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.     

Fluorescent Probes to Characterise FK506‐Binding Proteins

Talented all‐rounders : Fluorescence polarisation assays were developed for members of the FK506‐binding protein family by using fluorescent rapamycin analogues (demonstrated in the figure). These tracers retain medium to high affinity to all tested proteins (FKBP12, ‐12.6, ‐13, ‐25, ‐51, ‐52). They can be used for active‐site titrations, competition assays with unlabelled ligands and enable a robust, miniaturized assay adequate for high‐throughput screening.

     

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.     

A New Fluorogenic Probe for the Detection of endo‐β‐N‐Acetylglucosaminidase

We developed a fluorescence‐quenching‐based assay system to determine the hydrolysis activity of endo‐β‐N‐acetylglucosaminidases (ENGases). The pentasaccharide derivative 1 was labeled with an N‐methylanthraniloyl group as a reporter dye at the non‐reducing end and with a 2,4‐dinitrophenyl group as a quencher molecule at the reducing end. This derivative is hydrolyzed by ENGase, resulting in an increase in fluorescence intensity. Thus, the fluorescence signal is directly proportional to the amount of the tetrasaccharide derivative, hence allowing ENGase activity to be evaluated easily and quantitatively. Using this system, we succeeded in measuring the hydrolysis activities of ENGases and thus the inhibitory activities of known inhibitors. We confirmed that this assay system is suitable for high‐throughput screening for potential inhibitors of human ENGase that might serve as therapeutic agents for the treatment of N‐glycanase 1 (NGLY1) deficiency.     

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.     

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.     

Integrin‐Targeting Fluorescent Proteins: Exploration of RGD Insertion Sites

The potential of the fluorescent protein scaffold to control peptide sequence functionality is illustrated by an exploration of fluorescent proteins as novel probes for targeting integrins. A library of fluorescent mCitrine proteins with RGD motifs incorporated at several positions in loops within the protein main chain was generated and characterized. Amino acid mutations to RGD as well as RGD insertions were evaluated: both led to constructs with typical mCitrine fluorescent properties. Screening experiments against four human integrin receptors revealed two strong‐binding constructs and two selective integrin binders. The effect of the site of RGD incorporation illustrates the importance of the protein scaffold on RGD sequence functionality, leading to fluorescent protein constructs with the potential for selective integrin targeting.     

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.     

Activity‐Based Probes for HECT E3 Ubiquitin Ligases

Activity‐based probes (ABPs) have been used to dissect the biochemical/structural properties and cellular functions of deubiquitinases. However, their utility in studying cysteine‐based E3 ubiquitin ligases has been limited. In this study, we evaluate the use of ubiquitin‐ABPs (Ub‐VME and Ub‐PA) and a novel set of E2–Ub‐ABPs on a panel of HECT E3 ubiquitin ligases. Our in vitro data show that ubiquitin‐ABPs can label HECT domains. We also provide the first evidence that, in addition to the RBR E3 ubiquitin ligase Parkin, E2–Ub‐ABPs can also label the catalytic HECT domains of NEDD4, UBE3C, and HECTD1. Importantly, the endogenous proteasomal E3 ligase UBE3C was also successfully labelled by Ub‐PA and His‐UBE2D2–Ub‐ABP in lysate of cells grown under basal conditions. Our findings provide novel insights into the use of ABPs for the study of HECT E3 ubiquitin ligases.     

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.     

Long Residence Times Revealed by Aurora A Kinase‐Targeting Fluorescent Probes Derived from Inhibitors MLN8237 and VX‐689

We report the development of three fluorescent probes for protein kinase Aurora A that are derived from the well‐known inhibitors MLN8237 and VX‐689 (MK‐5108). Two of these probes target the ATP site of Aurora A, and one targets simultaneously the ATP and substrate sites of the kinase. The probes were tested in an assay with fluorescence polarisation/anisotropy readout, and we demonstrated slow association kinetics and long residence time of the probes (k_on 10⁵–10⁷ M ^?1 s^?1, k_off 10^?3–10^?4 s^?1; residence time 500–3000 s). The presence of the Aurora A activator TPX2 caused a significant reduction in the on‐rate and increase in the off‐rate of fluorescent probes targeting ATP site. These observations were supported by Aurora A inhibition assays with MLN8237 and VX‐689. Overall, our results emphasise the importance of rational design of experiments with these compounds and correct interpretation of the obtained data.     

A Fluorogenic AggTag Method Based on Halo- and SNAP-Tags to Simultaneously Detect Aggregation of Two Proteins in Live Cells

Protein aggregation involves the assembly of partially misfolded proteins into oligomeric and higher-order structures that have been associated with several neurodegenerative diseases. However, numerous questions relating to protein aggregation remain unanswered due to the lack of available tools for visualization of these species in living cells. We recently developed a fluorogenic method named aggregation tag (AggTag), and presented the AggTag probe P1 , based on a Halo-tag ligand, to report on the aggregation of a protein of interest (POI) in live cells. However, the Halo-tag-based AggTag method only detects the aggregation of one specific POI at a time. In this study, we have expanded the AggTag method by using SNAP-tag technology to enable fluorogenic and biorthogonal detection of the aggregation of two different POIs simultaneously in live cells. A new AggTag probe— P2 , based on a SNAP-tag ligand bearing a green solvatochromic fluorophore—was synthesized for this purpose. Using confocal imaging and chemical crosslinking experiments, we confirmed that P2 can also report both on soluble oligomers and on insoluble aggregates of a POI fused with SNAP-tag in live cells. Ultimately, we showed that the orthogonal fluorescence of P1 and P2 allows for simultaneous visualization of two different pathogenic protein aggregates in the same cell.     

Design,Synthesis, and Biological Evaluation of Novel Fluorescent Probes Targeting the 18-kDa Translocator Protein

A series of fluorescent probes from the 6-chloro-2-phenylimidazo[1,2-a]pyridine-3-yl acetamides ligands featuring the 7-nitro-2-oxa-1,3-diazol-4-yl (NBD) moiety has been synthesized and biologically evaluated for their fluorescence properties and for their binding affinity to the 18-kDa translocator protein (TSPO). Spectroscopic studies including UV/Vis absorption and fluorescence measurements showed that the synthesized fluorescent probes exhibit favorable spectroscopic properties, especially in nonpolar environments. In vitro fluorescence staining in brain sections from lipopolysaccharide (LPS)-injected mice revealed partial colocalization of the probes with the TSPO. The TSPO binding affinity of the probes was measured on crude mitochondrial fractions separated from rat brain homogenates in a [¹¹C]PK11195 radioligand binding assay. All the new fluorescent probes demonstrated moderate to high binding affinity to the TSPO, with affinity (K_i) values ranging from 0.58 nM to 3.28 μM. Taking these data together, we propose that the new fluorescent probes could be used to visualize the TSPO.     

Peptide‐Based Fluorescent Probes for Deacetylase and Decrotonylase Activity: Toward a General Platform for Real‐Time Detection of Lysine Deacylation

Histone deacetylases regulate the acetylation levels of numerous proteins and play key roles in physiological processes and disease states. In addition to acetyl groups, deacetylases can remove other acyl modifications on lysines, the roles and regulation of which are far less understood. A peptide‐based fluorescent probe for single‐reagent, real‐time detection of deacetylase activity that can be readily adapted for probing broader lysine deacylation, including decrotonylation, is reported. Following cleavage of the lysine modification, the probe undergoes rapid intramolecular imine formation that results in marked optical changes, thus enabling convenient detection of deacylase activity with good statistical Z′ factors for both absorption and fluorescence modalities. The peptide‐based design offers broader isozyme scope than that of small‐molecule analogues, and is suitable for probing both metal‐ and nicotinamide adenine dinucleotide (NAD⁺)‐dependent deacetylases. With an effective sirtuin activity assay in hand, it is demonstrated that iron chelation by Sirtinol, a commonly employed sirtuin inhibitor, results in an enhancement in the inhibitory activity of the compound that may affect its performance in vivo.     

Development of Fluorescent Protein Probes Specific for Parallel DNA and RNA G‐Quadruplexes

We have developed fluorescent protein probes specific for parallel G‐quadruplexes by attaching cyan fluorescent protein to the G‐quadruplex‐binding motif of the RNA helicase RHAU. Fluorescent probes containing RHAU peptide fragments of different lengths were constructed, and their binding to G‐quadruplexes was characterized. The selective recognition and discrimination of G‐quadruplex topologies by the fluorescent protein probes was easily detected by the naked eye or by conventional gel imaging.