Acta Pharmacologica Sinica最新文献

RNA m⁶A methylation, as the most prevalent modification in mRNA, is a dynamic and reversible process primarily regulated by m⁶A methyltransferases ("writers"), m⁶A demethylases ("erasers"), and m⁶A recognition proteins ("readers"). It has been shown that N6-methyladenosine (m⁶A) plays a pivotal role in hepatocellular carcinoma (HCC). In this study we investigated the contribution of the m⁶A eraser AlkB homolog 5 (ALKBH5) to hepatocarcinogenesis, particularly during the early stages of liver cancer development. We found that liver-specific Alkbh5 conditional knockout (Alkbh5-cKO) profoundly suppressed DEN/CCl₄-induced HCC tumorigenesis and development in mice. We further showed that exogenous ALKBH5 expression drove the malignant transformation of immortal normal hepatocytes (HHL5, BNL), whereas ALKBH5 depletion in HCC cells restored hepatocyte-specific functions and suppressed malignancy. By conducting integrated MeRIP-seq/RNA-seq analyses, we identified STAT1 as a key target of ALKBH5-mediated m⁶A demethylation. ALKBH5 directly bound to STAT1 mRNA and reduced its m⁶A modification, thereby decreasing mRNA stability and suppressing STAT1 expression. Downregulated STAT1 inactivated the hepatocyte nuclear factor FOXA3, blocking hepatic differentiation and promoting malignancy. In 42 pairs of clinical HCC samples analyzed, STAT1 was negatively correlated with ALKBH5, and HCC patients with high ALKBH5 and low STAT1 expression exhibited worse clinical outcomes. We conclude that ALKBH5 is a critical oncogene in hepatocarcinogenesis. These results provide novel insights into the epigenetic regulation of hepatocarcinogenesis.

N-methyl-D-aspartate receptors (NMDARs) are glutamate-gated ion channels essential for synaptic transmission and plasticity in the central nervous system. GluN1/GluN3A, an unconventional NMDAR subtype functioning as an excitatory glycine receptor, has been implicated in mood regulation, with high expression in brain regions governing emotional and motivational states. However, therapeutic exploration has been significantly hindered by a lack of potent and selective modulators, limited structural data and the intrinsic complexity of ion channels. Here, we introduce a compound virtual screening pipeline that combines artificial intelligence and physical models, integrating two sequence-based deep learning prediction models (TEFDTA and ESMLigSite) with a molecular docking approach. This approach was employed to identify potential inhibitors against GluN1/GluN3A by screening a commercial database containing 18 million compounds. The strategy resulted in an impressive hit rate of 50% for discovering inhibitors, with the most promising compound exhibiting strong inhibitory activity (IC₅₀ = 1.26 ± 0.23 μM) and remarkable target specificity (>23-fold selectivity over the GluN1/GluN2A receptor). These findings highlight the effectiveness of AI-assisted strategies in addressing challenges related to unconventional ion channels and pave the way for new therapeutic exploration.

Increasing evidence shows that microRNAs (miRNAs) are functionally associated with cardiac remodeling. Functionally, some cytoplasmic circRNAs may act as miRNA sponges to inhibit functions of the combined miRNAs. Our previous study showed that miR-199a-5p and -3p promote cardiac hypertrophy and fibrosis. In this study, we designed and synthesized a novel circularized RNA sponge, circSP199a, and evaluated the therapeutic effects of circSP199a against cardiac hypertrophy and fibrosis. The synthesized circSP199a included 6 repeats of reverse complements of seed sequences of miR-199a-5p and -3p with 515 nt in length. We showed that the synthesized circSP199a and expression vector-mediated circSP199a expression inhibited cardiomyocyte hypertrophy and mitigated the fibrotic phenotypes in neonatal mouse cardiac fibroblasts and human cardiac organoid fibrosis via the combination of miR-199a-5p and -3p. Furthermore, intravenous injection of AAV9-circSP199a for 21 days in advance significantly ameliorated transverse aortic constriction-induced cardiac injury and remodeling in mice. We demonstrated that circSP199a blocked the functions of miR-199a-5p and -3p to enhance the expression of target genes of PGC-1α, Rb1, Sirt1 and Smad1 both in vitro and in vivo. These results provide new insights into the development of RNA sponge-based therapies for cardiac hypertrophy and fibrosis. The artificial circSP199a functions as a novel inhibitor of miR-199a-5p and -3p, exogenous provision of circSP199a specifically sponges miR-199a-5p and -3p to block their functions with subsequent upregulations of the target genes, including PGC-1α, Rb1, SIRT1 and Smad1, to mitigate the pathological cardiac hypertrophy and fibrosis.

Recent investigations into the rapid antidepressant effects of ketamine, along with studies on schizophrenia-related susceptibility genes, have highlighted the GluN2A subunit as a critical regulator of both emotion and cognition. However, the specific impacts of acute pharmacological inhibition of GluN2A-containing NMDA receptors on brain microcircuits and the subsequent behavioral consequences remain poorly understood. In this study, we first examined the effects of MPX-004, a selective GluN2A NMDA receptor inhibitor, on behavior within the dorsomedial prefrontal cortex (dmPFC). Local administration of MPX-004 in the dmPFC led to a reduced immobility duration in the forced swim test, an acute antidepressant-like effect, impairments in sensorimotor gating, and a schizophrenia-like phenotype. In vivo multiple-channel recordings and c-Fos staining revealed that MPX-004 decreases the activity of parvalbumin-expressing interneurons (PV-INs) and increases the activity of pyramidal neurons (PYNs). In vivo patch-clamp recordings further confirmed that PV-IN inactivation leads to an elevated PYN firing rate in the PFC. In vitro whole-cell recordings demonstrated that PV-INs receive stronger excitatory synaptic input and respond more robustly to presynaptic stimulation than do somatostatin-expressing interneurons (SST-INs) and PYNs, rendering them susceptible to GluN2A inhibition. Finally, the specific knockdown of GluN2A in prefrontal PV-INs abolished the behavioral effects of MPX-004, underscoring a critical role of the GluN2A-mediated modulation of PV-INs in these phenotypes. Together, these findings reveal that PV-INs are particularly vulnerable to GluN2A inhibition, leading to disinhibition of prefrontal circuits and resulting in both antidepressant-like and schizophrenia-like behaviors.

Progressive loss of vascular smooth muscle cells (VSMCs) is the pathophysiological basis for aortic aneurysm and dissection (AAD), a life-threatening disease, but the underlying mechanisms are largely unknown. Sirtuin 6 (SIRT6), a class III histone deacetylase, is critical for maintenance of VSMC homeostasis and prevention of vascular remodeling-related diseases. In this study, we investigated the role of VSMC SIRT6 in AAD and the molecular mechanism. We showed that the expression levels of SIRT6 were significantly reduced in VSMCs of the thoracic aorta in AAD patients. We constructed a VSMC-specific Sirt6 deficient mouse line and found that loss of Sirt6 in VSMCs dramatically accelerated angiotensin II (Ang II)-induced AAD formation and rupture, even without an Apoe-deficient background. In human aortic smooth muscle cells (HASMCs), knockdown of SIRT6 led to mitochondrial dysfunction and accelerated VSMC senescence. We revealed that SIRT6 bound to and deacetylated NRF2, a key transcription factor for mitochondrial biogenesis. However, Sirt6 deficiency inhibited NRF2 and reduced mRNAs encoding mitochondrial complex proteins. Notably, MDL-811, a newly developed small-molecule SIRT6 agonist, effectively reversed Ang II-induced mitochondrial dysfunction in HASMCs. In a BAPN-induced TAAD mouse model, administration of MDL-811 (20 mg/kg, i.p., every other day for 28 d) effectively mitigated AAD progression and reduced mortality. These results suggest that SIRT6 plays a protective role against AAD development, and targeting SIRT6 with small-molecule activators such as MDL-811 could represent a promising therapeutic strategy for AAD.

N-methyl-D-aspartate receptors (NMDARs) are critical mediators of excitatory neurotransmission and are composed of seven subunits (GluN1, GluN2A-D, and GluN3A-B) that form diverse receptor subtypes. While GluN1/GluN2 subtypes have been extensively characterized and have led to approved therapeutics, the GluN1/GluN3A subtype remains underexplored despite emerging evidence of its involvement in neuropsychiatric disorders. Efficient identification of modulators requires accurate prediction of drug-target affinity (DTA), particularly for challenging targets such as GluN1/GluN3A. In this study, we applied the ImageDTA model, which is a multiscale 2D convolutional neural network (CNN), to virtually screen 18 million small molecules for GluN1/GluN3A inhibitors. This artificial intelligence (AI)-driven approach prioritized 12 compounds, three of which demonstrated potent inhibitory activity (IC₅₀ < 30 µM) in experimental validation. The most potent hit, with an IC₅₀ of 4.16 ± 0.65 µM, revealed a novel structural scaffold, thus highlighting the potential of AI in accelerating drug discovery for underexplored receptor subtypes. These findings establish a robust framework for advancing GluN1/GluN3A-targeted therapeutics.

Enteric glial cells (EGCs) play an important role in the pathogenesis of irritable bowel syndrome (IBS). Phosphodiesterase-4 (PDE4) functions as a catalyzing enzyme targeting hydrolyzation of intracellular cyclic adenosine monophosphate (cAMP). Increased PDE4 activity promotes excessive production of pro-inflammatory cytokines and chemokines in various immune and epithelial cells, exacerbating immune cell activation and infiltration in inflamed tissues, inhibition of PDE4 has been proven to be an important strategy for inflammatory and autoimmune diseases. In this study we investigated the pathological role of PDE4 and the therapeutic effects of a PDE4 inhibitor apremilast in IBS. 2,4,6-Trinitrobenzenesulfonic acid (TNBS)-induced IBS model was established in mice, the mice were treated with apremilast (50 mg/kg, i.g.) for 7 days. After treatment, the intestinal motility and visceral sensitivity were assessed. At the end of the study, the mice were euthanized and the blood and colon tissues were collected for analyses. We showed that apremilast treatment significantly ameliorated IBS symptoms in the mice, evidenced by improvement on delayed intestinal motility and visceral hypersensitivity. We found that EGCs were activated in the colon of IBS mice. We then demonstrated that apremilast (10 μM) significantly suppressed TNF-α/IFN-γ stimulated activation of rat EGC cell line CRL-2690 and primary EGCs in vitro, as well as the secretion of EGCs-derived pain mediators and inflammatory factors while ameliorating oxidative stress. These effects depended on the activation of the nuclear factor erythroid 2-related factor 2 (Nrf-2) signaling pathway, which was validated in Nrf-2 knockout EGCs. These results suggest that inhibition of PDE4 by apremilast suppresses EGCs activation by activating the Nrf-2 signaling pathway, leading to decreased expression of pain mediators and inflammatory factors while ameliorating oxidative stress, ultimately alleviating IBS.

Methotrexate (MTX) is frequently administered with the proton pump inhibitor omeprazole (OPZ) to relieve gastrointestinal adverse reactions of MTX, but the coadministration increases the risk of kidney injury. In this study, we investigated the mechanisms of combined OPZ and MTX-induced acute kidney injury (OPZ + MTX-AKI), which was induced in rats or mice by administration of OPZ plus MTX for 14 days. Analysis of the FAERS database revealed that AKI was the principal form of kidney injury when OPZ was administered with MTX. We showed that coadministration of OPZ and MTX to rats resulted in the development of AKI. We found that OPZ and MTX, by inhibiting the expression and activity of SERCA2 and IP3R, respectively, jointly disrupted Ca²⁺ homeostasis, thereby causing cell damage. Transcriptomic analysis of clinical samples revealed that G protein-coupled receptor kinase 2 (GRK2) served as a key protein in OPZ + MTX-AKI. In Grk2^+/- mice and in mice with renal tubular epithelial cell (RTEC)-specific Grk2 knockdown, the manifestations of kidney injury, along with the levels of oxidative stress and apoptosis in the context of OPZ + MTX-AKI, were notably ameliorated. Conversely, in mice with RTEC-specific Grk2 overexpression, the kidney injury was markedly aggravated. Administration of GRK2 inhibitor CP-25 (17.5, 35, 70 mg/kg/d, i.g.) for 14 days dose-dependently alleviated OPZ + MTX-AKI in mice with RTEC-specific Grk2 overexpression. This study elucidates a novel mechanism of AKI induced by the combination of OPZ and MTX and identifies potential therapeutic targets. We provide an essential theoretical foundation for the rational clinical application of OPZ and MTX, as well as for prevention and treatment of the related kidney injury.

Histamine H3 receptor (H3R) and H4 receptor (H4R) are key members of the histamine receptor family, with H3R as a potential target for narcolepsy treatments and H4R as a candidate for next-generation antihistamines for inflammatory and allergic diseases. Although progress has been made in understanding the structure of histamine receptors, the detailed mechanisms of ligand recognition and receptor antagonism for H3R and H4R remain unclear. In this study, using cryo-electron microscopy, we present an inactive structure of H4R bound to a selective antagonist, adriforant, and two Gi-coupled structures of H3R and H4R in complex with histamine. Our structural and mutagenesis analyses provide insights into the selective binding of adriforant to H4R and the recognition of histamine across histamine receptors. Our findings also uncovered distinct antagonistic mechanisms for H3R and H4R and identified the role of aromatic amino acids on extracellular loop 2 in modulating the constitutive activity of H3R and H4R. These findings advance our knowledge of the functional modulation of histamine receptors, providing a foundation for the development of targeted therapeutics for neurological and immune-related disorders.

The global obesity epidemic and its associated metabolic disorders urgently require more effective therapeutic interventions, particularly multi-pathway targeting therapies. Cagrilintide (Cagri), functioning as a dual amylin receptor (AMYRs) and calcitonin receptor (CTR) agonist (DACRA), demonstrates significant efficacy in obesity treatment, although its structural activation mechanism remains unclear. This study elucidates the non-selective activation mechanism by determining cryo-EM structures of Cagri bound to AMY₁R-G_s and CTR-G_s complexes. Cagri adopts similar "bypass" binding modes in both receptors, which is distinct from other existing DACRAs that primarily achieve extended half-life through N-terminal lipid modification. Key molecular features include the F23^Cagri residue anchoring the peptide at the receptor transmembrane (TM) bundle level and the micelle, an E14-R17 intramolecular salt bridge enhancing helical stability, and C-terminal P37^Cagri interaction with the receptor ECD. These features collectively enable non-specific binding and activation across different receptors. Both structural and functional analyses revealed Cagri's non-selective activation of G_s signaling pathways through CTR and AMY₁R. These findings provide a comprehensive structural framework for developing next-generation anti-obesity drugs based on dual receptor activation mechanisms.