Cell-permeable Probe Research Articles

Cell permeable affinity- and activity-based probes.

Chemical biology has a significant role to play in the discovery and validation of new therapeutic targets. Activity- and affinity-based probes have demonstrated considerable promise in the drug discovery setting as they provide a chemoproteomic means to confirm and quantify target engagement and selectivity of small molecule drug candidates. Many of these technologies have been developed using cell lysate (through the use of resin-immobilized enzyme inhibitors for example), but this does not represent the biology of an intact cell. This review highlights recent advances made in the design and application of cell-permeable probes that report on target activity and drug-target occupancy in living cells, thus providing a means to decipher molecular pharmacology and pathology in a more physiologically relevant manner.

Abstract 3528: The SMARCA2/4 catalytic activity, but not the bromodomain, is a drug target in SWI/SNF mutant cancers

Abstract The SWI/SNF multi-subunit complex modulates chromatin structure through the activity of two mutually exclusive catalytic subunits, SMARCA2 and SMARCA4, which both contain a bromodomain and an ATPase domain. Using RNAi, cancer-specific vulnerabilities have been identified in SWI/SNF mutant tumors, including SMARCA4-deficient lung cancer, however, the contribution of conserved, druggable protein domains to this anticancer phenotype is unknown. Here, we functionally deconstructed the SMARCA2/4 paralog dependence of cancer cells using bioinformatics, genetic and pharmacological tools. We evaluated a potent and selective SMARCA2/4 bromodomain inhibitor (PFI-3) and characterized its activity in chromatin-binding and cell-functional assays focusing on cells with altered SWI/SNF status (e.g. Lung, Synovial Sarcoma, Leukemia, and Rhabdoid tumors). We demonstrated that PFI-3 is a cell-permeable probe capable of displacing ectopically expressed, GFP-tagged SMARCA2-bromodomain from chromatin, yet contrary to target knockdown, the inhibitor failed to display an antiproliferative phenotype. Mechanistically, the lack of pharmacological efficacy was reconciled by the failure of bromodomain inhibition to displace endogenous, full-length SMARCA2 from chromatin as determined by in situ cell extraction, chromatin immunoprecipitation (ChIP) and target gene expression and promoter occupancy studies. Using RNAi and cDNA complementation (bromodomain and ATPase-dead constructs), we identified the catalytic ATPase domain, and not the bromodomain of SMARCA2, as a relevant therapeutic target. Taken together, our complementary genetic and pharmacological studies exemplify a general strategy for bromodomain drug-target validation and in case of SMARCA2/4 highlight the requirement for drugging the more challenging helicase/ATPase domain affording potential synthetic-lethal treatment options to cancer patients with genetically defined alterations in SWI/SNF. Citation Format: Bhavatarini Vangamudi, Thomas Paul, Parantu K. Shah, Maria K. Alimova, Lisa Nottebaum, Xi Shi, Yanai Zhan, Elisabetta Leo, Harshad S. Mahadeshwar, Alexei Protopopov, Andrew Futreal, Trang N. Tieu, Mike Peoples, Alessia Petrocchi, Joseph R. Marszalek, Carlo Toniatti, Timothy P. Heffernan, Dominique Verhelle, Giulio Draetta, Dafydd Owen, Philip Jones, Wylie Palmer, Shikhar Sharma, Jannik N. Andersen. The SMARCA2/4 catalytic activity, but not the bromodomain, is a drug target in SWI/SNF mutant cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3528. doi:10.1158/1538-7445.AM2015-3528

BioSpotlight / Citations

BioTechniquesVol. 58, No. 4 BioSpotlight / CitationsOpen AccessBioSpotlight / CitationsNathan S. Blow & Nijsje DormanNathan S. BlowSearch for more papers by this author & Nijsje DormanSearch for more papers by this authorPublished Online:3 Apr 2018https://doi.org/10.2144/000114271AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail Crispr cloningCloning via restriction enzymes or PCR is a mainstay technique in molecular biology labs around the globe. The challenge when working with restriction enzymes is that they are specific, cleaving in only certain locations, while PCR approaches can lead to unintended sequence changes and are limited by possible amplicon size. In this issue, Wang et al. demonstrate for the first time that direct, seamless cloning of DNA fragments into any sequence location can be achieved through the use of CRISPR/Cas9 nuclease cleavage along with subsequent Gibson assembly. The authors demonstrate their combination technique by linearizing a 22 kb plasmid with the Cas9 enzyme and a specific single guide RNA (sgRNA) in vitro and then inserting a DNA fragment using Gibson assembly. This new technique opens doors for researchers interested in working with larger vectors such as cosmids, adenoviral vectors, and bacterial artificial chromosome (BAC) plasmids, or modifying existing vector constructs when the existing methods are not suitable or sufficient.See “CRISPR/Cas9 nuclease cleavage combined with Gibson assembly for seamless cloning”Reagents to explore genome architectureIt's become clear in recent years that the three-dimensional organization of genomic DNA is critical when it comes to determining gene expression patterns and genome function. Much of this knowledge has been gained through the use of chromosome conformation capture (3C, 4C, 5C, Hi-C, etc.) techniques that measure long-range interactions between different regions of the genome. While these techniques have provided unique insights into genome regulation, there has yet to be a systematic evaluation and optimization of the two critical enzymes used in chromosome conformation capture assays: T4 DNA ligase and DNA polymerase. In this issue, Schwartz et al. perform a comparison of different commercially available ligases and polymerases. The authors tested 4 different T4 DNA ligases and 13 different DNA polymerases, identifying the most efficient and specific combinations of each and thereby providing researchers lists of well-validated reagents, which should facilitate more robust, reproducible, and less costly chromosome conformation capture analysis in the future.See “Comparative analysis of ligases and polymerases used in chromosome conformation capture assays”Cell-permeable probe to fluorescently label his-tagsThough best known for affinity purification, the His-tag can also be conjugated with small-molecule fluorescent labels. However, no fluorophore has been shown capable of rapid intracellular labeling of His-tagged proteins. Lai et al. change all that with the Ni-NTA-AC probe, comprising a nitrilotriacetate moiety conjugated with a coumarin fluorophore and an attached arylazide. Although binding is relatively weak, UV irradiation photoactivates the arylazide, which covalently binds the protein and enhances fluorescence 13-fold. In tests in HeLa cells with a His-tagged cytosolic protein, adding Ni-NTA-AC to the medium led to intracellular blue fluorescence within 2 minutes, indicating rapid transmembrane movement and protein labeling. No toxicity from the fluorophore or UV irradiation was observed. Furthermore, in studies with a 15-kDa DNA binding domain, the method gave the expected nuclear localization, unlike labeling via an RFP fusion. The new label also works in plant tissue, and other colors of His-tag–binding fluorophore are in development.Y.T. Lai et al. Rapid labeling of intracellular His-tagged proteins in living cells. Proc Natl Acad Sci U S A. [Epub ahead of print, Feb 23, 2015; doi: 10.1073/pnas.1419598112]Whole-mount single-molecule fish in a vertebrate embryoAnalyzing gene expression at the single-cell level reveals stochastic variations thought to underlie adaptability. Quantitative RT-PCR and RNA-Seq measure these fluctuations, but lack the spatial resolution afforded by single-molecule FISH (smFISH). Although smFISH has been performed in tissue from vertebrates, to date whole-mount smFISH protocols are limited to invertebrates. To fill this gap, Oka and Sato took elements of smFISH methods for cultured cells and Drosophila embryos and combined them with features of traditional in situ hybridization in whole mounts of zebrafish embryos. By optimizing pretreatment, fixation, and washes, the authors could detect transcripts just 720 nucleotides long (close to the theoretical minimum length of 700 nucleotides in this system) and genes that are expressed at fewer than 50 copies per cell. Experiments in which in vitro–transcribed RNA was injected and then probed gave confidence that each smFISH dot corresponds to one transcript. The authors expect their method will also be applicable to other vertebrate model systems.Y. Oka and T.N. Sato. 2015. Whole-mount single molecule FISH method for zebrafish embryo. Sci Rep. 5:8571.FiguresReferencesRelatedDetails Vol. 58, No. 4 Follow us on social media for the latest updates Metrics History Published online 3 April 2018 Published in print April 2015 Information© 2015 Author(s)PDF download

Target profiling of zerumbone using a novel cell-permeable clickable probe and quantitative chemical proteomics.

Zerumbone is a phytochemical with diverse biological activities ranging from anti-inflammatory to anti-cancer properties; however, to date the cellular targets of this important compound have remained elusive. Here we report the global protein target spectrum of zerumbone in living cancer cells using competitive activity-based protein profiling of a novel cell-permeable clickable probe, combined with quantitative mass spectrometry.

Consistent Upregulation of Oxidative Stress and DNA Damage Amongst Dyskeratosis Congenita Genotypes: Ameliorative Effects of Antioxidants

Introduction:Dyskeratosis congenita (DC) is an inherited multisystem disorder characterized by the classical clinical triad of leukoplakia, skin dyspigmentation and nail dystrophy with concomitant premature aging, cancer predisposition, and bone marrow failure. Conventional treatments for marrow failure, including transplantation, have been poorly tolerated for DC patients. Several mutations within telomeres/telomerase-associated genes have been attributed to DC, resulting in shortened telomeres. Growing evidence suggests that shortened telomeres are linked to DNA damage responses and subsequent elevation of reactive oxygen species (ROS). We have previously reported increased intracellular ROS and induction of DDR protein expression in TERC-deficient cells. This study was undertaken to confirm these findings in other DC genotypes (TERT, TINF2, DKC1) and provide additional evidence supporting a relationship among telomere shortening, DDR and dysregulation in oxidative pathways.Methods:Somatic cells from DC subjects with TERC (5 patients), TERT (4), DKC1 (1) and TINF2 (1) mutations and age-matched controls were used in these studies. Cell types included lymphocytes and fibroblasts from skin and lung. DC genotypes were confirmed by DNA sequencing, and all DC cells displayed shortened telomeres. T lymphocytes were expanded in vitro using CD3/CD28 beads. Ionizing radiation was used to induce DNA damage. Cell proliferation was assessed on a Nexelom automatic counter. Western blotting was used to measure basal and radiation-induced expression of DNA damage response (DDR) proteins, including total p53 and its activated form (serine 15 phosphorylated; p53S15), ATM, BRCA2 and phosphorylated H2AX (gH2AX). Levels of oxidative stress were determined by FACS using the cell-permeable fluorogenic probe DCFH and dihydroethedium (DHE) detecting reactive oxygen species (ROS). Antioxidant culture conditions, such as N acetyl cysteine (NAC) or low oxygen, were used in vitro to modulate levels of oxidative stress in control and radiated cells.Results:We investigated ROS levels and several parameters indicative of DNA damage in DC lymphocytes and fibroblasts with mutations in TERC, TERT, DKC1 or TINF2. In cells from all DC genotypes a consistent increase in p53 expression and ROS levels was noted. Basal expression of p53 was increased by at least 50% in DC lymphocytes compared to controls. Additionally, basal ROS levels from TERC and TINF2 mutated lymphocytes were significantly increased compared to controls. While the degree of radiation-induced DDR protein expression was similar in DC compared to control cells, ROS remained significantly higher in DC-irradiated lymphocytes. Lung fibroblasts from a DKC1 patient demonstrated increased ROS and reduced proliferative capacity compared to controls. Importantly, NAC decreased DDR in DC lymphocytes while a low oxygen environment markedly improved the proliferative capacity of both DC lymphocytes and lung fibroblasts.Conclusions:DC is an important human disease model for studying the effects of telomere shortening on pathways involved in cell senescence and aging. Our findings indicate elevated basal and irradiation-induced levels of p53 expression and ROS in DC cells regardless of the underlying mutation. This suggests a role for telomerase deficiency and/or shortened telomeres in regulating oxidative and DNA damage response pathways. Furthermore, reducing ROS may serve as an adjunctive therapeutic approach to limit toxicity of bone marrow transplant regimens. Clinical trials are needed to evaluate the impact of antioxidant therapies in this disease. DisclosuresNo relevant conflicts of interest to declare.

Corrigendum: Fluorescent Mimetics of CMP-Neu5Ac Are Highly Potent, Cell-Permeable Polarization Probes of Eukaryotic and Bacterial Sialyltransferases and Inhibit Cellular Sialylation

In this Communication, reference compound 1-Ac and intermediate 5-p were used. While both compounds were reported initially in reference 12b as cited, the detailed experimental protocols and spectroscopic data were published subsequently in reference 12c, which has to be added. The application of chiral oxaziridines for the stereoselective oxidation of phosphonates was published originally in reference 15a as cited. The method has been applied to allyl phosphonates in another reference 15b, which has to be added. References 12 and 15 should read as follows:

Imaging oxygen in neural cell and tissue models by means of anionic cell-permeable phosphorescent nanoparticles.

Cell-permeable phosphorescent probes enable the study of cell and tissue oxygenation, bioenergetics, metabolism, and pathological states such as stroke and hypoxia. A number of such probes have been described in recent years, the majority consisting of cationic small molecule and nanoparticle structures. While these probes continue to advance, adequate staining for the study of certain cell types using live imaging techniques remains elusive; this is particularly true for neural cells. Here we introduce novel probes for the analysis of neural cells and tissues: negatively charged poly(methyl methacrylate-co-methacrylic acid)-based nanoparticles impregnated with a phosphorescent Pt(II)-tetrakis(pentafluorophenyl)porphyrin (PtPFPP) dye (this form is referred to as PA1), and with an additional reference/antennae dye poly(9,9-diheptylfluorene-alt-9,9-di-p-tolyl-9H-fluorene) (this form is referred to as PA2). PA1 and PA2 are internalised by endocytosis, result in efficient staining in primary neurons, astrocytes, and PC12 cells and multi-cellular aggregates, and allow for the monitoring of local O(2) levels on a time-resolved fluorescence plate reader and PLIM microscope. PA2 also efficiently stains rat brain slices and permits detailed O(2) imaging experiments using both one and two-photon intensity-based modes and PLIM modes. Multiplexed analysis of embryonic rat brain slices reveals age-dependent staining patterns for PA2 and a highly heterogeneous distribution of O(2) in tissues, which we relate to the localisation of specific progenitor cell populations. Overall, these anionic probes are useful for sensing O(2) levels in various cells and tissues, particularly in neural cells, and facilitate high-resolution imaging of O(2) in 3D tissue models.

Deprotonation and chelation synergically triggered near infrared fluorescence for selective detection of Hg(II)

Abstract A convenient strategy that does not need the tedious modification of the π-conjugation moiety to construct near infrared (NIR) sensors is in high demand. In this study, we present the design and synthesis of a NIR Hg(II) turn-on fluorescent sensor BODIPY-SAN , which shows several advantages including (1) simple to be synthesized by only a predictable modification of the core structure of 6-hydroxyindole-based BODIPY, (2) giving a turn-on NIR emission synergically triggered by chelation and deprotonation, (3) exhibiting Hg(II)-specific fluorescence response with the detection limit to be 5 × 10 −7 M, (4) functioning as a novel cell-permeable NIR probe for intracellular Hg(II) in living HeLa cells.

Fluorescent mimetics of CMP-Neu5Ac are highly potent, cell-permeable polarization probes of eukaryotic and bacterial sialyltransferases and inhibit cellular sialylation.

Oligosaccharides of the glycolipids and glycoproteins at the outer membranes of human cells carry terminal neuraminic acids, which are responsible for recognition events and adhesion of cells, bacteria, and virus particles. The synthesis of neuraminic acid containing glycosides is accomplished by intracellular sialyl transferases. Therefore, the chemical manipulation of cellular sialylation could be very important to interfere with cancer development, inflammations, and infections. The development and applications of the first nanomolar fluorescent inhibitors of sialyl transferases are described herein. The obtained carbohydrate-nucleotide mimetics were found to bind all four commercially available and tested eukaryotic and bacterial sialyl transferases in a fluorescence polarization assay. Moreover, it was observed that the anionic mimetics intruded rapidly and efficiently into cells in vesicles and translocated to cellular organelles surrounding the nucleus of CHO cells. The new compounds inhibit cellular sialylation in two cell lines and open new perspectives for investigations of cellular sialylation.

The Organization of mitochondria in growing hyphae of Neurospora crassa

Details of mitochondrial system organization and behavior in the tip of growing N. crassa hyphae and hyphal fragments were studied by means of cell-permeable mitochondria-selective probes in glucose- and sorbitol-containing media. It was shown that filamentous mitochondria concentrate in the 30-μm apical zone of growing hyphae and hyphal fragments independently of the carbon source. The mitochondrial assemblies propagate forward with elongation of hypha, split upon apical branching, and are formed de novo when branches are formed away from the growing tip. These activities resemble microtubule behavior. Co-staining with Mitotracker Red and Mitotracker Green revealed possible functional heterogeneity in subpopulations of mitochondria. It was proposed that the observed features are governed by the microtubular network, while the primary function of mitochondria was to sustain the growth rate. The organizing role of electrical gradients on the growing tip and their influence on mitochondrial and microtubular networks are discussed.

A tuned affinity-based staurosporine probe for in situ profiling of protein kinases

A clickable and cell-permeable affinity-based probe (AfBP) was designed from staurosporine, by incorporating an electrophilic chloroacetamide warhead to facilitate in situ proteome labeling of a range of potential kinase targets.

Discovery of selective 2,4-diaminopyrimidine-based photoaffinity probes for glyoxalase I

L1-Bpyne was discovered as a potent inhibitor and cell permeable probe of glyoxalase I.

Use of Okadaic Acid to Identify Relevant Phosphoepitopes in Pathology: A Focus on Neurodegeneration

Protein phosphorylation is involved in the regulation of a wide variety of physiological processes and is the result of a balance between protein kinase and phosphatase activities. Biologically active marine derived compounds have been shown to represent an interesting source of novel compounds that could modify that balance. Among them, the marine toxin and tumor promoter, okadaic acid (OA), has been shown as an inhibitor of two of the main cytosolic, broad-specificity protein phosphatases, PP1 and PP2A, thus providing an excellent cell-permeable probe for examining the role of protein phosphorylation, and PP1 and PP2A in particular, in any physiological or pathological process. In the present work, we review the use of okadaic acid to identify specific phosphoepitopes mainly in proteins relevant for neurodegeneration. We will specifically highlight those cases of highly dynamic phosphorylation-dephosphorylation events and the ability of OA to block the high turnover phosphorylation, thus allowing the detection of modified residues that could be otherwise difficult to identify. Finally, its effect on tau hyperhosphorylation and its relevance in neurodegenerative pathologies such as Alzheimer’s disease and related dementia will be discussed.

ChemInform Abstract: Fluorescent Probes for G‐Quadruplex Structures

AbstractReview: 232 refs.

Extracellular Acidosis Is a Novel Danger Signal Alerting Innate Immunity via the NLRP3 Inflammasome

Local acidosis has been demonstrated in ischemic tissues and at inflammatory sites. Acidic extracellular pH triggers NLRP3 inflammasome activation and interleukin-1β secretion in human macrophages. Acidic pH represents a novel danger signal alerting the innate immunity. Local acidosis may promote inflammation at ischemic and inflammatory sites. Local extracellular acidification has been demonstrated at sites of ischemia and inflammation. IL-1β is one of the key proinflammatory cytokines, and thus, its synthesis and secretion are tightly regulated. The NLRP3 (nucleotide-binding domain leucine-rich repeat containing family, pyrin domain containing 3) inflammasome complex, assembled in response to microbial components or endogenous danger signals, triggers caspase-1-mediated maturation and secretion of IL-1β. In this study, we explored whether acidic environment is sensed by immune cells as an inflammasome-activating danger signal. Human macrophages were exposed to custom cell culture media at pH 7.5-6.0. Acidic medium triggered pH-dependent secretion of IL-1β and activation of caspase-1 via a mechanism involving potassium efflux from the cells. Acidic extracellular pH caused rapid intracellular acidification, and the IL-1β-inducing effect of acidic medium could be mimicked by acidifying the cytosol with bafilomycin A1, a proton pump inhibitor. Knocking down the mRNA expression of NLRP3 receptor abolished IL-1β secretion at acidic pH. Remarkably, alkaline extracellular pH strongly inhibited the IL-1β response to several known NLRP3 activators, demonstrating bipartite regulatory potential of pH on the activity of this inflammasome. The data suggest that acidic environment represents a novel endogenous danger signal alerting the innate immunity. Low pH may thus contribute to inflammation in acidosis-associated pathologies such as atherosclerosis and post-ischemic inflammatory responses.

A TR-FRET-based functional assay for screening activators of CARM1.

Epigenetics is an emerging field that demands selective cell-permeable chemical probes to perturb, especially in vivo, the activity of specific enzymes involved in modulating the epigenetic codes. Coactivator-associated arginine methyltransferase 1 (CARM1) is a coactivator of estrogen receptor α (ERα), the main target in human breast cancer. We previously showed that twofold overexpression of CARM1 in MCF7 breast cancer cells increased the expression of ERα-target genes involved in differentiation and reduced cell proliferation, thus leading to the hypothesis that activating CARM1 by chemical activators might be therapeutically effective in breast cancer. Selective, potent, cell-permeable CARM1 activators will be essential to test this hypothesis. Here we report the development of a cell-based, time-resolved (TR) FRET assay that uses poly(A) binding protein 1 (PABP1) methylation to monitor cellular activity of CARM1. The LanthaScreen TR-FRET assay uses MCF7 cells expressing GFP-PABP1 fusion protein through BacMam gene delivery system, methyl-PABP1 specific antibody, and terbium-labeled secondary antibody. This assay has been validated as reflecting the expression and/or activity of CARM1 and optimized for high throughput screening to identify CARM1 allosteric activators. This TR-FRET platform serves as a generic tool for functional screening of cell-permeable, chemical modulators of CARM1 for elucidation of its in vivo functions.

Fluorescent Probes for G‐Quadruplex Structures

Mounting evidence supports the presence of biologically relevant G-quadruplexes in single-cell organisms, but the existence of endogenous G-quadruplex structures in mammalian cells remains highly controversial. This is due, in part, to the common misconception that DNA and RNA molecules are passive information carriers with relatively little structural or functional complexity. For those working in the field, however, the lack of available tools for characterizing DNA structures in vivo remains a major limitation to addressing fundamental questions about structure-function relationships of nucleic acids. In this review, we present progress towards the direct detection of G-quadruplex structures by using small molecules and modified oligonucleotides as fluorescent probes. While most development has focused on cell-permeable probes that selectively bind to G-quadruplex structures with high affinity, these same probes can induce G-quadruplex folding, thereby making the native conformation of the DNA or RNA molecule (i.e., in the absence of probe) uncertain. For this reason, modified oligonucleotides and fluorescent base analogues that serve as "internal" fluorescent probes are presented as an orthogonal means for detecting conformational changes, without necessarily perturbing the equilibria between G-quadruplex, single-stranded, and duplex DNA. The major challenges and motivation for the development of fluorescent probes for G-quadruplex structures are presented, along with a summary of the key photophysical, biophysical, and biological properties of reported examples.

Cell-permeable probe for identification and imaging of sialidases

Alkyne-hinged 3-fluorosialyl fluoride (DFSA) containing an alkyne group was shown to be a mechanism-based target-specific irreversible inhibitor of sialidases. The ester-protected analog DFSA (PDFSA) is a membrane-permeable precursor of DFSA designed to be used in living cells, and it was shown to form covalent adducts with virus, bacteria, and human sialidases. The fluorosialyl–enzyme adduct can be ligated with an azide-annexed biotin via click reaction and detected by the streptavidin-specific reporting signals. Liquid chromatography-mass spectrometry/mass spectrometry analysis on the tryptic peptide fragments indicates that the 3-fluorosialyl moiety modifies tyrosine residues of the sialidases. DFSA was used to demonstrate influenza infection and the diagnosis of the viral susceptibility to the anti-influenza drug oseltamivir acid, whereas PDFSA was used for in situ imaging of the changes of sialidase activity in live cells.

Selective affinity-based probe for oncogenic kinases suitable for live cell imaging

Cell permeable probes to image the presence and localization of kinases are important in studying their function and as diagnostic tools. Despite the central role of kinases as therapeutic targets, there are remarkably few probes available. Herein we report the discovery of a probe to image two therapeutically relevant kinases: EGFR and ERBB2. The probe was identified from a library based on a scaffold derived from the resorcyclic acid lactones that form a covalent adduct by reacting specifically with an unconserved cysteine in the nucleotide-binding site of the kinases. We demonstrated the utility of the newly discovered probe by imaging of EGFR localization and ERBB2 inhibition in live cells.

The design and synthesis of novel “turn-on” fluorescent probes to visualize monoamine oxidase-B in living cells

We report the design and synthesis of a new class of fluorogenic probes based on a monoamine oxidase-B (MAO-B)-catalyzed α-carbon oxidation–hydrolysis mechanism. The cell-permeable probes exhibited good selectivity and sensitivity towards MAO-B, and a turn-on response in fluorescence intensity in living cells.