bluuebunny/crossref_metadata_2025_split huggingface dataset:
Antagonism of the Rate‐Decreasing Effects of Abused Psychostimulants and Bath Salt Component Mephedrone in Rats
Authors: Saber Iman A. , Walker Ellen A. 
Published: November 2023

To compare the potencies to decrease ongoing behavior among the monoamine alkaloid derivatives of the amphetamine class and the synthetic cathinone mephedrone, 12 food‐restricted male Sprague‐Dawley rats were trained to respond on a fixed ratio 5 schedule of reinforcement for banana‐flavored sucrose pellets. Dose‐response curves for the behavioral effects of d‐amphetamine, methamphetamine, 3,4‐methylenedioxy‐methamphetamine (MDMA), and mephedrone were determined in a multiple‐trial, cumulative dosing procedure. In addition, the rate‐decreasing effects of these psychomotor stimulants were examined in combination with dopamine (DA) antagonists to assess the role of subtypes of DA receptors in the rate‐decreasing effects of the psychostimulants.When multiple doses of DA1 antagonist SCH23390 were combined with a moderate dose of methamphetamine (1.0 mg/kg), an antagonism was observed. However, SCH23390 also appeared to have dose‐dependent, rate‐decreasing properties of its own potentially explaining the observed combined rate‐decreasing effects with the lower and higher doses of the stimulants.The DA2/3 antagonist, sulpiride, slightly attenuated the effects of low and moderate doses of both d‐amphetamine and methamphetamine. When combined with MDMA and mephedrone, attenuation occurred at low and moderate doses, respectfully.These results support previous work demonstrating the similarities and differences between these commonly abused CNS drugs in respects to dopamine activation. Furthermore, this dopaminergic comparison may apply to novel substituted amphetamines and provide insight for mechanisms underlying the pharmacological effects of novel cathinone derivatives such as mephedrone.
Effects of Intermittent or Ad Libitum Consumption of High‐Fat Diet on the Reinforcing Effects of Cocaine in Male Rats
Authors: Collins Gregory T , France Charles P 
Published: November 2023

Drug abuse and obesity are serious public health problems. Dopamine systems play a central role in mediating the reinforcing effects of drugs and food, and the prolonged use of drugs or consumption of foods high in fat is known to alter the function and/or sensitivity of neurotransmitter systems, including dopamine. The current studies evaluated the impact of ad libitum or intermittent (1‐hr per day; Mon, Wed, Fri) consumption of the high‐fat diet on the acquisition of cocaine self‐administration during late adolescence (PND 52–62), relative to rats that consumed a standard diet (n=10–12 male rats per condition). During adulthood (PND ≥70), dose‐response curves for cocaine self‐administration were obtained under both fixed ratio (FR) 5 and progressive ratio (PR) schedules of reinforcement. Although the rate of acquisition was comparable across the three dietary conditions, high levels of responding (≥65 infusions of 0.032 mg/kg cocaine per session) were observed more frequently (85%) in rats with intermittent access to the high‐fat food compared with rats eating only the standard (60%) or high‐fat diets (20%). Similarly, when dose‐response curves for cocaine self‐administration were evaluated under an FR 5 schedule of reinforcement, intermittent consumption of the high‐fat diet shifted the dose‐response curve leftward and/or upward relative to the dose‐response curve obtained in rats that consumed the standard diet; ad libitum consumption of the high‐fat diet did not alter the cocaine dose‐response curve. Although dose‐response curves obtained when cocaine was self‐administered under a PR schedule did not differ among the three groups of rats, a subset of rats from the intermittent high‐fat condition completed significantly more ratios than all other rats. In addition to self‐administering more cocaine under the FR 5 schedule and completing larger ratios to obtain cocaine under the PR schedule, this subset of rats also consistently consumed more high‐fat food during the 1‐hr access period than other rats from the intermittent high‐fat condition. Although it is unclear what factors account for these differential levels/patterns for consumption, the current results provide additional evidence that the abuse‐related effects of drugs, such as cocaine, can be influenced by dietary factors and suggest that patterns of food consumption (i.e., intermittent versus ad libitum) might have a greater impact than the type of foods that are consumed on vulnerability to drug abuse.Support or Funding InformationCPF is supported by a NIDA Senior Scientist Award (K05 DA017918).
Characterization of Mephedrone and its Bioactive Metabolites
Authors: Mayer Felix P , Partilla John S , Wimmer Laurin , Seddik Amir , Burchardt Nadine , Schmid Diethart , Bulling Simon , Ecker Gerhard F , Mihovilovic Marko D , Baumann Michael H , Sitte Harald H 
Published: November 2023

The psychostimulant 4‐methyl‐N‐methylcathinone (mephedrone, MEPH) is a major representative of former legal highs, which were sold as “bath salts”. Despite the ban of MEPH in 2011, the drug is still widely abused for recreational purposes and as doping agent. MEPH manipulates monoaminergic neurotransmission in an amphetamine‐like fashion. Therefore, MEPH acts as a substrate of the high‐affinity monoamine transporters (MATs) for dopamine (DAT), serotonin (SERT) and norepinephrine (NET) and triggers transporter‐mediated release of neurotransmitters. Interestingly, drug abuser reports state that the action of MEPH resembles the action of 3,4‐methylenedioxymethamphetamine (“ecstasy”, MDMA). However, MEPH acts as non‐selective substrate at MATs whereas MDMA clearly favors SERT over DAT and NET. We sought to test the phase‐1 metabolites of MEPH for their activities at MATs; which may be responsible for the observed impact on serotonergic neurotransmission.Radiotracer‐flux experiments were applied to determine the interactions between MATs and the tested metabolites in vitro and ex vivo. Single‐cell FRET measurements were performed to monitor test‐drug induced conformational changes in MATs in real‐time. Moreover, we assessed locomotion and stereotypy in vivo in rats and determined the extracellular concentrations of dopamine and serotonin by microdialysis in nucleus accumbens.The phase 1 metabolites were found to inhibit uptake mediated by MATs with appreciable affinities in the low micromolar range and induced efflux in a dose‐dependent manner. Real‐time FRET measurements revealed an amphetamine‐like mode of action at MATs. Consistent with in vitro findings, the test drugs mimicked the parental drug in vivo with pronounced effects on extracellular serotonin.The fact that MDMA‐like actions have been attributed to MEPH appeared to be inconsistent with its pharmacological profile. Our data unraveled the mode of action of MEPH and its phase‐1 metabolites on a molecular level and indicate that the metabolites essentially contribute to the action of MEPH.Support or Funding InformationAustrian Research Fund/FWF grants F3506 and W1232 to HHS Intramural Research Program of the NIDA, NIH, grant DA000523‐07 to MHB FWF‐DK MolTag, FWF W1232 to MDMAustrian Research Fund/FWF grants F3502 and W1232 to GFE FPM is a recipient of a DOC‐fellowship of the Austrian Academy of Sciences
Differential Antagonism and Nicotine‐Induced Tolerance/Cross‐Tolerance Among nAChR Agonists in Mice
Authors: de Moura Fernando Barreto , McMahon Lance Richard 
Published: November 2023

β2* nAChRs mediate the behavioral effects of nicotine; the extent to which β2* nAChRs also mediate the behavioral effects of other nAChR agonists including FDA approved smoking cessations aids (e.g., varenicline) has not been established. The goal of this study was to identify differences in nAChR mechanism(s) underlying the behavioral effects of nicotine, epibatidine, varenicline, and cytisine in male C57Bl/6J mice (n=7). Mice responded under an FR20 schedule of liquid food delivery; rectal temperature was assessed before and after operant conditioning sessions. The effects of nAChR agonists were tested alone, in combination with mecamylamine or DHβE, and after daily nicotine treatment consisting of 3 doses per day, 1.78 mg/kg per dose, 90 min apart. Nicotine, epibatidine, varenicline, and cytisine dose‐dependently decreased rate of FR responding; the respective ED50 values were 0.44 mg/kg, 0.0027 mg/kg, 2.8 mg/kg, and 4.0 mg/kg. Nicotine, epibatidine, varenicline, and cytisine dose‐dependently decreased rectal temperature; respective decreases in rectal temperature at the largest doses studied were −5.9°C, −3.4°C, −3.5°C, and −3.4°C. Mecamylamine (1 mg/kg) significantly antagonized the rate‐decreasing and hypothermic effects of each agonist. DHβE (3.2 mg/kg) antagonized the rate‐decreasing and hypothermic effects of nicotine and epibatidine, as evidenced by a maximum 3.5‐fold rightward shift in the nicotine and epibatidine dose‐response functions. In contrast, DHβE did not antagonize the effects of varenicline and cytisine. Daily nicotine treatment produced significant rightward shifts in the nicotine dose‐response functions (i.e., tolerance) of 4.1‐fold for rate‐decreasing effects and 4.0‐fold for hypothermic effects, as well as significant rightward shifts in the epibatidine dose‐response functions (i.e., cross‐tolerance) of 2.2‐ and 2.9‐fold, respectively. In contrast, daily nicotine treatment did not significantly modify the potency of varenicline and cytisine to produce either rate‐decreasing or hypothermic effects. To the extent that DHβE is a selective β2* nAChR antagonist, these results suggest that β2* nAChRs mediate the rate‐decreasing and hypothermic effects of nicotine and epibatidine, whereas non‐β2*, mecamylamine‐sensitive nAChRs mediate the effects of varenicline and cytisine. Chronic nicotine treatment appears to decrease the responsiveness of β2* nAChRs, but not the responsiveness of the non‐β2*, mecamylamine‐sensitive nAChRs that mediate the effects of varenicline and cytisine. These results suggest that nAChRs mediating the effects of varenicline and cytisine are different from the nAChRs mediating the effects of nicotine and epibatidine.Support or Funding InformationSupported by USPHS grant DA25267
Identification of LMAN1 as a Druggable Cellular Target for Reduced Secretion of Matrix Metalloproteinase‐9
Authors: Duellman Tyler , Burnett Jack , Yang Jay 
Published: November 2023

Matrix metalloproteinase‐9 (MMP‐9) is a secreted endopeptidase playing a major role in shaping the extracellular environment. Elevated levels of secreted MMP‐9 have been implicated in the pathogenesis of a number of diseases and thus a detailed understanding of the secretory mechanism of this enzyme could help identify novel cellular targets to prevent or reverse disease conditions that result from excess levels of secreted MMP‐9. Previous reports have characterized some of the early secretory events of MMP‐9 showing that this glycoprotein follows the canonical secretory pathway entering the quality control folding cycle in the endoplasmic reticulum (ER) before transport of the properly folded protein to the Golgi apparatus and beyond for secretion. Despite this knowledge there is limited information about the other secretory events including the mechanism by which MMP‐9 exits the ER. CRISPR‐mediated knockout of the ER resident carrier protein LMAN1 (Lectin, Mannose‐Binding 1) confirm its functional role in the efficient secretion of MMP‐9. Through a complementation assay we determined that LMAN1, a well‐studied lectin‐carrier protein, interacts with the secretion‐competent fully N‐glycosylated MMP‐9 in the ER. Our study suggests LMAN1 selectively interacts with the properly folded, fully N‐glycosylated MMP‐9 within the ER and shuttles it to the ER‐Golgi Intermediate Compartment (ERGIC). This study has identified a novel cellular target in LMAN1 that can be therapeutically targeted for inhibition of MMP‐9 secretion. Importantly the MMP‐9: LMAN1 complementation assay provides a valuable tool for the development of a fluorescence‐based high‐throughput drug screening assay aimed at finding small molecules capable of interfering with the LMAN1: MMP‐9 interaction and thus reducing the secretion of MMP‐9.Support or Funding InformationThis study was supported by the Clinical Translational Cardiovascular Training Grant T32 HL07936‐12 (TD), Bamforth Endowment Fund (JY) from the Department of Anesthesiology, UW Madison, and NIH RO1 GM107054 and GM105665 (JY).
Small Molecule Inhibitors of Constitutively Active Gsα Exert Varying Effects on Receptor‐Mediated Signaling
Authors: Rylaarsdam Robin Pals , Carroll Julie , Poch Mark , Schumacher Morgan 
Published: November 2023

McCune‐Albright Syndrome is a rare genetic disorder caused by mosaicism for a constitutively active allele of Gsα. Patients exhibit a broad range of hyperendcrinopathies, skin hyperpigmentation, and polyostotic fibrous dysplasia of bone. The bone disease is particularly difficult to manage clinically. We hypothesized that designing a molecule that preferentially binds to the mutated form of Gsα could provide the starting point for drug development and better therapy for the bone disease. Previous work in our lab identified the region near R231 as a potential druggable target site on the mutated version of the protein. In silico docking analysis provided a slate of candidate molecules to test for inhibition of the MAS protein's constitutive activation of adenylyl cyclase. Two molecules, 6‐oxo‐N‐{[2‐(pyrrolidin‐1‐yl)pyridin‐4‐yl]methyl}‐1,6‐dihydropyridazine‐3‐carboxamide and N‐{[3‐(3‐methylphenyl)phenyl]methyl}‐6‐oxo‐1,6‐dihydropyridazine‐3‐carboxamide were able to significantly and in a dose‐dependent manner reduce cAMP levels in HEK cells transiently transfected with the constitutively active Gs‐R201H allele.. Neither of these molecules significantly reduced basal cAMP levels in cells transfected with wild‐type Gs constructs. Cellular responses to receptor‐mediated adenylyl cyclase activity were unaffected by the first molecule, while the second drug reduced the maximum cAMP response to luteinizing hormone receptor activation but did not affect the EC50 of the response.Support or Funding InformationThis work was supported by NIH grant 1R15ED020190‐01 and the Scholl Foundation Endowed Chair in Natural Sciences.
A High Throughput Screening Campaign for Inhibitors of RGS6 and RGS17
Authors: Bodle Christopher , Hayes Michael , Wu Meng , Fisher Rory , Roman David 
Published: November 2023

G Protein Coupled Receptors (GPCRs) are highly targeted proteins for drug discovery, with over 50% of all drugs on the market targeting these receptors. Regulator of G Protein Signaling (RGS) Proteins regulate the signaling cascade of GPCRs by accelerating the intrinsic GTPase activity of G protein alpha subunits and returning the heterotrimeric G protein to its inactive state. Recently RGS proteins have been implicated as being important in a variety of disease states. RGS17 has been identified as being inappropriately upregulated in both lung and prostate cancer. Knockdown of RGS17 resulted in decreased cell proliferation in vitro, and reduced tumor mass and volume when compared to control when cell lines were injected subcutaneously in nude mice. RGS6 has been shown to play an essential role in doxorubicin mediated cardio toxicity, has been shown to have an important role in anxiolytic and depression phenotypes in mice, and has been implicated as a critical mediator of behavioral and pathological responses to alcohol. Here we present the screening of two RGS proteins (RGS6 and RGS17) using Alpha Screen technology against the NCI Next Diversity Library. Assay robustness was determined for both proteins, with RGS6 Z‐factors above 0.7 and RGS17 Z‐factors above 0.6. Approximately 20,000 compounds were screened against RGS6 resulting in identification of 20 hit compounds, and roughly 60,000 compounds (including the 20K also screened against RGS6) were screened against RGS17 resulting in the identification of 131 hit compounds. Tanimoto similarity analysis was performed on all hits to allow for small scale SAR analysis of confirmed lead compounds. Follow up experiments are under way including dose response analysis and structure based analysis utilizing NMR and molecular modeling.Support or Funding InformationSupported in part by RO1CA160470, in part by OVPRED internal funding initiative RAF‐DLR and in part by a Pre‐Doctoral Fellowship from the American Foundation of Pharmaceutical Education (AFPE)
RGS2 Modulation of Dopamine D2 Receptor Internalization and Recycling in Neuroblastoma Cells
Authors: Luessen Deborah Joyce , Hinshaw Tyler , Sun Haiguo , Marrs Glen , Chen Rong 
Published: November 2023

IntroductionAltered dopamine (DA) D2 receptor (D2R) expression and function are commonly implicated in the pathophysiology of drug addiction, Parkinson's disease and schizophrenia. Internalization and recycling of D2R play a key role in the regulation of D2R activity including desensitization and resensitization. Therefore, it is imperative to improve our understanding of the molecular mechanisms underlying D2R trafficking. Regulator of G‐protein signaling (RGS) proteins are known to modulate GPCR activity by terminating G protein signaling through acceleration of GTP hydrolysis. However, recent findings highlight non‐canonical roles of RGS proteins in modulation of various aspects of GPCR including trafficking.MethodsIn the present study, we utilized a neuroblastoma (N2A) cell model to assess the role of RGS2 (regulator of G‐protein signaling 2) in the modulation of D2R trafficking and signaling by knocking down RGS2 via siRNA. Immunocytochemistry and confocal microscopy were utilized to study both constitutive and receptor agonist (quinpirole) stimulated D2R internalization and recycling. The time courses of constitutive and quinpirole (1μM) stimulated D2R internalization and recycling were evaluated at numerous time points ranging from 0 to 90 minutes.ResultsOur data indicated that RGS2 knockdown increased constitutive D2R internalization and recycling when compared to the control cells treated with scrambled siRNA. When D2R was activated by the agonist quinpirole (1 μM), there was a reduction in surface D2R level in controls cells and an increase in surface D2R level in RGS2 knockdown cells. The reduced surface D2R level in control cells induced by quinpirole treatment was due to the accelerated D2R internalization which was both clathrin and dynamin‐dependent. Both dynasore, a dynamin inhibitor, and concanavalin A, a clathrin inhibitor, blocked quinpirole‐induced D2R internalization in control cells. In contrast, RGS2 knockdown significantly attenuated quinpirole‐induced D2R internalization. Furthermore, the D2R recycling rate following quinpirole treatment in RGS2 knockdown cells was 2.5 times greater than that in the control cells. The effect of quinpirole on D2R surface trafficking was G protein dependent because pertussis toxin treatment abolished induced D2R trafficking. We also examined whether the downstream signaling molecule pERK mediates D2R internalization and recycling in the RGS2 knockdown cells. These results add a novel insight to the role of RGS2 in modulation of D2R internalization and recycling and may highlight the novel mechanistic actions of RGS2 in mediation of D2R trafficking.Support or Funding InformationThis work is supported by the National Institute On Alcohol Abuse And Alcoholism of the National Institutes of Health T32AA007565 training grant, DA006634 and a pilot grant from Center for Molecular Communication and Signaling at Wake Forest University.
Covalent Inhibition Of Acetylcholinesterase Affects Quaternary Structure As Revealed By Small Angle X‐Ray Scattering
Authors: Blumenthal Donald K , Cheng Xiaolin , Fajer Mikolai , Gerlits Oksana , Kovalevsky Andrey , Taylor Palmer , Radić Zoran 
Published: November 2023

Small angle X‐ray scattering (SAXS) experiments were used to assess the influence of reversible and non‐reversible ligands on the solution structure of human acetylcholinesterase (hAChE; EC 3.1.1.7). SAXS measurements were performed using highly purified, monomeric human AChE in both non‐inhibited and organophosphate (OP) covalently inhibited states in 50 mM Tris/HCl, 100 mM NaCl, pH 7.4 at 22 °C. SAXS analysis of four OP‐hAChE conjugates (formed by VX, sarin and cyclosarin surrogates and paraoxon), as well as several reversible inhibitor‐hAChE complexes, reveals significant changes in the pair‐distance distribution function (P(r) vs r) curves in the 60 – 80 Å region for paraoxon and RP‐sarin covalent conjugates, but not for other covalent OP conjugates or reversible inhibitors of hAChE. Incubation of OP‐hAChE conjugates with the oxime reactivator antidotes 2PAM, RS‐194B, or HI‐6 resulted in reversal of the P(r) vs r profiles for paraoxon and RP‐sarin back to P(r) profiles that were similar to non‐inhibited hAChE. Analysis of the OP‐induced solution structural changes using ClusPro and FoXS indicate that “monomeric” hAChE actually consists of a reversible dimer‐monomer equilibrium ensemble that is shifted towards the monomer in paraoxon‐ and RP‐sarin‐conjugated complexes. This is the first observation of a quaternary structure modification in an AChE that results from covalent or non‐covalent binding in the AChE active center gorge. All‐atom molecular dynamics (MD) simulations at 285 K and 1 bar pressure are being used to interpret the OP‐induced conformational changes detected by SAXS. The OP‐induced changes in hAChE dimerization appear to result from distortion of the acyl pocket of the catalytic gorge that triggers changes in the dimer interface on the surface of hAChE. Analysis of OP‐induced conformational changes of hAChE by SAXS, MD simulation, and other structural methods will be used to aid the design of novel, more efficient oxime antidotes for treatment of OP intoxication.Support or Funding InformationThis work was supported by the CounterACT Program, National Institutes of Health Office of the Director, Grant 1U01NS083451 from NINDS.
Spontaneous Reactivation of Planarian Acetylcholinesterase after Inhibition by Organophosphorate Pesticides, Diazinon and Chlorpyrifos
Authors: Hirokawa Hideto , Collins Eva‐Maria , Hagstrom Danielle , Radic Zoran , Zhang Limin , Taylor Palmer 
Published: November 2023

Owing to its capacity to regenerate after amputation and a dominant cholinergic influence in motor activity, we have been studying the response of the freshwater planarian, Dugesia japonica, to cholinergic inhibitors such as organophosphate pesticides. In examining cholinesterase (ChE) and other carboxylesterase activities, we find that ~85% is acetylcholinesterase (AChE) activity, with slightly different specificities to the classic reversible inhibitors, such as edrophonium and BW286c51. Of particular interest in this planarian system is the capacity of diethylphosphoryl conjugates formed from Diazinon or other pesticide exposure to reactivate spontaneously, in vitro. A similar behavior has been noted in swine butyrylcholinesterase (BChE) as noted by Brazzolotto and colleagues (Abstracts XII Int. Cholinesterase Meeting), who have suggested that an altered tertiary structure in swine BChE may predispose ChE conjugates to spontaneous reactivation. Our studies show that spontaneous reactivation in planarians can be inhibited by classical cholinesterase inhibitors and by oximes that show limited nucleophilic activity, but can be enhanced by other oximes, particularly the zwitterionic oxime, RS194B. Further, spontaneous reactivation is enhanced at low pH, in contrast to the general base catalysis by oximes on small molecule alkylphosphorate esters and methylphosphonate esters. The observed reactivation kinetics suggest unique kinetic behaviors of H2O and various nucleophiles in the active center gorge of planarian AChE, perhaps dictated by hydrogen bonding and transient electric field influences in the AChE active center gorge.Support or Funding InformationSupported by USPHS grant GM 18360 and NS 058046.