TRAF6-mediated ubiquitination of AKT in the nucleus is a critical event underlying the desensitization of G protein-coupled receptors.

BACKGROUND: Desensitization of G protein-coupled receptors (GPCRs) refers to the attenuation of receptor responsiveness by prolonged or intermittent exposure to agonists. The binding of ß-arrestin to the cytoplasmic cavity of the phosphorylated receptor, which competes with the G protein, has been widely accepted as an extensive model for explaining GPCRs desensitization. However, studies on various GPCRs, including dopamine D2-like receptors (D2R, D3R, D4R), have suggested the existence of other desensitization mechanisms. The present study employed D2R/D3R variants with different desensitization properties and utilized loss-of-function approaches to uncover the mechanisms underlying GPCRs homologous desensitization, focusing on the signaling cascade that regulates the ubiquitination of AKT. RESULTS: AKT undergoes K8/14 ubiquitination by TRAF6, which occurs in the nucleus and promotes its membrane recruitment, phosphorylation and activation under receptor desensitization conditions. The nuclear entry of TRAF6 relies on the presence of the importin complex. Src regulates the nuclear entry of TRAF6 by mediating the interaction between TRAF6 and importin ß1. Ubiquitinated AKT translocates to the plasma membrane where it associates with Mdm2 to phosphorylate it at the S166 and S186 residues. Thereafter, phosphorylated Mdm2 is recruited to the nucleus, resulting in the deubiquitination of ß-Arr2. The deubiquitinated ß-Arr2 then forms a complex with Gßγ, which serves as a biomarker for GPCRs desensitization. Like in D3R, ubiquitination of AKT is also involved in the desensitization of ß2 adrenoceptors. CONCLUSION: Our study proposed that the property of a receptor that causes a change in the subcellular localization of TRAF6 from the cytoplasm to the nucleus to mediate AKT ubiquitination could initiate the desensitization of GPCRs.

Mdm2-mediated ubiquitination of PKCßII is responsible for insulin-induced heterologous desensitization of dopamine D3 receptor.

The insulin and dopaminergic systems in the brain are associated with schizophrenia and Parkinson's disease with respect to etiology and treatment. The present study investigated the crosstalk between the insulin receptor (IR) and dopamine receptor and found that insulin stimulation selectively inhibits signaling of D3 R in a PKCßII-dependent manner. Upon insulin stimulation, E3 ligase enzyme Mdm2 moves out of the nucleus to ubiquitinate PKCßII. Subsequently, ubiquitinated PKCßII translocates to the cell membrane and interacts with D3 R in a phosphorylation-dependent manner at S229/257, resulting in the attenuation of D3 R signaling and initiating clathrin-mediated endocytosis and downregulation. Considering that both IR and D3 R are closely related to some neuropsychosis, this study could provide new molecular insight into the etiology of the disorder.

Nanomedicine in cancer therapy.

Cancer remains a highly lethal disease in the world. Currently, either conventional cancer therapies or modern immunotherapies are non-tumor-targeted therapeutic approaches that cannot accurately distinguish malignant cells from healthy ones, giving rise to multiple undesired side effects. Recent advances in nanotechnology, accompanied by our growing understanding of cancer biology and nano-bio interactions, have led to the development of a series of nanocarriers, which aim to improve the therapeutic efficacy while reducing off-target toxicity of the encapsulated anticancer agents through tumor tissue-, cell-, or organelle-specific targeting. However, the vast majority of nanocarriers do not possess hierarchical targeting capability, and their therapeutic indices are often compromised by either poor tumor accumulation, inefficient cellular internalization, or inaccurate subcellular localization. This Review outlines current and prospective strategies in the design of tumor tissue-, cell-, and organelle-targeted cancer nanomedicines, and highlights the latest progress in hierarchical targeting technologies that can dynamically integrate these three different stages of static tumor targeting to maximize therapeutic outcomes. Finally, we briefly discuss the current challenges and future opportunities for the clinical translation of cancer nanomedicines.

Comparative study of the molecular mechanisms underlying the G protein and ß-arrestin-dependent pathways that lead to ERKs activation upon stimulation by dopamine D2 receptor.

Dopamine D2 receptor (D2 R) has been shown to activate extracellular signal-regulated kinases (ERKs) via distinct pathways dependent on either G-protein or ß-arrestin. However, there has not been a systematic study of the regulatory process of D2 R-mediated ERKs activation by G protein- versus ß-arrestin-dependent signaling since D2 R stimulation of ERKs reflects the simultaneous action of both pathways. Here, we investigated that differential regulation of D2 R-mediated ERKs activation via these two pathways. Our results showed that G protein-dependent ERKs activation was transient, rapid, reached maximum level at around 2 min, and importantly, the activated ERKs were entirely confined to the cytoplasm. In contrast, ß-arrestin-dependent ERKs activation was more sustained, slower, reached maximum level at around 10 min, and phosphorylated ERKs translocated into the nucleus. Src was found to be commonly involved in both the G protein- and ß-arrestin-dependent pathway-mediated ERKs activation. Pertussis toxin Gi/o inhibitor, GRK2-CT, AG1478 epidermal growth factor receptor inhibitor, and wortmannin phosphoinositide 3-kinase inhibitor all blocked G protein-dependent ERKs activation. In contrast, GRK2 and ß-Arr2 played a main role in ß-arrestin-dependent ERKs activation. Receptor endocytosis showed minimal effect on the activation of ERKs mediated by both pathways. Furthermore, we found that the formation of a complex composed of phospho-ERKs, ß-Arr2, and importinß1 promoted the nuclear translocation of activated ERKs. The differential regulation of various cellular components, as well as temporal and spatial patterns of ERKs activation via these two pathways, suggest the existence of distinct physiological outcomes.

A novel colchicine-myricetin heterozygous molecule: design, synthesis, and effective evaluations on the pathological models of acute lung injury in vitro and in vivo.

Acute lung injury (ALI) is an inflammatory condition and there are no effective treatments. A novel new compound----colchicine-myricetin hybrid (CMyrH) was herein designed and synthesized. To evaluate the activity of CMyrH in ALI, we used a bleomycin (BLM) induced BEAS-2B injury model in vitro and established a well-recognized rat model of BLM-induced lung injury in vivo. The results demonstrated that colchicine-myricetin hybrid protected BEAS-2B cells against BLM-induced cell injury in an increased dose manner, and reduced wet/dry weight ratio, histological scoring, and inflammation cytokines IL-1ß, IL-6, IL-18, and TNF-α levels of lung tissue of the rats. Furthermore, we found colchicine-myricetin hybrid inhibited caspase-1, ASC, GSDMD, and NLRP-3 expression in vivo. Meanwhile, we used molecular docking to analyze the binding mode of colchicine-myricetin hybrid and human neutrophil elastase (HNE), it revealed that colchicine-myricetin hybrid showed strong binding affinity toward human neutrophil elastase when compared to its parent molecules. In conclusion, It is suggested that colchicine-myricetin hybrid antagonized acute lung injury by focusing on multi-targets via multi-mechanisms, and might be served as a potential therapeutic agent for acute lung injury.

Design, synthesis and biological evaluation of a novel colchicine-magnolol hybrid for inhibiting the growth of Lewis lung carcinoma in Vitro and in Vivo.

Colchicine is a bioactive alkaloid originally from Colchicum autumnale and possesses excellent antiproliferative activity. However, colchicine-associated severe toxicity, gastrointestinal side effects in particular, limits its further therapeutic use. In the current study, we thus designed and synthesized a novel hybrid (CMH) by splicing colchicine and magnolol, a multifunctional polyphenol showing favorable gastrointestinal protection. The antitumor activity of CMH in Lewis lung carcinoma (LLC) was then evaluated in vitro and in vivo. Biologically, CMH inhibited the growth of LLC cells with an IC50 of 0.26 µM, 100 times more potently than cisplatin (26.05 µM) did. Meanwhile, the cytotoxicity of CMH was 10-fold lower than that of colchicine in normal human lung cells (BEAS-2B). In C57BL/6 mice xenograft model, CMH (0.5 mg/kg) worked as efficacious as colchicine (0.5 mg/kg) to inhibit tumor growth and 2 times more potently than cisplatin (1 mg/kg). In terms of mortality, 7 out of 10 mice died in colchicine group (0.75 mg/kg), while no death was observed in groups receiving CMH or cisplatin at 0.75 mg/kg. Mechanistic studies using Western blot revealed that CMH dose-dependently suppressed the protein expression of phosphorylated ERK. Molecular docking analysis further indicated that CMH was well fitted in the colchicine binding site of tubulin and formed several hydrogen bonds with tubulin protein. These results enable our novel hybrid CMH as a potential antineoplastic agent with lower toxicity, and provide perquisites for further investigation to confirm the therapeutic potentiality of this novel hybrid.

Licochalcone E improves insulin sensitivity in palmitic acid-treated HepG2 cells through inhibition of the NLRP3 signaling pathway.

Our previous research demonstrated that compound licochalcone E can reduce glucose tolerance and lipid metabolism in diabetic rats, although its mechanism remains unknown. Here, we used palmitic acid (PA) to establish a PA-treated HepG2 model, and then examined glucose uptake, glucose consumption, and blood lipids to evaluate the effects of licochalcone E within the safe dose range in the model. Polymerase chain reaction (PCR) was used to detect the expression levels of key genes associated with liver gluconeogenesis; enzyme-linked immunosorbent assay (ELISA) was deployed to evaluate the concentration of inflammatory factors; and laser confocal microscopy and western blot were used to determine the levels of reactive oxygen species (ROS) and NLRP3 inflammasome signaling pathway-related proteins, respectively. Finally, molecular simulations were exploited to validate the interaction between licochalcone E and the NLRP3 inflammasome. The results demonstrated that licochalcone E showed no toxicity in the dose range of 2.5-40 µM. In this dose range, licochalcone E substantially increased the uptake and consumption of glucose in the insulin resistance model and dose-dependently reduced the concentration of total cholesterol. The PCR results indicated that licochalcone E dose-dependently reduced the expression of Glucose-6-phosphatase (G6Pase) and Phosphoenolpyruvate carboxykinase (PEPCK) genes and increased the expression of Glucose Transporter 4 (Glut4) in PA-treated HepG2. Moreover, the ELISA results revealed that licochalcone E significantly reduced the expression of TNF-α, IL-1ß, and IL-18. Confocal microscopy results showed that licochalcone E dramatically reduced the generation of ROS and the expressions of NLRP3 and its downstream caspase-1 in PA-treated HepG2 model. Western blot results further indicated that licochalcone E significantly reduced the expression of NLRP3, caspase-1 and IL-1ß in the model. Additionally, molecular simulations demonstrated that licochalcone E has good binding affinity for the NLPR3 inflammasome. We concluded that licochalcone E has the potential to be used as an insulin sensitizer by reducing the release of ROS and inflammatory factors following inhibition of the NLPR3 signaling pathway.

Recombinant ling zhi-8 enhances Tregs function to restore glycemic control in streptozocin-induced diabetic rats.

OBJECTIVES: To explore the effect of recombinant LZ-8 (rLZ-8) on streptozocin (STZ)-induced diabetic rats and further illustrate its underlying mechanism. METHODS: Rats were intraperitoneally injected with single-dose STZ 50 mg/kg for induction of type 1 diabetes (T1D), and then, the diabetic rats were treated with rLZ-8 for 3 months. The clinical symptoms, fasting blood glucose, insulin, cytokines, histopathology, flow cytometry and immunofluorescence were used to evaluate the therapeutic effect and underlying mechanism of rLZ-8 on alleviating diabetes mellitus (DM). KEY FINDINGS: Treatment with rLZ-8 obviously alleviated the clinical symptoms of T1D and dose-dependently reduced the levels of blood glucose, blood lipid and haemoglobin A1c (HbA1c) in diabetic rat model. Meanwhile, rLZ-8 markedly increased insulin secretion and protected against STZ-induced pancreatic tissue injury. Additionally, rLZ-8 dramatically inhibited the levels of TNF-α and IL-1ß, and obviously increased the level of IL-10 in serum and pancreas. Further investigation indicated that rLZ-8 treatment significantly increased the number of regulatory T cells (Tregs) and up-regulated the expression of Foxp3 to restore balance between anti-inflammatory and inflammatory cytokines. CONCLUSIONS: These data suggest that rLZ-8 can antagonize STZ-induced T1D, and its mechanism may be related to inhibit inflammation and enhance Tregs generation.

Synthesis, biological evaluation, and molecular docking study of novel allyl-retrochalcones as a new class of protein tyrosine phosphatase 1B inhibitors.

We describe herein the design, synthesis, and biological evaluation of a series of novel protein tyrosine phosphatase 1B (PTP1B) inhibitor retrochalcones having an allyl chain at the C-5 position of their B ring. Biological screening results showed that the majority of these compounds exhibited an inhibitory activity against PTP1B. Thus, preliminary structure-activity relationship (SAR) and quantitative SAR analyses were conducted. Among the compounds, 23 was the most potent inhibitor, exhibiting the highest in vitro inhibitory activity against PTP1B with an IC50 of 0.57 µM. Moreover, it displayed a significant hepatoprotective property via activation of the IR pathway in type 2 diabetic db/db mice. In addition, the results of our docking study showed that 23, as a specific inhibitor of PTP1B, effectively transformed the WPD loop from "close" to "open" in the active site. These results may reveal suitable compounds for the development of PTP1B inhibitors.

Correction: Design, synthesis, and evaluation of bitopic arylpiperazine-phthalimides as selective dopamine D3 receptor agonists.

[This corrects the article DOI: 10.1039/C8MD00237A.].

Design, synthesis, and evaluation of bitopic arylpiperazine-phthalimides as selective dopamine D3 receptor agonists.

The dopamine D3 receptor (D3R) is a proven therapeutic target for the treatment of neurological and neuropsychiatric disorders. In particular, D3R-selective ligands that can eliminate side effects associated with dopamine D2 receptor (D2R) therapeutics have been validated. However, the high homology in signaling pathways and the sequence similarity between D2R and D3R have rendered the development of D3R-selective ligands challenging. Herein, we designed and synthesized a series of piperazine-phthalimide bitopic ligands based on a fragment-based and molecular docking inspired design. Compound 9i was identified as the most selective D3R ligand among these bitopic ligands. Its selectivity was improved compared to reference compounds 1 and 2 by 9- and 2-fold, respectively, and it was 21-fold more potent than compound 2. Molecular docking demonstrated that the orientation of Leu2.64 and Phe7.39 and the packing at the junction of helices may affect the specificity for D3R over D2R. Functional evaluation revealed that D3R-selective ligand 9i displayed a subpicomolar agonist activity at D3R with a 199-fold increase in potency compared to quinpirole. These results may be useful for the fragment-based design of bitopic compounds as selective D3R ligands.

Antidiabetic effect of SN158 through PPARα/γ dual activation in ob/ob mice.

In this study, we aimed to demonstrate the antidiabetic potential of (E)-N-(4-(3-(5-bromo-4-hydroxy-2-methoxyphenyl)acryloyl) phenyl)-4-tert-butylbenzamide (SN158) through peroxisome proliferator-activated receptor (PPAR)-α/γ dual activation. SN158 interacted with both PPARα and PPARγ, and increased their transcriptional activities. Simultaneously, SN158 treatment led to an increase in adipogenic differentiation of 3T3-L1 preadipocytes and fatty acid oxidation in hepatocytes. In addition, glucose uptake in myotubes was significantly increased by SN158 treatment. Finally, SN158 significantly lowered the plasma levels of glucose, triglycerides, and free fatty acids in ob/ob mice without severe weight gain and hepatomegaly. These results suggest that SN158 can be useful as a potential therapeutic agent against type 2 diabetes and related metabolic disorders by alleviating glucose and lipid abnormalities.

Design, synthesis and evaluation of bitopic arylpiperazinephenyl-1,2,4-oxadiazoles as preferential dopamine D3 receptor ligands.

The dopamine D3 receptor (D3R) was proposed as a therapeutic target for drug development to treat drug abuse and addiction and neuropsychiatric disorders. Several D3R-selective modulators over the dopamine D2 receptor (D2R) can avoid extrapyramidal symptoms (EPS) and hyperprolactinemia. However, few biased D3R ligands were identified or showed a narrow range of selectivity at the D3R over D2R because of their high sequence homology. Herein, we designed, synthesized and evaluated the binding affinity of a series of bitopic ligands: arypiperazine-phenyl-1,2,4-oxadiazoles. Compound 9e·HCl was the most potent and selective D3R modulator among these bitopic ligands. Molecular modeling revealed that D3R selectivity depends on the divergence of secondary binding pocket (SBP) in D3R and D2R. Specifically, non-conserved Tyr36, EL1 especially non-conserved Thr92 and Gly94, and EL2 Val180, Cys181 and Ser182 of D3R may contribute to D3R specificity over D2R.

Design and synthesis of 4-benzylpiperidine carboxamides as dual serotonin and norepinephrine reuptake inhibitors.

A series of 4-benzylpiperidine carboxamides were designed and synthesized, and tested for their dual (serotonin and norepinephrine) reuptake inhibition. The synthesis of 4-benzylpiperidine carboxamides involved two main steps: amidation and substitution. Derivatives with 3 carbon linker displayed better activity than with 2 carbon linker. 4-Biphenyl- and 2-naphthyl-substituted derivatives 7e and 7j showed greater dual reuptake inhibition than standard drug venlafaxine HCl.

Synthesis and evaluation of arylpiperazine-reverse amides as biased dopamine D3 receptor ligands.

The dopamine D3 receptor (D3R) preferential ligands have been universally adopted as a strategy for the treatment of drug addiction and other neuropsychiatric disorders due to fewer side effects. However, the high sequence homology between D3R and the D2 receptor (D2R) challenges the development of D3R-biased compounds. Herein, we design and synthesize a novel series of reverse amide-piperazine hybrid ligands and evaluate their biological affinities in vitro. Compound 4d was found to be the most potent D3R-selective ligand among these hybrid derivatives. Molecular modeling revealed that extracellular loop 1 (EL1) and loop 2 (EL2) of D3R together likely contribute to D3R selectivity over D2R. In particular, Gly94 in EL1 of D3R may act as a molecular determinant for D3R specificity.

Concise synthesis of licochalcone C and its regioisomer, licochalcone H.

Licochalone C (7a) is a retrochalcone isolated from Glycyrrhiza inflata, which shows potent antioxidant properties and inhibition of bacterial growth and cellular respiration. Biological studies have suggested that licochalcone C attenuates the lipopolysaccharide and interferon-gamma induced inflammatory response by decreasing the expression and activity of inducible nitric oxide synthase and modulating the antioxidant network activity of superoxide dismutase, catalase, and glutathione peroxidase activity. Licochalcone C also inhibits NADH-cytochrome C reductase in the membrane fraction of Micrococcus luteus. Since pharmacological activity studies of licochalcone C are ongoing and the yield of the compound is poor from natural product, we report a concise four step synthesis of licochalcone C (7a) and its regioisomer, tentatively called licochalcone H (7b), by employing acid-mediated Claisen-Schmidt condensation as a key step with 6 and 20 % overall yield, respectively.

[Studies on the chemical constituents of Callicarpa kochiana].

OBJECTIVE: To study on the chemical constituents of Callicarpa kochiana. METHODS: The chemical constituents were isolated by chromatographic methods and structurally elucidated by spectral analysis. RESULTS: Twelve compounds were obtained and identified as alpha-amyrin(I), 2beta, 3beta, 19alpha-trihydroxy-12-en-28-ursolic acid (II), oleanolic aicd (III), alpha-amyrin-3-0-beta-D-glucopyranoside (IV), ursolic acid (V), betulinic acid (VI), 2alpha, 3beta,23-trihydroxy-12-en-28-oic-0-beta-D-glucopyranoside (VII), 0-hydroxybenzoic acid (VI), pomolic acid (IX), myrianthic acid (X), beta-sitosterol (XI), dauricine (XII). CONCLUSION: All of these compounds are isolated from Callicarpa kochiana for the first time and compounds II, IV, VII, VIII, IX and X are reported for the first time from Callicarpa genus.