bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of a Novel Single‐Cell Assay to Interrogate a CRISPR/Cas9 Library for Regulators of MEK Kinase 2
Authors: Lotesto Krista , Cuevas Bruce
Published: November 2023

MEK Kinase 2 (MEKK2) controls activity of the mitogen‐activated protein kinase ERK5 and in vivo models strongly suggest that MEKK2 activity is an important contributing factor in diseases ranging from cardiomyopathy to cancer. Thus, MEKK2 and the signaling proteins that control MEKK2 activity represent potential therapeutic targets. However, pharmacological MEKK2 inhibitors are not currently available and MEKK2 regulation is poorly understood. CRISPR/Cas9‐based genetic screening represents a powerful technology for defining regulatory networks at the single‐cell level, but single‐cell assays of MEKK2 required for CRISPR/Cas9 screening have not been described. Herein, we report our development of a Fluorescence Activated Cell Sorting (FACS)‐based MEKK2 signaling assay that can be used to screen a CRISPR library to identify MEKK2 regulators. We examined the utility of MEKK2‐dependent ERK5 phosphorylation as a surrogate indicator of MEKK2 activity in several human cell lines by quantifying ERK5 activation loop phosphorylation (pERK5) with indirect immunofluorescence. We determined the pERK5 fluorescence signal intensity induced by multiple reported ERK5‐activating stimuli in multiple human cell lines, and performed z′ factor analysis to establish the preferred conditions for a FACS‐based separation of cells with active MEKK2 from cells with inactive MEKK2. We discovered that anti‐pERK5 staining of 293T cells stimulated with MEKK2 activator pervanadate provides a means to effectively separate cells with activated MEKK2 from non‐treated cells, and allows us to distinguish and separate the two populations by FACS. Our data suggest that fluorochrome‐linked anti‐pERK5 can be used to sort cells that express CRISPR gRNA that inhibit MEKK2 activity, thereby identifying MEKK2 regulators. Therefore, we conclude that anti‐pERK5‐based immunofluorescence coupled with FACS analysis provides a new tool for performing unbiased genetic screens utilizing CRISPR/Cas9 library‐expressing cells to identify MEKK2 regulators in order to identify novel pharmacological targets for the treatment of human disease.

Support or Funding Information

ASPET “Discover Pharmacology” Summer Undergraduate Research Fellowship
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 Information

NIH/NCI ‐ Grant PO1CA125066‐07

NIH NCCIH ‐ Grant T32AT007533

Science 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 Information

LIU 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

Background

Although 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.

Hypothesis

PARP‐1 interacts with IL‐4‐activated STAT6 and modifies it for maintaining STAT6 integrity leading, ultimately, to expression of Th2 cytokines.

Methods

A 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.

Results

PARP‐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.

Conclusion

Modification 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 Information

Louisiana Cancer Research Center Cancer Center
Menthol exhibits stereospecific inhibition of phase II UGT enzymes
Authors: Kozlovich Shannon , Chen Gang , Lazarus Philip
Published: November 2023

Mint flavoring (menthol) is often included as a flavor additive with many consumer products including candy, toothpaste and medications as well as various brands of tobacco. Menthol is a chiral aliphatic alcohol, with each enantiomer called D‐ or L‐menthol and the racemic mixture being referred to as DL‐menthol. Menthol can be added to tobacco products as either L‐menthol or DL‐menthol in both menthol and certain non‐menthol tobacco cigarette brands. Tobacco‐specific nitrosamines (TSNAs) are among the most potent carcinogens found in tobacco, and 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK) is the most potent and one of the most abundant. NNK is extensively metabolized to the equally carcinogenic chiral 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanol (NNAL). (S)‐NNAL exhibits higher stereoselective tissue retention in mice and humans and has been shown to be more carcinogenic in mice than its counterpart (R)‐NNAL. NNAL is primarily detoxified by six UDP‐glucuronyltransferase (UGT) enzymes [UGTs: 1A4, 1A9, 1A10, 2B7, 2B10 and 2B17]; each of these UGTs have been shown to have different stereospecificity for the formation of one NNAL glucuronide (NNAL‐gluc) over the other. DL‐Menthol has been shown to inhibit NNAL‐gluc formation in human liver microsomes. This differential carcinogenic potential and stereospecific metabolism makes it increasingly important to know which UGT enzymes are being inhibited by each of the menthol enantiomers. The goal of the present studies was to examine differences in the glucuronidation of menthol enantiomers and the inhibitory effects of these enantiomers on the glucuronidation of NNAL. Menthol exhibits the strongest inhibition of NNAL‐gluc formation for UGT2B7 with L‐ and D‐menthol exhibiting similar IC50 values of ~100 μM. This is in strong contrast to UGT1A9, where each menthol enantiomer exhibits a significantly (p < 0.001) different IC50 value, with D‐menthol exhibiting an IC50 (~1400 μM) that was more than double that of L‐menthol (~600 μM). For UGT2B7, there was a significant (p < 0.05) difference between the L‐ and D‐menthol inhibition effect for the formation of (R)‐NNAL‐gluc, with IC50's ~100 μM and >2500 μM, respectively, but no inhibition difference was observed for the formation of (S)‐NNAL‐gluc. These data indicate that the different menthol enantiomers could have a significant and stereospecific effect on the clearance of the tobacco carcinogen, NNAL in vivo.
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 Information

This 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 Information

Supported 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 Information

This 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 Information

Research 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

Objectives

The effect of opicapone, a third generation COMT inhibitor, on metabolic enzymes and drug transporters was evaluated in vitro to anticipate potential drug‐drug interactions.

Methods

in vitro studies using human liver microsomes and cell‐lines overexpressing a panel of selected drug‐transporters.

Results

When 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).

Conclusions

When 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

Introduction

Alcohol 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.

Methods

HIV‐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.

Results

Ethanol 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.

Conclusions

Overall, 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 Information

This research was supported by grant from the National Institute of Health to Santosh Kumar (NIAAA/NIH AA‐022063)
Novel Polymer Micelle and Nanocrystal Formulations for the Proteasome Inhibitor Drug Carfilzomib: Pharmacokinetic and Pharmacodynamic Studies in Human Lung and Breast Cancer Models
Authors: Park Ji Eun , Chun Se‐Eun , Reichel Derek , Park Joonyoung , Min Jee Sun , Ryoo Gongmi , Oh Yunseok , Bae Soo Kyung , Yeo Yoon , Bae Younsoo , Lee Wooin
Published: November 2023

Carfilzomib (CFZ) is a peptide epoxyketone proteasome inhibitor with much improved efficacy and safety profiles over bortezomib and has become a mainstay of multiple myeloma (MM) therapy. Despite the promising preclinical data of CFZ against solid cancers, the results from clinical trials in patients with solid cancers were rather disappointing. Rapid degradation of CFZ and its poor penetration to solid tumor sites have been considered to be the main culprits for the lack of efficacy. Thus, we explored nanoparticle‐based systems, in particular, polymer micelle and nanocrystal formulations, as new CFZ delivery strategies for the treatment of human lung and breast cancer. We first developed CFZ‐loaded polymer micelle (CFZ‐PM) composed of block copolymers poly‐(ethylene glycol) and poly‐(caprolactone). While CFZ‐PM improved the metabolic stability of CFZ in vitro, CFZ‐PM did not improve in vivo performances (in terms of anticancer efficacy and pharmacokinetic profiles) over the clinically used cyclodextrin‐based CFZ formulation. The proteasome activities in excised human lung cancer xenograft tissues from mice treated with CFZ‐PM displayed an incomplete inhibition, suggesting that CFZ‐PM did not effectively enhance the penetration of active CFZ to solid cancer tissues. As an alternative strategy to further improve in vivo stability and active targeting of CFZ to solid cancer cells, we designed albumin‐coated nanocrystal formulations containing CFZ (Alb‐CFZ‐NC). With optimized processing methods and surface stabilizers, Alb‐CFZ‐NC displayed the average particle size of ~260 nm and ~70% drug content as well as albumin conformation close to the native protein (with favorable uptake profiles by cancer cells). Ongoing investigations are focused on the assessment of Alb‐CFZ‐NC in terms of pharmacokinetic/biodistribution profiles and anticancer efficacy in human breast cancer models. Our current efforts may provide effective CFZ formulations to validate and harness the potential of CFZ‐based therapy for solid cancer patients.
Fluorescence Imaging Reveals a Potential Association of Laminin Receptor with SENP Machinery
Authors: Robinson Michael , Umbaugh Charles Samuel , Figueiredo Marxa Leao
Published: November 2023

Laminin receptor (LR) is a protein overexpressed in cancer. LR contributes to tumor‐cell migration and proliferation, and restructures laminins to increase aggressiveness and gene expression of tumor cells. Thus, the abundance of the receptor is a reliable determinant of tumor severity. Little is understood about how LR enhances aggressiveness of tumor cells.

To gain insight into the behavior of laminin receptor, we genetically fused a red fluorescent protein to LR (LR‐mCherry2). We confirmed the orientation and insertion of the gene via restriction digests. Using high‐throughput live cell imaging, we determined peak expression time and number of positively transfected cells for LR‐mCherry2 and mCherry.

Because there is evidence that laminin receptor is associated with SUMO (Small Ubiquitin‐Like MOdifying) proteins, we explored the hypothesis that SUMO proteins and SUMO machinery were associated with LR in the cell. A second set of proteins, SENPs (SUMO‐Sentrin Specific Proteases), are responsible for removing SUMO when their addition is no longer required. We cotransfected PC‐3 cells with LR‐mCherry2 or mCherry2 and one of four EGFP fused SENPs (SENP 2, 3, 5, or 6).

After live cell imaging, consecutive images were compiled into movies that revealed localization patterns for SENPs and LR‐mCherry2. Interestingly, we found that LR‐mCherry2 was expressed throughout the cell but it was strongly associated with SENPs in the nucleus. This suggests that the LR may interact with SUMO machinery in the nuclei of prostate cells.

Support or Funding Information

Supported by Summer Research Program (SROP) (MR) and start up funds from the BMS department (MF).
Rapid progression of breast cancer cell proliferation in diabetes: Role of polyamines
Authors: Morwitzer Melody Jane , Chandra Surabhi
Published: November 2023

Diabetes and cancer are comorbid conditions with a heavy toll on human life worldwide. Several cancer subtypes (pancreatic, breast, liver, and colorectal) rapidly progress to advanced stages and even metastasize in diabetic states. Though high blood glucose levels have been considered as fuel for growth of cancer cells, pathways leading to this condition are still under investigation. Cellular polyamines can modulate normal and cancer cell growth, and can be synthesized from multiple sources including endogenous amino acid precursors, diet and through degradation. Inhibition of polyamines can provide protection against diabetes in mouse models. It is not known however whether elevation of polyamines in diabetes is linked to cancer advancement. Thus, we hypothesized that high glucose stimulated polyamine production causes increased proliferation of breast cancer cells. Proliferation studies were performed with varying concentrations of glucose (5mM–25mM) treatment on MCF‐7 (early stage) and MDA‐MB‐231 cells (advanced stage) breast cancer cells. Though there was a significant increase in cell proliferation with HG (25mM) in both cell lines, MDA‐MB‐231 cells showed this effect at 72hr compared to longer incubation time with MCF7 cells (96–120hr). Inhibitor of polyamine synthesis (difluoromethylornithine, DFMO, 5mM) significantly suppressed cell proliferation observed with HG (25 mM). Protein levels of ornithine decarboxylase (ODC), the enzyme required for polyamine synthesis, was also elevated with HG treatment but the levels were restored to normal glucose (5mM) conditions upon treatment with DFMO. Our findings indicate that advanced stage breast cancer cells proliferate rapidly in hyperglycemic states compared to early stage breast cancer cells, and that polyamines are involved in the transition to advance stage of cancer.

Support or Funding Information

University of Nebraska‐Kearney start‐up funds (SC)
“Kill two birds with one stone”: Bioengineering of single RNA molecule to target miR‐21a and introduce miR‐34a in NSCLC cells
Authors: Petrek Hannah Elizabeth , Ho Pui Yan , Duan Zhijian , Batra Neelu , Jilek Joseph , Yu Ai‐Ming
Published: November 2023

Lung cancer is the second most common cancer among both men and women in the United States. The majority of lung cancer cases are classified as non‐small cell lung cancer (NSCLC). Current NSCLC treatments include resection surgery, radiation, and pharmacotherapy which all provide some benefits and have limitations. As a result, lung cancer caused deaths account for about 27% of all cancer deaths in the US. Therefore, new therapeutic agents are highly demanded for NSCLC. MicroRNAs (miRNA or miR) are a class of genome‐derived, small noncoding RNAs in cells that govern target gene expression through mRNA degradation or translational inhibition. MiR‐34a is commonly downregulated in NSCLC and targets various oncogenes such as c‐MYC, c‐MET, BCL‐2 and SIRT. On the other hand, miR‐21a is upregulated in NSCLC and reduces the expression of many tumor suppressors such as PTEN and PDCD4. Aiming at targeting oncogene miR‐21a and introducing tumor suppressor miR‐34a simultaneously in NSCLC cells, we employed our recently developed RNA bioengineering technology to produce a single RNA molecule containing both miR‐34a and a siRNA against miR‐21a (si‐miR21a). Our data showed this si‐miR21a/miR‐34a agent was highly expressed in E. coli cells. After purification by FPLC method, si‐miR21a/miR‐34a agent was transfected into human NSCLC cells. We further defined the levels of cellular si‐miR21a, miR‐21a and miR‐34a levels, the effects on miR‐21a and miR‐34a target gene expression, and the impact on NSCLC cell proliferation. Using a single RNA agent for dual targeting represents a new strategy for the development of cancer therapeutics.

Support or Funding Information

This study was supported by grants R01GM133888 and U01CA175315 from NIH.
The interaction of GIV with Galpha‐i is a druggable protein‐protein interaction
Authors: DiGiacomo Vincent , de Opakua Alain Ibanez , Merino Nekane , Blanco‐Canosa Juan B. , Blanco Francisco J. , Garcia‐Marcos Mikel
Published: November 2023

GIV (a.k.a Girdin) is a non‐receptor Guanine‐nucleotide Exchange Factor (GEF) for heterotrimeric G proteins. Dysregulation of signaling networks dependent on such G proteins is a contributing factor in many human pathologies from cancer to psychiatric disorders. Therapeutic development in this area has been very successful by targeting G protein‐coupled receptors (GPCRs) – the targets of greater than 30% of marketed drugs – but structural and mechanistic insights into regulators such as GIV have opened novel routes to pharmacological intervention. GIV itself is overexpressed in a variety of human cancers and promotes metastatic properties in tumor cells. Moreover, disrupting GIV binding to Gαi blunts this metastatic behavior. These findings situate the GIV‐Gαi interaction as a promising therapeutic target. Despite protein‐protein interactions (PPIs) generally being considered poorly “druggable,” here we provide structural details about the Gαi‐GIV interface and demonstrate its chemical tractability. In testing a collection of >1,000 bioactive compounds with two high‐throughput screening approaches (structure‐guided virtual screening and a fluorescence polarization assay), we discovered two common micromolar potency hits, which were confirmed as true inhibitors by orthogonal assays. We mapped the binding site of the top hit by NMR spectroscopy and biochemical assays, showing that it overlaps with the GIV engagement site on Gαi, thereby validating its mode of action. Moreover, this inhibitor did not block Gβγ binding to Gαi, indicating that GIV binding can be specifically inhibited. This work empirically confirms the Gαi‐GIV PPI as a druggable target and outlines a discovery approach for anti‐metastatic small molecules. We will present our progress to date in screening a larger library of 200,000 compounds.

Support or Funding Information

Funded by NIH R01GM112631 and the Hartwell Foundation
Synthetic 8‐hydroxygdeoxyguanosine paradoxically inhibited metastasis of pancreatic cancer through concerted inhibitions of ERM and Rho‐GTPase
Authors: Han Young‐Min , Park Jong‐Min , Chung Myung Hee , Kwon Chang Il , Ko Kwang Hyun , Hahm Ki Baik
Published: November 2023

8‐hydroxydeoxyguanosine (8‐OHdG) is generated consequent to oxidative stress, but its paradoxical anti‐oxidative, anti‐inflammatory, and anti‐mutagenic effects via Rho‐GTPase inhibition were noted in various models of inflammation and cancer. Metastasis occurs through cell detachment, epithelial–mesenchymal transition (EMT), and cell migration; during these processes, changes in cell morphology are initiated through Rho‐GTPase‐dependent actin cytoskeleton polymerization. In this study, we explored the anti‐metastatic mechanisms of 8‐OHdG in Panc‐1 pancreatic cancer cells. 8‐OHdG inhibits cell migration by inactivating ERM and Rho‐GTPase proteins, and inhibiting focal adhesion kinase (FAK) and matrix metalloproteinases (MMPs). At 15 min, 8‐OHdG significantly inactivated ERM (p < 0.05) and led to a significant retardation of wound healing; siERM and H1152 (ROCK inhibitor) had similar effects (p < 0.05). However, FAK inhibitor 14, DPI (NOX inhibitor), and NAC (antioxidant) significantly delayed wound healing without inhibiting ERM or CD44 (p < 0.05). In the experiments on cell migration, siERM, siCD44, DPI, and 8‐OHdG significantly inhibited MMPs. 8‐OHdG significantly decreased DCF‐DA activation in Panc‐1 pancreatic cancer cells and down‐regulated NOXs (nox‐1, nox‐2, and nox‐3). Finally, all of these anti‐migration actions of 8‐OHdG resulted in significant inhibition of EMT, as evidenced by the up‐regulation of ZO‐1 and claudin‐1 and down‐regulation of vimentin. We found significant inhibition of lung metastasis of Panc‐1 cells by 8‐OHdG. In conclusion, exogenous 8‐OHdG had potent anti‐metastasis effects mediated by either ERM or Rho GTPase inhibition in metastasis‐prone pancreatic cancer cells.
Protective Role of Autophagy in NLRP3 Inflammasome Activation and Medial Thickening of Mouse Coronary Arteries
Authors: Bhat Owais , Meng Nan , Yuan Xinxu , Lohner Hannah , Li Pin‐Lan
Published: November 2023

NLRP3 inflammasome is activated by proatherogenic stimulations to result in vascular injury, inflammation, and ultimately atherosclerosis. It remains unknown how this inflammasome activation is endogenously regulated during atherogenesis. The present study hypothesized that autophagy and associated lysosome function serve as a critical modulator in NLRP3 inflammasome activation upon proatherogeneic stimuli. We first detected inflammasome activation in isolated and cultured mouse coronary arterial smooth muscle cells (CASMCs) from wild type (CD38+/+) mice and mice with deletion of CD38 gene (CD38−/−), a gene encoding ADP‐ribosylcyclase that regulates lysosome function and autophagic flux. Western blot analysis showed that cleaved caspase‐1 was much higher in CASMCs from CD38−/− than CD38+/+ mice, and that increased active caspase‐1 led to enhanced IL‐1β release to the cell culture media. In animal experiments, we found increased colocalization of NLRP3 vs ASC or vs Casp‐1 as well as enhanced IL‐1β accumulation in the coronary arterial wall in CD38−/− mice on the Western diet (WD) compared to CD38+/+ mice. This WD‐induced NRLP3 inflammasome formation and activation could be blocked by treatment of rapamycin, an autophagy enhancer. However, chloroquine, a lysosme function inhibitor and blocker of autophagic flux, markedly enhanced WD‐induced NRLP3 inflammasome formation and activation. Morphological examinations confirmed that the media of coronary arteries was significantly thicker in CD38−/− mice on the WD than CD38+/+ mice. This WD‐induced coronary arterial thickening was completely blocked by treatment of mice with rapamycin but enhanced by chloroquine. In conclusion, deficiency of autophagic flux due to CD38 gene deletion promotes NLRP3 inflammasome formation and activation in CASMCs, and rapamycin‐induced autophagy reverses the effects of proatherogeneic stimulation via inhibition of the NLRP3 inflammasome. The efficient autophagy is essential in the control of NLRP3 inflamamsome activation preventing the medial thickening of coronary arteries in response to proatherogenic stimuli.

Support or Funding Information

Supported by NIH grants HL057244, HL075316 and HL122937
Endothelial NLRP3 Inflammasome Activation and Neointima Formation in Mice by Gut Microbial Metabolite Trimethylamine‐N‐Oxide
Authors: Koka Saisudha , Li Pin‐Lan , Zhang Yang , Boini Krishna M
Published: November 2023

Recent studies have demonstrated that trimethylamine‐N‐oxide (TMAO), an intestinal microbial metabolite induces atherosclerosis and enhances the risk for cardiovascular disorders. However, the precise mechanisms of action of TMAO in cardiovascular pathophysiology are yet to be elucidated. In the current study, we examined whether TMAO induces inflammasome activation and thereby contributing to endothelial dysfunction and vascular injury. Confocal microscopic analysis demonstrated the formation of Nlrp3 inflammasomes in the intima of the carotid arteries of Nlrp3+/+ mice but not in Nlrp3−/− mice. Correspondingly, TMAO‐induced increases in IL‐1β production in the carotid intima were abolished in Nlrp3−/− mice. The endothelial tight junction protein, ZO‐1 was reduced by TMAO in Nlrp3+/+ mice, but not in Nlrp3−/− mice. It was also found that TMAO significantly increased neointimal formation in partially ligated carotid arteries of Nlrp3+/+ mice, but not in Nlrp3−/− mice. In in vitro studies, we also found that TMAO treatment significantly increased the colocalization of NLRP3 with Asc or NLRP3 with caspase‐1 in endothelial cells compared to control cells. Pretreatment with Nlrp3 siRNA transfection abolished the TMAO‐induced inflammasome formation in these cells. Correspondingly, TMAO treatment significantly increased the caspase‐1 activity (2.2 folds) and IL‐1β production (1.8 folds) compared to control cells but not in Nlrp3 siRNA transfected cells. Furthermore, western blot analysis showed that TMAO treatment significantly decreased tight junction protein ZO‐1 level compared to control cells. In addition, TMAO treatment significantly increased the cell permeability of EOMA cell layers compared to control cells (Control: 1 ± 0.1 vs TMAO: 2 ± 0.07). Prior treatment with Nlrp3 siRNA attenuated the TMAO‐induced cell permeability. Together, these results suggest that TMAO‐induced formation and activation of NLRP3 inflammasomes in vascular endothelium thereby leading to endothelial dysfunction and atherosclerosis in mice.

Support or Funding Information

DK104031
Differential Effects of Short Chain Fatty Acids on Endothelial NLRP3 Inflammasome Activation and Neointima Formation
Authors: Yuan Xinxu , Wang Lei , Bhat Owais , Lohner Hannah , Zhang Yang , Li Pin‐Lan
Published: November 2023

Short‐chain fatty acids (SCFA) as a family of gut microbial metabolites have been reported to promote preservation of endothelial function and thereby exert anti‐atherosclerotic action preventing cardiovascular diseases. However, the precise mechanisms mediating this protective action of SCFA remain unknown. The present study investigated the effects of SCFAs (acetate, propionate and butyrate) on the activation of Nod‐like receptor pyrin domain 3 (NLRP3) inflammasome in endothelial cells (ECs) and associated carotid neointima formation. In cultured ECs (EOMA), we observed that butyrate significantly decreased the formation and activation of NLRP3 inflammasomes induced by 7‐ketocholesterol (7‐Ket) and cholesterol crystals (CHC) and that propionate only blocked CHC‐induced inflammasome activation. Acetate had no significant inhibitory effects on the NLRP3 inflammasome activation induced by either 7‐Ket or CHC, but itself even activated NLRP3 inflammsomes. Using a mouse model with partial left carotid ligation (PLCA), we demonstrated that butyrate significantly decreased NRLP3 inflammasome formation and activation in carotid arterial wall as shown by reduced co‐localization of inflammasome components (assembling) and lowered active caspase‐1 activity and IL‐1β level in wild type mice (ASC+/+), which was comparable to the inhibition of this inflammasome in mice with knockout of the adaptor protein apoptosis‐associated speck‐like protein gene (ACS−/−). Neverthless, both acetate and propionate markedly enhanced the formation and activation of NLRP3 inflammasome in carotid arteries with PLCA in ASC+/+, but not ASC−/− mice. Correspondingly, byturate suppressed, but acetate and propionate enhanced carotid neointima formation during PLCA in ASC+/+ mice. These results indicate that SCFAs have differential effects on endothelial NRLP3 inflammasome activation and associated carotid neointima formation.

Support or Funding Information

Supported by NIH grants HL057244, HL075316 and HL122937