Molecular Pharmacology最新文献

Atypical chemokine receptor 3 (ACKR3), formerly referred to as CXCR7, is considered to be an interesting drug target. In this study, we report on the synthesis, pharmacological characterization and radiolabeling of VUF15485, a new ACKR3 small-molecule agonist, that will serve as an important new tool to study this β-arrestin-biased chemokine receptor. VUF15485 binds with nanomolar affinity (pIC₅₀ = 8.3) to human ACKR3, as measured in [¹²⁵I]CXCL12 competition binding experiments. Moreover, in a bioluminescence resonance energy transfer-based β-arrestin2 recruitment assay VUF15485 acts as a potent ACKR3 agonist (pEC₅₀ = 7.6) and shows a similar extent of receptor activation compared with CXCL12 when using a newly developed, fluorescence resonance energy transfer-based ACKR3 conformational sensor. Moreover, the ACKR3 agonist VUF15485, tested against a (atypical) chemokine receptor panel (agonist and antagonist mode), proves to be selective for ACKR3. VUF15485 labeled with tritium at one of its methoxy groups ([³H]VUF15485), binds ACKR3 saturably and with high affinity (K _d = 8.2 nM). Additionally, [³H]VUF15485 shows rapid binding kinetics and consequently a short residence time (<2 minutes) for binding to ACKR3. The selectivity of [³H]VUF15485 for ACKR3, was confirmed by binding studies, whereupon CXCR3, CXCR4, and ACKR3 small-molecule ligands were competed for binding against the radiolabeled agonist. Interestingly, the chemokine ligands CXCL11 and CXCL12 are not able to displace the binding of [³H]VUF15485 to ACKR3. The radiolabeled VUF15485 was subsequently used to evaluate its binding pocket. Site-directed mutagenesis and docking studies using a recently solved cryo-EM structure propose that VUF15485 binds in the major and the minor binding pocket of ACKR3. SIGNIFICANCE STATEMENT: The atypical chemokine receptor atypical chemokine receptor 3 (ACKR3) is considered an interesting drug target in relation to cancer and multiple sclerosis. The study reports on new chemical biology tools for ACKR3, i.e., a new agonist that can also be radiolabeled and a new ACKR3 conformational sensor, that both can be used to directly study the interaction of ACKR3 ligands with the G protein-coupled receptor.

The signal transduction protein, regulator of G protein signaling 4 (RGS4), plays a prominent role in physiologic and pharmacological responses by controlling multiple intracellular pathways. Our earlier work identified the dynamic but distinct roles of RGS4 in the efficacy of monoamine-targeting versus fast-acting antidepressants. Using a modified chronic variable stress (CVS) paradigm in mice, we demonstrate that stress-induced behavioral abnormalities are associated with the downregulation of RGS4 in the medial prefrontal cortex (mPFC). Knockout of RGS4 (RGS4KO) increases susceptibility to CVS, as mutant mice develop behavioral abnormalities as early as 2 weeks after CVS resting-state functional magnetic resonance imaging I (rs-fMRI) experiments indicate that stress susceptibility in RGS4KO mice is associated with changes in connectivity between the mediodorsal thalamus (MD-THL) and the mPFC. Notably, RGS4KO also paradoxically enhances the antidepressant efficacy of ketamine in the CVS paradigm. RNA-sequencing analysis of naive and CVS samples obtained from mPFC reveals that RGS4KO triggers unique gene expression signatures and affects several intracellular pathways associated with human major depressive disorder. Our analysis suggests that ketamine treatment in the RGS4KO group triggers changes in pathways implicated in synaptic activity and responses to stress, including pathways associated with axonal guidance and myelination. Overall, we show that reducing RGS4 activity triggers unique gene expression adaptations that contribute to chronic stress disorders and that RGS4 is a negative modulator of ketamine actions. SIGNIFICANCE STATEMENT: Chronic stress promotes robust maladaptation in the brain, but the exact intracellular pathways contributing to stress vulnerability and mood disorders have not been thoroughly investigated. In this study, the authors used murine models of chronic stress and multiple methodologies to demonstrate the critical role of the signal transduction modulator regulator of G protein signaling 4 in the medial prefrontal cortex in vulnerability to chronic stress and the efficacy of the fast-acting antidepressant ketamine.

Prodigiosin (PG) is a naturally occurring polypyrrole red pigment produced by numerous microorganisms including some Serratia and Streptomyces strains. PG has exhibited promising anticancer activity; however, the molecular mechanisms of action of PG on malignant cells remain ambiguous. Transforming growth factor-β (TGF-β) is a multifunctional cytokine that governs a wide array of cellular processes in development and tissue homeostasis. Malfunctions of TGF-β signaling are associated with numerous human cancers. Emerging evidence underscores the significance of internalized TGF-β receptors and their intracellular trafficking in initiating signaling cascades. In this study, we identified PG as a potent inhibitor of the TGF-β pathway. PG blocked TGF-β signaling by targeting multiple sites of this pathway, including facilitating the sequestering of TGF-β receptors in the cytoplasm by impeding the recycling of type II TGF-β receptors to the cell surface. Additionally, PG prompts a reduction in the abundance of receptors on the cell surface through the disruption of the receptor glycosylation. In human Caucasian lung carcinoma cells and human hepatocellular cancer cell line cells, nanomolar concentrations of PG substantially diminish TGF-β-triggered phosphorylation of Smad2 protein. This attenuation is further reflected in the suppression of downstream target gene expression, including those encoding fibronectin, plasminogen activator inhibitor-1, and N-cadherin. SIGNIFICANCE STATEMENT: Prodigiosin (PG) emerges from this study as a potent TGF-β pathway inhibitor, disrupting receptor trafficking and glycosylation and reducing TGF-β signaling and downstream gene expression. These findings not only shed light on PG's potential therapeutic role but also present a captivating avenue towards future anti-TGF-β strategies.

Purpose: Long noncoding RNAs (lncRNAs) have been confirmed related to the occurrence and progress of multiple cancers, including cervical cancer nasopharyngeal carcinoma (NPC). This study focused on assessing GUSBP11 effects on NPC progression and exploring possible mechanisms. Materials and Methods: RT-qPCR was conducted for assessing GUSBP11 levels within NPC tissues and cells. CCK-8, colony formation, and Transwell were adopted for examining GUSBP11 impacts on NPC cell proliferation and cell metastasis. RT-qPCR analysis and dual-luciferase reporter assay were conducted for judging the expression interrelation of GUSBP11 and its potential target miR-1226-3p. The same methods were carried out for verifying the inhibiting influences of miR-1226-3p upregulation and its potential target TM9SF4. Results: GUSBP11 levels were upregulated within NPC tissues and cells. GUSBP11 downregulation repressed NPC cell proliferation and cell metastasis. In addition, GUSBP11 targeted and negatively regulated miR-1226-3p. Furthermore, miR-1226-3p targeted TM9SF4 and mediated GUSBP11's impacts on TM9SF4 levels. At last, the authors proved the critical role of the GUSBP11/miR-1226-3p/TM9SF4 axis in regulating NPC progression. Conclusion: These findings indicate that downregulation of GUSBP11 alleviates NPC development by regulating the miR-1226-3p/TM9SF4 axis.

Multiple sclerosis is an inflammatory and degenerative disease characterized by different clinical courses including relapsing multiple sclerosis (RMS) and primary progressive multiple sclerosis (PPMS). A hallmark of patients with multiple sclerosis (pwMS) includes a putative autoimmune response, which results in demyelination and neuroaxonal damage in the central nervous system. Sphingolipids in cerebrospinal fluid (CSF) have been proposed as potential biomarkers reflective of disease activity in pwMS. Hence, sensitive methods to accurately quantify sphingolipids in CSF are needed. In this study, we report the development of a sensitive high-throughput multiplexed liquid chromatography coupled to a tandem mass spectrometry method to perform quantitation on 14 species of sphingolipids in human CSF. We applied this method to measure CSF sphingolipids in healthy controls (n = 10), PPMS (n = 27), and RMS (n = 17) patients before and after ocrelizumab treatment. The median CSF levels of the 14 sphingolipids measured herein was higher in PPMS (17.2 ng/mL) and RMS (17.6 ng/mL) when compared with the healthy controls (13.8 ng/mL). Levels of sphingolipids were decreased by 8.6% at week 52 after treatment with ocrelizumab in RMS patients but not in PPMS patients. Specifically, C16 glucosylceramide (-26%; P = 0.004) and C18 ceramides (-13%; P = 0.042) decreased from baseline in RMS patients. Additionally, in PPMS patients C16 glucosylceramide levels correlated with CSF neurofilament heavy levels at baseline (Rho =0.532; P = 0.004) and after treatment (Rho =0.424; P = 0.028). Collectively, these results indicate that CSF sphingolipid levels are altered in pwMS and treatment with ocrelizumab results in significant shifts in the sphingolipid profile that may reflect a reduction in disease activity supporting further investigation into sphingolipids as tools to monitor disease state. SIGNIFICANCE STATEMENT: This study describes the development of a new method to measure 14 sphingolipid species in CSF. These results demonstrate that sphingolipids levels are elevated in CSF from pwMS compared to healthy controls. Distinct sphingolipid signatures were observed between patients with different clinical disease courses, and these lipid signatures changed after treatment with ocrelizumab, especially in RMS patients. This method enables further investigation into the role of sphingolipids as candidate biomarkers in pwMS and other central nervous system disorders.

The circadian clock is an endogenous biochemical timing system that coordinates the physiology and behavior of organisms to earth's ∼24-hour circadian day/night cycle. The central circadian clock synchronized by environmental cues hierarchically entrains peripheral clocks throughout the body. The circadian system modulates a wide variety of metabolic signaling pathways to maintain whole-body metabolic homeostasis in mammals under changing environmental conditions. Endocrine fibroblast growth factors (FGFs), namely FGF15/19, FGF21, and FGF23, play an important role in regulating systemic metabolism of bile acids, lipids, glucose, proteins, and minerals. Recent evidence indicates that endocrine FGFs function as nutrient sensors that mediate multifactorial interactions between peripheral clocks and energy homeostasis by regulating the expression of metabolic enzymes and hormones. Circadian disruption induced by environmental stressors or genetic ablation is associated with metabolic dysfunction and diurnal disturbances in FGF signaling pathways that contribute to the pathogenesis of metabolic diseases. Time-restricted feeding strengthens the circadian pattern of metabolic signals to improve metabolic health and prevent against metabolic diseases. Chronotherapy, the strategic timing of medication administration to maximize beneficial effects and minimize toxic effects, can provide novel insights into linking biologic rhythms to drug metabolism and toxicity within the therapeutical regimens of diseases. Here we review the circadian regulation of endocrine FGF signaling in whole-body metabolism and the potential effect of circadian dysfunction on the pathogenesis and development of metabolic diseases. We also discuss the potential of chrononutrition and chronotherapy for informing the development of timing interventions with endocrine FGFs to optimize whole-body metabolism in humans. SIGNIFICANCE STATEMENT: The circadian timing system governs physiological, metabolic, and behavioral functions in living organisms. The endocrine fibroblast growth factor (FGF) family (FGF15/19, FGF21, and FGF23) plays an important role in regulating energy and mineral metabolism. Endocrine FGFs function as nutrient sensors that mediate multifactorial interactions between circadian clocks and metabolic homeostasis. Chronic disruption of circadian rhythms increases the risk of metabolic diseases. Chronological interventions such as chrononutrition and chronotherapy provide insights into linking biological rhythms to disease prevention and treatment.

Opioid analgesics are frequently associated with gastrointestinal side effects, including constipation, nausea, dysphagia, and reduced gastric motility. Though it has been shown that stimulation of opioid receptors expressed in enteric motor neurons contributes to opioid-induced constipation, it remains unclear whether activation of opioid receptors in gastric-projecting nodose ganglia neurons contributes to the reduction in gastric motility and emptying associated with opioid use. In the present study, whole-cell patch-clamp recordings were performed to determine the mechanism underlying opioid receptor-mediated modulation of Ca²⁺ currents in acutely isolated gastric vagal afferent neurons. Our results demonstrate that Ca_V2.2 channels provide the majority (71% ± 16%) of Ca²⁺ currents in gastric vagal afferent neurons. Furthermore, we found that application of oxycodone, U-50488, or deltorphin II on gastric nodose ganglia neurons inhibited Ca²⁺ currents through a voltage-dependent mechanism by coupling to the Gα _i/o family of heterotrimeric G-proteins. Because previous studies have demonstrated that the nodose ganglia expresses low levels of δ-opioid receptors, we also determined the deltorphin II concentration-response relationship and assessed deltorphin-mediated Ca²⁺ current inhibition following exposure to the δ-opioid receptor antagonist ICI 174,864 (0.3 µM). The peak mean Ca²⁺ current inhibition following deltorphin II application was 47% ± 24% (EC₅₀ = 302.6 nM), and exposure to ICI 174,864 blocked deltorphin II-mediated Ca²⁺ current inhibition (4% ± 4% versus 37% ± 20%). Together, our results suggest that analgesics targeting any opioid receptor subtype can modulate gastric vagal circuits. SIGNIFICANCE STATEMENT: This study demonstrated that in gastric nodose ganglia neurons, agonists targeting all three classical opioid receptor subtypes (μ, δ, and κ) inhibit voltage-gated Ca²⁺ channels in a voltage-dependent mechanism by coupling to Gα_i/o. These findings suggest that analgesics targeting any opioid receptor subtype would modulate gastric vagal circuits responsible for regulating gastric reflexes.

This study describes the localization and computational prediction of a binding site for the A₃ adenosine receptor (A₃AR) positive allosteric modulator 2-cyclohexyl-1H-imidazo[4,5-c]quinolin-4-(3,4-dichlorophenyl)amine (LUF6000). The work reveals an extrahelical lipid-facing binding pocket disparate from the orthosteric binding site that encompasses transmembrane domain (TMD) 1, TMD7, and Helix (H) 8, which was predicted by molecular modeling and validated by mutagenesis. According to the model, the nearly planar 1H-imidazo[4,5-c]quinolinamine ring system lies parallel to the transmembrane segments, inserted into an aromatic cage formed by π-π stacking interactions with the side chains of Y284^7.55 in TMD7 and Y293^8.54 in H8 and by π-NH bonding between Y284^7.55 and the exocyclic amine. The 2-cyclohexyl group is positioned "upward" within a small hydrophobic subpocket created by residues in TMDs 1 and 7, while the 3,4-dichlorophenyl group extends toward the lipid interface. An H-bond between the N-1 amine of the heterocycle and the carbonyl of G29^1.49 further stabilizes the interaction. Molecular dynamics simulations predicted two metastable intermediates, one resembling a pose determined by molecular docking and a second involving transient interactions with Y293^8.54; in simulations, each of these intermediates converges into the final bound state. Structure-activity-relationships for replacement of either of the identified exocyclic or endocyclic amines with heteroatoms lacking H-bond donating ability were consistent with the hypothetical pose. Thus, we characterized an allosteric pocket for 1H-imidazo[4,5-c]quinolin-4-amines that is consistent with data generated by orthogonal methods, which will aid in the rational design of improved A₃AR positive allosteric modulators. SIGNIFICANCE STATEMENT: Orthosteric A₃AR agonists have advanced in clinical trials for inflammatory conditions, liver diseases, and cancer. Thus, the clinical appeal of selective receptor activation could extend to allosteric enhancers, which would induce site- and time-specific activation in the affected tissue. By identifying the allosteric site for known positive allosteric modulators, structure-based drug discovery modalities can be enabled to enhance the pharmacological properties of the 1H-imidazo[4,5-c]quinolin-4-amine class of A₃AR positive allosteric modulators.