bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Multifaceted Approach to Improve Upon the Freshwater Cyanobacteria Drug Lead Discovery Pipeline
Authors: Sullivan Peter , May Daniel , Crnkovic Camila , Orjala Jimmy 
Published: November 2023

Natural products have long been an excellent drug lead source. Our drug discovery workflow has resulted in the isolation of many cyanobacterial compounds active in a wide range of antibacterial, antiviral, and anticancer bioassays. While the present bioassay‐guided drug lead discovery has proven successful, we are currently developing a multifaceted strategy to improve upon this process in order to maximize the isolation and elucidation of novel, active cyanobacterial secondary metabolites. The first aspect to this approach is to expand our strain collection process by isolating cyanobacteria directly from the soil. Utilizing a pair of cyanobacterial specific 16S rRNA primers we have been able to identify cyanobacteria in the soil without macroscopic growth. Then, using inorganic media and light, we are able to culture the cyanobacteria directly from the soil for addition into our strain library. In addition, we are in the process of optimizing the culture media in order to maximize production of metabolites of interest. Using a comparative metabolomics approach, we were able to assess the influence of media components on the overall metabolic profile of cyanobacterial strains, assisting in the selection of best culture conditions for the purpose of drug discovery. The third feature to modifying our drug discovery pipeline is the incorporation of phylogenetics. Because metabolic production has shown to correlate with taxonomy, we can systematically prioritize which strains to culture and ultimately test against a particular bioassay. Overall, these measures have the capacity to significantly improve our lab's drug lead output.Support or Funding InformationNIH/NCI ‐ Grant PO1CA125066‐07NIH NCCIH ‐ Grant T32AT007533Science without Borders ‐ 13055‐13‐5
Evaluating In Vitro Canine Kidney Slices as a Renal Toxicity Model Using Nephrotoxic Agents Cisplatin and Cadmium Chloride
Authors: Kowalkowski Kenneth , Klapczynski Marcin , Blomme Eric , Buck Wayne , Liguori Michael 
Published: November 2023

Precision‐cut tissue slices retain the multicellular, structural, and functional features of their original organ and may potentially be a more relevant approach to interrogate toxicity compared to traditional cell‐based in vitro systems. Before testing newly developed compounds, we investigated the ability of this system to detect kidney toxicity using prototypical kidney tubular toxicants cadmium chloride (CdCl2) and cisplatin using a variety of endpoints. Kidneys from male beagle dogs were cut into approximately 300 mm slices. Primary experiments optimized the slicing conditions using ice cold Krebs‐Henseleit buffer with O2 bubbled through the chamber to maintain viability. Slices were cultured in 95% O2/5% CO2 atmosphere at 37°C for 24 to 48 hours. The slices were treated with vehicle or multiple doses of CdCl2 and cisplatin. Historical toxicity endpoints evaluated were H&E stained sections, intracellular ATP content, LDH release, and RNA integrity. In vivo studies have proven kidney injury molecule‐1 (Kim‐1) is a valuable tool for detecting kidney toxicity, so mRNA levels via in situ hybridization were examined as a potential novel endpoint. LDH leakage was significantly elevated at all doses tested for both compounds. The intracellular levels of ATP also declined significantly at 100 mM CdCl2 and 100mM cisplatin, indicative of substantial cellular damage. Mild to marked degeneration and necrosis of renal tubules was evident beginning at 24 h upon microscopic examination. Kim‐1, which is not expressed in normal tissue, was clearly overexpressed in CdCl2 treated slices, especially along the tubular epithelium, but not in vehicle treated slices. Kim‐1 was undetectable in severely necrotic slices, likely due to RNA destabilization, later confirmed using integrity assessments. These data demonstrate that a renal slice in vitro model can be used to detect potent nephrotoxicants and that Kim‐1 should be further explored as a novel in vitro biomarker to monitor toxicity in renal slice systems.Disclosures:All authors are employees of AbbVie. The design, study conduct, and financial support for this research was provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication.
Apelin Increases Airway Reactivity and Inflammation in a Mouse Model of Asthma
Authors: Dholakia Kshiti , Siddique Armaan , Kurade Mangesh , Narke Deven , Ponnoth Dovenia S. 
Published: November 2023

There is evidence that inflammatory exacerbations in asthma may be mediated by specific endogenous peptides. Apelin, an endogenous hypotensive and angiogenic peptide, is increased in atopic asthma. However, it is unclear how apelin affects allergic inflammation observed in asthma. We studied the role of exogenously administered apelin in airway hyperreactivity and inflammation in allergic mice. Mice were divided into control (CON) and allergen sensitized‐challenged (SEN) groups. Mice were sensitized (i.p.) on days 1, 6 with 0.2μg ovalbumin (OVA) followed by 5% OVA aerosol challenges on days 11–13. Apelin was given i.p. as a single bolus dose (4mg/kg) on day 14 to SEN mice. Whole body plethysmography (measuring airway responsiveness as enhanced pause, Penh) and bronchoalveolar lavage (BAL) studies were then performed. Airway responsiveness to MCh (48mg/ml) was highest in SEN+APELIN group (149.64±6116 vs. 95.16±30.48% in SEN, p>0.05, n=3). Differential BAL cell analysis showed that treatment with apelin increased eosinophils (68.67±1.86% in SEN vs 80.67 ±1.20% in SEN+APELIN, p<0.05) and neutrophils (8.33±0.88% in SEN vs 13.33±0.88% in SEN+APELIN, p<0.05) in sensitized mice, thus potentiating the allergic inflammation. Although further studies are needed to evaluate detailed cellular mechanisms, the role of apelin and apelinergic system appears to be proinflammatory in this model of asthma.Support or Funding InformationLIU start‐up funds (DSP)
Poly(ADP‐ribosyl)ation of Signal Transducer And Activator of Transcription (STAT)6 by Poly(ADP‐ribose) Polymerase (PARP)‐1 Is Critical for Its Integrity And Subsequent Regulation of Th2 Cytokines during Asthma
Authors: Wang Jeffrey , Ghonim Mohamed , Ibba Salome Valentina , Pyakurel Kusma , Lammi Matthew , Boulares Hamid 
Published: November 2023

BackgroundAlthough asthma is often controlled by corticosteroids and β2‐adrenergic agonists, many individuals cannot control the disease with these therapies. Identifying the key molecular mechanisms the disease is key to establishing strategies to improve the quality of life of asthmatics. Our laboratory performed a series of pioneering studies revealing the critical role of PARP‐1, classically regarded as a DNA repair enzyme, in asthma pathogenesis by controlling NF‐kB nuclear retention and STAT6 integrity. PARP‐1 inhibition destabilizes STAT6, a transcription factor central to Th2 cytokine signaling, upon IL‐4 or allergen exposure, potentially through a calpain‐dependent mechanism.HypothesisPARP‐1 interacts with IL‐4‐activated STAT6 and modifies it for maintaining STAT6 integrity leading, ultimately, to expression of Th2 cytokines.MethodsA cell‐free recombinant protein‐based enzymatic reactions, in vitro stimulation of primary splenocytes with IL‐4, histidine‐tag pulldown, and co‐immunoprecipitation with subsequent immunoblot were utilized to conduct the studies.ResultsPARP‐1 was activated in lungs and PBMCs of human asthmatics. STAT6 interacted with and was poly(ADP‐ribosyl)ated (PARylated) by PARP‐1 in both the cell‐free system and in IL‐4‐treated mouse splenocytes. PARylation of STAT6 was enhanced by JAK kinase‐3. Phosphorylation rendered STAT6 more susceptible to degradation by mu‐calpain but PARylation protected it from such degradation. Intriguingly, these events occurred in the complete absence of damaged DNA. Consistent with these results, IL‐4 stimulation‐induced PARylation of STAT6 by PARP‐1 was not accompanied with a DNA damage response as assessed by p53 phosphorylation.ConclusionModification of STAT6 by PARP‐1 through a DNA damage‐independent mechanism is critical in Th2 responses and overall asthma pathogenesis. Based on the prediction that the IL4/STAT6 pathway is upregulated in asthma, concomitant with elevated phosphorylation of STAT6, activated STAT6 in cells of patients with the disease should be highly susceptible to degradation upon PARP inhibition, leading to an extensive reduction in the expression of Th2 genes.Support or Funding InformationLouisiana Cancer Research Center Cancer Center
Expression and Regulation of SULT1C4 in Human Cells and Tissues
Authors: Dubaisi Sarah , Fang Hailin , Kocarek Thomas A , Runge‐Morris Melissa 
Published: November 2023

Cytosolic sulfotransferases (SULTs) are a superfamily of conjugating enzymes that catalyze the transfer of a sulfonate group from the cofactor 3′‐phosphoadenosine‐5′‐phosphosulfate to a wide range of endogenous and xenobiotic substrates. Human SULTs are classified into four gene families and eight subfamilies that include thirteen genes, including three in the SULT1C subfamily that appear to be preferentially expressed during prenatal development. We used 5′ Rapid Amplification of cDNA Ends (5′‐RACE) and RT‐PCR analysis to characterize the SULT1C4 transcript that is expressed in human intestinal and hepatic cell lines as well as in human liver specimens from different developmental stages. 5′‐RACE analysis indicated that SULT1C4 transcription begins in the first coding exon, consistent with previous findings. Because the NCBI nucleotide database contains SULT1C4 reference sequences for two transcript variants, a full‐length sequence containing seven exons (TV1) and a variant lacking exons 3 and 4 (TV2), we also evaluated the SULT1C4 transcript by RT‐PCR and sequencing. This analysis indicated that TV1 and TV2 as well as a third transcript variant lacking only exon 3 are co‐expressed in Caco‐2 cells, the hepatocyte‐like HepaRG cell line, and human liver specimens. We also evaluated the regulation of SULT1C4 by lipid‐ and xenobiotic‐sensitive transcriptional regulators as well as by the pro‐inflammatory cytokine interleukin‐6 (IL‐6) in HepaRG cells. Treatment with the pregnane X receptor agonist rifampicin increased SULT1C4 expression while treatment with the aryl hydrocarbon receptor agonist 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin or IL‐6 decreased SULT1C4 expression. These findings indicate that SULT1C4 expression is sensitive to xenobiotic and pro‐inflammatory stimuli and that multiple transcript variants are co‐expressed, which together have implications for the level of conjugating activity that is functional during human development.Support or Funding InformationThis research was supported by grants R01 ES022606 and P30 ES020957.
Development and Characterization of SULT4A1 Mutant Mouse Models
Authors: Garcia Patrick L. , Falany Charles N. 
Published: November 2023

Sulfotransferase 4A1 (SULT4A1) belongs to the cytosolic sulfotransferase gene superfamily of phase II conjugation enzymes. Initially cloned and characterized and by our lab, SULT4A1 was found to be predominately expressed in neuronal tissue. SULT4A1 demonstrates high homology between vertebrate species suggesting an evolutionarily conserved function in brain homeostasis; however, no known function or substrate has yet to be described. Relevant to SULT4A1 function, Phelan‐McDermid syndrome (PMS), or 22q13.3 deletion syndrome, is characterized by global developmental delay, intellectual disability, and autism spectrum disorder; approximately 30% of patients with PMS have a deletion encompassing SULT4A1. To further clarify the function of SULT4A1 in a mammalian system, CRISPR/Cas9 gene editing technology was used to generate SULT4A1 mutant mice. Two SULT4A1 mutant founder mice were selected for breeding: one mouse with a 28 bp (SULT4A1‐Δ28) deletion located on exon 1 that resulted in a frame shift mutation with a premature stop codon at 58 amino acids and another mouse with a 12 bp (SULT4A1‐Δ12) in‐frame deletion of the 4 amino acids immediately preceding the active site histidine on exon 3. For both mutant lines, heterozygotes appeared to develop normally and were used to breed homozygotes. Unexpectedly, homozygous mutants of both lines exhibited severe developmental defects including failure to thrive, difficulty feeding, abnormal gait, poor coordination, and absence seizures. Symptoms readily appear between post‐natal day 10–14 with animals continuing to decline until death around weaning. The similarity of symptoms in both mutant lines suggests that a fully functional SULT4A1 (including an intact active site) is essential for normal murine development, and the timing of death indicates a critical role for SULT4A1 in post‐natal development. Currently, our lab is focused on elucidating the functional role of SULT4A1 in wild‐type (WT), heterozygous, and homozygous SULT4A1‐Δ12 and ‐Δ28 mice. This analysis will include examining gene expression changes between SULT4A1 WT, heterozygous, and homozygous mice by RNA‐Seq to identify critical signal transduction pathways potentially altered in the brains of mutant mice, behavioral testing to quantify cognitive and locomotor deficits, and molecular biology assays to further characterize the role of SULT4A1 in the post‐natal mouse brain.Support or Funding InformationSupported by NIH grant GM113980 to CNF.
Physiologically Based Pharmacokinetic Model Verification and Improvement for CYP2D6‐mediated Drug‐drug Interaction Prediction
Authors: Ning Miaoran , Posada Maria M. , Hall Stephen D. , Dickinson Gemma L. 
Published: November 2023

Using physiologically based pharmacokinetic (PBPK) models to quantitatively predict drug‐drug interaction (DDI) is becoming common practice in drug development. To accurately assess DDI risk, it is important to verify the existing PBPK models of known substrates and inhibitors before applying the models for DDI prediction. Cytochrome P450 (CYP) 2D6 is a major drug metabolizing enzyme responsible for eliminating ~25% of clinically used drugs. Paroxetine is a strong mechanism‐based inhibitor of CYP2D6 and desipramine is a sensitive substrate of the enzyme. Here we verified and improved the Simcyp® models of paroxetine and desipramine for their application in CYP2D6‐mediated DDI prediction. We first examined the performance of existing Simcyp® models in describing drug exposure of paroxetine and desipramine alone. The Simcyp® paroxetine model predicted paroxetine drug exposure within 2 fold difference comparing with clinical data. However, when daily dose was 20 mg or lower, the model under‐predicted paroxetine drug accumulation at steady state, with observed/predicted Cmax and AUC ratio over 1.5 fold. Related parameters, KI and kinact for CYP2D6 mechanism‐based inhibition, were changed from 0.315 μM and 10.2 1/h to 0.067 μM and 11.55 1/h respectively, based on recently published in vitro data1. The new model showed improved prediction of paroxetine exposure at steady state under lower doses. On the other hand, the Simcyp® desipramine model under‐predicted drug clearance in CYP2D6 poor metabolizers (PMs), where renal clearance was the only clearance pathway. Additional systematic clearance of 13.5 L/h was added to the model based on observed clearance in PM subjects. The improved desipramine PBPK model was able to predict desipramine PK in both CYP2D6 extensive metabolizers and PMs. To evaluate the performance of the improved PBPK models of paroxetine and desipramine in predicting CYP2D6‐mediated DDI, we simulated paroxetine‐desipramine interaction in different scenarios and compared the results with observed data. For all simulations, the predicted AUC ratio of desipramine with or without interaction was within 1.25 fold difference with clinical observations. In summary, the improved PBPK models of paroxetine and desipramine can predict drug exposure of the drugs and DDI between paroxetine and desipramine when combined together. This increases our confidence in using the two models to predict CYP2D6‐mediated DDI in other circumstances.Support or Funding InformationThis work is part of a project funded by Eli Lilly & Company.
Modeling the Metabolism and Subsequent Reactivity of Drugs
Authors: Swamidass S. Joshua , Matlock Matthew , Le Dang Na , Hughes Tyler , Barnette Dustyn A , Miller Grover P. 
Published: November 2023

Adverse drug reactions (ADRs) are dangerous and expensive. Idiosyncratic ADRs, especially the difficult to predict, rare and severe hypersensitivity‐driven ADRs, are the leading cause of medicine withdrawal and termination of clinical development. At the same time, a large proportion of drugs are not associated with hypersensitivity driven ADRs, offering hope that new medicines could avoid them entirely with reliable predictors of risk. Hypersensitivity driven ADRs are caused by the formation of chemically reactive metabolites by metabolic enzymes. These reactive metabolites covalently attach to proteins to become immunogenic and provoke an ADR.Unfortunately, current computational and experimental approaches do not reliably identify drug candidates that form reactive metabolites. These approaches are limited because they inadequately model metabolism, which can both render toxic molecules safe and safe molecules toxic. Mathematical models of both metabolism and reactivity overcome this limitation. The models are constructed using machine‐learning algorithms that quantitatively summarize the knowledge from thousands of published studies.This approach more accurately models whether metabolism renders drugs toxic or safe. For example, it predicts the mechanisms of drug toxicity much more accurately than structural alerts. Applying this modeling approach to the literature, it identifies errors and corrects errors in literature‐reported bioactivation pathways, and can identify bioactivation events that are missed by standard experimental approaches. This demonstrates how modeling can be used alongside biochemical studies to more reliably determine how and when reactive metabolites are generated by drug candidates.Support or Funding InformationResearch reported in this publication was supported by the National Library Of Medicine of the National Institutes of Health under Award Numbers R01LM012222 and R01LM012482. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Computations were performed using the facilities of the Washington University Center for High Performance Computing, which were partially funded by NIH grants nos. 1S10RR022984‐01A1 and 1S10OD018091‐01. We also thank both the Department of Immunology and Pathology at the Washington University School of Medicine and the Washington University Center for Biological Systems Engineering for their generous support of this work.
POTENTIAL FOR DRUG INTERACTION OF THE NOVEL COMT INHIBITOR OPICAPONE
Authors: Loureiro Ana Isabel , Fernandes‐Lopes Carlos , Soares‐da‐Silva Patrício 
Published: November 2023

ObjectivesThe effect of opicapone, a third generation COMT inhibitor, on metabolic enzymes and drug transporters was evaluated in vitro to anticipate potential drug‐drug interactions.Methodsin vitro studies using human liver microsomes and cell‐lines overexpressing a panel of selected drug‐transporters.ResultsWhen tested against P450 cytochromes (CYP), opicapone was found to be reversible inhibitor of CYP2C8 (IC50 = 3.6 μg/ml and IC50 = 6.7 μg/ml, respectively) and CYP2C9 (IC50 = 9.6 μg/ml, and IC50 = 20.7 μg/ml, respectively). No metabolism dependent inhibition by opicapone was observed for all tested CYPs. Opicapone was a substrate for the efflux transporters breast cancer resistance protein (BCRP) and P‐glycoprotein (P‐gp) and for the organic anion transporting polypeptide (OATP1B3). Opicapone showed moderate inhibition of bile salt export pump (BSEP), and organic anionic transporters (OAT1 and OAT3), but inhibited OATP1B1 and OATP1B3 at lower concentrations (IC50 values of 0.45 and 1.4 μg/ml, respectively).ConclusionsWhen the 99.9% binding of opicapone to human serum proteins is considered, the 1.55 μg/mL Cmax in humans receiving 50 mg/day of opicapone equates to 0.002 μg/mL of unbound material. This is 225‐fold lower than the lowest IC50 against transporters (OATP1B1, OATP1B3) and metabolic enzymes (CYP2C8 and CYP2C9) measured in the present study. As such, the observed in vitro effect upon these biological targets is highly unlikely to have any clinical impact.
Alcohol induced changes in cellular pharmacokinetics and pharmacodynamics of elvitegravir and darunavir in HIV‐1 infected monocytic cells
Authors: Midde Narasimha M , Sinha Namita , Gong Yuqing , Meibohm Bernd , Kumar Santosh 
Published: November 2023

IntroductionAlcohol consumption is negatively associated with antiretroviral therapy (ART) adherence1. Maintenance of optimal ART levels in monocytes and/or macrophages, the known HIV viral reservoirs, is essential to suppress HIV viral replication. Previously, we reported ethanol mediated alterations in metabolism of elvitegravir (EVG) in human liver microsomes2. The objective of this study was to investigate whether the ethanol exposure alters the intracellular concentration of EVG in HIV‐infected monocytic cells.MethodsHIV‐infected U937 (U1) cells were treated with EVG (5 μM), EVG+Cobicistat (COBI) (2 μM), EVG+ethanol (20 mM), and EVG+COBI+ethanol combinations and collected cells at different time intervals up to 24 hours. EVG concentration was measured using LC MS/MS. HIV p24 levels were assessed by ELISA and cytochrome P450 (CYP) 3A4, multidrug resistance‐associated protein 1 (MRP1), and multidrug resistance protein 1 (MDR1) levels by In‐Cell Western (ICW) technique. One way ANOVA and student t‐test were employed wherever appropriate and the p‐value ≤ 0.05 was considered significant.ResultsEthanol treatment demonstrated a significant decrease of area under the concentration curve (AUC) and maximum concentration (Cmax) values of EVG, and increased the HIV p24 levels despite the presence of ART. Moreover, exposure of MRP1 inhibitor along with EVG and COBI reduced ethanol influence on EVG concentration but did not affect the p24 levels. Finally, our ICW data suggest that ethanol induces the expression of CYP3A4, MRP1 and MDR1 proteins.ConclusionsOverall, these findings suggest that ethanol reduces EVG intracellular availability in in vitro conditions, at least partially, by modifying CYP3A4, MRP1 and MDR1 protein levels. This evidence warrants further ex vivo investigation of the effects of alcohol on the availability of antiretroviral drugs at the cellular level.Support or Funding InformationThis research was supported by grant from the National Institute of Health to Santosh Kumar (NIAAA/NIH AA‐022063)