Specificity fingerprinting of retaining beta-1,4-glycanases in the Cellulomonas fimi secretome using two fluorescent mechanism-based probes

Functional proteomics methods are crucial for activity- and mechanism-based investigation of enzymes in biological systems at a post-translational stage. Glycosidases have central roles in cellular metabolism and its regulation, and their dysfunction can have detrimental effects. These enzymes also play key roles in biomass conversion. A functional profiling methodology was developed for direct, fluorescence-based, in-gel analysis of retaining beta-glycosidases. Two spectrally nonoverlapping fluorescent, mechanism-based probes containing different recognition elements for retaining cellulases and xylanases were prepared. The specificity-based covalent labelling of retaining glycanases by the two probes was demonstrated in model enzyme mixtures. Using the two probes and mass spectrometry, the secretomes of the biomass-converting bacterium Cellulomonas fimi, under induction by different polyglycan growth substrates, were analysed to obtain a specificity profile of the C. fimi retaining beta-glycanases. This is a facile strategy for the analysis of glycosidases produced by biomass-degrading organisms.     

Development of SNAP-tag fluorogenic probes for wash-free fluorescence imaging

The ability to specifically attach chemical probes to individual proteins represents a powerful approach to the study and manipulation of protein function in living cells. It provides a simple, robust and versatile approach to the imaging of fusion proteins in a wide range of experimental settings. However, a potential drawback of detection using chemical probes is the fluorescence background from unreacted or nonspecifically bound probes. In this report we present the design and application of novel fluorogenic probes for labeling SNAP-tag fusion proteins in living cells. SNAP-tag is an engineered variant of the human repair protein O(6)-alkylguanine-DNA alkyltransferase (hAGT) that covalently reacts with benzylguanine derivatives. Reporter groups attached to the benzyl moiety become covalently attached to the SNAP tag while the guanine acts as a leaving group. Incorporation of a quencher on the guanine group ensures that the benzylguanine probe becomes highly fluorescent only upon labeling of the SNAP-tag protein. We describe the use of intramolecularly quenched probes for wash-free labeling of cell surface-localized epidermal growth factor receptor (EGFR) fused to SNAP-tag and for direct quantification of SNAP-tagged β-tubulin in cell lysates. In addition, we have characterized a fast-labeling variant of SNAP-tag, termed SNAP(f), which displays up to a tenfold increase in its reactivity towards benzylguanine substrates. The presented data demonstrate that the combination of SNAP(f) and the fluorogenic substrates greatly reduces the background fluorescence for labeling and imaging applications. This approach enables highly sensitive spatiotemporal investigation of protein dynamics in living cells.     

Targeted Delivery of an Activatable Fluorescent Probe for the Detection of Furin Activity in Living Cells

Furin, a protein convertase, plays a crucial role in the progression of tumors. In this work, a new fluorescent probe consisting of a peptide, Arg‐Val‐Arg‐Arg (RVRR), and an aminoluciferin fluorophore was designed and prepared for the responsive and activatable detection of furin activity in vitro and in living cells. We demonstrated that this probe could be responsive toward furin with an “off–on” florescence signal and generated an approximately 3.58‐fold enhancement in the fluorescence intensity in vitro. Fluorescence imaging in MDA‐MB‐468 and LoVo cells showed that the probe could be cleaved by overexpressed furin with fluorescence turn‐on in MDA‐MB‐468 cells, and this probe was also found to be capable of discriminating between furin‐overexpressing and furin‐deficient cell lines. Our research indicates that this probe has great potential for the detection of furin activity in living cells.     

Rational Design,Synthesis and Biological Evaluation of Modular Fluorogenic Substrates with High Affinity and Selectivity for PTP1B

Protein‐tyrosine phosphatase 1B (PTP1B) is a key regulatory enzyme in several signal transduction pathways, and its upregulation has been associated with type‐2 diabetes, obesity and cancer. Selective determination of the functional significance of PTP1B remains a major challenge because the activity of this crucial enzyme is currently evaluated through the use of fluorescent probes that lack selectivity and are limited to biochemical assays. Here we describe the rational design, synthesis and biological evaluation of new modular PTP1B fluorogenic substrates. The self‐immolative 4‐hydroxybenzyl alcohol has been used as a key component for the design of phosphotyrosine mimics linked to a latent chromophore, which is released through an enzyme‐initiated domino reaction. Preliminary biological investigations showed that, by optimising the stereoelectronic properties and the binding interactions at the enzyme active site, it is possible to achieve substrates with high affinity and promising selectivity. Due to their modular nature, the synthesised fluorogenic probes represent versatile tools; customisation of the different subunits could widen the scope of these probes to a broader range of in vitro assays. Finally, these studies elucidate the critical role played by Asp181 in the PTP1B‐catalysed dephosphorylation mechanism: disruption of the native conformation of this key amino acid residue on the WDP loop yields fluorogenic inhibitors, rather than substrates. For this reason, our studies also represent a step forward for the development of improved PTP1B noncovalent inhibitors.     

Mixed Carbonates as Useful Substrates for a Fluorogenic Assay for Lipases and Esterases

Mixed carbonates of 7‐hydroxy‐4‐methylcoumarin were shown to be a new class of probe for fluorogenic assays. They are promising substrates for fingerprinting enzyme hydrolytic activity, and proved particularly useful because of the low level of nonspecific degradation and ease of synthesis. They are highly relevant for screening lipase and esterase libraries. These advantages make umbelliferyl carbonates highly suitable substrates for high‐throughput screening. Moreover, we report the use of chiral fluorogenic carbonates as enantiopreference probes.     

A Fluorescent sp2-iminosugar with pharmacological chaperone activity for gaucher disease: synthesis and intracellular distribution studies

Gaucher disease (GD) is the most prevalent lysosomal-storage disorder, it is caused by mutations of acid β-glucosidase (β-glucocerebrosidase; β-Glu). Recently, we found that bicyclic nojirimycin (NJ) derivatives of the sp(2)-iminosugar type, including the 6-thio-N'-octyl-(5N,6S)-octyliminomethylidene derivative (6S-NOI-NJ), behaved as very selective competitive inhibitors of the lysosomal β-Glu and exhibited remarkable chaperone activities for several GD mutations. To obtain information about the cellular uptake pathway and intracellular distribution of this family of chaperones, we have synthesized a fluorescent analogue that maintains the fused piperidine-thiazolidine bicyclic skeleton and incorporates a dansyl group in the N'-substituent, namely 6-thio-(5N,6S)-[4-(N'-dansylamino)butyliminomethylidene]nojirimycin (6S-NDI-NJ). This structural modification does not significantly modify the biological activity of the glycomimetic as a chemical chaperone. Our study showed that 6S-NDI-NJ is mainly located in lysosome-related organelles in both normal and GD fibroblasts, and the fluorescent intensity of 6S-NDI-NJ in the lysosome is related to the β-Glu concentration level. 6S-NDI-NJ also can enter cultured neuronal cells and act as a chaperone. Competitive inhibition studies of 6S-NDI-NJ uptake in fibroblasts showed that high concentrations of D-glucose have no effect on chaperone internalization, suggesting that it enters the cells through glucose-transporter-independent mechanisms.     

Utilizing functionalized bromomaleimides for fluorogenic conjugation and PEGylation of enzymes

Two efficient enzyme conjugation techniques have been explored by exploiting the reactions of bromomaleimides. The conjugations utilize monobromo‐ and dibromomaleimides, which have been reacted with reduced disulfide bonds or terminal amines in α‐chymotrypsin and human lysozyme. These reactions allow the formation of dithio‐, monoamino‐ and aminobromomaleimides, which are solvent‐dependent fluorophores and have a handle for further functionalization, which allowed fluorogenic PEGylation via this technique. In this work, the efficiency of these maleimide conjugations was monitored and the fluorescence of the resulting conjugates was examined. The quantum yields of the small‐molecule maleimide conjugates were calculated, but are low due to solvent quenching effects. Catalytic activities of the conjugate enzymes were compared to those of their respective native enzymes, which show no discernible effect of the modifications on enzymatic activity or stability at room temperature. © 2018 Society of Chemical Industry     

Design and synthesis of conformationally constrained cyclophilin inhibitors showing a cyclosporin-A phenotype in C. elegans

Cyclophilin A (CypA) is a member of the immunophilin family of proteins and receptor for the immunosuppressant drug cyclosporin A (CsA). Here we describe the design and synthesis of a new class of small-molecule inhibitors for CypA that are based upon a dimedone template. Electrospray mass spectrometry is utilised as an initial screen to quantify the protein affinity of the ligands. Active inhibitors and fluorescently labelled derivatives are then used as chemical probes for investigating the biological role of cyclophilins in the nematode Caenorhabditis elegans.     

Design and Expeditious Synthesis of Quinoline-Pyrene-Based Ratiometric Fluorescent Probes for Targeting Lysosomal pH

Intracellular pH plays a significant role in many pathological and physiological processes. A series of quinoline-pyrene probes were synthesized in one-step fashion through an oxonium-ion-triggered alkyne carboamination sequence involving C−C, C−O and C−N bond formation for intracellular pH sensing. The quinoline-pyrenes showed significant red shifts at low pH. Fluorescence lifetime decay measurements of the probes showed decreases in lifetime at pH 4. The probes showed excellent selectivity in the presence of various potential interfering agents such as amino acids and cations/anions. Furthermore, the probes were found to show completely reversible emission behaviour in the window between pH 4 and 7. A morpholine-substituted quinoline-pyrene probe efficiently stained lysosomes with high Pearson correlation coefficients (0.86) with Lysotracker Deep Red DND-99 as a reference. A co-localization study of the probe with Lysotracker DND-99 showed selective intracellular targeting and a shift in fluorescence emission due to acidic lysosomal pH.     

In Vitro Fluorogenic Real‐Time Assay of the Repair of Oxidative DNA Damage

The repair of oxidative damage to DNA is essential to avoid mutations that lead to cancer. Oxidized DNA bases, such as 8‐oxoguanine, are a main source of these mutations, and the enzyme 8‐oxoguanine glycosylase 1 (OGG1) is the chief human enzyme that excises 8‐oxoguanine from DNA. The activity of OGG1 has been linked to human inflammation responses and to cancer, and researchers are beginning to search for inhibitors of the enzyme. However, measuring the activity of the enzyme typically requires laborious gel‐based measurements of radiolabeled DNAs. Here we report the design and properties of fluorogenic probes that directly report on the activity of OGG1 (and its bacterial homologue Fpg) in real time as the oxidized base is excised. The probes are short, modified DNA oligomers containing fluorescent DNA bases and are designed to utilize 8‐oxoguanine itself as a fluorescence quencher. Screening of combinations of fluorophores and 8‐oxoguanine revealed two fluorophores, pyrene and tCo, that are strongly quenched by the damaged base. We tested 42 potential probes containing these fluorophores: the optimum probe, OGR1, yields a 60‐fold light‐up signal in vitro with OGG1 and Fpg. It can report on oxidative repair activity in mammalian cell lysate and with bacterial cells overexpressing a repair enzyme. Such probes might prove useful in quantifying enzyme activity and performing competitive inhibition assays.     

2′‐Bispyrene‐Modified 2′‐O‐Methyl RNA Probes as Useful Tools for the Detection of RNA: Synthesis,Fluorescent Properties,and Duplex Stability

The synthesis and properties two series of new 2′‐O‐methyl RNA probes, each containing a single insertion of a 2′‐bispyrenylmethylphosphorodiamidate derivative of a nucleotide (U, C, A, and G), are described. As demonstrated by UV melting studies, the probes form stable complexes with model RNAs and DNAs. Significant increases (up to 21‐fold) in pyrene excimer fluorescence intensity were observed upon binding of most of the probes with complementary RNAs, but not with DNAs. The fluorescence spectra are independent of the nature of the modified nucleotides. The nucleotides on the 5′‐side of the modified nucleotide have no effect on the fluorescence spectra, whereas the natures of the two nucleotides on the 3′‐side are important: CC, CG, and UC dinucleotide units on the 3′‐side of the modified nucleotide provide the maximum increases in excimer fluorescence intensity. This study suggests that these 2′‐bispyrene‐labeled 2′‐O‐methyl RNA probes might be useful tools for detection of RNAs.     

New methods for the generation of carbohydrate arrays on glass slides and their evaluation

Glycosides, having spacers functionalized with an aldehyde or a carboxylic group, were immobilized through reductive amination or amidation, respectively, onto amino-functionalized glass slides. Hybridization experiments with lectins exhibited very little nonspecific protein binding, hence precluding the necessity for the blocking of unreacted functional groups on the glass slide. The covalency and the concentration dependency of the sugar ligation to the glass slide were demonstrated; the reversibility and the selectivity of lectin-carbohydrate interactions were shown.     

Activity-based probes for the study of proteases: recent advances and developments

Proteases are important targets for the treatment of human disease. Several protease inhibitors have failed in clinical trials due to a lack of in vivo specificity, indicating the need for studies of protease function and inhibition in complex, disease-related models. The tight post-translational regulation of protease activity complicates protease analysis by traditional proteomics methods. Activity-based protein profiling is a powerful technique that can resolve this issue. It uses small-molecule tools-activity-based probes-to label and analyze active enzymes in lysates, cells, and whole animals. Over the last twelve years, a wide variety of protease activity-based probes have been developed. These synthetic efforts have enabled techniques ranging from real-time in vivo imaging of protease activity to high-throughput screening of uncharacterized proteases. This Review introduces the general principles of activity-based protein profiling and describes the recent advancements in probe design and analysis techniques, which have increased the knowledge of protease biology and will aid future protease drug discovery.     

Responsive Fluorescent PNA Analogue as a Tool for Detecting G‐quadruplex Motifs of Oncogenes and Activity of Toxic Ribosome‐Inactivating Proteins

Fluorescent oligomers that are resistant to enzymatic degradation and report their binding to target oligonucleotides (ONs) by changes in fluorescence properties are highly useful in developing nucleic‐acid‐based diagnostic tools and therapeutic strategies. Here, we describe the synthesis and photophysical characterization of fluorescent peptide nucleic acid (PNA) building blocks made of microenvironment‐sensitive 5‐(benzofuran‐2‐yl)‐ and 5‐(benzothiophen‐2‐yl)‐uracil cores. The emissive monomers, when incorporated into PNA oligomers and hybridized to complementary ONs, are minimally perturbing and are highly sensitive to their neighboring base environment. In particular, benzothiophene‐modified PNA reports the hybridization process with significant enhancement in fluorescence intensity, even when placed in the vicinity of guanine residues, which often quench fluorescence. This feature was used in the turn‐on detection of G‐quadruplex‐forming promoter DNA sequences of human proto‐oncogenes (c‐myc and c‐kit). Furthermore, the ability of benzothiophene‐modified PNA oligomer to report the presence of an abasic site in RNA enabled us to develop a simple fluorescence hybridization assay to detect and estimate the depurination activity of ribosome‐inactivating protein toxins. Our results demonstrate that this approach with responsive PNA probes will provide new opportunities to develop robust tools to study nucleic acids.     

Conjugated polyelectrolytes--conformation-sensitive optical probes for staining and characterization of amyloid deposits

Specific markers for diseases associated with protein aggregate depositions are of great interest. Here we report the use of conjugated polyelectrolytes as conformation-sensitive optical probes for histological labeling of amyloid deposits in ex vivo tissue samples-amyloid light chains in primary systemic amyloidosis, islet amyloid polypeptide in human pancreas, and Abeta amyloid in Alzheimer's disease. Under suitable conditions, these probes bind specifically to amyloid deposits, and this is seen as an orange-red emission from the polyelectrolyte. Furthermore, the probes emit light of different colors when bound to different amyloid deposits or other intracellular structures. This phenomenon is most probably due to differences in the protein conformation in these structures. Hence, different protein conformations will generate geometric alterations of the bound polyelectrolyte backbone, affording different emissions from the bound probe. Conformation-sensitive probes thus provide a direct link between spectral signal and protein conformation. Finally, the probes also proved useful for ex vivo fluorescence imaging by multiphoton excitation.     

Solid-Phase Synthesis and Application of a Clickable Version of Epoxomicin for Proteasome Activity Analysis

Degradation of proteins by the proteasome is an essential cellular process and one that many wish to study in a variety of disease types. There are commercially available probes that can monitor proteasome activity in cells, but they typically contain common fluorophores that limit their simultaneous use with other activity-based probes. In order to exchange the fluorophore or incorporate an enrichment tag, the proteasome probe likely has to be synthesized which can be cumbersome. Here, we describe a simple synthetic procedure that only requires one purification step to generate epoxomicin, a selective proteasome inhibitor, with a terminal alkyne. Through a copper-catalyzed cycloaddition, any moiety containing an azide can be incorporated into the probe. Many fluorophores are commercially available that contain an azide that can be “clicked”, allowing this proteasome activity probe to be included into already established assays to monitor both proteasome activity and other cellular activities of interest.     

Chemical Probes for Profiling of MALT1 Protease Activity

The paracaspase MALT1 is a key regulator of the human immune response. It is implicated in a variety of human diseases. For example, deregulated protease activity drives the survival of malignant lymphomas and is involved in the pathophysiology of autoimmune/inflammatory diseases. Thus, MALT1 has attracted attention as promising drug target. Although many MALT1 inhibitors have been identified, molecular tools to study MALT1 activity, target engagement and inhibition in complex biological samples, such as living cells and patient material, are still scarce. Such tools are valuable to validate MALT1 as a drug target in vivo and to assess yet unknown biological roles of MALT1. In this review, we discuss the recent literature on the development and biological application of molecular tools to study MALT1 activity and inhibition.     

Novel fluorescent phosphonic acid esters for discrimination of lipases and esterases

Lipases and esterases are responsible for carboxylester hydrolysis inside and outside cells and are useful biocatalysts for (stereo)selective modification of synthetic substrates. Here we describe novel fluorescent suicide inhibitors that differ in structure and polarity for screening and discrimination of lipolytic enzymes in enzyme preparations. The inhibitors covalently react with the enzymes to form fluorescent lipid-protein complexes that can be resolved by gel electrophoresis. The selectivities of the inhibitors were determined by using different (phospho)lipase, esterase and cholesterol esterase preparations. The results indicate that formation of an inhibitor-enzyme complex is highly dependent on the chemical structure of the inhibitor. We identified inhibitors with very low specificity, and other derivatives that were highly specific for certain subgroups of lipolytic enzymes such as lipases and cholesterol esterases. A combination of these substrate-analogous activity probes represents a useful toolbox for rapid identification and classification of serine hydrolase enzymes.