Chlorquinaldol Alleviates Lung Fibrosis in Mice by Inhibiting Fibroblast Activation through Targeting Methionine Synthase ReductaseClick to copy article linkArticle link copied!
- Xiangyu YangXiangyu YangThe Fifth Affiliated Hospital, Guangzhou Municipal and Guangdong Provincial Key Laboratory of Molecular Target & Clinical Pharmacology, the NMPA and State Key Laboratory of Respiratory Disease, School of Pharmaceutical Sciences, Guangzhou Medical University, Guangzhou 511436, ChinaMore by Xiangyu Yang
- Geng LinGeng LinThe Fifth Affiliated Hospital, Guangzhou Municipal and Guangdong Provincial Key Laboratory of Molecular Target & Clinical Pharmacology, the NMPA and State Key Laboratory of Respiratory Disease, School of Pharmaceutical Sciences, Guangzhou Medical University, Guangzhou 511436, ChinaMore by Geng Lin
- Yitong ChenYitong ChenThe Fifth Affiliated Hospital, Guangzhou Municipal and Guangdong Provincial Key Laboratory of Molecular Target & Clinical Pharmacology, the NMPA and State Key Laboratory of Respiratory Disease, School of Pharmaceutical Sciences, Guangzhou Medical University, Guangzhou 511436, ChinaMore by Yitong Chen
- Xueping LeiXueping LeiThe Fifth Affiliated Hospital, Guangzhou Municipal and Guangdong Provincial Key Laboratory of Molecular Target & Clinical Pharmacology, the NMPA and State Key Laboratory of Respiratory Disease, School of Pharmaceutical Sciences, Guangzhou Medical University, Guangzhou 511436, ChinaMore by Xueping Lei
- Yitao OuYitao OuThe Fifth Affiliated Hospital, Guangzhou Municipal and Guangdong Provincial Key Laboratory of Molecular Target & Clinical Pharmacology, the NMPA and State Key Laboratory of Respiratory Disease, School of Pharmaceutical Sciences, Guangzhou Medical University, Guangzhou 511436, ChinaMore by Yitao Ou
- Yuyun YanYuyun YanThe Fifth Affiliated Hospital, Guangzhou Municipal and Guangdong Provincial Key Laboratory of Molecular Target & Clinical Pharmacology, the NMPA and State Key Laboratory of Respiratory Disease, School of Pharmaceutical Sciences, Guangzhou Medical University, Guangzhou 511436, ChinaMore by Yuyun Yan
- Ruiwen WuRuiwen WuThe Fifth Affiliated Hospital, Guangzhou Municipal and Guangdong Provincial Key Laboratory of Molecular Target & Clinical Pharmacology, the NMPA and State Key Laboratory of Respiratory Disease, School of Pharmaceutical Sciences, Guangzhou Medical University, Guangzhou 511436, ChinaMore by Ruiwen Wu
- Jie YangJie YangThe Fifth Affiliated Hospital, Guangzhou Municipal and Guangdong Provincial Key Laboratory of Molecular Target & Clinical Pharmacology, the NMPA and State Key Laboratory of Respiratory Disease, School of Pharmaceutical Sciences, Guangzhou Medical University, Guangzhou 511436, ChinaMore by Jie Yang
- Yiming LuoYiming LuoThe Fifth Affiliated Hospital, Guangzhou Municipal and Guangdong Provincial Key Laboratory of Molecular Target & Clinical Pharmacology, the NMPA and State Key Laboratory of Respiratory Disease, School of Pharmaceutical Sciences, Guangzhou Medical University, Guangzhou 511436, ChinaMore by Yiming Luo
- Lixin ZhaoLixin ZhaoThe Fifth Affiliated Hospital, Guangzhou Municipal and Guangdong Provincial Key Laboratory of Molecular Target & Clinical Pharmacology, the NMPA and State Key Laboratory of Respiratory Disease, School of Pharmaceutical Sciences, Guangzhou Medical University, Guangzhou 511436, ChinaMore by Lixin Zhao
- Xiuxiu ZhangXiuxiu ZhangThe Fifth Affiliated Hospital, Guangzhou Municipal and Guangdong Provincial Key Laboratory of Molecular Target & Clinical Pharmacology, the NMPA and State Key Laboratory of Respiratory Disease, School of Pharmaceutical Sciences, Guangzhou Medical University, Guangzhou 511436, ChinaMore by Xiuxiu Zhang
- Zhongjin YangZhongjin YangThe Fifth Affiliated Hospital, Guangzhou Municipal and Guangdong Provincial Key Laboratory of Molecular Target & Clinical Pharmacology, the NMPA and State Key Laboratory of Respiratory Disease, School of Pharmaceutical Sciences, Guangzhou Medical University, Guangzhou 511436, ChinaMore by Zhongjin Yang
- Aiping QinAiping QinThe Fifth Affiliated Hospital, Guangzhou Municipal and Guangdong Provincial Key Laboratory of Molecular Target & Clinical Pharmacology, the NMPA and State Key Laboratory of Respiratory Disease, School of Pharmaceutical Sciences, Guangzhou Medical University, Guangzhou 511436, ChinaMore by Aiping Qin
- Ping Sun*Ping Sun*E-mail: [email protected]The Fifth Affiliated Hospital, Guangzhou Municipal and Guangdong Provincial Key Laboratory of Molecular Target & Clinical Pharmacology, the NMPA and State Key Laboratory of Respiratory Disease, School of Pharmaceutical Sciences, Guangzhou Medical University, Guangzhou 511436, ChinaMore by Ping Sun
- Xi-Yong Yu*Xi-Yong Yu*E-mail: [email protected]The Fifth Affiliated Hospital, Guangzhou Municipal and Guangdong Provincial Key Laboratory of Molecular Target & Clinical Pharmacology, the NMPA and State Key Laboratory of Respiratory Disease, School of Pharmaceutical Sciences, Guangzhou Medical University, Guangzhou 511436, ChinaMore by Xi-Yong Yu
- Wenhui Hu*Wenhui Hu*E-mail: [email protected]The Fifth Affiliated Hospital, Guangzhou Municipal and Guangdong Provincial Key Laboratory of Molecular Target & Clinical Pharmacology, the NMPA and State Key Laboratory of Respiratory Disease, School of Pharmaceutical Sciences, Guangzhou Medical University, Guangzhou 511436, ChinaMore by Wenhui Hu
Abstract
Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease with limited treatment options. Thus, it is essential to investigate potential druggable targets to improve IPF treatment outcomes. By screening a curated library of 201 small molecules, we have identified chlorquinaldol, a known antimicrobial drug, as a potential antifibrotic agent. Functional analyses have demonstrated that chlorquinaldol effectively inhibits the transition of fibroblasts to myofibroblasts in vitro and mitigates bleomycin-induced pulmonary fibrosis in mice. Using a mass spectrometry-based drug affinity responsive target stability strategy, we revealed that chlorquinaldol inhibited fibroblast activation by directly targeting methionine synthase reductase (MTRR). Decreased MTRR expression was associated with IPF patients, and its reduced expression in vitro promoted extracellular matrix deposition. Mechanistically, chlorquinaldol bound to the valine residue (Val-467) in MTRR, activating the MTRR-mediated methionine cycle. This led to increased production of methionine and s-adenosylmethionine, counteracting the fibrotic effect. In conclusion, our findings suggest that chlorquinaldol may serve as a novel antifibrotic medication, with MTRR-mediated methionine metabolism playing a critical role in IPF development. Therefore, targeting MTRR holds promise as a therapeutic strategy for pulmonary fibrosis.
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License Summary*
You are free to share(copy and redistribute) this article in any medium or format and to adapt(remix, transform, and build upon) the material for any purpose, even commercially within the parameters below:
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Synopsis
We identified MTRR as a novel target of Chlorquinaldol, which inhibits fibroblast activation and mitigates pulmonary fibrosis by binding to the valine residue (Val-467) in MTRR.
Introduction
Results and Discussion
Chlorquinaldol Suppress the Proliferation and Differentiation of Fibroblasts In Vitro
Figure 1
Figure 1. Inhibitory effects of chlorquinaldol on the proliferation and differentiation of pulmonary fibroblasts. (A) Schematic of a phenotypic screening procedure for antifibrotic agent discovery. Normal murine fibroblasts (NIH/3T3) were seeded in 96-well plates and pretreated with 10 ng/mL transforming growth factor-β1 (TGFβ1) for 48 h, followed by 24 h of exposure to 10 μM test compounds or 0.1% DMSO. Proliferation was measured via CCK8 assay to assess the antifibrotic potential of the compounds. (B) The CCK-8 assay illustrates the cell viability in response to compound exposure. Notably, four compounds significantly reduced TGFβ1-stimulated fibroblast proliferation by at least 50%. (C) The chemical structure of chlorquinaldol (CQD), characterized as 5,7-dichloro-2-methyl-8-hydroxyquinoline, a known antibacterial agent, is depicted. (D, E) Both quiescent and TGFβ1-activated MRC5 and NIH/3T3 cells were incubated with a range of CQD concentrations or DMSO vehicles for 72 h. Cell proliferation was then evaluated using the CCK-8 assay. (F, G) Following a 24 h treatment with DMSO or CQD, MRC5 cells were subjected to Western blot analysis to determine the protein expression levels of fibronectin, collagen I, and α-SMA, with GAPDH serving as the loading standard. Experiments were performed in triplicate (n = 3). (H, I) Western blot analysis was similarly conducted to assess the protein expression of fibronectin, collagen I, and α-SMA in NIH/3T3 cells, using GAPDH as the internal control. Each condition was replicated three times (n = 3). Data are represented as the mean ± standard error of the mean (SEM). Statistical significance is indicated for comparisons against unstimulated control with * for p < 0.05, ** for p < 0.01, and *** for p < 0.001 and against TGFβ1 alone with # for p < 0.05, ## for p < 0.01, and ### for p < 0.001.
Chlorquinaldol Prevents Lung Fibrosis in BLM-Induced Lung Remodeling
Figure 2
Figure 2. Protective effect of chlorquinaldol on bleomycin-induced pulmonary fibrosis. (A) Schematic of the preventive treatment protocol for lung fibrosis in animal models. (B) Survival curve for mice over 21 days following BLM challenge (n = 6–10). (C) HE stained lung tissue sections from mice on day 21 after BLM exposure. The inset displays a zoomed area (200× magnification), with a 200 μm scale bar (n = 4–6). (D) Representative images of Masson’s trichrome staining in lung tissue; the inset zooms in on a detailed area (200 × ), with a 200 μm scale bar (n = 4–6). (E) Assessment of the collagenous area ratio from Masson’s trichrome-stained sections. (F) Quantification of hydroxyproline levels in the right lung lobes. Data presented as mean ± SEM for 4–7 animals per group. Significance is indicated by *p < 0.05, **p < 0.01, and ***p < 0.001 for comparisons with the control and #p < 0.05, ##p < 0.01, and ###p < 0.001 for other group comparisons as indicated.
Chlorquinaldol Reverses Lung Fibrosis in BLM-Induced Lung Remodeling
Figure 3
Figure 3. Chlorquinaldol ameliorates pulmonary fibrosis and lung ventilation. (A) Treatment protocol for the pulmonary fibrosis mouse model, including the administration timeline for CQD or nintedanib. The first 9 days of post-BLM induction represent the inflammatory phase, followed by the fibrotic phase. (B) The survival rates of mice within the 21-day BLM model (n = 6–12). (C) Representative micro-CT images of the whole lung on the 21st day, featuring axial, coronal, and sagittal views, as well as three-dimensional reconstructions. All images of the right mainstem bronchus bifurcation were selected to ensure consistent anatomical comparison. (D) Analysis of the lung volume ventilation fraction in mice, with green indicating normally aerated areas (−860 to −435 HU), yellow representing poorly aerated areas (−434 to −121 HU), and red signifying nonaerated regions (−120 to +121 HU). Data are shown as mean ± SEM, with n = 3–5 per group. (E, F) The dynamic changes in the airway constriction index Penh and the midexpiratory flow rate EF50, each presented as mean ± SEM (n = 3–12). Statistical significance is denoted by *p < 0.05, **p < 0.01, and ***p < 0.001 for comparisons with the control group and #p < 0.05, ##p < 0.01, and ###p < 0.001 for comparisons with the BLM group.
Figure 4
Figure 4. Chlorquinaldol improved alveolar architecture and reduced interstitial collagen deposition. (A) Representative images of whole lung HE staining on the 21st day. (B) Pulmonary fibrosis scores based on the Ashcroft scoring system. (C) Representative Masson’s trichrome staining of entire lung tissue from the mice. (D) Quantitative analysis of collagen content as determined by Masson’s staining. Scale bar: 100 μM. Data is expressed as mean ± SEM (n = 3). Significance is denoted by *p < 0.05, **p < 0.01, ***p < 0.001 compared to the control and #p < 0.05, ##p < 0.01, ###p < 0.001 for comparisons with the BLM group.
Methionine Synthase Reductase Is a Novel Target of Chlorquinaldol
Figure 5
Figure 5. Chlorquinaldol directly targets MTRR protein. (A) Schematic of the DARTS/MS strategy for discovering potential chlorquinaldol binding proteins. (B) Heatmap of 18 candidate targets with differential expression levels, as determined by mass spectrometry. (C) SPR measures the binding affinity of chlorquinaldol to MTRR protein. (D, E) Immunoblots of MTRR levels in TGFβ1-activated NIH/3T3 cells with chlorquinaldol treatment and subsequent Pronase digestion. (F, G) CETSA melt response and related curves to assess the thermostability between chlorquinaldol and MTRR. (H, I) Isothermal dose response (ITDR) and its curve indicating the binding thermodynamics of chlorquinaldol. Data are mean ± SEM (n = 3), with statistical significance marked by *p < 0.05, **p < 0.01, ***p < 0.001 versus control. Abbreviations: MTRR, methionine synthase reductase; DARTS, drug affinity responsive target stability assay; SPR, surface plasmon resonance; CETSA, cellular thermal shift assay; CQD, chlorquinaldol.
Chlorquinaldol Interacts with the FAD-Binding Domain of Methionine Synthase Reductase
Figure 6
Figure 6. Chlorquinaldol exerts antifibrotic activity by directly interacting with the FAD domain of the MTRR protein. (A) Cartoon depiction of the structure of murine methionine synthase reductase (MTRR), featuring FMN and FAD domains connected by a flexible hinge. (B) Three-dimensional representation demonstrating chlorquinaldol’s top three binding sites within the MTRR protein architecture. (C) Detailed 3D interaction map of chlorquinaldol with the MTRR protein, alongside a local secondary structure binding interaction diagram. (D–G) SPR analysis detecting the binding of four peptides (peptide 1, 2, and 3 and peptide 1 mutant V467A) to chlorquinaldol. (H) Molecular docking simulation revealing the chlorquinaldol–MTRR binding interface, with interactions indicated by purple arrows and noncovalent distance interactions indicated by green dashed lines. Abbreviation: FMN, flavin mononucleotide; FAD, flavin adenine dinucleotide.
Chlorquinaldol Activates MTRR to Suppress Fibroblast Activation through Methionine Cycle Regulation
Figure 7
Figure 7. Chlorquinaldol promotes methionine and s-adenosyl methionine (SAM) accumulation via MTRR to inhibit fibrosis. (A) A violin plot illustrates the expression levels of methionine synthase reductase (MTRR) in lung tissues from idiopathic pulmonary fibrosis (IPF) patients, with data obtained from the GEO data set GSE213001. (B) qPCR analysis assesses the efficiency of Mtrr knockdown in NIH/3T3 cells. (C–F) Western blot (WB) analysis measures the protein expression levels of fibronectin, collagen I, and α-smooth muscle actin (α-SMA) in Mtrr knockdown cells following TGFβ1 stimulation. (G–J) HPLC/MS was utilized to quantify intracellular methionine, SAM, and SAH levels. (K–L) NIH/3T3 cells are pretreated with varying concentrations of SAM for 24 h before TGFβ1 stimulation, and WB is used to assess the protein expression levels of fibronectin, collagen I, and α-SMA. Data are presented as mean ± SEM (n = 3). Significance is denoted by *p < 0.05, **p < 0.01, and ***p < 0.001 compared to the control. Abbreviations: SAM, s-adenosylmethionine; SAH, s-adenosylhomocysteine.
Conclusion
Materials and Methods
Cell Culture
Cell Viability Assay
Western Blot Analysis
Quantitative Real-Time PCR (qRT-PCR)
Animal Models and Designs
Pulmonary Function Measurement
In Vivo Micro-CT Imaging
Measurement of Hydroxyproline Levels
HE and Masson’s Trichrome Staining
Cytokine Measurements by ELISA
Immunofluorescence Staining
DARTS Assay
Quantitive Proteomic Analysis by LC/MS
SPR Analysis
CETSA
Molecular Docking
Molecular Dynamics Simulation
Determination of cellular SAM, SAH, and Methionine Levels by HPLC/MS Assay
Knockdown of Mtrr by shRNA
MTRR Activity Detection
Statistic and Analysis
Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acscentsci.4c00798.
Effects of chlorquinaldol treatment, bioinformatics and SPR analysis of chlorquinaldol’s candidate targets, molecular dynamics analysis, myofibroblast differentiation and fibroblast activation analysis, pharmacokinetic parameters of CQD, small molecule compounds used in the screening experiment, potential target compounds of clorquinaldol, PCR primer sequences, and ShRNA target sequences (PDF)
Terms & Conditions
Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.
Acknowledgments
We would like to express our gratitude to BioRender.com for creating the schematic diagrams. We also extend our thanks to Guangzhou Raybio Medical Technology Co., Ltd. for their generous supply of the HALO software. This study was supported by the National Key Research and Development Program of China (Grant 2022YFE0209700 to X.-Y.Y.), the Innovative Team Project of Ordinary Universities in Guangdong Province (Grant 2022KCXTD022 to W.H.), and Plan on enhancing scientific research in GMU (Grant 02-410-2405018 to P.S.).
α-SMA | α-smooth muscle actin |
BLM | bleomycin |
CETSA | cell thermal shift assay |
CQD | chlorquinaldol |
DARTS | drug affinity responsive target stability assay |
ECM | extracellular matrix |
ELISA | enzyme-linked immunosorbent assay |
FMT | fibroblast-to-myofibroblast transition |
GARS1 | glycyl-tRNA Synthetase 1 |
HE | hematoxylin and eosin |
INPPL1 | inositol polyphosphate-specific phosphatase 1 |
IPF | idiopathic pulmonary fibrosis |
Ka | association rate constant |
Kd | dissociation rate constant |
KD | equilibrium dissociation constant |
MD | molecular dynamics |
MTRR | methionine synthase reductase |
OSM | oncostatin M |
SPR | surface plasmon resonance |
SAH | s-adenosylhomocysteine |
SAM | s-adenosylmethionine |
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- 15Wang, L.; Deng, K.; Gong, L.; Zhou, L.; Sayed, S.; Li, H.; Sun, Q.; Su, Z.; Wang, Z.; Liu, S.; Zhu, H.; Song, J.; Lu, D. Chlorquinaldol targets the β-catenin and T-cell factor 4 complex and exerts anti-colorectal cancer activity. Pharmacol. Res. 2020, 159, 104955, DOI: 10.1016/j.phrs.2020.104955Google ScholarThere is no corresponding record for this reference.
- 16Chen, Y.; Chen, X.; Liang, S.; Ou, Y.; Lin, G.; Hua, L.; Wu, X.; Zhou, Y.; Liu, Z.; Cai, H.; Yang, Z.; Hu, W.; Sun, P. Chlorquinaldol inhibits the activation of nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing protein 3 inflammasome and ameliorates imiquimod-induced psoriasis-like dermatitis in mice. Chem. Biol. Interact. 2022, 365, 110122, DOI: 10.1016/j.cbi.2022.110122Google ScholarThere is no corresponding record for this reference.
- 17Bidossi, A.; Bottagisio, M.; De Grandi, R.; Drago, L.; De Vecchi, E. Chlorquinaldol, a topical agent for skin and wound infections: anti-biofilm activity and biofilm-related antimicrobial cross-resistance. Infect. Drug Resist. 2019, 12, 2177– 2189, DOI: 10.2147/IDR.S211007Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXjsFaqurg%253D&md5=c194ca81f0e844b5d187d9f1f264885fChlorquinaldol, a topical agent for skin and wound infections: anti-biofilm activity and biofilm-related antimicrobial cross-resistanceBidossi, Alessandro; Bottagisio, Marta; De Grandi, Roberta; Drago, Lorenzo; De Vecchi, ElenaInfection and Drug Resistance (2019), 12 (), 2177-2189CODEN: IDRNAV; ISSN:1178-6973. (Dove Medical Press Ltd.)Purpose: Persistence of skin and wound infections is nowadays accepted being linked to bacterial biofilms, which are highly recalcitrant to treatments and contribute to maintain a const. inflammation state and prevent a correct healing. Topical antimicrobials are the most common first-line self-medications; however, treatment failure is not uncommon and emerging resistance to antibiotics is alarming. Chlorquinaldol is an antimicrobial with a wide spectrum of activity and desirable characteristics for topical application. Aim of this study was to evaluate the efficacy of chlorquinaldol to prevent or eradicate S. aureus and P. aeruginosa biofilms, in comparison to classic topical antibiotics like gentamicin and fusidic acid. Methods: Min. inhibitory concns. (MIC) were assessed for each strain and subinhibitory concns. (1/2 and 1/4 MIC) were used in the biofilm assay. Antimicrobial assays were performed during biofilm formation or were applied on mature biofilms and were evaluated by means of crystal violet assay and confocal laser scan microscopy. Results: Chlorquinaldol and gentamicin were the most effective antimicrobials in both eradicating and preventing pathogens biofilm; however, resistance to methicillin and impermeability to carbapenems impaired chlorquinaldol effect. In addn., similarly to other hydroxyquinolines, aspecific metal chelation is here proposed as chlorquinaldol mode of action. Conclusion: Relying on an acceptable antibiofilm and a wide spectrum of activity, an aspecific mode of action and consequent absence of resistance development, chlorquinaldol proved to be a good antimicrobial for topical use.
- 18Chen, X.; Guo, H. Comparison of Chlorquinaldol-Promestriene Vaginal Tablets and Opin Suppositories Effect on Inflammatory Factors and Immune Function in Chronic HPV Cervicitis. J. Coll. Physicians Surg. Pak. 2019, 29 (2), 115– 118, DOI: 10.29271/jcpsp.2019.02.115Google ScholarThere is no corresponding record for this reference.
- 19Darby, C. M.; Nathan, C. F. Killing of non-replicating Mycobacterium tuberculosis by 8-hydroxyquinoline. J. Antimicrob. Chemother. 2010, 65 (7), 1424– 1427, DOI: 10.1093/jac/dkq145Google ScholarThere is no corresponding record for this reference.
- 20Scotton, C. J.; Hayes, B.; Alexander, R.; Datta, A.; Forty, E. J.; Mercer, P. F.; Blanchard, A.; Chambers, R. C. Ex vivo micro-computed tomography analysis of bleomycin-induced lung fibrosis for preclinical drug evaluation. Eur. Respir. J. 2013, 42 (6), 1633– 1645, DOI: 10.1183/09031936.00182412Google ScholarThere is no corresponding record for this reference.
- 21Ruscitti, F.; Ravanetti, F.; Donofrio, G.; Ridwan, Y.; van Heijningen, P.; Essers, J.; Villetti, G.; Cacchioli, A.; Vos, W.; Stellari, F. F. A Multimodal Imaging Approach Based on Micro-CT and Fluorescence Molecular Tomography for Longitudinal Assessment of Bleomycin-Induced Lung Fibrosis in Mice. J. Vis. Exp. 2018, (134), e56443 DOI: 10.3791/56443Google ScholarThere is no corresponding record for this reference.
- 22Lai, R.; Zhao, C.; Guo, W.; Xiao, Y.; Li, R.; Liu, L.; Pan, H. The longitudinal and regional analysis of bleomycin-induced pulmonary fibrosis in mice by microcomputed tomography. Heliyon 2023, 9 (5), e15681 DOI: 10.1016/j.heliyon.2023.e15681Google ScholarThere is no corresponding record for this reference.
- 23Song, S.; Fu, Z.; Guan, R.; Zhao, J.; Yang, P.; Li, Y.; Yin, H.; Lai, Y.; Gong, G.; Zhao, S.; Yu, J.; Peng, X.; He, Y.; Luo, Y.; Zhong, N.; Su, J. Intracellular hydroxyproline imprinting following resolution of bleomycin-induced pulmonary fibrosis. Eur. Respir. J. 2022, 59 (5), 2100864, DOI: 10.1183/13993003.00864-2021Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XhsVyltLbL&md5=7d4c9c624ed1ab9586bc9475ccea960eIntracellular hydroxyproline imprinting following resolution of bleomycin-induced pulmonary fibrosisSong, Shengren; Fu, Zhenli; Guan, Ruijuan; Zhao, Jie; Yang, Penghui; Li, Yang; Yin, Hang; Lai, Yunxin; Gong, Gencheng; Zhao, Simin; Yu, Jiangtian; Peng, Xiaomin; He, Ying; Luo, Yumei; Zhong, Nanshan; Su, JinEuropean Respiratory Journal (2022), 59 (5), 2100864CODEN: ERJOEI; ISSN:1399-3003. (European Respiratory Society)Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease with few treatment options. The poor success in developing anti-IPF strategies has impelled researchers to reconsider the importance of the choice of animal model and assessment methodologies. Currently, it is still not settled whether the bleomycin-induced lung fibrosis mouse model finally returns to resoln. This study aimed to follow the dynamic fibrotic features of bleomycin-treated mouse lungs over extended durations through a combination of the latest technologies (micro-computed tomog. imaging and histol. detection of degraded collagens) and traditional methods. In addn., we also applied immunohistochem. to explore the distribution of all hydroxyproline-contg. mols. As detd. by classical biochem. methods, total lung hydroxyproline contents reached a peak at 4 wk after bleomycin injury and maintained a steady high level thereafter until the end of the expts. (16 wk). This result seemed to partially contradict with the changes of other fibrosis evaluation parameters, which indicated a gradual degrdn. of collagens and a recovery of lung aeration after the fibrosis peak. This inconsistency was well reconciled by our data from immunostaining against hydroxyproline and fluorescent peptide staining against degraded collagen, together showing large amts. of hydroxyproline-rich degraded collagen fragments detained and enriched within the intracellular regions at 10 or 16 wk rather than at 4 wk after bleomycin treatment. Our present data not only offer respiratory researchers a new perspective towards the resoln. nature of mouse lung fibrosis, but also remind them to be cautious when using the hydroxyproline content assay to evaluate the severity of fibrosis.
- 24Mecozzi, L.; Mambrini, M.; Ruscitti, F.; Ferrini, E.; Ciccimarra, R.; Ravanetti, F.; Sverzellati, N.; Silva, M.; Ruffini, L.; Belenkov, S.; Civelli, M.; Villetti, G.; Stellari, F. F. In-vivo lung fibrosis staging in a bleomycin-mouse model: a new micro-CT guided densitometric approach. Sci. Rep. 2020, 10, 18735, DOI: 10.1038/s41598-020-71293-3Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXit1Orur%252FP&md5=5484858297710c92f6540f42aa0a5102In-vivo lung fibrosis staging in a bleomycin-mouse model: a new micro-CT guided densitometric approachMecozzi, Laura; Mambrini, Martina; Ruscitti, Francesca; Ferrini, Erica; Ciccimarra, Roberta; Ravanetti, Francesca; Sverzellati, Nicola; Silva, Mario; Ruffini, Livia; Belenkov, Sasha; Civelli, Maurizio; Villetti, Gino; Stellari, Fabio FrancoScientific Reports (2020), 10 (1), 18735CODEN: SRCEC3; ISSN:2045-2322. (Nature Research)Abstr.: Although increasing used in the preclin. testing of new anti-fibrotic drugs, a thorough validation of micro-computed tomog. (CT) in pulmonary fibrosis models has not been performed. Moreover, no attempts have been made so far to define d. thresholds to discriminate between aeration levels in lung parenchyma. In the present study, a histogram-based anal. was performed in a mouse model of bleomycin (BLM)-induced pulmonary fibrosis by micro-CT, evaluating longitudinal d. changes from 7 to 21 days after BLM challenge, a period representing the progression of fibrosis. Two discriminative densitometric indexes (i.e. 40th and 70th percentiles) were extd. from Hounsfield Unit d. distributions and selected for lung fibrosis staging. The strong correlation with histol. findings (rSpearman = 0.76, p < 0.01) confirmed that variations in 70th percentile could reflect a pathol. lung condition and est. the effect of antifibrotic treatments. This index was therefore used to define lung aeration levels in mice distinguishing in hyper-inflated, normo-, hypo- and non-aerated pulmonary compartments. A retrospective anal. performed on a large cohort of mice confirmed the correlation between the proposed preclin. d. thresholds and the histol. outcomes (rSpearman = 0.6, p < 0.01), strengthening their suitability for tracking disease progression and evaluating antifibrotic drug candidates.
- 25Olteanu, H.; Banerjee, R. Human Methionine Synthase Reductase, a Soluble P-450 Reductase-like Dual Flavoprotein, Is Sufficient for NADPH-dependent Methionine Synthase Activation*. J. Biol. Chem. 2001, 276 (38), 35558– 35563, DOI: 10.1074/jbc.M103707200Google ScholarThere is no corresponding record for this reference.
- 26Leal, N. A.; Olteanu, H.; Banerjee, R.; Bobik, T. A. Human ATP:Cob(I)alamin Adenosyltransferase and Its Interaction with Methionine Synthase Reductase*. J. Biol. Chem. 2004, 279 (46), 47536– 47542, DOI: 10.1074/jbc.M405449200Google ScholarThere is no corresponding record for this reference.
- 27Wolthers, K. R.; Scrutton, N. S. Protein Interactions in the Human Methionine Synthase-Methionine Synthase Reductase Complex and Implications for the Mechanism of Enzyme Reactivation. Biochemistry 2007, 46 (23), 6696– 6709, DOI: 10.1021/bi700339vGoogle Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXkvVCltrc%253D&md5=a6c4ebd9eb15ca67c7e27e4d4e8d304cProtein Interactions in the Human Methionine Synthase-Methionine Synthase Reductase Complex and Implications for the Mechanism of Enzyme ReactivationWolthers, Kirsten R.; Scrutton, Nigel S.Biochemistry (2007), 46 (23), 6696-6709CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Methionine synthase (MS) is a cobalamin-dependent enzyme. It transfers a Me group from methyltetrahydrofolate to homocysteine forming methionine and tetrahydrofolate. On the basis of sequence similarity with Escherichia coli cobalamin-dependent MS (MetH), human MS comprises four discrete functional modules that bind from the N- to C-terminus, resp., homocysteine, methyltetrahydrofolate, cobalamin, and S-adenosylmethionine (AdoMet). The C-terminal activation domain also interacts with methionine synthase reductase (MSR), a NADPH-dependent diflavin oxidoreductase required for the reductive regeneration of catalytically inert cob(II)alamin (which is formed every 200-1000 catalytic cycles of MS) to cob(I)alamin. We have investigated complex formation between the (i) MS activation domain and MSR and (ii) MS activation domain and the isolated FMN-binding domain of MSR. We show that the MS activation domain interacts directly with the FMN-binding domain of MSR. Binding is weakened at high ionic strength, emphasizing the importance of electrostatic interactions at the protein-protein interface. Mutagenesis of conserved lysine residues (Lys1071 and Lys987) in the human activation domain weakens this protein interaction. Chem. crosslinking demonstrates complex formation mediated by acidic residues (FMN-binding domain) and basic residues (activation domain). The activation domain and isolated FMN-domain form a 1:1 complex, but a 1:2 complex is formed with activation domain and MSR. The midpoint redn. potentials of the FAD and FMN cofactors of MSR are not perturbed significantly on forming this complex, implying that electron transfer to cob(II)alamin is endergonic. The kinetics of electron transfer in MSR and the MSR-activation domain complex are similar. Our studies indicate (i) conserved binding determinants, but differences in protein stoichiometry, between human MS and bacterial MetH in complex formation with redox partners; (ii) a substantial endergonic barrier to electron transfer in the reactivation complex; and (iii) a lack of control on the thermodn. and kinetics of electron transfer in MSR exerted by complex formation with activation domain. The structural and functional consequences of complex formation are discussed in light of the known crystal structure of human activation domain and the inferred conformational heterogeneity of the multidomain MSR-MS complex.
- 28Wolthers, K. R.; Lou, X.; Toogood, H. S.; Leys, D.; Scrutton, N. S. Mechanism of Coenzyme Binding to Human Methionine Synthase Reductase Revealed through the Crystal Structure of the FNR-like Module and Isothermal Titration Calorimetry. Biochemistry 2007, 46 (42), 11833– 11844, DOI: 10.1021/bi701209pGoogle Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtVOnsL%252FO&md5=2631bf19a5cea1919774001656d22b4fMechanism of Coenzyme Binding to Human Methionine Synthase Reductase Revealed through the Crystal Structure of the FNR-like Module and Isothermal Titration CalorimetryWolthers, Kirsten R.; Lou, Xiaodong; Toogood, Helen S.; Leys, David; Scrutton, Nigel S.Biochemistry (2007), 46 (42), 11833-11844CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Human methionine synthase reductase (MSR) is a 78 kDa flavoprotein that regenerates the active form of cobalamin-dependent methionine synthase (MS). MSR contains one FAD and one FMN cofactor per polypeptide and functions in the sequential transfer of reducing equiv. from NADPH to MS via its flavin centers. We report the 1.9 Å crystal structure of the NADP+-bound FNR-like module of MSR that spans the NADP(H)-binding domain, the FAD-binding domain, the connecting domain, and part of the extended hinge region, a feature unique to MSR. The overall fold of the protein is similar to that of the corresponding domains of the related diflavin reductase enzymes cytochrome P 450 reductase and neuronal nitric oxide synthase (NOS). However, the extended hinge region of MSR, which is positioned between the NADP(H)/FAD- and FMN-binding domains, is in an unexpected orientation with potential implications for the mechanism of electron transfer. Compared with related flavoproteins, there is structural variation in the NADP(H)-binding site, in particular regarding those residues that interact with the 2'-phosphate and the pyrophosphate moiety of the coenzyme. The lack of a conserved binding determinant for the 2'-phosphate does not weaken the coenzyme specificity for NADP(H) over NAD(H), which is within the range expected for the diflavin oxidoreductase family of enzymes. Isothermal titrn. calorimetry (ITC) reveals a binding const. of 37 and 2 μM for binding of NADP+ and 2',5'-ADP, resp., for the ligand-protein complex formed with full-length MSR or the isolated FNR module. These values are consistent with Ki values (36 μM for NADP+ and 1.4 μM for 2',5'-ADP) obtained from steady-state inhibition studies. The relatively weaker binding of NADP+ to MSR compared with other members of the diflavin oxidoreductase family might arise from unique electrostatic repulsive forces near the 5'-pyrophosphate moiety and/or increased hydrophobic stacking between Trp697 and the re face of the FAD isoalloxazine ring. Small structural permutations within the NADP(H)-binding cleft have profound affects on coenzyme binding, which likely retards catalytic turnover of the enzyme in the cell. The biol. implications of an attenuated mechanism of MS reactivation by MSR on methionine and folate metab. are discussed.
- 29Gherasim, C. G.; Zaman, U.; Raza, A.; Banerjee, R. Impeded Electron Transfer From a Pathogenic FMN Domain Mutant of Methionine Synthase Reductase and Its Responsiveness to Flavin Supplementation. Biochemistry 2008, 47 (47), 12515– 12522, DOI: 10.1021/bi8008328Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhtlaiu7vO&md5=6fbcc1dbf7d76af328a5a8802f7ff5caImpeded Electron Transfer From a Pathogenic FMN Domain Mutant of Methionine Synthase Reductase and Its Responsiveness to Flavin SupplementationGherasim, Carmen G.; Zaman, Uzma; Raza, Ashraf; Banerjee, RumaBiochemistry (2008), 47 (47), 12515-12522CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Methionine synthase reductase (MSR) is a diflavin oxidoreductase that transfers electrons from NADPH to oxidized cobalamin and plays a vital role in repairing inactive cobalamin-dependent methionine synthase. MSR deficiency is a recessive genetic disorder affecting folate and methionine metab. and is characterized by elevated levels of plasma homocysteine. In this study, we have examd. the mol. basis of MSR dysfunction assocd. with a patient mutation, A129T, which is housed in the FMN binding domain and is adjacent to a cluster of conserved acidic residues found in diflavin oxidoreductases. We show that the substitution of alanine with threonine destabilizes FMN binding without affecting the NADPH coenzyme specificity or affinity, indicating that the mutation's effects may be confined to the FMN module. The A129T MSR mutant transfers electrons to ferricyanide as efficiently as wild type MSR but the rate of cytochrome c, 2,6-dichloroindophenol, and menadione redn. is decreased 10-15 fold. The mutant is depleted in FMN and reactivates methionine synthase with 8% of the efficiency of wild type MSR. Reconstitution of A129T MSR with FMN partially restores its ability to reduce cytochrome c and to reactivate methionine synthase. Hydrogen-deuterium exchange mass spectrometric studies localize changes in backbone amide exchange rates to peptides in the FMN-binding domain. Together, our results reveal that the primary biochem. penalty assocd. with the A129T MSR mutant is its lower FMN content, provide insights into the distinct roles of the FAD and FMN centers in human MSR for delivering electrons to various electron acceptors, and suggest that patients harboring the A129T mutation may be responsive to riboflavin therapy.
- 30Yamada, K.; Chen, Z.; Rozen, R.; Matthews, R. G. Effects of common polymorphisms on the properties of recombinant human methylenetetrahydrofolate reductase. Proc. Natl. Acad. Sci. U. S. A. 2001, 98 (26), 14853– 14858, DOI: 10.1073/pnas.261469998Google ScholarThere is no corresponding record for this reference.
- 31Zhang, X.; Wang, J.; Chen, X.; Yu, M.; Yu, S.; Sun, Y.; Duan, J.; Sun, H.; Yuan, P. Short-term immunogenicity of standard and accelerated hepatitis B virus vaccination schedules in healthy adults: a comparative field study in China. Biosci. Rep. 2018, 38 (5), BSR20180846, DOI: 10.1042/BSR20180846Google ScholarThere is no corresponding record for this reference.
- 32Chen, J.; Wang, Q.; Yin, F. Q.; Zhang, W.; Yan, L. H.; Li, L. MTRR silencing inhibits growth and cisplatin resistance of ovarian carcinoma via inducing apoptosis and reducing autophagy. Am. J. Transl. Res. 2015, 7 (9), 1510– 1527Google ScholarThere is no corresponding record for this reference.
- 33Yoon, S.-Y.; Hong, G. H.; Kwon, H.-S.; Park, S.; Park, S. Y.; Shin, B.; Kim, T.-B.; Moon, H.-B.; Cho, Y. S. S-adenosylmethionine reduces airway inflammation and fibrosis in a murine model of chronic severe asthma via suppression of oxidative stress. Exp. Mol. Med. 2016, 48 (6), e236 DOI: 10.1038/emm.2016.35Google ScholarThere is no corresponding record for this reference.
- 34Yu, G.; Tzouvelekis, A.; Wang, R.; Herazo-Maya, J. D.; Ibarra, G. H.; Srivastava, A.; de Castro, J. P. W.; DeIuliis, G.; Ahangari, F.; Woolard, T.; Aurelien, N.; Arrojo e Drigo, R.; Gan, Y.; Graham, M.; Liu, X.; Homer, R. J.; Scanlan, T. S.; Mannam, P.; Lee, P. J.; Herzog, E. L.; Bianco, A. C.; Kaminski, N. Thyroid hormone inhibits lung fibrosis in mice by improving epithelial mitochondrial function. Nat. Med. 2018, 24 (1), 39– 49, DOI: 10.1038/nm.4447Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhvFWgs7%252FN&md5=5d89b0faa601044f72a09eae3f9c30aeThyroid hormone inhibits lung fibrosis in mice by improving epithelial mitochondrial functionYu, Guoying; Tzouvelekis, Argyris; Wang, Rong; Herazo-Maya, Jose D.; Ibarra, Gabriel H.; Srivastava, Anup; de Castro, Joao Pedro Werneck; De Iuliis, Giuseppe; Ahangari, Farida; Woolard, Tony; Aurelien, Nachelle; Arrojo e Drigo, Rafael; Gan, Ye; Graham, Morven; Liu, Xinran; Homer, Robert J.; Scanlan, Thomas S.; Mannam, Praveen; Lee, Patty J.; Herzog, Erica L.; Bianco, Antonio C.; Kaminski, NaftaliNature Medicine (New York, NY, United States) (2018), 24 (1), 39-49CODEN: NAMEFI; ISSN:1078-8956. (Nature Research)Thyroid hormone (TH) is crit. for the maintenance of cellular homeostasis during stress responses, but its role in lung fibrosis is unknown. Here we found that the activity and expression of iodothyronine deiodinase 2 (DIO2), an enzyme that activates TH, were higher in lungs from patients with idiopathic pulmonary fibrosis than in control individuals and were correlated with disease severity. We also found that Dio2-knockout mice exhibited enhanced bleomycin-induced lung fibrosis. Aerosolized TH delivery increased survival and resolved fibrosis in two models of pulmonary fibrosis in mice (intratracheal bleomycin and inducible TGF-β1). Sobetirome, a TH mimetic, also blunted bleomycin-induced lung fibrosis. After bleomycin-induced injury, TH promoted mitochondrial biogenesis, improved mitochondrial bioenergetics and attenuated mitochondria-regulated apoptosis in alveolar epithelial cells both in vivo and in vitro. TH did not blunt fibrosis in Ppargc1a- or Pink1-knockout mice, suggesting dependence on these pathways. We conclude that the antifibrotic properties of TH are assocd. with protection of alveolar epithelial cells and restoration of mitochondrial function and that TH may thus represent a potential therapy for pulmonary fibrosis.
- 35Roque, W.; Romero, F. Cellular metabolomics of pulmonary fibrosis, from amino acids to lipids. Am. J. Physiol. Cell Physiol. 2021, 320 (5), C689– C695, DOI: 10.1152/ajpcell.00586.2020Google ScholarThere is no corresponding record for this reference.
- 36Ung, C. Y.; Onoufriadis, A.; Parsons, M.; McGrath, J. A.; Shaw, T. J. Metabolic perturbations in fibrosis disease. Int. J. Biochem. Cell Biol. 2021, 139, 106073, DOI: 10.1016/j.biocel.2021.106073Google ScholarThere is no corresponding record for this reference.
- 37Lee, J.-U.; Song, K. S.; Hong, J.; Shin, H.; Park, E.; Baek, J.; Park, S.; Baek, A.-R.; Lee, J.; Jang, A. S.; Kim, D. J.; Chin, S. S.; Kim, U. J.; Jeong, S. H.; Park, S.-W. Role of lung ornithine aminotransferase in idiopathic pulmonary fibrosis: regulation of mitochondrial ROS generation and TGF-β1 activity. Exp. Mol. Med. 2024, 56 (2), 478– 490, DOI: 10.1038/s12276-024-01170-wGoogle ScholarThere is no corresponding record for this reference.
- 38Henderson, N. C.; Rieder, F.; Wynn, T. A. Fibrosis: from mechanisms to medicines. Nature 2020, 587 (7835), 555– 566, DOI: 10.1038/s41586-020-2938-9Google Scholar38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXisVeqt77N&md5=9993852a8cfbbbd72d0c85727f4be799Fibrosis: from mechanisms to medicinesHenderson, Neil C.; Rieder, Florian; Wynn, Thomas A.Nature (London, United Kingdom) (2020), 587 (7835), 555-566CODEN: NATUAS; ISSN:0028-0836. (Nature Research)Abstr.: Fibrosis can affect any organ and is responsible for up to 45% of all deaths in the industrialized world. It has long been thought to be relentlessly progressive and irreversible, but both preclin. models and clin. trials in various organ systems have shown that fibrosis is a highly dynamic process. This has clear implications for therapeutic interventions that are designed to capitalize on this inherent plasticity. However, despite substantial progress in our understanding of the pathobiol. of fibrosis, a translational gap remains between the identification of putative antifibrotic targets and conversion of this knowledge into effective treatments in humans. Here we discuss the transformative exptl. strategies that are being leveraged to dissect the key cellular and mol. mechanisms that regulate fibrosis, and the translational approaches that are enabling the emergence of precision medicine-based therapies for patients with fibrosis.
- 39Bae, D.-H.; Lane, D. J. R.; Jansson, P. J.; Richardson, D. R. The old and new biochemistry of polyamines. Biochim. Biophys. Acta Gen. Subj. 2018, 1862 (9), 2053– 2068, DOI: 10.1016/j.bbagen.2018.06.004Google Scholar39https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtlentrnK&md5=7b4865b049c3d381ba5c039276e649a3The old and new biochemistry of polyaminesBae, Dong-Hun; Lane, Darius J. R.; Jansson, Patric J.; Richardson, Des R.Biochimica et Biophysica Acta, General Subjects (2018), 1862 (9), 2053-2068CODEN: BBGSB3; ISSN:0304-4165. (Elsevier B.V.)A review. Polyamines are ubiquitous pos. charged amines found in all organisms. These mols. play a crucial role in many biol. functions including cell growth, gene regulation and differentiation. The three major polyamines produced in all mammalian cells are putrescine, spermidine and spermine. The intracellular levels of these polyamines depend on the interplay of the biosynthetic and catabolic enzymes of the polyamine and methionine salvage pathway, as well as the involvement of polyamine transporters. Polyamine levels are obsd. to be high in cancer cells, which contributes to malignant transformation, cell proliferation and poor patient prognosis. Considering the crit. roles of polyamines in cancer cell proliferation, numerous anti-polyaminergic compds. have been developed as anti-tumor agents, which seek to suppress polyamine levels by specifically inhibiting polyamine biosynthesis, activating polyamine catabolism, or blocking polyamine transporters. However, in terms of the development of effective anti-cancer therapeutics targeting the polyamine system, these efforts have unfortunately resulted in little success. Recently, several studies using the iron chelators, O-trensox and ICL670A (Deferasirox), have demonstrated a decline in both iron and polyamine levels. Since iron levels are also high in cancer cells, and like polyamines, are required for proliferation, these latter findings suggest a biochem. integrated link between iron and polyamine metab.
- 40Sanderson, S. M.; Gao, X.; Dai, Z.; Locasale, J. W. Methionine metabolism in health and cancer: a nexus of diet and precision medicine. Nat. Rev. Cancer. 2019, 19 (11), 625– 637, DOI: 10.1038/s41568-019-0187-8Google Scholar40https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhslKiu7bK&md5=8286901f7dd03a0637e87b792aaf1503Methionine metabolism in health and cancer: a nexus of diet and precision medicineSanderson, Sydney M.; Gao, Xia; Dai, Ziwei; Locasale, Jason W.Nature Reviews Cancer (2019), 19 (11), 625-637CODEN: NRCAC4; ISSN:1474-175X. (Nature Research)A review. Methionine uptake and metab. is involved in a host of cellular functions including methylation reactions, redox maintenance, polyamine synthesis and coupling to folate metab., thus coordinating nucleotide and redox status. Each of these functions has been shown in many contexts to be relevant for cancer pathogenesis. Intriguingly, the levels of methionine obtained from the diet can have a large effect on cellular methionine metab. This establishes a link between nutrition and tumor cell metab. that may allow for tumor-specific metabolic vulnerabilities that can be influenced by diet. Recently, a no. of studies have begun to investigate the mol. and cellular mechanisms that underlie the interaction between nutrition, methionine metab. and effects on health and cancer.
- 41Parkhitko, A. A.; Jouandin, P.; Mohr, S. E.; Perrimon, N. Methionine metabolism and methyltransferases in the regulation of aging and lifespan extension across species. Aging Cell 2019, 18 (6), e13034 DOI: 10.1111/acel.13034Google Scholar41https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhs1CmsLbF&md5=dff592c1c5f532b83766085513cd1cb3Methionine metabolism and methyltransferases in the regulation of aging and lifespan extension across speciesParkhitko, Andrey A.; Jouandin, Patrick; Mohr, Stephanie E.; Perrimon, NorbertAging Cell (2019), 18 (6), e13034CODEN: ACGECQ; ISSN:1474-9718. (Wiley-Blackwell)A review. Methionine restriction (MetR) extends lifespan across different species and exerts beneficial effects on metabolic health and inflammatory responses. In contrast, certain cancer cells exhibit methionine auxotrophy that can be exploited for therapeutic treatment, as decreasing dietary methionine selectively suppresses tumor growth. Thus, MetR represents an intervention that can extend lifespan with a complementary effect of delaying tumor growth. Manipulation of each of these branches extends lifespan; however, the interplay between MetR and these branches during regulation of lifespan is not well understood. In addn., a potential mechanism linking the activity of methionine metab. and lifespan is regulation of prodn. of the Me donor S-adenosylmethionine, which, after transferring its Me group, is converted to S-adenosylhomocysteine. Although the exact mechanisms of lifespan extension by MetR or methionine metab. reprogramming are unknown, it may act via reducing the rate of translation, modifying gene expression, inducing a hormetic response, modulating autophagy, or inducing mitochondrial function, antioxidant defense, or other metabolic processes. Here, we review the mechanisms of lifespan extension by MetR and different branches of methionine metab. in different species and the potential for exploiting the regulation of methyltransferases to delay aging.
- 42Li, Z.; Wang, F.; Liang, B.; Su, Y.; Sun, S.; Xia, S.; Shao, J.; Zhang, Z.; Hong, M.; Zhang, F.; Zheng, S. Methionine metabolism in chronic liver diseases: an update on molecular mechanism and therapeutic implication. Signal Transduct. Target. Ther. 2020, 5, 280, DOI: 10.1038/s41392-020-00349-7Google ScholarThere is no corresponding record for this reference.
- 43Ramani, K.; Donoyan, S.; Tomasi, M. L.; Park, S. Role of Methionine Adenosyltransferase α2 and β Phosphorylation and Stabilization in Human Hepatic Stellate Cell Trans-Differentiation. J. Cell. Physiol. 2015, 230 (5), 1075– 1085, DOI: 10.1002/jcp.24839Google ScholarThere is no corresponding record for this reference.
- 44Wang, K.; Fang, S.; Liu, Q.; Gao, J.; Wang, X.; Zhu, H.; Zhu, Z.; Ji, F.; Wu, J.; Ma, Y.; Hu, L.; Shen, X.; Gao, D.; Zhu, J.; Liu, P.; Zhou, H. TGF-β1/p65/MAT2A pathway regulates liver fibrogenesis via intracellular SAM. EBioMedicine 2019, 42, 458– 469, DOI: 10.1016/j.ebiom.2019.03.058Google Scholar44https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3cbps1WlsA%253D%253D&md5=dfb702e80ee1028d16b4e7a5ac7ef675TGF-β1/p65/MAT2A pathway regulates liver fibrogenesis via intracellular SAMWang Kuifeng; Ji Feihong; Fang Shanhua; Liu Qian; Gao Jing; Zhu Hongwen; Zhu Zhenyun; Wang Xiaoning; Wu Jiasheng; Ma Yueming; Hu Lihong; Shen Xu; Gao Daming; Zhu Jiansheng; Liu Ping; Zhou HuEBioMedicine (2019), 42 (), 458-469 ISSN:.BACKGROUND: Hepatic stellate cell (HSC) activation induced by transforming growth factor β1 (TGF-β1) plays a pivotal role in fibrogenesis, while the complex downstream mediators of TGF-β1 in such process are largely unknown. METHODS: We performed pharmacoproteomic profiling of the mice liver tissues from control, carbon tetrachloride (CCl4)-induced fibrosis and NPLC0393 administrated groups. The target gene MAT2A was overexpressed or knocked down in vivo by tail vein injection of AAV vectors. We examined NF-κB transcriptional activity on MAT2A promoter via luciferase assay. Intracellular SAM contents were analyzed by LC-MS method. FINDINGS: We found that methionine adenosyltransferase 2A (MAT2A) is significantly upregulated in the CCl4-induced fibrosis mice, and application of NPLC0393, a known small molecule inhibitor of TGF-β1 signaling pathway, inhibits the upregulation of MAT2A. Mechanistically, TGF-β1 induces phosphorylation of p65, i.e., activation of NF-κB, thereby promoting mRNA transcription and protein expression of MAT2A and reduces S-adenosylmethionine (SAM) concentration in HSCs. Consistently, in vivo and in vitro knockdown of MAT2A alleviates CCl4- and TGF-β1-induced HSC activation, whereas in vivo overexpression of MAT2A facilitates hepatic fibrosis and abolishes therapeutic effect of NPLC0393. INTERPRETATION: This study identifies TGF-β1/p65/MAT2A pathway that is involved in the regulation of intracellular SAM concentration and liver fibrogenesis, suggesting that this pathway is a potential therapeutic target for hepatic fibrosis. FUND: This work was supported by National Natural Science Foundation of China (No. 81500469, 81573873, 81774196 and 31800693), Zhejiang Provincial Natural Science Foundation of China (No. Y15H030004), the National Key Research and Development Program from the Ministry of Science and Technology of China (No. 2017YFC1700200) and the Key Program of National Natural Science Foundation of China (No. 8153000502).
- 45Mato, J. M.; Martinez-Chantar, M. L.; Lu, S. C. S-adenosylmethionine metabolism and liver disease. Ann. Hepatol. 2013, 12 (2), 183– 9, DOI: 10.1016/S1665-2681(19)31355-9Google Scholar45https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhtVant7vM&md5=3bbff53ca1f23ac05d7474d81ed7c0d0S-adenosylmethionine metabolism and liver diseaseMato, Jose M.; Luz Martinez-Chantar, M.; Lu, Shelly C.Annals of Hepatology (2013), 12 (2), 183-189CODEN: AHNEAQ; ISSN:1665-2681. (Mexican Association of Hepatology)A review. Methionine is an essential amino acid that is metabolized mainly by the liver where it is converted to S-adenosylmethionine (SAMe) by the enzyme methionine adenosyltransferase. Although all mammalian cells synthesize SAMe, the liver is where the bulk of SAMe is generated as it is the organ where about 50% of all dietary methionine is metabolized. SAMe is mainly needed for methylation of a large variety of substrates (DNA, proteins, lipids and many other small mols.) and polyamine synthesis, so if the concn. of SAMe falls below a certain level or rises too much the normal function of the liver will be also affected. There are physiol. conditions that can affect the hepatic content of SAMe. Consequently, to control these fluctuations, the rate at which the liver both synthesizes and catabolizes SAMe is tightly regulated. In mice, failure to do this can lead to fatty liver disease and to the development of hepatocellular carcinoma (HCC). Therefore, maintaining SAMe homeostasis may be a therapeutic target in nonalcoholic steatohepatitis, alc.- and non-alc. liver cirrhosis, and for the chemoprevention of HCC formation.
- 46Casini, A.; Banchetti, E.; Milani, S.; Maggioni Moratti, E.; Surrenti, C. S-adenosylmethionine inhibits collagen synthesis by human fibroblasts in vitro. Methods Find. Exp. Clin. Pharmacol. 1989, 11 (5), 331– 334Google ScholarThere is no corresponding record for this reference.
- 47Karaa, A.; Thompson, K. J.; McKillop, I. H.; Clemens, M. G.; Schrum, L. W. S-adenosyl-L-methionine attenuates oxidative stress and hepatic stellate cell activation in an ethanol-LPS-induced fibrotic rat model. Shock 2008, 30 (2), 197– 205, DOI: 10.1097/SHK.0b013e318160f417Google ScholarThere is no corresponding record for this reference.
- 48Jubinville, É.; Milad, N.; Maranda-Robitaille, M.; Lafrance, M.-A.; Pineault, M.; Lamothe, J.; Routhier, J.; Beaulieu, M.-J.; Aubin, S.; Laplante, M.; Morissette, M. C. Critical importance of dietary methionine and choline in the maintenance of lung homeostasis during normal and cigarette smoke exposure conditions. Am. J. Physiol. Lung Cell. Mol. Physiol. 2020, 319 (2), L391– L402, DOI: 10.1152/ajplung.00353.2019Google ScholarThere is no corresponding record for this reference.
- 49Gao, J.; Cahill, C. M.; Huang, X.; Roffman, J. L.; Lamon-Fava, S.; Fava, M.; Mischoulon, D.; Rogers, J. T. S-Adenosyl Methionine and Transmethylation Pathways in Neuropsychiatric Diseases Throughout Life. Neurotherapeutics 2018, 15 (1), 156– 175, DOI: 10.1007/s13311-017-0593-0Google ScholarThere is no corresponding record for this reference.
- 50Cavallaro, R. A.; Fuso, A.; Nicolia, V.; Scarpa, S. S-adenosylmethionine prevents oxidative stress and modulates glutathione metabolism in TgCRND8 mice fed a B-vitamin deficient diet. J. Alzheimers Dis. 2010, 20 (4), 997– 1002, DOI: 10.3233/JAD-2010-091666Google ScholarThere is no corresponding record for this reference.
- 51Huang, X.; Sun, P.; Qin, Y.; Wang, X.-j.; Wang, M.; Lin, Y.; Zhou, R.; Hu, W.; Liu, Q.; Yu, X.; Qin, A. Disulfiram attenuates MCMV-Induced pneumonia by inhibition of NF-κB/NLRP3 signaling pathway in immunocompromised mice. Int. Immunopharmacol. 2022, 103, 108453, DOI: 10.1016/j.intimp.2021.108453Google ScholarThere is no corresponding record for this reference.
- 52Pai, M. Y.; Lomenick, B.; Hwang, H.; Schiestl, R.; McBride, W.; Loo, J. A.; Huang, J. Drug affinity responsive target stability (DARTS) for small-molecule target identification. Methods Mol. Biol. 2015, 1263, 287– 98, DOI: 10.1007/978-1-4939-2269-7_22Google Scholar52https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xls12murk%253D&md5=17d9d8199173653f814d80e0945c94adDrug Affinity Responsive Target Stability (DARTS) for Small-Molecule Target IdentificationPai, Melody Y.; Lomenick, Brett; Hwang, Heejun; Schiestl, Robert; McBride, William; Loo, Joseph A.; Huang, JingMethods in Molecular Biology (New York, NY, United States) (2015), 1263 (Chemical Biology), 287-298CODEN: MMBIED; ISSN:1940-6029. (Springer)Drug affinity responsive target stability (DARTS) is a relatively quick and straightforward approach to identify potential protein targets for small mols. It relies on the protection against proteolysis conferred on the target protein by interaction with a small mol. The greatest advantage of this method is being able to use the native small mol. without having to immobilize or modify it (e.g., by incorporation of biotin, fluorescent, radioisotope, or photoaffinity labels). Here we describe in detail the protocol for performing unbiased DARTS with complex protein lysates to identify binding targets of small mols. and for using DARTS-Western blotting to test, screen, or validate potential small-mol. targets. Although the ideas have mainly been developed from studying mols. in areas of biol. that are currently of interest to us and our collaborators, the general principles should be applicable to the anal. of all mols. in nature.
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Abstract
Figure 1
Figure 1. Inhibitory effects of chlorquinaldol on the proliferation and differentiation of pulmonary fibroblasts. (A) Schematic of a phenotypic screening procedure for antifibrotic agent discovery. Normal murine fibroblasts (NIH/3T3) were seeded in 96-well plates and pretreated with 10 ng/mL transforming growth factor-β1 (TGFβ1) for 48 h, followed by 24 h of exposure to 10 μM test compounds or 0.1% DMSO. Proliferation was measured via CCK8 assay to assess the antifibrotic potential of the compounds. (B) The CCK-8 assay illustrates the cell viability in response to compound exposure. Notably, four compounds significantly reduced TGFβ1-stimulated fibroblast proliferation by at least 50%. (C) The chemical structure of chlorquinaldol (CQD), characterized as 5,7-dichloro-2-methyl-8-hydroxyquinoline, a known antibacterial agent, is depicted. (D, E) Both quiescent and TGFβ1-activated MRC5 and NIH/3T3 cells were incubated with a range of CQD concentrations or DMSO vehicles for 72 h. Cell proliferation was then evaluated using the CCK-8 assay. (F, G) Following a 24 h treatment with DMSO or CQD, MRC5 cells were subjected to Western blot analysis to determine the protein expression levels of fibronectin, collagen I, and α-SMA, with GAPDH serving as the loading standard. Experiments were performed in triplicate (n = 3). (H, I) Western blot analysis was similarly conducted to assess the protein expression of fibronectin, collagen I, and α-SMA in NIH/3T3 cells, using GAPDH as the internal control. Each condition was replicated three times (n = 3). Data are represented as the mean ± standard error of the mean (SEM). Statistical significance is indicated for comparisons against unstimulated control with * for p < 0.05, ** for p < 0.01, and *** for p < 0.001 and against TGFβ1 alone with # for p < 0.05, ## for p < 0.01, and ### for p < 0.001.
Figure 2
Figure 2. Protective effect of chlorquinaldol on bleomycin-induced pulmonary fibrosis. (A) Schematic of the preventive treatment protocol for lung fibrosis in animal models. (B) Survival curve for mice over 21 days following BLM challenge (n = 6–10). (C) HE stained lung tissue sections from mice on day 21 after BLM exposure. The inset displays a zoomed area (200× magnification), with a 200 μm scale bar (n = 4–6). (D) Representative images of Masson’s trichrome staining in lung tissue; the inset zooms in on a detailed area (200 × ), with a 200 μm scale bar (n = 4–6). (E) Assessment of the collagenous area ratio from Masson’s trichrome-stained sections. (F) Quantification of hydroxyproline levels in the right lung lobes. Data presented as mean ± SEM for 4–7 animals per group. Significance is indicated by *p < 0.05, **p < 0.01, and ***p < 0.001 for comparisons with the control and #p < 0.05, ##p < 0.01, and ###p < 0.001 for other group comparisons as indicated.
Figure 3
Figure 3. Chlorquinaldol ameliorates pulmonary fibrosis and lung ventilation. (A) Treatment protocol for the pulmonary fibrosis mouse model, including the administration timeline for CQD or nintedanib. The first 9 days of post-BLM induction represent the inflammatory phase, followed by the fibrotic phase. (B) The survival rates of mice within the 21-day BLM model (n = 6–12). (C) Representative micro-CT images of the whole lung on the 21st day, featuring axial, coronal, and sagittal views, as well as three-dimensional reconstructions. All images of the right mainstem bronchus bifurcation were selected to ensure consistent anatomical comparison. (D) Analysis of the lung volume ventilation fraction in mice, with green indicating normally aerated areas (−860 to −435 HU), yellow representing poorly aerated areas (−434 to −121 HU), and red signifying nonaerated regions (−120 to +121 HU). Data are shown as mean ± SEM, with n = 3–5 per group. (E, F) The dynamic changes in the airway constriction index Penh and the midexpiratory flow rate EF50, each presented as mean ± SEM (n = 3–12). Statistical significance is denoted by *p < 0.05, **p < 0.01, and ***p < 0.001 for comparisons with the control group and #p < 0.05, ##p < 0.01, and ###p < 0.001 for comparisons with the BLM group.
Figure 4
Figure 4. Chlorquinaldol improved alveolar architecture and reduced interstitial collagen deposition. (A) Representative images of whole lung HE staining on the 21st day. (B) Pulmonary fibrosis scores based on the Ashcroft scoring system. (C) Representative Masson’s trichrome staining of entire lung tissue from the mice. (D) Quantitative analysis of collagen content as determined by Masson’s staining. Scale bar: 100 μM. Data is expressed as mean ± SEM (n = 3). Significance is denoted by *p < 0.05, **p < 0.01, ***p < 0.001 compared to the control and #p < 0.05, ##p < 0.01, ###p < 0.001 for comparisons with the BLM group.
Figure 5
Figure 5. Chlorquinaldol directly targets MTRR protein. (A) Schematic of the DARTS/MS strategy for discovering potential chlorquinaldol binding proteins. (B) Heatmap of 18 candidate targets with differential expression levels, as determined by mass spectrometry. (C) SPR measures the binding affinity of chlorquinaldol to MTRR protein. (D, E) Immunoblots of MTRR levels in TGFβ1-activated NIH/3T3 cells with chlorquinaldol treatment and subsequent Pronase digestion. (F, G) CETSA melt response and related curves to assess the thermostability between chlorquinaldol and MTRR. (H, I) Isothermal dose response (ITDR) and its curve indicating the binding thermodynamics of chlorquinaldol. Data are mean ± SEM (n = 3), with statistical significance marked by *p < 0.05, **p < 0.01, ***p < 0.001 versus control. Abbreviations: MTRR, methionine synthase reductase; DARTS, drug affinity responsive target stability assay; SPR, surface plasmon resonance; CETSA, cellular thermal shift assay; CQD, chlorquinaldol.
Figure 6
Figure 6. Chlorquinaldol exerts antifibrotic activity by directly interacting with the FAD domain of the MTRR protein. (A) Cartoon depiction of the structure of murine methionine synthase reductase (MTRR), featuring FMN and FAD domains connected by a flexible hinge. (B) Three-dimensional representation demonstrating chlorquinaldol’s top three binding sites within the MTRR protein architecture. (C) Detailed 3D interaction map of chlorquinaldol with the MTRR protein, alongside a local secondary structure binding interaction diagram. (D–G) SPR analysis detecting the binding of four peptides (peptide 1, 2, and 3 and peptide 1 mutant V467A) to chlorquinaldol. (H) Molecular docking simulation revealing the chlorquinaldol–MTRR binding interface, with interactions indicated by purple arrows and noncovalent distance interactions indicated by green dashed lines. Abbreviation: FMN, flavin mononucleotide; FAD, flavin adenine dinucleotide.
Figure 7
Figure 7. Chlorquinaldol promotes methionine and s-adenosyl methionine (SAM) accumulation via MTRR to inhibit fibrosis. (A) A violin plot illustrates the expression levels of methionine synthase reductase (MTRR) in lung tissues from idiopathic pulmonary fibrosis (IPF) patients, with data obtained from the GEO data set GSE213001. (B) qPCR analysis assesses the efficiency of Mtrr knockdown in NIH/3T3 cells. (C–F) Western blot (WB) analysis measures the protein expression levels of fibronectin, collagen I, and α-smooth muscle actin (α-SMA) in Mtrr knockdown cells following TGFβ1 stimulation. (G–J) HPLC/MS was utilized to quantify intracellular methionine, SAM, and SAH levels. (K–L) NIH/3T3 cells are pretreated with varying concentrations of SAM for 24 h before TGFβ1 stimulation, and WB is used to assess the protein expression levels of fibronectin, collagen I, and α-SMA. Data are presented as mean ± SEM (n = 3). Significance is denoted by *p < 0.05, **p < 0.01, and ***p < 0.001 compared to the control. Abbreviations: SAM, s-adenosylmethionine; SAH, s-adenosylhomocysteine.
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- 6Amati, F.; Stainer, A.; Polelli, V.; Mantero, M.; Gramegna, A.; Blasi, F.; Aliberti, S. Efficacy of Pirfenidone and Nintedanib in Interstitial Lung Diseases Other than Idiopathic Pulmonary Fibrosis: A Systematic Review. Int. J. Mol. Sci. 2023, 24 (9), 7849, DOI: 10.3390/ijms24097849There is no corresponding record for this reference.
- 7Raghu, G.; Selman, M. Nintedanib and Pirfenidone. New Antifibrotic Treatments Indicated for Idiopathic Pulmonary Fibrosis Offer Hopes and Raises Questions. Am. J. Respir. Crit. Care Med. 2015, 191 (3), 252– 254, DOI: 10.1164/rccm.201411-2044EDThere is no corresponding record for this reference.
- 8Galli, J. A.; Pandya, A.; Vega-Olivo, M.; Dass, C.; Zhao, H.; Criner, G. J. Pirfenidone and nintedanib for pulmonary fibrosis in clinical practice: Tolerability and adverse drug reactions. Respirology 2017, 22 (6), 1171– 1178, DOI: 10.1111/resp.130248https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1czosFOmtw%253D%253D&md5=c130cb9a8a630a30a7380bb310bac5f0Pirfenidone and nintedanib for pulmonary fibrosis in clinical practice: Tolerability and adverse drug reactionsGalli Jonathan A; Pandya Aloknath; Vega-Olivo Michelle; Dass Chandra; Zhao Huaqing; Criner Gerard JRespirology (Carlton, Vic.) (2017), 22 (6), 1171-1178 ISSN:.BACKGROUND AND OBJECTIVE: The real-world tolerability of pirfenidone and nintedanib in non-clinical trial patients is unknown. Many patients with pulmonary fibrosis have significant medical co-morbidities or baseline characteristics that exclude them from clinical trial participation. METHODS: We conducted a retrospective chart review study on subjects prescribed nintedanib or pirfenidone for pulmonary fibrosis treatment (any aetiology) from September 2014 to February 2016. A total of 186 subjects were included: 129 received pirfenidone and 57 were prescribed nintedanib and followed up for mean observation periods of 52 ± 17 weeks for pirfenidone and 41 ± 15 weeks for nintedanib. The primary outcome was drug discontinuation as a result of an adverse event. RESULTS: Subjects had significant respiratory impairment at baseline, 63% required home oxygen therapy and mean diffusion capacity of carbon monoxide (DLCO) was 36 ± 14% predicted. Drug discontinuation as a result of an adverse event occurred in 20.9% of subjects on pirfenidone and 26.3% on nintedanib. Drug discontinuation rates for both pirfenidone and nintedanib did not significantly differ from corresponding large clinical trials (ASCEND/CAPACITY and INPULSIS 1 and 2, respectively). Adverse events that occurred with highest frequency on pirfenidone were nausea (26.4%), rash/photosensitivity (14.7%) and dyspepsia/gastroesophageal reflux disease (GERD) (12.4%). Diarrhoea (52.6%) and nausea (29.8%) were reported most often with nintedanib therapy. CONCLUSION: Patients with pulmonary fibrosis treated with nintedanib or pirfenidone in routine clinical practice had drug tolerability and adverse event profiles comparable with subjects enrolled in clinical trials despite having a greater degree of respiratory impairment and a high prevalence of co-morbid medical conditions.
- 9Pardo, A.; Selman, M. Lung Fibroblasts, Aging, and Idiopathic Pulmonary Fibrosis. Ann. Am. Thorac. Soc. 2016, 13 (Supplement_5), S417– S421, DOI: 10.1513/AnnalsATS.201605-341AWThere is no corresponding record for this reference.
- 10Misharin, A. V.; Budinger, G. R. S. Targeting the Myofibroblast in Pulmonary Fibrosis. Am. J. Respir. Crit. Care Med. 2018, 198 (7), 834– 835, DOI: 10.1164/rccm.201806-1037EDThere is no corresponding record for this reference.
- 11Zhang, J.-x.; Huang, P.-j.; Wang, D.-p.; Yang, W.-y.; Lu, J.; Zhu, Y.; Meng, X.-x.; Wu, X.; Lin, Q.-h.; Lv, H.; Xie, H.; Wang, R.-l. m6A modification regulates lung fibroblast-to-myofibroblast transition through modulating KCNH6 mRNA translation. Mol. Ther. 2021, 29 (12), 3436– 3448, DOI: 10.1016/j.ymthe.2021.06.008There is no corresponding record for this reference.
- 12Hamanaka, R. B.; Mutlu, G. M. Metabolic requirements of pulmonary fibrosis: role of fibroblast metabolism. FEBS J. 2021, 288 (22), 6331– 6352, DOI: 10.1111/febs.15693There is no corresponding record for this reference.
- 13Yu, W.-C.; Yeh, T.-Y.; Ye, C.-H.; Chong, P. C. T.; Ho, Y.-H.; So, D. K.; Yap, K. Y.; Peng, G.-R.; Shao, C.-H.; Jagtap, A. D.; Chern, J.-W.; Lin, C.-S.; Lin, S.-P.; Lin, S.-L.; Yu, S.-H.; Yu, C.-W. Discovery of HDAC6, HDAC8, and 6/8 Inhibitors and Development of Cell-Based Drug Screening Models for the Treatment of TGF-β-Induced Idiopathic Pulmonary Fibrosis. J. Med. Chem. 2023, 66 (15), 10528– 10557, DOI: 10.1021/acs.jmedchem.3c00644There is no corresponding record for this reference.
- 14Heukels, P.; Moor, C. C.; von der Thüsen, J. H.; Wijsenbeek, M. S.; Kool, M. Inflammation and immunity in IPF pathogenesis and treatment. Respir. Med. 2019, 147, 79– 91, DOI: 10.1016/j.rmed.2018.12.01514https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3cjnsFyitA%253D%253D&md5=244456fa676911536e075c1d31d5458aInflammation and immunity in IPF pathogenesis and treatmentHeukels P; Moor C C; Wijsenbeek M S; von der Thusen J H; Kool MRespiratory medicine (2019), 147 (), 79-91 ISSN:.Idiopathic pulmonary fibrosis (IPF) is a progressive, and ultimately fatal, chronic interstitial lung disease characterized by enhanced extracellular matrix deposition. Repetitive alveolar epithelial injury triggers the early development of fibrosis. These injuries, in combination with dysregulated wound repair and fibroblast dysfunction, lead to ongoing tissue remodelling and fibrosis seen in end-stage pulmonary fibrosis. Although the exact etiology in IPF is unknown and probably diverse, all stages of fibrosis are accompanied by innate and adaptive immune responses. The role of inflammation as an important component in IPF etiology is controversial and sometimes seen as an epiphenomenon of fibrosis. This view is partly the result of negative multicenter trials of anti-inflammatory drugs for IPF treatment. However, new insights on the role of macrophages, the loss of T-cell and B-cell tolerance leading auto-immune responses in IPF, and the interaction of immune cells with (myo)fibroblasts have led to a slow change of this opinion. Clearly, more insight is needed to integrate basic immune mechanisms into translational research and finally new IPF therapies. In this concise review, we will focus on the role of our innate and adaptive immune system in the initiation and perpetuation of IPF pathobiology. Next, we will discuss how immune responses are influenced by current anti-fibrotic treatments, such as pirfenidone and nintedanib and end with an overview of recent and upcoming therapeutic trials that target and modulate our immune system in patients with IPF.
- 15Wang, L.; Deng, K.; Gong, L.; Zhou, L.; Sayed, S.; Li, H.; Sun, Q.; Su, Z.; Wang, Z.; Liu, S.; Zhu, H.; Song, J.; Lu, D. Chlorquinaldol targets the β-catenin and T-cell factor 4 complex and exerts anti-colorectal cancer activity. Pharmacol. Res. 2020, 159, 104955, DOI: 10.1016/j.phrs.2020.104955There is no corresponding record for this reference.
- 16Chen, Y.; Chen, X.; Liang, S.; Ou, Y.; Lin, G.; Hua, L.; Wu, X.; Zhou, Y.; Liu, Z.; Cai, H.; Yang, Z.; Hu, W.; Sun, P. Chlorquinaldol inhibits the activation of nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing protein 3 inflammasome and ameliorates imiquimod-induced psoriasis-like dermatitis in mice. Chem. Biol. Interact. 2022, 365, 110122, DOI: 10.1016/j.cbi.2022.110122There is no corresponding record for this reference.
- 17Bidossi, A.; Bottagisio, M.; De Grandi, R.; Drago, L.; De Vecchi, E. Chlorquinaldol, a topical agent for skin and wound infections: anti-biofilm activity and biofilm-related antimicrobial cross-resistance. Infect. Drug Resist. 2019, 12, 2177– 2189, DOI: 10.2147/IDR.S21100717https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXjsFaqurg%253D&md5=c194ca81f0e844b5d187d9f1f264885fChlorquinaldol, a topical agent for skin and wound infections: anti-biofilm activity and biofilm-related antimicrobial cross-resistanceBidossi, Alessandro; Bottagisio, Marta; De Grandi, Roberta; Drago, Lorenzo; De Vecchi, ElenaInfection and Drug Resistance (2019), 12 (), 2177-2189CODEN: IDRNAV; ISSN:1178-6973. (Dove Medical Press Ltd.)Purpose: Persistence of skin and wound infections is nowadays accepted being linked to bacterial biofilms, which are highly recalcitrant to treatments and contribute to maintain a const. inflammation state and prevent a correct healing. Topical antimicrobials are the most common first-line self-medications; however, treatment failure is not uncommon and emerging resistance to antibiotics is alarming. Chlorquinaldol is an antimicrobial with a wide spectrum of activity and desirable characteristics for topical application. Aim of this study was to evaluate the efficacy of chlorquinaldol to prevent or eradicate S. aureus and P. aeruginosa biofilms, in comparison to classic topical antibiotics like gentamicin and fusidic acid. Methods: Min. inhibitory concns. (MIC) were assessed for each strain and subinhibitory concns. (1/2 and 1/4 MIC) were used in the biofilm assay. Antimicrobial assays were performed during biofilm formation or were applied on mature biofilms and were evaluated by means of crystal violet assay and confocal laser scan microscopy. Results: Chlorquinaldol and gentamicin were the most effective antimicrobials in both eradicating and preventing pathogens biofilm; however, resistance to methicillin and impermeability to carbapenems impaired chlorquinaldol effect. In addn., similarly to other hydroxyquinolines, aspecific metal chelation is here proposed as chlorquinaldol mode of action. Conclusion: Relying on an acceptable antibiofilm and a wide spectrum of activity, an aspecific mode of action and consequent absence of resistance development, chlorquinaldol proved to be a good antimicrobial for topical use.
- 18Chen, X.; Guo, H. Comparison of Chlorquinaldol-Promestriene Vaginal Tablets and Opin Suppositories Effect on Inflammatory Factors and Immune Function in Chronic HPV Cervicitis. J. Coll. Physicians Surg. Pak. 2019, 29 (2), 115– 118, DOI: 10.29271/jcpsp.2019.02.115There is no corresponding record for this reference.
- 19Darby, C. M.; Nathan, C. F. Killing of non-replicating Mycobacterium tuberculosis by 8-hydroxyquinoline. J. Antimicrob. Chemother. 2010, 65 (7), 1424– 1427, DOI: 10.1093/jac/dkq145There is no corresponding record for this reference.
- 20Scotton, C. J.; Hayes, B.; Alexander, R.; Datta, A.; Forty, E. J.; Mercer, P. F.; Blanchard, A.; Chambers, R. C. Ex vivo micro-computed tomography analysis of bleomycin-induced lung fibrosis for preclinical drug evaluation. Eur. Respir. J. 2013, 42 (6), 1633– 1645, DOI: 10.1183/09031936.00182412There is no corresponding record for this reference.
- 21Ruscitti, F.; Ravanetti, F.; Donofrio, G.; Ridwan, Y.; van Heijningen, P.; Essers, J.; Villetti, G.; Cacchioli, A.; Vos, W.; Stellari, F. F. A Multimodal Imaging Approach Based on Micro-CT and Fluorescence Molecular Tomography for Longitudinal Assessment of Bleomycin-Induced Lung Fibrosis in Mice. J. Vis. Exp. 2018, (134), e56443 DOI: 10.3791/56443There is no corresponding record for this reference.
- 22Lai, R.; Zhao, C.; Guo, W.; Xiao, Y.; Li, R.; Liu, L.; Pan, H. The longitudinal and regional analysis of bleomycin-induced pulmonary fibrosis in mice by microcomputed tomography. Heliyon 2023, 9 (5), e15681 DOI: 10.1016/j.heliyon.2023.e15681There is no corresponding record for this reference.
- 23Song, S.; Fu, Z.; Guan, R.; Zhao, J.; Yang, P.; Li, Y.; Yin, H.; Lai, Y.; Gong, G.; Zhao, S.; Yu, J.; Peng, X.; He, Y.; Luo, Y.; Zhong, N.; Su, J. Intracellular hydroxyproline imprinting following resolution of bleomycin-induced pulmonary fibrosis. Eur. Respir. J. 2022, 59 (5), 2100864, DOI: 10.1183/13993003.00864-202123https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XhsVyltLbL&md5=7d4c9c624ed1ab9586bc9475ccea960eIntracellular hydroxyproline imprinting following resolution of bleomycin-induced pulmonary fibrosisSong, Shengren; Fu, Zhenli; Guan, Ruijuan; Zhao, Jie; Yang, Penghui; Li, Yang; Yin, Hang; Lai, Yunxin; Gong, Gencheng; Zhao, Simin; Yu, Jiangtian; Peng, Xiaomin; He, Ying; Luo, Yumei; Zhong, Nanshan; Su, JinEuropean Respiratory Journal (2022), 59 (5), 2100864CODEN: ERJOEI; ISSN:1399-3003. (European Respiratory Society)Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease with few treatment options. The poor success in developing anti-IPF strategies has impelled researchers to reconsider the importance of the choice of animal model and assessment methodologies. Currently, it is still not settled whether the bleomycin-induced lung fibrosis mouse model finally returns to resoln. This study aimed to follow the dynamic fibrotic features of bleomycin-treated mouse lungs over extended durations through a combination of the latest technologies (micro-computed tomog. imaging and histol. detection of degraded collagens) and traditional methods. In addn., we also applied immunohistochem. to explore the distribution of all hydroxyproline-contg. mols. As detd. by classical biochem. methods, total lung hydroxyproline contents reached a peak at 4 wk after bleomycin injury and maintained a steady high level thereafter until the end of the expts. (16 wk). This result seemed to partially contradict with the changes of other fibrosis evaluation parameters, which indicated a gradual degrdn. of collagens and a recovery of lung aeration after the fibrosis peak. This inconsistency was well reconciled by our data from immunostaining against hydroxyproline and fluorescent peptide staining against degraded collagen, together showing large amts. of hydroxyproline-rich degraded collagen fragments detained and enriched within the intracellular regions at 10 or 16 wk rather than at 4 wk after bleomycin treatment. Our present data not only offer respiratory researchers a new perspective towards the resoln. nature of mouse lung fibrosis, but also remind them to be cautious when using the hydroxyproline content assay to evaluate the severity of fibrosis.
- 24Mecozzi, L.; Mambrini, M.; Ruscitti, F.; Ferrini, E.; Ciccimarra, R.; Ravanetti, F.; Sverzellati, N.; Silva, M.; Ruffini, L.; Belenkov, S.; Civelli, M.; Villetti, G.; Stellari, F. F. In-vivo lung fibrosis staging in a bleomycin-mouse model: a new micro-CT guided densitometric approach. Sci. Rep. 2020, 10, 18735, DOI: 10.1038/s41598-020-71293-324https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXit1Orur%252FP&md5=5484858297710c92f6540f42aa0a5102In-vivo lung fibrosis staging in a bleomycin-mouse model: a new micro-CT guided densitometric approachMecozzi, Laura; Mambrini, Martina; Ruscitti, Francesca; Ferrini, Erica; Ciccimarra, Roberta; Ravanetti, Francesca; Sverzellati, Nicola; Silva, Mario; Ruffini, Livia; Belenkov, Sasha; Civelli, Maurizio; Villetti, Gino; Stellari, Fabio FrancoScientific Reports (2020), 10 (1), 18735CODEN: SRCEC3; ISSN:2045-2322. (Nature Research)Abstr.: Although increasing used in the preclin. testing of new anti-fibrotic drugs, a thorough validation of micro-computed tomog. (CT) in pulmonary fibrosis models has not been performed. Moreover, no attempts have been made so far to define d. thresholds to discriminate between aeration levels in lung parenchyma. In the present study, a histogram-based anal. was performed in a mouse model of bleomycin (BLM)-induced pulmonary fibrosis by micro-CT, evaluating longitudinal d. changes from 7 to 21 days after BLM challenge, a period representing the progression of fibrosis. Two discriminative densitometric indexes (i.e. 40th and 70th percentiles) were extd. from Hounsfield Unit d. distributions and selected for lung fibrosis staging. The strong correlation with histol. findings (rSpearman = 0.76, p < 0.01) confirmed that variations in 70th percentile could reflect a pathol. lung condition and est. the effect of antifibrotic treatments. This index was therefore used to define lung aeration levels in mice distinguishing in hyper-inflated, normo-, hypo- and non-aerated pulmonary compartments. A retrospective anal. performed on a large cohort of mice confirmed the correlation between the proposed preclin. d. thresholds and the histol. outcomes (rSpearman = 0.6, p < 0.01), strengthening their suitability for tracking disease progression and evaluating antifibrotic drug candidates.
- 25Olteanu, H.; Banerjee, R. Human Methionine Synthase Reductase, a Soluble P-450 Reductase-like Dual Flavoprotein, Is Sufficient for NADPH-dependent Methionine Synthase Activation*. J. Biol. Chem. 2001, 276 (38), 35558– 35563, DOI: 10.1074/jbc.M103707200There is no corresponding record for this reference.
- 26Leal, N. A.; Olteanu, H.; Banerjee, R.; Bobik, T. A. Human ATP:Cob(I)alamin Adenosyltransferase and Its Interaction with Methionine Synthase Reductase*. J. Biol. Chem. 2004, 279 (46), 47536– 47542, DOI: 10.1074/jbc.M405449200There is no corresponding record for this reference.
- 27Wolthers, K. R.; Scrutton, N. S. Protein Interactions in the Human Methionine Synthase-Methionine Synthase Reductase Complex and Implications for the Mechanism of Enzyme Reactivation. Biochemistry 2007, 46 (23), 6696– 6709, DOI: 10.1021/bi700339v27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXkvVCltrc%253D&md5=a6c4ebd9eb15ca67c7e27e4d4e8d304cProtein Interactions in the Human Methionine Synthase-Methionine Synthase Reductase Complex and Implications for the Mechanism of Enzyme ReactivationWolthers, Kirsten R.; Scrutton, Nigel S.Biochemistry (2007), 46 (23), 6696-6709CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Methionine synthase (MS) is a cobalamin-dependent enzyme. It transfers a Me group from methyltetrahydrofolate to homocysteine forming methionine and tetrahydrofolate. On the basis of sequence similarity with Escherichia coli cobalamin-dependent MS (MetH), human MS comprises four discrete functional modules that bind from the N- to C-terminus, resp., homocysteine, methyltetrahydrofolate, cobalamin, and S-adenosylmethionine (AdoMet). The C-terminal activation domain also interacts with methionine synthase reductase (MSR), a NADPH-dependent diflavin oxidoreductase required for the reductive regeneration of catalytically inert cob(II)alamin (which is formed every 200-1000 catalytic cycles of MS) to cob(I)alamin. We have investigated complex formation between the (i) MS activation domain and MSR and (ii) MS activation domain and the isolated FMN-binding domain of MSR. We show that the MS activation domain interacts directly with the FMN-binding domain of MSR. Binding is weakened at high ionic strength, emphasizing the importance of electrostatic interactions at the protein-protein interface. Mutagenesis of conserved lysine residues (Lys1071 and Lys987) in the human activation domain weakens this protein interaction. Chem. crosslinking demonstrates complex formation mediated by acidic residues (FMN-binding domain) and basic residues (activation domain). The activation domain and isolated FMN-domain form a 1:1 complex, but a 1:2 complex is formed with activation domain and MSR. The midpoint redn. potentials of the FAD and FMN cofactors of MSR are not perturbed significantly on forming this complex, implying that electron transfer to cob(II)alamin is endergonic. The kinetics of electron transfer in MSR and the MSR-activation domain complex are similar. Our studies indicate (i) conserved binding determinants, but differences in protein stoichiometry, between human MS and bacterial MetH in complex formation with redox partners; (ii) a substantial endergonic barrier to electron transfer in the reactivation complex; and (iii) a lack of control on the thermodn. and kinetics of electron transfer in MSR exerted by complex formation with activation domain. The structural and functional consequences of complex formation are discussed in light of the known crystal structure of human activation domain and the inferred conformational heterogeneity of the multidomain MSR-MS complex.
- 28Wolthers, K. R.; Lou, X.; Toogood, H. S.; Leys, D.; Scrutton, N. S. Mechanism of Coenzyme Binding to Human Methionine Synthase Reductase Revealed through the Crystal Structure of the FNR-like Module and Isothermal Titration Calorimetry. Biochemistry 2007, 46 (42), 11833– 11844, DOI: 10.1021/bi701209p28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtVOnsL%252FO&md5=2631bf19a5cea1919774001656d22b4fMechanism of Coenzyme Binding to Human Methionine Synthase Reductase Revealed through the Crystal Structure of the FNR-like Module and Isothermal Titration CalorimetryWolthers, Kirsten R.; Lou, Xiaodong; Toogood, Helen S.; Leys, David; Scrutton, Nigel S.Biochemistry (2007), 46 (42), 11833-11844CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Human methionine synthase reductase (MSR) is a 78 kDa flavoprotein that regenerates the active form of cobalamin-dependent methionine synthase (MS). MSR contains one FAD and one FMN cofactor per polypeptide and functions in the sequential transfer of reducing equiv. from NADPH to MS via its flavin centers. We report the 1.9 Å crystal structure of the NADP+-bound FNR-like module of MSR that spans the NADP(H)-binding domain, the FAD-binding domain, the connecting domain, and part of the extended hinge region, a feature unique to MSR. The overall fold of the protein is similar to that of the corresponding domains of the related diflavin reductase enzymes cytochrome P 450 reductase and neuronal nitric oxide synthase (NOS). However, the extended hinge region of MSR, which is positioned between the NADP(H)/FAD- and FMN-binding domains, is in an unexpected orientation with potential implications for the mechanism of electron transfer. Compared with related flavoproteins, there is structural variation in the NADP(H)-binding site, in particular regarding those residues that interact with the 2'-phosphate and the pyrophosphate moiety of the coenzyme. The lack of a conserved binding determinant for the 2'-phosphate does not weaken the coenzyme specificity for NADP(H) over NAD(H), which is within the range expected for the diflavin oxidoreductase family of enzymes. Isothermal titrn. calorimetry (ITC) reveals a binding const. of 37 and 2 μM for binding of NADP+ and 2',5'-ADP, resp., for the ligand-protein complex formed with full-length MSR or the isolated FNR module. These values are consistent with Ki values (36 μM for NADP+ and 1.4 μM for 2',5'-ADP) obtained from steady-state inhibition studies. The relatively weaker binding of NADP+ to MSR compared with other members of the diflavin oxidoreductase family might arise from unique electrostatic repulsive forces near the 5'-pyrophosphate moiety and/or increased hydrophobic stacking between Trp697 and the re face of the FAD isoalloxazine ring. Small structural permutations within the NADP(H)-binding cleft have profound affects on coenzyme binding, which likely retards catalytic turnover of the enzyme in the cell. The biol. implications of an attenuated mechanism of MS reactivation by MSR on methionine and folate metab. are discussed.
- 29Gherasim, C. G.; Zaman, U.; Raza, A.; Banerjee, R. Impeded Electron Transfer From a Pathogenic FMN Domain Mutant of Methionine Synthase Reductase and Its Responsiveness to Flavin Supplementation. Biochemistry 2008, 47 (47), 12515– 12522, DOI: 10.1021/bi800832829https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhtlaiu7vO&md5=6fbcc1dbf7d76af328a5a8802f7ff5caImpeded Electron Transfer From a Pathogenic FMN Domain Mutant of Methionine Synthase Reductase and Its Responsiveness to Flavin SupplementationGherasim, Carmen G.; Zaman, Uzma; Raza, Ashraf; Banerjee, RumaBiochemistry (2008), 47 (47), 12515-12522CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)Methionine synthase reductase (MSR) is a diflavin oxidoreductase that transfers electrons from NADPH to oxidized cobalamin and plays a vital role in repairing inactive cobalamin-dependent methionine synthase. MSR deficiency is a recessive genetic disorder affecting folate and methionine metab. and is characterized by elevated levels of plasma homocysteine. In this study, we have examd. the mol. basis of MSR dysfunction assocd. with a patient mutation, A129T, which is housed in the FMN binding domain and is adjacent to a cluster of conserved acidic residues found in diflavin oxidoreductases. We show that the substitution of alanine with threonine destabilizes FMN binding without affecting the NADPH coenzyme specificity or affinity, indicating that the mutation's effects may be confined to the FMN module. The A129T MSR mutant transfers electrons to ferricyanide as efficiently as wild type MSR but the rate of cytochrome c, 2,6-dichloroindophenol, and menadione redn. is decreased 10-15 fold. The mutant is depleted in FMN and reactivates methionine synthase with 8% of the efficiency of wild type MSR. Reconstitution of A129T MSR with FMN partially restores its ability to reduce cytochrome c and to reactivate methionine synthase. Hydrogen-deuterium exchange mass spectrometric studies localize changes in backbone amide exchange rates to peptides in the FMN-binding domain. Together, our results reveal that the primary biochem. penalty assocd. with the A129T MSR mutant is its lower FMN content, provide insights into the distinct roles of the FAD and FMN centers in human MSR for delivering electrons to various electron acceptors, and suggest that patients harboring the A129T mutation may be responsive to riboflavin therapy.
- 30Yamada, K.; Chen, Z.; Rozen, R.; Matthews, R. G. Effects of common polymorphisms on the properties of recombinant human methylenetetrahydrofolate reductase. Proc. Natl. Acad. Sci. U. S. A. 2001, 98 (26), 14853– 14858, DOI: 10.1073/pnas.261469998There is no corresponding record for this reference.
- 31Zhang, X.; Wang, J.; Chen, X.; Yu, M.; Yu, S.; Sun, Y.; Duan, J.; Sun, H.; Yuan, P. Short-term immunogenicity of standard and accelerated hepatitis B virus vaccination schedules in healthy adults: a comparative field study in China. Biosci. Rep. 2018, 38 (5), BSR20180846, DOI: 10.1042/BSR20180846There is no corresponding record for this reference.
- 32Chen, J.; Wang, Q.; Yin, F. Q.; Zhang, W.; Yan, L. H.; Li, L. MTRR silencing inhibits growth and cisplatin resistance of ovarian carcinoma via inducing apoptosis and reducing autophagy. Am. J. Transl. Res. 2015, 7 (9), 1510– 1527There is no corresponding record for this reference.
- 33Yoon, S.-Y.; Hong, G. H.; Kwon, H.-S.; Park, S.; Park, S. Y.; Shin, B.; Kim, T.-B.; Moon, H.-B.; Cho, Y. S. S-adenosylmethionine reduces airway inflammation and fibrosis in a murine model of chronic severe asthma via suppression of oxidative stress. Exp. Mol. Med. 2016, 48 (6), e236 DOI: 10.1038/emm.2016.35There is no corresponding record for this reference.
- 34Yu, G.; Tzouvelekis, A.; Wang, R.; Herazo-Maya, J. D.; Ibarra, G. H.; Srivastava, A.; de Castro, J. P. W.; DeIuliis, G.; Ahangari, F.; Woolard, T.; Aurelien, N.; Arrojo e Drigo, R.; Gan, Y.; Graham, M.; Liu, X.; Homer, R. J.; Scanlan, T. S.; Mannam, P.; Lee, P. J.; Herzog, E. L.; Bianco, A. C.; Kaminski, N. Thyroid hormone inhibits lung fibrosis in mice by improving epithelial mitochondrial function. Nat. Med. 2018, 24 (1), 39– 49, DOI: 10.1038/nm.444734https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhvFWgs7%252FN&md5=5d89b0faa601044f72a09eae3f9c30aeThyroid hormone inhibits lung fibrosis in mice by improving epithelial mitochondrial functionYu, Guoying; Tzouvelekis, Argyris; Wang, Rong; Herazo-Maya, Jose D.; Ibarra, Gabriel H.; Srivastava, Anup; de Castro, Joao Pedro Werneck; De Iuliis, Giuseppe; Ahangari, Farida; Woolard, Tony; Aurelien, Nachelle; Arrojo e Drigo, Rafael; Gan, Ye; Graham, Morven; Liu, Xinran; Homer, Robert J.; Scanlan, Thomas S.; Mannam, Praveen; Lee, Patty J.; Herzog, Erica L.; Bianco, Antonio C.; Kaminski, NaftaliNature Medicine (New York, NY, United States) (2018), 24 (1), 39-49CODEN: NAMEFI; ISSN:1078-8956. (Nature Research)Thyroid hormone (TH) is crit. for the maintenance of cellular homeostasis during stress responses, but its role in lung fibrosis is unknown. Here we found that the activity and expression of iodothyronine deiodinase 2 (DIO2), an enzyme that activates TH, were higher in lungs from patients with idiopathic pulmonary fibrosis than in control individuals and were correlated with disease severity. We also found that Dio2-knockout mice exhibited enhanced bleomycin-induced lung fibrosis. Aerosolized TH delivery increased survival and resolved fibrosis in two models of pulmonary fibrosis in mice (intratracheal bleomycin and inducible TGF-β1). Sobetirome, a TH mimetic, also blunted bleomycin-induced lung fibrosis. After bleomycin-induced injury, TH promoted mitochondrial biogenesis, improved mitochondrial bioenergetics and attenuated mitochondria-regulated apoptosis in alveolar epithelial cells both in vivo and in vitro. TH did not blunt fibrosis in Ppargc1a- or Pink1-knockout mice, suggesting dependence on these pathways. We conclude that the antifibrotic properties of TH are assocd. with protection of alveolar epithelial cells and restoration of mitochondrial function and that TH may thus represent a potential therapy for pulmonary fibrosis.
- 35Roque, W.; Romero, F. Cellular metabolomics of pulmonary fibrosis, from amino acids to lipids. Am. J. Physiol. Cell Physiol. 2021, 320 (5), C689– C695, DOI: 10.1152/ajpcell.00586.2020There is no corresponding record for this reference.
- 36Ung, C. Y.; Onoufriadis, A.; Parsons, M.; McGrath, J. A.; Shaw, T. J. Metabolic perturbations in fibrosis disease. Int. J. Biochem. Cell Biol. 2021, 139, 106073, DOI: 10.1016/j.biocel.2021.106073There is no corresponding record for this reference.
- 37Lee, J.-U.; Song, K. S.; Hong, J.; Shin, H.; Park, E.; Baek, J.; Park, S.; Baek, A.-R.; Lee, J.; Jang, A. S.; Kim, D. J.; Chin, S. S.; Kim, U. J.; Jeong, S. H.; Park, S.-W. Role of lung ornithine aminotransferase in idiopathic pulmonary fibrosis: regulation of mitochondrial ROS generation and TGF-β1 activity. Exp. Mol. Med. 2024, 56 (2), 478– 490, DOI: 10.1038/s12276-024-01170-wThere is no corresponding record for this reference.
- 38Henderson, N. C.; Rieder, F.; Wynn, T. A. Fibrosis: from mechanisms to medicines. Nature 2020, 587 (7835), 555– 566, DOI: 10.1038/s41586-020-2938-938https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXisVeqt77N&md5=9993852a8cfbbbd72d0c85727f4be799Fibrosis: from mechanisms to medicinesHenderson, Neil C.; Rieder, Florian; Wynn, Thomas A.Nature (London, United Kingdom) (2020), 587 (7835), 555-566CODEN: NATUAS; ISSN:0028-0836. (Nature Research)Abstr.: Fibrosis can affect any organ and is responsible for up to 45% of all deaths in the industrialized world. It has long been thought to be relentlessly progressive and irreversible, but both preclin. models and clin. trials in various organ systems have shown that fibrosis is a highly dynamic process. This has clear implications for therapeutic interventions that are designed to capitalize on this inherent plasticity. However, despite substantial progress in our understanding of the pathobiol. of fibrosis, a translational gap remains between the identification of putative antifibrotic targets and conversion of this knowledge into effective treatments in humans. Here we discuss the transformative exptl. strategies that are being leveraged to dissect the key cellular and mol. mechanisms that regulate fibrosis, and the translational approaches that are enabling the emergence of precision medicine-based therapies for patients with fibrosis.
- 39Bae, D.-H.; Lane, D. J. R.; Jansson, P. J.; Richardson, D. R. The old and new biochemistry of polyamines. Biochim. Biophys. Acta Gen. Subj. 2018, 1862 (9), 2053– 2068, DOI: 10.1016/j.bbagen.2018.06.00439https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtlentrnK&md5=7b4865b049c3d381ba5c039276e649a3The old and new biochemistry of polyaminesBae, Dong-Hun; Lane, Darius J. R.; Jansson, Patric J.; Richardson, Des R.Biochimica et Biophysica Acta, General Subjects (2018), 1862 (9), 2053-2068CODEN: BBGSB3; ISSN:0304-4165. (Elsevier B.V.)A review. Polyamines are ubiquitous pos. charged amines found in all organisms. These mols. play a crucial role in many biol. functions including cell growth, gene regulation and differentiation. The three major polyamines produced in all mammalian cells are putrescine, spermidine and spermine. The intracellular levels of these polyamines depend on the interplay of the biosynthetic and catabolic enzymes of the polyamine and methionine salvage pathway, as well as the involvement of polyamine transporters. Polyamine levels are obsd. to be high in cancer cells, which contributes to malignant transformation, cell proliferation and poor patient prognosis. Considering the crit. roles of polyamines in cancer cell proliferation, numerous anti-polyaminergic compds. have been developed as anti-tumor agents, which seek to suppress polyamine levels by specifically inhibiting polyamine biosynthesis, activating polyamine catabolism, or blocking polyamine transporters. However, in terms of the development of effective anti-cancer therapeutics targeting the polyamine system, these efforts have unfortunately resulted in little success. Recently, several studies using the iron chelators, O-trensox and ICL670A (Deferasirox), have demonstrated a decline in both iron and polyamine levels. Since iron levels are also high in cancer cells, and like polyamines, are required for proliferation, these latter findings suggest a biochem. integrated link between iron and polyamine metab.
- 40Sanderson, S. M.; Gao, X.; Dai, Z.; Locasale, J. W. Methionine metabolism in health and cancer: a nexus of diet and precision medicine. Nat. Rev. Cancer. 2019, 19 (11), 625– 637, DOI: 10.1038/s41568-019-0187-840https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhslKiu7bK&md5=8286901f7dd03a0637e87b792aaf1503Methionine metabolism in health and cancer: a nexus of diet and precision medicineSanderson, Sydney M.; Gao, Xia; Dai, Ziwei; Locasale, Jason W.Nature Reviews Cancer (2019), 19 (11), 625-637CODEN: NRCAC4; ISSN:1474-175X. (Nature Research)A review. Methionine uptake and metab. is involved in a host of cellular functions including methylation reactions, redox maintenance, polyamine synthesis and coupling to folate metab., thus coordinating nucleotide and redox status. Each of these functions has been shown in many contexts to be relevant for cancer pathogenesis. Intriguingly, the levels of methionine obtained from the diet can have a large effect on cellular methionine metab. This establishes a link between nutrition and tumor cell metab. that may allow for tumor-specific metabolic vulnerabilities that can be influenced by diet. Recently, a no. of studies have begun to investigate the mol. and cellular mechanisms that underlie the interaction between nutrition, methionine metab. and effects on health and cancer.
- 41Parkhitko, A. A.; Jouandin, P.; Mohr, S. E.; Perrimon, N. Methionine metabolism and methyltransferases in the regulation of aging and lifespan extension across species. Aging Cell 2019, 18 (6), e13034 DOI: 10.1111/acel.1303441https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhs1CmsLbF&md5=dff592c1c5f532b83766085513cd1cb3Methionine metabolism and methyltransferases in the regulation of aging and lifespan extension across speciesParkhitko, Andrey A.; Jouandin, Patrick; Mohr, Stephanie E.; Perrimon, NorbertAging Cell (2019), 18 (6), e13034CODEN: ACGECQ; ISSN:1474-9718. (Wiley-Blackwell)A review. Methionine restriction (MetR) extends lifespan across different species and exerts beneficial effects on metabolic health and inflammatory responses. In contrast, certain cancer cells exhibit methionine auxotrophy that can be exploited for therapeutic treatment, as decreasing dietary methionine selectively suppresses tumor growth. Thus, MetR represents an intervention that can extend lifespan with a complementary effect of delaying tumor growth. Manipulation of each of these branches extends lifespan; however, the interplay between MetR and these branches during regulation of lifespan is not well understood. In addn., a potential mechanism linking the activity of methionine metab. and lifespan is regulation of prodn. of the Me donor S-adenosylmethionine, which, after transferring its Me group, is converted to S-adenosylhomocysteine. Although the exact mechanisms of lifespan extension by MetR or methionine metab. reprogramming are unknown, it may act via reducing the rate of translation, modifying gene expression, inducing a hormetic response, modulating autophagy, or inducing mitochondrial function, antioxidant defense, or other metabolic processes. Here, we review the mechanisms of lifespan extension by MetR and different branches of methionine metab. in different species and the potential for exploiting the regulation of methyltransferases to delay aging.
- 42Li, Z.; Wang, F.; Liang, B.; Su, Y.; Sun, S.; Xia, S.; Shao, J.; Zhang, Z.; Hong, M.; Zhang, F.; Zheng, S. Methionine metabolism in chronic liver diseases: an update on molecular mechanism and therapeutic implication. Signal Transduct. Target. Ther. 2020, 5, 280, DOI: 10.1038/s41392-020-00349-7There is no corresponding record for this reference.
- 43Ramani, K.; Donoyan, S.; Tomasi, M. L.; Park, S. Role of Methionine Adenosyltransferase α2 and β Phosphorylation and Stabilization in Human Hepatic Stellate Cell Trans-Differentiation. J. Cell. Physiol. 2015, 230 (5), 1075– 1085, DOI: 10.1002/jcp.24839There is no corresponding record for this reference.
- 44Wang, K.; Fang, S.; Liu, Q.; Gao, J.; Wang, X.; Zhu, H.; Zhu, Z.; Ji, F.; Wu, J.; Ma, Y.; Hu, L.; Shen, X.; Gao, D.; Zhu, J.; Liu, P.; Zhou, H. TGF-β1/p65/MAT2A pathway regulates liver fibrogenesis via intracellular SAM. EBioMedicine 2019, 42, 458– 469, DOI: 10.1016/j.ebiom.2019.03.05844https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3cbps1WlsA%253D%253D&md5=dfb702e80ee1028d16b4e7a5ac7ef675TGF-β1/p65/MAT2A pathway regulates liver fibrogenesis via intracellular SAMWang Kuifeng; Ji Feihong; Fang Shanhua; Liu Qian; Gao Jing; Zhu Hongwen; Zhu Zhenyun; Wang Xiaoning; Wu Jiasheng; Ma Yueming; Hu Lihong; Shen Xu; Gao Daming; Zhu Jiansheng; Liu Ping; Zhou HuEBioMedicine (2019), 42 (), 458-469 ISSN:.BACKGROUND: Hepatic stellate cell (HSC) activation induced by transforming growth factor β1 (TGF-β1) plays a pivotal role in fibrogenesis, while the complex downstream mediators of TGF-β1 in such process are largely unknown. METHODS: We performed pharmacoproteomic profiling of the mice liver tissues from control, carbon tetrachloride (CCl4)-induced fibrosis and NPLC0393 administrated groups. The target gene MAT2A was overexpressed or knocked down in vivo by tail vein injection of AAV vectors. We examined NF-κB transcriptional activity on MAT2A promoter via luciferase assay. Intracellular SAM contents were analyzed by LC-MS method. FINDINGS: We found that methionine adenosyltransferase 2A (MAT2A) is significantly upregulated in the CCl4-induced fibrosis mice, and application of NPLC0393, a known small molecule inhibitor of TGF-β1 signaling pathway, inhibits the upregulation of MAT2A. Mechanistically, TGF-β1 induces phosphorylation of p65, i.e., activation of NF-κB, thereby promoting mRNA transcription and protein expression of MAT2A and reduces S-adenosylmethionine (SAM) concentration in HSCs. Consistently, in vivo and in vitro knockdown of MAT2A alleviates CCl4- and TGF-β1-induced HSC activation, whereas in vivo overexpression of MAT2A facilitates hepatic fibrosis and abolishes therapeutic effect of NPLC0393. INTERPRETATION: This study identifies TGF-β1/p65/MAT2A pathway that is involved in the regulation of intracellular SAM concentration and liver fibrogenesis, suggesting that this pathway is a potential therapeutic target for hepatic fibrosis. FUND: This work was supported by National Natural Science Foundation of China (No. 81500469, 81573873, 81774196 and 31800693), Zhejiang Provincial Natural Science Foundation of China (No. Y15H030004), the National Key Research and Development Program from the Ministry of Science and Technology of China (No. 2017YFC1700200) and the Key Program of National Natural Science Foundation of China (No. 8153000502).
- 45Mato, J. M.; Martinez-Chantar, M. L.; Lu, S. C. S-adenosylmethionine metabolism and liver disease. Ann. Hepatol. 2013, 12 (2), 183– 9, DOI: 10.1016/S1665-2681(19)31355-945https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhtVant7vM&md5=3bbff53ca1f23ac05d7474d81ed7c0d0S-adenosylmethionine metabolism and liver diseaseMato, Jose M.; Luz Martinez-Chantar, M.; Lu, Shelly C.Annals of Hepatology (2013), 12 (2), 183-189CODEN: AHNEAQ; ISSN:1665-2681. (Mexican Association of Hepatology)A review. Methionine is an essential amino acid that is metabolized mainly by the liver where it is converted to S-adenosylmethionine (SAMe) by the enzyme methionine adenosyltransferase. Although all mammalian cells synthesize SAMe, the liver is where the bulk of SAMe is generated as it is the organ where about 50% of all dietary methionine is metabolized. SAMe is mainly needed for methylation of a large variety of substrates (DNA, proteins, lipids and many other small mols.) and polyamine synthesis, so if the concn. of SAMe falls below a certain level or rises too much the normal function of the liver will be also affected. There are physiol. conditions that can affect the hepatic content of SAMe. Consequently, to control these fluctuations, the rate at which the liver both synthesizes and catabolizes SAMe is tightly regulated. In mice, failure to do this can lead to fatty liver disease and to the development of hepatocellular carcinoma (HCC). Therefore, maintaining SAMe homeostasis may be a therapeutic target in nonalcoholic steatohepatitis, alc.- and non-alc. liver cirrhosis, and for the chemoprevention of HCC formation.
- 46Casini, A.; Banchetti, E.; Milani, S.; Maggioni Moratti, E.; Surrenti, C. S-adenosylmethionine inhibits collagen synthesis by human fibroblasts in vitro. Methods Find. Exp. Clin. Pharmacol. 1989, 11 (5), 331– 334There is no corresponding record for this reference.
- 47Karaa, A.; Thompson, K. J.; McKillop, I. H.; Clemens, M. G.; Schrum, L. W. S-adenosyl-L-methionine attenuates oxidative stress and hepatic stellate cell activation in an ethanol-LPS-induced fibrotic rat model. Shock 2008, 30 (2), 197– 205, DOI: 10.1097/SHK.0b013e318160f417There is no corresponding record for this reference.
- 48Jubinville, É.; Milad, N.; Maranda-Robitaille, M.; Lafrance, M.-A.; Pineault, M.; Lamothe, J.; Routhier, J.; Beaulieu, M.-J.; Aubin, S.; Laplante, M.; Morissette, M. C. Critical importance of dietary methionine and choline in the maintenance of lung homeostasis during normal and cigarette smoke exposure conditions. Am. J. Physiol. Lung Cell. Mol. Physiol. 2020, 319 (2), L391– L402, DOI: 10.1152/ajplung.00353.2019There is no corresponding record for this reference.
- 49Gao, J.; Cahill, C. M.; Huang, X.; Roffman, J. L.; Lamon-Fava, S.; Fava, M.; Mischoulon, D.; Rogers, J. T. S-Adenosyl Methionine and Transmethylation Pathways in Neuropsychiatric Diseases Throughout Life. Neurotherapeutics 2018, 15 (1), 156– 175, DOI: 10.1007/s13311-017-0593-0There is no corresponding record for this reference.
- 50Cavallaro, R. A.; Fuso, A.; Nicolia, V.; Scarpa, S. S-adenosylmethionine prevents oxidative stress and modulates glutathione metabolism in TgCRND8 mice fed a B-vitamin deficient diet. J. Alzheimers Dis. 2010, 20 (4), 997– 1002, DOI: 10.3233/JAD-2010-091666There is no corresponding record for this reference.
- 51Huang, X.; Sun, P.; Qin, Y.; Wang, X.-j.; Wang, M.; Lin, Y.; Zhou, R.; Hu, W.; Liu, Q.; Yu, X.; Qin, A. Disulfiram attenuates MCMV-Induced pneumonia by inhibition of NF-κB/NLRP3 signaling pathway in immunocompromised mice. Int. Immunopharmacol. 2022, 103, 108453, DOI: 10.1016/j.intimp.2021.108453There is no corresponding record for this reference.
- 52Pai, M. Y.; Lomenick, B.; Hwang, H.; Schiestl, R.; McBride, W.; Loo, J. A.; Huang, J. Drug affinity responsive target stability (DARTS) for small-molecule target identification. Methods Mol. Biol. 2015, 1263, 287– 98, DOI: 10.1007/978-1-4939-2269-7_2252https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xls12murk%253D&md5=17d9d8199173653f814d80e0945c94adDrug Affinity Responsive Target Stability (DARTS) for Small-Molecule Target IdentificationPai, Melody Y.; Lomenick, Brett; Hwang, Heejun; Schiestl, Robert; McBride, William; Loo, Joseph A.; Huang, JingMethods in Molecular Biology (New York, NY, United States) (2015), 1263 (Chemical Biology), 287-298CODEN: MMBIED; ISSN:1940-6029. (Springer)Drug affinity responsive target stability (DARTS) is a relatively quick and straightforward approach to identify potential protein targets for small mols. It relies on the protection against proteolysis conferred on the target protein by interaction with a small mol. The greatest advantage of this method is being able to use the native small mol. without having to immobilize or modify it (e.g., by incorporation of biotin, fluorescent, radioisotope, or photoaffinity labels). Here we describe in detail the protocol for performing unbiased DARTS with complex protein lysates to identify binding targets of small mols. and for using DARTS-Western blotting to test, screen, or validate potential small-mol. targets. Although the ideas have mainly been developed from studying mols. in areas of biol. that are currently of interest to us and our collaborators, the general principles should be applicable to the anal. of all mols. in nature.
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Effects of chlorquinaldol treatment, bioinformatics and SPR analysis of chlorquinaldol’s candidate targets, molecular dynamics analysis, myofibroblast differentiation and fibroblast activation analysis, pharmacokinetic parameters of CQD, small molecule compounds used in the screening experiment, potential target compounds of clorquinaldol, PCR primer sequences, and ShRNA target sequences (PDF)
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