bluuebunny/crossref_metadata_2025 huggingface dataset:
PAR1 and p38 MAPK Regulation of Endothelial Pro‐inflammatory Responses
Authors: Rada Cara , Grimsey Neil , Canto Isabel , Trejo JoAnn
Published: November 2023

Endothelial cells line the lumen of blood vessels and create a semi‐permeable barrier that is disrupted during vascular inflammation resulting in increased permeability, tissue edema, and subsequent organ failure. Endothelial dysfunction also results in cytokine production and leukocyte recruitment. Thrombin, a coagulant protease, is generated during tissue injury and inflammation and triggers inflammatory responses including endothelial barrier disruption and cytokine production through activation of protease‐activated receptor‐1 (PAR1), a G protein‐coupled receptor. We recently discovered that thrombin activation of PAR1 stimulates p38 MAPK activation through a non‐canonical pathway mediated by TAB1 to promote endothelial permeability. PAR1‐induced p38 activation has been linked to vascular inflammation; however, the mechanism as to how p38 may be integrated into major signaling pathways that promote endothelial inflammatory responses is not known. We found that thrombin‐stimulated p38 functions independent of the classic RhoA activation and MLC phosphorylation pathways but how p38 controls endothelial inflammatory responses is not known. We found that thrombin‐stimulated p38 signals through activation of MAPKAPK2 (MK2), which mediates phosphorylation of HSP27. Interestingly, thrombin/PAR1 activation of MK2 is dependent on p38 MAPK and results in differential phosphorylation of HSP27. The mechanisms by which the p38‐MK2‐HSP27 signaling pathway regulates endothelial pro‐inflammatory responses will be further investigated.

Support or Funding Information

T32 Pharmacological Sciences Training Grant (NIGMS T32 GM007752)
Structural and Functional Studies of Latrophilin‐Family Adhesion G‐Protein Coupled Receptors
Authors: Leon Katherine , Lu Yue Christine , Nazarko Olha , Sando Richard , Salzman Gabriel , Südhof Thomas , Araç Demet
Published: November 2023

Adhesion G‐protein coupled receptors (aGPCRs) contain uniquely large, autoproteolyzed extracellular regions that are involved in cell adhesion and may couple cellular adhesion to receptor signaling. The latrophilin (Lphn/ADGRL) family of aGPCRs has important roles in brain development, such as mediating interactions with fibronectin leucine‐rich repeat transmembrane proteins (FLRTs) in order to form synaptic junctions. There is growing interest in Lphns as promising drug targets because mutations in Lphns are associated with neurological disorders and cancers. To aid in this effort, we used structural and functional approaches to characterize Lphn ligand‐binding and signaling. We present the crystal structure of an extracellular fragment of Lphn3 in complex with FLRT3 and a model of the Lphn3/FLRT3/Uncoordinated‐5 trimeric complex, which provide insight into the role of cell‐adhesion proteins in synapse function. In addition, we developed a signaling assay to better understand the role of autoproteolysis in receptor function.
Adhesion G Protein‐Coupled Receptor Activation by Novel Small Molecule Modulators
Authors: Stoveken Hannah Marie , Smrcka Alan V , Tall Gregory G.
Published: November 2023

Adhesion G protein‐coupled receptors (aGPCRs) are constitutively cleaved during biosynthesis into two‐part receptors that remain non‐covalently bound at the plasma membrane. Self‐cleavage occurs within the GPCR Autoproteolysis Inducing (GAIN) domain, a unit of the large extracellular domain (ECDs), which is closely apposed to the 7‐transmembrane (7TM) domain. The auto‐cleavage generates a tethered agonist that remains concealed within the hydrophobic GAIN domain core. We demonstrated that operative dissociation of the ECD of the aGPCR GPR56 / ADGRG1 resulted in enhanced G protein 13 activation kinetics and that the proposed tethered agonist region is both necessary and sufficient to activate the 7TM. Proposed aGPCR ligands are primarily extracellular matrix (ECM) and anchored cell surface proteins. In vivo receptor activation likely requires cell‐movement mediated shear forces to dissociate the ECDs to permit receptor activation. Understanding the physiological roles these receptors play in development, immunity, and aberrantly in cancer growth and metastasis has been fettered by an inability to recapitulate authentic receptor pharmacology using dilute, soluble matrix proteins. We hypothesized, therefore, that small molecule modulators could be identified to activate or inhibit aGPCRs independent of protein ligand engagement of the ECD. Using the G13‐coupled GPR56 receptor with a truncated tethered agonist, we screened a 2,000 compound library using the Serum Response Element (SRE)‐luciferase gene reporter assay for compounds that functionally replicate the tethered agonist. From this pilot screen we identified two related classes of natural compounds that were capable of activating a subset of aGPCRs but did not active two Class A GPCRs that were tested. The most potent compound identified was a gedunin‐derivative, 3α‐acetoxydihydrodeoxygedunin, which activated the tethered agonist truncated GPR56 in two independent assay platforms with an EC50 of ~5 μM. By examining similar compounds, we were able to perform a structure‐activity relationship to narrow down functional groups that were essential for receptor activation. The aGPCR activators identified and characterized in our studies will serve as invaluable research tools to understand this unique‐class of GPCRs.

Support or Funding Information

RO1GM120110 Mechanism of Adhesion GPCR Action
Chlorpromazine hydrochloride suppresses visceral hypersensitivity to colorectal distension in a rat model of irritable bowel syndrome
Authors: Asano Teita , Takenaga Mistuko
Published: November 2023

Background and Aims

Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder characterized by chronic and recurrent abdominal pain and discomfort associated with altered bowel habits. Although IBS markedly reduces the patient's quality of life, pharmacological therapy has not been established. Visceral hypersensitivity plays an important role in pathogenesis of IBS. In the present study, we screened a compound library consisting of clinically used drugs for their ability to prevent visceral hypersensitivity to colorectal distension (CRD) in rats.

Methods

Visceral sensitivity was monitored by the measurement of the electrical activity of the abdominal external oblique muscle in response to CRD (visceromotor response) in rats using a pressure‐volume control device, barostat. Two hundred nine clinically used drugs including bronchodilators, anticonvulsants, antibiotics, anti‐hypertensives and anti‐allergy drugs were selected for drug screen in rats. The drugs were screened for their ability to suppress visceral sensitivity to repeated CRD. Visceral hypersensitivity to CRD was induced by intra‐colonic administration of sodium butyrate in adulthood or 0.5% acetic acid administration in the early neonatal period.

Results

Chlorpromazine, a typical antipsychotic drug, was identified as a compound that is able to suppress the visceromotor response to repetitive CRD. Oral administration of a 4 mg/kg of chlorpromazine restored visceral hypersensitivity to CRD induced by sodium butyrate or acetic acid to normal condition. This dose of chlorpromazine did not affect visceral sensitivity to CRD and colonic compliance in non‐stressed rats. We also found that quetiapine and risperidone (atyptical psychotic drugs) but not sulpiride and haloperidol (other typical psychotics drugs) suppressed butyrate‐induced colonic hypersensitivity. Chlorpromazine has antagonist action at dopamine D2 receptors and serotonin 2A (5‐HT2A) receptors. From analysis with selective antagonists, kentanserin (a 5‐HT2A receptor antagonist) but not L‐741,626 (a dopamine D2 receptor antagonist) suppressed colonic hypersensitivity induced by butyrate. Furthermore, a peripherally active 5‐HT2A agonist, AL‐34662 produced colonic hypersensitivity in normal rats.

Conclusions

Taken together, our results suggest that chlorpromazine ameliorates visceral hypersensitivity to CRD through the inhibition of 5‐HT2A receptors. Then, the activation of 5‐HT2A receptors may be involved in the development of visceral hypersensitivity. 5‐HT2A receptors could be a molecular target for IBS treatment.

Support or Funding Information

This research received no specific grant from any funding agency in the public, commercial, or not‐for‐profit sectors.
Biological Activity of Resveratrol‐Hydroxycinnamic Acid Ester Conjugates
Authors: Urbaniak Alicja Joanna , Kacprzak Karol , Senol Fatma Sezer , Orhan Ilkay Erdogan , Radominska‐Pandya Anna
Published: November 2023

Resveratrol (3,5,4′‐trihydroxy‐trans‐stilbene, tRV) is one of the most biologically active polyphenols of natural origin. It exhibits many beneficial properties namely antioxidant, anti‐inflammatory, cancer preventing, and cardio‐protective. Despite that, the wide application of tRV, is limited by its poor water solubility, low stability, and consequently, weak bioavailability when administered orally. To overcome this deficiency, our group has developed novel resveratrol‐based derivatives by conjugation with other bioactive compounds such as hydroxycinnamic acid derivatives (p‐, m‐coumaric, ferulic and sinapinic). It is anticipated that conjugation of tRV will result in derivatives with lowered metabolism, higher stability, and altered molecular targets. The developed synthetic method is a four step procedure and leads to the high‐yield production of compounds with high purity. In this work, tRV esters and their unconjugated counterparts were tested in vitro for their biological activity. Experiments were carried out to check their potency to inhibit cholinoesterases, tyrozynaze, elastaze, and collagenase; moreover, their ability as antioxidants and metal chelators were investigated. Resveratryl tri‐p‐coumarate, resveratryl tri‐m‐coumarate, and resveratryl triferulate express significant inhibitory activity towards butyrylcholinesterase, IC50= 29.57 ± 3.33 μg/ml, IC50=32.40±1.29 μg/ml, IC50=36.89 ± 1.05 μg/ml respectively. These values were similar to the IC50 values of reference compound galanthamine HBr. Inhibitory activity towards acetylcholinesterase were slightly lower. tRV conjugates with p‐coumaric and ferulic acid were the most potent in elastase inhibition assay; however, the tyrosynase inhibition study showed more of an effect by resveratryl tri‐p‐coumarate and resveratryl trisinapinate. The unconjugated tRV exhibited the strongest collagenase inhibiting property. tRV ester with sinapinic acid exhibited the highest metal chelating activity. Unconjugated tRV, sinapinic, and ferulic acids exhibited the highest antioxidant activity in both 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) and Ferric‐Reducing Antioxidant Power (FRAP) assays. The properties of free radical scavengers were diminished with the conjugated compounds; however, synthetic esters of those compounds may still be considered as good free radical scavengers.

Support or Funding Information

[NIH/NIDA DA039143 to ARP, Kosciuszko Foundation's Exchange Program to the US to AU]
The ProS Peptide of Porcine Cathelicidins Is an Active Component of Swine Concentrated Animal Feeding Operations (CAFOs) Dust that Mobilizes Calcium and Stimulates Proliferation in Human Airway Smooth Muscle Cells
Authors: Toews Myron L. , Schulte Nancy A. , Tu Yaping , Panettieri Reynold A. , Romberger Debra J. , Tian Geng
Published: November 2023

Components in the dust from swine concentrated animal feeding operations (CAFOs) can induce inflammatory lung disease with features of chronic obstructive lung disease. An aqueous hogbarn dust extract (HDE) induces pro‐inflammatory responses in both airway cells in culture and in mouse airways in vivo, thus providing a starting point for identifying the dust molecules that are involved. Proteomics analysis of an EGF receptor‐activating peak from gel filtration of HDE revealed multiple fragments from the ProS peptide precursor of a subset of porcine anti‐bacterial cathelicidins. Antibodies against ProS confirmed its presence in HDE. The full‐length ProS peptide, without the N‐terminal signal peptide and the C‐terminal anti‐bacterial peptide, stimulated proliferation of human airway smooth muscle (HASM) cells assessed as [3H]thymidine incorporation. Stimulation was concentration‐dependent, with a maximal stimulation of 2‐ to 3‐fold, greater than that with EGF and somewhat less than with the G protein‐coupled receptor (GPCR) mitogen lysophosphatidic acid (LPA). ProS stimulation was blocked by the EGF receptor inhibitor AG1478 and the phospholipase C inhibitor U73122, but not by the Gq inhibitor YM‐254890 or the matrix metalloprotease inhibitor GM6001. ProS also stimulated a concentration‐dependent increase in intracellular calcium that was not blocked by AG1478 but was blocked by U73122 and by the Gq inhibitor YM‐254890. These data indicate that ProS is likely to be a novel Gq‐coupled GPCR agonist that mobilizes calcium in HASM cells via Gq activation but stimulates HASM cell proliferation through a Gq‐independent pathway involving EGF receptor activation. Whether and how the phospholipase C‐dependent calcium response may be related to the phospholipase C‐dependent proliferation response remains to be established. Further studies are in progress to identify the specific receptor and signaling pathways involved. The simultaneous formation of both an anti‐bacterial peptide to fight infection and the ProS peptide to activate EGF receptors to initiate cell proliferation and repair suggests a unique dual role for the cathelicidin precursor protein.

Support or Funding Information

Funded by NIOSH 1RO1‐0H008539‐09 to DJR and UNMC Institutional Funds to MLT
DIFFERENTIAL CARDIOVASCULAR PHYSIOLOGY AND PATHOLOGY IN SELECTED LINEAGES OF MINIATURE SWINE AND COMPARISON TO HUMAN
Authors: Stricker‐Krongrad Alain M , Brocksmith Derek , Bouchard Guy M
Published: November 2023

The miniature swine has been increasingly recognized as a valid alternative to canine and non‐human primates in regulatory toxicity. This poster presents the results of cardiovascular assessments in the Yucatan, Hanford, and Sinclair miniature swine conducted during clinical investigations and control toxicity testing. Anatomic parameters were obtained at necropsy. Blood vessels diameter, velocity, and flow were obtained by Doppler ultrasonography. Cardiac electrophysiology was obtained using clinical ECG and surgical monitor units. Macroscopic lesions and histopathology assessments were conducted on heart and kidneys. Data were compared to published measurements of adult human illustrating similarities or differences (for practicality, male data are reported here). Across the three lineages, heart‐to‐body weights ratio ranged from 0.41 to 0.50 and were higher than human (0.42). The geometric corrections for heart rate adjustment to body size ranged from 215 to 297 and were comparable to human (241), indicating that heart volume and function were well adjusted to the reduction in body size. The miniswine hearts showed a coronary artery distribution comparable to human. The right coronary internal diameters ranged from 1.44 to 1.79 mm and were comparable to human (3.9 mm) when adjusted to body surface area (weight range: 10–30 kg). External femoral blood flows at rest averaged 93 mL/min and were slightly lower than human (260 mL/min) when adjusted to body size. Electrophysiological heart segments duration (e.g. RR ranged from 360 to 662 msec) and their ratio (QT/RR) were proportional to human and well‐adjusted to body size. Macroscopic lesions were nonexistent. Histopathology findings were rare and limited to sub‐level myocardial inflammation with low incidence in the Hanford lineage. In conclusion, the similarities between the cardiovascular systems make these three lineages of miniature swine suitable animals to model the human counterpart. In addition, the differences will aid investigators select a relevant lineage of miniature swine if specific cardiovascular parameters are required.
Modulation of the Aryl Hydrocarbon Receptor Protein Levels by p23 is Hsp90‐Independent
Authors: Pappas Beverly , Cheung Michael , Ngo Katherine , Chang Tiffany , Ng Cory , Chan William K
Published: November 2023

The aryl hydrocarbon receptor (AHR) is a signaling molecule involved in many disease states such as cancers, autoimmune diseases, diabetes, and asthma. AHR is widely known as a xenobiotic‐sensitive transcription factor which is responsible for the cellular effects of numerous environmental toxins and carcinogens such as dioxins and halogenated hydrocarbons. While the majority of our knowledge on regulation of the AHR protein levels is limited to its ligand‐bound state, we are limited in understanding how the cellular AHR protein levels are regulated in the absence of ligand. Previously, our lab has shown that reduction of the p23 protein, which is a component of the AHR cytoplasmic complex, causes an increase in degradation of the AHR protein. Exogenous p23 restores this phenotype, clearly showing that p23 is important in controlling AHR protein degradation. It is well established that p23 interacts directly with heat shock protein 90 (Hsp90). We investigated whether the mechanism of how p23 regulates AHR protein degradation is Hsp90‐dependent. Several p23 mutants (W8A, W86A, W106A, W109A, D108A, E110A, D111A, and D112A) were generated to reduce the interaction between p23 and Hsp90 via site‐directed mutagenesis. Of the mutations, W8A, W86A, and E110A were unable to be purified via affinity chromatography. Mutations D108A (13.31%) and D111A (16.87%) exhibited minimal binding to Hsp90 compared to p23 WT (100%). D108A and D112A (38.95%) were transfected into p23KD HeLa stable cell line. Similar to exogenous transfected p23 WT, these mutants with decreased Hsp90 binding affinity were still able to restore AHR protein levels, suggestive that p23 protects AHR from degradation is a Hsp90‐independent process.
Investigation of Pyrrolizidine Alkaloid‐Induced Pulmonary Toxicity
Authors: Lian Wei , Ma Jiang , Wong Chi‐Wai , Zhu Lin , Lin Ge
Published: November 2023

Introduction

Pyrrolizidine alkaloids (PAs) are widely present in plants, and about half of them had been reported to induce toxicity in different organs. The previous study has proved that PAs exert their toxicity by metabolic activation to form dehydro‐PAs (DHPAs), which interact with proteins to form pyrrole‐protein adducts leading to toxicity. Monocrotaline (MCT) and retrorsine (RTS) are two representative toxic PAs. MCT is known to cause pneumotoxicity, while other PAs, including RTS, induced pneumotoxicity is unknown. The present study aimed to investigate whether other PAs can also cause pneumotoxicity and the mechanism initiating PA‐induced pneumotoxicity.

Methods

MCT and RTS in rat liver and lung microsomal incubation were tested to compare the metabolic site of these two PAs. Then, the transport of DHPAs in blood after a single dose of MCT or RTS in rats was studied by using LC‐MS/MS analysis. DHPAs in blood fractions were detected by addition of glutathione (GSH) to form pyrrole‐GSH conjugate, and the addition of saline was used as the control. The biomarker of PA‐induced toxicity, pyrrole‐protein adducts in liver and lung of MCT or RTS treated rats were also determined. The histological study was performed to examine RTS‐induced pneumotoxicity in rats.

Results

The microsomal incubation results showed that DHPAs were primarily generated in liver. Comparing with the control group, significant increase in the formation of pyrrole‐GSH conjugate was found in GSH‐added red blood cells (RBC) and plasma of both PA‐treated rats. Further, hepatic pyrrole‐protein adducts level in RTS‐treated rats was found about 3‐folds of that in MCT‐treated rats, while pyrrole‐protein adducts levels in lung were similar in both PA‐treated groups. Mild to severe vascular medial hypertrophy was observed along with the time after RTS treatment from 2 days to 4 weeks.

These findings proved that RTS could cause pneumotoxicity. Furthermore, it provided, for the first time, that for both PAs, 1) DHPAs were present in the blood circulation; 2) DHPAs were transported by not only RBC as demonstrated previously but also by plasma; and 3) the transport of DHPAs by both RBC and plasma may be common for different toxic PAs rather than solely for MCT.

Conclusions

Our study delineated the mechanism initiating PA‐induced pneumotoxicity via migration of DHPAs in blood circulation, and also revealed a potential risk of PA‐induced pulmonary toxicity.

Support or Funding Information

This study was supported by RGC HKSAR (GRF Project No. 469712 and 14111816), CUHK Direct Grant (No. 4054215 and 4054302) and CUHK One‐off Funding for Joint Lab/Research Collaboration (Project Code: 3132968)
Global Gene Expression Changes between Human Prenatal and Pediatric Liver
Authors: Vyhlidal Carrie A. , Xiong Min , Heruth Daniel P. , Ye Shui Q.
Published: November 2023

The liver is the largest internal organ in humans and provides multiple functions including bile production, metabolism and detoxification of xenobiotic compounds, glycogen storage, glucose regulation, and production of serum proteins (e.g. albumin, fibrinogen). In adults, the liver is primarily comprised of hepatocytes (70–80% liver mass). However, during prenatal development, the liver serves as the primary site of hematopoiesis and is comprised of hematopoietic stem cells as well as hepatic progenitor cells. Human fetal liver is generally accepted to have limited capacity for the metabolism of xenobiotics. However, some enzymes show preferential expression in fetal liver versus postnatal liver as exemplified by CYP3A7. The objective of this study was to further understand differences in prenatal versus pediatric liver functions utilizing whole‐genome transcriptome analyses. RNA‐seq was performed on 10 prenatal (14.7–16.4 weeks EGA) and 52 pediatric human liver samples that were separated into 4 groups by age (<1 year; 1–<6 years; 6–<12 years; and 12–18 years). Comparisons of differential gene expression between two adjacent age groups were made for transcripts that were expressed in 5 or more samples. Overall, 20355 transcripts were differentially expressed in prenatal or pediatric liver samples of which 9507 (46.7%) transcripts exhibited consistent expression across age groups. The largest number of gene expression changes (13.2% of genes) were observed between prenatal and pediatric liver samples from children <1year of age with 2084 and 606 genes decreasing or increasing between these two groups, respectively. As expected, drug/xenobiotic metabolizing enzymes were among genes differentially expressed between prenatal and postnatal liver including FMO1, ALDH3B1, members of the GST family (GSTA4, GSTP1, GSTO2, HPGDS), and MGST3 that are expressed higher in prenatal liver versus postnatal liver (corrected p<0.05). Pathway analysis with Ingenuity reveals that the top gene network with decreasing expression is DNA Replication, Recombination, and Repair, Cell Cycle, Developmental Disorder, and is typified by the expression of TGFB1 (score=37). The top network with increased expression is Cellular Function and Maintenance, Inflammatory Response, Cellular Development (score=36) and is typified by the gene encoding the glucocorticoid receptor. Global changes in gene expression, including for genes associated with xenobiotic metabolism, are likely due to changes in the function of the liver during the transition from the in utero environment.
Downregulation of intestinal P450 enzymes in a mouse model of ulcerative colitis and the impact on P450‐mediated drug metabolism
Authors: Fan Xiaoyu , Ding Xinxin , Zhang Qing‐Yu
Published: November 2023

Intestinal cytochrome P450 enzymes (P450s) play an essential role in the first‐pass metabolism of orally administered drugs and other xenobiotics. The expression of intestinal P450s can be regulated by many environmental or pathophysiological factors, including tissue inflammation. In the present study, we have explored the impact of inflammation on the expression of intestinal P450s in a mouse model of dextran sulfate sodium (DSS)‐induced colitis. Several P450 enzymes, including CYP1A1, CYP2B, CYP2C, and CYP3A, were downregulated at both mRNA and protein levels in mice treated with DSS (2.5% in drink water for 7 days), along with increased cytokine levels in colon epithelium. The impact of this downregulation on P450‐mediated drug metabolism was also studied. In addition, the expression of several nuclear receptors that are potentially involved in the regulation of intestinal P450 expression was compared between control and DSS‐treated groups. Whereas no difference was detected in the expression of PXR, CAR, or FXR, the expression of several FXR target genes was significantly decreased by the DSS treatment. Interestingly, the expression of VDR and its target genes was also suppressed by the DSS treatment. Thus, both FXR and VDR may be involved in the downregulation of intestinal P450s in DSS‐induced colitis. Further studies on the mechanism of P450 downregulation in the intestine by DSS‐induced colitis are warranted.

Support or Funding Information

Supported in part by NIH grant GM082978
Transcriptional Regulation of Disease‐Drug Interactions
Authors: Xie Wen
Published: November 2023

Drug metabolism and disposition are critical in maintaining the chemical and functional homeostasis of xenobiotics and endobiotics. Accumulating evidence suggests that many hepatic and systemic diseases can affect drug metabolism and disposition by regulating the expression and/or activity of DMEs and transporters in the liver. This presentation will focus on the recent progress in describing and understanding the hepatic injury and sepsis responsive regulation of DMEs in animal models. Liver ischemia and reperfusion (I/R) will be used as a typical example of a hepatic injury model, whereas lipopolysaccharides (LPS) and cecal ligation and puncture (CLP) will be used as the sepsis models. Our results showed that liver I/R and sepsis can affect liver metabolism through the transcriptional regulation of DMEs in the liver. In the meantime, the expression and/or activity of DMEs can affect the disease manifestations of I/R and sepsis. It is hoped that understanding the disease effect on drug metabolism will facilitate the efficient and safe use of drugs in the clinic.

Support or Funding Information

This work is supported in part by the NIH grant ES023438.
Comparison of expression profiles of drug‐metabolizing enzymes between human liver and the hepatic cell lines HepG2, Hep3B, and HuH7: a parallel reaction monitoring (PRM)‐based targeted proteomics study
Authors: Shi Jian , Wang Xinwen , Zhu Hao‐Jie
Published: November 2023

Background

Several human hepatic cell lines have been widely used as an in vitro model for drug metabolism related studies because of their availability and immortalization. However, the validity of this model is a subject of debate as the expression patterns of drug‐metabolizing enzymes (DMEs) in these cells could differ significantly from that of human livers. In the present study, we systematically quantified the basal protein expression of 61 clinically important DMEs in the human liver‐derived cell lines HepG2, Hep3B, and HuH7, and compared the expression profiles to that in human livers.

Methods

With high resolution and high mass accuracy, parallel reaction monitoring (PRM) is a new powerful quantitative proteomics tool, providing wider dynamic range and higher selectivity than selected reaction monitoring (SRM). A PRM‐based targeted proteomics assay was developed for relative quantification of DMEs in the microsomes prepared from human livers and the hepatic cell lines HepG2, Hep3B, and HuH7. The 45 targeted enzymes include 14 cytochromes P450 enzymes (CYPs), 9 UDP‐glucuronosyltransferases (UGTs), and 38 other enzymes relevant to clinical drug metabolism.

Results

With the exception of aldehyde dehydrogenase 1 family member A1 (ALDH1A1), glutathione s‐transferase omega‐1 (GSTO1), and catechol‐O‐methyltransferase (COMT), most of the DMEs are expressed at a substantially lower level relative to that in human livers. In addition, the expression levels of different enzymes vary markedly among the three hepatic cell lines.

Conclusions

Protein expression profiles of DMEs differ significantly between human livers and the hepatic cell lines HepG2, Hep3B, and HuH7. Appropriate caution must be exercised when considering these cell lines for drug metabolism studies, and interpreting the data from the studies based on these cell lines.
Physical Interactions of Cytochrome b5 with Human Cytochrome P450 Enzymes
Authors: Bart Aaron G , Scott Emily E
Published: November 2023

Human cytochrome P450 enzymes metabolize drugs, detoxify environmental compounds, and synthesize many endogenous biomolecules. Binding of the small heme protein cytochrome b5 (b5) can increase, inhibit, or have no effect on P450 catalysis, depending on the specific P450 and/or substrate. Although some evidence points to allosteric modulation of P450 conformations, it is unclear how b5 produces disparate outcomes for different P450 enzymes. Solution NMR was used as a high‐resolution approach to systematically map the surfaces of b5 interacting with several different human P450 enzymes and ascertain the relative affinities of these protein/protein interactions.

NMR analysis of b5 interactions with the xenobiotic‐metabolizing cytochrome P450 enzymes CYP2A6, CYP2D6, and CYP3A4 suggests they interact with varying b5 surfaces. Addition of CYP2A6 caused dramatic differential line‐broadening of resonances for b5 residues in alpha helices 3 and 4, while titrating with CYP3A4 resulted in differential broadening corresponding to residues in alpha helices 2 and 4. In contrast, titrations with CYP2D6 caused minimal line‐broadening of b5 resonances, suggesting a weaker interaction. Only when an excess of CYP2D6 was present were line‐broadening effects observed, primarily for residues in b5 alpha helix 4. To evaluate the importance of these different b5 surfaces in complex formation, key anionic b5 residues were mutated. Consistent with the above observations, the D65N mutation in b5 helix 3 prevented formation of the b5/CYP2A6 complex, but not the b5/CYP3A4 complex. Conversely, the E49Q mutation in b5 alpha helix 2 significantly reduced formation of the b5/CYP3A4 complex, but not the b5/CYP2A6 complex. Ongoing studies include additional human P450 enzymes, but the data already in hand demonstrate that three different P450 enzymes interact in distinct ways with cytochrome b5. These varying P450/b5 interaction modes and relative affinities may underlie or contribute to the differential effects b5 has on P450 catalysis. Thus, functional studies are used to validate the relevance of these P450/b5 interactions using parallel evaluation of substrate oxidation.

Overall, this study provides new insights on protein/protein interactions that modulate the function of a clinically important enzyme superfamily.

Support or Funding Information

National Institutes of Health R37 GM076343, T32 GM08545 Dynamic Aspects of Chemical Biology
Tissue Specific Regulation of CYP26 Enzymes
Authors: Tripathy Sasmita , Isoherranen Nina
Published: November 2023

all‐trans‐retinoic acid (atRA) regulates many biological processes via RAR and PPAR mediated pathways. The metabolism and clearance of atRA is mediated by the CYP26A1 and CYP26B1 enzymes, that both appear to be critical retinoic acid hydroxylases during fetal development and adult life contributing to tissue specific regulation of atRA concentrations and signaling. CYP26A1 and CYP26B1 have different tissue specific expression both in adults and during development, and distinct switches occur between fetal expression patterns and adult tissues for both CYP26A1 and CYP26B1. In human fetal tissues CYP26A1 is exclusively expressed in brain, whereas CYP26B1 is not present in brain but found in other tissues. In adults, CYP26A1 is predominantly expressed in liver, whereas CYP26B1 is predominantly found in cerebellum and ubiquitously expressed in many other tissues such as adipose, testes, lymphocytes and kidney. While both CYP26A1 and CYP26B1 expression is believed to be regulated by atRA, the mechanisms that lead to distinct tissue specific expression and regulation of the CYP expression are not known. The aim of this study was to examine the tissue specific regulation of CYP26A1 and CYP26B1 by RAR and PPAR using various models of human liver, adipose and immune cells that reflect tissues specifically expressing CYP26A1 or CYP26B1. Our data revealed that constitutively CYP26A1 is predominantly expressed in liver cells, whereas CYP26B1 is found in adipocytes and lymphocytes. Similarly, CYP26A1 was highly inducible by atRA in liver cells while CYP26B1 was induced in adipocytes and lymphocytes. siRNA mediated knock down of nuclear receptors showed that CYP26A1 mRNA expression is solely regulated by RARα/β, whereas CYP26B1 expression is mainly regulated by PPARγ. These data suggest that the tissue specific expression of CYP26 enzymes is regulated by different nuclear receptors acting on the CYP26 promoter and that the two CYP26 enzymes will respond differently to endogenous and xenobiotic regulators of CYP expression. In conclusion, these findings may explain the different phenotypes observed after loss of CYP26A1 or CYP26B1 activity.

Support or Funding Information

Funding: NIH R01 GM111772
Physical complexes formed by CYP1A2 and CYP2B4 are associated with altered P450 activity
Authors: Connick John Patrick , Reed James Robert , Backes Wayne L
Published: November 2023

It has been demonstrated that the presence of one P450 can affect the activity of another. Previous studies have shown, for example, that when CYP1A2, CYP2B4, and stoichiometrically‐limiting concentrations of NADPH‐cytochrome P450 reductase (CPR) were incorporated into phospholipid vesicles, CYP1A2 activity was higher and CYP2B4 activity was lower than observed in single‐P450 systems. These data are inconsistent with a model of simple competition among P450s for CPR binding but could be caused by physical interactions among the pair of P450s. The goal of this work was to determine how physical interactions between CYP1A2 and CYP2B4 might play a role in modulating the P450‐CPR interaction and P450 activity. Various pairs of P450 and CPR constructs were tagged with one each of GFP and Renilla luciferase and transfected into HEK293T cells. Complexes between these proteins were detected by measuring energy transfer between the tags. A complex between CYP1A2 and CYP2B4 was detected both in the presence and absence of cotransfected, untagged CPR. Interactions of both P450s with CPR were detected, and untagged CYP1A2 was shown to disrupt the CPR‐CYP2B4 interaction while untagged CYP2B4 had no effect on the CPR‐CYP1A2 interaction. These data are consistent with a model in which CPR preferentially binds to the CYP1A2 moiety of a CYP1A2‐CYP2B4 complex. Previous work has also shown—using BRET, crosslinking, and kinetic measurements—that CYP1A2 forms homomeric complexes associated with inhibition of CYP1A2 activity at subsaturating CPR. Here it is shown using BRET that these complexes were disrupted by the presence of either CPR or CYP2B4. The activity of GFP‐tagged CYP1A2 and CYP2B4 co‐expressed in HEK293T cells was measured and shown to be consistent with data from the prior experiments on purified proteins. These findings explain the observed effects of CYP1A2 and CYP2B4 on each other's activity.

Support or Funding Information

NIH ES004344

NIH ES013648

Louisiana Board of Regents
Induction of Apoptosis and Generation of Reactive Oxygen Species by Novel Chalcone Derivatives
Authors: Stang Christopher R.T. , Adams Bryce R. , Faler Ashton D. , Schuler Brian R. , Eichel Amanda M. , Dick Matt J. , Orahood Cassandra B. , Whited Tawna L. , Rao P.S.S. , Dudley Richard W. , Khupse Rahul S. , Schneider Ryan A.
Published: November 2023

The presence of alpha‐beta unsaturated systems in naturally occurring flavonoids has been attributed to a diverse array of physiological activities. We previously reported on the anti‐cancer activity of several chalcone and flavanone derivatives from our small flavonoid library. Here we present the anti‐cancer effects of our two newest chalcone derivatives, RK15 and RK19, in four diverse human cancer cell lines: A‐172 glioblastoma, A375 melanoma, HT‐29 colon carcinoma, and MCF7 breast adenocarcinoma. The XTT cell proliferation assay revealed that RK15 was the most potent chalcone derivative in our library with IC50 values of 3, 4, and 6 μM respectively in A375, A‐172, and HT‐29 cells. Interestingly, RK15 was much less potent in MCF7 cells, with an IC50 of 85 μM. The second chalcone derivative, RK19, was much less potent than RK15, with IC50 values ranging from 56 to 95 μM in these four cell lines.

We investigated the mechanism of cell death elicited by these compounds using the Caspase‐Glo 3/7 assay. RK19 induced executioner caspase‐3/7 activity in A‐172, A375, and HT‐29 cells. An increase in caspase‐3/7 activity was also noted following RK15 treatment in A‐172 cells but interestingly not in HT‐29 cells. A reversal experiment using the pan‐caspase inhibitor Z‐VAD‐FMK was utilized to confirm the results from the Caspase‐Glo 3/7 assay. Since others have shown that chalcones can generate reactive oxygen species (ROS), we hypothesized that RK15 may lead to apoptosis through ROS generation. A DCFDA plate‐based assay was utilized to measure ROS generation following RK15 treatment. RK15 modestly increased ROS levels in A375 and HT‐29 cells, but not in A‐172 cells. These results suggest that though RK15 may increase ROS in some instances, an alternate mechanism of action is likely involved in the observed apoptotic effect.
Autophagy Activation by Leptin Mediates the Growth of Breast Cancer Cells via Estrogen Receptor Signaling: Involvement of AMPK/FoxO3A Axis
Authors: Raut Pawan Kumar , Pun Nirmala Tilija , Kim Eun Hye , Khakurel Amrita , Oh Hye‐Jin , Shrestha Aastha , Park Pil‐Hoon
Published: November 2023

Leptin, a hormone predominantly produced in adipose tissue, induces growth of cancer cells via inhibition of apoptosis and/or induction of the cell cycle progression. Previous studies demonstrated that estrogen receptor signaling plays an important role in the growth of breast cancer cells. Similarly, autophagy induction by leptin plays a critical role in the growth cancer cells. However, the role of estrogen receptor signaling in relation to leptin‐induced autophagy activation, mediating the growth of breast cancer cells, has not been explored yet. In this study, we examined the role of estrogen receptor signaling in leptin‐induced autophagy activation, which mediates the growth of breast cancer cells, and the potential underlying mechanisms. In this study, we found that leptin significantly induced the growth and proliferation of estrogen receptor positive breast cancer cells (MCF‐7), whereas no significant effects were observed in estrogen receptor negative breast cancer cells (MDA‐MB‐231). Furthermore, leptin‐induced growth of MCF‐7 cells was significantly inhibited by pretreatment with tamoxifen, a selective estrogen receptor modulator, and gene silencing of estrogen receptor alpha, indicating that estrogen receptor signaling plays an important role in leptin‐induced growth of MCF‐7 cells. Moreover, pretreatment with tamoxifen and gene silencing of estrogen receptor alpha significantly suppressed leptin‐induced increase in the expression of autophagy‐related genes, including microtubule‐associated protein light chain 3‐II (LC3 II), Atg5, beclin‐1, and autophagosome formation and induction of autophagic flux, indicating the critical role of estrogen receptor signaling in leptin‐induced autophagy activation in MCF‐7 cells. In addition, leptin‐induced growth of MCF‐7 cells was significantly inhibited, on pretreatment with 3‐Methyladenine (3‐MA), a pharmacological inhibitor of autophagy, implicating the role of leptin‐induced autophagy activation in the growth of breast cancer cells. Moreover, leptin‐induced phosphorylation of AMPK was significantly inhibited by tamoxifen pretreatment and leptin‐induced FoxO3A expression was significantly suppressed by pretreatment with Compound C, a pharmacological inhibitor of AMPK, indicating that estrogen receptor signaling modulates AMPK/FoxO3A axis, which has been shown to play a critical role in leptin‐induced autophagy induction, in breast cancer cells. Based on these data, it can be concluded that estrogen receptor signaling plays a critical role in leptin‐induced autophagy activation via modulation of AMPK/FoxO3A axis in breast cancer cells.
Modulation of Autophagy Proteins in Response to a Compound Designed as a Sigma‐2 Receptor Agonist Derived from Tamoxifen
Authors: Stang Christopher R.T. , Adams Bryce R. , Faler Ashton D. , Schuler Brian R. , Kinder David H. , Sabol Patrick , Carlston Leah , Schneider Ryan A.
Published: November 2023

The selective estrogen receptor modulator tamoxifen has been shown to have anti‐cancer effects in cells that lack estrogen receptor expression. It is thought that these effects are mediated through sigma‐2 receptor agonism. We previously reported the anti‐cancer effects of a novel tamoxifen derivative, SP‐1, against three diverse human cancer cell lines. SP‐1 was the first in a small library of compounds designed to target sigma‐2 receptors while lacking structural characteristics required for estrogen receptor binding. The cytotoxic effects of SP‐1 were determined to be caspase‐independent. Here we present further data on the anti‐cancer effects of SP‐1 in MCF7 breast adenocarcinoma cells along with further pharmacological investigation in HT‐29 colon adenocarcinoma, A375 melanoma, and A‐172 glioblastoma cells. The IC50 of SP‐1 in MCF7 cells was determined to be 31 μM using the XTT cell proliferation assay. This represents a fourth cancer cell line in which SP‐1 displays anti‐cancer activity with an IC50 in the 25 to 40 μM range. Western blotting using lysates from MCF7 cells was negative for cleavage of caspase‐7 and poly[ADP‐ribose]polymerase (PARP), suggesting yet again a caspase‐independent mechanism for SP‐1. Additionally, the pan‐caspase inhibitor, Z‐VAD‐FMK, failed to prevent cell death following SP‐1 treatment in all four cell lines.

Given the lack of caspase involvement, we sought to determine whether autophagy was involved. Treatment with SP‐1 at 50 μM modulated several autophagy‐related proteins including phospho‐AKT (serine 473), light chain 3A/B (LC3A/B) and p62. Phosphorylation of the pro‐survival protein AKT was noted in the A‐172, HT‐29, and MCF7 cell lines at 24 hours following SP‐1 treatment. SP‐1 also elicited an increase in p62 expression in all four cell lines while conjugation of cytosolic LC3A/B I to autophagosome‐associated LC3A/B II was noted in three of the four cell lines. These results suggest the involvement of autophagy‐related machinery in the cell's response to SP‐1 treatment. Further investigations into autophagic flux and whether the autophagic response induced by SP‐1 is cytotoxic or cytoprotective are underway.
A constitutive androstane receptor (CAR) inhibitor CINPA1 as a tool to understand receptor structure and function
Authors: Cherian Milu T , Chai Sergio C , Casal Morgan A , Chen Taosheng
Published: November 2023

This study aims to use CINPA1, a recently discovered small molecule inhibitor of the xenobiotic receptor CAR (constitutive androstane receptor), for understanding the binding modes of CAR and to guide CAR‐mediated gene expression profiling studies in human primary hepatocytes.

CAR and PXR are xenobiotic sensors that respond to drugs and endobiotics by modulating the expression of metabolic genes that enhance detoxification and elimination. Elevated levels of drug metabolizing enzymes and efflux transporters resulting from CAR activation promote the elimination of chemotherapeutic agents leading to reduced therapeutic effectiveness. Multidrug resistance in tumors after chemotherapy could be associated with errant CAR activity, as shown in the case of neuroblastoma. CAR inhibitors used in combination with existing chemotherapeutics could be utilized to attenuate multidrug resistance and resensitize chemo‐resistant cancer cells. CAR and PXR have many overlapping modulating ligands as well as many overlapping target genes which confounded attempts to understand and regulate receptor‐specific activity. Through a directed screening approach we previously identified a new CAR inhibitor, CINPA1, which is novel in its ability to inhibit CAR function without activating PXR.

The cellular mechanisms by which CINPA1 inhibits CAR function were also extensively examined along with its pharmacokinetic properties. CINPA1 binding was shown to change CAR‐coregulator interactions as well as modify CAR recruitment at DNA response elements of regulated genes. CINPA1 was shown to be broken down in the liver to form two, mostly inactive, metabolites. We used the structure‐activity differences of CINPA1 and its metabolites to guide computational modeling using the CAR‐LBD structure. To rationalize how ligand binding may lead to different CAR pharmacology we performed an analysis of the docked poses of human CAR bound to an activator (CITCO) vs our inhibitor or the metabolites. From our modeling, we predicted strong hydrogen bonding of CINPA1 with N165 and H203 in the CAR‐LBD. We validated these residues to be important for CINPA1 binding using CAR single amino‐acid mutants in a functional reporter assay. Also predicted were residues making key hydrophobic interactions with CINPA1 but not the inactive metabolites. We identified some of these hydrophobic amino‐acids and additionally, determined the differential coregulator interactions of these mutants in mammalian two‐hybrid systems.

We demonstrate that CINPA1 represents an excellent starting point for future optimization into highly relevant probe molecules to study the function of the CAR receptor in normal‐ and patho‐physiology, and possible development as therapeutics (e.g. for resensitizing chemoresistant neuroblastoma cells.)

Support or Funding Information

St. Jude Children's Research Hospital, ALSAC, and the NIH.