Acta Pharmacologica Sinica最新文献

The activation of voltage-gated potassium Kv7/M channels is an attractive therapeutic strategy for chronic pain. Galangin, the principal active component of the medicinal herb Alpinia officinarum Hance, has exhibited analgesic effects in mice. In this study, we investigated the antinociceptive effects of galangin in the treatment of various types of chronic pain and the underlying mechanisms. Using whole-cell recordings of CHO cells expressing Kv7.2/Kv7.3 channels, we showed that galangin enhanced Kv7.2/Kv7.3 currents in a concentration-dependent manner, with an EC₅₀ value of 8.8 ± 1.6 μM, and shifted the voltage-dependent activation curve of the channels toward depolarization. We demonstrated that galangin selectively and potently activated the Kv7.2, Kv7.4, and Kv7.5 channels while reducing the Kv7.1 current and exerting no effect on the Kv7.3 current. Notably, galangin no longer increased the current amplitude and slightly shifted the voltage-dependent activation of the Kv7.2 (E322A) mutant, suggesting that Glu-322 in Kv7.2 is important for galangin activation of the channels. Moreover, we showed that galangin (100 μM) significantly enhanced the M-current and consequently reduced the excitability of DRG neurons in SNI mice. In multiple chronic pain mouse models, the administration of galangin (15 mg/kg, i.p.) significantly increased the threshold for mechanical stimuli and the withdrawal latency to thermal stimuli, which were reversed by the Kv7/M channel blocker XE991. Taken together, the results of this study demonstrated that galangin exerts its antinociceptive effects mainly through the activation of Kv7/M channels, representing a novel approach for treating neuronal excitatory diseases.

To date, monthly oral bisphosphonates have not been available in China. In this randomized, double blind, positive-controlled, multicenter phase III clinical trial, we compared the efficacy and safety of monthly minodronate versus weekly alendronate in the treatment of Chinese postmenopausal women with osteoporosis. A total of 548 participants were screened across 31 study centers, of which 330 participants were randomized into two groups: the experimental group (n = 165) received oral minodronate (50 mg/tablet once every four weeks) and alendronate placebo (once weekly), while the positive control group (n = 165) received oral alendronate (70 mg/tablet once weekly) and minodronate placebo (once every four weeks) for a duration of 48 weeks. The bone mineral density (BMD) of the lumbar spine, femoral neck and total hip were measured using dual-energy X-ray absorptiometry (DXA) at baseline and at 24 and 48 weeks. At the end of treatments, the experimental group exhibited a mean increase (SD) in BMD above the baseline at the lumbar spine, femoral neck and total hip of 4.61% (4.613%), 3.04% (4.034%) and 3.40% (3.569%), respectively, compared with those of 4.55% (3.753%), 1.86% (3.592%) and 2.30% (4.838%) in the control group. All improvements from the baseline in the two groups were statistically significant. The monthly minodronate did not cause new safety risks compared with alendronate. This study demonstrates that monthly minodronate administration is non-inferior to weekly alendronate in terms of therapeutic efficacy, while maintaining a comparable safety profile. Furthermore, the monthly dosing schedule of minodronate may significantly enhance medication adherence among osteoporosis patients, potentially improving long-term treatment outcomes.

Inhibition of autophagy has been considered as a promising strategy for tumor therapy, discovery of small-molecule autophagy inhibitors suitable for clinical use would be of great significance. Since cysteine protease ATG4B plays a key role in the autophagy machinery by processing pro-LC3 and lipidated LC3 to drive the autophagy progress, inhibition of ATG4B may serve as a potential therapeutic strategy. We previously found that copper ions instead of other ions efficiently inhibited ATG4B activity, which was more potent than other ATG4B inhibitors reported. As copper ions are easily chelated, copper complexes may develop into novel ATG4B inhibitors. In this study we identified a copper complex antifouling agent, copper pyrithione (CuPT), which effectively inhibited ATG4B activity and blocked autophagy flux. By combining FRET-based assay in vitro and cell-based assays, we showed that CuPT effectively inhibited ATG4B activity with an IC₅₀ of 250.9 nM. CuPT (0.5, 1, 2 µM) dose-dependently promoted the formation of insoluble ATG4B and p62 aggregates in HeLa cells, which was similar to copper ions. Importantly, CuPT exhibited potent anticancer activities in vitro and in vivo: it potently suppressed the cell viability of 8 different cancer cell lines with IC₅₀ values less than 1 µM; administration of CuPT (1 mg/kg; i.p. every three days) significantly inhibited the tumor growth in colorectal xenograft mouse model without obvious organs damage. CuPT-induced cytotoxicity in HCT116 cells could be reversed by enhancing autophagy using rapamycin or Earle's Balanced Salt Solution (EBSS). Besides, we demonstrated that CuPT induced a novel copper-dependent cell death, cuproptosis, of cancer cells. Together, this study presents the first copper complex ATG4B inhibitor CuPT, a copper compound that can be further developed for the treatment of a wide range of cancers.

The anti-HER2 antibody‒drug conjugate (ADC) DS-8201 presents new hope for patients with advanced HER2-positive tumors. Its clinical application, however, is hindered by serious adverse reactions and reduced efficacy following long-term treatment. In this study, we investigated the factors influencing the sensitivity of DS-8201 and developed effective combination regimens to optimize its therapeutic efficacy. We showed that HER3 upregulation diminished the sensitivity of HER2-positive tumor cells to DS-8201. We found that DS-8201 treatment activated DNA damage repair responses in BT-474 cells, in which the ATR kinase pathway induced the expression of the HER3 transcription factor FoxO1, leading to increased HER3 levels. This process was triggered by the payload component of DS-8201, the topoisomerase I inhibitor DXd, rather than the antibody. Based on this finding, we showed that combining DS-8201 with either a HER3-targeting antibody (SIBP-03) or an ATR inhibitor (BAY1895344) resulted in significant synergistic antitumor efficacy without substantial toxicity in vitro or in vivo. Overall, this study revealed that the ATR/FoxO1/HER3 pathway plays a critical role in modulating the efficacy of DS-8201, suggesting that combining DS-8201 with ATR or HER3 inhibition represents a promising therapeutic strategy for HER2-positive cancers.

Pathological cardiac hypertrophy as a major contributor to heart failure is characterized by complicated mechanisms. Fumarate hydratase (FH) is a crucial enzyme in the tricarboxylic acid cycle. FH mutations and dysfunction have been implicated in various pathological processes including hereditary leiomyomatosis and renal cell cancer, neurodegenerative diseases, metabolic syndrome and cardiovascular diseases. In this study we investigated the role of FH in cardiac hypertrophy. Cardiac hypertrophy was induced in mice by transverse aortic constriction (TAC) surgery as well as in neonatal rat cardiomyocytes (NRCMs) by phenylephrine (PE) stimulation. We showed that the expression levels of FH were gradually increased with development of cardiac hypertrophy in TAC mice. Cardiomyocyte-specific overexpression of FH by intravenous injection of recombinant adeno-associated virus serotype 9 (AAV9) carrying FH two weeks before TAC surgery prevented the morphological changes, cardiac dysfunction and remodeling in TAC mice; FH overexpression also significantly attenuated PE-induced hypertrophy in NRCMs along with suppressed expression of hypertrophic markers ANP, BNP and β-MHC. We demonstrated that FH overexpression alleviated TAC-induced mitochondrial structural damage in cardiomyocytes and facilitated metabolic remodeling. RNA sequencing and untargeted metabolomics revealed that FH overexpression mitigated myocardial remodeling and mitochondrial metabolism dysfunction in TAC mice mainly by suppressing the transcription factor SREBP and reducing the gene expression of elongation of very long chain fatty acids protein 7 (Elovl7). Overexpression of Elovl7 reversed the protective effects of FH in both TAC mice and PE-stimulated NRCMs. Knockdown of the transcription factor SREBP reduced Elovl7 expression, thereby exerting cardioprotective effects. In conclusion, we demonstrate that FH overexpression prevents cardiac hypertrophy in mice by regulating glucose and lipid metabolism through the malate-SREBP-Elovl7 pathway.

Antibody-drug conjugate (ADC) represents a promising paradigm for tumor-targeted delivery of chemotherapy. Trastuzumab deruxtecan (T-Dxd/DS-8201), a second-generation HER2-ADC, has significantly improved treatment outcomes for breast cancer patients. But due to the large molecular weight, the performance of ADC is still limited by lower tumor penetration, insufficient BBB permeability, and prolonged systemic exposure to normal tissues. In this study, we generated novel anti-HER2 nanobodies (VHH2, VHH3) that exhibited outstanding target affinity and tumor inhibition. After i.v. injection, VHH3-Fc fusion distributed 4 to 5-fold higher in subcutaneous tumor and intracranial tumor compared with trastuzumab. VHH3-Fc and VHH3-ABD were also more penetrant in an in vitro BBB permeability assay. Site-specific conjugation of VHH3-Fc or VHH3-ABD fusions with anti-microtubule MMAE or anti-topoisomerase-1 Dxd payload produced nanobody-drug conjugates (NDCs) with highly potent and durable antitumor efficacy. When evaluated on the same linker-payload (GGFG-Dxd) dosages, VHH3-Fc-Dxd (DAR3.9) outperformed T-Dxd (DAR8) in both the subcutaneous and intracranial tumor models. Moreover, IHC staining and RNA-seq analysis of the treated tumor tissues revealed the involvement of the cGAS-STING-IFNs pathway in mediating the drug activity. Gene expression and protein function were more profoundly modulated by VHH3-Fc-Dxd than T-Dxd. Unlike the higher tumor distribution, the mouse serum PK study revealed a faster clearance (T_1/2), reduced exposure (AUC), and higher volume distribution (Vz) for VHH3-Fc-Dxd relative to T-Dxd. Our results provide an example for the next generation HER2-NDC with substantially differentiated pharmacokinetics and pharmacodynamics profiles that will further benefit treatment outcomes and therapeutic windows.

Protein-peptide interactions (PpIs) play a critical role in major cellular processes. Recently, a number of machine learning (ML)-based methods have been developed to predict PpIs, but most of them rely heavily on sequence data, limiting their ability to capture the generalized molecular interactions in three-dimensional (3D) space, which is crucial for understanding protein-peptide binding mechanisms and advancing peptide therapeutics. Protein-peptide docking approaches provide a feasible way to generate the 3D models of PpIs, but they often suffer from low-precision scoring functions (SFs). To address this, we developed DeepPpIScore, a novel SF for PpIs that employs unsupervised geometric deep learning coupled with a physics-inspired statistical potential. Trained solely on curated experimental structures without binding affinity data or classification labels, DeepPpIScore exhibits broad generalization across multiple tasks. Our comprehensive evaluations in bound and unbound peptide bioactive conformation prediction, binding affinity prediction, and binding pair identification reveal that DeepPpIScore outperforms or matches state-of-the-art baselines, including popular protein-protein SFs, ML-based methods, and AlphaFold-Multimer 2.3 (AF-M 2.3). Notably, DeepPpIScore achieves superior results in peptide binding mode prediction compared to AF-M 2.3. More importantly, DeepPpIScore offers interpretability in terms of hotspot preferences at protein interfaces, physics-informed noncovalent interactions, and protein-peptide binding energies.

Non-small cell lung cancer (NSCLC) is an aggressive malignancy with a poor prognosis. Abnormal expression of focal adhesion kinase (FAK) is closely linked to NSCLC progression, highlighting the need for effective FAK inhibitors in NSCLC treatment. In this study we conducted high-throughput virtual screening combined with cellular assays to identify potential FAK inhibitors for NSCLC treatment. Fosaprepitant (FOS), a clinical antiemetic drug, exhibited a high affinity for FAK with a K_D value of 4.35 × 10⁻⁵ M. The direct interaction between FOS and FAK was confirmed by molecular docking, molecular dynamics, drug affinity responsive target stability and surface plasmon resonance analysis. We showed that FOS (15, 25 μM) dose-dependently inhibited the proliferation, migration and invasion of A549 and H1299 cells by targeting FAK. The IC₅₀ values in inhibiting the cell viability at 24 h were 73.05 and 126.1 μM, respectively. Knockdown FAK reversed the inhibitory effects of FOS on A549 cells. Using RNA sequencing and Western blotting analysis, we demonstrated that FOS treatment led to downregulation of the AKT and JNK/c-Jun signaling pathways in A549 and H1299 cells. Importantly, point mutation analyses revealed that FOS primarily targeted the Y925 phosphorylation site on FAK. In A549 cells xenograft nude mouse model, administration of FOS (20, 60 mg/kg, i.p. every 2 d for 2 weeks) dose-dependently suppressed the tumor growth. Collectively, FOS exhibits significant anti-NSCLC activity both in vitro and in vivo by binding to FAK and inhibiting its phosphorylation, thereby blocking the AKT and JNK/c-Jun signaling pathways. These results suggest FOS as a novel FAK inhibitor for NSCLC treatment.

Mitochondria are not only the most important organelles in eukaryotic cells that participate in energy metabolism, signal transduction, cell apoptosis and other physiological processes, but also essential regulators of neurodevelopment, neuroplasticity, survival and adult neurogenesis. The mitochondria-localized hydroxylase Clk-1 is involved in ubiquinone biosynthesis. Recent evidence shows that Clk1^+/- mutant mice are resistant to morphine- and methamphetamine-induced conditioned place preference. Given the critical role of learning and memory in drug dependence, we herein explored whether and how Clk1 deficiency affected the cognitive processes in mice. We found that mutant Clk1 mice (Clk1^+/-) exhibited recognition memory impairment in novel object recognition (NOR) and novel arm recognition (NAR) tests. In addition, we observed in Clk1^+/- mutant mice a selective reduction in dendritic spine density in prefrontal cortex (PFC) but not in the hippocampus (HIP). The expression of brain-derived neurotrophic factor (BDNF) was also decreased in PFC but not in HIP. Furthermore, Clk1^+/- mutant mice displayed impairment in the ERK/CREB signaling pathway in PFC that might underlie Clk1^+/- mutation-induced changes in BDNF and dendritic morphology. Administration of antipsychotic drugs aripiprazole (0.3 mg·kg^-1·d^-1, i.p.) or risperidone (1 mg·kg^-1·d^-1, i.p.) for 7 days fully rescued Clk1 mutation-induced recognition memory deficits. This study provides primary evidence highlighting the role of mitochondrial Clk1 in the regulation of recognition memory and presents an informative model for investigating mitochondrial function in learning and memory.

Hypoxia is a common phenomenon in the microenvironment of solid tumors; mitochondria, as the site of cellular oxidative respiration, are among the first organelles to be affected under hypoxic conditions. Mitochondrial cristae organizing protein 19 (MIC19), a core component of the mitochondrial contact site and cristae organizing system (MICOS), is essential for preserving mitochondrial activity. In this study, we investigated the effects of hypoxia on MIC19 and its regulatory mechanisms in non-small cell lung cancer (NSCLC). We showed that the expression levels of MIC19 were significantly increased in NSCLC, which were associated with advanced stages and a poor prognosis in patients with NSCLC. We demonstrated that MIC19 promoted the proliferation and invasion of A549 and PC9 cells in vitro, and MIC19 played a crucial role in maintaining mitochondrial function. We revealed that USP3 mediated the hypoxia-induced upregulation of MIC19 expression in A549 and PC9 cells. In the hypoxic microenvironment, HIF-1α bound to the USP3 promoter region and promoted USP3 expression, which in turn stabilized MIC19 through K48-linked deubiquitination, thereby driving NSCLC progression. The role of MIC19 in NSCLC growth and progression was confirmed in nude mice bearing A549 xenograft tumors in vivo. In conclusion, under hypoxic conditions, USP3 stabilizes MIC19 through deubiquitination, thereby promoting NSCLC progression. This study reveals the HIF1α-USP3-MIC19 axis in NSCLC progression, providing a theoretical basis for future therapeutic strategies.