bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of GPCRs and G‐αs in the antidepressant action of ketamine
Authors: Wray Nathan , Schappi Jeffrey , Rasenick Mark M
Published: November 2023

Ketamine produces rapid and robust antidepressant effects in depressed patients within hours of administration, often when traditional antidepressant compounds have failed to alleviate symptoms even after 2 months of treatment. We hypothesized that ketamine would translocate Gas from lipid rafts to non‐raft microdomains, similarly to other antidepressants but with a distinct, abbreviated treatment duration. C6 glioma and C6 cells stably transfected with G‐αs‐GFP were treated with 10uM ketamine for 15 minutes, which translocated Gas from lipid raft domains to non‐raft domains. Other NMDA antagonist did not translocate Gas from lipid raft to non‐raft domains. The ketamine induced Gas plasma membrane redistribution allows increased functional coupling of Gas and adenylyl cyclase to increase intracellular cyclic adenosine monophosphate (cAMP). Furthermore, increased intracellular cAMP increased phosphorylation of cAMP response element‐binding protein (CREB), which, in turn, increased BDNF expression. These results reveal a novel antidepressant mechanism mediated by acute ketamine treatment in glial cells that may contribute to ketamine's powerful antidepressant effect.
Leveraging a Novel ITIM Motif in GPCRs for Targeted Antibody Design
Authors: Belcastro Lili T. , Jancina Anastasia , Adams Christina , Paulukinas Ryan D. , Moore Catherine C.
Published: November 2023

CXCR4, a chemokine GPCR, is essential for migration of neuronal, hematopoietic, and breast cancer cells during metastasis whereby CXCR4 dysregulation promotes migration and invasion. Following SDF stimulation, CXCR4 is phosphorylated on Ser/Thr residues which initiates adaptor recruitment, receptor desensitization, and trafficking to endocytic sites. Here we show that stimulation with gradient SDF, delays receptor phosphorylation and trafficking, leading to sustained signaling to a novel CXCR4‐SHP2‐ERK pathway. SHP2 is a tyrosine phosphatase implicated in HER2(+) and triple‐negative breast cancers, whereby it transduces mitogenic and migratory signals driving hyperproliferation and invasion. SHP2 is recruited to tyrosine phosphorylated ITIM motifs (immmunoreceptor tyrosine‐based inhibitory consensus motifs), a hallmark found in inhibitory immune receptors with little evidence in GPCRs. Here we identify an ITIM motif in CXCR4 that regulates both SHP2 binding and signaling. Specifically, we assessed if gradient SDF stimulation of CXCR4 1) delays receptor phosphorylation and trafficking, 2) sustains signaling to SHP2‐ERK, 3) induces SHP2‐dependent migration; and if CXCR4 Tyr mutation within the ITIM motif 4) maintains SDF gradient sensing ability, and 5) disrupts interaction with and signaling to SHP2. Our data demonstrate that gradient SDF delays receptor Ser/Thr phosphorylation and internalization thereby sustaining signaling to SHP2‐ERK and driving SHP2‐dependent migration. Furthermore, the ITIM mutant maintains SDF gradient sensing ability, but disrupts interaction with and signaling to SHP2. Our data support a working model that CXCR4 contains a functional ITIM motif which we are currently leveraging for targeted antibody design for use in migration studies of aggressive breast cancer cells with dysregulated CXCR4.

Support or Funding Information

These studies were supported by NIH grant GM‐097718, PA Department of Health grant SAP4100057688, and the Milton Lev Memorial Faculty Research Fund.
Optimization of a Novel D2 Dopamine Receptor Antagonist Scaffold Reveals Exceptionally Selective and Potent Lead Molecules
Authors: Day Marilyn M. , Dulcey Andres E. , Bayron Diane , Stang Christopher R.T. , Moritz Amy E. , Doyle Trevor , Javitch Jonathan A. , Southall Noel T. , Ferrer Marc , Marugan Juan J. , Sibley David R. , Free R. Benjamin
Published: November 2023

FDA‐approved antipsychotics are primarily D2 dopamine receptor (D2R) antagonists, however they generally lack receptor selectivity and interact with other GPCRs, including D3Rs. Despite its clear clinical importance, there are few compounds that selectively modulate the D2R. This is due to structural similarities in the orthosteric binding sites of the D2‐like receptors (D2R, D3R, and D4R), as well as other biogenic amine receptors. To find better therapeutic candidates, as well as probe compounds to investigate D2R pathophysiology, we sought to identify highly selective D2R antagonists by implementing a high‐throughput screening (HTS) campaign using more than 400,000 unique compounds in the Molecular Libraries Screening Center Network library. Most of the hit compounds that were identified as antagonists of the D2R were also antagonists of the closely related D3R. However, several compounds were found to exhibit high affinity for the D2R with low affinity for the D3R. One such compound (MLS6916) served as our lead scaffold for optimization. MLS6916 is ~700 fold selective for the D2R vs. D3R as determined in functional β‐arrestin recruitment assays and radioligand binding competition assays. Importantly, MLS6916 was also examined for functional activity at 168 different GPCRs and showed no agonist activity on any GPCR tested, and only exhibited antagonist activity at the D2‐like receptors. Preliminary investigations, however, revealed that this initial hit compound was metabolically unstable. To optimize this hit scaffold, we conducted an iterative medicinal chemistry campaign with the goals of increasing selectivity, potency, and engendering metabolic stability. Over 70 analogs of the MLS6916 parent compound were synthesized to dissect the influence of various constituents of the molecule and to construct structure‐activity relationships. Analogs were analyzed for activity on D2R, D3R, and D4R using both radioligand binding and functional assays to determine affinities and potencies for the D2‐like receptors. In addition, analogs were tested for solubility, permeability, and stability. Several lead compounds were identified with Ki values of < 50 nM for the D2R with > 1,000‐fold selectivity vs. the D3R. These selectivity trends were confirmed in functional β‐arrestin recruitment assays. Importantly, lead compounds with enhanced metabolic stability and permeability were identified. Further analysis and optimization of these lead compounds will provide new insight into highly selective D2R antagonists for the treatment and understanding of dopamine‐related disorders.

Support or Funding Information

NINDS Intramural Research Program
Priming GPCR Signaling Through the Synergistic Effect of Two G Proteins
Authors: Gupte Tejas M. , Malik Rabia U. , Sommese Ruth F. , Ritt Michael , Sivaramakrishnan Sivaraj
Published: November 2023

While individual GPCRs are known to activate one or more G proteins, the GPCR‐G protein interaction is viewed as a bi‐molecular event involving the formation of a ternary ligand‐GPCR‐G protein complex. We have designed a FRET‐based sensor to monitor and modulate GPCR – G protein interactions in live cells. Using this systematic protein affinity strength modulation (SPASM) approach we present evidence that individual GPCR‐G protein interactions can reinforce each other to enhance signaling through canonical downstream second messengers, a phenomenon we term GPCR priming. Specifically, we find that the presence of non‐cognate Gq protein enhances cAMP stimulated by two Gs coupled receptors, β2‐adrenergic receptor (β2‐AR) and D1 dopamine receptor (D1‐R). The C‐terminus of either the Gαs or Gαq subunit is sufficient to enhance Gα subunit activation and cAMP levels. Interaction of Gαs or Gαq C‐termini with the GPCR increases signaling potency suggesting an altered GPCR conformation as the underlying basis for GPCR priming. We propose three parallel mechanisms involving (1) sequential G protein interactions at the orthosteric site, (2) simultaneous allosteric and orthosteric G protein interactions, and (3) asymmetric GPCR dimers. GPCR priming suggests another layer of regulation in the classic GPCR ternary complex model, with broad implications for the multiplicity inherent in signaling networks.

Support or Funding Information

Research was funded by the American Heart Association Scientist Development Grant (13SDG14270009) & the NIH (1DP2 CA186752‐01 & 1‐R01‐GM‐105646‐01‐A1) to S.S. T.M.G is an NCBS‐inStem Career Development Fellow. R.F.S is a Life Sciences Research Foundation postdoctoral fellow.
Upregulation of RGS2: A New Mechanism for Pirfenidone Amelioration of Pulmonary Fibrosis
Authors: Xie Yan , Jiang Haihong , Zhang Qian , Mehrotra Suneet , Abel Peter W. , Toews Myron L. , Wolff Dennis W. , Rennard Stephen , Panettieri Reynold A. , Casale Thomas B. , Tu Yaping
Published: November 2023

Background

Pirfenidone was recently approved for treatment of idiopathic pulmonary fibrosis. However, the therapeutic dose of pirfenidone is very high, causing side effects that limit its doses and therapeutic effectiveness. Understanding the molecular mechanisms of action of pirfenidone could improve its safety and efficacy. Because activated fibroblasts are critical effector cells associated with the progression of fibrosis, this study investigated the genes that change expression rapidly in response to pirfenidone treatment of pulmonary fibroblasts and explored their contributions to the anti‐fibrotic effects of pirfenidone.

Methods

We used the GeneChip microarray to screen for genes that were rapidly up‐regulated upon exposure of human lung fibroblast cells to pirfenidone, with confirmation for specific genes by real‐time PCR and western blots. Biochemical and functional analyses were used to establish their anti‐fibrotic effects in cellular and animal models of pulmonary fibrosis.

Results

We identified Regulator of G‐protein Signaling 2 (RGS2) as an early pirfenidone‐induced gene. Treatment with pirfenidone significantly increased RGS2 mRNA and protein expression in both a human fetal lung fibroblast cell line and primary pulmonary fibroblasts isolated from patients without or with idiopathic pulmonary fibrosis. Pirfenidone treatment or direct overexpression of recombinant RGS2 in human lung fibroblasts inhibited the profibrotic effects of thrombin, whereas loss of RGS2 exacerbated bleomycin‐induced pulmonary fibrosis and mortality in mice. Pirfenidone treatment reduced bleomycin‐induced pulmonary fibrosis in wild‐type but not RGS2 knockout mice.

Conclusions

Endogenous RGS2 exhibits anti‐fibrotic functions. Upregulated RGS2 contributes significantly to the anti‐fibrotic effects of pirfenidone.

Support or Funding Information

This work was supported by NIH grant R01HL116849 to Y. Tu and T.B. Casale; R01HL097796, P01 HL114471 and P30 ES013508 to R.A. Panettieri; Nebraska State LB595 grant to Y. Tu and P.W. Abel; and the NIH National Center for Research Resources G20‐RR024001.
Functional Mechanisms of Novel G Protein Inhibitor Ligands As Vasodilators
Authors: Meleka Matthew M , Medcalf Matthew , Moeller Kevin D , Osei‐Owusu Patrick
Published: November 2023

G‐protein coupled receptor (GPCR) signaling plays an essential role in physiologic homeostasis and cardiovascular health. Dysfunction of GPCR pathway is implicated in hypertension, a leading risk factor for cardiovascular diseases. Here, we examined the functional properties of novel Gq inhibitor ligands, YM‐254890, FR900359, and WU‐07047, and determined their effectiveness at blocking GPCR signaling in the resistance vasculature. Under ex‐vivo pressurized vessel preparation, we measured vascular reactivity of small mesenteric arteries isolated from 3–4 month old wild type mice. Vasoconstriction of mesenteric arteries was evoked by applying increasing concentrations of the α1‐adrenergic receptor agonist, phenylephrine (PE), in the presence or absence of Gq inhibitors. We found that WU‐07047 had the least effect on maximal contractile response to PE (FR[0.01 μM]: 0.8 ± 0.6% vs. YM[0.01 μM]: 12.8 ± 2.8% vs. WU[10 μM]: 34.8 ± 3.2% %), whereas FR was the most potent (FR[0.01uM]: LogIC50 −0.008 ± 0 vs. YM[0.01uM]: LogIC50 −0.49 ± 0 vs. WU: LogIC50 −64.95 ± 6.4). WU‐07047 and YM‐254890 inhibited vasoconstriction through G protein‐independent pathway by blocking L‐type calcium channels (LTCCs). Bioinformatics analysis revealed a high sequence similarity between binding sites for dihydropyridines in the pore‐forming subunit of LTCCs and the binding pocket for the novel Gq inhibitor ligands in Gqα subunit. We conclude that the novel Gq inhibitors decrease vascular tone by blocking G protein‐dependent and G protein‐independent signaling mechanisms mediating resistance artery vasoconstriction. These findings suggest that targeting signaling downstream of GPCRs may be a more effective therapeutic approach to managing hypertension and associated cardiovascular diseases.

Support or Funding Information

Pennsylvania Dept. of Health (CURE) Grant (PO)

Margaret Q. Landenberger Research Grant (PO)

AHA Scientist Development Grant (PO)

National Science Foundation (CBET 1262176) (KDM)
Molecular Characteristics of FBXO44‐Mediated RGS2 Protein Degradation
Authors: Sjogren Benita
Published: November 2023

Hypertension and heart failure are major health issues and there is a need to identify more effective treatments. Regulator of G protein Signaling 2 (RGS2) is highly expressed throughout the cardiovascular system and negatively regulates signaling via several G protein‐coupled receptors known to mediate vasoconstriction and hypertrophic responses. As a result, RGS2−/− mice are hypertensive, show enhanced responses to vasoconstrictors and lower tolerability to pressure overload. Conversely, over‐expression of RGS2 reduces cardiac hypertrophy and we have shown that pharmacologically enhanced RGS2 protein expression has functional effects on G protein signaling both in vitro and in vivo. Therefore, we hypothesize that enhancing RGS2 protein expression is a novel route in treating cardiovascular disease.

RGS2 has a short protein half‐life due to rapid degradation through the ubiquitin‐proteasomal pathway. Thus, identifying the specific molecular mechanisms of this intricate system involved in RGS protein degradation will enable rational drug discovery efforts to develop small molecule stabilizers of RGS2 protein. In our previous work, we identified a multi‐component Cullin 4B/DNA Damage Binding protein 1/F‐box protein 44 (CUL4B/DDB1/FBXO44) E3 ligase responsible for mediating RGS protein degradation through high‐throughput siRNA screening. This work is now continuing by defining the molecular characteristics of this complex using biochemical approaches. FBXO44 belongs to a family of over 50 F‐box proteins that confer substrate recognition in Cullin‐RING E3 ligases. In our previous work, we demonstrated that FBXO44 associates with CUL4B and DDB1 through its conserved F‐box domain, in agreement with the canonical action of F‐box proteins. FBXO44 associates with RGS2 through its F‐box Associated (FBA) domain, which is much more variable.

FBXO44 is closely related to FBXO2, however it does not bind the same substrates. Based on the homology between FBXO44 and FBXO2, as well as the known substrate recognition sites in FBXO2, we used site‐directed mutagenesis to identify the substrate recognition sites for RGS2 in FBXO44. Mutations in non‐conserved regions of the FBA domain revealed key interaction sites with RGS2. Ongoing work includes defining the corresponding residues in RGS2, responsible for FBXO44 binding, as well as investigating the role of posttranslational modifications and other mechanisms responsible for targeting RGS2 for protein degradation. These studies will aid in rational design of small molecule stabilizers of RGS2 protein that could serve as novel therapeutics in disease states associated with low RGS2 protein levels.

Support or Funding Information

American Heart Association (AHA 15SDG21630002)
Computational Prediction of Off‐Target Pharmacology for Discontinued Drugs
Authors: Rao Mohan , Liguori Michael , Mantena Srinivasa , Mittelstadt Scott , Blomme Eric , Van Vleet Terry
Published: November 2023

Most small molecule drugs interact with unintended, often unknown targets that lead to a wide range of undesirable adverse effects in both pre‐clinical and clinical development and that result in safety‐related attrition. Undesired off‐target interactions are generally weak and quite often not detected using current drug discovery assays. Thus, improvement in the early identification of off‐target interactions is critical to reduce the drug attrition rates related to toxicities. In order to better identify potential off‐target interactions that could be linked to predictable safety issues, we have evaluated and designed a novel computational process to predict safety‐relevant low and high‐affinity interactions that are currently not covered in experimental polypharmacology screens. The result of these analyses termed Off Target Safety Assessment (OTSA) assesses more than 1500 mechanisms of action (target + actions). This approach exploits a highly curated training set of about 10 million compounds, with known in‐vitro activities, derived from patents, journals and internally installed external databases. To evaluate the tool, we used a panel of computational tools to predict both primary and secondary pharmacology for 1279 discontinued drug molecules. A total of 33,513 possible interactions were predicted for these drugs, of which 19,513 interactions (i.e. 15 interactions/compound) were associated with known safety warnings/issues at various levels. The top common safety associated off‐targets for these discontinued drugs are i) Adenosine receptor's ii) Opioid receptor's iii) PPAR's iv) PDE's v) Adrenoceptor's and vi) Potassium channel's. The computational OTSA's most often identified the right pharmacological targets, but also predicted a significant number of off‐targets that may be relevant to the observed in‐vivo effects.

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.
Sub Acute Effect of Pirimiphos Methyl (Actellic 2% Dust) On Oxidative Stress in Albino Rats
Authors: Nosiri Chidi Ijeoma , Anyanwu Chukwuma , Ibeh Anderline Ogochukwu
Published: November 2023

The subacute effect of Pirimiphos Methyl on oxidative stress in albino rats was evaluated after two weeks of treatment in albino rats. This is an organophosphate insecticide (OPIs) used in the preservation of grains in Nigeria. Occupational exposure is associated with acute intoxication. Populations can be exposed through food and water. (OPIs) may induce oxidative stress leading to generation of Reactive Oxygen Species (ROS) and alteration in antioxidant system The animals were divided into 4 groups of 5 rats each, group 1 serving as the control (n‐saline), group 2(10mg/kg per os Actellic 2% dust), groups 3 and 4 given 20mg/kg and 40mg/kg per os of the test sample respectively. The antioxidant status of the stress enzymes Superoxide Dismutase (SOD), Glutathione (GSH), Catalase (CAT) and Glutathione Peroxidase(GPx) were evaluated. The results showed a dose dependent decrease in GSH, CAT and GPx levels while the SOD showed a dose dependent increase in activity level when compared with the control. The 20 and 40mg/kg of Pirimiphos methyl were statistically significant (P≤0.05) with the control in the activity levels of GSH and GPx. It may be concluded that in vivo oral administration of Pirimiphos methyl altered the antioxidant enzyme activity or defense system of the gastro intestinal tract in albino rats suggesting that ROS may be involved in its sub acute toxicity.
THE ANTI‐APOPTOTIC EFFECT OF METHANOL LEAF EXTRACT OF AZADIRACHTA INDICA ON LEAD ACETATE‐INDUCED APOPTOSIS IN CARDIAC AND RENAL TISSUES OF MALE WISTAR RATS
Authors: Adeyeoluwa Temitayo Esther , Oyagbemi Ademola Adetokunbo , Adedapo Adeolu Alex , Omobowale Temidayo Olutayo
Published: November 2023

Lead toxicity is an existing and ongoing challenge. The apoptotic effects of lead have been extensively studied and several herbal remedies have been proposed. In this study, the anti‐apoptotic effect of the methanol extract of Azadirachta indica (MEAI) was investigated.

70 adult male Wistar rats were divided into 7 groups. Groups B, C and D were exposed orally to 1.0 mg/ml lead acetate and groups E, F and G exposed to 2.0 mg/ml for 6‐week duration, after which groups C and D were given 100 mg/kg and 200 mg/kg Azadirachta indica methanol leaf extract respectively, groups F and G given 100 mg/kg and 200 mg/kg Azadirachta indica methanol leaf extract respectively, and groups B and E withdrawn from lead treatment for another 6 weeks. Group A, the control group, was given clean drinking water for the duration of 12 weeks.

Exposure to 1.0 mg/ml and 2 mg/ml lead (Pb) resulted in an increased expression of caspase 3 and cytochrome C, which were seen to increase with increased concentration of exposure while there was a reduction in the expression of Bcl‐2 in both the cardiac and renal tissues.

At 100 mg/Kg treatment, MEAI induced a higher expression of Bcl‐2 in both the cardiac and renal tissues while at 200 mg/kg treatment, there was a reduction in the expressions of Bcl‐2 in the kidney tissues in the 1 mg/ml Lead (Pb) exposed groups. The treatment of rats that were exposed to 2.0 mg/ml Lead (Pb) with 100 mg/kg and 200 mg/kg MEAI, resulted in an increased expression of Bcl‐2 in the kidney tissues but a down regulation in the heart tissues. MEAI at both 100 mg/kg and 200 mg/kg treatment resulted in a reduced expression of cytochrome C in both tissues

The results of this study show that at 100 mg/kg and 200 mg/kg concentrations of MEAI, there was an inhibition/reversal of the lead induced apoptosis observed in both the cardiac and renal tissues.
Efavirenz Treatment Activates XBP1 Splicing in Primary Hepatocytes: a Comparison Across Species and an Investigation into the Role of PXR
Authors: Heck Carley J.S. , Bumpus Namandje N.
Published: November 2023

The non‐nucleoside reverse transcriptase inhibitor efavirenz (EFV) is used to treat human immunodeficiency virus (HIV) despite adverse effects, including hepatoxicity in some individuals. The mechanisms underlying this hepatotoxicity remain largely unknown, although the formation of the primary metabolite, 8OH‐EFV, has been linked to the activation of certain cell death pathways. In light of this, the present study tested whether EFV activates the endoplasmic reticulum (ER) stress response in primary hepatocytes. Splicing of X‐box‐binding protein 1 (XBP1) (a process activated by the ER stress response), was monitored using reverse transcriptase PCR following treatment of primary human and mouse hepatocytes with EFV (10 μM and 50 μM). Incubation of primary hepatocytes isolated from male C57Bl/6 mice with 50 μM EFV resulted in a ratio of spliced to unspliced XBP1 that was 8.8‐fold greater than the vehicle control. In human primary hepatocytes, an increase of 2.6‐fold was observed in response to EFV. In order to gain an understanding of the structural characteristics of EFV that render it a stimulator of XBP1 splicing, treatments were performed using a panel of EFV analogs as well as the 8OH‐EFV. Interestingly, an analog of EFV that differed from EFV in that it contains a trans‐alkene in place of the alkyne exhibited augmented XBP1 splicing response as compared to EFV, with a ratio of spliced to unspliced increased 29.4‐fold over vehicle control in hepatocytes isolated from mice. Several other analogs, including one in which the cyclopropyl ring is broken and the 8OH‐EFV metabolite, did not stimulate splicing of XBP1. Similar patterns of structural dependence were observed between human and mouse primary hepatocytes. Since EFV has been previously reported to activate the nuclear receptor pregnane X receptor (PXR), we tested whether PXR was required for the XBP1 splicing in response to EFV. Interestingly, we observed that XBP1 splicing following EFV treatment was increased in PXR null mice as compared to wild‐type C57Bl/6 mice (76.2‐fold in PXR null versus 8.8‐fold in wild‐type). Similarly, the above mentioned trans‐alkene analog also exhibited enhanced splicing of XBP1 in the PXR null mice as compared to that of wild‐type mice. Viability was measured in primary mouse hepatocytes during the above treatments. All analogs that increased XBP1 splicing also displayed a commensurate decrease in viability, except one in which the oxazinone carbonyl is replaced with a methyl group. In conclusion, EFV stimulates XBP1 splicing in mouse and human primary hepatocytes in a manner that does not appear to be dependent on metabolism to the primary 8OH‐EFV metabolite.

Support or Funding Information

NIH R01GM103853 and NSF‐GRFP DGE‐1232825
Structural differences in gut microbiota lead to altered hepatic expression of Drug Disposition Genes (DDG) in mice
Authors: Guo Yukuang , Cho Sung‐joon , Chung Eunah , Park Joo‐Seop , Jeong Hyunyoung
Published: November 2023

Hepatic drug metabolism is a major route of drug elimination, and it is mediated by multiple DDG. Varied expression and activity of DDG leads to inter‐individual variability in drug response. Results from previous studies comparing conventional and germ‐free mice showed that the presence of gut microbiota is associated with the differential hepatic DDG expression. The purpose of this study was to examine whether structural differences in gut microbiota lead to altered DDG expression in the liver. Male C57BL/6 mice from two different vendors [Jackson Laboratory (JAX) and Taconic Laboratory (TAC)] were cohoused with mice from the same or the other vendor for 4 weeks, creating the following 4 groups (n=6–7/group): JAX, TAC, coJAX (JAX mice cohoused with TAC), and coTAC (TAC mice cohoused with JAX). Mice were sacrificed and RNAs were extracted from the liver tissues. RNA sequencing was performed (n=3 mice/group), followed by bioinformatics analysis of 225 DDG. Principle component analysis of RNA‐sequencing data revealed that overall expression profiles of DDG were distinct in JAX mice while those for the other groups were similar to each other. A similar pattern was observed in results from previous gut microbiome analysis (i.e., 16S rRNA sequencing of fecal samples collected from the same group of mice), suggesting that the differential expression of DDG may be due to structural differences in gut microbiota. To identify DDG whose expression is influenced by gut microbiota, the RNA‐sequencing results of 225 DDG were further examined in livers from the 4 groups of mice. Preliminary statistical analysis revealed that 30 genes (14 phase I; 12 phase II; and 4 transporters) showed significantly different expression between JAX and TAC. The majority (i.e., 27) of these 30 genes showed no difference between coJAX and coTAC, suggesting that expression of these genes may be determined by differential gut microbiota. To further verify the RNA‐sequencing results, quantitative real‐time PCR was performed (n=6–7/group) for 9 genes that were selected based on biological significance and previously reported effects of gut microbiota on their expression. Among them, Cyp4a10 and Cyp4a14 showed 6.0‐ and 33.8‐fold higher expression in JAX, respectively, and Fmo5 showed 1.4‐fold lower expression in JAX. Our study suggests that structural differences in gut microbiota may underlie differential expression of DDG in the liver, potentially contributing to the inter‐individual variability in drug response.

Support or Funding Information

This study was supported by Chicago Biomedical Consortium.
Cytochrome P450 1A2 N‐terminal Site Directed Mutagenesis: Effects on Lipid Microdomain Localization
Authors: Lott Charles S. , Park Ji Won , Reed James R. , Backes Wayne L.
Published: November 2023

The protein components of the cytochrome P450 monooxygenase system consist of cytochromes P450 (CYPs), NADPH‐dependent cytochrome P450 reductase (CPR), and cytochrome b5. They form functional complexes that catalyze the oxidation of exogenous and endogenous molecules, rendering them more water soluble. In eukaryotes, these membrane bound proteins are found in many tissues and are located primarily in the endoplasmic reticulum. The lipid milieu into which the P450 system proteins reside is heterogenous, consisting of ordered and disordered domains. Ordered domains have a higher content of saturated fatty acids, cholesterol, and sphingomyelin than disordered domains, whereas disordered domains have a higher content of phospholipids with more unsaturated fatty acid chains. When a bilayer membrane is treated with non‐ionic detergent such as Brij 98, the disordered domains readily solubilize whereas ordered regions remain intact. Our lab has shown that various P450 system proteins reside in different lipid microdomains. For example, despite their high level of amino acid sequence similarity, CYP1A2 and CYP1A1 reside in ordered and disordered regions, respectively. Furthermore, our lab has shown that the N‐terminal amino acid sequences of CYP1A2 and CYP1A1 contribute to their specific localization in the ER membrane. The goal of this study is to use site directed mutagenesis to identify which N‐terminal amino acids may be responsible in the lipid raft localization of CYP1A2 and how this effects CYP1A2 enzymatic activity. CYP1A2 N‐terminal amino acids were mutated to their CYP1A1 counterparts and expressed in HEK293‐T cells, and their microdomain localization determined. Although substitution of the NH2‐terminal amino acids of CYP1A2 with that of CYP1A1 leads to changes in their microdomain localization, the results show that Serine4Aspartic Acid and Proline5Phenylalanine mutations alone do not alter microdomain localization of the CYP1A proteins.

Support or Funding Information

This work was supported, in whole or in part, by National Institutes of Health, NIEHS, United StatesPublic Health Service Grants ES004344, and ES013648.
Effect of Genotype, Age and Gender on Hepatic Abundance of Flavin Monooxigenase 3
Authors: Xu Meijuan , Bhatt Deepak Kumar , Chaw Katrina , Yeung Catherine , Prasad Bhagwat
Published: November 2023

Background and Purpose

Hepatic flavin monooxigenase 3 (FMO3) metabolizes a broad array of nucleophilic heteroatom‐containing drugs (e.g., amphetamine, sulindac, benzydamine, ranitidine, tamoxifen and ethioniamide), endogenous and exogenous chemicals (e.g., catecholamine, trimethylamine and nicotine) and pesticides. To predict the effect of genotype, age and gender on the hepatic metabolism of FMO3 substrates, we quantified FMO3 protein abundance in human liver microsomes by LC‐MS/MS proteomics.

Methods

Human liver microsomes were isolated from the tissue samples procured from University of Washington (UW, n=39) and St. Jude Children's Hospital (SJLB, n=249). Two peptides, i.e., VAIIGAGVSGLASIR (quantifier) and NNLPTAISDWLYVK (qualifier) were selected for LC‐MS/MS quantification. Total protein concentration was determined and the samples were denatured, reduced, alkylated, desalted and digested as previously reported (1, 2). FMO3 was quantified by AB Sciex 6500 triple quadrupole MS/MS using optimum conditions of reproducibility (1). Genotyping and mRNA expression analyses were performed using previously reported methods (3). The functional activity of FMO3 in selected human liver microsomes (UW samples) was quantified by using benzydamine as a probe substrate.

Results

Genotype and age significantly affect FMO3 protein abundance and activity. Out of the common twenty‐one FMO3 SNPs, six SNPs (rs2064074, rs28363536, rs2266782, rs909530, rs2266780 and rs909531) were associated with significantly decreased protein abundance. However, there was no correlation of these SNPs on the mRNA expression of FMO3 perhaps due to the effect of these SNPs on post‐translational modification, e.g., proteosomal degradation. Based on the activity data on representative samples, human livers harboring variant allele of rs2266780 showed no in vitro metabolic activity toward benzydamine, indicating effect of this SNP on both substrate affinity (Km) and FMO3 protein abundance. Consistent with our previous data (4), infants and children have significantly lower FMO3 abundance as compared to adults. FMO3 abundance was not associated with gender.

Conclusions

FMO3 protein abundance is highly affected by genotype and age but is independent of gender. These quantitative data are important in predicting FMO3 mediated hepatic N‐oxidation or S‐oxidation of endogenous biomolecules and xenobiotics.

Support or Funding Information

NICHD R01HD081299‐02
Nicotine metabolism by the flavin monooxygenase (FMO) family of enzymes.
Authors: Perez‐Paramo Yadira , Watson Christy , Chen Gang , Lazarus Philip
Published: November 2023

Nicotine is the addictive tobacco‐specific alkaloid present in cigarettes. Nicotine is metabolized in vivo primarily by hydroxylation by CYP2A6, but is also glucuronidated by UDP‐glucuronosyltransferases (UGTs) and oxidized by FMOs. Nicotine‐N‐oxide has been detected in smoker's urine, accounting for 4–8% of total urinary nicotine metabolites. While previous studies have suggested that the hepatic FMO3 is the principal enzyme involved in nicotine‐N‐oxidation, a comprehensive analysis of nicotine oxidation by all FMOs has not previously been performed. To characterize the catalytic activity of FMOs in the formation of nicotine‐N‐oxide, wild‐type FMOs 1 through 5 were individually overexpressed in HEK293 cells and used in oxidation reactions containing between 0.1 – 5000 μM of (−)‐nicotine and NADPH‐regenerative system; nicotine‐N‐oxide formation was monitored by UPLC‐MS/MS. While all five FMOs exhibited oxidation activity against nicotine, only FMO1 exhibited a KM of less than 1 mM (0.76 mM), with FMOs 3, 4 and 5 all exhibiting a KM of >40 mM. While FMO2 is expressed primarily in the lung and possibly brain, this enzyme is functional only in people of African ancestry due to a SNP in the FMO2 stop codon (codon 472; MAF=0.15); most of the remaining human population expresses a truncated non‐functional enzyme. Functional FMO2427Gln also exhibited low activity against nicotine, with a KM = 71 mM. Non‐synonymous polymorphic variants for FMO1 and FMO3 have been overexpressed in HEK293 cells and are currently being screened for activity against nicotine in vitro. These data are consistent with FMO1 being the principal enzyme involved in nicotine‐N‐oxidation and with previous studies demonstrating that functional genetic variants in FMO1 are associated with differences in nicotine dependence.

Support or Funding Information

National Institutes of Health, National Institutes of Environmental Health Sciences [Grant R01‐ES025460]

Health Sciences and Services Authority of Spokane, WA [Grant WSU002292]
ecoAO: A Simple System for the Study of Drug Metabolism by Human Aldehyde Oxidase
Authors: Paragas Erickson Mesina , Humphreys Sara , Min Joshua , Joswig‐Jones Carolyn , Jones Jeffrey
Published: November 2023

Aldehyde oxidase (AO) is a cytosolic enzyme responsible for the metabolism of some drugs and drug candidates. AO catalyzes the oxidative hydroxylation of substrates including several aliphatic and aromatic aldehydes, and nitrogen‐containing heterocyclic compounds. Although AO plays a significant role in human Phase I drug metabolism, its role in drug clearance is not well understood. Major reasons for its recalcitrance to examination are two‐fold. First, established in vitro systems used to study AO, including liver cytosol, pure enzyme, S9 fractions, and primary and cultured hepatocytes, display hyper‐variability in their AO expression and activity profiles. Second, AO metabolite standards are often difficult to chemically synthesize due to the chemo‐ and region‐selectivity of the enzyme. In this study, we describe a simple, economical, Escherichia coli system – ecoAO – that addresses both issues simultaneously. ecoAO contains pTHco‐hAOX1 (Silke Leimkühler, University of Potsdam), a codon‐optimized and lactose‐inducible human aldehyde oxidase‐containing plasmid, expressed in Moco‐producing E. coli strain TP1017 (Tracy Palmer, University of Dundee). Our direct cell paste assays exhibit specific AO activity with a range of known substrates (DACA, zaleplon, zoniporide and O6‐benzylguanine) and candidate substrates (quinoline series) against a non‐mammalian background. We also demonstrate an important application of the ecoAO system, the biotransformation of AO substrates, yielding significant quantities of metabolite (~30% yield) upon purification.

Support or Funding Information

This work was supported by the National Institutes of Health National Institute of General Medical Sciences grant GM100874.
Comparison of Cytochrome P450 3A4 and 3A7 with Azole Inhibitors
Authors: Godamudunage Malika P. , Lampe Jed N. , Scott Emily E.
Published: November 2023

Cytochrome P450 (CYP) 3A enzymes metabolize up to 50% of human drugs. While CYP3A4 is the major adult enzyme, CYP3A7 is the major form through 6–12 months of development. Though 87% identical and frequently assumed to be functionally very similar, it has recently become clear that there are some significant differences between CYP3A4 and CYP3A7 that may affect fetal and infant drug metabolism. This knowledge gap motivates a direct study of CYP3A7 metabolism and inhibition to better inform drug usage in very young patients.

Azoles can be effective for common fungal infections in infants, but can also be rather nonselective CYP inhibitors. In addition to the clinical relevance of these compounds, azoles constitute a useful series of CYP3A active site probes. This study systematically evaluated the interactions of a series of azoles with purified, recombinant human CYP3A4 and CYP3A7. Binding studies revealed that each of the following azoles demonstrated spectral features typical for direct coordination to the heme iron (type II binding) with parallel rank order affinities for the two enzymes: from subnanomolar Kd values for clotrimazole, to moderate affinity for liarozole and ketoconazole, and low affinity for imidazole. In contrast, fluconazole and fosfluconazole demonstrated more unusual binding characteristics that differed between the two CYP3A enzymes. Fluconazole binding to CYP3A7 resulted in only increasing absorbance, migrating at higher concentrations to wavelengths associated with heme coordination. In contrast, fluconazole binding to CYP3A4 was characteristic of water displacement from the heme (type I) at low concentrations, but converted to heme coordination at high concentrations, suggesting that CYP3A4 may bind fluconazole in different orientations, perhaps depending on whether one fluconazole molecule or two are bound in the active site simultaneously. This would be consistent with the established capacity of CYP3A4 to bind more than one azole molecule simultaneously. Fosfluconazole, a more water‐soluble phosphorylated version of fluconazole, demonstrated a red‐shifted type I spectrum with CYP3A4, but with CYP3A7 yielded only increasing absorbance consistent with water displacement from the iron. Overall, these observations establish differences between CYP3A7 and CYP3A4 interactions with fluconazole and fosfluconazole, prompting further evaluation by comparative inhibition studies and structural studies. Such differences may underlie differential metabolism of common drugs at different life stages and inform dosing in infants vs. adults.

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National Institutes of Health R37GM076343 (EES)
Benzop(a)pyrene in Cigarette Smoke Enhances HIV‐1 Replication in Monocytic Cells: Potential Role of Cytochrome P450s and Oxidative Stress
Authors: Ranjit Sabina , Midde Narasimha M. , Sinha Namita , Kumar Santosh
Published: November 2023

Smoking aggravates HIV‐1 pathogenesis and leads to decreased responses to antiretroviral therapy. However, the exact mechanism by which smoking enhances HIV‐1 pathogenesis is unknown. Benzo(a)pyrene (BaP) is a major carcinogen in cigarette smoke. It requires metabolic activation through cytochrome P450s (CYPs) to exert its toxic effects. We hypothesize that BaP is metabolized by CYP‐mediated pathway and produce reactive oxygen species (ROS). Oxidative stress resulting from increased ROS would aggravate HIV‐1 replication. Recently, we have shown that chronic exposure of BaP to U937 monocytic cells results in the significant induction of CYPs (CYP 1A1 and 3A4) and antioxidants (catalase and SOD1), and increase in the generation of ROS and cellular toxicity. We also confirmed these results in human primary macrophages. Next, we examined the chronic effect of BaP in U1 cells (HIV‐1 infected U937 monocytic cells). When U1 cells were exposed to BaP for seven days, we observed ~4 fold increase in HIV‐1 replication. In addition, there was a significant increase in the expression of CYP1A1 at mRNA level as well as there was an increase in CYP1A1 enzymatic activity. Elevated ROS and massive cell death were also observed. In order to confirm the role of CYP1A1 and 1B1 in BaP‐induced oxidative stress pathway and HIV‐1 replication, we knocked down the gene expression of CYP1A1 and 1B1 in HIV‐1 infected human primary macrophages, using SiRNA. Our results showed decrease in the viral replication in CYP1A1 and 1B1 knock‐out cells upon BaP exposure. We will determine oxidative DNA damage in untreated, and CYP1A1 and 1B1 knock‐out cells using TUNNEL assay. We are now in the process of determining the role of oxidative stress mediated through CYP pathway in HIV‐1 replication. For this, we will expose HIV‐infected primary macrophages with general and specific antioxidants, followed by exposure to BaP. Finally, we will quantify BaP metabolites in U1 cells and/or HIV‐1 infected human primary macrophages using Liquid Chromatography‐Mass Spectrometry. The results from the present work are clinically relevant as they would help to find a novel therapeutic target to improve drug therapy outcomes in HIV positive smokers.

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Department of Pharmaceutical Sciences, University of Tennessee Health Science Center
Globular Adiponectin Induces Endoplasmic Reticulum Stress and Autophagy in Macrophages
Authors: Oh Hye‐Jin , Pun Nirmala Tilija , Raut Pawan Kumar , Kim Eun‐Hye , Khakurel Amrita , Shrestha Aastha , Park Pil‐Hoon
Published: November 2023

Adiponectin, a kind of adipokine predominantly secreted from adipose tissue, is known to possess various biological functions. We have previously shown that globular adiponectin induces autophagy in macrophages, which is implicated in the suppression of inflammatory cytokines production and acts as a cell survival mechanism rather than cell death. Endoplasmic reticulum (ER) is a membranous cell organelle and carries out the process of protein folding and modifying to maintain cellular homeostasis. ER stress triggered by ER malfunction and excessive demand for protein folding induces cellular oxidative stress. It has been shown that ER stress is a potent trigger of autophagy, which is implicated in cell survival pathway. In the present study, we investigated the effect of globular adiponectin on ER stress and further its role in autophagy induction in RAW 264.7 macrophages. We found that globular adiponectin treatment induces phosphorylation of protein kinase RNA‐like endoplasmic reticulum kinase (PERK) and increase in expression of CCAAT/‐enhancer‐binding protein homologous protein (CHOP), implying that adiponectin stimulates ER stress. Given that ER stress is one of the critical mechanisms leading to autophagy induction, these results suggest a possibility that adiponectin induces autophagy via ER stress. In addition, interestingly, treatment with moderate dose of globular adiponectin significantly enhanced cell viability determined by MTS assay and decreased caspase‐3 activity in RAW 264.7 macrophages, indicating that adiponectin treatment causes growth and survival of macrophages. Taken together, these findings suggest a hypothesis that adiponectin protects macrophages from various extracellular stresses such as lipopolysaccharide (LPS) via autophagy activation mediated by ER stress. Further studies would provide better insights into the mechanisms underlying adiponectin‐induced protection of macrophages, particularly in relation to the role of ER stress and autophagy.
Evaluation of Seahorse XF Cell Mito Stress Test for Detection of Mitochondrial Dysfunction Using Prototypical Mitochondrial Toxicants in the PC‐3 Cell Line
Authors: Zhang Jie L , Krause Caitlin , Ciurlionis Rita , Sharapova Tatiana L , Wilsbacher Julie , Ackler Scott , Van Vleet Terry , Liguori Michael J
Published: November 2023

Mitochondrial dysfunction has been frequently implicated in some drug‐induced toxicities. Various methods for the detection of mitochondrial toxicity are available such as an oxygen consumption assay to measure mitochondrial basal respiration, assays to measure direct effects of drugs on the five complexes in the mitochondrial electron transport chain (ETC), and assays to detect changes in mitochondrial DNA (mtDNA) copy number or changes in mitochondrial membrane potential. The Seahorse technology platform has emerged as a new technique in assessing mitochondrial respiration in cells, where basal, ATP‐linked, maximal and non‐mitochondrial respiration can be measured in the same experiment. Spare respiratory capacity (SRC) is calculated as the difference between maximal and basal respiration. In this study, Seahorse XF Cell Mito Stress Test was evaluated in the PC‐3 cell line using tool compounds known to directly inhibit the complex or complexes in the ETC. Cells were acutely treated with amiodarone (5–80 μM), simvastatin (5–75 μM), tamoxifen (0.625–20 μM), troglitazone (2.5–50 μM), penicillin (12.5–100 μM, negative control), and vehicle (0.1% DMSO). Exposure of these tool compounds revealed an immediate and pronounced decrease in SRC with a ranking of (greatest to lowest inhibition) troglitazone (IC50< 2.5 μM)> amiodarone (IC50<5 μM) >tamoxifen (IC50 = 17.7 μM) >simvastatin (IC50 = 20.7μM).

Tool compounds known to cause mitochondrial toxicity through an indirect mechanism were also explored in the Seahorse platform. 2′, 3′‐dideoxycytidine (ddC), an inhibitor of mtDNA synthesis, and chloramphenicol (CAM), an inhibitor of mitochondrial protein synthesis, were tested. A 3‐day pre‐treatment of ddC (0.12–30 μM) and CAM (0.04–10 μM) showed a decrease of SRC in PC‐3 cells with IC50=0.39 μM and 2.7 μM respectively. The decrease of SRC was further supported by an in‐cell ELISA assay where a set of proteins in the ETC complexes was measured. A decrease of mitochondrial encoded COX‐1 to nuclear encoded SDH‐A protein ratio was observed after a 5‐day treatment of ddC and CAM in PC‐3 cells with IC50=0.016 μM and <0.1 μM respectively. In the dose ranges tested, the 3 or 5 day pre‐treatment of ddC and CAM showed minimal cytotoxicity in the PC‐3 cells. In conclusion, the Seahorse XF Cell Mito Stress Test is a sensitive assay to evaluate mitochondrial dysfunction using a set of tool compounds, and this new method combined with traditional methods will be helpful to better understand the mechanism of mitochondrial toxicity in the development of new drugs.

Support or Funding Information

The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication.