Hijacking the Hydrogen Sulfide Axis: A Novel 4‑Trifluoromethylquinoline Derivative Suppresses Glioblastoma via Cystathionine γ‑Lyase Suppression

Abstract

Cystathionine γ-lyase (CTH) is markedly enriched in glioblastoma (GBM) and is associated with poor patient survival, enhanced temozolomide (TMZ) resistance, and aggressive phenotypes; however, effective CTH inhibitors for GBM therapy are currently lacking. Using click chemistry-based target identification, we identified cystathionine γ-lyase (CTH) as the direct molecular target of a novel 4-trifluoromethylquinoline derivative, TKL002. TKL002 exhibits strong antitumor activity both in vitro and in vivo, inducing late-stage apoptosis and G₂/M cell cycle arrest. Mechanistically, TKL002 inhibits CTH activity, reduces hydrogen sulfide (H₂S) production, suppresses NF-κB phosphorylation, and downregulates pro-inflammatory cytokine expression. In addition, TKL002 inhibits GBM cell migration and invasion by upregulating E-cadherin and downregulating N-cadherin and vimentin. Collectively, these findings demonstrate that TKL002 exerts potent antiglioblastoma activity via modulation of the CTH/H₂S/NF-κB/EMT signaling axis, highlighting its potential as a quinoline-based therapeutic candidate to overcome intrinsic GBM resistance and invasiveness.

Introduction

Gliomas are primary brain tumors believed to originate from neural stem or glial progenitor cells. They account for nearly 30% of all primary brain tumors, of which approximately 80% are malignant, representing the leading cause of mortality among patients with primary brain tumors. Among them, glioblastoma (GBM), classified as a World Health Organization grade IV glioma, is the most aggressive and lethal subtype, accounting for 70–77% of all primary malignant brain tumors. The current standard-of-care for GBM consists of maximal safe surgical resection followed by concurrent radiotherapy and temozolomide (TMZ)-based chemotherapy. However, therapeutic outcomes remain poor owing to the highly infiltrative growth pattern, pronounced tumor microenvironment heterogeneity, intrinsic chemoresistance, and limited permeability of the blood–brain barrier (BBB). As a result, the median overall survival of patients is only 14–18 months, with a five-year survival rate below 10%. , TMZ and bevacizumaba monoclonal antibody targeting vascular endothelial growth factor (VEGF)are first-line chemotherapeutic agents for GBM. Nevertheless, over half of patients eventually develop resistance to TMZ, and antiangiogenic therapies such as bevacizumab merely delay disease progression without significantly improving overall survival. These limitations highlight an urgent unmet need for new therapeutic agents capable of effectively penetrating the BBB while simultaneously targeting key survival pathways in GBM.

Cystathionine γ-lyase (CTH/CSE) is a pyridoxal-5′-phosphate (PLP)-dependent enzyme that catalyzes the terminal step of the transsulfuration pathway, converting cystathionine into cysteine, α-ketobutyrate, and ammonia. Under physiological conditions, CTH provides cysteine for glutathione (GSH) biosynthesis, thereby maintaining cellular redox homeostasis, and participates in sulfur amino acid metabolism, mitochondrial bioenergetics, and protein persulfidation. Moreover, CTH is a major enzymatic source of endogenous hydrogen sulfide (H₂S) in mammalian tissues. , By modulating multiple signaling cascadesincluding NF-κB, STAT3, PI3K/Akt, and MAPK/ERK1/2H₂S promotes endothelium-dependent vasodilation, angiogenesis, metabolic activation, oxidative stress resistance, and inflammatory regulation. , Recent studies have shown that CTH is enriched in invasive glioma cells derived from biomimetic 3D hydrogel invasion systems and patient-derived samples, where it plays a pivotal role in tumor infiltration. Genetic ablation of CTH significantly attenuates GBM invasion in vivo, whereas supplementation with exogenous cysteine restores the invasive phenotype of CTH-deficient cells. , Maria Peleli et al. further demonstrated that glioblastoma formation was markedly reduced in immunocompetent CTH-knockout mice bearing CTH-expressing tumors. In human GBM specimens, CTH expression correlated positively with SOX2 mRNA levels, suggesting that CTH may facilitate glioma stem-like characteristics. In addition, the brain-permeable CTH inhibitor propargylglycine (PAG) significantly suppressed GBM cell proliferation, inhibited TGF-β-induced cell migration, and reduced NOX4-derived reactive oxygen species through an H₂S-dependent mechanism. Notably, CTH expression is negatively correlated with key immune cell subsets, including regulatory T cells, monocytes, and resting dendritic cells, implying an immunosuppressive function. Collectively, these findings identify CTH as a promising therapeutic target for GBM; however, no potent or selective CTH inhibitors have yet been reported.

Quinoline (1-azanaphthalene, C₉H₇N) is a weakly basic nitrogen-containing heterocycle whose distinctive electronic configuration facilitates both electrophilic and nucleophilic substitutions, enabling flexible structural modification and optimization of drug-like properties. , Quinoline derivatives exhibit a broad range of pharmacological activities, including sedative, analgesic, anticonvulsant, anti-inflammatory, antioxidant, antimicrobial, antiviral, and anticancer effects. Several U.S. Food and Drug Administration-approved quinoline-based anticancer agentssuch as bosutinib (a BCR-ABL inhibitor), lenvatinib (a multikinase inhibitor), and topotecan (a topoisomerase inhibitor)demonstrate the therapeutic versatility of this scaffold. − Our previous work found that the compound FKL-296 exhibited potent anti-GBM activity. Guided by its molecular structure, we synthesized a series of novel 4-trifluoromethylquinoline derivatives and systematically evaluated their antiproliferative activity in prostate cancer cells. In this study, a click-chemistry-based screening strategy was employed to identify potential CTH-targeting 4-trifluoromethylquinoline derivatives for GBM therapy, and their underlying mechanisms were systematically investigated.

Results

Chemistry

Compounds FKL-296 and TKL001–TKL012 (Figure S1A) were prepared according to our previously reported procedure. The synthesis of TKL120 is outlined in Scheme . Initially, selective demethylation of the 6-methoxy group in compound 1 under hydrobromic acid (HBr) conditions afforded the 6-hydroxy compound 2. Chlorination of the corresponding hydroxyl group in 2 with phosphorus oxychloride (POCl₃) then gave chloride 3. The 6-hydroxy group of 3 was subsequently protected via benzylation to yield the benzyl-protected derivative 4. Next, a Buchwald–Hartwig coupling reaction between compound 4 and a specific aniline fragment provided compound 5. Deprotection of the benzyl group in 5 furnished intermediate 6, which was alkylated with 1-bromo-3-chloropropane to introduce the chloropropyl side chain, yielding key precursor 7. Nucleophilic substitution of 7 with piperazine then afforded compound 8. Finally, introduction of a diazirine fragment onto 8, followed by acidic removal of the N-Boc protecting group, delivered the target compound TKL120.

1. Reagents and Conditions: (i) HBr (48 wt % in H₂O), 135 °C; (ii) POCl₃, DMF, 90 °C; (iii) BnBr, Cs₂CO₃, DMF, r.t; (iv) Pd₂(dba)₃, Xantphos, Cs₂CO₃, 1,4-Dioxane, 80 °C; (v) H₂, Pd­(OH)₂/C, THF, r.t; (vi) 1-Bromo-3-chloropropane, K₂CO₃, DMF, r.t; (vii) Piperazine, K₂CO₃, DMF, 80 °C; (viii) 3-(But-3-yn-1-yl)-3-(2-iodoethyl)-3H-diazirine, K₂CO₃, DMF, r.t; (ix) HCl (4 M in 1,4-Dioxane), DCM, 0 °C.

TKL002 Inhibited GBM Cell Proliferation with Relatively Low Toxicity

MTT-based preliminary screening of 13 synthesized 4-trifluoromethylquinoline derivatives revealed that compound TKL002 exhibited the highest inhibitory activity against U87MG, U118MG, and U251MG glioma cells, with growth inhibition rates of 67.51%, 77.59%, and 75.89%, respectively. In contrast, its inhibitory effect on normal LX2 cells was comparatively lower (74.58%) (Figure S1A,B). Therefore, TKL002 was selected for subsequent mechanistic investigations. Figure A shows the structure of TKL002.

1.

Inhibitory activity of TKL002 on GBM cell proliferation. (A) The molecular structure formula of compound TKL002. (B–D) Inhibition curves of U87MG, U118MG and U251MG cells treated with TKL002 at different concentrations (0.625, 1.25, 2.5, 5, and 10 μmol/L). (E) Cell morphology observation of different cell lines of GBM after treatment with different concentrations of TKL002 for 48 h. Scale bar = 50 μm. The representative images were shown. The data were represented as mean ± SD (n = 3).

Treatment of GBM cells with increasing concentrations of TKL002 (0.625, 1.25, 2.5, 5, and 10 μmol/L) for 24, 48, and 72 h resulted in a dose- and time-dependent suppression of cell viability. The half-maximal inhibitory concentration (IC₅₀) values decreased with prolonged exposure, and at 48 h were calculated as 5.091 μmol/L for U87MG, 4.586 μmol/L for U118MG, 6.326 μmol/L for U251MG, and 13.788 μmol/L for LX2 (Table and Figure B–D). The SI coefficients for the U87MG, U118MG, and U251MG cell lines are 2.71, 3.01, and 2.18 respectively. Morphological assessment under an inverted microscope following 48 h treatment demonstrated a marked reduction in cell number, accompanied by cellular rounding, shrinkage, membrane blebbing, and fragmentation, all of which increased in severity with higher drug concentrations (Figure E). Based on these findings, U87MG and U118MG cell lines were selected for subsequent in vitro and in vivo experiments.

1. Summary of IC₅₀ Values (μmol/L) and SI Coefficients for Compound TKL002 .

cell line	time (h)			SI
	24	48	72
U87MG	5.486 ± 0.522	5.091 ± 0.898	5.007 ± 1.160	2.71
U118MG	5.567 ± 0.480	4.586 ± 0.558	4.141 ± 0.431	3.01
U251MG	6.506 ± 0.045	6.326 ± 0.156	4.727 ± 0.165	2.18
LX2		13.788 ± 1.954

TKL002 Induced Late Apoptosis in GBM Cells via Bcl-2/Bax/Caspase-3 Modulation

Flow cytometry analysis of U87MG cells treated with TKL002 (2, 4, and 6 μmol/L) for 48 h revealed that 2 and 4 μmol/L treatments did not significantly alter early or late apoptosis rates compared to the control (0.1% DMSO). However, 6 μmol/L TKL002 increased late apoptosis to 22.63% (P < 0.01) (Figure A). Western blot analysis showed that TKL002 treatment dose-dependently reduced the expression of antiapoptotic proteins Bcl-2 (P < 0.001) and active-caspase-3 (P < 0.05), while increasing pro-apoptotic proteins Bax (P < 0.01) and caspase-3 (P < 0.05) (Figure B,C).

2.

TKL002 induced late apoptosis in GBM cells and blocked GBM cell cycle at G₂/M phase. (A) Flow cytometry was used to detect apoptosis after different concentrations (2, 4, and 6 μmol/L) of TKL002 acted on U87MG cells for 48 h and its statistical analysis. (B,C) WB assay was performed to detect the effect of TKL002 on the expression of apoptosis-related proteins and its statistical analysis. α-Tubulin was used as an internal reference. (D–E) Flow cytometry and PI fluorescent probes were used to detect the effect of TKL002 on the cell cycle of U87MG and U118MG cells. The data were represented as mean ± SD (n = 3). *P < 0.05, **P < 0.01 vs control (0 μmol/L).

TKL002 Induced G₂/M Cell Cycle Arrest in GBM Cells

Flow cytometry analysis indicated that, following 24 h treatment with TKL002 (2, 4, and 6 μmol/L), the proportion of cells in the G₂/M phase increased in a concentration-dependent manner in both U87MG and U118MG cell lines. Compared to controls (U87MG: 13.13%, U118MG: 15.36%), TKL002 increased the G₂/M fraction in U87MG cells to 17.01%, 20.21%, and 22.68% (P < 0.001) and in U118MG cells to 18.16% (P < 0.05), 24.04% (P < 0.001), and 30.31% (P < 0.001) for 2, 4, and 6 μmol/L, respectively (Figure D,E).

TKL002 Suppressed Subcutaneous GBM Tumor Growth In Vivo

Acute toxicity tests in mice indicated that TKL002 had minimal in vivo toxicity (Figure S2). Using U87MG cells, a subcutaneous GBM xenograft model in nude mice was established. Tumor volume during the treatment period (Figure B,C), body weight of nude mice (Figure D), and tumor weight at the end of treatment (Figure A) were used to evaluate in vivo therapeutic efficacy and toxicity. At the end of treatment, the mean subcutaneous tumor weights were 1.458 g (vehicle), 1.117 g (5 mg/kg TKL002), 0.530 g (20 mg/kg TKL002), and 0.493 g (5 mg/kg TMZ). No significant differences in tumor weight or volume were observed between the 20 mg/kg TKL002 and 5 mg/kg TMZ groups. H&E staining revealed minimal pathological changes in liver, spleen, and kidney in TKL002 groups, whereas 5 mg/kg TMZ treatment disrupted splenic architecture and hepatic cell arrangement (Figure F). Relative organ index analysis showed dose-dependent reductions in splenic index for TKL002 and 5 mg/kg TMZ, with no significant changes in heart, liver, lung, or kidney indices (Figure E).

3.

TKL002 suppressed subcutaneous GBM tumor growth in vivo. (A) The tumor weight after 14 days of TKL002 treatment. (B) The tumor images were across different groups after 14 days of TKL002 treatment. (C) The tumor volume during the TKL002 administration. (D) Body weight during the TKL002 administration. (E) The relative organ weight after 14 days of TKL002 treatment. (F) The HE staining of the spleen, kidney and liver of mice in each group after 14 days of TKL002 treatment. Scale bar = 250 μm. *P < 0.05, **P < 0.01 vs vehicle.

TKL002 Selectively Inhibited Orthotopic Glioblastoma Growth

Considering the heterogeneity of the glioblastoma (GBM) tumor microenvironment, an orthotopic xenograft GBM model was established using U87MG-Luc cells to evaluate the tumor-targeting and antitumor efficacy of TKL002 (Figure H). TKL002 was administered via tail vein injection at doses of 10 or 20 mg/kg, and tumor burden was longitudinally monitored using bioluminescence imaging (BLI). On days 15 and 20, both TKL002-treated groups exhibited significantly reduced bioluminescent signal intensity compared with the vehicle-treated model group (P < 0.01). The in vivo BLI signal intensities were 25.21, 13.62, and 8.52 × 10⁴ photons/s, respectively, while the ex vivo tumor BLI signal intensities were 63.88, 21.48, and 9.20 × 10⁴ photons/s in the model, 10 mg/kg TKL002, and 20 mg/kg TKL002 groups, respectively (Figure A,B,I,J). Mice in the model group exhibited weight loss, reduced activity, and cachexia, whereas 20 mg/kg TKL002-treated mice maintained normal body condition (Figure C,F,G). Kaplan–Meier survival analysis showed that the treatment groups, particularly the 20 mg/kg TKL002-treated group, exhibited a higher survival rate compared with the model group; however, the difference did not reach statistical significance (P = 0.17) (Figure E). No significant differences in organ indices were observed among the groups (Figure D). To further investigate the in vivo effect of TKL002 on CTH expression, immunohistochemical analysis was performed on orthotopic glioblastoma tissues. CTH staining was markedly reduced in tumors from TKL002-treated mice compared with the model group, with a more pronounced decrease observed in the 20 mg/kg group (Figure K,L).

4.

TKL002 selectively inhibited orthotopic glioblastoma growth. (A,B) Tumor fluorescence intensity on days 7 and 15 of the loaded tumor and its statistical analysis. (C) Body weight changes in tumor-bearing nude mice. (D) The relative organ weight after 20 days of tumor loading. (E) Kaplan–Meier survival analysis (P = 0.17). (F) Status of nude mice in 20 mg/kg tail vein administration group. (G) Status of nude mice in the model group. (H) Appearance of brain tissue (red arrows showed the longitudinal column of the brain shifted to the left, blue arrows showed the tumor bulge). (I,J) Ex vivo imaging of brain tissue at the end of treatment and its statistical analysis. (K) Representative immunohistochemical staining of CTH in orthotopic glioblastoma tissues from the model, 10 mg/kg, and 20 mg/kg TKL002-treated groups (red asterisks denote peritumoral tissue; blue triangles denote GBM orthotopic xenografts). Scale bar = 200 μm. (L) Quantification of CTH-positive area in tumor sections. ^# P < 0.05, ^## P < 0.01 vs model.

Click Chemistry, Network Pharmacology, and Bioinformatics Predicted CTH as a Key Target of TKL002

Click chemistry-based target enrichment and mass spectrometry identified candidate small-molecule interacting proteins. TKL120 (Figure A), a photoreactive alkyne-modified derivative of TKL002, demonstrated enhanced inhibitory activity against U118MG cells with IC₅₀ values of 1.695 ± 0.022, 1.318 ± 0.093, and 1.346 ± 0.086 μmol/L at 24, 48, and 72 h, respectively (Table and Figure B–D), serving as the probe for subsequent experiments. Following the determination of the optimal labeling concentration of TKL120 and the optimal competitive concentration of TKL002 (Figure F,G), proteomic analyses were conducted to compare the control versus probe group and the probe versus competition group (Figure S3). This workflow enabled the identification of ten overlapping target proteins, among which CTH and SRM (spermidine synthase) were prominently enriched (Figures E and A–F). Molecular docking indicated TKL002 binds CTH with the lowest binding energy (−8.7 kcal/mol), forming a hydrogen bond with TYR-213 (2.8 Å) (Figure I and Table ). Pan-cancer and differential expression analysis showed elevated CTH expression in GBM (P < 0.05) (Figure G,H), with higher expression correlating with poorer patient survival (P < 0.05).

5.

Click chemistry has been instrumental in predicting the key target of the anti-GBM action of TKL002. (A) Molecular formula of the probe compound TKL120. (B–D) Inhibition curves of U87MG, U118MG and U251MG cells treated with TKL120 at different concentrations (0.625, 1.25, 2.5, 5, and 10 μmol/L). (E) Differential protein screening. (F) Probe labeling optimal concentration screening. (G) Proto-small molecule optimal competitive concentration screening.

2. Summary of IC₅₀ Values for Biotin Probe-Labelled Compound TKL120 (μmol/L).

cells	time (h)
	24	48	72
U87MG	2.139 ± 0.104	1.462 ± 0.030	1.362 ± 0.089
U118MG	1.695 ± 0.022	1.318 ± 0.093	1.346 ± 0.086
U251MG	2.171 ± 0.353	2.094 ± 0.028	1.539 ± 0.065

6.

Network pharmacology and bioinformatics predicted CTH as a key target of TKL002. (A) COG analysis. (B) KOG analysis. (C) GO analysis. (D) KEGG analysis. (E) Venn diagram screening for overlapping genes. (F) PPI analysis. (G) Differential expression analysis of CTH. (H) Pan-cancer analysis of CTH. (I) Visualization of molecular docking of TKL002 with CTH proteins.

3. Molecular Docking Results.

target symbol	PDB ID	affinity (kcal/mol)
CTH	5TT2	–8.7
SRM	2O05	–7.6

CETSA Confirmed TKL002 Directly Binds CTH and Enhances Its Thermal Stability

Cellular thermal shift assay (CETSA) analysis demonstrated that TKL002 treatment increased CTH thermal stability in U118MG cells in a dose-dependent manner. At 10 and 25 μmol/L, CTH thermal stability was significantly increased (P < 0.01) (Figure A–D). In untreated cells, CTH degraded with rising temperature, whereas TKL002-treated cells maintained higher CTH levels, starting at 52 °C, with 5 μmol/L TKL002 showing significant stabilization compared to control (P < 0.001) (Figure E,F).

7.

CETSA confirmed TKL002 directly binds CTH and enhances its thermal stability. (A–D) Differences in CTH protein expression and its statistical analysis after treatment of U87MG and U118MG cells with different concentrations (0.1, 0.5, 1, 5, 10, and 25 μmol/L) of TKL002 for 3 h at 52 °C. (E,F) Differences in CTH protein expression between U118MG cells treated with 5 μmol/L TKL002 for 3 h and the control group (0.1% DMSO) at different temperatures (46, 49, 52, 55, 58, 61 °C) and their statistical analysis. *P < 0.05, **P < 0.01 vs control.

CTH Overexpression Partially Reversed the Inhibitory Effect of TKL002 on GBM Cells

Western blot analysis showed that CTH expression was lower in U251MG cells than in U118MG and U87MG cells (Figure A,B). Stable overexpression in U251MG cells markedly increased CTH protein levels compared with controls (P < 0.001; Figure C,D). MTT assays demonstrated that TKL002 reduced cell viability in a dose-dependent manner in both groups, whereas OE-CTH cells retained higher viability than OE-control cells at equivalent concentrations (P < 0.05; Figure E). Transwell assays further revealed that CTH overexpression enhanced cell migration (P < 0.001), and although 5 μmol/L TKL002 suppressed migration in both groups, the inhibitory effect was attenuated in OE-CTH cells (P < 0.05; Figure F,G). These results indicate that CTH overexpression partially counteracts the antiproliferative and antimigratory actions of TKL002 in GBM cells.

8.

CTH overexpression and knockdown differentially regulate the antiproliferative and antimigratory effects of TKL002 in glioblastoma cells. (A,B) Differences in CTH protein expression in different GBM cell lines and their statistical analysis. *P < 0.05 is considered significant. (C,D) Western blot to verify the effect of CTH overexpression assay and its statistical analysis. (E) MTT assay to detect the effects of different concentrations of TKL002 on the viability of GBM cells in OE control and OE-CTH groups. (F,G) 5 μmol/L TKL002 or 0.1% DMSO was used to treat the GBM cells in OE control group and OE-CTH group, and the difference in the number of migrated cells between groups was detected and statistically analyzed by Transwell assay. Scale bar = 250 μm. ^# P < 0.05, ^## P < 0.01 vs OE-control. (H,I) The knockdown efficiency of CTH by three independent shRNAs (sh1–sh3) was evaluated by Western blotting; sh1 was selected for subsequent experiments. (J) Inhibition rates of shC and sh1 cells following treatment with increasing concentrations of TKL002. (K,L) Representative images and quantitative analysis of migrated shC and sh1 cells in the absence or presence of TKL002 (5 μmol/L). Scale bar = 100 μm. ^& P < 0.05, ^&& P < 0.01 vs shC.

CTH Knockdown Attenuated the Antiproliferative and Antimigratory Effects of TKL002 in GBM Cells

Three independent CTH-targeting shRNAs were screened by Western blotting. sh1 achieved the most efficient reduction of CTH protein levels and was selected for subsequent experiments (Figure H,I). MTT assays showed that TKL002 decreased cell viability in a concentration-dependent manner in both shC and sh1 cells. However, at equivalent concentrations, sh1 cells consistently exhibited lower inhibition rates than shC cells (P < 0.05, Figure J). Transwell assays demonstrated that CTH knockdown significantly reduced number of migrated cells compared with shC cells. Treatment with TKL002 (5 μmol/L) markedly suppressed migration in shC cells (P < 0.01), whereas only a limited additional reduction was observed in sh1 cells, with no statistically significant difference (Figure K,L).

TKL002 Reduced Cysteine and H₂S Levels and Suppressed H₂S-Dependent NF-κB Signaling, Migration, and Inflammatory Responses in GBM Cells

CTH catalyzes cystathionine to cysteine, generating GSH and H2S, which regulate redox balance, angiogenesis, tumor invasion, and inflammation (Figure A). ELISA results showed dose-dependent reductions of CTH, cysteine, and H2S levels in U87MG and U118MG supernatants (P < 0.001), whereas GSH levels moderately increased, with 6 μmol/L TKL002-treated U87MG (P < 0.001) and U118MG (P < 0.05) showing significant differences vs control (Figure B–F). Transwell assays indicated decreased cell migration and invasion with increasing TKL002 concentration (P < 0.05) (Figure A,B). Western blot analysis demonstrated increased E-cadherin (P < 0.05) and decreased N-cadherin (P < 0.01) and vimentin (P < 0.01), suggesting inhibition of EMT (Figure C–E). Moreover, TKL002 suppressed NF-κB expression and phosphorylation (P < 0.01), as well as TNF-α (P < 0.001) and IL-6 (P < 0.05) levels in U87MG cells. In U118MG cells, 6 μmol/L TKL002 reduced NF-κB expression and phosphorylation (P < 0.05), along with TNF-α (P < 0.05) and IL-6 (P < 0.05) (Figure G–I). Importantly, supplementation with the exogenous H₂S donor NaHS partially reversed the TKL002-induced suppression of NF-κB activation, as evidenced by restored NF-κB phosphorylation and increased nuclear localization of NF-κB detected by Western blot (Figure A,B) and immunofluorescence analysis (Figure C,D).

9.

TKL002 reduced cysteine and H₂S production in GBM cells and inhibited the inflammatory response through modulation of the NF-κB signaling pathway. (A) Mechanism of action of CTH proteins diagram. (B) Effect of TKL002 on CTH level in the supernatants of U87MG and U118MG cells. (C–F) Effect of TKL002 on cystathionine, cysteine, H₂S and GSH levels in the supernatants of U87MG and U118MG cells. (G–I) Effect of TKL002 on the expression of NF-κB proteins and inflammation-related proteins and its statistical analysis. *P < 0.05, **P < 0.01 vs control (0 μmol/L).

10.

TKL002 inhibited GBM cell migration and invasion via modulation of EMT markers. (A,B) Effect of TKL002 on migration and invasion capacity of U87MG and U118MG cells. (C–E) Effect of TKL002 on the expression of EMT-related proteins and its statistical analysis. *P < 0.05, **P < 0.01 vs control (0 μmol/L).

11.

Exogenous H₂S donor partially restores NF-κB activation suppressed by TKL002. (A) Western blot analysis of total NF-κB (p65), phosphorylated NF-κB (p-NF-κB) in U87MG cells treated with TKL002 (5 μmol/L) in the absence or presence of NaHS (50 μmol/L). (B) Quantitative analysis of the p-NF-κB/NF-κB ratio normalized to the control group. (C) Quantification of nuclear NF-κB mean fluorescence intensity (MFI) determined by immunofluorescence analysis. Each dot represents one cell. (D) Representative immunofluorescence images showing NF-κB (red), nuclei stained with DAPI (blue), and merged images. Scale bar = 250 μm. *P < 0.05, **P < 0.01.

Discussion and Conclusions

Glioblastoma (GBM) remains the most aggressive and lethal primary tumor of the central nervous system, accounting for nearly half of all intracranial malignancies. Despite substantial progress in microsurgical techniques, radiotherapy, and TMZ-based chemotherapy, the median overall survival of patients remains below 18 months. This disappointing outcome primarily arises from the tumor’s diffuse infiltration, pronounced molecular heterogeneity, and the restrictive nature of the BBB, all of which collectively limit drug efficacy and render recurrence virtually inevitable. These clinical challenges highlight the urgent need for therapeutic agents that not only penetrate the BBB efficiently but also target essential survival pathways of GBM cells.

Among the emerging molecular targets, cystathionine γ-lyase (CTH) has attracted growing attention due to its dual function at the intersection of cellular metabolism and redox signaling. CTH catalyzes the final step of the transsulfuration pathway, generating cysteine for glutathione (GSH) synthesis and serving as a major enzymatic source of hydrogen sulfide (H₂S). − This enzyme thereby links sulfur metabolism to oxidative stress regulation and inflammatory control. Recent studies have correlated elevated CTH expression in GBM with poor patient survival, enhanced TMZ resistance, and an invasive phenotype, suggesting that aberrant H₂S signaling supports tumor persistence and adaptation. Consistent with these findings, genetic silencing or pharmacologic inhibition of CTH significantly suppresses glioma formation, while supplementation with cysteine restores invasive capacity. − These data collectively support the concept that CTH functions as a metabolic checkpoint promoting GBM progression and immune tolerance. Nevertheless, potent and brain-penetrant CTH inhibitors remain unavailable.

In this context, our study identifies TKL002a novel 4-trifluoromethylquinoline derivativeas a promising CTH-targeting compound whose molecular target was elucidated using click-chemistry-based − approaches. TKL002 exhibited potent in vitro cytotoxicity against glioblastoma cells, concomitantly inducing apoptosis and G₂/M-phase cell-cycle arrest. Notably, the selectivity index (SI) exceeded 2, indicating preferential cytotoxicity toward glioblastoma cells. In vivo, TKL002 significantly suppressed tumor growth in both subcutaneous and orthotopic xenograft models at doses without overt systemic toxicity and was associated with a prolongation of overall survival. Although the survival benefit did not reach statistical significance (P > 0.05), this may be attributable to the limited sample size. These findings collectively position TKL002 as a pharmacologically favorable lead for CTH-targeted therapy.

Importantly, click-chemistry-enabled proteomic profiling, together with molecular docking analyses, identified CTH as the direct molecular target of TKL002, a conclusion further validated by CETSA and genetic overexpression and knockdown experiments. Consistent with these in vitro findings, immunohistochemical analysis of orthotopic glioblastoma xenografts revealed a pronounced reduction in CTH expression following TKL002 treatment, supporting effective target engagement in vivo and reinforcing the translational relevance of CTH inhibition in glioblastoma.

Mechanistically, TKL002 suppresses CTH activity, resulting in decreased intracellular cysteine and H₂S levels, accompanied by a compensatory increase in intracellular GSH (which may be attributed to a compensatory redox adaptation, whereby inhibition of CTH-driven H₂S production shifts intracellular sulfur flux toward the transsulfuration–glutathione axis, leading to activation of Nrf2-dependent antioxidant responses and enhanced glutathione biosynthesis as a cellular defense mechanism against oxidative stress). Given that H₂S functions as an endogenous gasotransmitter regulating signaling pathways such as NF-κB, STAT3, Wnt/β-catenin, and ERK1/2, the suppression of H₂S biosynthesis disrupts both metabolic and inflammatory homeostasis within tumor cells. − We observed that TKL002 treatment markedly decreased NF-κB phosphorylation and downregulated its proinflammatory cytokine targets, including IL-6 and TNF-α. Furthermore, TKL002 reversed EMT by restoring E-cadherin and suppressing N-cadherin and vimentin expression, thereby impairing GBM cell motility and invasiveness. These results delineate a coherent molecular framework in which TKL002 attenuates GBM progression through concerted inhibition of CTH-mediated metabolic adaptation and NF-κB-driven inflammatory signaling.

From a medicinal chemistry standpoint, the trifluoromethylquinoline scaffold of TKL002 may underlie its excellent BBB permeability, metabolic stability, and multitarget potentialpharmacokinetic traits often critical for CNS-active agents. Quinoline derivatives, owing to their lipophilicity, hydrogen-bonding versatility, and electronic tunability, have long been recognized as privileged scaffolds in anticancer drug design. The favorable balance between potency and tolerability observed here supports the further development of TKL002 and related analogs as next-generation, brain-penetrant CTH inhibitors.

Collectively, these findings propose a mechanistically distinct therapeutic paradigm: by targeting the metabolic–inflammatory axis of GBM through CTH inhibition, TKL002 impairs both tumor energy metabolism and the inflammatory circuitry sustaining its invasive behavior. This dual-action mechanism provides a conceptual framework for designing combination therapies that address the intrinsic resistance of GBM.

Despite establishing TKL002 as a promising prototype for CTH-targeted therapy in GBM, several limitations should be acknowledged. Notably, the blood–brain barrier (BBB) permeability of TKL002 was not directly evaluated, and no pharmacokinetic or brain distribution data were obtained, which limits conclusions regarding its intracranial bioavailability. In addition, the current evidence is restricted to preclinical in vitro and in vivo models, and the long-term safety, metabolic stability, and potential off-target toxicities of TKL002 remain undefined. Future studies should prioritize systematic assessment of BBB penetration, pharmacokinetics, and pharmacodynamics in clinically relevant models, together with rational scaffold optimization to improve brain retention and target selectivity. Furthermore, comprehensive interrogation of the immunometabolic effects of TKL002 within the tumor microenvironment and its therapeutic potential in combination with TMZ or immune checkpoint inhibitors may facilitate its translation from an experimental compound to a clinically viable therapeutic candidate for glioblastoma.

Experimental Section

General Chemistry

Unless otherwise noted, all reactions were performed under an argon atmosphere. Reagents and solvents, purchased from Sigma-Aldrich, Energy-Chemical, and J&K Scientific, were used as received. The progress of reactions was monitored by TLC on precoated silica gel GF254 plates (0.25 mm). Spots were visualized under UV light at 254 nm or by treatment with suitable staining reagents. Purification by column chromatography was carried out on silica gel 60 (200–300 mesh). NMR spectra were acquired on a Bruker AVANCE NEO 600 spectrometer using deuterated solvents. Chemical shifts are reported in δ (ppm) and refer to ¹H (600 MHz), ¹³C (150 MHz), and ¹⁹F (564 MHz) nuclei. The following abbreviations are used for signal multiplicities: s (singlet), d (doublet), dd (double doublet), t (triplet), q (quartet), m (multiplet). The compounds TKL002 and TKL120 were obtained with purities exceeding 99%, as determined by high-performance liquid chromatography (HPLC). Analyses were performed on an Agilent 1260 series HPLC system equipped with a UV-DAD detector (set at 268 nm) and a CAPCELL PAK C18 MG II column (4.6 mm × 250 mm, 5 μm). The column temperature was maintained at 30.0 °C. The mobile phase consisted of acetonitrile, water, and trifluoroacetic acid (TFA). For compound TKL002, the elution program used 40% acetonitrile in water containing 0.2% TFA, while for compound TKL120, 30% acetonitrile in water containing 0.2% TFA was employed. The flow rate was 1.0 mL/min with an injection volume of 5 μL. Compound purity was calculated based on the peak area percentage, and retention times are reported in minutes. The Supporting Information includes HPLC purity analyses for compounds TKL002 and TKL120 (Figures S4 and S5), along with the complete NMR spectra (¹H, ¹³C, and ¹⁹F) for compounds 9 and TKL120 (Figures S6–S11).

General Procedure for the Preparation of Compound 1

6-Methoxy-2-hydroxy-4-(trifluoromethyl)­quinoline (1)

Compound 1 was prepared according to our previously reported procedure.

General Procedure for the Preparation of Compound 2

2,6-Dihydroxy-4-(trifluoromethyl)­quinoline (2)

A mixture of compound 1 (1.50 g, 6.19 mmol) and aqueous HBr (48 wt %, 30 mL) was heated under reflux at 135 °C for 2 h. After completion, the reaction mixture was poured into a biphasic system of ethyl acetate (200 mL) and saturated aqueous sodium bicarbonate (200 mL). The mixture was vigorously stirred and then allowed to settle for phase separation. The organic layer was collected, and the aqueous phase was further extracted with ethyl acetate (2 × 50 mL). The combined organic extracts were washed with water (100 mL) and brine (50 mL), dried over anhydrous MgSO₄, and filtered. The filtrate was concentrated under reduced pressure to afford the crude product. The crude material was sonicated in a mixture of petroleum ether and ethyl acetate (5:1, v/v) and then filtered. The collected solid was washed with petroleum ether and dried under vacuum to give compound 2 as a gray solid (930 mg, 66% yield).

General Procedure for the Preparation of Compound 3

2-Chloro-6-hydroxy-4-(trifluoromethyl)­quinoline (3)

Compound 2 (500 mg, 2.19 mmol) was dissolved in anhydrous DMF (5 mL) and heated to 90 °C under an argon atmosphere. To the solution was then added POCl₃ (0.6 mL, 6.57 mmol), and the resulting mixture was stirred at 90 °C for 1 h. After cooling to room temperature, the reaction mixture was poured into a biphasic system of ethyl acetate (100 mL) and water (100 mL). The mixture was stirred vigorously and allowed to settle for phase separation. The organic layer was collected, and the aqueous phase was further extracted with ethyl acetate (2 × 50 mL). The combined organic extracts were washed with water (50 mL) and brine (30 mL), dried over anhydrous MgSO₄, and filtered. The filtrate was concentrated under reduced pressure to afford crude compound 3, which was used directly in the next step without further purification.

General Procedure for the Preparation of Compound 4

6-Benzyloxy-2-chloro-4-(trifluoromethyl)­quinoline (4)

A mixture of compound 3 (540 mg, 2.19 mmol) and cesium carbonate (1.07 g, 3.28 mmol) was dissolved in DMF (10 mL) with stirring. Benzyl bromide (0.32 mL, 2.63 mmol) was added dropwise, and the resulting mixture was stirred at room temperature for 1 h. Upon completion, the reaction mixture was diluted with ethyl acetate and water. The organic layer was separated, and the aqueous phase was extracted with ethyl acetate (2 × 50 mL). The combined organic extracts were washed sequentially with water and brine, dried over anhydrous MgSO₄, and filtered. The filtrate was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate = 60/1) to afford compound 4 (670 mg, 90% yield) as a yellow solid.

General Procedure for the Preparation of Compound 5

N-(3-Fluoro-4-(4-(tert-butoxycarbonyl)­piperazin-1-yl)­phenyl)-6-benzyloxy-4-(trifluoromethyl)­quinolin-2-amine (5)

Under an argon atmosphere, a reaction tube was charged successively with compound 4 (670 mg, 1.98 mmol), aniline (703.14 mg, 2.38 mmol), Pd₂(dba)₃ (54.50 mg, 59.52 μmol), Xantphos (57.40 mg, 99.19 μmol), and cesium carbonate (969.57 mg, 2.98 mmol). The mixture was dissolved in 1,4-dioxane (20 mL) and stirred at 80 °C for 3 h. After the reaction was complete, the mixture was filtered while hot through a silica gel pad (200–300 mesh). The residue was washed with ethyl acetate (3 × 10 mL), and the combined filtrates were concentrated under reduced pressure. The resulting crude product was purified by silica gel column chromatography (petroleum ether/ethyl acetate, 6:1) to afford compound 5 (1.1 g, 93% yield) as a light-yellow solid.

General Procedure for the Preparation of Compound 6

N-(3-Fluoro-4-(4-(tert-butoxycarbonyl)­piperazin-1-yl)­phenyl)-6-hydroxy-4-(trifluoromethyl)­quinolin-2-amine (6)

To a solution of compound 5 (1.1 g, 1.84 mmol) in THF (60 mL) was added Pd­(OH)₂ (86.31 mg, 92.18 μmol). The reaction vessel was evacuated and backfilled with H₂ (three cycles) and stirred under a H₂ atmosphere (balloon) at room temperature. After the starting material was consumed (monitored by TLC), the reaction mixture was filtered through a silica gel pad (200–300 mesh), the pad was washed thoroughly with methanol, and the combined filtrates were concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate, 3:1) to afford compound 6 as a yellow solid (867.4 mg, 93% yield).

General Procedure for the Preparation of Compound 7

N-(3-Fluoro-4-(4-(tert-butoxycarbonyl)­piperazin-1-yl)­phenyl)-6-(3-chloropropoxy)-4-(trifluoromethyl)­quinolin-2-amine (7)

A mixture of compound 6 (500 mg, 0.98 mmol) and potassium carbonate (204.65 mg, 1.48 mmol) was suspended in anhydrous DMF (4 mL). To this suspension was added 1-bromo-3-chloropropane (196 μL, 1.97 mmol). The reaction mixture was stirred at room temperature for 12 h until completion (monitored by TLC). The mixture was then poured into a mixture of ethyl acetate and water (50 mL/50 mL). The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (2 × 20 mL). The combined organic extracts were washed with water and brine, dried over anhydrous MgSO₄, filtered, and concentrated under reduced pressure. The crude residue was purified by flash column chromatography on silica gel (eluent: petroleum ether/ethyl acetate = 5:1, v/v) to afford compound 7 (484.8 mg, 84% yield) as a yellow solid.

General Procedure for the Preparation of Compound 8

N-(3-Fluoro-4-(4-(tert-butoxycarbonyl)­piperazin-1-yl)­phenyl)-6-(3-(piperazin-1-yl)­propoxy)-4-(trifluoromethyl)­quinolin-2-amine (8)

Under an argon atmosphere, a reaction tube was charged with compound 7 (150 mg, 0.26 mmol), piperazine (88.65 mg, 1.03 mmol), and potassium carbonate (106.67 mg, 0.77 mmol). Anhydrous DMF (5 mL) was added to dissolve the solids, and the reaction mixture was stirred at 80 °C for 5 h. After completion, the mixture was poured into a biphasic system of ethyl acetate and water. The mixture was shaken and allowed to settle for phase separation. The organic layer was separated, and the aqueous phase was extracted with ethyl acetate (2 × 50 mL). The combined organic extracts were washed with water and saturated brine, dried over anhydrous MgSO₄, filtered, and concentrated under reduced pressure. The crude residue was purified by silica gel column chromatography (dichloromethane/methanol = 50:1) to afford compound 8 (111.0 mg, 68% yield).

General Procedure for the Preparation of Compound 9

N-(3-Fluoro-4-(4-(tert-butoxycarbonyl)­piperazin-1-yl)­phenyl)-6-(3-(4-(2-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)­ethyl)­piperazin-1-yl)­propoxy)-4-(trifluoromethyl)­quinolin-2-amine (9)

Compound 8 (90 mg, 0.14 mmol) and potassium carbonate (98.30 mg, 0.71 mmol) were dissolved in anhydrous DMF (1 mL) in a reaction vial. 3-(But-3-yn-1-yl)-3-(2-iodoethyl)-3H-diazirine (27 μL, 0.18 mmol) was added, and the resulting mixture was stirred at room temperature for 19 h. Upon completion, the reaction mixture was diluted with ethyl acetate and poured into water. The layers were separated, and the aqueous phase was extracted with ethyl acetate (2 × 10 mL). The combined organic layers were washed with water and brine, dried over anhydrous MgSO₄, filtered, and concentrated under reduced pressure. Purification of the crude compound by silica gel column chromatography (dichloromethane/methanol = 50:1) afforded compound 9 (70 mg, 65% yield) as yellow solid. ¹H NMR (600 MHz, CDCl₃): δ 7.78 (d, J = 9.2 Hz, 1H), 7.60 (dd, J = 13.9, 2.5 Hz, 1H), 7.32 (dd, J = 9.2, 2.6 Hz, 1H), 7.22 (s, 1H), 7.20–7.14 (m, 2H), 6.93 (t, J = 9.0 Hz, 1H), 6.79 (s, 1H), 4.10 (t, J = 6.3 Hz, 2H), 3.61 (t, J = 4.9 Hz, 4H), 3.00 (t, J = 5.0 Hz, 4H), 2.70–2.35 (m, 9H), 2.21 (t, J = 7.7 Hz, 2H), 2.11–1.90 (m, 6H), 1.65 (t, J = 7.5 Hz, 2H), 1.58 (t, J = 7.7 Hz, 2H), 1.49 (s, 9H); ¹³C NMR (151 MHz, CDCl₃): δ 156.00 (d, J = 246.0 Hz), 155.85, 154.92, 151.41, 144.25, 135.63 (d, J = 14.6 Hz), 135.63 (d, J = 5.3 Hz), 134.96 (q, J = 31.5 Hz), 129.17, 123.46 (q, J = 274.4 Hz), 122.74, 119.85 (d, J = 4.1 Hz), 119.47, 115.84 (d, J = 3.1 Hz), 110.27 (q, J = 5.5 Hz), 109.02 (d, J = 25.0 Hz), 104.12, 82.96, 80.03, 69.23, 66.69, 55.28, 53.32, 53.11, 52.75, 51.03, 32.58, 30.55, 28.58, 27.45, 26.79, 13.45; ¹⁹F NMR (565 MHz, CDCl₃): δ −62.64, −120.87.

General Procedure for the Preparation of Compound TKL120

N-(3-Fluoro-4-(piperazin-1-yl)­phenyl)-6-(3-(4-(2-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)­ethyl)­piperazin-1-yl)­propoxy)-4-(trifluoromethyl)­quinolin-2-amine (TKL120)

A solution of compound 9 (32 mg, 38.23 μmol) in anhydrous dichloromethane (DCM, 1 mL) was prepared and cooled to 0 °C under an argon atmosphere. Hydrochloric acid (0.5 mL, 4 M in 1,4-dioxane) was added dropwise at this temperature. The reaction was stirred at 0 °C and monitored by thin-layer chromatography (TLC). After completion, the mixture was carefully neutralized with an aqueous potassium carbonate solution and extracted with ethyl acetate (3 × 20 mL). The combined organic layers were washed with water and brine, dried over anhydrous MgSO₄, filtered, and concentrated in vacuo. The resulting crude material was purified by silica gel column chromatography (dichloromethane/methanol = 50:1) to afford compound TKL120 (20 mg, 71% yield). ¹H NMR (600 MHz, CD₃OD): δ 8.00 (dd, J = 15.0, 2.5 Hz, 1H), 7.73 (d, J = 9.2 Hz, 1H), 7.49–7.39 (m, 1H), 7.31 (dd, J = 9.2, 2.7 Hz, 1H), 7.19 (t, J = 2.3 Hz, 1H), 7.03 (t, J = 9.2 Hz, 1H), 4.06 (t, J = 6.1 Hz, 2H), 3.24–2.97 (m, 8H), 2.71–2.15 (m, 13H), 2.12–1.96 (m, 4H), 1.78–1.47 (m, 4H); ¹³C NMR (151 MHz, CD₃OD): δ 155.77 (d, J = 243.1 Hz), 155.44, 151.64, 143.82, 137.44 (d, J = 11.1 Hz), 133.79 (d, J = 31.5 Hz), 133.53 (d, J = 9.9 Hz), 128.65, 126.59–120.50 (m), 121.64, 119.51 (d, J = 3.9 Hz), 118.43, 114.17 (d, J = 3.0 Hz), 111.73 (q, J = 5.5 Hz), 106.77 (d, J = 26.1 Hz), 103.79, 82.32, 68.98, 66.13, 54.83, 52.49, 52.17 (d, J = 3.5 Hz), 50.19 (d, J = 2.6 Hz), 44.67, 32.10, 29.68, 26.78, 26.01, 12.46 (d, J = 4.3 Hz); ¹⁹F NMR (565 MHz, CD₃OD): δ −64.08, −123.57.

Cell Lines and Culture

Human glioblastoma cell lines (U87MG, U118MG, U251MG), human hepatic stellate LX2 cells, and human embryonic kidney 293T cells were obtained from the American Type Culture Collection (ATCC, USA). Luciferase-expressing U87MG cells (U87MG-Luc) were purchased from Shanghai Fuheng Biotechnology Co., Ltd. Cells were maintained in Dulbecco’s modified Eagle medium (DMEM, Gibco, USA) supplemented with 10% fetal bovine serum (FBS) at 37 °C in a humidified incubator with 5% CO₂.

MTT Assay

U87MG, U118MG, U251MG, and LX2 cells were seeded in 96-well plates at a density of 4 × 10³ cells per well and allowed to adhere for 6–8 h in a humidified incubator. Compounds were prepared at the desired concentrations for initial screening (5 μmol/L) and for subsequent dose–response assays using TKL002 at 0.625, 1.25, 2.5, 5, and 10 μmol/L, with 0.1% DMSO as the vehicle control. After 24, 48, or 72 h of treatment, 5 mg/mL MTT solution (Solarbio, China) was added, followed by DMSO to dissolve formazan crystals. Absorbance at 490 nm was measured using a microplate reader, and inhibition rates and IC₅₀ values were calculated using IBM SPSS Statistics 21.0.

Apoptosis Assay

U87MG cells were seeded in 6-well plates at a density of 2 × 10⁵ cells per well and treated with TKL002 at 2, 4, and 6 μmol/L for 48 h, with 0.1% DMSO as the control. Cells were harvested using 0.25% trypsin without EDTA, resuspended in 500 μL 1× binding buffer, and stained with Annexin V-FITC/PI according to the manufacturer’s instructions (Solarbio, China). Early and late apoptotic cells were quantified by flow cytometry (BD Biosciences, USA).

Cell Cycle Analysis

U87MG and U118MG cells were seeded in 6-well plates at 2 × 10⁵ cells per well and treated with TKL002 (2, 4, and 6 μmol/L) for 24 h; 0.1% DMSO served as the control. After treatment, cells were trypsinized, washed twice with PBS, and fixed in 70% ethanol at −20 °C overnight. Following fixation, cells were washed to remove ethanol, resuspended in PBS, and stained with DNA content detection reagents according to the kit protocol (Solarbio, China). Cell cycle distribution was analyzed by flow cytometry.

Western Blot

U87MG and U118MG cells were treated with TKL002 (2, 4, and 6 μmol/L) or 0.1% DMSO for 48 h. Cell pellets were lysed in RIPA buffer containing 1% PMSF (Beyotime, China) at 4 °C for 30 min, followed by centrifugation at 12,000 rpm for 15 min to collect supernatants. Protein concentrations were determined using a BCA assay (Beyotime, China), and 50 μg of total protein per sample was subjected to SDS-PAGE, followed by transfer to PVDF membranes (Millipore, USA). Membranes were blocked with 5% nonfat milk (Solarbio, China) for 2 h and incubated with primary antibodies at 4 °C overnight. After five washes with 1× TBST, membranes were incubated with HRP-conjugated secondary antibodies for 2 h at room temperature and washed again. Antibody details are listed in Table . Protein bands were visualized using ECL chemiluminescence (Beyotime, China) and captured with a Bio-Rad ChemiDoc MP system (Hercules, USA). Densitometric analysis was performed using ImageJ software.

4. Information of Antibodies.

antibody	catalogue	dilution	company
α-tubulin	11224-1-AP	1:10000	Proteintech, China
GAPDH	10494-1-AP	1:10000	Proteintech, China
Bcl2	12789-1-AP	1:1000	Proteintech, China
Bax	50599-2-Ig	1:1000	Proteintech, China
caspase 3	9662S	1:1000	Cell Signaling Technology (CST), USA
active-caspase 3	ER30804	1:1000	HUABIO, China
gamma cystathionase	12217-1-AP	1:5000	Proteintech, China
NF-κB P65	10745-1-AP	1:1000	Proteintech, China
Phospho-NF-κB p65 (Ser468)	82335-1-RR	1:1000	Proteintech, China
TNF-α	17590-1-AP	1:1000	Proteintech, China
IL-6	21865-1-AP	1:1000	Proteintech, China
N-cadherin	4061S	1:1000	Cell Signaling Technology (CST), USA
E-cadherin	3195T	1:1000	Cell Signaling Technology (CST), USA
vimentin	3932S	1:1000	Cell Signaling Technology (CST), USA

Acute Toxicity Study in Mice

Acute toxicity of TKL002 was evaluated in 36 Kunming mice (equal numbers of males and females; n = 3 per sex per group). Mice received a single intraperitoneal injection of TKL002 at 150, 200, 250, 300, 500 mg/kg, while control mice were administered an equivalent volume of saline. Animals were monitored daily for 14 days for clinical signs, including body weight changes, locomotor activity, respiratory function, gastrointestinal symptoms, neurological status, and mortality. On day 14, mice were euthanized by CO₂ inhalation. Heart, liver, spleen, lung, and kidney were collected for organ index calculation (organ weight/body weight × 100%), and blood samples were obtained via cardiac puncture for hematological analysis and serum biochemical evaluation of liver and kidney function. These assessments allowed comprehensive evaluation of the systemic safety profile of TKL002.

Subcutaneous GBM Xenograft Model

U87MG cells were resuspended at 1 × 10⁸ cells/mL and injected subcutaneously (100 μL per mouse) into the left axillary region of BALB/c nude mice. One week after inoculation, tumor-bearing mice were randomly assigned to four groups: vehicle control, TKL002 at 5 mg/kg, TKL002 at 20 mg/kg, and temozolomide (TMZ) at 5 mg/kg. Treatments were administered intraperitoneally every other day for a total of seven doses. Tumor volume and body weight were measured throughout the treatment period. Tumor volume (V, cm³) was calculated using the formula: V = 0.5 × length × width², where length and width were measured with calipers. After 14 days of treatment, mice were euthanized, and tumors and major organs were harvested for histopathological and toxicity assessments.

Orthotopic GBM Xenograft Model

U87MG-Luc cells were adjusted to 1 × 10⁷ cells/mL and kept on ice until use. Nude mice were anesthetized with isoflurane, and 3.5 mm deep intracranial injections were performed using a stereotactic apparatus (REWORD, China) at 0.5 mm anterior and 2 mm lateral to the bregma. Tumor establishment was confirmed by in vivo bioluminescence imaging (Thermo Fisher Scientific, USA) 7 days postimplantation. TKL002 was administered via tail vein at 10 or 20 mg/kg every 3 days for a total of five doses. Body weight, general condition, and tumor bioluminescence intensity were monitored throughout the treatment. On day 20, mice were anesthetized and perfused with saline, after which brain and major organs were collected for evaluation of TKL002 efficacy and systemic toxicity.

Hematoxylin and Eosin (H&E) Staining

Collected tissues were fixed in 4% paraformaldehyde, dehydrated through graded ethanol concentrations, embedded in paraffin, and sectioned at 5 μm thickness. Sections were deparaffinized in xylene for 20 min, rehydrated through descending ethanol concentrations, and stained with hematoxylin and eosin (Beyotime, China) following standard protocols. Histopathological changes were examined under a light microscope, and representative images were captured for analysis.

Immunohistochemistry

Tumor tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at a thickness of 4 μm. After deparaffinization and rehydration, antigen retrieval was performed by heating the sections in citrate buffer (pH 6.0). Endogenous peroxidase activity was quenched with 3% hydrogen peroxide, followed by blocking with 5% bovine serum albumin. Sections were incubated with primary antibodies against the indicated proteins at 4 °C overnight, followed by incubation with appropriate horseradish peroxidase-conjugated secondary antibodies. Immunoreactivity was visualized using 3,3′-diaminobenzidine (DAB) as the chromogenic substrate, and nuclei were counterstained with hematoxylin. Stained sections were examined under a light microscope, and staining intensity was evaluated in a blinded manner.

Click Chemistry Reaction

Based on the molecular structure of TKL002, a photoaffinity group and an alkyne moiety were introduced to generate the probe compound TKL120. Optimal probe concentration and small-molecule competition conditions were determined using in-gel fluorescence assays. Experimental groups included a control group, probe-only group, and competition group. U118MG cell lysates were incubated with different compounds, followed by UV cross-linking and click chemistry reactions. Affinity purification was performed to enrich small-molecule-interacting proteins. Protein samples were separated by SDS-PAGE for 3–5 mm into the resolving gel, the gel was excised into ∼1 mm³ pieces, transferred into 1.5 mL Eppendorf tubes, and processed by destaining, reduction, alkylation, and overnight enzymatic digestion. Peptides were then extracted and analyzed by mass spectrometry.

Bioinformatics Analysis

Mass spectrometry raw data obtained from the Thermo Q Exactive HF-X system were searched against corresponding protein databases for peptide and protein identification. Data quality was evaluated based on peptide and protein coverage. Identified proteins were filtered, annotated using functional databases (COG/KOG, GO, KEGG), and analyzed for protein–protein interactions to predict potential molecular targets. The 2D structure of TKL002 was drawn using ChemSketch software and converted into a 3D structure in ChemBio3D Ultra 14.0, followed by energy minimization. PDB IDs of potential target proteins were retrieved from UniProt, corresponding 3D structures downloaded from the PDB database, and water molecules and ligands removed using PyMOL 2.5. AutoDockTools 1.5.7 was used to add hydrogens and define active pockets, followed by molecular docking using AutoDock Vina 1.2.0. Docking results were visualized in PyMOL 2.5. Finally, UALCAN and TCGA databases were employed for pan-cancer, differential expression, and survival analyses of target proteins.

Cellular Thermal Shift Assay

U118MG cells were treated with 5 μmol/L TKL002 for 2–3 h (0.1% DMSO as control). Cell pellets were collected and equally divided into seven aliquots. Samples were heated at 46, 49, 52, 55, 58, and 61 °C for 3 min using a PCR instrument, followed by three cycles of 37 °C water bath and liquid nitrogen freeze–thaw. Supernatants were obtained by centrifugation at 12,000 rpm for 15 min at 4 °C. To evaluate concentration-dependent binding, cells were treated with 0.1, 0.5, 1, 5, 10, and 25 μmol/L TKL002 for 2–3 h, heated at 52 °C for 3 min, and processed as described above.

Gene Knockdown and Overexpression Assay

Short hairpin RNA (shRNA) plasmids targeting CTH (sh1, sh2, sh3), a nontargeting control (shControl), CTH-overexpression (OE-CTH), and corresponding vector control (OE-Control) plasmids (Guizhou Hejin Biotechnology, China) were packaged into viral particles via standard transfection protocols in 293T cells. Viral supernatants were collected 48 h post-transfection and titrated. U87MG and U251MG cells were seeded at 2 × 10⁵ cells per well in 6-well plates and transduced with viral particles at a 1:1 ratio with antibiotic-free DMEM containing 10% fetal bovine serum. After 24 h, the medium was replaced with fresh complete medium, and cells were cultured for an additional 48 h. Stable CTH-knockdown or CTH-overexpressing cell lines were established via puromycin selection (Beyotime, China). Prior to downstream functional assays, successful modulation of CTH expression was confirmed by total protein extraction and immunoblot analysis.

Enzyme-Linked Immunosorbent Assay

Culture supernatants from U87MG and U118MG cells treated with 2, 4, and 6 μmol/L TKL002 (0.1% DMSO as control) were collected and centrifuged at 2000 rpm for 20 min. Enzyme-linked immunosorbent assay (ELISA) assays were performed using kits for human cystathionine γ-lyase, cysteine, cystathionine, H₂S, and glutathione (GSH) (Shanghai Keqiao Biotechnology, China) according to the manufacturer’s instructions. Optical density (OD) at 450 nm was measured, and sample concentrations were calculated from standard curves and adjusted for dilution.

Cell Migration and Invasion Assays

U87MG and U118MG cells were resuspended in serum-free medium at 1 × 10⁴ cells/mL and seeded into transwell inserts with polycarbonate membranes. Cells were allowed to adhere, after which 200 μL of serum-free medium containing 2, 4, and 6 μmol/L TKL002 was added to the upper chamber, and 800 μL DMEM with 15% FBS was added to the lower chamber. After 48 h, cells on the membrane underside were stained with 0.1% crystal violet, observed under a microscope, and photographed. For invasion assays, inserts were precoated with Matrigel; otherwise, procedures were identical.

Immunofluorescence

Cells were seeded on coverslips, treated as indicated, and fixed with 4% paraformaldehyde. After permeabilization with 0.1% Triton X-100, nonspecific binding was blocked with 5% bovine serum albumin. Cells were then incubated with primary antibodies against the indicated proteins at 4 °C overnight, followed by incubation with species-specific fluorophore-conjugated secondary antibodies in the dark. Nuclei were counterstained with DAPI.

Statistical Analysis

All experiments were independently repeated three times. Data were expressed as mean ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism 8.0 and IBM SPSS Statistics 21.0. Differences between multiple groups were analyzed using one-way ANOVA, and comparisons between two groups were performed using student’s t-test. P < 0.05 was considered statistically significant.

Glossary

Abbreviations

BBB

blood–brain barrier

BLT

bioluminescence imaging

CTH

cystathionine γ-lyase

DMSO

dimethyl sulfoxide

EMT

epithelial-mesenchymal transition

GSH

glutathione

H₂S

hydrogen sulfide

IC₅₀

half-maximum inhibitory concentration

LD₅₀

dose that is lethal in 50% of test subjects

NF-κB

nuclear factor κB

NOX4

NADPH oxidase 4

PBS

phosphate buffered saline

RNA

ribonucleic acid

SOX2

SRY-box transcription factor 2

SRM

spermidine synthase

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.5c03477.

• Molecular formula strings (CSV)

• Inhibitory activity of trifluoromethylquinoline derivatives (Figure S1); acute toxicity study in mice (Figure S2); quality control analysis in proteomics (Figure S3); HPLC purity analysis of compound TKL002 and TKL120 (Figures S4 and S5); NMR spectrum of compound 9 (Figures S6–S8); NMR spectrum of compound TKL120 (Figures S9–S11); original WB gel image (Figures S12–S16) (PDF)

⊥.

Z.L. and G.X. contributed equally to this paper.

Male BALB/c nude mice (4–5 weeks old, 16–18 g) were obtained from Chongqing Tengxin Biotechnology Co., Ltd. Mice were housed under specific pathogen-free (SPF) conditions with a 12 h light–12 h dark cycle and an ambient temperature of 25 ± 1 °C. All experimental procedures complied with the Guidelines for the Care and Use of Laboratory Animals in China and were approved by the Animal Ethics Committee of Guizhou Medical University (Ethics Approval No. 2100293).

The authors declare no competing financial interest.