bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of Novel Biased KOR Agonist‐Mediated Cell Signaling
Authors: Ho Jo‐Hao , Lovell Kimberly , Zhou Lei , Frankowski Kevin , Aubé Jeffrey , Bohn Laura
Published: November 2023

Kappa opioid receptors (KORs) are widely expressed in the central nervous system and are involved in the regulation of pain perception. Therefore, KORs have been attractive drug targets for treating pain; however, KOR agonists also produce sedation and dysphoria. Recent evidence suggests that while G protein signaling may promote KOR‐mediated antinociception, βarrestin‐mediated signaling may lead to the dysphoric effects. In an effort to begin to determine whether KOR activation without βarrestin2 recruitment would maintain the therapeutic potency of KOR agonists while avoiding the aversive effects, we evaluated two classes of KOR agonists, triazole analogue and isoquinolinone analogue, both of which potently activate G protein coupling but weakly recruit βarrestin2. Canonically, KOR couples to inhibitory G proteins that suppress adenylyl cyclase. However, the biased KOR agonists have very weak potency for inhibiting adenylyl cyclase in cell‐based signaling assays. Therefore, we hypothesize that these novel KOR agonists preferentially transduce signaling through different types of Gα or Gβγ proteins. In this study, we compare these novel KOR agonists to the reference compound, U69,593, for their abilities to activate three different KOR‐mediated G protein signaling events: adenylyl cyclase activity by cAMP HTRF technology, G protein‐coupled inwardly‐rectifying potassium (GIRK) channel activity by FLIPR potassium technology, and intracellular calcium mobilization by FLIPR calcium technology. These novel KOR agonists display weak potency in regulating adenylyl cyclase activity and stimulating calcium response; however, they potently activate GIRK channels in cell‐based assays. Together, these results suggest that the novel KOR agonists display different signatures in KOR‐mediated G protein signaling. This study provides insights into the novel KOR agonist‐mediated signaling pathways which will inform future behavioral studies.

Support or Funding Information

This work was supported by NIH/NIDA R01 DA031927 to LMB and JA.
Pharmacological Properties of Chromobacterium violaceum Violacein at the Human Serotonin 2C Receptor
Authors: Fears Letimicia , Curtis Mary , Johnson Terrance , Fentress Hugh
Published: November 2023

Serotonin (5‐HT), a neurotransmitter, plays a role in many physiological responses by binding to 16 receptor subtypes. The 5‐HT2C receptor subtype is a 7‐transmembrane, G protein‐coupled receptor (GPCR) that is involved in neuronal excitability, spatial learning, mood, and appetite. The microorganism Chromobacterium violaceum produces a purple pigment, violacein, which can be extracted from liquid cultures. Violacein has antibiotic, antileishmanial, antiviral and antitumoral effects in various cancer cell lines. Violacein is similar in structure to 5‐HT, however, no one has ever reported the activity of violacein at 5‐HT receptors. To investigate the effect of violacein on 5‐HT2C receptor trafficking, Human Embryonic Kidney (HEK) 293 cells expressing fluorescently‐tagged 5‐HT2C receptor were treated with 5‐HT, violacein, water or vehicle, fixed and visualized with fluorescent microscopy. Violacein treatment did not cause receptor internalization. Given our recent discovery that the 5‐HT2C receptor can activate the JAK/STAT pathway, we examined if violacein could impact this activation. HEK 293 cells expressing 5‐HT2C receptor were treated with violacein or pretreated with violacein followed by incubation with 5‐HT. Phosphorylation states of JAK2 and STAT3 were examined by immunoblotting. Preliminary data suggests that violacein may hinder activation of STAT3 but further study is required. We will also investigate the effect of violacein on G‐protein activation by measuring phosphoinositide hydrolysis. Affinity for violacein to the 5‐HT2Creceptor will be investigated by competition binding.

Support or Funding Information

Department of Education Title III HBGI Grant No. P031B141010
Semen Characteristics And Sperm Morphology Of Pistia stratiotes Linn. (Araceae) Protected Male Albino Rats (wistar strain) Exposed To Sodium Arsenite
Authors: Ola‐Davies Olufunke Eunice , Olawuwo Olasunkanmi Stephen , Ajani Olumide Samuel , Oyeyemi Olugbenga Matthew
Published: November 2023

The sperm protective potential of Pistia stratiotes Linn (Aracea) was carried out in arsenic‐exposed rats using twenty‐four male albino rats (225 to 228g) aged between 14 and 16 weeks old. They were grouped into 4 (A–D), each group containing 6 rats. Group A animals were orally treated with 100mg/kg ethanol leaf extract of Pistia stratiotes Linn. daily for 14 days. B (sodium arsenite at 2.5 mg/kg body weight‐ positive control), C (Pistia stratiotes extract for 14 days and single dose of sodium arsenite on the 14th day, D (0.1 ml Propylene glycol‐ negative control/vehicle).

Group B had a significantly lower (P<0.05) percentage sperm motility (26.7±6.67%) while group A had a significantly (P<0.05) higher mean value (63.3±3.33%) when compared across the groups. The sperm motility of rats in group D was significantly higher (P<0.05) than groups B and C. This implies that P. stratiotes extract had no adverse effect on sperm motility. The presence of P. stratiotes with sodium arsenite alleviated its harmful effect on sperm motility. The mean value obtained for sperm viability, semen volume and sperm count followed a similar pattern although, the difference was not significant (P>0.05) for semen volume and the sperm count of rats across the groups. Total sperm abnormality was 10.44 and 14.27% with the (SA) treated group having the highest value when compared with groups A treated with Pistia stratiotes extract and D treated with Propylene, although, the differences were not significant (P>0.05). The study concluded that ethanol leaf extract of Pistia stratiotes has no negative effect on sperm motility, viability and morphology and also protected spermatozoa against arsenic induced reproductive toxicity in wistar strain albino rats. Therefore, it may play an important role in the protection of populations with chronic sodium arsenite exposure.
Neuroprotective activity of Macamides on Manganese Chloride Induced Toxicity in U‐87 MG Glioblastoma Cells
Authors: Gugnani Kuljeet , Bohlke Mark , Pino‐Figueroa Alejandro
Published: November 2023

Lepidium meyenii, known as Maca, is a Peruvian herbaceous biennial plant used by indigenous Andean societies as a source of nourishment and healing. Macamides, a distinct class of secondary metabolites which are structurally similar to the endocannabinoids, have neuroprotective effects. The purpose of this study was to evaluate the neuroprotective effects of some of the most prominent macamides: N‐(3‐methoxybenzyl) oleamide, N‐(3‐methoxybenzyl) linoleamide and N‐(3‐methoxybenzyl) linolenamide, at concentrations of 0.1 to 50uM, on U87MG glioblastoma cells exposed to a neurotoxic concentration (10 μM) of MnCl2. A cell viability assay utilizing 3‐(4, 5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium (MTS) was performed to evaluate the neuroprotective effects. The results demonstrated that the macamides counteracted the toxicity of MnCl2, showing significant increases in cell viability in response to N‐(3‐methoxybenzyl) oleamide and N‐(3‐methoxybenzyl) linoleamide at all concentrations studied (21% and 25% respectively). A dose dependent increase in cell viability was observed for N‐(3‐methoxybenzyl) linolenamide (P≤ 0.001). Moreover, glutathione levels and other markers were measured to evaluate the neuroprotective effects of macamides. Further assays will be performed in order to determine the mechanism of neuroprotective activity demonstrated by these macamides.
The Marine Pharmacology and Pharmaceuticals Pipeline in 2015
Authors: Mayer Alejandro M , Nguyen Michelle , Newman David J , Glaser Keith B
Published: November 2023

In October 2015 there were six FDA‐approved marine‐derived drugs: for cancer, cytarabine (Cytosar‐U®, Depocyt®, FDA‐approved 1969), eribulin mesylate (Halaven®, FDA‐approved 2010), trabectedin (Yondelis®, FDA‐orphan drug approval 2005) being EU‐registered, and brentuximab vedotin (Adcetris®, FDA‐approved 2011); for pain, ziconotide (Prialt®, FDA‐approved 2004); for hypertriglyceridemia, omega‐3‐acid ethyl esters (Lovaza®, FDA‐approved 2004). The clinical marine pharmaceutical pipeline (Mayer et al. TIPS 31:255–265, 2010) consisted of 10 marine‐derived compounds in clinical development in 2010, including three monoclonal antibodies conjugated to monomethyl auristatin E, a synthetic analog of the marine compound dolastatin, in either Phase I, Phase II or Phase III clinical trials. The current clinical marine pharmaceutical pipeline consists of 9 marine‐derived compounds, and at least 16 other auristatin‐containing antibody drug conjugates in clinical development. Updated information on the clinical marine pharmaceutical pipeline is available at http://marinepharmacology.midwestern.edu/clinPipeline.htm. Finally, the global preclinical marine pharmacology pipeline was reviewed in Mayer et al. MARINE DRUGS 11:2510–2573, 2013). In late 2015, both the marine pharmacology preclinical and clinical pharmaceutical pipelines remained very active.

Support or Funding Information

Supported by Midwestern University
EFFECT OF NIMESULIDE ON BLOOD SUGAR LEVEL AND HAEMATOLOGICAL INDICES OF ALBINO RATS
Authors: Agbasi Patrick Ugochukwu , Chukwu Leo Clinton , Unekwe Prince
Published: November 2023

Background

Nimesulide is relatively new drug that belong to the family of non steroidal anti inflammatory drugs (NSAIDs) and is a selective cyclo‐oxygenase 11 inhibitor that is gastro intestinal tract protective as against other non specific NSAIDs cyclo‐oxygenase inhibitors like indomethacin which usually cause stomach ulcer. NSAIDs are used as pain killers against post operative, menstrual, arthritic, cancer pains etc.

Purpose

This study looked at the possible toxic effects of chronic administration of selecctive cyclo‐oxygenase 11 inhibitor like nimesulide on the haematological and fasting blood sugar level of Albino rats. The chronic use of selective cyclooxygenase 11 inhibitors in the treatment of musculo‐skeletal and joint pains of various origins necessitated this research.

Methods

Albino rats aged between four to five weeks were used. The animals were divided into three groups of five rats each. The first group received 5mg/kg/day, the second group also received 7.5mg/kg/day nimesulide respectively while the third received no drug and served as control. The drugs were orally administered for fifty six days during which the animals were given free access to feed and water. At the end of the study, the animals were anesthetized and blood samples were collected for biochemical and hematological parameters while spleen and pancreas were isolated for histological analysis.

Results

It showed that the fasting blood sugar levels were not significantly increased p<0.05 when compared with control. The pancreas histopathology result produced no appreciable pathological changes in the two dose levels when compared with control. Also spleen histopathological result showed mild lymphoid reactive hyperplasia and moderate necrosis. The white blood cell (WBC) indices showed that total WBC count in 5mg/kg group were significantly increased p<0.05 when compared with control while the WBC level in 7.5mg/kg were significantly reduced p>0.05 when compared to both 5mg/kg rats and those of control. We recorded significant increase p<0.05 in lymphocyte level in both 5 and 7.5mg/kg group and significant reduction in mean monocyte level for both 5 and 7.5mg/kg rats when compared to control. The mean red blood cell (RBC) count values showed statistical significant increase P<0.05 in 7.5mg/kg Nimesulide group. Packed cell volume (PCV) values were not significantly increased p<0.05 in 5mg/kg group while the rats in 7.5mg/kg category were not significantly reduced when compared with control. Platelet values were statistically significantly increased p<0.05 in 5mg/kg group while those in 7.5mg/kg category were not significantly decreased p>0.05 when compared with control.

Guiding Principles for the use and care of animals were strictly adhered to in this study.

The Protocol for this research was approved by our institution's ethical committee.

Support or Funding Information

Support/Funding NON
Cholesterol 7α‐hydroxylase (CYP7A1) plays a critical role in preventing hepatic inflammation and fibrosis
Authors: Liu Hailiang , Ferrell Jessica , Boehme Shannon , Chiang John
Published: November 2023

Introduction

Cholesterol 7α‐hydroxylase (CYP7A1) is the rate‐limiting enzyme in the bile acid biosynthetic pathway in the liver and controls cholesterol and bile acid homeostasis. Accumulation of toxic bile acids in liver causes cholestasis and inflammatory liver injury. The mechanism of bile acid homeostasis in hepatic inflammation and fibrosis remains unclear.

Methods

We investigated the roles of CYP7A1 in hepatic inflammation and fibrosis using lipopolysaccharides (LPS)‐induced acute liver injury and methionine/choline deficient (MCD) diet‐induced liver injury models. Cyp7a1−/− mice in pure C57BL6 background, bile acid‐activated nuclear receptor farnesoid X receptor deficient (Fxr−/−) mice, and G protein‐coupled bile acid receptor deficient (Tgr5−/−) mice, and adenovirus‐mediated transduction of Cyp7a1 were used to study the roles of CYP7A1 and bile acid receptors in hepatic inflammation and fibrosis. Real‐time PCR, western blot, immunohistochemistry, luciferase report and chromatin immunoprecipitation (ChIP) assays were used in the study.

Results

Adenovirus‐mediated overexpression of CYP7A1 ameliorated LPS‐induced inflammatory cell infiltration and pro‐inflammatory cytokines production in WT and Tgr5−/−, but not Fxr−/− mice suggesting that FXR played a key role in protection against LPS‐induced hepatic inflammation. Luciferase report assay and real‐time PCR showed that FXR agonists, GW4064 or INT‐747 significantly inhibited NF‐κB activation and pro‐inflammatory cytokine mRNA expression in HepG2 cells and human primary hepatocytes. Mammalian two‐hybrid assay, co‐immunoprecipitation and ChIP assays showed that activation of FXR promotes FXR interaction with p65, which in turn blocked p65 binding to NF‐κB response elements on the TNF‐α and IL‐1β gene promoters. Serum AST and ALT levels were significantly increased in the liver of Cyp7a1−/− mice fed with MCD diet compared with WT mice. Pro‐inflammatory cytokine mRNA expression and immunohistochemistry of α‐SMA showed that Cyp7a1−/− mice fed with MCD diet developed more severe hepatic inflammation and fibrosis than WT mice. Cholesterol is aggravated in Cyp7a1−/− mice fed with MCD diet, and may increase reactive oxidizing species and cell death in the liver.

Conclusion

These data suggest that increased bile acids ameliorated hepatic inflammation through activation of FXR but not TGR5 signaling. On the other hand, reduced bile acid pool increased susceptibility to MCD diet‐induced hepatic inflammation and fibrosis. Maintaining bile acid homeostasis is important in protection against liver injury and non‐alcoholic fatty liver disease.
Effect Of Ethanol On The Metabolism Of Antiretroviral Drugs Elvitegravir And Darunavir In The Presence And Absence Of Their Pharmacoenhancers In Microsomes
Authors: Midde Narasimha M , Rahman Mohammad A , Rathi Chetan , Li Junhao , Meibohm Bernd , Li Weihua , Kumar Santosh
Published: November 2023

Alcohol consumption is highly prevalent in HIV positive individuals (50–70%) compared to the general population (14–24%). This drinking behavior is significantly associated with increased viral replication and decreased response to antiretroviral therapy (ART). Previously, we have shown that ethanol alters ART‐CYP3A4 interactions with protease inhibitors, which is likely to alter their metabolisms. In this study, we characterized Elvitegarvir (EVG), cobicistat (cobi)‐boosted EVG, Darunavir (DRV), and Ritonavir (RTV)‐boosted DRV metabolism in human microsomes followed by ethanol‐EVG/cobi and ethanol‐DRV/RTV interactions with CYP3A4. First, we developed and validated a simple, sensitive, and robust liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method for the quantification of EVG and DRV in the human liver microsomes. The lower limit of quantification for the EVG and DRV were at 0.003 μM (1.34ng/ml) and 0.023 μM (12.61ng/ml), respectively. Time kinetics data showed that 20 mM ethanol decreases the apparent half‐life of EVG metabolism by ~50%. Our substrate kinetic results revealed that ethanol slightly decreases the catalytic efficiency for EVG metabolism. Inhibition studies demonstrated that 20 mM ethanol causes a decrease in the IC50 of EVG. However, these parameters for EVG+cobi combination could not be determined because of strong inhibitory nature of cobi on CYP3A4. Furthermore, although ethanol did not show any appreciable effects on DRV kinetics with or without RTV, it robustly increased the IC50 of DRV in the presence of RTV. Finally, molecular docking studies predicted a shift of EVG or DRV binding parameters to the active site of CYP3A4 in the presence of ethanol. More specifically, the binding orientation of these substrates with CYP3A4 were altered in the presence of ethanol suggesting formation of new metabolite(s), which could be toxic. Taken together, these results suggest that ethanol interacts with microsomal CYP3A4 and alter ART metabolism, and their inhibition to CYP3A4. These findings have clinical implications with respect to adjustments of the regimens of these anti‐HIV drugs in alcohol users.

Support or Funding Information

This research was supported by grants from the National Institute of Health to Santosh Kumar (NIAAA/NIH AA‐022063) and the National Natural Science Foundation of China to Weihua Li (Grant 81373328)
Implications of In Vitro System Conditions for CYP2C9 and CYP3A4: Impact of pH on Clearance and Enzyme Inhibition
Authors: Rougee Luc R.A. , Mohutsky Michael A , Bedwell David W , Ruterbories Kenneth J , Hall Stephen D
Published: November 2023

Common practice in drug discovery is to use buffer systems witha pH of 7.4 for drug metabolism and drug‐drug interaction studies despite evidence that the intracellular pH of hepatocytes is approximately 7.0. Systemp H will govern the ratio of ionized (charged) and non‐ionized (non‐charged)species for ionizable drugs. As a result of the pH gradient between the extracellular plasma and the intracellular cytosol of the hepatocyte, anunbound drug gradient, dependent on the physiochemical properties of the drug exists. An ionization correction factor [Berezhkovskiy (2011)]was proposed to account for this phenomenon when scaling in vitro findings at pH 7.4 to in vivo predictions of hepatic clearance. However, this correction ignores the potential changes in enzyme function at the lower pH environment.

In the current study we evaluated, in human liver microsomes, the impact of buffer pH on the unbound fraction, Km, Vmax and intrinsic clearance (CLint=Vmax/Km) of probe substrates for two CYP enzymes, CYP2C9 (diclofenac and S‐warfarin) and CYP3A4 (midazolam, dextromethorphan, testosterone for CYP3A4) over a pH range of 6.0 to 9.0. Variable changes in the enzyme kinetic factors were observed over the pH range. These changes did not coincide with a particular species, ionizedor non‐ionized, for any of the substrates. When comparing the physiologically relevant pHs (7.0 and 7.4) for CYP2C9, a consistent trend was observed for both substrates, with lower Km, higher Vmax and higher CLint observed at 7.0 compared to 7.4. For the CYP3A4 enzyme, the inverse trend for observed, with higher Km, lower Vmax (Vmax was unchanged for midazolam) and lower CLint observed at 7.0 compared to7.4 for the dextromethorphan and midazolam substrates only. When testosterone was used as the probe substrate, both Km and Vmax were lower at pH 7.0 compared to pH 7.4, resulting in only a slight decrease in CLint at pH 7.0.

Changes in the inhibition parameter, Ki, for the CYP2C9 reversible inhibitors amiodarone, desethylamiodarone, clozapine and nicardipine were consistent between the two probe substrates, with pH 7.0 Ki values consistently higher (less potent) than pH 7.4. Fold changes between pH 7.0 and pH 7.4 varied between the inhibitors and ranged from 1.25 to2.46 for the probe substrate diclofenac and 1.4 to 1.65 for S‐warfarin. These results suggest that changes as a result of pH vary from enzyme, compound and inhibitor combinations. In order to improve predictive performance of hepatic clearance, we recommend that in vitro parameters be determined at pH 7.0 to provide appropriate starting inputs for in vitro to in vivo extrapolations for ionizable drugs.
Identification of Novel Substrates and Substrate‐Dependent Interactions of Human Organic Cation Transporter 1
Authors: Boxberger Kelli H , Hagenbuch Bruno , Lampe Jed N
Published: November 2023

The human organic cation transporter 1 (OCT1) is a polyspecific transporter involved in the uptake of positively‐charged and neutral small molecules in the liver. The clinical relevance of OCT1 has been established for several drugs, including the anti‐diabetic agent metformin. It has been demonstrated that OCT1 can contribute to drug efficacy, as well as to clinically relevant drug‐drug interactions (DDIs). Currently, the FDA recommends that the pharmaceutical industry screen new drug candidates for potential transporter‐associated DDIs using one or two probe substrates. Previous work from our laboratory suggests that inhibition of OCT1 may depend on the transported substrate. To study this further, we employed the competitive counterflow (CCF) efflux assay developed by Harper and Wright (Am J Physiol Renal Physiol. 2013;304(1):F56–67.) which tests a compound's ability to exchange with a known substrate. We have demonstrated that metformin and imatinib do not exchange with the probe substrate 1‐methyl‐4‐phenylpyridinium (MPP+) through OCT1. Because metformin and imatinib are known OCT1 substrates, these results indicate the possibility of different substrate binding domains and/or translocation pathways. Additionally, using the CCF assay, we have identified the negatively‐charged liver function test dye bromosulfophthalein (BSP) as a novel substrate of OCT1. These observed substrate‐dependent interactions may provide important insight into transporter structure and function. Furthermore, substrate‐dependent interactions can have important consequences when trying to identify DDIs for new drug candidates. Because regulatory guidelines only recommend one or two probe substrates to be tested, important DDIs may be missed. Therefore, these results may have important implications for revising guidelines for preclinical studies.

Support or Funding Information

This work was supported by the National Institutes of Health Institute of General Medical Sciences [Grant NIH R01‐GM077335] and [Grant P20‐GM103549‐07], National Center for Research Resources [Grant P20‐RR021940], National Institute of Environmental Health and Sciences [Grant T32‐ES007079], and Kansas IDeA Network of Biomedical Research Excellence [Grant QH846868‐K‐INBRE].
miR‐186 suppresses cell proliferation and anchorage‐independence in a metastatic prostate cancer cell line
Authors: Jones Dominique Z. , Schmidt M. Lee , Hobbing Katharine R. , Clark Geoffrey , Kidd LaCreis R.
Published: November 2023

MicroRNA (miR) dysregulation alters the expression of cancer related genes and contributes to disease states in many cancers. For example, ectopic miR‐186 expression leads to enhanced cell proliferation and migration in pancreatic cancer. However, the role of miR‐186 in prostate cancer (PCa) remains unclear. Previously, we observed significant upregulation of miR‐186‐5p in PCa patient serum (stage III/IV) relative to non‐cancerous controls. Furthermore, miR‐186 was significantly up‐regulated in metastatic PCa (PC‐3) cells compared to normal prostate epithelial cells (RWPE1). We hypothesized miR‐186 inhibition will reduce aggressive PCa in vitro.

Consequently, miR‐186 was transiently and stably inhibited in PC‐3 cells. Cell proliferation and colony formation were evaluated for 7 and 21 days via Trypan Blue exclusion and soft agar assays. Aberrant gene expression was evaluated in transfected cells to identify miR‐186 targets. Candidate miR‐186 targets were selected using published reports on ‘miR‐186 and cancer’, availability of robust antibodies, and statistical filtering (false discovery ≥0.05 and ±1.2 fold change).

Mir‐186 inhibition in PC‐3 cells significantly repressed proliferation and colony formation by 34–64%. Following miR‐186 inhibition in PC‐3 cells, 2,343 identified mRNA targets were differentially expressed compared to scramble controls (p < 0.05). The target list was reduced to 11 up‐regulated candidates. Predicted miR‐186 targets are undergoing validation via qRT‐PCR, western blots, and luciferase reporter assays. In addition, other studies are in progress to evaluate the impact of miR‐186 on cellular migration and invasion. Such efforts may lead to the identification of novel biomarkers to improve detection and clinical management strategies for aggressive PCa.

Support or Funding Information

Research & Fellowship Support: National Cancer Institute Grant R25‐CA‐134283 to DWH; Our Highest Potential Endowment in Cancer Research to LRK
Novel ShetA2 Analog, SL‐01‐18, Triggers Apoptosis and Targets Androgen Receptors in Human Prostate Cancer Cells
Authors: Rajagopalan Vanishree , Nguyen Tran , Naizghi Girmay , Liu Shengquan
Published: November 2023

SHetA2 (NSC 726189), a “Flexible Heteroarotinoid” compound that is presently in preclinical development for cancer prevention through the National Cancer Institute (NCI) Rapid Access to Intervention Development (RAID) program has shown to exert its anti‐cancer activity by regulating apoptosis, cell growth, differentiation and angiogenesis. However, SHetA2 may not be an ideal drug candidate due to the following two limitations: its high lipophilicity (Log P=7.9) and cumbersome six step synthesis. We have tried to address these limitations by designing over ten different analogs of SHetA2 by modifying the thiochromane ring of ShetA2 while keeping the nitrophenyl group and the thiourea linker intact. These analogs were tested for growth inhibition in three human prostate cancer cell‐lines. SL‐01‐18 was found to be the most potent growth inhibitor of prostate cancer cells. Further, SL‐01‐18 inhibited the growth of androgen dependent prostate cancer cell line, LNCaP, while minimally affecting the growth of androgen independent prostate cancer cell lines such as DU145 and PC‐3.

The aim of this project was to further characterize the growth inhibitory effects of SL‐01‐18 on LNCaPs grown in media conditioned for androgen dependent growth. Within 24 hours, Hoechst 33342 live nuclear staining showed chromatin condensation and nuclear fragmentation, the hallmarks of apoptosis, in LNCaPs treated with SL‐01‐18. In vitro androgen receptor (AR) binding studies with SL‐01‐18 suggested that SL‐01‐18 might act as an antagonist to AR with an IC50 value of 2640nM. Since androgen receptor (AR) activation induces expression and secretion of prostate specific antigen (PSA), ELISA assays for both secreted and cellular PSA levels in LNCaPs revealed significantly lowered PSA levels after SL‐01‐18 treatment. Finally, a significant reduction in cellular AR levels in LNCaP cells was observed after treatment with SL‐01‐18 by western blot.

These results confirm that SL‐01‐18 selectively inhibits growth of androgen dependent prostate cancer cells and eventually induces cell death via apoptosis. Our preliminary investigations into the mechanism of action of SL‐01‐18 in LNCaP cells reveal in vitro binding of SL‐01‐18 to AR and blocking it, potentially resulting in modulation of AR signaling by SL‐01‐18 as indicated by diminished AR protein as well as PSA levels upon treatment. Further mechanism directed studies will continue in future as will the design and synthesis of newer analogs SL‐01‐18.

Support or Funding Information

This research was supported by funds from TU‐COP MSMHS program.
The Repurposing of Ritanserin for the Inhibition of Diacylglycerol Kinase Alpha (DGKα), a Novel Therapeutic Target in Glioblastoma Multiforme (GBM)
Authors: Boroda Salome , Harris Thurl E. , Purow Benjamin W.
Published: November 2023

Diacylglycerol Kinase α (DGKα) catalyzes the conversion of diacylglycerol (DAG) to phosphatidic acid (PA). Recent work has demonstrated that DGKα activity is at a central tumorigenic node and the attenuation of its activity by a small molecule inhibitor, R59022, has shown promising results in countering glioblastoma multiforme (GBM) growth both in vitro and in vivo (Dominguez, et al, 2013). Until recently, R59022 and R59949 have been the only described inhibitors of DGKα. We have identified a serotonin receptor antagonist, Ritanserin, which is almost structurally identical to R59022. Our data show that Ritanserin is a more potent inhibitor of DGKα activity than R59022 in vitro. We have additionally investigated the specificity of Ritanserin for DGKα over other mammalian DGKs, and the affinity of R59022 for serotonin receptors. Since Ritanserin has previously been used clinically, and shown to have a low incidence of adverse side‐effects, we believe that it could be a viable therapeutic agent for treatment of GBM. Additionally, our work sets the stage for the development for more potent DGKα inhibitors.

Support or Funding Information

R01 1R01DK101946 to T.E.H, Wanger Fellowship Award, N.I.H Pharmacology graduate student training grant
Identification and Evaluation in silico and in vitro of Small Molecules in the KRas4B‐PDEδ Molecular Complex in Pancreatic Cancer
Authors: Aguirre Diana Casique , Gutiérrez Ponciano García , de León Bautista Mercedes Piedad , Mejía Miguel Ángel Vargas
Published: November 2023

Pancreatic cancer (PDAC) is the most lethal of all cancer types, characterized by an average survival of 6 months and a five‐years survival rate of less than 5%. To increase the survival rate of pancreatic cancer patients is necessary to search for improved tumor markers for an earliest diagnosis and new molecular targets for drug development. In most cases, PDAC is initiated by the mutant of GTPase KRas4B, which has been shown to drive pancreatic neoplasia. Furthermore, it has been demonstrated that nearly 95% of PDAC shown mutational activated KRas4B. Certainly, KRas4B protein has been used as a principal target in anticancer drugs development. Therefore, the identification of new organic molecules that recognize the KRas4B‐PDEδ molecular complex is of great interest for its stabilization for avoiding the activation of KRas4B in the cytoplasmic membrane. Thus, the objective of this work is to identify and evaluate new small molecules that stabilize the KRas4B‐PDEδ complex by using docking techniques. First, we constructed a homology model of KRas4B‐PDEδ complex and this revealed a set of exposed non‐conserved residues at the interface, which are considered an important target for ligand identification. Then, we carried out a docking simulation between the extensive database of drug‐like compounds from ENAMINE and the non‐conserved site at the Kras4B‐PDEδ model using a previous reported protocol. The in vitro evaluation of fourteen compounds with high scores from docking showed a significant specific effect in three of them on the viability of pancreatic cancer cell line MIAPaCa‐2 but not in the pancreatic cell line hTERT‐HPNE used as a control. Interestingly, the compounds decreased Ras‐GTP concentration in the cell line MIAPaCa‐2 in more than 50%, indicating a reduced activity. In conclusion, our results open the possibility to develop drugs against to PDCA; however, we need to perform more experiments in order to establish the specific mechanism of action of the compounds developed in this work.

Support or Funding Information

This work was sponsored by the CONACYT and ISSSTE project number: 002.2015 ISSSTE
A Retrospective Study of Semen Quality in Infertile Men with Varicoceles: Urological Expertise in Designing and Conducting a Testicular Safety Evaluation Study
Authors: Yono Makoto , Inoue Yumi , Ito Kazuya , Kimura Megumi , Furukawa Kohichiro , Hori Masaharu , Tsuji Shigeki , Tanaka Takanori , Sakata Yukikuni , Irie Shin , Latifpour Jamshid
Published: November 2023

Objectives

Designing and conducting a phase 1 study to evaluate the potential for testicular toxicity of a new drug requires urological expertise. Because varicoceles are present in 15–20% of the normal male population and associated with progressive testicular damage and a consequent reduction in fertility, we evaluated semen quality in infertile men with varicoceles, in comparison with control subjects. Information from this study may provide insight into the design and conduct of semen analyses in clinical trials of new drugs.

Methods

In a retrospective study, the records of 12 infertile patients with clinical varicoceles (mean age, 32.6 years) and 8 fertile men without clinical varicoceles (mean age, 32.0 years) between October 2005 and March 2015 were evaluated. Serum follicle‐stimulating hormone (FSH), luteinizing hormone (LH) and testosterone levels and semen parameters (semen volume, sperm concentration, total sperm number and total motility) were assessed in all the subjects. Two semen samples were collected at 4‐week intervals for initial evaluation and semen analyses were performed according to the WHO laboratory manual. Sperm concentration was measured by the same urologist using Makler counting chamber.

Results

At initial evaluation, inter‐ and intra‐individual variations in semen quality were observed in both the infertile men with grade 1 (palpable during Valsalva maneuver, but not otherwise) varicoceles and the fertile men, and both groups had similar profiles of hormone levels and semen parameters. On the other hand, the infertile men with grade 2 (palpable at rest, but not visible) or grade 3 (visible and palpable at rest) varicoceles had significantly higher FSH and LH levels and lower testosterone levels and semen quality compared with the fertile men. There was a significant increase in FSH levels and a decrease in semen quality in the infertile men with grade 1 varicoceles over a 4‐year period.

Conclusions

Our data indicate the presence of large biological variations in semen quality in the normal male population. These findings are considered to have consequences for the interpretation of semen analyses in clinical trials to evaluate testicular safety. Because a significant decline in semen quality over time was observed in the infertile men with grade 1 varicoceles and these small varicoceles could not be detected by hormone levels and semen parameters, men with varicoceles should be excluded from testicular safety evaluation studies by palpation and Valsalva maneuver.
Real‐time, continuous electrochemical monitoring of therapeutic drugs in vivo
Authors: Vieira Philip A , Arroyo‐Curras Netzahualcoyotl , Ploense Kyle L , Somerson Jacob , Plaxco Kevin W , Kippin Tod E
Published: November 2023

Personalized medicine takes into account individual patient factors that impact the efficacy of pharmaceutical therapies. Measuring the pharmacokinetic/toxicokinetic profile of a specific drug for each patient requires a cost‐effective, selective, robust and minimally invasive sensor. To that end, we have developed an electrochemical aptamer‐based indwelling sensor that achieves continuous, real‐time pharmacokinetic measurements of therapeutic drugs in living animals. Our preliminary experiments demonstrate that these sensors are selective to specific drug targets, durable and stable. We tested sensors selective to aminoglycosides, doxorubicin and cocaine, both in vitro and in vivo, and successfully measured fluctuating concentrations of therapeutics in real‐time. We carried out continuous measurements in Sprague‐Dawley rats (n=6) treated intravenously with separate drugs to create metabolic profiles specific to each animal. We found that our sensors are sensitive to therapeutic doses of antibiotic aminoglycosides (including kanamycin and gentamicin), the antitumor chemotherapy drug doxorubicin, and cocaine. The sensors are stable for up to 6hrs (in vivo) and 12hrs (in vitro), with minimal signal decay over time. This work is a first step in achieving personalized medicine by tracking the pharmacokinetics of selective agents in vivo.

Support or Funding Information

This work was supported by the Keck Foundation (to TEK) and the Otis Williams Fellowship (to NAC).
T‐type Voltage‐gated Ca2+ Channels Do Not Contribute To The Negative Feedback Regulation Of Myogenic Tone In Murine Superior Epigastric Arteries
Authors: Mullan Brendan , Pettis Jessica , Jackson William F
Published: November 2023

T‐type voltage gated Ca2+ channels (VGCC) have been hypothesized to control spontaneous transient outward currents (STOCs) through large‐conductance Ca2+‐activated K+ channels (BKCa) and contribute to the negative‐feedback regulation of myogenic tone. We tested this hypothesis in superior epigastric arteries (SEAs) isolated from male C57BL‐6 mice. SEAs were isolated and enzymatically dissociated to obtain single smooth muscle cells for whole‐cell recording of paxilline‐sensitive (PAX, 1 μM) STOCs at −30 mV, or cannulated and studied by pressure myography (80 cm H2O, 37°C). The T‐type blocker Ni2+ (30 μM) had no effect on STOC amplitude (20.1±1.7 pA vs. 20.6±1.7 pA; n = 12, p > 0.05), but increased STOC frequency (0.79±0.15 Hz vs. 1.21±0.22 Hz; n = 12, p < 0.05). While Ni2+ produced concentration‐dependent constriction of isolated, pressurized SEAs (logEC50 = −5.6±0.1; Emax = 40±3% constriction), block of BKCa with PAX had no effect on vasoconstriction induced by 30 μM Ni2+ (before PAX = 44±11% constriction vs. in the presence of 1μM PAX = 47±11% constriction; n = 6, p > 0.05). In contrast to Ni2+, the non‐selective T‐type blocker, mibefradil, produced vasodilation (logEC50‐ = −6.9±0.2; Emax = 74±8% dilation), whereas the putative T‐type blocker, ML218, had no significant effect on myogenic tone between 10nM and 10μM (n=6–7, p > 0.05). Our data do not support a role for T‐type VGCC in the negative‐feedback regulation of myogenic tone and suggest that Ni2+ may constrict murine SEAs by means other than block of T‐type VGCC.

Support or Funding Information

Supported by PO1‐HL070687 and ASPET‐SURF to B. Mullan
Time to Blood Pressure Control According to Drug Class in Patients with Hypertension
Authors: Johnson Micah William , Sprissler Ryan , Olson Thomas P , Beenken Greg William , Snyder Eric M
Published: November 2023

Introduction

Hypertension is one of the most important preventable contributors to disease and death worldwide and represents the most common condition seen in the primary care setting. Approximately one in three adults living in the US has hypertension. Despite the prevalence and impact of hypertension, little data exists on the average time to blood pressure (BP) control in US patients and time to control according to common BP medication classes.

Purpose

Therefore, the purpose of this study was to assess the average time to BP control in adults with controlled hypertension, according to drug class.

Methods

For this study we assessed how long (in months) and how many clinic visits were needed in order to control BP according to common medication classes in patients with existing hypertension. The study was performed at two clinical sites where patients came in for one visit for an in‐clinic assessment of BP and for completion of a patient survey regarding their history of BP. The primary care physician performed a chart review for a detailed objective BP history.

Results

Ninety‐nine patients completed all aspects of the study (age=58±0.8yrs., %female=46, height=169±1.0cm, weight=86±1.4kg, BMI=29.9±0.4m/kg2). The average initial BP was 151±2mmHg systolic (SBP), 91±1mmHg diastolic, and 111±1mmHg mean arterial (MAP). Patients demonstrated a significant drop in BP with therapy (SBP=16mmHg, DBP=10mmHg, MAP=12mmHg). Patients were prescribed an average of 1.8 BP medications (62% on ACE‐inhibitor, 42% on diuretic, 33% on β‐blocker, 27% on angiotensin receptor blocker, and 16% on Ca+ channel blocker). The average time to BP control was 14.9 months or 3.6 clinic visits. Patients on a diuretic had the longest time to BP control, while patients on a Ca+ channel blocker had the shortest time to control (diuretic=19.5±10.4, angiotensin receptor blocker=22.8±13.1, ACE‐inhibitor=22.2±11.4, β‐blocker=24.5±16.9, Ca+ channel blocker=9.9±4.5 months, pANOVA<0.05). In contrast, patients on a Ca+ channel blocker had significantly more clinic visits to attain BP control (diuretic=4.5±0.6, ACE‐inhibitor=3.1±0.4, angiotensin receptor blocker=3.9±0.6, β‐blocker=4.9±0.7, Ca+ channel blocker=5.1±0.7 visits).

Conclusion

These results suggest important differences in the time to BP control, and the number of office visits necessary for BP control according to common classes of hypertension therapies. Funding was provided by Geneticure, LLC

Support or Funding Information

Funded by Geneticure
ROMK Inhibitor Actions in the Nephron Probed with Diuretics
Authors: Kharade Sujay V. , Flores Daniel , Lindsley Craig , Satlin Lisa , Denton Jerod S.
Published: November 2023

More than a billion people worldwide are afflicted with essential hypertension and diuretics are usually the first line of treatment. Diuretics acting on specific nephron segments to inhibit Na+ reabsorption have been used clinically for decades; however, drug interactions, tolerance, and derangements in serum K+ complicate their use to achieve target blood pressure. ROMK is an attractive new diuretic target, in part, because its inhibition is postulated to indirectly inhibit the bumetanide‐sensitive Na+‐K+‐2Cl− co‐transporter, NKCC2, and the amiloride‐sensitive epithelial Na+ channel, ENaC. ROMK inhibition is also postulated to prevent urinary K+ wasting caused by NKCC2 inhibition. The development of small‐molecule ROMK inhibitors has created opportunities for exploring the physiological responses to ROMK inhibition. The present study evaluated how ROMK inhibition alone or in combination with NKCC2, ENaC, or the hydrochlorothiazide (HCTZ) target NCC, alters fluid and electrolyte transport in the nephron. The ROMK inhibitor VU591 failed to induce diuresis when administered orally to rats. However, another inhibitor, termed Compound A, induced a robust natriuretic diuresis without kaliuresis. Compound A produced additive effects on urine output and Na+ excretion when combined with HCTZ, amiloride or benzamil but not when co‐administered with bumetanide, suggesting that the major diuretic target site is the thick ascending limb (TAL). Interestingly, Compound A inhibited the kaliuretic response to NKCC2 inhibition, an effect we attribute to inhibition of ROMK‐mediated K+ secretion in the TAL and CD. Compound A had no effect on heterologously expressed flow‐sensitive BK channels (Slo1/b1). In conclusion, Compound A represents an important new pharmacological tool for investigating the renal consequences of ROMK inhibition and therapeutic potential of ROMK as a diuretic target.

Support or Funding Information

DK082884
PDI via Nrf2 Contributes to the Regulation of Renal AT1 Receptor Function and Blood Pressure
Authors: Wang Xitao , Asghar Mohammad
Published: November 2023

Renal angiotensin II AT1 receptor (AT1R) plays a vital role in regulation of blood pressure (BP). We recently report that oxidative stress causes exaggerated renal AT1R function and high BP in an aging rat model. This indicates that redox balance is crucial for normal AT1R function and BP. Nrf2 transcription factor is a master regulator of redox‐homeostasis. We hypothesized that protein disulfide isomerase (PDI), an enzyme involved in diverse biological reaction, by regulating Nrf2, regulates redox homeostasis, renal AT1R function and BP. To test the hypothesis, we treated adult Sprague‐Dawley rats with bacitracin, an PDI inhibitor (100mg/kg) for 14 days followed by determination of different parameters. We found that bacitracin treated rats, compared to the saline treated adult rats, showed exaggerated AT1R function, in terms of increased natriuresis and diuresis with AT1R blocker candesartan, and higher systolic BP. Bacitracin treated rats also exhibited increased oxidative stress marker in the renal tissues such as protein carbonyls. Kidney injury marker KIM‐1 level was also elevated in the urine of bacitracin treated rats. Interestingly, bacitracin treatment caused decreased accumulation of nuclear Nrf2 levels suggesting its impaired activity. These data suggest that PDI via Nrf2 pathway plays a critical role in redox‐homeostasis, renal AT1R function and BP.

Support or Funding Information

Source of Research Support: NIH/NIA, AG039836
Identification of a biased agonist for P2Y2 receptor to suppress pro‐thrombotic gene expression in human coronary artery endothelial cells
Authors: Zhang Lingxin
Published: November 2023

Recently we reported that in human coronary artery endothelial cells (HCAEC), the P2Y2 nucleotide receptor (P2Y2R) regulates the pro‐thrombotic gene tissue factor (TF) expression through positive and negative signaling mechanism. Here, we report the identification of a P2Y2R ligand JZS0312 which selectively activates the negative, but not the positive pathways, leading to decreased TF expression. Unlike the endogenous agonist UTP, JZS0312 alone had no effect on TF mRNA transcription, but it unexpectedly inhibited UTP‐induced TF expression. Mechanistic study showed that JZS0312 induced intracellular Ca2+ mobilization in HCAEC which exhibited the same efficacy as UTP and was inhibited by AR‐C118925, a P2Y2R‐selective antagonist. However, when evaluated in their activation of the MAPK pathways, JZS0312 only activated the ERK1/2, but not JNK and p38, whereas UTP activated all the three, suggesting JZS0312 as a biased ligand for the P2Y2R. This notion was supported by the fact that JZS0312‐induced ERK1/2 activation was inhibited by AR‐C1189258 and it had no effect on ERK1/2 in P2Y2R‐null cells. In addition, JZS0312 selectively activated the TF gene repressor Fra‐1, but not the positive AP‐1 subunits c‐Jun and ATF‐2, whereas UTP activated all of them. Furthermore, luciferase activity assay indicates that JZS0312 suppressed TF gene promoter activity via ERK1/2 activation, while UTP increased overall TF promoter activity. These findings reveal that JZS0312 acts as a biased agonist in human endothelial P2Y2R, in which it selectively activates the ERK1/2‐Fra1 pathway, leading to suppression of TF gene transcription. Thus, JZS0312 represents the first identified biased ligand in the P2Y receptor family, opening new avenue for potential drug discovery in relevant inflammatory and thrombotic diseases.

Support or Funding Information

Scientist Development Grant, AHA