Molecular Pharmacology最新文献

Trace amine-associated receptor 1 (TAAR1) is a G protein-coupled receptor stimulated by trace amines and amphetamine-like psychostimulants. The TAAR1 agonists RO5166017 and RO5256390 have antidepressant- and anxiolytic-like effects in preclinical models, and the TAAR1/partial 5HT_1A agonist ulotaront has been evaluated for its clinical utility as an antipsychotic. Early clinical investigations of ulotaront for treating psychosis in schizophrenia yielded positive endpoints. However, results from phase III clinical trials showed that ulotaront had the same efficacy as placebo. One concern arising from these results is that investigational TAAR1 agonists could be diverse in the mechanisms by which they influence dopamine homeostasis. Thus, we evaluated the pharmacology of TAAR1 agonists RO5166017, RO5256390, and ulotaront at the dopamine transporter (DAT) and TAAR1 to test our hypothesis that there would be differences among agonists in their effects on DAT. We found that RO5166017 and RO5256390 directly bind DAT and inhibit dopamine uptake, while ulotaront did not. In cultured cells and rodent synaptosomes, pretreatment with ulotaront and RO5256390 reduced dopamine uptake by approximately half, while RO5166017 pretreatment increased dopamine uptake by a similar magnitude. In cells, the effects of TAAR1 agonist pretreatment on dopamine uptake were TAAR1-dependent. RO5166017, but not RO5256390 or ulotaront, increased amphetamine-induced dopamine efflux in a TAAR1-dependent manner. Surface biotinylation experiments indicated that RO5166017 pretreatment increased cell-surface DAT by approximately 15% via a TAAR1-dependent mechanism. These findings demonstrate clinically relevant differences in the effects of 3 TAAR1 agonists on DAT. SIGNIFICANCE STATEMENT: This study evaluated the direct and heterologous TAAR1-dependent effects of the TAAR1 agonists RO5166017, RO5256390, and ulotaront at DAT. All 3 affected DAT transport and/or trafficking, with each exhibiting a unique profile of direct and heterologous effects, some of which were TAAR1-dependent. These issues should be considered with therapeutic design and clinical use of TAAR1 agonists.

Pirin is a nonheme iron-binding protein with a variety of proposed functions, including serving as a coactivator of p65 NFκB and quercetinase activity. We report here, failure to confirm pirin's primary proposed mechanism, binding of Fe(III)-pirin and p65. Analytical size exclusion chromatography and fluorescence polarization studies did not detect an interaction. We also found no effects of pirin on tumor necrosis factor α-activated p65-regulated gene transcription using mouse embryonic fibroblasts from a pirin knockout mouse and a pirin knockdown NIH3T3 fibroblast cell line. Tumor necrosis factor α-activated p65 response gene mRNA was neither increased nor decreased in cells with loss of pirin compared to wild-type (WT) cells. Furthermore, pirin immunofluorescence in NIH3T3 fibroblasts showed primarily a cytoplasmic localization, not nuclear, as in most previous studies. This was confirmed by cell fractionation analysis. Pirin did show colocalization with the endoplasmic reticulum (ER) marker protein disulfide isomerase as well as cyotoplasmic labeling. We confirmed pirin's quercetinase activity in biochemical assays and demonstrated competitive inhibition by the pirin inhibitor, CCG-257081. Cellular quercetin levels in cells exposed to quercetin in vitro were increased by knockdown of pirin or by treatment with pirin inhibitors. Because pirin is localized to the ER and flavanols are protective of ER stress, we investigated whether pirin knockdown altered ER stress signaling, but did not find any effect of pirin knockdown on ER stress response genes. Our results challenge the dominant model of pirin's function (NFκB regulation) but confirm its quercetinase activity with implications for the mechanisms of pirin binding small molecules. SIGNIFICANCE STATEMENT: Pirin has multiple proposed functions and plays an important role in cancer (melanoma, colon, and breast) and inflammatory diseases. Small molecule pirin-binding compounds have been identified, but pirin's functional mechanism remains poorly understood. This study raises doubts about the primary description of pirin as a nuclear regulator of p65 NFκB function but validates pirin's role as a quercetinase. This study shows that pirin-binding compounds can raise cellular quercetin levels. Further studies will be required to fully understand pirin's biological mechanisms.

The hepatic P450 hemoproteins CYPs 4A are typical N-terminally anchored type I endoplasmic reticulum (ER) proteins, inducible by many hypolipidemic drugs and peroxisome proliferators. They are engaged in the ω-/ω-1-oxidation of various fatty acids including arachidonic acid, prostaglandins, and leukotrienes and in the biotransformation of some therapeutic drugs. Because the proteolytic turnover of the mammalian liver CYPs 4A remains obscure, we have characterized it. We report that of these proteins, human CYP4A11 and mouse Cyp4a12a are preferential targets of the ER-lysosome-associated degradation. Consequently, these proteins are stabilized 2- to 3-fold both as 1%Triton X100-soluble and insoluble species in mouse hepatocytes and HepG2 cells deficient in the autophagic initiation ATG5 gene. Despite exhibiting surface microtubule-associated protein light chain 3-interacting regions that could target them directly to the autophagosome, they nevertheless interact intimately with the autophagic receptor SQSTM1/p62. Through structural deletion analyses and site-directed mutagenesis, we have identified the CYP4A-interacting p62 subdomain to lie between residues 170 and 233, which include its Traf6-binding and LIM-binding subdomains. Mice carrying a liver-specific genetic deletion of p62 residues 69-251 (p62Mut) that includes the CYP4A-interacting subdomain also exhibit Cyp4a-protein stabilization as 1% Triton X100-soluble and insoluble species. Consistently, p62Mut mouse liver microsomes exhibit 1.5- to 2-fold enhanced ω- and ω-1-arachidonic acid hydroxylation to its physiologically active metabolites 19 and 20-HETEs relative to the corresponding wild-type mouse liver microsomes. Collectively, our findings suggest that disruption of CYP4A ER-lysosome-associated degradation results in functionally active P450 protein stabilization and consequent proinflammatory metabolite generation along with insoluble CYP4A aggregates, which may contribute to pathological aggregates, ie, Mallory-Denk bodies/inclusions, hallmarks of many liver diseases. SIGNIFICANCE STATEMENT: Human CYP4A11 and mouse Cyp4a12a, liver P450 enzymes engaged in ω-/ω-1-oxidation of arachidonic acid, prostaglandins, and leukotrienes, are documented to physiologically turn over via endoplasmic reticulum-lysosome-associated autophagic degradation, which involves their intimate association with the autophagic receptor SQSTM1/p62. Genetic endoplasmic reticulum-lysosome-associated autophagic degradation disruption or deletion of their hepatic p62-interaction subdomain in mice results in Cyp4a-protein stabilization as functionally active solubilizable species with consequently enhanced proinflammatory 20-HETE arachidonate metabolite generation and insoluble Cyp4a aggregates, potential contributors to pathologic liver inclusions.

Although multiparameter cellular morphological profiling methods and three-dimensional (3D) biological model systems can potentially provide complex insights for pharmaceutical discovery campaigns, there have been relatively few reports combining these experimental approaches. In this study, we used the U87 glioblastoma cell line grown in a 3D spheroid format to validate a multiparameter cellular morphological profiling screening method. The steps of this approach include 3D spheroid treatment, cell staining, fully automated digital image acquisition, image segmentation, numerical feature extraction, and multiple machine learning approaches for cellular profiling. For comparison, we measured the same samples after live-cell microscopy with an endpoint CellTiter-Glo cell viability assay. The combined method characterized 7 reference compounds with previously reported anticancer/cytotoxic properties that induce quantifiably different spheroid morphologies in this assay. The method was then used to screen a library of 925 compounds that are related to kinase signaling pathways. Both unsupervised and supervised machine learning approaches identified compounds that induced morphologies similar to those induced by the reference compounds. We performed a follow-up 16-point concentration response experiment for 3 of these compounds selected from our profiling pipeline and confirmed their phenotype. The morphology-based concentration response for these compounds was also correlated with the CellTiter-Glo endpoint assay. Additionally, the measured morphological phenotypes displayed different enrichment levels of commonly annotated mechanisms of action. Our analysis was able to identify selected mechanisms of action associated with specific phenotypic signatures. Overall, the presented screening and analysis method can distinguish between different spheroid structural changes that are caused by specific candidate anticancer compounds. SIGNIFICANCE STATEMENT: Morphological profiling has become a powerful tool in the field of microscopy for finding distinct mechanisms of action groups and small molecule screening to identify new phenotypes. This study presents new potential mechanisms of action groups for known glioblastoma candidates from the screening library, which is believed to help advance the search for more effective glioblastoma therapies.

Metabolic dysfunction-associated steatohepatitis (MASH) is a progressive liver disease characterized by steatosis, inflammatory responses, and fibrosis. Peroxisome proliferator-activated receptors (PPARs), master regulators of glucolipid homeostasis and inflammatory pathways, have emerged as promising therapeutic targets for MASH. PPAR agonists have demonstrated therapeutic potential in MASH by ameliorating hepatic lipid deposition, normalizing dyslipidemia, enhancing insulin sensitivity, and suppressing proinflammatory signaling. In this study, we reported that NCPC-626, a natural fungal metabolite, was discovered as a novel potent pan-PPAR agonist through high-throughput screening. In an in vitro model of MASH, NCPC-626 inhibited lipid accumulation, fibrosis, and inflammation. Moreover, NCPC-626 treatment reduced body weight, liver triglyceride levels, and improved glucose tolerance in db/db mice by regulating glucolipid metabolism. Additionally, NCPC-626 exhibited preventive and therapeutic effects against fibrosis in a CCl₄-induced fibrosis model and ameliorated MASH progression in a diet-induced model. This study highlights NCPC-626 as a pan-PPAR agonist with a novel chemical scaffold for MASH treatment. SIGNIFICANCE STATEMENT: This study identifies a novel structural compound, NCPC-626, demonstrating specific and balanced activation of peroxisome proliferator-activated receptors. NCPC-626 demonstrates therapeutic efficacy against metabolic dysfunction-associated steatohepatitis in experimental models.

μ-Opioid receptor (MOR) agonists are a mainstay in acute pain management. However, they also produce adverse effects and are frequently misused, increasing susceptibility for opioid use disorder. Thus, a strategy for improving the safety of opioid analgesics is needed. Gallein-mediated inhibition of Gβγ signaling to a subset of inhibitory effector feedback systems, such as phospholipase C (PLC) β3 and G protein-coupled receptor kinase 2, potentiates the antinociceptive effects of morphine without altering its rewarding effects in vivo. In this study, we examined effectiveness of gallein in the context of persistent pain using a nitroglycerin (NTG)-induced thermal hypersensitivity assay. In the warm water tail withdrawal assay, NTG (10 mg/kg, i.p.) decreased tail withdrawal latencies by 90%, from 30 to 3 seconds, indicating a hyperalgesic state. Gallein alone fully reversed NTG-induced decreases in withdrawal latencies. Furthermore, a low dose of gallein (3.2 mg/kg, i.p.) that was ineffective alone was sufficient to potentiate the antihyperalgesic effects of morphine. Pretreatment with the nonselective opioid antagonist naloxone (1.0 mg/kg, i.p.) attenuated both the antihyperalgesic effects of gallein alone and gallein-mediated potentiation of morphine. NTG did not decrease tail withdrawal latencies in PLCβ3^-/- mice compared with wild-type littermates, and this apparent antihyperalgesia was also reversed by naloxone. Taken together, the Gβγ inhibitor gallein alone produced antihyperalgesic effects mediated by endogenous opioid receptor activation. These data suggest that inhibiting Gβγ effectors, such as PLCβ3, downstream of MOR activation improves the analgesic effects of both endogenous and exogenous MOR agonists. SIGNIFICANCE STATEMENT: This study demonstrates the potential of small molecule-mediated modulation of Gβγ signaling to enhance the analgesic effects of endogenous opioid peptides suggesting a novel strategy for safer pain management, reducing the risk of opioid use disorder while maintaining effective pain relief.

Metabotropic glutamate receptors (mGlus) are obligate dimer G protein-coupled receptors that can all homodimerize and heterodimerize in select combinations. Responses of mGlu heterodimers to selective ligands, including orthosteric agonists and allosteric modulators, are largely unknown. The pharmacological properties of each group II and III mGlu homodimer (except the exclusively retinally expressed mGlu6) and several heterodimers were examined when stochastically assembled in HEK293T cells, or specifically measured using an improved G protein-mediated bioluminescence resonance energy transfer assay employing complemented fragments of Nanoluciferase. Stochastically assembled receptors adopted unique signaling characteristics. Some favored the potency, efficacy, or signaling kinetics of a dominant subunit, whereas others exhibited blended profiles reflective of a combination of homo- and heterodimers at various ratios of expressed receptor. Finally, group II and III mGlu dimers were examined for responses to selective agonists and allosteric modulators. Effects of glutamate and selective group II and III orthosteric agonists were found to result in unique concentration response profiles when examining each combination of group II and III mGlu. Effects of select allosteric modulators were examined for each mGlu2-containing dimer as well as several group III dimer pairs. Likewise, allosteric modulator effects were often unique across dimers containing the targeted subunit of the ligand being tested. The results demonstrate that mGlu dimers respond uniquely to ligands selective for just one subunit, even when they are ostensibly pharmacologically similar. SIGNIFICANCE STATEMENT: This study demonstrates novel pharmacological responses to ligands acting on metabotropic glutamate receptor heterodimers in isolation. To our knowledge, it also shows for the first time the kinetics and pharmacological properties of group II and III metabotropic glutamate receptors when expressed in pairs and assembled stochastically.

The myristoylated preS1 domain (myr-preS1) of the hepatitis B virus (HBV) large surface protein is essential for binding to the receptor protein, Na⁺/taurocholate co-transporting polypeptide (NTCP), and for the subsequent internalization of the virus-receptor complex. NTCP, which is expressed in hepatocytes, plays a physiological role in hepatic bile acid transport. Recent cryo-electron microscopy structures of the myr-preS1-NTCP complex were used to analyze virus-receptor interactions at the molecular level. Several interaction domains with high binding energies have been identified, including the interaction between myr-preS1 tryptophan 41 (W41) and the NTCP residues tyrosine 146 and phenylalanine 274 (Y146/F274), which are located at a considerable distance from the physiological bile acid binding sites of NTCP. The Y146A/F274A mutant of NTCP retained bile acid transport function but showed a reduced myr-preS1 binding. The W41G myr-preS1 mutant exhibited reduced binding to wild-type NTCP. The Y146A/F274A NTCP mutant did not support in vitro HBV infection, and the W41G myr-preS1 peptide was less effective in blocking infection compared with the wild-type myr-preS1 peptide. In conclusion, the myr-preS1-W41/NTCP-Y146/F274 interaction site, characterized by high binding energy, is essential for HBV entry into hepatocytes. Because this domain is spatially distinct from the bile acid binding and translocation sites of NTCP, it presents an attractive receptor target site for structure-based development of virus-selective HBV entry inhibitors that preserve the physiological bile acid transport function of NTCP. SIGNIFICANCE STATEMENT: This study identified and validated a high energy interaction site between the hepatitis B virus and its receptor Na⁺/taurocholate co-transporting polypeptide that can be used for structure-based drug design of virus entry inhibitors.

Gastric cancer (GC) is a leading cause of cancer-related deaths globally, with metastasis critically impacting prognosis. Splicing factors are key regulators of tumorigenesis, particularly in metastasis. In this exploratory study, we investigated the role and mechanism of heterogeneous nuclear ribonucleoprotein A/B (HNRNPAB) in GC cell invasion and migration. We detected a 3.45-fold increase in HNRNPAB protein levels in highly metastatic MKN45 cells compared with low metastatic MKN7 cells (P < .01) and marked upregulation in human GC tissues (n = 408) versus normal tissues (n = 211, P < .05, predicted by gene expression profiling interactive analysis). HNRNPAB overexpression in MKN45 and MKN7 cells substantially enhanced proliferation by 50% (P < .001, 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2-H-tetrazolium bromide assay), migration by 60% (P < .001, wound healing assay), and invasion by 70% (P < .001, Transwell assay), whereas knockdown reduced these by similar magnitudes. Mechanistically, HNRNPAB decreased E-cadherin (0.5 ± 0.1-fold, P < .001) and increased N-cadherin (1.8 ± 0.2-fold), Vimentin (2.0 ± 0.2-fold), and Snail levels (1.7 ± 0.2-fold, P < .001), promoting epithelial-mesenchymal transition, which was associated with Akt-GSK3β-Wnt pathway modulation by elevating phosphorylated Akt (Ser473, 2.0 ± 0.2-fold) and phosphorylated glycogen synthase kinase 3β (Ser9, 1.8 ± 0.2-fold, P < .001). These findings suggest that HNRNPAB is a potential diagnostic and therapeutic target for GC. SIGNIFICANCE STATEMENT: This exploratory study reveals that heterogeneous nuclear ribonucleoprotein A/B is associated with gastric cancer progression and epithelial-mesenchymal transition through its potential modulation of the Akt-GSK3β-Wnt signaling pathway. Targeting heterogeneous nuclear ribonucleoprotein A/B could offer novel therapeutic approaches for improving treatment outcomes in gastric cancer, highlighting its potential as a biomarker for disease prognosis.