Activity-based Fluorescent Probes Research Articles

Activity-Based Fluorescent Probes for Hydrogen Sulfide and Related Reactive Sulfur Species.

Hydrogen sulfide (H2S) is not only a well-established toxic gas but also an important small molecule bioregulator in all kingdoms of life. In contemporary biology, H2S is often classified as a "gasotransmitter," meaning that it is an endogenously produced membrane permeable gas that carries out essential cellular processes. Fluorescent probes for H2S and related reactive sulfur species (RSS) detection provide an important cornerstone for investigating the multifaceted roles of these important small molecules in complex biological systems. A now common approach to develop such tools is to develop "activity-based probes" that couple a specific H2S-mediated chemical reaction to a fluorescent output. This Review covers the different types of such probes and also highlights the chemical mechanisms by which each probe type is activated by specific RSS. Common examples include reduction of oxidized nitrogen motifs, disulfide exchange, electrophilic reactions, metal precipitation, and metal coordination. In addition, we also outline complementary activity-based probes for imaging reductant-labile and sulfane sulfur species, including persulfides and polysulfides. For probes highlighted in this Review, we focus on small molecule systems with demonstrated compatibility in cellular systems or related applications. Building from breadth of reported activity-based strategies and application, we also highlight key unmet challenges and future opportunities for advancing activity-based probes for H2S and related RSS.

A rapid access to activity-based and mitochondria targeting rhodol-like chromenoxanthene dyes for selective bioimaging

Chromenoxanthenes are colorful dyes which do not suffer from any fluorescence turn-off induced by spirocyclic forms as usually observed for conventional rhodamine and fluorescein xanthene analogues. In that way, wide variety of applications in particular in bioimaging can be foreseen for those underexplored fluorophores. Herein, a one-pot synthesis of rhodol-like chromenoxanthene dyes is reported, which are further modified at the periphery of the core structure to tune the optical properties. Resulting neutral derivatives are bright fluorophores in the orange-red spectral window, while cationic counterparts are almost non-emissive. These optical properties are fully characterized and are also rationalized via first-principles calculations. In particular, the occurrence of twisted intramolecular charge transfers is shown to account for the dark-state quenching occurring in the charged compounds. This feature is harvested in the construction of an activity-based fluorescent probe for mitochondria imaging. Excellent organelle localization is achieved, while the emission signal is proportional to the hydrogen peroxide concentration in the mitochondria. Furthermore, the absence of cytotoxicity bestows rhodol-like chromenoxanthene dyes a bright future in cellular imaging applications.

Recent Progress of Activity-Based Fluorescent Probes for Imaging Leucine Aminopeptidase.

Leucine aminopeptidase (LAP) is an important protease that can specifically hydrolyze Leucine residues. LAP occurs in microorganisms, plants, animals, and humans and is involved in a variety of physiological processes in the human body. In the physiological system, abnormal levels of LAP are associated with a variety of diseases and pathological processes, such as cancer and drug-induced liver injury; thus, LAP was chosen as the early biochemical marker for many physiological processes, including cancer. Considering the importance of LAP in physiological and pathological processes, it is critical that high-efficiency and dependable technology be developed to monitor LAP levels. Herein, we summarize the organic small molecule fluorescence/chemiluminescence probes used for LAP detection in recent years, which can image LAP in cancer, drug-induced liver injury (DILI), and bacteria. It can also reveal the role of LAP in tumors and differentiate the serum of cirrhotic, drug-induced liver injury and normal models.

Combining activity-based protein profiling and flow cytometry to elucidate the role of lipid-metabolizing enzymes in cell heterogeneity

Abstract Cell metabolism is mediated by intracellular enzymes that supply essential and specific molecular cues needed to initiate the differentiation of homogenous cell populations into heterogenous cell populations. Intracellular enzymes involved in the metabolism of lipids are of particular interest as lipids play key roles in cell signaling and differing lipid-metabolizing enzyme activity may produce varying signaling profiles, and thus, a varying cell phenotype. To understand how these enzymes regulate function at the single-cell level, we aim to develop methodology that combines activity-based protein profiling (ABPP) and flow cytometry to allow for the separation of heterogenous cells into new populations based on enzyme activity. Our initial studies have focused on targeting serine hydrolases, as this family of enzymes includes numerous intracellular lipases involved with the metabolism of lipids. We have synthesized a modified activity-based fluorescent probe containing a triazole urea warhead known to label active serine hydrolases in live cells. Based on the fluorescent profile generated by measuring single cell fluorescence using flow cytometry, the probe targeted a select population of cells when compared to a control probe and unlabeled cells. This probe-labeled population was concentration dependent as higher concentration of probe labeled more enzyme, suggesting that a different population of cells could be collected between two different concentrations. After these cells are sorted based on the differing fluorescent signals, these new subsets will be analyzed via targeted lipidomics and proteomics methods to quantity any differences in the probed intracellular enzyme expression. This work was supported by the National Science Foundation CAREER Award no. 1942467.

Observing hydrogen sulfide in the endoplasmic reticulum of cancer cells and zebrafish by using an activity-based fluorescent probe.

A crucial endogenous signaling chemical, hydrogen sulfide, is involved in many physiological actions. In this work, we created the fluorescent probe ER-Nap-NBD using a naphthalimide fluorophore as the signal reporter, a 7-nitro-1,2,3-benzoxadiazole amine as the responsive moiety, and a sulfonamide part for endoplasmic reticulum targeting. ER-Nap-NBD could be detected the H2S levels in solution and in living systems (cells and zebrafish).

Activity-Based Imaging of Macrophage Migration Inhibitory Factor with a Two-Photon Fluorescent Probe.

Macrophage migration inhibitory factor (MIF), as a cytokine, plays an important role in the pathogenesis of cancer and some other diseases, and it is also one of the potential drug targets for disease treatment. However, due to the lack of simple and effective MIF imaging detection tools, the fluctuation and distribution of MIF in living cells or at lesion sites remain difficult to track precisely and in real time. Here, we report activity-based fluorescent probes, named MIFP1-MIFP3, which are used for real-time imaging and tracking of intracellular MIF, thus establishing a relationship between the fluctuation of MIF and the change of fluorescence signal during the cancer disease process. With the excellent optical properties of two-photon probe imaging, we can easily distinguish multiple cancer cells from normal cells with the representative probe, MIFP3. Moreover, MIFP3 has also been successfully used to directly identify the pathological tissues of patients with clinical liver cancer. These potential MIF probes could provide powerful tools for further study of the physiological function of MIF and will be helpful to promote the accurate diagnosis and therapeutic evaluation of MIF-associated malignancies.

Activity-Based Fluorescent Probes Based on Hemicyanine for Biomedical Sensing.

In recent years, fluorescent probes, as an analytical tool that can target and rapidly detect analytes, have been increasingly used for applications related to medical treatment, detection, and bioimaging. Researchers are interested in hemicyanine-based fluorescent probes because of their high quantum yield, tunable spectrum characteristics, absorption and emission in the near-infrared (NIR) region, and good photo-stability. The development of these dyes and their derivatives as NIR fluorescent probes for biological applications has advanced significantly in the last ten years. This review introduces processes for making hemicyanine dyes and the methodology for creating functional activity-based fluorescent probes. A variety of hemicyanine-based probes have been systematically developed for the detection of small biomolecules in various illnesses. Finally, the potential drawbacks of hemicyanine-based functional probes, and the prospects for future research and translation into clinical medicine, are also discussed. This study is intended to provide strategies for the development and design of novel fluorescence probes.

“Domain Directional Optimization” strategy for the development of UGT1A1-activated fluorescent probe and the application in living systems

Activity-based fluorescent probes have emerged as the powerful tool for directly monitoring enzyme activity in living systems. Whereas, constructing high isoform-specific fluorescent probes for a target isoenzyme with multiple subtypes is still facing challenge. UGT1A1, as an isoform of UGTs, is a clear target for neonatal jaundice, irinotecan-induced toxicity, and fatal Crigler-Najjar syndrome type I. Herein, a “Domain Directional Optimization” strategy was proposed based on the systematic computational chemistry and synchronously experimental verification. Finally, an isoform-specific “switch-on” fluorescent probe (BUHE) was developed for UGT1A1 based on the boron-dipyrromethene (BODIPY) skeleton. BUHE exhibited the high selectivity and sensitivity toward UGT1A1 and the catalytic progress followed typical Michaelis-Menten kinetic. Moreover, BUHE had a good biocompatibility and chemical stability which was successfully applied to the activity evaluation and in situ imaging of UGT1A1 in complicated bio-systems including living cells, tissue slices and whole organs. In summary, the “Domain Directional Optimization” strategy could serve as a powerful method for developing the isoform-specific fluorescent probe.

Ultrasensitive Fibroblast Activation Protein-α-Activated Fluorogenic Probe Enables Selective Imaging and Killing of Melanoma In Vivo.

Melanoma is a malignant cancer with a high risk of metastasis and continued increase in death rates over the past decades, and its prognosis is highly related to the disease's stage, while early detection and treatment of melanoma are significant to the improvement of its therapy outcome. Different from the traditional methods for disease diagnosis, enzyme-activated fluorescent probes were developed rapidly due to their high sensitivity and temporal-spatial ratio and have been widely applied in tumor detection, surgical navigation, and cancer-related research. Fibroblast activation protein-α (FAPα), a serine-type cell surface protease that plays important roles in cell invasion and extracellular matrix degradation, is widely involved in tumor progression such as malignant melanoma, so developing a FAPα activity-based molecular tool would be of great potential for the early diagnosis and therapy of melanoma. However, few fluorescent probes targeting FAPα have been applied in melanoma-related studies, and thus, the construction of FAPα activity-based fluorescent probes for melanoma detection is in urgent need. By incorporating the selective recognition unit with a red-emission fluorophore, cresyl violet, we herein report an ultrasensitive (limit of detection = 5.3 ng/mL) fluorogenic probe for FAPα activity sensing, named CV-FAP; the acquired probe showed a significantly higher binding affinity (15.7-fold) and overall catalytic efficiency (2.6-fold) when compared with those of the best reported FAPα probes. The good performance of CV-FAP made it possible to discriminate malignant melanoma cells and tumor-bearing mice from normal cells and mice with high contrast. More importantly, CV-FAP showed significant antitumor activity toward melanoma in cultured cells and tumor-bearing nude mice (over 95% inhibited tumor growth) with good safety, which made it an ideal theranostic agent for melanoma.

Development and Application of Activity-based Fluorescent Probes for High-Throughput Screening.

High-throughput screening facilitates the rapid identification of novel hit compounds; however, it remains challenging to design effective high-throughput assays, partially due to the difficulty of achieving sensitivity in the assay techniques. Among the various analytical methods that are used, fluorescence-based assays dominate due to their high sensitivity and ease of operation. Recent advances in activity-based sensing/imaging have further expanded the availability of fluorescent probes as monitors for high-throughput screening of result outputs. In this study, we have reviewed various activity-based fluorescent probes used in high-throughput screening assays, with an emphasis on their structure-related working mechanisms. Moreover, we have explored the possibility of developing additional and better probes to boost hit identification and drug development against various targets.

Isolation of New Colibactin Metabolites from Wild-Type Escherichia coli and In Situ Trapping of a Mature Colibactin Derivative.

Colibactin is a polyketide-nonribosomal peptide hybrid secondary metabolite that can form interstrand cross-links in double-stranded DNA. Colibactin-producing Escherichia coli has also been linked to colorectal oncogenesis. Thus, there is a strong interest in understanding the role colibactin may play in oncogenesis. Here, using the high-colibactin-producing wild-type E. coli strain we isolated from a clinical sample with the activity-based fluorescent probe we developed earlier, we were able to identify colibactin 770, which was recently identified and proposed as the complete form of colibactin, along with colibactin 788, 406, 416, 420, and 430 derived from colibactin 770 through structural rearrangements and solvolysis. Furthermore, we were able to trap the degrading mature colibactin species by converting the diketone moiety into quinoxaline in situ in the crude culture extract to form colibactin 860 at milligram scale. This allowed us to determine the stereochemically complex structure of the rearranged form of an intact colibactin, colibactin 788, in detail. Furthermore, our study suggested that we were capturing only a few percent of the actual colibactin produced by the microbe, providing a crude quantitative insight into the inherent instability of this compound. Through the structural assignment of colibactins and their degradative products by the combination of LC-HRMS and NMR spectroscopies, we were able to elucidate further the fate of inherently unstable colibactin, which could help acquire a more complete picture of colibactin metabolism and identify key DNA adducts and biomarkers for diagnosing colorectal cancer.

Activation of the executioner caspases-3 and -7 promotes microglial pyroptosis in models of multiple sclerosis

BackgroundPyroptosis is a type of proinflammatory regulated cell death (RCD) in which caspase-1 proteolytically cleaves gasdermin D (GSDMD) to yield a cytotoxic pore-forming protein. Recent studies have suggested that additional cell death pathways may interact with GSDMD under certain circumstances to execute pyroptosis. Microglia/macrophages in the central nervous system (CNS) undergo GSDMD-associated pyroptosis in multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE) but the contribution of other cell death pathways to this phenomenon is unknown. Herein, we tested the hypothesis that multiple RCD pathways underlie microglial pyroptosis in the context of neuroinflammation.MethodsA siRNA screen of genes with known RCD functions was performed in primary human microglia to evaluate their role in nigericin-induced pyroptosis using supernatant lactate dehydrogenase activity as a read-out of cell lysis. Activation of apoptotic executioner proteins and their contribution to pyroptosis was assessed using semi-quantitative confocal microscopy, high-sensitivity ELISA, immunoblot, cell lysis assays, and activity-based fluorescent probes. Quantification of pyroptosis-related protein expression was performed in CNS lesions from patients with progressive MS and mice with MOG35-55-induced EAE, and in matched controls.ResultsAmong progressive MS patients, activated caspase-3 was detected in GSDMD immunopositive pyroptotic microglia/macrophages within demyelinating lesions. In the siRNA screen, suppression of caspase-3/7, caspase-1, or GSDMD expression prevented plasma membrane rupture during pyroptosis. Upon exposure to pyroptotic stimuli (ATP or nigericin), human microglia displayed caspase-3/7 activation and cleavage of caspase-3/7-specific substrates (e.g., DFF45, ROCK1, and PARP), with accompanying features of pyroptosis including GSDMD immunopositive pyroptotic bodies, IL-1β release, and membrane rupture. Pyroptosis-associated nuclear condensation and pyroptotic body formation were suppressed by caspase-3/7 inhibition. Pharmacological and siRNA-mediated inhibition of caspase-1 diminished caspase-3/7 activation during pyroptosis. In mice with EAE-associated neurological deficits, activated caspase-3 colocalized with GSDMD immunopositivity in lesion-associated macrophages/microglia.ConclusionsActivation of executioner caspases-3/7, widely considered key mediators of apoptosis, contributed to GSDMD-associated microglial pyroptosis under neuroinflammatory conditions. Collectively, these observations highlight the convergence of different cell death pathways during neuroinflammation and offer new therapeutic opportunities in neuroinflammatory disease.

Multiplexed Enzyme Activity-Based Probe Display via Hybridization.

Emulsions offer the means to miniaturize and parallelize high-throughput screening but require a robust method to localize activity-based fluorescent probes in each droplet. Multiplexing probes in droplets is impractical, though highly desirable for identifying library members that possess very specific activity. Here, we present multiplexed probe immobilization on library beads for emulsion screening. During library bead preparation, we quantitated ∼106 primers per bead by fluorescence in situ hybridization, however emulsion PCR yielded only ∼103 gene copies per bead. We leveraged the unextended bead-bound primers to hybridize complementary probe-oligonucleotide heteroconjugates to the library beads. The probe-hybridized bead libraries were then used to program emulsion in vitro transcription/translation reactions and analyzed by FACS to perform multiplexed activity-based screening of trypsin and chymotrypsin mutant libraries for novel proteolytic specificity. The approach's modularity should permit a high degree of probe multiplexing and appears extensible to other enzyme classes and library types.

Two Step Mechanism of an Activity-Based Fluorescent Probe for Cyclooxygenase-2

Two Step Mechanism of an Activity-Based Fluorescent Probe for Cyclooxygenase-2

An activity-based fluorescent probe and its application for differentiating alkaline phosphatase activity in different cell lines.

Herein, a new fluorescent probe, AE-Phos, is reported for detecting the ALP activity with the combined advantages of aggregation-induced emission (AIE) and excited state intramolecular proton transfer (ESIPT). Further detailed fluorescence experiments demonstrated that AE-Phos exhibited excellent selectivity and sensitivity, a large Stokes shift, and a fast response towards ALP. Furthermore, AE-Phos was applied to imaging the ALP activity in different cell lines quantitatively.

Asymmetric and Reduced Xanthene Fluorophores: Synthesis, Photochemical Properties, and Application to Activatable Fluorescent Probes for Detection of Nitroreductase.

Xanthene fluorophores, including fluorescein, rhodol, and rhodamines, are representative classes of fluorescent probes that have been applied in the detection and visualization of biomolecules. “Turn on” activatable fluorescent probes, that can be turned on in response to enzymatic reactions, have been developed and prepared to reduce the high background signal of “always-on” fluorescent probes. However, the development of activity-based fluorescent probes for biological applications, using simple xanthene dyes, is hampered by their inefficient synthetic methods and the difficulty of chemical modifications. We have, thus, developed a highly efficient, versatile synthetic route to developing chemically more stable reduced xanthene fluorophores, based on fluorescein, rhodol, and rhodamine via continuous Pd-catalyzed cross-coupling. Their fluorescent nature was evaluated by monitoring fluorescence with variation in the concentration, pH, and solvent. As an application to activatable fluorescent probe, nitroreductase (NTR)-responsive fluorescent probes were also developed using the reduced xanthene fluorophores, and their fluorogenic properties were evaluated.

Fluorescent Probes for Single-Step Detection and Proteomic Profiling of Histone Deacetylases.

Histone deacetylases (HDACs) play important roles in regulating various physiological and pathological processes. Developing fluorescent probes capable of detecting HDAC activity can help further elucidate the roles of HDACs in biology. In this study, we first developed a set of activity-based fluorescent probes by incorporating the Kac residue and the O-NBD group. Upon enzymatic removal of the acetyl group in the Kac residue, the released free amine reacted intramolecularly with the O-NBD moiety, resulting in turn-on fluorescence. These designed probes are capable of detecting HDAC activity in a continuous fashion, thereby eliminating the extra step of fluorescence development. Remarkably, the amount of turn-on fluorescence can be as high as 50-fold, which is superior to the existing one-step HDAC fluorescent probes. Inhibition experiments further proved that the probes can serve as useful tools for screening HDAC inhibitors. Building on these results, we moved on and designed a dual-purpose fluorescent probe by introducing a diazirine photo-cross-linker into the probe. The resulting probe was not only capable of reporting enzymatic activity but also able to directly identify and capture the protein targets from the complex cellular environment. By combining a fluorometric method and in-gel fluorescence scanning technique, we found that epigenetic readers and erasers can be readily identified and differentiated using a single probe. This is not achievable with traditional photoaffinity probes. In light of the prominent properties and the diverse functions of this newly developed probe, we envision that it can provide a robust tool for functional analysis of HDACs and facilitate future drug discovery in epigenetics.

Fluorescent probes designed for detecting human serum albumin on the basis of its pseudo-esterase activity

We developed activity-based fluorescent probes for detecting human serum albumin (HSA) on the basis of its pseudo-esterase activity. These probes could also detect HSA in blood-contaminated tissue samples.

Activity-based fluorescent probes for monitoring sulfatase activity

A small-molecule probe for sulfatase is developed that shows a significant change in fluorescence upon reaction with sulfatase in an activity-based manner. As this probe is free from interference from background fluorescence caused by an unreacted probe, it could be a simple and efficient tool for the study of sulfatase activity.

TcdB from hypervirulent Clostridium difficile exhibits increased efficiency of autoprocessing

TcdB, an intracellular bacterial toxin that inactivates small GTPases, is a major Clostridium difficile virulence factor. Recent studies have found that TcdB produced by emerging/hypervirulent strains of C. difficile is more potent than TcdB from historical strains, and in the current work, studies were performed to investigate the underlying mechanisms for this change in TcdB toxicity. Using a series of biochemical analyses we found that TcdB from a hypervirulent strain (TcdB(HV) ) was more efficient at autoprocessing than TcdB from a historical strain (TcdB(HIST) ). TcdB(HV) and TcdB(HIST) were activated by similar concentrations of IP6; however, the overall efficiency of processing was 20% higher for TcdB(HV) . Using an activity-based fluorescent probe (AWP19) an intermediate, activated but uncleaved, form of TcdB(HIST) was identified, while only a processed form of TcdB(HV) could be detected under the same conditions. Using a much higher concentration (200 µM) of the probe revealed an activated uncleaved form of TcdB(HV) , indicating a preferential and more efficient engagement of intramolecular substrate than TcdB(HIST) . Furthermore, a peptide-based inhibitor (Ac-GSL-AOMK) was found to block the cytotoxicity of TcdB(HIST) at a lower concentration than required to inhibit TcdB(HV) . These findings suggest that TcdB(HV) may cause increased cytotoxicity due to more efficient autoprocessing.