Search Results (81)

Background: The amylin receptor is a receptor for the peptide hormone amylin, and its activation is known to reduce body weight. The amylin receptor functions as a heterodimer complex that consists of the calcitonin receptor for peptide hormone calcitonin and an accessary protein. Although the structural information of amylin receptors is currently available, receptor–ligand binding studies that support the peptide binding mode for amylin receptors remain incomplete. Methods: Here, we introduced mutagenesis to the amylin receptor 1 extracellular domain and examined mutational effects on peptide binding affinity. We focused on several residues mainly from the peptide-binding pocket (D97, D101, E123, N124, and N135 of the calcitonin receptor). Two well-known peptide ligands for amylin receptors were used for this study: a salmon calcitonin fragment and an antagonist amylin analog AC413 fragment with Y25P mutation. Results: Among the introduced mutations, D101A and N135A mutations abolished peptide ligand binding, suggesting that these residues are critical for peptide interaction. The N124A mutation also significantly decreased the peptide binding affinity by more than 8-fold. Intriguingly, the N124D mutation restored the decreased affinity of the salmon calcitonin fragment, while it failed to restore the decreased affinity of the AC413 fragment. Structural analyses suggested that there was a potential role of salmon calcitonin serine 29 in the interaction with aspartate of the N124D mutation. Conclusions: This study validates the critical residues of the amylin receptor 1 extracellular domain for the interaction with C-terminal fragments of peptide ligands. This study also suggests that modulating receptor–ligand interaction is feasible by the modification of receptor amino acids near an interacting peptide ligand. Full article

Background and Objectives: Most genetic studies have focused on catecholamine system genes to identify etiology in attention-deficit/hyperactivity disorder (ADHD), and there is growing evidence that the interaction of several genes may synergistically or antagonistically affect disease outcomes. We investigated the interaction between the alpha-2 adrenergic receptor (ADRA2A) and its transporter (SLC6A2) to determine the etiology and treatment outcomes for ADHD. Materials and Methods: Children with ADHD (age 8.3 ± 2.0 y, 72 boys and 11 girls) were assessed using the Kiddie Schedule for Affective Disorders-Present and Lifetime (K-SASD-PL), ADHD rating scale-IV (ARS), Clinical Global Impressions-Improvement (CGI-I), and Clinical Global Impressions-Severity (CGI-S) scales. Neuropsychological assessments were performed using a continuous performance test (CPT). Methylphenidate was titrated based on the CGI-I and CGI-S scales for 8 weeks. We assessed two polymorphisms, ADRA2A rs553668 and SLC6A2 rs998424, for their association with disease outcomes. Results: The ADRA2A polymorphism had a significant effect on visual/auditory commission errors in the CPT. The CC genotype for ADRA2A combined with the GG genotype for SLC6A2 showed more commission errors than the other combinations of genotypes. Treatment outcome assessment using the CGI-S showed that the SLC6A2 GG genotype had more favorable treatment outcome (p < 0.05) and significant gene × dose interaction on ARS score across 8 weeks (p < 0.01). Conclusions: Our findings provide preliminary evidence for the effect of ADR2A and SLC6A2 gene–gene interactions on the attention system and treatment response in children with ADHD. Although these findings require future replication, our study contributes to the understanding of the genetic basis of ADHD. Full article

The tumor microenvironment and healing wounds both contain extremely high concentrations of adenosine triphosphate (ATP) compared to normal tissue. The P2Y2 receptor, an ATP-activated purinergic receptor, is typically associated with pulmonary, endothelial, and neurological cell signaling. Here, we examine ATP-dependent signaling in breast epithelial cells and how it is altered in metastatic breast cancer. Using rapid imaging techniques, we show how ATP-activated P2Y2 signaling causes an increase in intracellular Ca²⁺ in non-tumorigenic breast epithelial cells, approximately 3-fold higher than their tumorigenic and metastatic counterparts. The non-tumorigenic cells respond to increased Ca²⁺ with actin polymerization and localization to the cell edges after phalloidin staining, while the metastatic cells remain unaffected. The increase in intracellular Ca²⁺ after ATP stimulation was blunted to control levels using a P2Y2 antagonist, which also prevented actin mobilization and significantly increased cell dissemination from spheroids in non-tumorigenic cells. Furthermore, the lack of Ca²⁺ changes and actin mobilization in metastatic breast cancer cells could be due to the reduced P2Y2 expression, which correlates with poorer overall survival in breast cancer patients. This study elucidates the rapid changes that occur after elevated intracellular Ca²⁺ in breast epithelial cells and how metastatic cancer cells have adapted to evade this cellular response. Full article

The peptidergic systems are involved in neuroblastoma. Peptides (angiotensin II, neuropeptide Y, neurotensin, substance P) act as oncogenic agents in neuroblastoma, whereas others (adrenomedullin, corticotropin-releasing factor, urocortin, orexin) exert anticancer effects against neuroblastoma. This plethora of peptidergic systems show the functional complexity of the mechanisms regulated by peptides in neuroblastoma. Peptide receptor antagonists act as antineuroblastoma agents since these compounds counteracted neuroblastoma cell growth and migration and the angiogenesis promoted by oncogenic peptides. Other therapeutic approaches (signaling pathway inhibitors, focal adhesion kinase inhibitors, peptide receptor knockdown, acetic acid analogs) that also counteract the beneficial effects mediated by the oncogenic peptides in neuroblastoma are discussed, and future research lines to be developed in neuroblastoma (interactions between oncogenic and anticancer peptides, combination therapy using peptide receptor antagonists and chemotherapy/radiotherapy) are also suggested. Although the data regarding the involvement of the peptidergic systems in neuroblastoma are, in many cases, fragmentary or very scarce for a particular peptidergic system, taken together, they are quite promising with respect to potentiating and developing this research line with the aim of developing new therapeutic strategies to treat neuroblastoma in the future. Peptidergic systems are potential and promising targets for the diagnosis and treatment of neuroblastoma. Full article

Thrombosis is one of the most prevalent and serious health issues amongst humans. A key component of thrombotic events is the activation and aggregation of platelets, of which the P2Y₁ and P2Y₁₂ receptors play a crucial role in this process. Despite a breadth of knowledge on thrombosis and its mechanisms and treatment in various disorders in humans, there is less of an understanding of the expression and exact role of these receptors in companion animals such as dogs and cats. Therefore, this study aimed to investigate P2Y₁ and P2Y₁₂ receptors on dog and cat platelets in platelet-rich plasma and compare them to human platelets. Immunoblotting revealed the presence of P2Y₁ and P2Y₁₂ receptor proteins on dog and cat platelets, although relative amounts of each receptor appeared to contrast those of human platelets, with increased amounts of P2Y₁ compared to P2Y₁₂ receptors in dogs and cats. Using a modified 384-well plate aggregation assay, designed for use with small volumes, the human P2Y₁ and P2Y₁₂ receptor agonists adenosine 5′-diphosphate and 2-methylthio-adenosine 5′-diphosphate caused aggregation of dog and cat platelets. This aggregation was near-completely inhibited by the selective P2Y₁₂ antagonist ticagrelor. Aggregation of dog and cat platelets was partly inhibited by the human P2Y₁ receptor antagonist MRS2179. The agonist and antagonist responses in dog and cat platelets were like those of human platelets. In contrast, the aggregation of dog platelets in the absence of added nucleotides was two-fold greater than that of cats and humans. This study indicates that platelets of cats and dogs possess functional P2Y₁ and P2Y₁₂ receptors that can be inhibited by human antagonists. The data presented suggest differing roles or responses of the platelet P2Y receptors in dogs and cats compared to humans but also highlight the potential of using currently available P2Y₁ or P2Y₁₂ antiplatelet drugs such as ticagrelor for the treatment of thrombosis in these companion animals. Full article

Ticagrelor, a reversible platelet P2Y₁₂ receptor antagonist, exerts various pleiotropic actions, some of which are at least partially mediated through adenosine. We studied the ticagrelor and adenosine effect on the angiogenic properties of progenitor CD34⁺-derived endothelial colony-forming cells (ECFCs). Angiogenesis studies were performed in vitro using capillary-like tube formation and spheroid-based angiogenesis assays. The effects of adenosine receptor antagonists, including DPCPX (A₁ antagonist), SCH58621 (A_2A antagonist), MRS1706 (A_2B inverse agonist and antagonist), MRS1220 (A₃ antagonist) and adenosine deaminase (ADA), were also investigated. Ticagrelor, adenosine, and their combination increased capillary-like tube formation and spheroid sprout formation by ECFCs in a dose-dependent manner. This effect was significantly reduced by SCH58621, MRS1706, and their combination, as well as by ADA. By contrast, DPCPX and MRS1220 did not exhibit any inhibitory effects. Similar results were obtained when mature human umbilical vein endothelial cells (HUVECs) were studied. These results show that ticagrelor stimulates angiogenesis by progenitor and mature endothelial cells in an adenosine-dependent pathway in which the adenosine receptors A_2A and A_2B play major roles. The significance of these results at the clinical level in patients with atherothrombotic events and treated with ticagrelor needs to be investigated. Full article

In the second edition of this Special Issue, several promising antitumor strategies have been presented in addition to those reported in the first edition, in which several compounds (acetylcorynoline, BaP1, sarco/endoplasmic reticulum calcium ATPase inhibitors, neuropeptide Y, neuropeptide Y antagonists, neurokinin-1 receptor antagonists) exerting antitumor effects against colorectal cancer, papillary thyroid carcinoma, cholangiocarcinoma, Ewing sarcoma, liver cancer, and breast cancer were reported [...] Full article

Dual antiplatelet therapy (DAPT), which is essential in AMI management, combines aspirin with a P2Y12 receptor antagonist. This study compared the effectiveness of potent P2Y12 inhibitors versus clopidogrel in AMI patients treated with percutaneous coronary intervention (PCI). Methods: 65,986 AMI patients included in a nationwide prospective registry who underwent PCI and received DAPT were studied. In total, 9,014 patients received potent P2Y12 inhibitors, and 56,074 received clopidogrel. This study focused on mortality, recurrent myocardial infarction, stroke, repeat revascularization, and major adverse cardiovascular events (MACE) over seven years. The analysis utilized unadjusted models and inverse probability of treatment weighting (IPTW) to compare prognosis, and decision curve analyses were constructed to aid clinical decision making. Results: Potent P2Y12 inhibitors significantly reduced mortality risk (unadjusted hazard ratio (HR): 0.58; IPTW HR: 0.68) and MACE (unadjusted HR: 0.66; IPTW HR: 0.78). Diabetic patients showed greater benefits (HR:0.45). In patients at high bleeding risk, the mortality rate was 13% (HR: 0.87, p = 0.08). For patients aged 75–79, the HR for mortality was 0.82, whereas for those aged >80 years, it was 0.79, indicating significant mortality risk reduction. Similar trends were observed for MACE. Conclusion: This study demonstrated that potent P2Y12 inhibitors are more effective than clopidogrel in reducing mortality and MACE in patients with AMI and underscored their potential role in improving outcomes across diverse patient subgroups. The trend was consistent even during the COVID-19 pandemic. These findings highlight the need for personalized DAPT strategies, particularly for high-bleeding-risk patients, and challenge current guidelines favoring clopidogrel use in older patients. Full article

Background/Objectives: Brick dust molecules exhibit high melting points and ultralow solubility. Overcoming this solubility issue is challenging. Previously, we formulated a co-amorphous system for a neuropeptide Y5 receptor antagonist (NP) as a brick dust drug using sodium taurocholate (ST) to improve its dissolution profile. In this study, we have designed a ternary amorphous system involving polymer addition to further improve a co-amorphous system. Methods: The amorphous samples were prepared by the ball milling. The thermal and spectroscopic analyses were performed, and the isothermal crystallization and dissolution profiles were evaluated. Results: The ball milling of NPs, ST, and each of the three types of polymers successfully converted crystalline NPs to amorphous NPs. Thermal analysis confirmed the formation of a single amorphous phase. The infrared spectra revealed a specific interaction between an NP and ST in the co-amorphous system. Moreover, the intermolecular interactions of NP-ST were maintained in the ternary amorphous systems, suggesting the miscible dispersion of the co-amorphous system into the polymer via weak interactions as co-amorphous solid dispersions. The dissolution profile of co-amorphous NP-ST was 4.1- and 6.7-fold higher than that of crystalline NPs in pH 1.2 and 6.8 buffers, respectively. The drug concentration in the ternary amorphous system in pH 1.2 and 6.8 buffers became 1.1–1.2- and 1.4–2.7-fold higher than that seen in the co-amorphous system, respectively. Conclusions: Co-amorphous solid dispersion is a promising method for enhancing the solubility of brick dust molecules. Full article

The P2Y₆ receptor (P2Y₆R), a G_q-coupled receptor, is a potential drug discovery target for various inflammatory and degenerative conditions. Antagonists have been shown to attenuate colitis, acute lung injury, etc. In the search for competitive antagonists, we have investigated the SAR of 3-nitro-2-(trifluoromethyl)-2H-chromene derivatives, although high affinity is lacking. We now reveal that long-chain amino-functionalized congeners display greatly enhanced affinity in the antagonism of UDP-induced Ca²⁺ mobilization in human (h) P2Y₆R-transfected 1321N1 astrocytoma cells. A 6-(Boc-amino-n-heptylethynyl) analogue 30 (MRS4940) had an IC₅₀ of 162 nM, which was a 123-fold greater affinity than the corresponding unprotected primary alkylamine, 107-fold greater than the corresponding pivaloyl derivative 30, and 132-fold selective compared to the P2Y₁₄R. However, similar Boc-amino chains attached at the 8-position produced weak µM affinity. Thus, the P2Y₆R affinity depended on the chain length, attachment point, and terminal functionality. Off-target activities, at 45 sites, were tested for acylamino derivatives 20, 24, 26, 30, 31, and 37, which showed multiple interactions, particularly at the biogenic amine receptors. The more potent analogues may be suitable for evaluation in inflammation and cancer models, which will be performed in future studies. Full article

Myocardial necrosis following the successful reperfusion of a coronary artery occluded by thrombus in a patient presenting with ST-elevation myocardial infarction (STEMI) continues to be a serious problem, despite the multiple attempts to attenuate the necrosis with agents that have shown promise in pre-clinical investigations. Possible reasons include confounding clinical risk factors, the delayed application of protective agents, poorly designed pre-clinical investigations, the possible effects of routinely administered agents that might unknowingly already have protected the myocardium or that might have blocked protection, and the biological differences of the myocardium in humans and experimental animals. A better understanding of the pathobiology of myocardial infarction is needed to stem this reperfusion injury. P2Y₁₂ receptor antagonists minimize platelet aggregation and are currently part of the standard treatment to prevent thrombus formation and propagation in STEMI protocols. Serendipitously, these P2Y₁₂ antagonists also dramatically attenuate reperfusion injury in experimental animals and are presumed to provide a similar protection in STEMI patients. However, additional protective agents are needed to further diminish reperfusion injury. It is possible to achieve additive protection if the added intervention protects by a mechanism different from that of P2Y₁₂ antagonists. Inflammation is now recognized to be a critical factor in the complex intracellular response to ischemia and reperfusion that leads to tissue necrosis. Interference with cardiomyocyte inflammasome assembly and activation has shown great promise in attenuating reperfusion injury in pre-clinical animal models. And the blockade of the executioner protease caspase-1, indeed, supplements the protection already seen after the administration of P2Y₁₂ antagonists. Importantly, protective interventions must be applied in the first minutes of reperfusion, if protection is to be achieved. The promise of such a combination of protective strategies provides hope that the successful attenuation of reperfusion injury is attainable. Full article

P2X7 is an ATP-activated purinergic receptor implicated in pro-inflammatory responses. It is associated with the development of several diseases, including inflammatory and neurodegenerative conditions. Although several P2X7 receptor antagonists have recently been reported in the literature, none of them is approved for clinical use. However, the structure of the known antagonists can serve as a scaffold for discovering effective compounds in clinical therapy. This study aimed to propose an improved virtual screening methodology for the identification of novel potential P2X7 receptor antagonists from natural products through the combination of shape-based and docking approaches. First, a shape-based screening was performed based on the structure of JNJ-47965567, a P2X7 antagonist, using two natural product compound databases, MEGx (~5.8 × 10³ compounds) and NATx (~32 × 10³ compounds). Then, the compounds selected by the proposed shape-based model, with Shape–Tanimoto score values ranging between 0.624 and 0.799, were filtered for drug-like properties. Finally, the compounds that met the drug-like filter criteria were docked into the P2X7 allosteric binding site, using the docking programs GOLD and DockThor. The docking poses with the best score values were submitted to careful visual inspection of the P2X7 allosteric binding site. Based on our established visual inspection criteria, four compounds from the MEGx database and four from the NATx database were finally selected as potential P2X7 receptor antagonists. The selected compounds are structurally different from known P2X7 antagonists, have drug-like properties, and are predicted to interact with key P2X7 allosteric binding pocket residues, including F88, F92, F95, F103, M105, F108, Y295, Y298, and I310. Therefore, the combination of shape-based screening and docking approaches proposed in our study has proven useful in selecting potential novel P2X7 antagonist candidates from natural-product-derived compounds databases. This approach could also be useful for selecting potential inhibitors/antagonists of other receptors and/or biological targets. Full article

Numerous studies suggest the involvement of adenosine-5′-triphosphate (ATP) and similar nucleotides in the pathophysiology of asthma. Androgens, such as testosterone (TES), are proposed to alleviate asthma symptoms in young men. ATP and uridine-5′-triphosphate (UTP) relax the airway smooth muscle (ASM) via purinergic P2Y₂ and P2Y₄ receptors and K⁺ channel opening. We previously demonstrated that TES increased the expression of voltage-dependent K⁺ (K_V) channels in ASM. This study investigates how TES may potentiate ASM relaxation induced by ATP and UTP. Tracheal tissues treated with or without TES (control group) from young male guinea pigs were used. In organ baths, tracheas exposed to TES (40 nM for 48 h) showed enhanced ATP- and UTP-evoked relaxation. Tetraethylammonium, a K⁺ channel blocker, annulled this effect. Patch-clamp experiments in tracheal myocytes showed that TES also increased ATP- and UTP-induced K⁺ currents, and this effect was abolished with flutamide (an androgen receptor antagonist). K_V channels were involved in this phenomenon, which was demonstrated by inhibition with 4-aminopyridine. RB2 (an antagonist of almost all P2Y receptors except for P2Y₂), as well as N-ethylmaleimide and SQ 22,536 (inhibitors of G proteins and adenylyl cyclase, respectively), attenuated the enhancement of the K⁺ currents induced by TES. Immunofluorescence and immunohistochemistry studies revealed that TES did not modify the expression of P2Y₄ receptors or COX-1 and COX-2, while we have demonstrated that this androgen augmented the expression of K_V1.2 and K_V1.5 channels in ASM. Thus, TES leads to the upregulation of P2Y₄ signaling and K_V channels in guinea pig ASM, enhancing ATP and UTP relaxation responses, which likely limits the severity of bronchospasm in young males. Full article

(1) Background: Modulators of the Neuropeptide Y (NPY) system are involved in energy metabolism, but the effect of NPY receptor antagonists on metabolic-dysfunction-associated steatotic liver disease (MASLD), a common obesity-related comorbidity, are largely unknown. In this study, we report on the effects of antagonists of the NPY-2 receptor (Y2R) in comparison with empagliflozin and semaglutide, substances that are known to be beneficial in MASLD. (2) Methods: Diet-induced obese (DIO) male Wistar rats were randomized into the following treatment groups: empagliflozin, semaglutide ± PYY_3-36, the Y2R antagonists JNJ 31020028 and a food-restricted group, as well as a control group. After a treatment period of 8 weeks, livers were weighed and histologically evaluated. QrtPCR was performed to investigate liver inflammation and de novo lipogenesis (in liver and adipose tissue). Serum samples were analysed for metabolic parameters. (3) Results: Semaglutide + PYY_3-36 led to significant weight loss, reduced liver steatosis (p = 0.05), and decreased inflammation, insulin resistance, and leptin levels. JNJ-31020028 prevented steatosis (p = 0.03) without significant weight loss. Hepatic downregulation of de novo lipogenesis-regulating genes (SREBP1 and MLXIPL) was observed in JNJ-31020028-treated rats (p ≤ 0.0001). Food restriction also resulted in significantly reduced weight, steatosis, and hepatic de novo lipogenesis. (4) Conclusions: Body weight reduction (e.g., by food restriction or drugs like semaglutide ± PYY_3-36) is effective in improving liver steatosis in DIO rats. Remarkably, the body-weight-neutral Y2R antagonists may be effective in preventing liver steatosis through a reduction in de novo lipogenesis, making this drug class a candidate for the treatment of (early) MASLD. Full article

Purine nucleosides (adenosine) and nucleotides such as adenosine mono/di/triphosphate (AMP/ADP/ATP) may produce complex cardiovascular responses. For example, adenosine-5′-(β-thio)-diphosphate (ADPβS; a stable synthetic analogue of ADP) can induce vasodilatation/vasodepressor responses by endothelium-dependent and independent mechanisms involving purinergic P2Y receptors; however, the specific subtypes participating in these responses remain unknown. Therefore, this study investigated the receptor subtypes mediating the blood pressure changes induced by intravenous bolus of ADPβS in male Wistar rats in the absence and presence of central mechanisms with the antagonists MRS2500 (P2Y₁), PSB0739 (P2Y₁₂), and MRS2211 (P2Y₁₃). For this purpose, 120 rats were divided into 60 anaesthetised rats and 60 pithed rats, and further subdivided into four groups (n = 30 each), namely: (a) anaesthetised rats, (b) anaesthetised rats with bilateral vagotomy, (c) pithed rats, and (d) pithed rats continuously infused (intravenously) with methoxamine (an α₁-adrenergic agonist that restores systemic vascular tone). We observed, in all four groups, that the immediate decreases in diastolic blood pressure produced by ADPβS were exclusively mediated by peripheral activation of P2Y₁ receptors. Nevertheless, the subsequent increases in systolic blood pressure elicited by ADPβS in pithed rats infused with methoxamine probably involved peripheral activation of P2Y₁, P2Y₁₂, and P2Y₁₃ receptors. Full article