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.

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.

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 20mg·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.

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.

Nav1.5 is the main sodium channel subtype in the heart, playing a crucial role in maintaining regular cardiac electrical activity. It is a well-established therapeutic target for class I antiarrhythmic drugs used to treat both inherited and acquired arrhythmias. In this study, we report a highly effective (IC50 = 1.38 ± 0.28 μM) and novel Nav1.5 inhibitor, KH2, identified through an integrated drug discovery approach. Molecular dynamics (MD) simulations and experimental findings reveal that, unlike traditional class I antiarrhythmic drugs, KH2 shows a completely novel binding mechanism. Moreover, using electrophysiological mapping systems on rat isolated hearts, we found that KH2 significantly reduced cardiac conduction, highlighting its potential as a therapeutic agent for arrhythmias. Our finding of KH2 provided a valuable reference for designing drugs targeting Nav1.5 to treat arrhythmias.

The management of rheumatoid arthritis (RA) has advanced into the realm of targeted therapies; however, these therapies often lack tissue specificity and cause systemic adverse effects. Fibroblast-activating protein α (FAPα+) expressing fibroblast-like synoviocytes (FLSs) are critical pathogenic cell components in RA and are particularly abundant in inflamed joints, whereas they are minimal in other tissues. Consequently, FAPα+ FLSs are emerging as promising therapeutic targets for treating RA. However, strategies to specifically target FAPα+ FLSs in RA remain underdeveloped. To bridge this gap, we developed a novel compound, FAPI-Gly-Pro-MTX (FM), which integrates a FAPα+ tracer, FAPα inhibitor (FAPI), with the traditional drug methotrexate (MTX) via a glycine-proline dipeptide that can be cleaved by the dipeptidyl peptidase activity of FAPα. In an arthritis mouse model, FM selectively targeted FAPα+ FLSs in inflamed joints, facilitating the localized release of MTX and resulting in the significant alleviation of arthritis symptoms while minimizing systemic toxicity. Importantly, the presence of FAPI ensured that FM induced cell death specifically when FAPα+ FLSs were presented, thereby enhancing safety. Consequently, FM demonstrated considerable clinical potential as a safe and effective off-the-shelf therapeutic option for targeting FAPα+ FLSs in patients with RA. a FAPα+ FLSs are induced by various inflammatory cytokines in inflamed joints and aggravate inflammation and bone destruction; b FM selectively delivers MTX to FAPα+ FLSs in RA-inflamed joints and minimizes off-target effects; c Conventional MTX administration lacks cell specificity, leading to systemicadverse effects.

Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by the selective loss of nigral dopaminergic neurons and abnormal accumulation of α-synuclein. Our recent study has shown that α-synuclein induces cellular senescence prior to the loss of dopaminergic neurons and the onset of motor dysfunction. Microglia are known to contribute to dopaminergic neurodegeneration, primarily through NLRP3-mediated neuroinflammatory mechanism or by facilitating the propagation of α-synuclein. In this study, we identified the cell type susceptible to α-synuclein-induced cellular senescence in the substantia nigra and investigated the specific role of microglia with a particular focus on the NLRP3 inflammasome. PD mouse model was established by bilateral microinjection of viaAAV2/9 vectors encoding human α-syn-A53T into the SNpc to overexpress human mutant α-synuclein-A53T. We showed that overexpression of α-synuclein-A53T (α-syn-A53T) for 1 week not only induced a pro-inflammatory phenotype in nigral microglia but also led to the acquisition of a senescent state in a subset of microglial cells. Depletion of microglia by administration of the CSF1R inhibitor PLX5622 (1200 ppm) in diet for 1 week significantly attenuated α-synuclein aggregation, iron dysregulation and cellular senescence in the substantia nigra of PD mouse model. Transcriptomic and immunostaining analyses revealed that α-syn-A53T promoted senescence in nigral dopaminergic neurons via the SATB1/DNA damage/p21 signaling pathway, evidenced by reduced SATB1 expression along with increased levels of γ-H2A.X and p21 in TH-positive dopaminergic neurons within the substantia nigra. Moreover, genetic knockout of NLRP3 effectively mitigated α-syn-A53T-induced cellular senescence in these neurons by suppressing the SATB1/DNA damage/p21 signaling pathway. These results highlight the critical role of microglia in promoting dopaminergic neuronal senescence and suggest that NLRP3 may serve as a promising therapeutic target for early intervention in PD to mitigate neuronal senescence and subsequent neurodegeneration.

In the etiology of cancer, p62 is a well-known autophagic receptor and signaling adapter. High p62 expression is known to accelerate hepatocellular carcinoma (HCC) growth by activating various downstream signaling pathways. In this study, we investigated the activity of elevated p62 and its associated regulatory mechanisms during HCC progression. By conducting immunohistochemical staining on a human liver tissue microarray including 10 liver cancer tissues and 10 paracancerous tissues, we found that the expression levels of p62 and oncoprotein LAMTOR5 were markedly increased in HCC tissues compared with noncancerous tissues; LAMTOR5 was positively associated with p62 expression, and high LAMTOR5 or p62 expression predicted reduced overall and release-free survival. Transcriptomic analysis revealed that LAMTOR5 overexpression inhibited autophagy in HepG2 cells. We demonstrated that LAMTOR5 interacted with the LC3-interacting region domain of p62 and inhibited autophagy caused by the binding of p62 to LC3, thereby leading to the accumulation of p62 protein in HCC. Moreover, LAMTOR5 blocked p62 ubiquitination-mediated proteasome degradation, which increased the stability of p62. Functionally, p62 overexpression reversed LAMTOR5 deficiency-reduced hepatoma cell proliferation in vitro and in vivo. Lenvatinib, a multi-receptor tyrosine kinase inhibitor, significantly suppressed HCC growth in vitro and in vivo by downregulating LAMTOR5 and p62 expression. We conclude that LAMTOR5-mediated p62 stabilization is a novel HCC growth mechanism, targeting this axis as a promising therapeutic strategy.