ABSTRACT
BACKGROUND: Aptamers are screened via the systematic evolution of ligands by exponential enrichment (SELEX) and are widely used in molecular diagnostics and targeted therapies. The development of efficient and convenient SELEX technology has facilitated rapid access to high-performance aptamers, thereby advancing the aptamer industry. Graphene oxide (GO) serves as an immobilization matrix for libraries in GO-SELEX, making it suitable for screening aptamers against diverse targets. RESULTS: This review summarizes the detailed steps involved in GO-SELEX, including monitoring methods, various sublibrary acquisition methods, and practical applications from its inception to the present day. In addition, the potential of GO-SELEX in the development of broad-spectrum aptamers is explored, and its current limitations for future development are emphasized. This review effectively promotes the application of the GO-SELEX technique by providing valuable insights and assisting researchers interested in conducting related studies. SIGNIFICANCE AND NOVELTY: To date, no review on the topic of GO-SELEX has been published, making it challenging for researchers to initiate studies in this area. We believe that this review will broaden the SELEX options available to researchers, ensuring that they can meet the growing demand for molecular probes in the scientific domain.
Subject(s)
Aptamers, Nucleotide , Graphite , Molecular Probes , SELEX Aptamer Technique , Graphite/chemistry , SELEX Aptamer Technique/methods , Aptamers, Nucleotide/chemistry , Molecular Probes/chemistry , HumansABSTRACT
Natural products-based screening of active ingredients and their interactions with target proteins is an important ways to discover new drugs. Assessing the binding capacity of target proteins, particularly when multiple components are involved, presents a significant challenge for sensors. As far as we know, there is currently no sensor that can accomplish high-throughput quantitative analysis of natural product-target protein binding capacity based on Raman spectroscopy. In this study, a novel sensor model has been developed for the quantitative analysis of binding capacity based on Surface-Enhanced Raman Spectroscopy (SERS) and Photocrosslinked Molecular Probe (PCMP) technology. This sensor, named SERS-PCMP, leverages the high throughput of molecular probe technology to investigate the active ingredients in natural products, along with the application of SERS labelling technology for target proteins. Thus it significantly improves the efficiency and accuracy of target protein identification. Based on the novel strategy, quantitative analysis of the binding capacity of 20 components from Shenqi Jiangtang Granules (SJG) to α-Glucosidase were completed. Ultimately, the binding capacity of these active ingredients was ranked based on the detected Raman Intensity. The compounds with higher binding capacity were Astragaloside IV (Intensity, 138.17), Ginsenoside Rh2 (Intensity, 87.46), Ginsenoside Rg3 (Intensity, 73.92) and Ginsenoside Rh1 (Intensity, 64.37), which all exceeded the binding capacity of the positive drug Acarbose (Intensity, 28.75). Furthermore, this strategy also performed a high detection sensitivity. The limit of detection for the enzyme using 0.1 mg of molecular probe magnetic nanoparticles (MP MNPs) was determined to be no less than 0.375 µg/mL. SERS-PCMP sensor integrating SERS labeling and photocrosslinked molecular probes which offers a fresh perspective for future drug discovery studies. Such as high-throughput drug screening and the exploration of small molecule-target protein interactions in vitro.
Subject(s)
Biological Products , Molecular Probes , Spectrum Analysis, Raman , Spectrum Analysis, Raman/methods , Biological Products/chemistry , Biological Products/analysis , Molecular Probes/chemistry , alpha-Glucosidases/metabolism , alpha-Glucosidases/chemistry , Protein Binding , Photochemical Processes , Cross-Linking Reagents/chemistry , Silver/chemistryABSTRACT
Chemical proteomics using biotin probes of natural products have significantly advanced our understanding of molecular targets and therapeutic potential. This review highlights recent progress in the application of biotin probes of homoisoflavonoids for identifying binding proteins and elucidating mechanisms of action. Notably, homoisoflavonoids exhibit antiangiogenic, anti-inflammatory, and antidiabetic effects. A combination of biotin probes, pull-down assays, mass spectrometry, and molecular modeling has revealed how natural products and their derivatives interact with several proteins such as ferrochelatase (FECH), soluble epoxide hydrolase (sEH), inosine monophosphate dehydrogenase 2 (IMPDH2), phosphodiesterase 4 (PDE4), and deoxyhypusine hydroxylase (DOHH). These target identification approaches pave the way for new therapeutic avenues, especially in the fields of oncology and ophthalmology. Future research aimed at expanding the repertoire of target identification using biotin probes of homoisoflavonoids promises to further elucidate the complex mechanisms and develop new drug candidates.
Subject(s)
Angiogenesis Inhibitors , Anti-Inflammatory Agents , Biotin , Humans , Biotin/chemistry , Anti-Inflammatory Agents/pharmacology , Anti-Inflammatory Agents/chemistry , Angiogenesis Inhibitors/pharmacology , Angiogenesis Inhibitors/chemistry , Animals , Isoflavones/pharmacology , Isoflavones/chemistry , Molecular Probes/chemistryABSTRACT
The proton-sensing heterotrimeric guanine nucleotide-binding protein-coupled receptor GPR65 is expressed in immune cells and regulates tissue homeostasis in response to decreased extracellular pH, which occurs in the context of inflammation and tumorigenesis. Genome-wide association studies linked GPR65 to several autoimmune and inflammatory diseases such as multiple sclerosis and inflammatory bowel disease (IBD). The loss-of-function GPR65 I231L IBD risk variant alters cellular metabolism, impairs protective tissue functions, and increases proinflammatory cytokine production. Hypothesizing that a small molecule designed to potentiate GPR65 at subphysiological pH could decrease inflammatory responses, we found positive allosteric modulators of GPR65 that engage and activate both human and mouse orthologs of the receptor. We observed that the chemical probe BRD5075 alters cytokine and chemokine programs in dendritic cells, establishing that immune signaling can be modulated by targeting GPR65. Our investigation offers improved chemical probes to further interrogate the biology of human GPR65 and its clinically relevant genetic variants.
Subject(s)
Cytokines , Inflammatory Bowel Diseases , Receptors, G-Protein-Coupled , Signal Transduction , Receptors, G-Protein-Coupled/metabolism , Receptors, G-Protein-Coupled/genetics , Humans , Inflammatory Bowel Diseases/metabolism , Inflammatory Bowel Diseases/genetics , Animals , Mice , Allosteric Regulation , Cytokines/metabolism , Dendritic Cells/metabolism , Dendritic Cells/drug effects , Molecular Probes/chemistryABSTRACT
Seed germination of a parasitic plant Striga hermonthica is elicited by strigolactones which are exuded from roots of host plants. Here, we describe a high-throughput germination assay and a method for visualizing in vivo strigolactone receptor functions with a fluorogenic probe.
Subject(s)
Germination , Lactones , Seeds , Striga , Striga/physiology , Striga/growth & development , Striga/drug effects , Seeds/growth & development , Lactones/metabolism , Lactones/pharmacology , Plant Roots/growth & development , Plant Roots/parasitology , Molecular Probes/chemistry , Fluorescent Dyes/chemistryABSTRACT
Activin receptor-like kinases 1-7 (ALK1-7) regulate a complex network of SMAD-independent as well as SMAD-dependent signaling pathways. One of the widely used inhibitors for functional investigations of these processes, in particular for bone morphogenetic protein (BMP) signaling, is LDN-193189. However, LDN-193189 has insufficient kinome-wide selectivity complicating its use in cellular target validation assays. Herein, we report the identification and comprehensive characterization of two chemically distinct highly selective inhibitors of ALK1 and ALK2, M4K2234 and MU1700, along with their negative controls. We show that both MU1700 and M4K2234 efficiently block the BMP pathway via selective in cellulo inhibition of ALK1/2 kinases and exhibit favorable in vivo profiles in mice. MU1700 is highly brain penetrant and shows remarkably high accumulation in the brain. These high-quality orthogonal chemical probes offer the selectivity required to become widely used tools for in vitro and in vivo investigation of BMP signaling.
Subject(s)
Activin Receptors, Type II , Animals , Humans , Mice , Activin Receptors, Type II/metabolism , Activin Receptors, Type II/antagonists & inhibitors , Activin Receptors, Type I/antagonists & inhibitors , Activin Receptors, Type I/metabolism , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/chemistry , Structure-Activity Relationship , Signal Transduction/drug effects , Drug Discovery , Molecular Probes/chemistry , Bone Morphogenetic Proteins/metabolism , Pyrazoles/chemistry , Pyrazoles/pharmacology , Pyrazoles/chemical synthesisABSTRACT
Background: Biomarker-driven molecular imaging has emerged as an integral part of cancer precision radiotherapy. The use of molecular imaging probes, including nanoprobes, have been explored in radiotherapy imaging to precisely and noninvasively monitor spatiotemporal distribution of biomarkers, potentially revealing tumor-killing mechanisms and therapy-induced adverse effects during radiation treatment. Methods: We summarized literature reports from preclinical studies and clinical trials, which cover two main parts: 1) Clinically-investigated and emerging imaging biomarkers associated with radiotherapy, and 2) instrumental roles, functions, and activatable mechanisms of molecular imaging probes in the radiotherapy workflow. In addition, reflection and future perspectives are proposed. Results: Numerous imaging biomarkers have been continuously explored in decades, while few of them have been successfully validated for their correlation with radiotherapeutic outcomes and/or radiation-induced toxicities. Meanwhile, activatable molecular imaging probes towards the emerging biomarkers have exhibited to be promising in animal or small-scale human studies for precision radiotherapy. Conclusion: Biomarker-driven molecular imaging probes are essential for precision radiotherapy. Despite very inspiring preliminary results, validation of imaging biomarkers and rational design strategies of probes await robust and extensive investigations. Especially, the correlation between imaging biomarkers and radiotherapeutic outcomes/toxicities should be established through multi-center collaboration involving a large cohort of patients.
Subject(s)
Biomarkers, Tumor , Molecular Imaging , Neoplasms , Humans , Neoplasms/radiotherapy , Neoplasms/diagnostic imaging , Molecular Imaging/methods , Animals , Biomarkers, Tumor/metabolism , Molecular Probes/chemistry , Radiotherapy/methods , Radiotherapy/adverse effects , Biomarkers/metabolismABSTRACT
The postsynaptic density (PSD) comprises numerous scaffolding proteins, receptors, and signaling molecules that coordinate synaptic transmission in the brain. Postsynaptic density protein 95 (PSD-95) is a master scaffold protein within the PSD and one of its most abundant proteins and therefore constitutes a very attractive biomarker of PSD function and its pathological changes. Here, we exploit a high-affinity inhibitor of PSD-95, AVLX-144, as a template for developing probes for molecular imaging of the PSD. AVLX-144-based probes were labeled with the radioisotopes fluorine-18 and tritium, as well as a fluorescent tag. Tracer binding showed saturable, displaceable, and uneven distribution in rat brain slices, proving effective in quantitative autoradiography and cell imaging studies. Notably, we observed diminished tracer binding in human post-mortem Parkinson's disease (PD) brain slices, suggesting postsynaptic impairment in PD. We thus offer a suite of translational probes for visualizing and understanding PSD-related pathologies.
Subject(s)
Brain , Disks Large Homolog 4 Protein , Post-Synaptic Density , Animals , Humans , Disks Large Homolog 4 Protein/metabolism , Brain/metabolism , Brain/diagnostic imaging , Rats , Post-Synaptic Density/metabolism , Molecular Imaging/methods , Fluorine Radioisotopes/chemistry , Parkinson Disease/metabolism , Parkinson Disease/diagnostic imaging , Peptides/chemistry , Peptides/metabolism , Molecular Probes/chemistry , Male , Autoradiography , Rats, Sprague-Dawley , Tritium , Pyridines , PyrrolidinonesABSTRACT
Gangliosides, sialic acid bearing glycosphingolipids, are components of the outer leaflet of plasma membranes of all vertebrate cells. They contribute to cell regulation by interacting with proteins in their own membranes (cis) or their extracellular milieu (trans). As amphipathic membrane constituents, gangliosides present challenges for identifying their ganglioside protein interactome. To meet these challenges, we synthesized bifunctional clickable photoaffinity gangliosides, delivered them to plasma membranes of cultured cells, then captured and identified their interactomes using proteomic mass spectrometry. Installing probes on ganglioside lipid and glycan moieties, we captured cis and trans ganglioside-protein interactions. Ganglioside interactomes varied with the ganglioside structure, cell type, and site of the probe (lipid or glycan). Gene ontology revealed that gangliosides engage with transmembrane transporters and cell adhesion proteins including integrins, cadherins, and laminins. The approach developed is applicable to other gangliosides and cell types, promising to provide insights into molecular and cellular regulation by gangliosides.
Subject(s)
Click Chemistry , Gangliosides , Gangliosides/chemistry , Gangliosides/metabolism , Humans , Photoaffinity Labels/chemistry , Photoaffinity Labels/chemical synthesis , Molecular Probes/chemistry , Molecular Probes/chemical synthesis , Cell Membrane/metabolism , Cell Membrane/chemistryABSTRACT
The overexpression of PDIA1 in cancer has spurred the quest for effective inhibitors. However, existing inhibitors often bind to only one active site, limiting their efficacy. In our study, we developed a PROTAC-mimetic probe dPA by combining PACMA31 (PA) analogs with cereblon-directed pomalidomide. Through protein profiling and analysis, we confirmed dPA's specific interaction with PDIA1's active site cysteines. We further synthesized PROTAC variants with a thiophene ring and various linkers to enhance degradation efficiency. Notably, H4, featuring a PEG linker, induced significant PDIA1 degradation and inhibited cancer cell proliferation similarly to PA. The biosafety profile of H4 is comparable to that of PA, highlighting its potential for further development in cancer therapy. Our findings highlight a novel strategy for PDIA1 inhibition via targeted degradation, offering promising prospects in cancer therapeutics. This approach may overcome limitations of conventional inhibitors, presenting new avenues for advancing anti-cancer interventions.
Subject(s)
Antineoplastic Agents , Cell Proliferation , Humans , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/chemical synthesis , Cell Line, Tumor , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Molecular Probes/chemistry , Molecular Probes/pharmacology , Molecular Probes/chemical synthesis , Molecular Structure , Procollagen-Proline Dioxygenase , Protein Disulfide-Isomerases/antagonists & inhibitors , Protein Disulfide-Isomerases/metabolism , Structure-Activity Relationship , Peptides/chemistry , Peptides/pharmacologyABSTRACT
Kidney diseases have become an important global health concern due to their high incidence, inefficient diagnosis, and poor prognosis. Devising direct methods, especially imaging means, to assess renal function is the key for better understanding the mechanisms of various kidney diseases and subsequent development of effective treatment. Herein, we developed a fluorinated ferrous chelate-based sensitive probe, 1,7-DO2A-Fe(II)-F18 (Probe 1), for 19F magnetic resonance imaging (MRI). This highly fluorinated probe (containing 18 chemically equivalent 19F atoms with a fluorine content at 35 wt %) achieves a 15-time enhancement in signal intensity compared with the fluorine-containing ligand alone due to the appropriately regulated 19F relaxation times by the ferrous ion, which significantly increases imaging sensitivity and reduces acquisition time. Owing to its high aqueous solubility, biostability, and biocompatibility, this probe could be rapidly cleared by kidneys, which provides a means for monitoring renal dysfunction via 19F MRI. With this probe, we accomplish in vivo imaging of the impaired renal dysfunction caused by various kidney diseases including acute kidney injury, unilateral ureteral obstruction, and renal fibrosis at different stages. Our study illustrates the promising potential of Probe 1 for in vivo real-time visualization of kidney dysfunction, which is beneficial for the study, diagnosis, and even stratification of different kidney diseases. Furthermore, the design strategy of our probe is inspiring for the development of more high-performance 19F MRI probes for monitoring various biological processes.
Subject(s)
Halogenation , Animals , Mice , Molecular Probes/chemistry , Kidney/diagnostic imaging , Kidney/pathology , Coordination Complexes/chemistry , Coordination Complexes/chemical synthesis , Ferrous Compounds/chemistry , Magnetic Resonance Imaging , Kidney Diseases/diagnostic imaging , Fluorine-19 Magnetic Resonance Imaging/methods , Fluorine/chemistryABSTRACT
Uropathogenic Escherichia coli (UPECs) is a leading cause for urinary tract infections (UTI), accounting for 70-90 % of community or hospital-acquired bacterial infections owing to high recurrence, imprecision in diagnosis and management, and increasing prevalence of antibiotic resistance. Current methods for clinical UPECs detection still rely on labor-intensive urine cultures that impede rapid and accurate diagnosis for timely UTI therapeutic management. Herein, we developed a first-in-class near-infrared (NIR) UPECs fluorescent probe (NO-AH) capable of specifically targeting UPECs through its collaborative response to bacterial enzymes, enabling locoregional imaging of UTIs both in vitro and in vivo. Our NO-AH probe incorporates a dual protease activatable moiety, which first reacts with OmpT, an endopeptidase abundantly present on the outer membrane of UPECs, releasing an intermediate amino acid residue conjugated with a NIR hemicyanine fluorophore. Such liberated fragment would be subsequently recognized by aminopeptidase (APN) within the periplasm of UPECs, activating localized fluorescence for precise imaging of UTIs in complex living environments. The peculiar specificity and selectivity of NO-AH, facilitated by the collaborative action of bacterial enzymes, features a timely and accurate identification of UPECs-infected UTIs, which could overcome misdiagnosis in conventional urine tests, thus opening new avenues towards reliable UTI diagnosis and personalized antimicrobial therapy management.
Subject(s)
Fluorescent Dyes , Urinary Tract Infections , Urinary Tract Infections/microbiology , Urinary Tract Infections/diagnosis , Fluorescent Dyes/chemistry , Uropathogenic Escherichia coli/enzymology , Animals , Mice , Optical Imaging , Humans , Molecular Probes/chemistryABSTRACT
Small molecular tool compounds play an essential role in the study of G protein-coupled receptors (GPCRs). However, tool compounds most often occupy the orthosteric binding site, hampering the study of GPCRs upon ligand binding. To overcome this problem, ligand-directed labeling techniques have been developed that leave a reporter group covalently bound to the GPCR, while allowing subsequent orthosteric ligands to bind. In this work, we applied such a labeling strategy to the adenosine A2B receptor (A2BAR). We have synthetically implemented the recently reported N-acyl-N-alkyl sulfonamide (NASA) warhead into a previously developed ligand and show that the binding of the A2BAR is not restricted by NASA incorporation. Furthermore, we have investigated ligand-directed labeling of the A2BAR using SDS-PAGE, flow cytometric, and mass spectrometry techniques. We have found one of the synthesized probes to specifically label the A2BAR, although detection was hindered by nonspecific protein labeling most likely due to the intrinsic reactivity of the NASA warhead. Altogether, this work aids the future development of ligand-directed probes for the detection of GPCRs.
Subject(s)
Receptor, Adenosine A2B , Sulfonamides , Ligands , Sulfonamides/chemistry , Humans , Receptor, Adenosine A2B/metabolism , Receptor, Adenosine A2B/chemistry , Molecular Probes/chemistry , Binding Sites , Receptors, G-Protein-Coupled/metabolism , Receptors, G-Protein-Coupled/chemistry , HEK293 Cells , Protein BindingABSTRACT
Carbonic anhydrase IX (CAIX), a zinc metal transmembrane protein, is highly expressed in 95% of clear cell renal cell carcinomas (ccRCCs). A positron emission tomography (PET) probe designed to target CAIX in nuclear medicine imaging technology can achieve precise positioning, is noninvasive, and can be used to monitor CAIX expression in lesions in real time. In this study, we constructed a novel acetazolamide dual-targeted small-molecule probe [68Ga]Ga-LF-4, which targets CAIX by binding to a specific amino acid sequence. After attenuation correction, the radiolabeling yield reached 66.95 ± 0.57% (n = 5) after 15 min of reaction and the radiochemical purity reached 99% (n = 5). [68Ga]Ga-LF-4 has good in vitro and in vivo stability, and in vivo safety and high affinity for CAIX, with a Kd value of 6.62 nM. Moreover, [68Ga]Ga-LF-4 could be quickly cleared from the blood in vivo. The biodistribution study revealed that the [68Ga]Ga-LF-4 signal was concentrated in the heart, lung, and kidney after administration, which was the same as that observed in the micro-PET/CT study. In a ccRCC patient-derived xenograft (PDX) model, the signal significantly accumulated in the tumor after administration, where it was retained for up to 4 h. After competitive blockade with LF-4, uptake at the tumor site was significantly reduced. The SUVmax of the probe [68Ga]Ga-LF-4 at the ccRCC tumor site was three times greater than that in the PC3 group with low CAIX expression at 30 min (ccRCC vs PC3:1.86 ± 0.03 vs 0.62 ± 0.01, t = 48.2, P < 0.0001). These results indicate that [68Ga]Ga-LF-4 is a novel small-molecule probe that targets CAIX and can be used to image localized and metastatic ccRCC lesions.
Subject(s)
Carbonic Anhydrase IX , Carcinoma, Renal Cell , Gallium Radioisotopes , Kidney Neoplasms , Animals , Carbonic Anhydrase IX/metabolism , Carbonic Anhydrase IX/antagonists & inhibitors , Humans , Mice , Carcinoma, Renal Cell/diagnostic imaging , Carcinoma, Renal Cell/metabolism , Kidney Neoplasms/diagnostic imaging , Kidney Neoplasms/metabolism , Tissue Distribution , Cell Line, Tumor , Radiopharmaceuticals/pharmacokinetics , Radiopharmaceuticals/chemistry , Mice, Nude , Antigens, Neoplasm/metabolism , Molecular Probes/pharmacokinetics , Molecular Probes/chemistry , Positron Emission Tomography Computed Tomography/methods , Acetazolamide/pharmacokinetics , Female , Mice, Inbred BALB C , Positron-Emission Tomography/methods , Male , Xenograft Model Antitumor AssaysABSTRACT
Super-resolution molecular probes (SRMPs) are essential tools for visualizing drug dynamics within cells, transcending the resolution limits of conventional microscopy. In this review, we provide an overview of the principles and design strategies of SRMPs, emphasizing their role in accurately tracking drug molecules. By illuminating the intricate processes of drug distribution, diffusion, uptake, and metabolism at a subcellular and molecular level, SRMPs offer crucial insights into therapeutic interventions. Additionally, we explore the practical applications of super-resolution imaging in disease treatment, highlighting the significance of SRMPs in advancing our understanding of drug action. Finally, we discuss future perspectives, envisioning potential advancements and innovations in this field. Overall, this review serves to inform and practitioners about the utility of SRMPs in driving innovation and progress in pharmacology, providing valuable insights for drug development and optimization.
Subject(s)
Molecular Probes , Molecular Probes/chemistry , Humans , Pharmaceutical Preparations/metabolism , Pharmaceutical Preparations/chemistry , Pharmaceutical Preparations/administration & dosage , Animals , Molecular Imaging/methodsABSTRACT
In recent years, organic fluorophores-based molecular probes with dual-fluorescence ratiometric responses to in-vitro/in-vivo pH (DFR-MPs-pH) have been attracting much interest in fundamental application research fields. More and more scientific publications have reported the exploration of various DFR-MPs-pH systems that have unique dual-fluorescence ratiometry as the signal output, in-built and signal self-calibration functions to improve precise detection of targets. DFR-MPs-pH systems possess high-performance applications in biosensing, bioimaging and biomedicine fields. This review has comprehensively summarized recent advances of DFR-MPs-pH for the first time. First of all, the compositions and types of DFR-MPs-pH are introduced by summarizing different organic fluorophores-based molecule systems. Then, construction strategies are analyzed based on specific components, structures, properties and functions of DFR-MPs-pH. Afterward, biosensing and bioimaging applications are discussed in detail, primarily referring to pH sensing and imaging detection at the levels of living cells and small animals. Finally, biomedicine applications are fully summarized, majorly involving bio-toxicity evaluation, bio-distribution, biomedical diagnosis and therapeutics. Meanwhile, the current status, challenges and perspectives are rationally commented after detailed discussions of representative and state-of-the-art studies. Overall, this present review is comprehensive, in-time and in-depth, and can facilitate the following further exploration of new and versatile DFR-MPs-pH systems toward rational design, facile preparation, superior properties, adjustable functions and highly efficient applications in promising fields.
Subject(s)
Biosensing Techniques , Fluorescent Dyes , Optical Imaging , Biosensing Techniques/methods , Hydrogen-Ion Concentration , Fluorescent Dyes/chemistry , Humans , Animals , Optical Imaging/methods , Molecular Probes/chemistryABSTRACT
Given its pivotal role in modulating various pathological processes, precise measurement of nitric oxide (âNO) levels in physiological solutions is imperative. The key techniques include the ozone-based chemiluminescence (CL) reactions, amperometric âNO sensing, and Griess assay, each with its advantages and drawbacks. In this study, a hemin/H2O2/luminol CL reaction was employed for accurately detecting âNO in diverse solutions. We investigated how the luminescence kinetics was influenced by âNO from two donors, nitrite and peroxynitrite, while also assessing the impact of culture medium components and reactive species quenchers. Furthermore, we experimentally and theoretically explored the mechanism of hemin oxidation responsible for the initiation of light generation. Although both hemin and âNO enhanced the H2O2/luminol-based luminescence reactions with distinct kinetics, hemin's interference with âNO/peroxynitrite- modulated their individual effects. Leveraging the propagated signal due to hemin, the âNO levels in solution were estimated, observing parallel changes to those detected via amperometric detection in response to varying concentrations of the âNO-donor. The examined reactions aid in comprehending the mechanism of âNO/hemin/H2O2/luminol interactions and how these can be used for detecting âNO in solution with minimal sample size demands. Moreover, the selectivity across different solutions can be improved by incorporating certain quenchers for reactive species into the reaction.
Subject(s)
Hemin , Molecular Probes , Nitric Oxide , Hemin/chemistry , Hydrogen Peroxide/chemistry , Hydrogen Peroxide/analysis , Kinetics , Luminescent Measurements , Luminol/chemistry , Molecular Probes/chemistry , Nitric Oxide/analysis , Oxidation-Reduction , Peroxynitrous Acid/analysis , Peroxynitrous Acid/chemistry , SolutionsABSTRACT
Pyroptosis is a recently discovered process of programmed cell death that is linked with tumor progression and potential treatment strategies. Unlike other forms of programmed cell death, such as apoptosis or necrosis, pyroptosis is associated with pore-forming proteins gasdermin D (GSDMD), which are cleaved by caspase enzymes to form oligomers. These oligomers are then inserted into the cell surface membrane, causing pores to consequently result in rapid cell death. Pyroptosis, in conjunction with immunotherapy, represents a promising avenue for prognostication and antitumor therapy, providing a more precise direction for disease treatment. To gain deeper insight into the mechanisms underlying pyroptosis in real-time, non-invasive and live cell imaging techniques are urgently needed. Non-invasive imaging techniques can enhance future diagnostic and therapeutic approaches for inflammatory diseases, including different types of tumors. This review article discusses various non-invasive molecular probes for detecting pyroptosis, including genetic reporters and nanomaterials. These strategies can enhance scientists' understanding of pyroptosis and help discover personalized and effective ways to treat inflammatory diseases, particularly tumors.
Subject(s)
Molecular Probes , Neoplasms , Pyroptosis , Humans , Molecular Probes/chemistry , Molecular Probes/metabolism , Animals , Neoplasms/therapy , Neoplasms/metabolism , Neoplasms/diagnostic imaging , Neoplasms/diagnosis , Neoplasms/genetics , Neoplasms/pathology , Molecular Imaging/methods , Theranostic Nanomedicine/methods , Phosphate-Binding Proteins/metabolism , Phosphate-Binding Proteins/genetics , GasderminsABSTRACT
Glycosphingolipids (GSLs) are abundant glycolipids on cells and essential for cell recognition, adhesion, signal transduction, and so on. However, their lipid anchors are not long enough to cross the membrane bilayer. To transduce transmembrane signals, GSLs must interact with other membrane components, whereas such interactions are difficult to investigate. To overcome this difficulty, bifunctional derivatives of II3-ß-N-acetyl-D-galactosamine-GA2 (GalNAc-GA2) and ß-N-acetyl-D-glucosamine-ceramide (GlcNAc-Cer) were synthesized as probes to explore GSL-interacting membrane proteins in live cells. Both probes contain photoreactive diazirine in the lipid moiety, which can crosslink with proximal membrane proteins upon photoactivation, and clickable alkyne in the glycan to facilitate affinity tag addition for crosslinked protein pull-down and characterization. The synthesis is highlighted by the efficient assembly of simple glycolipid precursors followed by on-site lipid remodeling. These probes were employed to profile GSL-interacting membrane proteins in HEK293 cells. The GalNAc-GA2 probe revealed 312 distinct proteins, with GlcNAc-Cer probe-crosslinked proteins as controls, suggesting the potential influence of the glycan on GSL functions. Many of the proteins identified with the GalNAc-GA2 probe are associated with GSLs, and some have been validated as being specific to this probe. The versatile probe design and experimental protocols are anticipated to be widely applicable to GSL research.
Subject(s)
Cell Membrane , Glycosphingolipids , Membrane Proteins , Humans , Glycosphingolipids/metabolism , Glycosphingolipids/chemistry , HEK293 Cells , Cell Membrane/metabolism , Membrane Proteins/metabolism , Membrane Proteins/chemistry , Molecular Probes/chemistry , Molecular Probes/metabolism , Diazomethane/chemistry , Diazomethane/metabolism , Acetylgalactosamine/metabolism , Acetylgalactosamine/chemistryABSTRACT
Accurate visualization of tumor microenvironment is of great significance for personalized medicine. Here, we develop a near-infrared (NIR) fluorescence/photoacoustic (FL/PA) dual-mode molecular probe (denoted as NIR-CE) for distinguishing tumors based on carboxylesterase (CE) level by an analyte-induced molecular transformation (AIMT) strategy. The recognition moiety for CE activity is the acetyl unit of NIR-CE, generating the pre-product, NIR-CE-OH, which undergoes spontaneous hydrogen atom exchange between the nitrogen atoms in the indole group and the phenol hydroxyl group, eventually transforming into NIR-CE-H. In cellular experiments and in vivo blind studies, the human hepatoma cells and tumors with high level of CE were successfully distinguished by both NIR FL and PA imaging. Our findings provide a new molecular imaging strategy for personalized treatment guidance.