Selective ligand recognition and activation of somatostatin receptors SSTR1 and SSTR3.

Somatostatin receptors (SSTRs) exert critical biological functions such as negatively regulating hormone release and cell proliferation, making them popular targets for developing therapeutics to treat endocrine disorders, especially neuroendocrine tumors. Although several panagonists mimicking the endogenous ligand somatostatin are available, the development of more effective and safer somatostatinergic therapies is limited due to a lack of molecular understanding of the ligand recognition and regulation of divergent SSTR subtypes. Here, we report four cryoelectron microscopy structures of Gi-coupled SSTR1 and SSTR3 activated by distinct agonists, including the FDA-approved panagonist pasireotide as well as their selective small molecule agonists L-797591 and L-796778. Our structures reveal a conserved recognition pattern of pasireotide in SSTRs attributed to the binding with a conserved extended binding pocket, distinct from SST14, octreotide, and lanreotide. Together with mutagenesis analyses, our structures further reveal the dynamic feature of ligand binding pockets in SSTR1 and SSTR3 to accommodate divergent agonists, the key determinants of ligand selectivity lying across the orthosteric pocket of different SSTR subtypes, as well as the molecular mechanism underlying diversity and conservation of receptor activation. Our work provides a framework for rational design of subtype-selective SSTR ligands and may facilitate drug development efforts targeting SSTRs with improved therapeutic efficacy and reduced side effects.

Revealing the signaling of complement receptors C3aR and C5aR1 by anaphylatoxins.

The complement receptors C3aR and C5aR1, whose signaling is selectively activated by anaphylatoxins C3a and C5a, are important regulators of both innate and adaptive immune responses. Dysregulations of C3aR and C5aR1 signaling lead to multiple inflammatory disorders, including sepsis, asthma and acute respiratory distress syndrome. The mechanism underlying endogenous anaphylatoxin recognition and activation of C3aR and C5aR1 remains elusive. Here we reported the structures of C3a-bound C3aR and C5a-bound C5aR1 as well as an apo-C3aR structure. These structures, combined with mutagenesis analysis, reveal a conserved recognition pattern of anaphylatoxins to the complement receptors that is different from chemokine receptors, unique pocket topologies of C3aR and C5aR1 that mediate ligand selectivity, and a common mechanism of receptor activation. These results provide crucial insights into the molecular understanding of C3aR and C5aR1 signaling and structural templates for rational drug design for treating inflammation disorders.

Lysosome Targeting Chimaeras for Glut1-Facilitated Targeted Protein Degradation.

Targeted protein degradation technology holds great potential in biomedicine, particularly in treating tumors and other protein-related diseases. Research on intracellular protein degradation using molecular glues and PROTAC technology is leading, while research on the degradation of membrane proteins and extracellular proteins through the lysosomal pathway is still in the preclinical stage. The scarcity of useful targets is an immense limitation to technological advancement, making it essential to explore novel, potentially effective approaches for targeted lysosomal degradation. Here, we employed the glucose transporter Glut1 as an innovative lysosome-targeting receptor and devised the Glut1-Facilitated Lysosomal Degradation (GFLD) strategy. We synthesized potential Glut1 ligands via reversible addition-fragmentation chain transfer (RAFT) polymerization and acquired antibody-glycooligomer conjugates through bioorthogonal reactions as lysosome-targeting protein degradation molecules, utilized in the management of PD-L1 high-expressing triple-negative breast cancer. The glucose transporter Glut1 as a lysosome-targeting receptor exhibits potential for the advancement of a broader array of medications in the future.

The Role of Introgression During the Radiation of Endemic Fishes Adapted to Living at Extreme Altitudes in the Tibetan Plateau.

Recent genomic analyses of evolutionary radiations suggest that ancient introgression may facilitate rapid diversification and adaptive radiation. The loach genus Triplophysa, a genus with most species endemic to Tibetan Plateau, shows ecological diversity and rapid evolution and represents a potential example of adaptive radiation linked to the uplift of the Tibetan Plateau. Here, we interrogate the complex evolutionary history of Triplophysa fishes through the analysis of whole-genome sequences. By reconstructing the phylogeny of Triplophysa, quantifying introgression across this clade, and simulating speciation and migration processes, we confirm that extensive gene flow events occurred across disparate Triplophysa species. Our results suggest that introgression plays a more substantial role than incomplete lineage sorting in underpinning phylogenetic discordance in Triplophysa. The results also indicate that genomic regions affected by ancient gene flow exhibit characteristics of lower recombination rates and nucleotide diversity and may associate with selection. Simulation analysis of Triplophysa tibetana suggests that the species may have been affected by the Gonghe Movement in the third uplift of the Tibetan Plateau, resulting in founder effects and a subsequent reduction in Ne.

Molecular recognition of two endogenous hormones by the human parathyroid hormone receptor-1.

Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) are two endogenous hormones recognized by PTH receptor-1 (PTH1R), a member of class B G protein- coupled receptors (GPCRs). Both PTH and PTHrP analogs including teriparatide and abaloparatide are approved drugs for osteoporosis, but they exhibit distinct pharmacology. Here we report two cryo-EM structures of human PTH1R bound to PTH and PTHrP in the G protein-bound state at resolutions of 2.62 Å and 3.25 Å, respectively. Detailed analysis of these structures uncovers both common and unique features for the agonism of PTH and PTHrP. Molecular dynamics (MD) simulation together with site-directed mutagenesis studies reveal the molecular basis of endogenous hormones recognition specificity and selectivity to PTH1R. These results provide a rational template for the clinical use of PTH and PTHrP analogs as an anabolic therapy for osteoporosis and other disorders.

C=C Bond Oxidative Cleavage of BODIPY Photocages by Visible Light.

Photocages for protection and the controlled release of bioactive compounds have been widely investigated. However, the vast majority of these photocages employ the cleavage of single bonds and high-energy ultraviolet light. The construction of a photoactivation system that uses visible light to cleave unsaturated bonds still remains a challenge. Herein, we report a regioselective oxidative cleavage of C=C bonds from a boron-dipyrrolemethene (BODIPY)-based photocage by illumination at 630 nm, resulting in a free aldehyde and a thiol fluorescent probe. This strategy was demonstrated in live HeLa cells, and the generated α-formyl-BODIPY allowed real-time monitoring of aldehyde release in the cells. In particular, it is shown that a mannose-functionalized photocage can target HepG2 cells.

LSD2/KDM1B and its cofactor NPAC/GLYR1 endow a structural and molecular model for regulation of H3K4 demethylation.

Dynamic regulation of histone methylation represents a fundamental epigenetic mechanism underlying eukaryotic gene regulation, yet little is known about how the catalytic activities of histone demethylases are regulated. Here, we identify and characterize NPAC/GLYR1 as an LSD2/KDM1b-specific cofactor that stimulates H3K4me1 and H3K4me2 demethylation. We determine the crystal structures of LSD2 alone and LSD2 in complex with the NPAC linker region in the absence or presence of histone H3 peptide, at resolutions of 2.9, 2.0, and 2.25 Å, respectively. These crystal structures and further biochemical characterization define a dodecapeptide of NPAC (residues 214-225) as the minimal functional unit for its cofactor activity and provide structural determinants and a molecular mechanism underlying the intrinsic cofactor activity of NPAC in stimulating LSD2-catalyzed H3K4 demethylation. Thus, these findings establish a model for how a cofactor directly regulates histone demethylation and will have a significant impact on our understanding of catalytic-activity-based epigenetic regulation.

Correction: Zhao, X.W., et al. Dioscin Induces the Apoptosis of Human Cervical Carcinoma HeLa and SiHa Cells Through ROS-mediated DNA Damage, Cell Cycle Arrest and Mitochondrial Signaling Pathways. Molecules 2016, 21, 730.

During the course of a review of our publications, an error in the title paper [...].

Soluplus®/TPGS mixed micelles for co-delivery of docetaxel and piperine for combination cancer therapy.

In this study, mixed micelles of Soluplus® and TPGS were developed for co-administering docetaxel (DTX) and piperine (PIP) for exerting the synergistic effect, which increased the cytotoxicity and improved the anti-cancer activity in HepG2 cell lines compared to free DTX. These in vitro (MTT assay, intracellular uptake of micelles) and in vivo (pharmacokinetic study, immunostaining, TUNEL analysis) studies exhibited the advantages of co-delivery of anticancer drugs with Soluplus®/TPGS by mixed micelles and furthermore established that co-delivery of DTX and PIP via the mixed micelles of Soluplus®/TPGS could be a promising strategy for the treatment of liver cancer.

Mixed micelles of TPGS and Soluplus® for co-delivery of paclitaxel and fenretinide: in vitro and in vivo anticancer study.

Fenretinide (4-HPR), as a semi-synthetic retinoid, has apoptosis-promoting effects as a single agent and chemotherapy synergist in vitro. When a human ovarian cancer cells line (A2780s) was treated with both PTX and 4-HPR, there was a synergistic anti-cancer effect demonstrated with a average combination index of 0.44. In this research, a new TPGS-Soluplus® mixed micelles were developed which encapsulation efficiencies of paclitaxel (PTX) and fenretinide (4-HPR) were as high as 98%, and the average diameter of the micelles was 66.26 nm. Cytotoxicity of the mixed micelles co-delivered with PTX and 4-HPR reduced significantly 7.3 and 25.1 times compared with free drug respectively in A2780s cells. More importantly, in vivo pharmacokinetic study, the loaded drugs in mixed micelles exhibited higher AUC and t1/2 values than free drugs. Furthermore, in vivo antitumor efficacy experiments demonstrated that PF-TS exhibited superior in vivo antitumor activity on the inhibition rate of tumor growth than other treatment groups (77.8% corresponding tumor growth inhibition in PF-TS treated group vs 19.9, 12.5, and 26.0% of tumor growth inhibition rate in Taxol®, 4-HPR, and Taxol®+4-HPR, respectively). Therefore, the mixed micelles of co-deliver PTX and 4-HPR successfully constructed may hopefully be applied to the cancer combination treatment with less toxic effect and more antitumor activity.

Fenretinide-polyethylene glycol (PEG) conjugate with improved solubility enhanced cytotoxicity to cancer cell and potent in vivo efficacy.

Fenretinide (4-HPR), a synthetic retinoid, has shown its antitumor activity in many tumor types with low cytotoxicity to normal cells and high clinical safety. However, the low water solubility limits its further biological applications. To increase solubility, 4-HPR was conjugated with methoxy polyethylene glycol carboxylic acid (mPEG2K-COOH) by an ester linkage between the phenol hydroxyl of 4-HPR and the carboxyl of mPEG2K-COOH. The 4-HPR-PEG2K conjugate micelles had mean size of 76.70 ± 1.248 nm with a narrow distribution and a low critical micelle concentration. In vitro cytotoxicity studies showed the micelles have higher cytotoxicity to A2780s and MCF-7 cells. Its IC50 was 4.7 and 4.1-fold lower than the free 4-HPR, respectively. Importantly, in vivo pharmacokinetic studies, the AUC of 4-HPR was found to be 2.3-fold higher in 4-HPR-PEG2K micelles compared to free 4-HPR. And the 4-HPR-PEG2K micelles had higher antitumor activity. Meanwhile, the histopathology analysis exhibited that the micellar treatment decreased the viability of A2780s cells and increased the level of induced apoptosis. Therefore, the enhanced activity of 4-HPR by the method of conjugation with mPEG2K-COOH could hopefully provide new insights into the matter of ovarian cancer and breast cancer treatment.

Dioscin ameliorates intestinal ischemia/reperfusion injury via adjusting miR-351-5p/MAPK13-mediated inflammation and apoptosis.

Inflammatory reaction and cell apoptosis are two important processes in intestinal ischemia/reperfusion (II/R) injury, and exploration of effective lead compounds against II/R injury via regulating inflammation and apoptosis is critical important. In this paper, the results indicated that dioscin significantly increased cell viability, and inhibited inflammation and apoptosis caused by hypoxia-reoxygenation (H/R) injury in IEC-6 cells. in vivo II/R injury, dioscin markedly suppressed inflamma- tion and apoptosis, improved pathological changes, and depressed chiu' score in rats. Mechanistic studies indicated that dioscin notably up-regulated the expression level of MAPK13 through decreasing miR-351-5p level, and thereby decreased the expression levels of p-PKD1, NF-κB, Apaf-1, cleaved Caspase-3 and cleaved Caspase-9. Furthermore, miR-351-5p mimic and inhibitor experiments in IEC-6 cells further proved that dioscin up-regulated MAPK13 expression by decreasing miR-351-5p level to inhibit inflammation and apoptosis. Therefore, dioscin showed protective effect against II/R injury via adjusting miR-351-5/MAPK13-mediated inflammation and apoptosis. Dioscin should be considered as one potent candidate and miR-351-5/ MAPK13 should be one effective drug target for the treatment of II/R injury.

Correction to: Protective effects of dioscin against alcohol­induced liver injury.

During the course of writing and revision of this paper, the band of GAPDH.

Neuroprotective Effect of Dioscin on the Aging Brain.

Our previous works have shown that dioscin, a natural product, has various pharmacological activities, however, its role in brain aging has not been reported. In the present study, in vitro H2O2-treated PC12 cells and in vivo d-galactose-induced aging rat models were used to evaluate the neuroprotective effect of dioscin on brain aging. The results showed that dioscin increased cell viability and protected PC12 cells against oxidative stress through decreasing reactive oxygen species (ROS) and lactate dehydrogenase (LDH) levels. In vivo, dioscin markedly improved the spatial learning ability and memory of aging rats, reduced the protein carbonyl content and aging cell numbers, restored the levels of superoxide dismutase (SOD), glutathione (GSH), glutathione peroxidase (GSH-Px), malondialdehyde (MDA) and nitric oxide synthase (NOS) in brain tissue, and reversed the histopathological structure changes of nerve cells. Mechanism studies showed that dioscin markedly adjusted the MAPK and Nrf2/ARE signalling pathways to decrease oxidative stress. Additionally, dioscin also significantly decreased inflammation by inhibiting the mRNA or protein levels of TNF-α, IL-1ß, IL-6, CYP2E1 and HMGB1. Taken together, these results indicate that dioscin showed neuroprotective effect against brain aging via decreasing oxidative stress and inflammation, which should be developed as an efficient candidate in clinical to treat brain aging in the future.

Correction: Liu, M., et al. Potent Effects of Flavonoid-Rich Extract from Rosa laevigata Michx Fruit against Hydrogen Peroxide-Induced Damage in PC12 Cells via Attenuation of Oxidative Stress, Inflammation and Apoptosis. Molecules 2014, 19, 11816-11832.

During the course of a review of our publication, an error in the title paper [...].

Mn-Catalyzed Dehydrocyanative Transannulation of Heteroarenes and Propargyl Carbonates through C-H Activation: Beyond the Permanent Directing Effects of Pyridines/Pyrimidines.

Pyridines/pyrimidines are common and effective directing groups in C-H activation. However, they are poorly functionalizable heteroarenes. Reported in this work is Mn-catalyzed dehydrocyanative transannulation between three classes of heteroarenes and propargyl carbonates. The pyridyl/pyrimidyl groups in the heteroarenes initially function as directing groups to enable C-H allenylation; they then undergo intramolecular Diels-Alder/retro-Diels-Alder reactions with the allene moiety to afford fused carbo/heterocycles. Three key intermediates at different stages of the reaction were isolated.

Dioscin alleviates non-alcoholic fatty liver disease through adjusting lipid metabolism via SIRT1/AMPK signaling pathway.

Dioscin, one natural product, has active effect against non-alcoholic fatty liver disease (NAFLD) in our previous work. However, the pharmacological data are insufficient and the mechanisms have not been reported. Thus, this study aims to comprehensively investigate the effects and molecular mechanisms of dioscin against NAFLD. The primary cultured hepatocytes, AML-12 and HepG-2 cells were treated with palmic acid (PA) after dioscin treatment. The mice and rats were induced by high fat diet to establish the in vivo models of NAFLD. Dioscin obviously alleviated liver lipid accumulation symptoms and improved the levels of serum and hepatic biochemical parameters in vitro and in vivo. Further investigations revealed that dioscin significantly attenuated lipid metabolism via adjusting SIRT1/AMPK signal pathway to regulate the expression levels of SREBP-1c, CPT, FAS, SCD, FoxO1 and ATGL. In addition, suppression of SIRT1 by Nicotinamide or abrogation of AMPK by Compound C eliminated the inhibitory effects of dioscin on lipid metabolism. Therefore, our findings further demonstrated that dioscin markedly prevented NAFLD through adjusting lipid metabolism via SIRT1/AMPK signal pathway, which should be developed as a new candidate for NAFLD.

Development and evaluation of a novel drug delivery: Soluplus®/TPGS mixed micelles loaded with piperine in vitro and in vivo.

BACKGROUND: Although piperine can inhibit cells of tumors, the poor water solubility restricted its clinical application. This paper aimed to develop mixed micelles based on Soluplus® and D-α-tocopherol polyethylene glycol succinate (TPGS) to improve the aqueous solubility and anti-cancer effect. METHODS: Piperine-loaded mixed micelles were prepared using a thin-film hydration method, and their physicochemical properties were characterized. The cellular uptake of the micelles was confirmed by confocal laser scanning microscopy in A549 lung cancer cells and HepG2 liver cancer cells. In addition, cytotoxicity of the piperine mixed micelles was studied in A549 lung cancer cells and HepG2 liver cancer cells. Free piperine or piperine-loaded Soluplus®/TPGS mixed micelles were administered at an equivalent dose of piperine at 3.2 mg/kg via a single intravenous injection in the tail vain for the pharmacokinetic study in vivo. RESULTS: The diameter of piperine-loaded Soluplus®/TPGS (4:1) mixed micelles was about 61.9 nm and the zeta potential -1.16 ± 1.06 mV with 90.9% of drug encapsulation efficiency and 4.67% of drug-loading efficiency. Differential scanning calorimetry (DSC) studies confirmed that piperine is encapsulated by the Soluplus®/TPGS. The release results in vitro showed that the piperine-loaded Soluplus®/TPGS mixed micelles presented sustained release behavior compared to the free piperine. The mixed micelles exhibited better antitumor efficacy compared to free piperine and physical mixture against in A549 and HepG2 cells by MTT assay. The pharmacokinetic study revealed that the AUC of piperine-loaded mixed micelles was 2.56 times higher than that of piperine and the MRT for piperine-loaded mixed micelles was 1.2-fold higher than piperine (p < .05). CONCLUSION: The results of the study suggested that the piperine-loaded mixed micelles developed might be a potential nano-drug delivery system for cancer chemotherapy. These results demonstrated that piperine-loaded Soluplus®/TPGS mixed micelles are an effective strategy to deliver piperine for cancer therapy.