) and serotonin (5-HT) levels. Subsequent study suggested that treatment of CCI animals with 40 and 80 mg/kg FA also inhibited spinal MAO-A levels. FA's effects on mechanical allodynia or thermal hyperalgesiawas blocked by 6-hydroxydopamine (6-OHDA) or p-chlorophenylalanine (PCPA) via pharmacological depletion of spinal noradrenaline or serotonin. Moreover, the anti-allodynic action of FA on mechanical stimuli was prevented by pre-treatment with beta2-adrenoceptor antagonist ICI 118,551, or by the delta-opioid receptor antagonist naltrindole. While the anti-hyperalgesia on thermal stimuli induced by FA was blocked by pre-treatment with 5-HT1A receptor antagonist WAY-100635, or with the irreversible mu-opioid receptor antagonist beta-funaltrexamine. These results suggest that the effect of FA on neuropathic pain is potentially mediated via amelioration of the descending monoaminergic system that coupled with spinal beta2- and 5-HT1A receptors and the downstream delta- and mu-opioid receptors differentially.

A1 Adenosine Receptor-Mediated Inhibition of Parasympathetic Neuromuscular Transmission in Human and Murine Urinary Bladder.

The potential role of A1 adenosine receptors in modulating neuromuscular transmission in the detrusor muscle of the urinary bladder has been tested in human and murine preparations with the intent to determine the viability of using adenosine receptor agonists as adjuncts to treat overactive bladder. In human detrusor muscle preparations, contractile responses to electrical field stimulation were inhibited by the selective A1 adenosine receptor agonists 2-chloro-N(6)-cyclopentyladenosine, N(6)-cyclopentyladenosine (CPA), and adenosine (rank order of potency: 2-chloro-N(6)-cyclopentyladenosine > CPA > adenosine). Pretreatment with 8-cyclopentyl-3-[3-[[4(fluorosulphonyl)benzoyl]oxy]propyl]-1-propylxanthine, an irreversible A1 antagonist, blocked the effects of CPA, thus confirming the role of A1 receptors in human detrusor preparations. In murine detrusor muscle preparations, contractions evoked by electrical field stimulation were reduced by CPA or adenosine. Amplitudes of the P2X purinoceptor-mediated excitatory junctional potentials (EJPs) recorded with intracellular microelectrodes were reduced in amplitude by CPA and adenosine with no effect on the spontaneous EJP amplitudes, confirming the prejunctional action of these agents. 8-Cyclopentyltheophylline, a selective A1 receptor antagonist, reversed the effects of CPA on EJP amplitudes with no effect of spontaneous EJPs, confirming the role of A1 receptors in mediating these effects.

Identification of a New Type of Covalent PPARγ Agonist using a Ligand-Linking Strategy.

Peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-activated transcription factor that plays an important role in adipogenesis and glucose metabolism. The ligand-binding pocket (LBP) of PPARγ has a large Y-shaped cavity with multiple subpockets where multiple ligands can simultaneously bind and cooperatively activate PPARγ. Focusing on this unique property of the PPARγ LBP, we describe a novel two-step cell-based strategy to develop PPARγ ligands. First, a combination of ligands that cooperatively activates PPARγ was identified using a luciferase reporter assay. Second, hybrid ligands were designed and synthesized. For proof of concept, we focused on covalent agonists, which activate PPARγ through a unique activation mechanism regulated by a covalent linkage with the Cys285 residue in the PPARγ LBP. Despite their biological significance and pharmacological potential, few covalent PPARγ agonists are known except for endogenous fatty acid metabolites. With our strategy, we determined that plant-derived cinnamic acid derivatives cooperatively activated PPARγ by combining with GW9662, an irreversible antagonist. GW9662 covalently reacts with the Cys285 residue. A docking study predicted that a cinnamic acid derivative can bind to the open cavity in GW9662-bound PPARγ LBP. On the basis of the putative binding mode, structures of both ligands were linked successfully to create a potent PPARγ agonist, which enhanced the transactivation potential of PPARγ at submicromolar levels through covalent modification of Cys285. Our approach could lead to the discovery of novel high-potency PPARγ agonists.

The Synergistic Enhancement of Cloning Efficiency in Individualized Human Pluripotent Stem Cells by Peroxisome Proliferative-activated Receptor-γ (PPARγ) Activation and Rho-associated Kinase (ROCK) Inhibition

Although human pluripotent stem cells (hPSCs) provide valuable sources for regenerative medicine, their applicability is dependent on obtaining both suitable up-scaled and cost effective cultures. The Rho-associated kinase (ROCK) inhibitor Y-27632 permits hPSC survival upon dissociation; however, cloning efficiency is often still low. Here we have shown that pioglitazone, a selective peroxisome proliferative-activated receptor-γ agonist, along with Y-27632 synergistically diminished dissociation-induced apoptosis and increased cloning efficiency (2-3-fold versus Y-27632) without affecting pluripotency of hPSCs. Pioglitazone exerted its positive effect by inhibition of glycogen synthase kinase (GSK3) activity and enhancement of membranous β-catenin and E-cadherin proteins. These effects were reversed by GW-9662, an irreversible peroxisome proliferative-activated receptor-γ antagonist. This novel setting provided a step toward hPSC manipulation and its biomedical applications.

Opioid Receptor: Its structure and how it works

The First and Second Opium Wars were two conflicts caused by the the reaction of China over the illegal exportation of the drug opium by the British government. Opium, also known as poppy tears, is a narcotic drug extracted from the plant Papaver somniferum. This drug causes a sensation of euphoria and pain relief by binding to opioid receptors that exist on cell membranes of neurons and the digestive tract. Opioid receptors (OR) are G‐­protein‐coupled receptors (GPCRs) with three main variables, μ‐­OR, δ‐OR and κ­‐OR. The μ­‐OR is the most important opioid receptor for the management of pain. To better understand this receptor's structure and how it can impact function, the Worcester Academy SMART Team has modeled the μ­‐OR using 3D printing technology. Using the irreversible antagonist β‐­FNA, the μ‐OR has a complex shape, consisting of seven transmembrane α‐­helices connected by three extracellular loops and three intracellular loops; transmembrane helices 5 and 6 are involved in receptor dimerization. The binding pocket is relatively open which causes a greater exposure to the extracellular surface, providing evidence as to why potent opioids like buprenorphine and etorphine have a rapid dissociation. β‐­FNA interacts with 14 residues, of which nine have more interaction with the ligand and are conserved in the δ­‐OR and the κ‐­OR. The μ‐­OR contains 11 identical amino acid residues with the the other two ORs. The only differences are the positions E229, K303 and W318, which are Asp, Trp and Leu. Further understanding of opioid receptors and their structure can lead to a way of treating pain without side effects.

Ferulic Acid Alleviates Neuropathic Pain: Involvement of Descending Monoaminergic System and Opioid Receptors

Neuropathic pain can be considered as a form of chronic stress that may share common neuropathological mechanism between pain and stress‐related depression and respond to similar treatment. FA is a major active component of angelica sinensis and has been reported to exert antidepressant‐like effects; however, it remains unknown whether resveratrol ameliorate CCI‐induced neuropathic pain. Chronic treatment with FA (10, 20, 40 and 80 mg/kg) twice daily for 3 weeks ameliorated mechanical allodynia and thermal hyperalgesia in von Frey hair and hot plate tasks, accompanied by increasing spinal monoamine levels. Pharmacological depletion of spinal noradrenaline (NA) or serotonin (5‐HT) by 6‐hydroxydopamine (6‐OHDA) or p‐chlorophenylalanine (PCPA) completely blocked FA's effects on mechanical allodynia or thermal hyperalgesia, respectively. The anti‐allodynic action of FA on mechanical stimuli was prevented by co‐treatment with beta2‐adrenoceptor antagonist ICI 118,551, or by co‐treatment with delta‐opioid receptor antagonist naltrindole. Moreover, the anti‐hyperalgesia on thermal stimuli induced by FA was blocked by co‐treatment with 5‐HT1A receptor antagonist WAY‐100635, or with the irreversible mu‐opioid receptor antagonist beta‐funaltrexamine. These results suggest that the effect of FA on neuropathic pain is potentially mediated via modulation of descending monoaminergic system that coupled with spinal b2‐ and 5‐HT1A receptors and the downstream opioid receptors.

Potent Irreversible Inhibitors of LasR Quorum Sensing in Pseudomonas aeruginosa.

Antagonism of quorum sensing represents a promising new antivirulence approach for the treatment of bacterial infection. The development of a novel series of non-natural irreversible antagonists of P. aeruginosa LasR is described. The lead compounds identified (25 and 28) display potent LasR antagonist activity and inhibit expression of the P. aeruginosa virulence factors pyocyanin and biofilm formation in PAO1 and PA14.

PPAR-γ Agonists Induce Growth Arrest of Kit/AML1-ETO Leukemia Cells Acting on Diverse Pathways

Translocation 8;21, t(8;21), with an incidence of up to 12% is among the most common genetic aberrations found in Acute Myeloid leukemias (AMLs). t(8;21) rearranges the AML1 gene on chromosome 21 and the ETO gene on chromosome 8, generating an AML1-ETO fusion protein. It acts as dominant-negative repressor of genes involved in cell cycle control and myeloid differentiation. Despite its critical role in tumor initiation, AML1-ETO is not sufficient, per se, to cause an overt leukemia. It has to cooperate with other genetic alterations, such as those involving receptor tyrosine kinases. KIT tyrosine kinase gain-of-function mutations have been detected in about 50% of t(8;21) AMLs.Peroxisome Proliferator-Activated Receptor-γ (PPAR-γ) agonists are known to inhibit proliferation and induce apoptosis in cancer cells, including those from hematologic malignancies. Despite several promising pre-clinical studies, results of clinical trials are not conclusive so far.We investigated the effects and mechanisms of action of some PPAR-γ agonists in an AML1-ETO/Kit activated leukemia cell line: Kasumi-1. Troglitazone (TGZ) and rosiglitazone (RGZ) reduced proliferation rate and clonogenic potential of Kasumi-1, inducing G1 arrest and apoptosis in a dose and time dependent manner. Activated Kit induces proliferation mostly through the Src kinases/Ras/MAPK pathway. Erk1/2 is constitutively phosphorylated in Kasumi-1 cells and exposure to troglitazone (50μM) significantly reduced its phosphorylated state even if not as much as imatinib. The Kit signaling cascades include activation of PI3-kinase/AKT pathway, but surprisingly TGZ treatment did not affect AKT phosphorylation.It has been shown that PPAR agonists 2-cyano-3,12-dioxo-oleana-1,9(11)-dien-28-oic acid (CDDO) and 15-Deoxy-Δ12,14-ProstaglandinJ2 (15d-PGJ2) are able to suppress TGFβ-induced phosphorylation of AktS473, leading an anti-fibrotic activity in the lung. CDDO and 15d-PGJ2 inhibition effect have been demonstrated direct, PPAR-γindependent and mostly related to electrophilic carbons present in their structures. In the same cellular context rosiglitazone was found relatively poorly effective. We compared, in Kasumi-1 cells, activity of 15d-PGJ2 and CAY10410, two PPAR agonists with similar structure but while 15d-PGJ2 has an electrophilic center CAY10410 does not. CAY10410 is a strong PPAR agonist but was unable to induce any reduction in Kasumi-1 cells proliferation rate or induce apoptosis whereas 15d-PGJ2 strongly inhibited Kasumi-1 growth and induced apoptosis even more efficiently than troglitazone. Molecular characterization of such functional effect is of extreme interest and is ongoing.TGZ and RGZ induced up-regulation of p27Kip1 and p21cip1. We found that it occurs by reduced proteosomal degradation and accumulation of p27kip1 due to up-regulation of Connexin 43 (Cx43) and consequent downregulation of Skp2. AML1-ETO has been reported as Cx43 transcriptional inducer. Upregulation of p21Waf1 seems to be, in preliminary experiments, mostly transcriptional and AML1-ETO direct induction of p21Waf1has been described as well. Interestingly TGZ seems to restore AML1-ETO functions counteracted upon Kit constitutive activation.Some, if not all, the effects might be receptor independent since they were just minimally lessened by a pre-treatment with high dose of GW9662, an irreversible PPAR-γ antagonist that covalently binds to the active site of PPAR-γ.The overall prognosis of AML patients remains severe and AML incidence is expected to increase with the aging population, forcing the need for less toxic regimens in patients with co-morbid conditions precluding intensive chemotherapy. PPAR agonists are promising candidates as specific anticancer drugs within low toxicity multidrug regimens. However, the effectiveness of PPAR ligands seems to vary as various compounds, targeting distinct leukemic cells, behavior differently, because different are the leading molecular mechanism evoked. Their precise clarification will define the real extent of PPAR agonists’ therapeutic potential. DisclosuresNo relevant conflicts of interest to declare.

Acute-phase protein α1-antitrypsin--a novel regulator of angiopoietin-like protein 4 transcription and secretion.

The angiopoietin-like protein 4 (angptl4, also known as peroxisome proliferator-activated receptor [PPAR]γ-induced angiopoietin-related protein) is a multifunctional protein associated with acute-phase response. The mechanisms accounting for the increase in angptl4 expression are largely unknown. This study shows that human α1-antitrypsin (A1AT) upregulates expression and release of angplt4 in human blood adherent mononuclear cells and in primary human lung microvascular endothelial cells in a concentration- and time-dependent manner. Mononuclear cells treated for 1 h with A1AT (from 0.1 to 4 mg/ml) increased mRNA of angptl4 from 2- to 174-fold, respectively, relative to controls. In endothelial cells, the maximal effect on angptl4 expression was achieved at 8 h with 2 mg/ml A1AT (11-fold induction versus controls). In 10 emphysema patients receiving A1AT therapy (Prolastin), plasma angptl4 levels were higher relative to patients without therapy (nanograms per milliliter, mean [95% confidence interval] 127.1 [99.5-154.6] versus 76.8 [54.8-98.8], respectively, p = 0.045) and correlated with A1AT levels. The effect of A1AT on angptl4 expression was significantly diminished in cells pretreated with a specific inhibitor of ERK1/2 activation (UO126), irreversible and selective PPARγ antagonist (GW9662), or genistein, a ligand for PPARγ. GW9662 did not alter the ability of A1AT to induce ERK1/2 phosphorylation, suggesting that PPARγ is a critical mediator in the A1AT-driven angptl4 expression. In contrast, the forced accumulation of HIF-1α, an upregulator of angptl4 expression, enhanced the effect of A1AT. Thus, acute-phase protein A1AT is a physiological regulator of angptl4, another acute-phase protein.

Desensitization of functional µ-opioid receptors increases agonist off-rate.

Desensitization of µ-opioid receptors (MORs) develops over 5-15 minutes after the application of some, but not all, opioid agonists and lasts for tens of minutes after agonist removal. The decrease in function is receptor selective (homologous) and could result from 1) a reduction in receptor number or 2) a decrease in receptor coupling. The present investigation used photolysis of two caged opioid ligands to examine the kinetics of MOR-induced potassium conductance before and after MOR desensitization. Photolysis of a caged antagonist, carboxynitroveratryl-naloxone (caged naloxone), blocked the current induced by a series of agonists, and the time constant of decline was significantly decreased after desensitization. The increase in the rate of current decay was not observed after partial blockade of receptors with the irreversible antagonist, β-chlornaltrexamine (β-CNA). The time constant of current decay after desensitization was never more rapid than 1 second, suggesting an increased agonist off-rate rather than an increase in the rate of channel closure downstream of the receptor. The rate of G protein-coupled K(+) channel (GIRK) current activation was examined using photolysis of a caged agonist, carboxynitrobenzyl-tyrosine-[Leu(5)]-enkephalin. After acute desensitization or partial irreversible block of MORs with β-CNA, there was an increase in the time it took to reach a peak current. The decrease in the rate of agonist-induced GIRK conductance was receptor selective and dependent on receptor number. The results indicate that opioid receptor desensitization reduced the number of functional receptor and that the remaining active receptors have a reduced agonist affinity.

An in silico pharmacokinetic/pharmacodynamic model of agonist self‐administration behavior in rats (1053.8)

Rats trained to maintain self‐administration of dopamine (DA) receptor agonists do so with mathematically predictable regularity. The goal of this research is to model a proposed receptor‐based mechanism underlying the satiety threshold model of maintained self‐administration. MATLAB Simbiology was used to construct a multi‐compartment pharmacokinetic/pharmacodynamic model with a receptor population that exhibits mutually exclusive binding with either an agonist or a competitive antagonist according to the law of mass action. This model also incorporates drug distribution and elimination rate constants. Self‐administration events are programmed to occur only if the amount of agonist‐receptor complexes is at or below a threshold above which no further self‐administration events occur. Simulations show that the inter‐event interval is proportional to the unit dose, competitive antagonists decrease the inter‐event interval until they are eliminated from the system, and reducing the population of receptors via modeling irreversible antagonism also decreases the inter‐event interval. These simulations are consistent with maintained self‐administration behavior in rats, and suggest that the proposed receptor‐based mechanism is a viable explanation for this behavior. This model provides a useful tool for investigating the potential of antagonist‐based pharmacotherapies for drug addiction.Grant Funding Source: NIH Grant DP1DA031386

Irreversible antagonism of receptors accelerates rat cocaine self‐administration behavior (848.4)

A pharmacological model of self‐administration can be explained by the equation T=ln(1+Du/Dst)/k, where T=time between self‐administrations, Du=unit dose, Dst=satiety threshold, and k=first order elimination rate constant, predicts that increasing the pharmacodynamic parameter Dst accelerates self‐administration. Competitive dopamine receptor antagonists temporarily increase cocaine intake implicating dopamine receptor fractional occupancy by dopamine as a mediator of the behavior. Competitive antagonists do not alter the number of receptors, so they cannot be used to study spare receptors or to differentiate between fractional occupancy of receptors and absolute numbers of receptors occupied. Rats that reliably self‐administered cocaine were injected with the non‐specific irreversible antagonist, N‐Ethoxycarbonyl‐2‐ethoxy‐1,2‐dihydroquinoline (1mg/kg i.v.). Attempts were made every 8 hours to reinstate self‐administration and once reinstated self‐administration was regular but was initially 2‐fold higher than baseline with a concomitant increase in the satiety threshold. Over 6 days the rate slowly declined and eventually returned to baseline presumably due to receptor repopulation. This indicates that satiety threshold corresponds to a specific number of receptors occupied. Changes in the number of dopamine receptors are key regulators of addictive behavior.Grant Funding Source: NIH Grant DP1DA031386