bluuebunny/crossref_metadata_2025_split huggingface dataset:
Antinociceptive effects of synthetic tetracycline compound and influence of Cx3cr1 in inflammatory pain
Authors: Guindon Josee , Bezboruah Christian , Blanton Henry , Sherfey Caitlyn , Marquardt Patrick C , Martinez Joseph M , Bergeson Susan E 
Published: November 2023

Tetracycline compounds are broad‐spectrum antibiotics used against different bacterial infectious diseases. Synthetic tetracycline compounds have been developed and are able to reduce infection as well as inflammation. However, the effect on alleviation of pain, and more specifically reduction of inflammatory pain, has not been investigated. The goal of the study is to evaluate the effect of synthetic tetracycline compounds using an inflammatory pain model and mechanical and cold allodynia responses. The formalin test (10 μl at 2.5 % intraplantar) was use to evaluate pain threshold differences between male wild‐type C57BL/6 mice and male GFP Cx3cr1 +/− mice. First, our preliminary data demonstrate that male GFP Cx3cr1 +/− mice show lower pain threshold in both (acute and inflammatory) phases of the formalin test. Second, we evaluated the effect of a synthetic tetracycline compound on inflammatory pain and on mechanical and cold allodynia. Our data indicate that mechanical and cold allodynia remains unchanged after administration of a synthetic tetracycline compound meaning values are similar to baseline levels. However, synthetic tetracycline compound lower pain threshold in both phases of the formalin test from 4 hours up to 20 hours after its administration and showed antinociceptive effects to mechanical and cold allodynia following the formalin test (60 minutes after injection of formalin). Moreover, mechanical and cold allodynia responses return to baseline levels 2 hours after the formalin test. These results demonstrate potential antinociceptive properties of synthetic tetracycline compound with long lasting effect up to 20 hours after its administration. Further studies are needed to investigate the mechanism underlying this antinociceptive effect of synthetic tetracycline compounds. We will also evaluate potential stronger antinociceptive effect of the synthetic tetracycline compound in Cx3cr1 +/− male mice and evaluate if there is any gender specific differences. Antinociceptive properties of synthetic tetracycline compound could be a great alternative avenue to relieve inflammatory pain and improve alleviation of pain in patients.Support or Funding InformationFunded by grant from NIAAA 021142 (SEB) and funded by Texas Tech University Health Sciences Center School of Medicine grant 121035 (JG).
Lysergic Acid Diethylamide (LSD) Modulates Circadian Activity Behavior And The Expression Level of Circadian Genes in Drosophila melanogaster in Both Sexes
Authors: Sherman Kelly Jean , Nichols Charles D. 
Published: November 2023

Circadian rhythms modulate biochemical and molecular pathways in living systems. In humans an endogenous clock mechanism is synchronized by a daily light‐dark cycle: sun up and sun down. These external cues from the environment regulate the core‐clock through auto‐regulating circadian genes’ expression levels. These oscillating gene products modulate physiological, biochemical, and molecular pathways that maintain homeostasis of cells and the overall homeostasis of a living organism. Drosophila and other model organisms are utilized to study the input pathways that modulate the core clock and its regulation of circadian rhythms.Our laboratory has previously used serotonin agonists to study behaviors in the fly, but the role of serotonin and its receptors in circadian behaviors have not been studied extensively. Therefore to study the impact of serotonin receptor linked pathways on circadian gene expression, we investigated the effects of the pharmacological agent lysergic acid diethylamide (LSD), a mixed serotonin receptor agonist, on circadian activity in the fly. In humans, studies have shown that LSD affects the sleep/wake circadian rhythms, but the mechanism is not known. Although, LSD has not been used to study circadian activity rhythms in the fly, previous work in the laboratory demonstrated that serotonin and its receptors play a role in modulating circadian related activity in the fly, We therefore hypothesize that LSD will alter clock gene expression levels and disrupt circadian activity in Drosophila.The Drosophila Activity Monitor (DAM) was used to record circadian activity in entrain (light/dark) and free run (dark/dark) environments. The Capillary Feeding (CAFE) assay was simultaneously used to measure food/drug consumption. Five concentrations (0, 0.01, 0.1, 1.0, 3.0, and 10.0 mM) of LSD were administered to the flies for seven days in a light/dark environment, during which time the flies were monitored for activity levels. Our data show that LSD affects circadian activity in the fly in a dose‐dependent manner in both males and females, with a more robust effect in females. Changes were measured in the RNA levels of circadian and P450 genes in the fly heads between LSD‐treated and control flies. In free run experiments three doses of LSD (0, 1.0 and 3.0mM) were tested, and these data show that LSD also disrupts the endogenous circadian rhythm in a dose dependent manner in both males and females. These behavioral experiments and molecular studies suggest that LSD affects circadian activity, potentially through modulating expression of circadian genes downstream of serotonin receptor activation.
Structural Basis for Allosteric Coupling Between G Protein and the Agonist‐Binding Pocket in GPCRs
Authors: Mahoney Jacob , DeVree Brian , Velez‐Ruiz Gisselle , Rasmussen Soren , Kuszak Adam , Edwald Elin , Manglik Aashish , Masureel Matthieu , Du Yang , Matt Rachel , Pardon Els , Steyaert Jan , Kobilka Brian , Sunahara Roger 
Published: November 2023

G protein‐coupled receptors (GPCRs) translate extracellular ligand binding into intracellular signaling responses via allosteric communication between two distant binding sites. For many GPCRs, allosteric communication between agonists and G proteins has been observed in the form of G protein‐mediated enhancement of agonist affinity. However, the mechanism by which G proteins allosterically regulate a receptor's orthosteric ligand binding site has remained unclear. Based on recent crystal structures of the β2‐adrenergic receptor (β2AR), we hypothesized that stabilization of an active receptor conformation by nucleotide‐free G protein leads to closing of the receptor's orthosteric site around a bound agonist, thus slowing agonist dissociation and enhancing affinity. Using purified receptors reconstituted into high‐density lipoprotein (rHDL) particles, we performed equilibrium and kinetic radioligand binding assays to characterize the allosteric effects of G protein and G protein‐mimetic nanobodies on the ligand‐binding properties of multiple GPCRs. We also investigated the contribution of individual residues within β2AR in mediating high‐affinity agonist binding. When β2AR was bound to nucleotide‐free Gs heterotrimer, we observed slowed association of the antagonist [3H]DHAP, which was reversible by GDP or GTPγS. A Gs‐mimetic nanobody (Nb80), like the nucleotide‐free G protein, slowed [3H]DHAP association but also slowed [3H]DHAP dissociation. However, this effect was not antagonist‐specific, as Nb80 also inhibited association of the full agonist [3H]formoterol, the partial agonist [3H]CGP‐12177, and the inverse agonist [3H]carvedilol. Mutation of tyrosine 308 to alanine in β2AR abolished the rate‐slowing effects of Nb80 on antagonist and agonist association. Together, these data suggest that transit into or out of the orthosteric site in the active β2AR conformation (stabilized by either Gs or Nb80) is restricted due to closing of the orthosteric site, and that Tyr308 of β2AR is an important residue for controlling access to the orthosteric site. Interestingly, the M2 muscarinic acetylcholine receptor and mu opioid receptor behaved similarly to β2AR when bound to nucleotide‐free G protein or G protein‐mimetic nanobodies, suggesting that G protein‐mediated closing of the orthosteric site may extend to GPCRs beyond β2AR and the amine receptor family.Support or Funding InformationThis work was supported by AHA 13PRE17110027 and NIH GM083118.
LPA1 Receptor Antagonists Exhibit Signaling Bias and Differential Efficacy in Models of Renal Fibrosis
Authors: Hazelwood Lisa A , Leys Laura , Lundgaard Greta , Putman C. Brent , McGaraughty Steve , Clapham Bruce , DeGoey David , Lee Lance 
Published: November 2023

The endogenous phospholipid, lysophosphatidic acid (LPA), is known to mediate an array of physiologic processes, including proliferation and cell migration. In addition, LPA signaling is implicated in the pathophysiology of fibrotic disease and evokes a pro‐inflammatory response. The various effects of LPA are mediated by interaction with one of six cognate receptors, termed LPA1–6. In particular, many of the pro‐fibrotic and pro‐inflammatory effects of LPA are attributed to interaction with the LPA1 receptor. LPA1 receptor stimulation promotes signaling through Gq, Gi/o, G12/13 and arrestin. Notably, while LPA species 18:1 is equipotent at evoking each of these downstream signals, we identified antagonists that exhibit biased inhibition of the various pathways. To better understand the molecular pharmacology of LPA signaling in renal fibrosis and inflammation, we studied the signaling pathways activated by 18:1 LPA in various primary human renal cell types, including proximal tubule epithelial cells and renal fibroblasts. These studies enabled mapping of the complex LPA signaling network that drives key pro‐fibrotic and pro‐inflammatory endpoints. Finally, we will present efficacy data comparing full antagonists against antagonists with various biased profiles in the mouse UUO model of renal fibrosis. In total, these data demonstrate the complexity of the LPA‐LPA1 signaling events that drive renal fibrosis, and highlight the requisite antagonist profiles to reverse renal fibrosis endpoints in vivo.Disclosures: All authors are employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication.Support or Funding InformationAll research was funded by AbbVie Inc.
Efficacy at the Mu Opioid Receptor: Insights from Orthosteric and Allosteric Ligands
Authors: Livingston Kathryn E , Mahoney Jacob , Manglik Aashish , Kobilka Brian , Sunahara Roger , Traynor John R 
Published: November 2023

The mu opioid receptor (MOPr) represents one of the most pharmacologically targeted G protein‐coupled receptors (GPCRs) worldwide. Activation by an orthosteric agonist like morphine causes robust pain relief but also results in unwanted effects including respiratory depression, constipation, and addiction liability. However, these actions have different agonist efficacy requirements. Consequently an understanding of the factors governing efficacy is required. In addition, we have recently described a series of positive allosteric modulators (PAMs), exemplified by the thiazolidine BMS‐986122 that control the affinity and/or efficacy of orthosteric MOPr ligands. Here we report that BMS‐986187, a delta opioid receptor (DOPr) PAM, although structurally very distinct from BMS‐986122 acts as a MOPr‐PAM with increased allosteric efficacy compared to BMS‐986122, but with the same probe dependence and the same mechanism of action: by allosterically disrupting Na+ binding. Furthermore, competition experiments suggest that BMS‐986187 binds to the same site as BMS‐986122 indicating a somewhat conserved allosteric binding site on MOPr and DOPr. Utilizing purified MOPr reconstituted into high density lipoprotein (HDL) particles we have studied binding of the MOPr state‐sensitive sensor nanobody39 (Nb39) by interferometry to monitor formation of MOPr active state. Differences in orthosteric ligand efficacy were seen to correlate with different kinetics of Nb39 association and dissociation. The PAMs alone enhanced the on‐rates of Nb39 but differences were seen reflective of distinct allosteric efficacies. Finally, we show that cooperativity between agonist and BMS‐986187 is unchanged in the absence or presence of G proteins suggesting that the allosteric modulators alone are able to stabilize of an active state of MOPr.Support or Funding InformationFunded by DA039997.
Investigation of a cyanobacterial collection from Curaçao for CNS activity
Authors: Hilton Edward , Staub Corinne , Kolber Benedict , Tidgewell Kevin 
Published: November 2023

Cyanobacteria are photosynthetic prokaryotes found in diverse terrestrial and aquatic habitats around the world. Secondary metabolites produced by marine cyanobacteria are known to exhibit pharmacological activity and resemble endogenous ligands to mammalian receptors1. Our lab is interested in discovering novel compounds from marine cyanobacteria that have activity at targets involved in CNS disorders. This project focuses on the serotonin and peripheral benzodiazepine receptors, which are involved in pain, depression, and anxiety in the central nervous system. A marine cyanobacterium, field classified as a paintbrush Symploca, was collected off the coast of Santa Barbara Beach, Curaçao and was extracted and fractioned following standard lab protocol. Extracted fractions of compounds will be sent to the Psychoactive Drug Screening Program run by the Roth Lab at the University of North Carolina Chapel Hill. Fractions are screened against a panel of GPCRs, ion channels, and transporters, which are linked to CNS disorders. Active fractions and compounds will be tested in behavioral assays using mouse models of depression and anxiety, such as the forced swim test and elevated zero maze. Concurrently, active compounds in these fractions will undergo structure elucidation using 1D‐ and 2D‐NMR and LC‐MS. Phylogenetic analysis will be carried out using 16S rRNA sequencing to definitively determine the species of cyanobacteria. Results to date will be presented.Support or Funding InformationR15AT008060 (NIH NCCIH)
Bioengineered miR‐1291prodrugas sensitizing agent for the control of pancreatic carcinoma cell proliferation and tumor growth
Authors: Tu Meijuan , Li Meimei , Yu Aiming 
Published: November 2023

MicroRNAs (miRs or miRNAs) are critical factors in the control of tumor initiation and progression via crosstalk with cancer signaling pathways. Our recent studies have demonstrated that miR‐1291 is able to sensitize pancreatic carcinoma cells to chemotherapy through downregulation of efflux transporter ABCC1/MRP1. We have also developed a novel biological method to produce large quantity ofmiR‐1291 prodrug for research and development. In this study, we aimed to assess the utility of bioengineered miR‐1291 prodrug for improving the efficacy of chemotherapy. Our data showed that miR‐1291 prodrug was readily processed to mature miR‐1291 in human carcinoma PANC‐1 cells and consequently reduced the protein levels of miR‐1291 targeted genes including efflux transporter ABCC1/MRP1. Furthermore, miR‐1291 prodrug significantly enhanced the sensitivity of ABCC1‐overexpressing PANC‐1 cells to doxorubicin, a substrate of ABCC1. In addition, in vivo‐jetPEI formulated miR‐1291 prodrug was distributed to PANC‐1 xenograft tumors in mouse models and enhanced the efficacy of chemotherapy. These results indicate that bioengineered miR‐1291prodrug may be utilized as sensitizing agent for the development of more effective cancer therapy.Support or Funding InformationNational Institutes of Health (1U01CA175315 to A.M.Y.)
A Dock‐Derived PEDF Mimic Targeting Laminin Receptor Downregulates VEGF Receptor
Authors: Umbaugh Charles S , Diaz‐Quiñones Adriana , Figueiredo Marxa 
Published: November 2023

Pigment epithelium derived factor (PEDF) is regarded as an exceptionally potent inhibitor of angiogenesis. Our lab and others have used PEDF as a novel prostate cancer therapy, as PEDF inhibits tumor establishment, vascularization, and alters malignant signaling. Recently, PEDF was identified as a binding partner for the cancer associated protein Laminin Receptor (LAMR1). LAMR1 overexpression is common in a variety of cancers including prostate, breast, and colon. In an attempt to mimic the protein‐protein interaction between PEDF and LAMR1, we used a structure based drug design approach. After screening 14,000 compounds in silico against the crystal structure of LAMR1 and screening 7 compounds in vitro against five cell types, we identified a hit compound with low micromolar anti‐proliferative capacity against the prostate cancer cell line PC3. Our hit compound markedly decreased VEGFR at the mRNA level, a finding that has been associated with PEDF expression. In an unbiased redock, our hit compound bound a laminin binding site on the crystal structure of LAMR1. This binding appears to be facilitated by a hydrogen bond between the compound and HIS169 of LAMR1. Next, we will confirm compound activity via a fluorescent polarization assay and endothelial tube formation.Support or Funding InformationWe acknowledge support from the Basic Medical Sciences Department in the College Of Veterinary Medicine at Purdue University.
Discovering potent PTP4A3 phosphatase small molecule inhibitors for colorectal cancer
Authors: McQueeney Kelley E , Salamoun Joseph M , Handing Kasia , Boulton Adam , Sharlow Elizabeth R , Bushweller John H , Minor Wladek , Wipf Peter , Lazo John S 
Published: November 2023

Although there has been significant progress in understanding colorectal cancer pathogenesis, the 5‐year patient survival rate in the United States of ~60% underlines the need for better treatment options when surgical resection and current therapies are not sufficient. In recent years, protein tyrosine phosphatase 4A3 (PTP4A3) has garnered significant interest and extensive validation as an anticancer target. The Oncomine database documents PTP4A3 is in the top 1% of the most highly expressed colorectal cancer genes with >650 tumor samples. Moreover, it has been noted that increased PTP4A3 expression correlates with increased tumor invasiveness and poor patient prognosis. We synthesized the thienopyridone JMS‐631‐050, which is the most potent published PTP4A3 small molecule inhibitor. Using 6,8‐difluoro‐4‐methylumbelliferyl phosphate as an artificial substrate, we found JMS‐631‐050 non‐competitively inhibited recombinant PTP4A3 in vitro phosphatase activity with an IC50 value of 138 nM. We have generated the first NMR spectra of PTP4A3 in complex with JMS‐631‐050 to more thoroughly characterize the nature of their interaction. Preliminary analysis of NMR data suggests that JMS‐631‐050 binds to a region of the phosphatase outside of the active site, which we hypothesize decreases protein flexibility and proximity of the general acid to the catalytic cysteine. This binding site is also consistent with molecular docking simulation studies. Recently, we discovered a new, more potent thienopyridone analog, JMS‐631‐053, which inhibited recombinant PTP4A3 in vitro phosphatase activity with an IC50 value of 8 nM and showed limited or no inhibition against other phosphatases at concentrations < 1 μM. Both JMS‐631‐050 and JMS‐631‐053, which computationally have drug‐like properties, reduced cellular migration of mouse colorectal tumor cells and prevented the formation of cohesive spheroids in an extracellular matrix phenocopying results observed with PTP4A3 null colorectal cancer cells. JMS‐631‐050 and JMS‐631‐053 should be valuable reagents for further validation of PTP4A3 as a cancer target as well as potential leads for drug discovery.Support or Funding InformationNIH R21CA191944, the Farrow Fellowship, and NCI Cancer Center Support Grant P30 CA44579
Evidence that aspirin blocks platelet‐induced growth, invasive activity and epithelial‐mesenchymal‐transition (EMT) of mouse colon cancer cells
Authors: Lichtenberger Lenard M , Fang Dexing , Dial Elizabeth , Vijayan K. Vinod 
Published: November 2023

BackgroundAspirin consumption is associated with a significant reduction in the incidence and mortality of colorectal cancer (CRC). The evidence was sufficiently compelling to lead a subcommittee of the US Preventive Services Task Force (USPSTF) this year to recommend that individuals aged 50–69 years of age take low‐dose aspirin for chemoprevention of CRC. There is also evidence that platelet number is increased in late‐stage cancer patients and that platelets may stimulate cancer cell growth and EMT.ObjectiveTo determine if aspirin's chemopreventive action against CRC may be due to the drugs ability to inhibit platelet activation (via irreversible COX‐1 acetylation) and thereby block platelet‐ induced effects on CRC cell growth, invasive activity and EMT.MethodsMC‐26 mouse colon cancer cells were incubated in cell culture on Transwell plates in the absence and presence of freshly isolated mouse platelets, at a cell : platelet ratio of 1:400 and 1:800, in medium containing aspirin from 0–1mM. Cell growth, invasive activity (translocation across the Matrigel‐coated membrane insert), and EMT (change in cell shape, and β‐catenin nuclear translocation) were assessed at different periods of culture. Medium Thromboxane B2 (TXB2) conc was measured by ELISA.ResultsCo‐culture of colon cancer cells with platelets induced a significant increase in MC‐26 cell number and invasive activity and an activation of EMT, as the cells transformed from an epithelial to a mesenchymal shape with the generation of intracellular and extracellular microfilaments and an increase in medium TXB2 conc. Aspirin at concentrations between 0.3–1.0 mM blocked platelet activation (as indicated by a >95% reduction in medium TXB2) and all of the above dysplastic platelet‐induced changes in the colon cancer cells in culture.ConclusionAspirin's chemopreventive action against colon cancer may be dependent on the drug's ability to irreversibly block platelet activation and the ability of platelets to promote cancer cell growth, invasion and EMT.Support or Funding InformationSupported by NIH grant R21 CA182798.