Molecular Pharmacology最新文献

The rise of drug-resistant influenza A virus (IAV) strains and severe lung injury caused by excessive inflammation highlight the need for new antivirals that combine antiviral and immunomodulatory effects. Adamantane, an established anti-IAV agent, targets the viral M2 matrix protein, but widespread mutations have limited its clinical utility. Previously, we identified that 2,5-dihydroxybenzylalcohol and its derivatives possess anti-IAV activity. In this study, we synthesized a series of adamantane-2,5-dihydroxy-benzylalcohol derivatives via reductive amination. Among these, Amt-1 demonstrated strong antiviral activity against both amantadine-sensitive (H1N1 PR8) and clinical isolates IAV strains (-log IC₅₀ = 6.12-6.51 M). Mechanistic analyses, including immunofluorescence, isothermal titration calorimetry, quantitative polymerase chain reaction, ELISA, luciferase assays, and western blotting, revealed that Amt-1 not only interacts with the peptide segment of M2 ion channel protein (-log Kd = 5.08 M) but also activates nuclear factor erythroid 2-related factor 2, leading to increased heme oxygenase-1 expression and reduced production of proinflammatory cytokines interleukin-6, interleukin-1β, and tumor necrosis factor-α. Additionally, Amt-1 inhibited IAV-induced nuclear factor kappa-B activation, thereby suppressing viral replication. In vivo, Amt-1 reduced lung viral titers and mitigated histopathological damage in mice, surpassing amantadine in controlling inflammation and increased survival to 33%. In summary, Amt-1 is the first adamantane derivative shown to exert anti-IAV effects by modulating nuclear factor erythroid 2-related factor 2/heme oxygenase-1-mediated anti-inflammatory pathways and nuclear factor kappa-B-driven antiviral responses, while retaining M2 ion channel inhibition. This host-directed mechanism addresses drug resistance and limits immunopathology, making Amt-1 a promising therapeutic candidate for resistant influenza infections. SIGNIFICANCE STATEMENT: Amt-1 exhibits a dual mechanism of action, including direct antiviral effects through residual M2 affinity and host-directed anti-inflammatory activity mediated by the nuclear factor erythroid 2-related factor 2/heme oxygenase-1 and nuclear factor kappa-B pathways. This balanced immunomodulatory and antiviral profile presents a distinct approach in adamantane-based drug development.

Transient receptor potential ankyrin 1 (TRPA1) is an ion channel known for its chemosensory function in neurons, causing pain and neurogenic inflammation. TRPA1 is activated by many noxious compounds including some inflammatory mediators. We and others have shown that TRPA1 is also expressed in epithelial cells, but its function in the epithelial barrier remains unclear. Here, we discovered in RNA-seq studies that inhibition of TRPA1 reduced the expression of a large number of antiviral and inflammatory genes under the influence of the key antiviral cytokine interferon beta in human A549 lung epithelial cells. In the gene ontology analysis, the terms most strongly affected by TRPA1 antagonists included many associated with antiviral defense such as "defense response to virus" and "antiviral innate immune response." To validate the RNA-seq results, selected antiviral genes such as myxovirus resistance protein 1 were further studied and found to be upregulated by TRPA1 by using reverse transcription quantitative polymerase chain reaction and western blotting, pharmacological TRPA1 inhibitors, TRPA1-targeting small interfering RNA, and ex vivo lung tissue cultures from TRPA1-deficient mice. Mechanistically, TRPA1 inhibitors partially reduced interferon beta-induced Ca²⁺ influx, phosphorylation of the transcription factor signal transducer and activator 1, and the interferon-sensitive response element-dependent transcription. These data suggest that TRPA1 mediates cellular signaling and biologically relevant changes in gene expression induced by type I interferons. The results offer TRPA1 as a novel treatment target for inflammatory conditions characterized by enhanced type I interferon activity such as hyperinflammatory states associated with viral infections and some autoimmune diseases, but TRPA1 inhibition may also influence interferon-induced antiviral immunity. SIGNIFICANCE STATEMENT: We found that the transient receptor potential ankyrin 1 channel can promote gene expression changes induced by type I interferons in human lung epithelial cells. Inhibiting transient receptor potential ankyrin 1 could decrease interferon-induced inflammation but could also influence the antiviral state.

Cyclic oligomeric depsipeptides represent a distinct structural class of naturally occurring compounds known for their wide-ranging biological activities. We previously reported that the unnatural form of verticilide (ent-verticilide) inhibits cardiac ryanodine receptor 2 (RyR2) and exhibits antiarrhythmic effects in mice, but its mechanism of action on the RyR2 channel is not known. Here, we collected single-channel recordings in artificial lipid bilayers to elucidate the mechanism of RyR2 modulation by ent-verticilide and its polar side chain analog activert. ent-Verticilide reduced RyR2 activity by increasing the RyR2 mean closed time without changing the RyR2 mean open time, suggesting that ent-verticilide functions as a closed-channel stabilizer. ent-Verticilide exhibited partial inhibition on RyR2 single channels with an IC₅₀ of ∼0.2 μM and a maximal inhibitory efficacy of ∼23%. To explore the effect of a charged residue on ent-verticilide-RyR2 binding, we introduced a terminal carboxylic acid on a single pentyl side chain. The resulting compound lost its inhibitory activity, increased RyR1 and RyR2 single-channel activity, and increased Ca spark frequency. Thus, we named this analog activert (1). Single-channel analysis showed that activert shortened mean closed time without changing mean open time, indicating closed-channel destabilization. Compared with ent-verticilide, activert was ∼100-fold less potent (EC₅₀ ∼ 30 μM) on RyR2 and had low membrane permeability. RyR2 activation was confirmed by [³H]-ryanodine binding and Ca spark assays. Although poor membrane permeability represents an obstacle for therapeutic development, activert serves as a proof-of-concept partial RyR2 activator and a promising scaffold for future structure-activity optimization. SIGNIFICANCE STATEMENT: This study reveals the dual modulatory potential of cyclooligomeric depsipeptides on ryanodine receptor 2, with ent-verticilide acting as a closed-channel stabilizer and its analog, activert, functioning as a closed-channel destabilizer. By leveraging nonnatural enantiomers and rational scaffold modifications, we highlight an underexplored approach to uncover important structure-activity relationships, advancing the development of novel ryanodine receptor 2-targeted therapeutics with potential applications in cardiac arrhythmia management.

The non-selective Ca²⁺ permeable transient receptor potential vanilloid 3 (TRPV3) ion channel is highly expressed in mouse keratinocytes, where its activation causes an accelerated cell migration and proliferation via an epidermal growth factor receptor-mediated mechanism in vitro. Therefore, TRPV3 has been proposed as a potential target to accelerate dermal wound healing. In this study, we provide an insight into the effects of TRPV3 activation on mouse keratinocytes and skin wound healing in vitro and in vivo using the newly identified activator of TRPV3 1 (AV3-1). To investigate a possible TRPV3-mediated effect on dermal wound closure in vivo, 2 circular wounds were excised on the back of wild-type and TRPV3 knockout mice and topically treated with AV3-1 or the corresponding vehicle. Unexpectedly, the comparison of neither the wound areas nor the histologic parameters yielded a statistically significant difference between wild-type and TRPV3 knockout wounds. Supporting this notion, AV3-1 treatment could not induce any further acceleration of the wound closure compared to vehicle-treated wounds. Therefore, the described TRPV3-mediated acceleration of keratinocyte migration and proliferation in vitro cannot be directly translated into an in vivo context. SIGNIFICANCE STATEMENT: We here show that deficiency of the transient receptor potential vanilloid 3 (TRPV3) channel impairs mouse keratinocyte migration in vitro. In vivo, however, neither TRPV3 deficiency nor TRPV3 activation by the novel activator of TRPV3 activator 1 (AV3-1) had statistically significant effects on healing rates or reepithelialization of skin wounds.

Non-small cell lung cancer remains the leading cause of cancer-related mortality worldwide. Cisplatin-based chemotherapy is a primary treatment strategy for non-small cell lung cancer, but acquired drug resistance limits its therapeutic efficacy. Emerging evidence suggests that microRNA-mediated competing endogenous RNA networks play a critical role in regulating anticancer drug resistance. In this study, we initially established a cisplatin sensitivity prediction model that enables the evaluation of cisplatin responsiveness based on patients' transcriptomic profiling. Integrative analysis of cisplatin sensitiveness and the transcriptomic data identified GDF15 and ZDHHC9 as essential drivers of cisplatin resistance. We further constructed the competing endogenous RNA regulatory network centered on these resistance-associated genes. Functional validation determined that hsa-miR-873-5p directly represses GDF15 expression, and its overexpression increased cellular sensitivity to cisplatin, as evidenced by a 35% reduction in half-maximal inhibitory concentration in PC9 cells and a 24% reduction in H226 cells. Our study provides a prediction model for cisplatin responsiveness evaluation and suggests the hsa-miR-873-5p/GDF15 axis as a promising therapeutic target for overcoming cisplatin resistance in non-small cell lung cancer. SIGNIFICANCE STATEMENT: This study develops a predictive model for cisplatin sensitivity in lung cancer and identifies key resistance genes via transcriptomic analysis. The study further reveals the critical hsa-miR-873-5p/GDF15 regulatory axis as a potential biomarker for personalized therapy in patients with non-small cell lung cancer.

Post-traumatic stress disorder is a highly prevalent and debilitating psychiatric condition characterized by symptoms such as depression, anxiety, and persistent fear. Although traumatic stress significantly affects both adolescents and adults, its behavioral and neurobiological impacts may vary substantially across developmental stages. In this study, inescapable foot shock (IFS), a well-established model of traumatic stress that recapitulates aspects of fear memory reactivation, was used to investigate the underlying molecular mechanisms associated with age-dependent responses. Our results demonstrated that adult mice exhibited greater susceptibility to defensive, depressive, and dysphoric behaviors than adolescent mice under identical IFS conditions, as evidenced by adult mice displaying approximately twice the freezing duration compared with adolescents in the conditioned fear test. Concomitant with these behavioral abnormalities, adult mice also showed more pronounced deficits in neuroplasticity and increased neuronal injury relative to their adolescent counterparts. Mechanistically, miR-34c-5p was up-regulated in the hippocampus of adult mice compared with adolescent mice under IFS exposure. Notably, phospholipase C β1, a predicted target of miR-34c-5p and a molecular mediator of neuroplasticity, was markedly upregulated upon miR-34c-5p knockdown, accompanied by activation of the phosphatase and tensin homolog/protein kinase B/cAMP-response element binding protein signaling pathway. Taken together, this study demonstrated that miR-34c-5p might serve as a risk susceptibility factor accompanied by age alterations in the traumatic stress mice model. By modulating the phospholipase C β1/phosphatase and tensin homolog/protein kinase B/cAMP-response element binding protein signaling cascade, miR-34c-5p contributes to the observed age-related differences in affective disorders after IFS exposure. Hence, miR-34c-5p may act as a potential therapeutic target for mitigating post-traumatic stress disorder susceptibility across different developmental stages. SIGNIFICANCE STATEMENT: This study provides evidence that age-related vulnerability discrepancies were markedly presented in mice under inescapable foot shock exposure. MiR-34c-5p might serve as a susceptibility factor with age alterations in traumatic stress mice models. These findings reveal mechanisms by which miR-34c-5p/phospholipase C β1 axis may regulate age-dependent susceptibility differences in mice exposed to inescapable foot shock.

The atypical chemokine receptor 3 (ACKR3) has emerged as a promising drug target for the treatment of cancer, cardiovascular, and autoimmune diseases. In this study, we present the pharmacological characterization of VUF16840, the first small-molecule inverse agonist of ACKR3. VUF16840 effectively displaces CXC chemokine ligand 12 binding to ACKR3 and inhibits chemokine-induced β-arrestin2 recruitment in a concentration-dependent manner. Furthermore, VUF16840 stabilizes the inactive conformation of ACKR3, as demonstrated by its ability to suppress constitutive recruitment of downstream effector proteins. This inverse agonism alters ACKR3 constitutive trafficking, leading to receptor enrichment at the plasma membrane and inhibition of intracellular CXC chemokine ligand 12 uptake. Importantly, VUF16840 exhibits high selectivity for ACKR3 over a broad panel of human chemokine receptors. These findings establish VUF16840 as a potent and selective ACKR3 inverse agonist capable of modulating constitutive and chemokine-induced signaling and internalization events. As such, VUF16840 represents a valuable pharmacological tool for exploring the molecular and translational roles of ACKR3 in both physiologic and pathologic contexts. SIGNIFICANCE STATEMENT: A small molecule inverse agonist of the atypical chemokine receptor 3 (ACKR3), named VUF16840, is characterized in this work. It was shown that VUF16840 was able to inhibit basal as well as ligand-induced ACKR3 activation and, moreover, inhibits the scavenging function of ACKR3.

Activation of most G protein-coupled receptors (GPCRs) initiates several branches of signaling. The consequences of some of these can be therapeutically beneficial, whereas others may cause unwanted side effects. Therefore, search for ligands biasing receptor signaling toward desired directions and away from undesired ones is increasingly popular. Currently, the field is focusing on G protein versus arrestin dichotomy. Arrestin binding to most GPCRs critically depends on receptor phosphorylation by GPCR kinases (GRKs). Existing data suggest that to bias signaling for or against arrestin-mediated branches one needs to enhance or reduce, respectively, the propensity of the receptor to recruit GRKs and become phosphorylated. As GRKs are gatekeepers for arrestin-mediated signaling, rational design of biased ligands should be based on the comparison of receptor complexes with G proteins with the structures of GPCRs bound to GRKs rather than arrestins. The same GPCR may engage distinct GRK subtypes differently, establishing an additional layer of bias. SIGNIFICANCE STATEMENT: Because arrestin binding to G protein-coupled receptors (GPCRs) requires prior receptor phosphorylation, GPCR-GPCR kinase complexes, not GPCR complexes with arrestins, should be compared to GPCR-G protein complexes to guide the design of G protein- and arrestin-biased ligands.

Metastasis remains a formidable challenge in the treatment of non-small cell lung cancer (NSCLC), necessitating novel therapeutic strategies targeting anchorage-independent growth (AIG). This study investigates the inhibitory effects of LZ-106, a quinolone analog and potential therapeutic candidate for NSCLC, on AIG and metastasis. Through a series of in vitro and in vivo experiments, LZ-106 is demonstrated to induce anoikis in NSCLC cells, inhibiting their AIG ability. Mechanistically, LZ-106 disrupts autophagic flux, leading to the accumulation of autophagosomes and the promotion of anoikis. Additionally, LZ-106 elevates reactive oxygen species (ROS) levels, further enhancing anoikis in NSCLC cells under AIG conditions. Notably, a positive feedback loop between ROS induction and autophagy flux blockage is identified, reinforcing LZ-106's inhibitory effects on AIG and metastasis. In vivo studies corroborate LZ-106's antimetastatic efficacy, highlighting its potential as a promising therapeutic agent for NSCLC. Together, these findings nominate LZ-106 as a promising antimetastatic candidate and underscore a targetable ROS-autophagy axis for therapeutic development in NSCLC. SIGNIFICANCE STATEMENT: LZ-106 inhibits anchorage-independent growth and metastasis of non-small cell lung cancer by triggering a positive feedback loop between reactive oxygen species overproduction and autophagic flux blockade, causing autophagosome accumulation and anoikis. In vitro and in vivo data nominate LZ-106 and the reactive oxygen species-autophagy axis as therapeutic targets.

Two human dopamine D2 receptor mutations cause dominant hyperkinetic movement disorders. The phenotype of carriers of the DRD2 variant c.634A>T, p.Ile212Phe (D2-I212F) is less severe than that of carriers of the variant c.1121 T>G, p.Met374Arg (D2-M^6.36R). Both are gain-of-function mutations with respect to G protein-mediated signaling; however, D2-M^6.36R exhibits greater gain-of-function than D2-I212F, suggesting that this is a major contributor to pathogenicity. Drd2^I212F knock-in mice exhibit altered D2 receptor function consistent with the clinical phenotype. We now report that mice carrying the mutation D2-M^6.36R, Drd2^M6.36R knock-in mice, exhibited gait abnormalities and a 2-fold increase in locomotor activity, and females fell more quickly from an accelerating rotarod. Electrically evoked inhibitory postsynaptic conductances in midbrain dopamine neurons from heterozygous Drd2^M6.36R mice were prolonged compared with both Drd2^I212F and wild-type (WT) mice. Electrically evoked release of acetylcholine and dopamine was similar in neostriatal slices from Drd2^M6.36R, Drd2^I212F, and WT mice. Inhibition of acetylcholine release by the D2 receptor agonist quinpirole was decreased by approximately 25% relative to WT mice in slices from either heterozygous knock-in mouse, whereas reversal by sulpiride was reduced by approximately 40% only in Drd2^M6.36R+/- mice. Although the postsynaptic current measured in dopamine cell bodies was dramatically prolonged in Drd2^M6.36R mice, quinpirole inhibition of dopamine release in the striatum was not altered. This could reflect differences between axonal and somatodendritic compartments or between responses proximal to and distal from the receptor. The results support a pathogenic role for these D2 receptor point mutations in mouse models of human hyperkinetic disorders. SIGNIFICANCE STATEMENT: Two dopamine receptor mutations cause movement disorders. Both are activating mutations; however, D2-M^6.36R is activated more than D2-I212F, paralleling the more severe phenotype of D2-M^6.36R in people. The phenotype of mice with D2-M^6.36R is also more severe. These mice will help develop drug therapy.