ABSTRACT
Bioluminescence is a process that converts biochemical energy to visible light. Bioluminescence-based imaging technology has been widely applicable in the imaging of process envisioned in life sciences. As one of the most popular bioluminescence system, the firefly luciferin-luciferase system is exceptionally suitable for deep tissue imaging in living animals owing to its long wavelength emission light. Herein, we report the experimental detail of bioluminogenic imaging of aminopeptidase N activity both in vitro and in vivo.
Subject(s)
CD13 Antigens/metabolism , Luminescent Measurements/methods , Molecular Imaging/methods , Animals , Cell Line , In Vitro Techniques , Luciferases, Firefly/metabolism , Luminescent Agents , MiceABSTRACT
α1-Adrenergic receptors (α1-ARs), as the essential members of G protein-coupled receptors (GPCRs), can mediate numerous physiological responses in the sympathetic nervous system. In the current research, a series of quinazoline-based small-molecule fluorescent probes to α1-ARs (1a-1e), including two parts, a pharmacophore for α1-AR recognition and a fluorophore for visualization, were well designed and synthesized. The biological evaluation results displayed that these probes held reasonable fluorescent properties, high affinity, accepted cell toxicity, and excellent subcellular localization imaging potential for α1-ARs.
ABSTRACT
Reactive oxygen species (ROS) often have significant roles in mediating redox modifications and other essential physiological processes, such as biological process regulation and signal transduction. Considering that H2O2 is a substantial member of ROS, detection and quantitation of H2O2 undertakes important but urgent responsibility. In this report, a bioluminescent probe for detecting H2O2 was well designed, synthesized, and evaluated. This probe was designed into three parts: a H2O2-sensitive aryl boronic acid, a bioluminescent aminoluciferin moiety, and a self-immolative linker. After extensive evaluation, this probe can selectively and sensitively react with H2O2 to release aminoluciferin. It should be pointed out that this probe is a potential bioluminescent sensor for H2O2 since it can provide a promising toolkit for real-time detection of the H2O2 level in vitro, in cellulo, and in vivo.
Subject(s)
Boronic Acids/chemistry , Firefly Luciferin/analogs & derivatives , Hydrogen Peroxide/analysis , Luminescent Agents/chemistry , Molecular Imaging/methods , Molecular Probes/chemistry , Animals , Cell Line, Tumor , Female , Humans , Luminescent Measurements , Male , Mice , Mice, Nude , Neoplasm Transplantation , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Oxidation-Reduction , Reactive Oxygen Species/metabolismABSTRACT
Peptidases, which can cleave specific peptide bonds in innumerable categories of substrates, usually present pivotal positions in protein activation, cell signaling and regulation as well as in the origination of amino acids for protein generation or application in other metabolic pathways. They are also involved in many pathological conditions, such as cancer, atherosclerosis, arthritis, and neurodegenerative disorders. This review article aims to conduct a wide-ranging survey on the development of small-molecule fluorescent probes for peptidases, as well as to realize the state of the art in the tailor-made probes for diverse types of peptidases.
Subject(s)
Fluorescent Dyes/chemistry , Molecular Probes/chemistry , Molecular Probes/pharmacology , Peptide Hydrolases/drug effects , Peptide Hydrolases/metabolism , Drug Design , Humans , Protein Binding/drug effects , Signal TransductionABSTRACT
α1-Adrenergic receptors (α1-ARs), as one of the most important members of G protein-coupled receptors (GPCRs), can mediate lots of physiological responses of the sympathetic nervous system. Until now, α1-ARs have been divided into at least three subtypes, α1A, α1B, and α1D, which distribute in various tissues and organs. In this research, we designed and synthesized several napthalimide-based small-molecule fluorescent probes for α1-ARs, which mainly contained two parts: the pharmacophore (quinazoline and phenylpiperazine) that binds to α1-ARs and the fluorophore (naphthalimide) that labels the receptors with fluorescent properties. Moreover, some of these compounds demonstrated potent affinity to α1-ARs and cell imaging potential.
Subject(s)
Fluorescent Dyes/metabolism , Naphthalimides/metabolism , Piperazines/metabolism , Quinazolines/metabolism , Receptors, Adrenergic, alpha-1/metabolism , Fluorescent Dyes/chemical synthesis , HEK293 Cells , Humans , Microscopy, Fluorescence , Molecular Structure , Naphthalimides/chemical synthesis , Piperazines/chemical synthesis , Protein Binding , Quinazolines/chemical synthesis , Receptors, Adrenergic, alpha-1/genetics , TransfectionABSTRACT
To find an approach that can image the hydrolysis activity of aminopeptidase N (APN) both in vitro and in vivo, three bioluminescent probes have been well designed and synthesized herein. All of them can be recognized and hydrolyzed by APN to produce bioluminescence emission in the presence of firefly luciferase. To the best of our knowledge, they are the first bioluminescent probes for imaging APN in deep tissues and living animals.
Subject(s)
CD13 Antigens/metabolism , Molecular Probes , LuminescenceABSTRACT
Striatal-enriched tyrosine phosphatase (STEP) is an important regulator of neuronal synaptic plasticity, and its abnormal level or activity contributes to cognitive disorders. One crucial downstream effector and direct substrate of STEP is extracellular signal-regulated protein kinase (ERK), which has important functions in spine stabilisation and action potential transmission. The inhibition of STEP activity toward phospho-ERK has the potential to treat neuronal diseases, but the detailed mechanism underlying the dephosphorylation of phospho-ERK by STEP is not known. Therefore, we examined STEP activity toward para-nitrophenyl phosphate, phospho-tyrosine-containing peptides, and the full-length phospho-ERK protein using STEP mutants with different structural features. STEP was found to be a highly efficient ERK tyrosine phosphatase that required both its N-terminal regulatory region and key residues in its active site. Specifically, both kinase interaction motif (KIM) and kinase-specific sequence of STEP were required for ERK interaction. In addition to the N-terminal kinase-specific sequence region, S245, hydrophobic residues L249/L251, and basic residues R242/R243 located in the KIM region were important in controlling STEP activity toward phospho-ERK. Further kinetic experiments revealed subtle structural differences between STEP and HePTP that affected the interactions of their KIMs with ERK. Moreover, STEP recognised specific positions of a phospho-ERK peptide sequence through its active site, and the contact of STEP F311 with phospho-ERK V205 and T207 were crucial interactions. Taken together, our results not only provide the information for interactions between ERK and STEP, but will also help in the development of specific strategies to target STEP-ERK recognition, which could serve as a potential therapy for neurological disorders. Regulation of phospho-ERK by STEP underlies important neuronal activities. A detailed enzymologic characterisation and cellular studies of STEP revealed that specific residues in KIM and active site mediated ERK recognition. Structural differences between the KIM-ERK interfaces and the active site among different ERK phosphatases could be targeted to develop specific STEP inhibitor, which has therapeutic potential for neurological disorders. PKA, protein kinase A & NGF, nerve growth factor.
Subject(s)
Mitogen-Activated Protein Kinase 1/metabolism , Protein Tyrosine Phosphatases, Non-Receptor/metabolism , Amino Acid Sequence , Animals , Catalytic Domain , Models, Molecular , Molecular Sequence Data , Mutation , PC12 Cells , Phosphorylation , Protein Tyrosine Phosphatases, Non-Receptor/genetics , RatsABSTRACT
A novel fluorogenic probe for tert-butoxy radicals based on the hydrazino-naphthalimide system is reported. Interestingly, different regioisomers exhibited significantly different optical properties toward ROS, which suggested 4-hydrazinyl naphthalimide as a potential new platform for the in vitro and in cellulo detection of alkoxyl radicals.
Subject(s)
Alcohols/analysis , Fluorescent Dyes/chemistry , Hydrazines/chemistry , Naphthalimides/chemistry , Free Radicals/analysis , Molecular StructureABSTRACT
Bioluminescent imaging (BLI) has been widely applicable in the imaging of process envisioned in life sciences. As the most conventional technique for BLI, the firefly luciferin-luciferase system is exceptionally functional in vitro and in vivo. The state-of-the-art strategy in such a system is to cage the luciferin, in which free luciferin is conjugated with distinctive functional groups, thus accommodating an impressive toolkit for exploring various biological processes, such as monitoring enzymes activity, detecting bioactive small molecules, evaluating the properties of molecular transporters, etc. This review article summarizes the rational design of caged luciferins towards diverse biotargets, as well as their applications in bioluminescent imaging. It should be emphasized that these caged luciferins can stretch out the applications of bioluminescence imaging and shed light upon understanding the pathogenesis of various diseases.
Subject(s)
Firefly Luciferin/chemistry , Luminescent Agents/chemistry , Luminescent Measurements/methods , Optical Imaging/methods , Animals , Enzyme Assays/methods , Fireflies/enzymology , Firefly Luciferin/metabolism , Humans , Luciferases, Firefly/metabolism , Luminescent Agents/metabolism , Models, MolecularABSTRACT
In the current paper, three activity-based colorimetric and ratiometric fluorescent probes based on a naphthalimide fluorophore were well designed and synthesized, which can be recognized and hydrolyzed by aminopeptidase N (APN) at both the enzymatic and cellular level by following the fluorescent emission wavelength change from blue to green light. As a result, these molecules were successfully identified as the first ratiometric fluorescent probes for APN cell imaging.
