Search Results (62)

Breast cancer is the most prevalent cancer in women worldwide and presents a major therapeutic challenge, particularly triple-negative breast cancer (TNBC), a subtype characterized by an aggressive clinical course but heightened sensitivity to chemotherapy. Natural products, such as triterpene glycosides derived from sea cucumbers, have emerged as promising candidates with high anticancer potential against TNBC. This study investigated the pathways of anticancer action of cucumarioside A₀-1 (Cuc A₀-1) and djakonovioside A (Dj A), isolated from the sea cucumber Cucumaria djakonovi, triggered and regulated in MDA-MB-231 cells (triple-negative breast cancer cell line). We employed functional assays (cAMP level, Ca²⁺ influx, control of cell proliferation and colony formation), Western blotting for mitogen-activated protein kinase MAPK) signaling, and in silico molecular docking. A_2B adenosine receptor (A_2BAR) was identified as a novel target for both glycosides. As antagonists, they reduced cAMP production and inhibited NECA (5-(N-ethylcarboxamido)adenosine)-induced Ca²⁺ influx. This A_2BAR blockade suppressed the MAPK pathway, profoundly inhibiting phospho-ERK1/2, p38, and JNK1/2, which led to the activation of the intrinsic apoptotic pathway and strong inhibition of cell proliferation and colony formation. Surprisingly, co-treatment with the NECA agonist enhanced the antiproliferative effects of the glycosides. It was supposed that the interaction of glycosides with the NECA-preactivated receptor may bias signaling toward the Gi and Gq/PLC/ERK1/2 pathways, underscoring the central role of the MAPK pathway in controlling cell growth. Molecular docking confirmed binding to the A_2BAR orthosteric site, revealing that Cuc A₀-1 and Dj A employ distinct interaction modes. To our knowledge, this is the first report to define A_2BAR as a target for sea cucumber glycosides. Their potent antitumor effects, mediated through the antagonism of A_2BAR and subsequent MAPK pathway inhibition, position them as promising lead compounds for cancer types with high expression A_2BAR. Full article

CB₁ agonist compounds may be potential drug candidates for the treatment of gliomas, as they have been shown to inhibit tumor cell proliferation, induce apoptosis, and reduce angiogenesis in various preclinical models. Their ability to modulate the endocannabinoid system suggests a promising therapeutic approach for targeting glioma growth and progression. Herein, we report the design, synthesis, biological studies, and bioinformatics assays of novel benzo[d]imidazole–naphthalen-arylmethanone regioisomers with affinity for the CB₁ receptor, as well as propose an indirect methodology to evaluate their presumed CB₁ agonist activity. Compounds that showed a propensity for binding to the CB₁ receptor were regioisomers 4d, 5b, 5e, 5f, and 5f′. Likewise, derivatives that displaced more than 50% of the radioligand [³H]CP-55940 at the CB₁ receptor were subjected to in vitro viability experiments. Compounds 4d, 5b, 5e, and 5f′ showed toxicity against U87MG cells (malignant glioma) in a considerable percentage. Notably, compound 5f′ showed CB₁ affinity, with a K_i of 2.12 µM, and was selectively toxic to U87MG cells, which highly express the CB₁ receptor, while exhibiting no toxicity toward the healthy HEK293 cell line, which expresses both cannabinoid receptors at negligible levels. Docking studies at the CB₁ orthosteric site indicate that 5f′ forms π-π interactions, a T-shaped interaction, and hydrogen bonding through the oxygen atom of the furan ring. Biologically, our experimental indirect model-based on a simple viability assay is supported by well-established evidence that activation of CB₁ and CB₂ receptors by agonists induces cell death and inhibits tumor cell growth. Structurally, we conclude that the presence of a furan ring at the 2-position of the benzo[d]imidazole core is beneficial for the development of new ligands with potential CB₁ agonist activity. Full article

TGR5 has emerged as a promising therapeutic target for obesity and metabolic disorders due to its regulatory roles in energy expenditure, glucose homeostasis, thermogenesis, and gut hormone secretion. This review summarizes the structural mechanisms of TGR5 activation, focusing on orthosteric and allosteric ligand interactions, toggle switch dynamics, and G protein coupling based on cryo-EM and docking-based models. A wide range of bioactive natural compounds including oleanolic acid, curcumin, betulinic acid, ursolic acid, quinovic acid, obacunone, nomilin, and 5β-scymnol are examined for their ability to modulate TGR5 signaling and elicit favorable metabolic effects. Molecular docking simulations using CB-Dock2 and PDB ID 7BW0 revealed key interactions within the orthosteric pocket, supporting their mechanistic potential as TGR5 agonists. Emerging strategies in TGR5-directed drug development are also discussed, including gut-restricted agonism to minimize gallbladder-related side effects, biased and allosteric modulation to fine-tune signaling specificity, and AI-guided optimization of natural product scaffolds. These integrated insights provide a structural and pharmacological framework for the rational design of safe and effective TGR5-targeted therapeutics. Full article

Activated protein C (APC) exerts anticoagulant and cytoprotective cell signaling activities. APC’s cell signaling requires protease-activated receptor (PAR) PAR1 and PAR3, and APC’s PAR cleavages generate peptides capable of agonizing biased G-protein coupled receptor (GPCR) cytoprotective signaling, resulting in anti-inflammatory and anti-apoptotic activities and endothelial barrier stabilization. The PAR-sequence-derived 34-residue “G10 peptide” comprising PAR1 residues 47–55 covalently attached by a 10-glycine linker to PAR3 residues 51–65 is an orthosteric/allosteric bivalent GPCR agonist that potently mimics APC’s anti-inflammatory activity and endothelial barrier stabilization activity. The objective of this study was to determine whether the G10 peptide mimics APC’s anti-apoptotic activity using cultured murine neurons challenged by N-methyl-d-aspartate that provokes neuronal apoptosis. In these new studies, the G10 peptide mimicked APC’s anti-apoptotic activity. Thus, the PAR-derived 34-residue G10 peptide mimics APC’s three major cytoprotective activities, namely anti-inflammatory and anti-apoptotic activities and endothelial barrier stabilization. Peptides that agonize GPCRs provide promising and currently approved drugs; e.g., semaglutide and tirzepatide that contain 31 and 39 amino acid residues, respectively. Thus, this new study adds to the rationale for pursuing further studies of the G10 peptide for potential therapeutic value for multiple pathologies where APC or signaling-selective APC variants are therapeutic in preclinical animal studies. Full article

Ionotropic glutamate receptors—including N-methyl-d-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and kainate receptors—play a pivotal role in excitatory signaling in the central nervous system (CNS), which is particularly important for learning and memory processes. Among them, AMPA and kainate receptors (known as ‘non-NMDA’ receptors) have gained increasing attention as therapeutic targets for various CNS disorders. Positive allosteric modulators (PAMs) of these receptors enhance their activity without directly activating them, offering a promising strategy to fine-tune glutamatergic signaling with potentially fewer side effects compared to orthosteric agonists. This review presents a comprehensive overview of recent advances in the development of AMPA and kainate receptor PAMs. We classify the most relevant modulators into main chemotype groups and discuss their binding modes, structure–activity relationships, and efficacy as determined through in vitro and in vivo studies. Additionally, we provide an overview of AMPA receptor PAMs that have entered into clinical trials over the past few decades. The increasing interest in kainate receptor PAMs is also mentioned, underlining their emerging role in future neuropharmacological strategies. Full article

The development of orally bioavailable non-peptidomimetic glucagon-like peptide-1 receptor agonists (GLP-1RAs) offers a promising therapeutic avenue for the treatment of type 2 diabetes mellitus (T2DM) and obesity. An extensive in silico approach combining structure-based drug design and ligand-based strategies together with pharmacokinetic properties and drug-likeness predictions is implemented to identify novel non-peptidic GLP-1RAs from the COCONUT and Marine Natural Products (CMNPD) libraries. More than 700,000 compounds were screened by shape-based similarity filtering in combination with precision docking against the orthosteric site of the GLP-1 receptor (PDB ID: 6X1A). The docked candidates were further assessed with the molecular mechanics MM-GBSA tool to check the binding affinities; the final list of candidates was validated by running a 500 ns long MD simulation. Twenty final hits were identified, ten from each database. The hits contained compounds with reported antidiabetic effects but with no evidence of GLP-1 agonist activity, including hits 1, 6, 7, and 10. These findings proposed a novel mechanism for these hits through GLP-1 activity and positioned the other hits as potential promising scaffolds. Among the studied compounds—especially hits 1, 5, and 9—possessed strong and stable interactions with critical amino acid residues such as TRP-203, PHE-381, and GLN-221 at the active site of the 6X1A-substrate along with favorable pharmacokinetic profiles. Moreover, the RMSF and RMSD plots further suggested the possibility of stable interactions. Specifically, hit 9 possessed the best docking score with a ΔG_bind value of −102.78 kcal/mol, surpassing even the control compound in binding affinity. The ADMET profiling also showed desirable drug-likeness and pharmacokinetic characteristics for hit 9. The pipeline of computational integration underscores the potential of non-peptidic alternatives in natural product libraries to pursue GLP-1-mediated metabolic therapy into advanced preclinical validation. Full article

Small-molecule glucagon-like peptide-1 receptor agonists (GLP-1RAs) represent an innovative advancement in oral therapeutics, addressing key limitations associated with injectable peptide-based incretin therapies. These nonpeptidic agents exert their actions primarily through non-canonical binding orthosteric sites within the GLP-1 receptor transmembrane domain, enabling selective G protein (Gs)-biased signaling with reduced β-arrestin-mediated adverse effects. Orforglipron has notably advanced through Phase 3 clinical development, demonstrating significant reductions in hemoglobin A1c and body weight (up to 7.9%) with favorable tolerability. Conversely, promising candidates such as danuglipron and lotiglipron were discontinued due to hepatotoxicity, underscoring critical safety concerns intrinsic to small-molecule GLP-1RA development. Current clinical candidates, including GSBR-1290, CT-996, and ECC5004, continue to offer substantial potential due to their oral bioavailability, simplified dosing regimens, and favorable gastrointestinal tolerability. Nevertheless, challenges persist regarding hepatic safety, pharmacodynamic variability, and limited long-term outcome data. This review integrates current structural, pharmacological, and clinical evidence, highlights key mechanistic innovations—including biased agonism, covalent binding strategies, and allosteric modulation—and discusses future directions for this rapidly evolving therapeutic class in metabolic disease management. Full article

The adenosine A1 receptor (A₁R) is a promising target for pain treatment. However, the development of therapeutic agonists is hampered by adverse effects, mainly including sedation, bradycardia, hypotension, or respiratory depression. Recently discovered molecules able to overcome this impediment are the positive allosteric modulator MIPS521 and the A1R-selective agonist BnOCPA, which are both potent and powerful analgesics with fewer side effects. While BnOCPA directly activates the A₁R from the canonical orthosteric site, MIPS521 binds to an allosteric site, acting in concert with orthosteric adenosine and tuning its pharmacology. Given their overlapping profile in pain models but distinct mechanisms of action, we combined pharmacology and microsecond molecular dynamics simulations to address MIPS521 and BnOCPA activity and their reciprocal influence when bound to the A1R. We show that MIPS521 changes adenosine and BnOCPA G protein selectivity in opposite ways and propose a structural model where TM7 dynamics are differently affected and involved in the G protein preferences of adenosine and BnOCPA. Full article

Human endocannabinoid signaling is primarily mediated by the cannabinoid receptors, CB1 and CB2, which are G protein-coupled receptors (GPCRs). These receptors have been linked to a variety of physiological processes and are being pursued as prospective drug targets due to their potential in treating pain and inflammation. However, because of their homology and shared signaling mechanisms, investigating the individual physiological roles of these receptors and designing subtype-selective ligands has been challenging. Using active-state CB1 and CB2 structures as guides, homologous residues within the orthosteric pocket of each receptor were mutated to alanine to test whether they equally impair CB1 and CB2 activity in response to two high-affinity, nonselective agonists (CP55,940 and AM12033). Interestingly, mutating the Y5.39 position impairs CB1 but not CB2 function. Conversely, mutating residue C6.47 improves CB1 but impairs CB2 signaling. The F7.35A mutation leads to a decrease in CP55,940 potency at CB1 and impairs internalization; however, AM12033 gains potency and promotes CB1 internalization. In CB2, mutation of F7.35A decreases the potency of CP55,940 and neither agonist induces internalization. These observations provide some insight into functional sensitivity of CB1 and CB2 to different agonists when conserved residues are mutated in the orthosteric pocket. Full article

Despite the availability of different treatments for type 2 diabetes (T2D), post-diagnosis complications remain prevalent; therefore, more effective treatments are desired. Glucagon-like peptide (GLP)-1-based drugs are currently used for T2D treatment. They act as orthosteric agonists for the GLP-1 receptor (GLP-1R). In this study, we analyzed in vitro how the GLP-1R orthosteric and allosteric agonists augment glucose-stimulated insulin secretion (GSIS) and intracellular cAMP production (GSICP) in INS-1E pancreatic beta cells under healthy, diabetic, and recovered states. The findings from this study suggest that allosteric agonists have a longer duration of action than orthosteric agonists. They also suggest that the GLP-1R agonists do not deplete intracellular insulin, indicating they can be a sustainable and safe treatment option for T2D. Importantly, this study demonstrates that the GLP-1R agonists variably augment GSIS through GSICP in healthy, diabetic, and recovered INS-1E cells. Furthermore, we find that INS-1E cells respond differentially to the GLP-1R agonists depending on both glucose concentration during and before treatment and/or whether the cells have been previously exposed to these drugs. In conclusion, the findings described in this manuscript will be useful in determining in vitro how pancreatic beta cells respond to T2D drug treatments in healthy, diabetic, and recovered states. Full article

Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter regulating numerous physiological functions, and its dysregulation is a crucial component of the pathological processes of schizophrenia, depression, migraines, and obesity. 5-HT interacts with 14 different receptors, of which 5-HT_1A-1FRs, 5-HT_2A-CRs, and 5-HT_4-7Rs are G protein-coupled receptors (GPCRs), while 5-HT₃R is a ligand-gated ion channel. Over the years, selective orthosteric ligands have been identified for almost all serotonin receptors, yielding several clinically relevant drugs. However, the high degree of homology between 5-HTRs and other GPCRs means that orthosteric ligands can have severe side effects. Thus, there has recently been increased interest in developing safer ligands of GPCRs, which bind to less conserved, more specific sites, distinct from that of the receptor’s natural ligand. The present review describes the identification of allosteric ligands of serotonin receptors, which are largely natural compounds (oleamide, cannabidiol, THC, and aporphine alkaloids), complemented by synthetic modulators developed in large part for the 5-HT_2C receptor. The latter are positive allosteric modulators sought after for their potential as drugs preferable over the orthosteric agonists as antiobesity agents for their potentially safer profile. When available, details on the interactions between the ligand and allosteric binding site will be provided. An outlook on future research in the field will also be provided. Full article

Understanding the role of biased G protein-coupled receptor (GPCR) agonism in receptor signaling may provide novel insights into the opposing effects mediated by cannabinoids, particularly in cancer and cancer metastasis. GPCRs can have more than one active state, a phenomenon called either ‘biased agonism’, ‘functional selectivity’, or ‘ligand-directed signaling’. However, there are increasing arrays of cannabinoid allosteric ligands with different degrees of modulation, called ‘biased modulation’, that can vary dramatically in a probe- and pathway-specific manner, not from simple differences in orthosteric ligand efficacy or stimulus-response coupling. Here, emerging evidence proposes the involvement of CB1 GPCRs in a novel biased GPCR signaling paradigm involving the crosstalk between neuraminidase-1 (Neu-1) and matrix metalloproteinase-9 (MMP-9) in the activation of glycosylated receptors through the modification of the receptor glycosylation state. The study findings highlighted the role of CB1 agonists AM-404, Aravnil, and Olvanil in significantly inducing Neu-1 sialidase activity in a dose-dependent fashion in RAW-Blue, PANC-1, and SW-620 cells. This approach was further substantiated by findings that the neuromedin B receptor inhibitor, BIM-23127, MMP-9 inhibitor, MMP9i, and Neu-1 inhibitor, oseltamivir phosphate, could specifically block CB1 agonist-induced Neu-1 sialidase activity. Additionally, we found that CB1 receptors exist in a multimeric receptor complex with Neu-1 in naïve, unstimulated RAW-Blue, PANC-1, and SW-620 cells. This complex implies a molecular link that regulates the interaction and signaling mechanism among these molecules present on the cell surface. Moreover, the study results demonstrate that CB1 agonists induce NFκB-dependent secretory alkaline phosphatase (SEAP) activity in influencing the expression of epithelial–mesenchymal markers, E-cadherin, and vimentin in SW-620 cells, albeit the impact on E-cadherin expression is less pronounced compared to vimentin. In essence, this innovative research begins to elucidate an entirely new molecular mechanism involving a GPCR signaling paradigm in which cannabinoids, as epigenetic stimuli, may traverse to influence gene expression and contribute to cancer and cancer metastasis. Full article

The α_2A adrenergic receptor (α_2A-AR) serves as a critical molecular target for sedatives and analgesics. However, α_2A-AR ligands with an imidazole ring also interact with an imidazoline receptor as well as other proteins and lead to undesirable effects, motivating us to develop more novel scaffold α_2A-AR ligands. For this purpose, we employed an ensemble-based ligand discovery strategy, integrating long-term molecular dynamics (MD) simulations and virtual screening, to identify new potential α_2A-AR agonists with novel scaffold. Our results showed that compounds SY-15 and SY-17 exhibited significant biological effects in the preliminary evaluation of protein kinase A (PKA) redistribution assays. They also reduced levels of intracellular cyclic adenosine monophosphate (cAMP) in a dose-dependent manner. Upon treatment of the cells with 100 μM concentrations of SY-15 and SY-17, there was a respective decrease in the intracellular cAMP levels by 63.43% and 53.83%. Subsequent computational analysis was conducted to elucidate the binding interactions of SY-15 and SY-17 with the α_2A-AR. The binding free energies of SY-15 and SY-17 calculated by MD simulations were −45.93 and −71.97 kcal/mol. MD simulations also revealed that both compounds act as bitopic agonists, occupying the orthosteric site and a novel exosite of the receptor simultaneously. Our findings of integrative computational and experimental approaches could offer the potential to enhance ligand affinity and selectivity through dual-site occupancy and provide a novel direction for the rational design of sedatives and analgesics. Full article

Glutamate (Glu) toxicity has been an important research topic in toxicology and neuroscience studies. In vitro and in vivo studies have shown that Group II metabotropic Glu2 (mGlu2) activators have cell viability effects. This study aims to determine a candidate ligand with high mGlu2 allosteric region activity among cytotoxicity-safe molecules using the in silico positioning method and to evaluate its cell viability effect in vitro. We investigated the candidate molecule’s cell viability effect on the SH-SY5Y human neuroblastoma cell line by MTT analysis. In the study, LY 379268 (agonist) and JNJ-46281222 (positive allosteric modulator; PAM) were used as control reference molecules. Drug bank screening yielded THRX-195518 (docking score being −12.4 kcal/mol) as a potential novel drug candidate that has a high docking score and has not been mentioned in the literature so far. The orthosteric agonist LY 379268 exhibited a robust protective effect in our study. Additionally, our findings demonstrate that JNJ-46281222 and THRX-195518, identified as activating the mGlu2 allosteric region through in silico methods, preserve cell viability against Glu toxicity. Therefore, our study not only emphasizes the positive effects of this compound on cell viability against Glu toxicity but also sheds light on the potential of THRX-195518, acting as a mGlu2 PAM, based on in silico absorption, distribution, metabolism, excretion, and toxicity (ADMET) data, as a candidate drug molecule. These findings underscore the potential utility of THRX-195518 against both neurotoxicity and Central Nervous System (CNS) disorders, providing valuable insights. Full article

Background: Although autophagy is a pro-survival process of tumor cells, it can stimulate cell death in particular conditions and when differently regulated by specific signals. We previously demonstrated that the selective stimulation of the M2 muscarinic receptor subtype (mAChR) negatively controls cell proliferation and survival and causes oxidative stress and cytotoxic and genotoxic effects in both GBM cell lines and GBM stem cells (GSCs). In this work, we have evaluated whether autophagy was induced as a downstream mechanism of the observed cytotoxic processes induced by M2 mAChR activation by the orthosteric agonist APE or the dualsteric agonist N8-Iper (N8). Methods: To assess the activation of autophagy, we analyzed the expression of LC3B using Western blot analysis and in LC3B-EGFP transfected cell lines. Apoptosis was assessed by measuring the protein expression of Caspases 3 and 9. Results: Our data indicate that activation of M2 mAChR by N8 promotes autophagy in both U251 and GB7 cell lines as suggested by the LC3B-II expression level and analysis of the transfected cells by fluorescence microscopy. Autophagy induction by M2 mAChRs is regulated by the decreased activity of the PI3K/AKT/mTORC1 pathway and upregulated by pAMPK expression. Downstream of autophagy activation, an increase in apoptosis was also observed in both cell lines after treatment with the two M2 agonists. Conclusions: N8 treatment causes autophagy via pAMPK upregulation, followed by apoptosis in both investigated cell lines. In contrast, the absence of autophagy in APE-treated GSC cells seems to indicate that cell death could be triggered by mechanisms alternative to those observed for N8. Full article