ABSTRACT
AIMS: Beinaglutide has been approved for glucose lowering in type 2 diabetes mellitus (T2DM) in China. In addition to glycemic control, significant weight loss is observed from real world data. This study is designed to investigate the pharmacological and pharmacokinetic profiles of beinaglutide in different models. METHODS: The pharmacological efficacy of beinaglutide was evaluated in C57BL/6 and ob/ob mice after single administration. Pharmacokinetic profiles in mice were investigated after single or multiple administration. Sub-chronic pharmacological efficacy was investigated in ob/ob mice for two weeks treatment and diet-induced ob/ob mice model of nonalcoholic steatohepatitis (NASH) for four weeks treatment. KEY FINDINGS: Beinaglutide could dose-dependently reduce the glucose levels and improve insulin secretion in glucose tolerance tests, inhibit food intake and gastric emptying after single administration. At higher doses, beinaglutide could inhibit food intake over 4 h, which results in weight loss in ob/ob mice after about two weeks treatment. No tachyphylaxis is observed for beinaglutide in food intake with repeated administration. In NASH model, beinaglutide could reduce liver weight and hepatic steatosis and improve insulin sensitivity. Signiant changes of gene levels were observed in fatty acid ß-oxidation (Ppara, Acadl, Acox1), mitochondrial function (Mfn1, Mfn2), antioxidation (Sod2), Sirt1, and et al. SIGNIFICANCE: Our results characterize the pharmacological and pharmacokinetic profiles of beinaglutide in mice and supported that chronic use of beinaglutde could lead to weight loss and reduce hepatic steatosis, which suggest beinaglutide may be effective therapy for the treatment of obesity and NASH.
Subject(s)
Diabetes Mellitus/metabolism , Glucagon-Like Peptide 1/analogs & derivatives , Hypoglycemic Agents/pharmacology , Non-alcoholic Fatty Liver Disease/drug therapy , Obesity/metabolism , Peptide Fragments/pharmacology , Animals , Antioxidants/pharmacology , Diabetes Complications/drug therapy , Diabetes Mellitus/drug therapy , Glucagon-Like Peptide 1/metabolism , Glucagon-Like Peptide 1/pharmacology , Hypoglycemic Agents/metabolism , Insulin/metabolism , Insulin Resistance , Leptin/metabolism , Liraglutide/pharmacology , Liver Cirrhosis/metabolism , Liver Cirrhosis/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Obese , Non-alcoholic Fatty Liver Disease/metabolism , Obesity/complications , Oxidation-Reduction , PPAR alpha/metabolism , Peptide Fragments/chemistry , Weight Loss/drug effectsABSTRACT
In recent years, bioorthogonal reactions have emerged as a powerful toolbox for specific labeling and visualization of biomolecules, even within the highly complex and fragile living systems. Among them, copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction is one of the most widely studied and used biocompatible reactions. The cytotoxicity of Cu(I) ions has been greatly reduced due to the use of Cu(I) ligands, which enabled the CuAAC reaction to proceed on the cell surface, as well as within an intracellular environment. Meanwhile, other transition metals such as ruthenium, rhodium and silver are now under development as alternative sources for catalyzing bioorthogonal cycloadditions. In this review, we summarize the development of CuAAC reaction as a prominent bioorthogonal reaction, discuss various ligands used in reducing Cu(I) toxicity while promoting the reaction rate, and illustrate some of its important biological applications. The development of additional transition metals in catalyzing cycloaddition reactions will also be briefly introduced.
Subject(s)
Transition Elements/chemistry , Alkynes/chemistry , Amino Acids/chemistry , Azides/chemistry , Catalysis , Cycloaddition Reaction , Light , Lipids/chemistry , Nucleic Acids/chemistry , Polysaccharides/chemistryABSTRACT
Heme plays pivotal roles in various cellular processes as well as in iron homeostasis in living systems. Here, we report a genetically encoded fluorescence resonance energy transfer (FRET) sensor for selective heme imaging by employing a pair of bacterial heme transfer chaperones as the sensory components. This heme-specific probe allows spatial-temporal visualization of intracellular heme distribution within living cells.
Subject(s)
Biosensing Techniques/methods , Fluorescence Resonance Energy Transfer/methods , Heme/analysis , Animals , Bacillus anthracis/genetics , Bacillus anthracis/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cell Line , Cell Line, Tumor , Cricetinae , Genetic Engineering , Heme/metabolism , Humans , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Mice , Optical Imaging/methodsABSTRACT
Bioorthogonal reactions, especially the Cu(I)-catalysed azide-alkyne cycloaddition, have revolutionized our ability to label and manipulate biomolecules under living conditions. The cytotoxicity of Cu(I) ions, however, has hindered the application of this reaction in the internal space of living cells. By systematically surveying a panel of Cu(I)-stabilizing ligands in promoting protein labelling within the cytoplasm of Escherichia coli, we identify a highly efficient and biocompatible catalyst for intracellular modification of proteins by azide-alkyne cycloaddition. This reaction permits us to conjugate an environment-sensitive fluorophore site specifically onto HdeA, an acid-stress chaperone that adopts pH-dependent conformational changes, in both the periplasm and cytoplasm of E. coli. The resulting protein-fluorophore hybrid pH indicators enable compartment-specific pH measurement to determine the pH gradient across the E. coli cytoplasmic membrane. This construct also allows the measurement of E. coli transmembrane potential, and the determination of the proton motive force across its inner membrane under normal and acid-stress conditions.
Subject(s)
Click Chemistry/methods , Escherichia coli/chemistry , Biocompatible Materials/chemistry , Copper/chemistry , Cytoplasm/chemistry , Electrochemistry , Escherichia coli Proteins/chemistry , Flow Cytometry , Fluorescent Dyes/chemistry , Green Fluorescent Proteins/chemistry , Hydrogen-Ion Concentration , Ions , Ligands , Membrane Potentials , Periplasm/chemistry , Plasmids , Proton-Motive Force , Reactive Oxygen Species/chemistryABSTRACT
Considerable attention has been focused on improving the biocompatibility of Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), a hallmark of bioorthogonal reaction, in living cells. Besides creating copper-free versions of click chemistry such as strain promoted azide-alkyne cycloaddition (SPAAC), a central effort has also been made to develop various Cu(I) ligands that can prevent the cytotoxicity of Cu(I) ions while accelerating the CuAAC reaction. Meanwhile, additional transition metals such as palladium have been explored as alternative sources to promote a bioorthogonal conjugation reaction on cell surface, as well as within an intracellular environment. Furthermore, transition metal mediated chemical conversions beyond conjugation have also been utilized to manipulate protein activity within living systems. We highlight these emerging examples that significantly enriched our protein chemistry toolkit, which will likely expand our view on the definition and applications of bioorthogonal chemistry.
Subject(s)
Proteins/chemistry , Transition Elements/chemistry , Alkynes/chemistry , Azides/chemistry , Bacteria/metabolism , Catalysis , Click Chemistry , Copper/chemistry , Cycloaddition Reaction , Palladium/chemistry , Proteins/metabolism , Transition Elements/metabolismABSTRACT
Employing small molecules or chemical reagents to modulate the function of an intracellular protein, particularly in a gain-of-function fashion, remains a challenge. In contrast to inhibitor-based loss-of-function approaches, methods based on a gain of function enable specific signalling pathways to be activated inside a cell. Here we report a chemical rescue strategy that uses a palladium-mediated deprotection reaction to activate a protein within living cells. We identify biocompatible and efficient palladium catalysts that cleave the propargyl carbamate group of a protected lysine analogue to generate a free lysine. The lysine analogue can be genetically and site-specifically incorporated into a protein, which enables control over the reaction site. This deprotection strategy is shown to work with a range of different cell lines and proteins. We further applied this biocompatible protection group/catalyst pair for caging and subsequent release of a crucial lysine residue in a bacterial Type III effector protein within host cells, which reveals details of its virulence mechanism.
Subject(s)
Palladium/chemistry , Proteins/metabolism , Bacteria/metabolism , Green Fluorescent Proteins/genetics , HeLa Cells , Humans , Mass Spectrometry , Proteins/genetics , Signal TransductionABSTRACT
Palladium, a key transition metal in advancing modern organic synthesis, mediates diverse chemical conversions including many carbon-carbon bond formation reactions between organic compounds. However, expanding palladium chemistry for conjugation of biomolecules such as proteins, particularly within their native cellular context, is still in its infancy. Here we report the site-specific protein labeling inside pathogenic Gram-negative bacterial cells via a ligand-free palladium-mediated cross-coupling reaction. Two rationally designed pyrrolysine analogues bearing an aliphatic alkyne or an iodophenyl handle were first encoded in different enteric bacteria, which offered two facial handles for palladium-mediated Sonogashira coupling reaction on proteins within these pathogens. A GFP-based bioorthogonal reaction screening system was then developed, allowing evaluation of both the efficiency and the biocompatibilty of various palladium reagents in promoting protein-small molecule conjugation. The identified simple compound-Pd(NO3)2 exhibited high efficiency and biocompatibility for site-specific labeling of proteins in vitro and inside living E. coli cells. This Pd-mediated protein coupling method was further utilized to label and visualize a Type-III Secretion (T3S) toxin-OspF in Shigella cells. Our strategy may be generally applicable for imaging and tracking various virulence proteins within Gram-negative bacterial pathogens.
Subject(s)
Bacterial Proteins/analysis , Escherichia coli/cytology , Palladium/chemistry , Shigella/cytology , Green Fluorescent Proteins/analysis , Staining and Labeling/methodsABSTRACT
Live-cell pH measurements: An environment-sensitive fluorophore (green) was site-specifically introduced on HdeA, an acid-resistant chaperone showing pH-mediated conformational changes under low pH conditions. A survey of the attachment sites led to the discovery of one position on HdeA at which the attached fluorophore showed a strong fluorescence increase upon acidification.
Subject(s)
Acids/analysis , Fluorescent Dyes/chemistry , Indicators and Reagents/chemistry , Proteins/chemistry , Animals , Cell Line , Escherichia coli/chemistry , Escherichia coli/cytology , Fluorescent Dyes/chemical synthesis , Hydrogen-Ion Concentration , Mice , Models, Molecular , Molecular Structure , Solvents/chemistry , Spectrometry, FluorescenceABSTRACT
Expressed protein ligation bridges the gap between synthetic peptides and recombinant proteins and thereby significantly increases the size and complexity of chemically synthesized proteins. Although the intein-based expressed protein ligation method has been extensively used in this regard, the development of new expressed protein ligation methods may improve the flexibility and power of protein semisynthesis. In this study a new alternative version of expressed protein ligation is developed by combining the recently developed technologies of hydrazide-based peptide ligation and genetic code expansion. Compared to the previous intein-based expressed protein ligation method, the new method does not require the use of protein splicing technology and generates recombinant protein α-hydrazides as ligation intermediates that are more chemically stable than protein α-thioesters. Furthermore, the use of an evolved mutant pyrrolysyl-tRNA synthetase(PylRS), ACPK-RS, from M. barkeri shows an improved performance for the expression of recombinant protein backbone oxoesters. By using HdeA as a model protein we demonstrate that the hydrazide-based method can be used to synthesize proteins with correctly folded structures and full biological activity. Because the PylRS-tRNACUAPyl system is compatible with both prokaryotic and eukaryotic cells,the strategy presented here may be readily expanded to manipulate proteins produced in mammalian cells. The new hydrazide-based method may also supplement the intein-based expressed protein ligation method by allowing for a more flexible selection of ligation site.
Subject(s)
Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Escherichia coli/genetics , Hydrazines/chemistry , Hydroxy Acids/chemistry , Protein Engineering/methods , Escherichia coli/chemistry , Escherichia coli/metabolism , Escherichia coli Proteins/metabolism , Gene Expression , Hydrazines/metabolism , Hydroxy Acids/metabolism , Models, Molecular , Protein Folding , Protein Stability , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolismABSTRACT
A concise route was developed for the facile synthesis of a cyclic pyrrolysine analogue bearing an azide handle. Directed evolution enabled the encoding of this non-natural amino acid in both prokaryotic and eukaryotic cells, which offers a highly efficient approach for the site-specific protein labeling using click chemistry.