Chlorquinaldol Alleviates Lung Fibrosis in Mice by Inhibiting Fibroblast Activation through Targeting Methionine Synthase Reductase

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.

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

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

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

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

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

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

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.