Acta Pharmacologica Sinica最新文献

The high relapse rate of drugs is currently a therapeutic dilemma in the treatment of substance use disorder (SUD). Emerging evidence from preclinical animal models demonstrates that pretreatment with selective dopamine D₂ receptor (D₂R) antagonists prevents reinstatement of drug-seeking. However, the role of D₂R downstream signaling in regulating relapse behavior remains unclear. In this study, we investigated the roles of G_αi-protein- and β-arrestin-dependent D₂R signaling pathways in cocaine-primed reinstatement using cocaine self-administration (SA) mouse model treated with biased ligands. We found that treatment of D₂R G_αi-protein antagonists, but not β-arrestin antagonists, significantly attenuated cocaine-primed reinstatement of drug seeking without affecting locomotor activity or anxiety levels. Administration of D₂R G_αi-protein antagonists, but not β-arrestin antagonists, increased cyclic adenosine monophosphate (cAMP) levels in the nucleus accumbens (NAc). Furthermore, treatment of D₂R G_αi-protein antagonists, but not β-arrestin antagonists, suppressed cocaine-induced neuronal activation in the NAc. Our results demonstrate that G_αi-protein-dependent D₂R signaling plays a crucial role in cocaine-primed reinstatement and suggest that D₂R G_αi-protein-biased ligands may be promising pharmacotherapeutic targets for SUD treatment.

Cerebral edema is a severe complication following ischemic stroke. Recent studies have highlighted the crucial role of the glymphatic system (GS) in the clearance of water and macromolecules. GS dysfunction involving the disorders of AQP4 polarization may be crucial in the pathophysiology of cerebral edema. β-Hydroxybutyrate (BHB), the main component of the ketone body, has been shown to alleviate neurological deficits by restoring GS function in subarachnoid hemorrhage models and to reduce Aβ deposition in Alzheimer's disease models. In this study we investigated the effects of BHB on cerebral edema following ischemic stroke and its mechanisms. The mice were fed a ketogenic diet (KD) or a normal diet for 4 weeks before transient middle cerebral artery occlusion (MCAO). Alternatively, the mice received BHB (5 g·kg^-1·d^-1) or vehicle post-MCAO. By using brain section analysis, transcranial macroimaging, two-photon in vivo imaging and MRI, we demonstrated that both KD and BHB treatment significantly enhanced GS function under normal and MCAO conditions. BHB reduced cerebral edema and infarct volume post-MCAO. Notably, delayed BHB treatment initiated 10 h post-MCAO still improved GS function, but did not influence infarct volume. Furthermore, we revealed that BHB increased α1-syntrophin expression and H3K27ac levels in α1-syntrophin (Snta1) enhancer, restoring AQP4 polarization. In addition, BHB also reduced HDAC3 expression and elevated p300 expression. These results suggest that a KD and BHB treatment enhance GS function in mice and that BHB also mitigates brain edema after MCAO. The potentiation of GS function by BHB is likely mediated by the inhibition of HDAC3 activity and the increase in p300 activity, which upregulate α1-syntrophin expression and restore AQP4 polarization.

Targeted covalent inhibitors (TCIs) are emerging as a new modality in drug discovery because of their strong binding affinity and prolonged target engagement. However, the rational design of TCIs remains a significant challenge and is hindered by the lack of methods that accurately predict the structures of covalent protein-ligand complexes. Recent advances in co-folding approaches have made substantial strides in modeling complex biomolecular structures. Despite significant progress, their performance profiles for predicting the structures of covalent protein-ligand complexes remain largely unexplored because of the absence of rigorous benchmarks. Here, we introduce CoFD-Bench, a comprehensive benchmark dataset comprising 218 recently resolved covalent complexes designed to systematically evaluate both classical docking methods (AutoDock-GPU, CovDock, and GNINA) and deep learning co-folding models (AlphaFold3 (AF3), Chai-1, and Boltz-1x). Our results demonstrate that co-folding methods achieve superior ligand RMSD accuracy and protein-ligand interaction recovery. However, their performance markedly declines for novel pocket-ligand pairs. In contrast, classical docking methods exhibit stable but modest performance, which is primarily limited by target conformations. Furthermore, computational efficiency evaluations show that co-folding methods are slower than classical approaches, posing challenges for large-scale predictions. We also reveal that AF3 has the potential to identify native covalent residues through noncovalent co-folding, with a ligand RMSD comparable to that of covalent co-folding. These findings offer a possible route to explore covalent binding without prior specification of reactive residues, which are often unknown in real-world scenarios. Our study provides crucial insights and new opportunities for future co-folding-based TCI design, informing future model applications and improvements. CoFD-Bench offers rigorous evaluation criteria, diverse docking scenarios, and various methodological baselines, positioning it as an important benchmark for future model development and assessment.

Mitochondrial DNA (mtDNA) mutations are the most common cause in aberrant mitochondrion-leading cancer, exploration of direct targeting mutated mtDNA still remains incomplete. Secoemestrin C (Sec C) is epitetrathiodioxopiperazine derived from the endophytic fungus, which exhibited a rapid and prominent anti-breast cancer effect in triple-negative breast cancer (TNBC). In this study we investigated the anticancer mechanism of Sec C, especially its effect on TNBC cells. We showed that Sec C potently inhibited the viability of both TNBC (MDA-MB-231, HS578T, BT-549) and non-TNBC (MCF-7, T47D, SK-BR-3) cells in vitro with IC₅₀ values of 1-2 μM. In MDA-MB-231 cells, treatment with Sec C (2 μM) induced DNA breakage and subsequent apoptosis. Furthermore, treatment with Sec C (2 μM) caused mtDNA damage, mitochondrial ubiquitination and subsequent mitophagy in MDA-MB-231 and MCF-7 cells. RNA-seq analysis revealed that Sec C mitigated YAP level in time and dose-dependent manner either in MDA-MB-231 and MCF-7 cells. By re-analyzing the Sec C-responsive gene network proteins, we identified SLX4 as an oncogene promoting breast cancer development, potentially by stabilizing mtDNA to suppress pathologic mitochondrion mitophagy. Specifically, Sec C initiated MDA-MB-231 cells to yield ROS that induced SLX4 ubiquitination and degradation, leading to mtDNA damage and exacerbated mitophagy and promoted YAP degradation bypassing YAP-driven DNA repair pathways. This study not only demonstrates that Sec C is a rapid and prominent anti-breast cancer drug for TNBC, but also reveals SLX4 as a novel mtDNA stabilizer supporting breast cancer progression, positioning it as both a prognostic biomarker and therapeutic target.

The adult human heart is incapable of regeneration after myocardial infarction (MI) injury. One potential therapeutic strategy is to enhance the proliferation of resident cardiomyocytes (CMs). In this study, we developed a high-content screening assay based on DNA synthesis in human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) to identify small molecules that could promote CM proliferation. In the primary screening, we found that L-type calcium channel (LTCC) blockers induced DNA synthesis of hPSC-CMs. Among the 6 clinically approved calcium channel blockers tested in secondary screening and confirmatory experiments, nimodipine (NM) consistently enhanced CM proliferation both in vitro and in vivo. RNA-Seq analysis revealed that NM activated the canonical Wnt signaling pathway, while inhibiting Wnt signaling blunted the proliferative effect of NM. Lrp5, a co-receptor for Wnt ligands known to interact with LTCC, was found to mediate the effect of NM to promote nuclear localization of β-catenin and CM proliferation. In the MI mouse model established by ligating the left anterior descending coronary artery, administration of NM (10 mg/kg, i.p.) for 7 consecutive days significantly improved cardiac contractile function and enhanced resident CM proliferation, which was attenuated by co-treatment with Wnt inhibitor Wnt-C59 (10 mg/kg, i.p.). Our data suggest that L-type calcium channel blockers that induce CM proliferation may be potentially used in the treatment of MI and heart failure to promote cardiac regeneration.

Metabolic dysfunction-associated steatohepatitis (MASH), an inflammatory subtype of metabolic dysfunction-associated fatty liver disease (MAFLD), drives hepatic dysfunction and poses a significant health burden. Lipophagy dysfunction disrupts lipid droplet degradation and induces lysosomal damage, which is closely linked to MASH progression; thus, targeting lipophagy-lysosomal activation has emerged as a promising therapeutic strategy for the therapy of MASH. β-Sitosterol (β-SIT) derived from Polygonum hydropiper L. is structurally similar to cholesterol, and exhibits neuroprotective, antidiabetic and anti-obesity bioactivities. In this study, we explored the therapeutic potential of β-SIT for MASH. The mouse models of MASH were established by feeding a choline-deficient, L-amino acid-defined high-fat diet (CDAHFD) for 10 weeks, or high-fat diet (HFD) for 12 weeks. For in vitro experiments, AML-12 cells were treated with FFA mixture (OA:PA molar ratio = 2:1) to mimic lipid overload condition. MASH mice were administered β-SIT (10 or 20 mg·kg^-1 d^-1, i.g.) for 10 weeks. We showed that β-SIT treatment dose-dependently alleviated MASH by enhancing the lipophagy-lysosomal pathway in vivo and in vitro. In FFA-stimulated AML-12 cells, we demonstrated that β-SIT (20 μM) activated autophagic flux, promoted lysosomal biogenesis, and enhanced lysosome-lipid droplet interactions, as revealed by transmission electron microscopy, multi-SIM real-time fluorescence monitoring, and lipophagy-related marker detection. By integrated approaches including bioinformatics, molecular dynamics, CETSA and functional assays, we found that β-SIT inhibited mTOR pathway activation by directly targeting Ras-related C3 botulinum toxin substrate 1 (RAC1) in MASH mice. By conducting imaging/3D reconstruction, co-immunoprecipitation, immunofluorescence colocalization, lysosomal fractionation, and biochemical analyses in FFA-stimulated AML-12 cells, we confirmed that β-SIT modulated RAC1/mTOR interactions on lysosomes to restore lipophagy function. Critically, β-SIT promoted transcription factor EB (TFEB) nuclear translocation by modulating the RAC1-mTOR axis, thereby repairing lipophagy-lysosomal defects and attenuating MASH progression. Our results suggest that targeting the RAC1-mTOR-TFEB axis is a novel mechanism of β-SIT-driven lipophagy-lysosomal regulation, and highlight β-SIT as a potential candidate for the treatment of MASH.

Targeting the IGF1 system holds promise as a therapeutic approach for breast cancer. However, the intricate nature of IGF1 signaling and suboptimal drug combinations have resulted in limited clinical success. This study demonstrates that silencing p90 ribosomal S6 kinase 2 (RSK2), a downstream effector of the Ras/ERK pathway, inhibits IGF1 signaling by upregulating the expression and secretion of IGFBP5, a potent inhibitor of the IGF1-IGF1R axis that competes with IGF1 for binding. Mechanistically, GATA3 is identified as a novel transcription factor for IGFBP5, and RSK2 promotes GATA3 degradation by directly binding and phosphorylating it at serine 308, thus suppressing IGFBP5 transcription. Moreover, combined treatment with the RSK2 inhibitor LJH685 and the IGF1R inhibitor PPP significantly reduces metastasis of triple-negative breast cancer (TNBC) in both in vitro and in vivo models. These findings uncover new targets for synergistic antitumor therapy in TNBC and suggest that concurrent inhibition of IGF1R and RSK2 may offer an effective combinatorial treatment strategy.

Chronic itch is a debilitating symptom of atopic dermatitis (AD). Current therapeutic approaches for managing this condition include topical and systemic pharmacological agents with inconsistent efficacy and potential adverse effects. Sophocarpine (SPC) is a quinolizidine alkaloid derived from Sophora flavescens and has a wide range of bioactive activities, including anticancer, anti-inflammatory, antiviral, and analgesic effects. Recent research has shown that SPC exerts anti-inflammatory, anti-pruritic, and analgesic effects primarily through the inhibition of TRPA1 and TRPV1 channels. In this study, we investigated the antipruritic effects of SPC in an AD mouse model of chronic itch. AD-like chronic itch was induced in mice by topical application of MC903 solution (2 nmol in ethanol) on the shaved nape skin once daily for 14 consecutive days. Spontaneous scratching behaviors were recorded on D8, D10, D12, and D14. AD mice were administered SPC (1, 5, 10, and 20 mg·kg^-1·d^-1, i.p.) from D8 to D14. SPC (500 ng/10 μL) was also intrathecally injected once a day for 7 days. We showed that SPC treatment dose-dependently mitigated scratching behavior and suppressed spinal astrocyte reactivity in AD mice. Histological and imaging analyses revealed that SPC treatment reversed epidermal thickening and attenuated dermal vasodilation. In LPS-stimulated astrocytes in vitro, SPC (20, 80 μM) dose-dependently downregulated the mRNA levels of the proinflammatory factors Tnf, Cxcl1, Ccl2, Il1b, Il6, and Lcn2. In IP3R2 knockout mice, disruption of spinal astrocytic calcium signaling also reduced chronic itch, thereby supporting the involvement of astroglial pathways. Collectively, these results demonstrate that SPC effectively alleviates chronic itch in the AD mouse model by suppressing astrocyte reactivity, likely through modulation of neuroinflammatory and calcium signaling pathways, supporting its potential as a promising therapeutic candidate for the treatment of AD-associated chronic itch. Schematic summary of the main findings illustrating that SPC alleviates chronic itch in AD by inhibiting spinal astrocyte reactivity and pro-inflammatory signaling. Specifically, SPC suppresses the activation of spinal astrocytes in the dorsal horn, reduces the expression of pro-inflammatory mediators, and thereby decreases scratching behavior in AD mice.

Atypical antipsychotics often cause hyperglycemia, with clozapine showing the highest risk. Metformin is a first-line medication for managing diabetes or prediabetes and is often used to control clozapine-induced hyperglycemia. Clinical studies, however, have reported metformin resistance in some clozapine-treated patients, but the underlying mechanism remains unclear. In this study, we investigated the mechanism by which clozapine impaired the hypoglycemic effect of metformin and developed a mechanism-based semi-PBPK-PD model to predict the effect of clozapine on the pharmacokinetics and hypoglycemic effect of metformin in rats. Rats received clozapine (50 mg·kg^-1·d^-1, i.g.) for 7 days. The rats received metformin (200 mg/kg, i.g.) at 0.5 h after the last dose on D7. IPGTT or pharmacokinetics study was performed at 0.5 h after the administration of metformin. We showed that clozapine impaired the hypoglycemic effect of metformin during the glucose tolerance test without altering the plasma exposure of metformin in the rats. The liver is the main target for the hypoglycemic effect of metformin. We showed that clozapine significantly reduced the hepatic distribution of metformin, inhibited metformin uptake in rat livers and rat primary hepatocytes, and inhibited the glucose consumption enhanced by metformin in rat primary hepatocytes. OCT1 mediates the hepatic uptake of metformin. We demonstrated that clozapine dose-dependently inhibited metformin uptake in HEK293-OCT1 cells with the IC₅₀ value of 8.9 μM. Silencing OCT1 in rat primary hepatocytes impaired metformin uptake and attenuated the enhanced glucose consumption by metformin, suggesting that clozapine impaired the hypoglycemic effect of metformin by inhibiting OCT1-mediated hepatic uptake. Subsequently, a semi-PBPK-PD model was constructed based on this mechanism. The model well predicted the decreased hepatic exposure and hypoglycemic effect of metformin in the rat co-administered with clozapine.

The sigma-1 receptor is an important new therapeutic drug target for Alzheimer's disease (AD). Here, we reported that SOMCL-668, a novel selective and potent sigma-1 receptor allosteric modulator, is neuroprotective in AD both in vitro and in vivo. SOMCL-668 promoted PC12 cells against Aβ-induced intracellular reactive oxygen species (ROS) accumulation, mitochondrial membrane potential hyperpolarization and neuronal apoptosis. Similar results were obtained in SH-SY5Y and primary cortical culture neurons. The mechanistic study showed that SOMCL-668 stimulated the phosphorylation of ERK and CREB, while pharmacological inhibition or knockout of ERK via CRISPR-Cas9 attenuated its protective effects. Further studies with the sigma-1 receptor agonists/antagonists and knockout of sigma-1 receptor via CRISPR-Cas9 indicated that the sigma-1 receptor is essential for the effect of SOMCL-668. In 3xTg-AD mice, SOMCL-668 improved the learning and memory deficits, inhibited neuronal apoptosis and oxidative stress, reduced Aβ deposition and tau protein phosphorylation via ERK/CREB pathway. Moreover, pretreatment with sigma-1 receptor antagonist BD1047 blocked the effect of SOMCL-668. These results demonstrated that SOMCL-668 provides neuroprotection in AD and its effect is mediated by the sigma-1 receptor/ERK/CREB pathway. Our findings support that SOMCL-668 can be utilized as a potential drug for the prevention and treatment of Alzheimer's disease.