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.     

Far‐Red Fluorogenic Probes for Esterase and Lipase Detection

Fluorogenic enzyme probes go from a dark to a bright state following hydrolysis and can provide a sensitive, real‐time readout of enzyme activity. They are useful for examining enzymatic activity in bacteria, including the human pathogen Mycobacterium tuberculosis. Herein, we describe two fluorogenic esterase probes derived from the far‐red fluorophore 7‐hydroxy‐9H‐(1,3‐dichloro‐9,9‐dimethylacridin‐2‐one) (DDAO). These probes offer enhanced optical properties compared to existing esterase probes because the hydrolysis product, DDAO, excites above 600 nm while retaining a good quantum yield (?=0.40). We validated both probes with a panel of commercially available enzymes alongside known resorufin‐ and fluorescein‐derived esterase substrates. Furthermore, we used these probes to reveal esterase activity in protein gel‐resolved mycobacterial lysates. These probes represent new tools for esterase detection and characterization and should find use in a variety of applications.     

Clickable 4‐Oxo‐β‐lactam‐Based Selective Probing for Human Neutrophil Elastase Related Proteomes

Human neutrophil elastase (HNE) is a serine protease associated with several inflammatory processes such as chronic obstructive pulmonary disease (COPD). The precise involvement of HNE in COPD and other inflammatory disease mechanisms has yet to be clarified. Herein we report a copper‐catalyzed alkyne–azide 1,3‐dipolar cycloaddition (CuAAC, or ′click′ chemistry) approach based on the 4‐oxo‐β‐lactam warhead that yielded potent HNE inhibitors containing a triazole moiety. The resulting structure–activity relationships set the basis to develop fluorescent and biotinylated activity‐based probes as tools for molecular functional analysis. Attaching the tags to the 4‐oxo‐β‐lactam scaffold did not affect HNE inhibitory activity, as revealed by the IC₅₀ values in the nanomolar range (56–118 nm ) displayed by the probes. The nitrobenzoxadiazole (NBD)‐based probe presented the best binding properties (ligand efficiency (LE)=0.31) combined with an excellent lipophilic ligand efficiency (LLE=4.7). Moreover, the probes showed adequate fluorescence properties, internalization in human neutrophils, and suitable detection of HNE in the presence of a large excess of cell lysate proteins. This allows the development of activity‐based probes with promising applications in target validation and identification, as well as diagnostic tools.     

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.     

Fluorescent Mechanism‐Based Probe for Aerobic Flavin‐Dependent Enzyme Activity

Diversity in non‐ribosomal peptide and polyketide secondary metabolism is facilitated by interactions between biosynthetic domains with discrete monomer loading and their cognate tailoring enzymes, such as oxidation or halogenation enzymes. The cooperation between peptidyl carrier proteins and flavin‐dependent enzymes offers a specialized strategy for monomer selectivity for oxidization of small molecules from within a complex cellular milieu. In an effort to study this process, we have developed fluorescent probes to selectively label aerobic flavin‐dependent enzymes. Here we report the preparation and implementation of these tools to label oxidase, monooxygenase, and halogenase flavin‐dependent enzymes.     

Ubiquitin‐Based Probes Prepared by Total Synthesis To Profile the Activity of Deubiquitinating Enzymes

Epitope‐tagged active‐site‐directed probes are widely used to visualize the activity of deubiquitinases (DUBs) in cell extracts, to investigate the specificity and potency of small‐molecule DUB inhibitors, and to isolate and identify DUBs by mass spectrometry. With DUBs arising as novel potential drug targets, probes are required that can be produced in sufficient amounts and to meet the specific needs of a given experiment. The established method for the generation of DUB probes makes use of labor‐intensive intein‐based methods that have inherent limitations concerning the incorporation of unnatural amino acids and the amount of material that can be obtained. Here, we describe the total chemical synthesis of active‐site‐directed probes and their application to activity‐based profiling and identification of functional DUBs. This synthetic methodology allowed the easy incorporation of desired tags for specific applications, for example, fluorescent reporters, handles for immunoprecipitation or affinity pull‐down, and cleavable linkers. Additionally, the synthetic method can be scaled up to provide significant amounts of probe. Fluorescent ubiquitin probes allowed faster, in‐gel detection of active DUBs, as compared to (immuno)blotting procedures. A biotinylated probe holding a photocleavable linker enabled the affinity pull‐down and subsequent mild, photorelease of DUBs. Also, DUB activity levels were monitored in response to overexpression or knockdown, and to inhibition by small molecules. Furthermore, fluorescent probes revealed differential DUB activity profiles in a panel of lung and prostate cancer cells.     

Novel and Selective Fluorescent σ2‐Receptor Ligand with a 3,4‐Dihydroisoquinolin‐1‐one Scaffold: A Tool to Study σ2 Receptors in Living Cells

Although sigma‐2 (σ₂) receptors are still enigmatic proteins, they are promising targets for tumor treatment and diagnosis. With the aim of clarifying their role in oncology, we developed a σ₂‐selective fluorescent tracer (compound 5 ) as a specific tool to study σ₂ receptors. By using flow cytometry with 5 , we performed competition binding studies on three different cell lines where we also detected the content of the σ₂ receptors, avoiding the inconvenient use of radioligands. Comparison with a previously developed mixed σ₁/σ₂ fluorescent tracer ( 1 ) also allowed for the detection of σ₁ receptors within these cells. Results obtained by flow cytometry with tracers 1 and 5 were confirmed by standard methods (western blot for σ₁, and Scatchard analysis for σ₂ receptors). Thus, we have produced powerful new tools for research on the σ whose reliability and adaptability to a number of fluorescence techniques will be useful to elucidate the roles of σ receptors in oncology.     

Probes for Photoaffinity Labelling of Kinases

Protein kinases, one of the largest enzyme superfamilies, regulate many physiological and pathological processes. They are drug targets for multiple human diseases, including various cancer types. Probes for the photoaffinity labelling of kinases are important research tools for the study of members of this enzyme superfamily. In this review, we discuss the design principles of these probes, which are mainly derived from inhibitors targeting the ATP pocket. Overall, insights from crystal structures guide the placement of photoreactive groups and detection tags. This has resulted in a wide variety of probes, of which we provide a comprehensive overview. We also discuss several areas of application of these probes, including the identification of targets and off-targets of kinase inhibitors, mapping of their binding sites, the development of inhibitor screening assays, the imaging of kinases, and identification of protein binding partners.     

Phosphoramidates as Novel Activity‐Based Probes for Serine Proteases

Activity‐based probes (ABPs) are small molecules that exclusively form covalent bonds with catalytically active enzymes. In the last decade, they have especially been used in functional proteomics studies of proteases. Here, we present phosphoramidate peptides as a novel type of ABP for serine proteases. These molecules can be made in a straightforward manner by standard Fmoc‐based solid‐phase peptide synthesis, allowing rapid diversification. The resulting ABPs covalently bind different serine proteases, depending on the amino acid recognition element adjacent to the reactive group. A reporter tag enables downstream gel‐based analysis or LC‐MS/MS‐mediated identification of the targeted proteases. Overall, we believe that these readily accessible probes will provide new avenues for the functional study of serine proteases in complex proteomes.     

A One‐Step Staining Probe for Phosphatidylethanolamine

Phosphatidylethanolamine (PE) is an abundant phospholipid in cellular membranes, but relatively little is known about the kinetics of PE in biological membrane systems. Characterizing PE on a cellular level has been challenging owing to a lack of proper molecular tools. The lantibiotic duramycin and its structural analogue, cinnamycin, are currently the only known polypeptides that have an established stereospecific structure for binding membrane PE with high affinity and high specificity. These lantibiotics are recognized for their potential as molecular probes for studying PE kinetics in various membranes. However, owing to their antibiotic nature, duramycin and cinnamycin exhibit appreciable levels of cytotoxicity at low micromolar concentrations in cultured mammalian cells by inducing membrane distortion and possible PE redistribution. These issues can potentially complicate study design and data interpretation. Here, we report the construction of a molecular probe consisting of duramycin attached to the C terminus of green fluorescent protein (GFP) by a PEG linker at a stoichiometry of 1. The construct retained specific binding toward PE and essentially no cytotoxicity compared to native duramycin. The biological utilities of this probe were demonstrated in a number of cellular staining studies involving PE dynamics. The availability of a one‐step, nontoxic molecular probe for PE will enable characterization of the biology of this important phospholipid.     

A Novel Photoaffinity‐Based Probe for Selective Detection of Cathepsin L Active Form

Detecting the active forms of proteases by using activity‐based probes in complex proteomes has become an intensively investigated field of research over the past years because many pathogenic conditions involve alterations in protease activities. The detection of lysosomal cysteine proteases, the cathepsins, has mostly relied on the use of probes that incorporate reactive electrophilic moieties to modify a cysteine in the active site covalently. Here we report the first example of an activity‐based probe that targets the cathepsins and incorporates a photoactivatable benzophenone group for covalent labelling. When tested on a set of five cathepsins (B, K, L, S and V), this probe selectively labelled the active site of cathepsin L. Furthermore, when tested on crude cell extracts, the probe specifically detected cathepsin L quantities as low as a few picomoles. This study suggests that photoaffinity labelling is a promising approach for developing highly selective and useful cathepsin L probes. In particular, this probe might allow the detection of small amounts of the secreted active cathepsin L form in the cellular microenvironment in vitro and ex vivo.     

Chemical Probes to Directly Profile Palmitoleoylation of Proteins

Palmitoleoylation is a unique fatty acylation of proteins in which a monounsaturated fatty acid, palmitoleic acid (C16:1), is covalently attached to a protein. Wnt proteins are known to be palmitoleoylated by cis‐Δ9 palmitoleate at conserved serine residues. O‐palmitoleoylation plays a critical role in regulating Wnt secretion, binding to the receptors, and in the dynamics of Wnt signaling. Therefore, protein palmitoleoylation is important in tissue homeostasis and tumorigenesis. Chemical probes based on saturated fatty acids, such as ω‐alkynyl palmitic acid (Alk‐14 or Alk‐C₁₆), have been used to study Wnt palmitoleoylation. However, such probes require prior conversion to the unsaturated fatty acid by stearoyl‐CoA desaturase (SCD) in cells, significantly decreasing their selectivity and efficiency for studying protein palmitoleoylation. We synthesized and characterized ω‐alkynyl cis‐ and trans‐palmitoleic acids (cis‐ and trans‐Alk‐14:1) as chemical probes to directly study protein palmitoleoylation. We found that cis‐Alk‐14:1 could more efficiently label Wnt proteins in cells. Interestingly, the DHHC family of palmitoyl acyltransferases can charge both saturated and unsaturated fatty acids, potentially using both as acyl donors in protein palmitoylation and palmitoleoylation. Furthermore, proteomic analysis of targets labeled by these probes revealed new cis‐ and trans‐palmitoleoylated proteins. Our studies provided new chemical tools and revealed new insights into palmitoleoylation in cell signaling.     

Inhibitor and protein microarrays for activity-based recognition of lipolytic enzymes

Protein and small-molecule microarrays are useful tools for high-throughput analysis of DNA-protein, protein-protein, and protein-small molecule interactions. Here we report on novel microarrays for activity screening of lipases and esterases based on phosphonic acid ester inhibitors. These compounds are activity recognition probes (ARPs) and bind to active serine hydrolases in a stoichiometric and irreversible manner. Protein microarrays were generated by spotting six different lipolytic enzymes onto hydrogel-coated glass slides. The activity of immobilized enzymes was determined after treatment with fluorescently labeled ARPs. Alternatively, biotinylated ARPs were bound to streptavidin slides in order to identify their affinity for enzymes in solution. Both systems, the protein- and ARP microarrays proved to be useful and versatile tools for the rapid identification and characterization of novel and known lipolytic enzymes.     

Highly Efficient Quencher‐Free Molecular Beacon Systems Containing 2‐Ethynyldibenzofuran‐ and 2‐Ethynyldibenzothiophene‐Labeled 2′‐Deoxyuridine Units

We have prepared two fluorescent DNA probes—U^DBF and U^DBT, containing 2‐ethynyldibenzofuran and 2‐ethynyldibenzothiophene moieties, respectively, covalently attached to the base dU—and incorporated them in the central positions of oligodeoxynucleotides (ODNs) so as to develop new types of quencher‐free linear beacon probes and investigate the effect of functionalization of the fluorene scaffold on the photophysical properties of the fluorescent ODNs. The ODNs containing adenine flanking bases (FBs) displayed a selective fluorescence “turn‐off” response to mismatched targets with guanine bases; this suggests that these probes could be used as base‐discriminating fluorescent nucleotides. On the other hand, we observed a “turn‐on” response to matched targets when the U^DBF and U^DBT units of ODNs containing pyrimidine‐based FBs were positioned opposite the four natural nucleobases. In particular, an ODN incorporating U^DBT and cytosine FBs has potential use in single‐nucleotide polymorphism typing.     

Progress in the Development of non‐BET Bromodomain Chemical Probes

The bromodomain and extra terminal (BET) family of bromodomains have been the focus of extensive research, leading to the development of many potent, selective chemical probes and recent clinical assets. The profound biology associated with BET bromodomain inhibition has provided a convincing rationale for targeting bromodomains for the treatment of disease. However, the BET family represents just eight of the at least 56 human bromodomains identified to date. Until recently, there has been significantly less interest in non‐BET bromodomains, leaving a vast area of research and the majority of this new target class yet to be thoroughly investigated. It has been widely reported that several non‐BET bromodomain containing proteins are associated with various diseases including cancer and HIV. Therefore, the development of chemical probes for non‐BET bromodomains will facilitate elucidation of their precise biological roles and potentially lead to the development of new medicines. This review summarises the progress made towards the development of non‐BET bromodomain chemical probes to date. In addition, we highlight the potential for future work in this new and exciting area.     

A Fluorescent Probe for Imaging Sirtuin Activity in Living Cells,Based on One‐Step Cleavage of the Dabcyl Quencher

Sirtuins (SIRTs) are a family of NAD⁺‐dependent histone deacetylases. In mammals, dysfunction of SIRTs is associated with age‐related metabolic diseases and cancers, so SIRT modulators are considered attractive therapeutic targets. However, current screening methodologies are problematic, and no tools for imaging endogenous SIRT activity in living cells have been available until now. In this work we present a series of simple and highly sensitive new SIRT activity probes. Fluorescence of these probes is activated by SIRT‐mediated hydrolytic release of a 4‐(4‐dimethylaminophenylazo)benzoyl (Dabcyl)‐based FRET quencher moiety from the ?‐amino group of lysine in a nonapeptide derived from histone H3K9 and bearing a C‐terminal fluorophore. The probe SFP3 detected activities of SIRT1, ‐2, ‐3, and ‐6, which exhibit deacylase activities towards long‐chain fatty acyl groups. We then truncated the molecular structure of SFP3 in order to improve both its stability to peptidases and its membrane permeability, and developed probe KST‐F, which showed specificity for SIRT1 over SIRT2 and SIRT3. We show that KST‐F can visualize endogenous SIRT1 activity in living cells.     

Glycan‐Mediated,Ligand‐Controlled Click Chemistry for Drug‐Target Identification

Membrane‐bound proteins are important pharmaceutical drug targets, yet few strategies exist for the identification of small‐molecule‐targeted membrane proteins in live‐cell systems. By exploiting metabolic glycan engineering of cell membrane proteins, we have developed an in situ glycan‐mediated ligand‐controlled click (“GLiCo‐Click”) chemistry methodology that enables the attachment of small‐molecule chemical probes to their receptor protein through glycans on live cells. In addition to enabling receptor enrichment from cell lysates, this strategy can be used to demonstrate target receptor engagement and enables the molecular characterization of receptors.     

Evaluation of a 7‐Methoxycoumarin‐3‐carboxylic Acid Ester Derivative as a Fluorescent,Cell‐Cleavable,Phosphonate Protecting Group

Cell‐cleavable protecting groups often enhance cellular delivery of species that are charged at physiological pH. Although several phosphonate protecting groups have achieved clinical success, it remains difficult to use these prodrugs in live cells to clarify biological mechanisms. Here, we present a strategy that uses a 7‐methoxycoumarin‐3‐carboxylic acid ester as a fluorescent protecting group. This strategy was applied to synthesis of an (E)‐4‐hydroxy‐3‐methyl‐but‐2‐enyl diphosphate (HMBPP) analogue to assess cellular uptake and human Vγ9Vδ2 T cell activation. The fluorescent ester displayed low cellular toxicity (IC₅₀>100 μm ) and strong T cell activation (EC₅₀=0.018 μm ) relative to the unprotected anion (EC₅₀=23 μm ). The coumarin‐derived analogue allowed no‐wash analysis of biological deprotection, which revealed rapid internalization of the prodrug. These results demonstrate that fluorescent groups can be applied both as functional drug delivery tools and useful biological probes of drug uptake.     

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.     

Red Fluorescent Turn‐On Ligands for Imaging and Quantifying G Protein‐Coupled Receptors in Living Cells

Classical fluorescence‐based approaches to monitor ligand–protein interactions are generally hampered by the background signal of unbound ligand, which must be removed by tedious washing steps. To overcome this major limitation, we report here the first red fluorescent turn‐on probes for a G protein‐coupled receptor (oxytocin receptor) at the surface of living cells. The peptide ligand carbetocin was conjugated to one of the best solvatochromic (fluorogenic) dyes, Nile Red, which turns on emission when reaching the hydrophobic environment of the receptor. We showed that the incorporation of hydrophilic octa(ethylene glycol) linker between the pharmacophore and the dye minimized nonspecific interaction of the probe with serum proteins and lipid membranes, thus ensuring receptor‐specific turn‐on response. The new ligand was successfully applied for background‐free imaging and quantification of oxytocin receptors in living cells.