Development and Characterization of a Highly Selective Turn-On Fluorescent Ligand for ß3-Adrenergic Receptor.

For the precise visualization of GPCR, subtype selectivity of turn-on fluorescent ligands is of major relevance. Although there are many thriving ß-adrenergic receptors (ß-ARs) probes, none of them are selective to the ß3-subtype, which severely limits the development of ß3-AR investigations. Using a polyethylene glycol (PEG) chain to conjugate the Py-5 fluorophore with mirabegron, we present here a highly selective fluorescent ligand, H2, for ß3-AR. It was established by the radioligand and NanoLuc-based bioluminescence resonance energy transfer (NanoBRET) binding experiments that molecule H2 has a substantially higher affinity for ß3-AR than the other two subtypes (1/3, 45-fold; 2/3, 16-fold). More crucially, when molecule H2 was incubated with ß3-AR, the turn-on fluorescent signals could be quickly released. The subsequent investigations, which included cell imaging, tissue imaging, and flow-cytometry analysis, proved that molecule H2 may make it possible to quickly and accurately fluorescently identify ß3-AR at different levels. We offer a prospective fluorescent turn-on ligand with exceptional selectivity for ß3-AR as a result of our combined efforts.

Photophosphatidylserine Guides Natural Killer Cell Photoimmunotherapy via Tim-3.

Natural killer (NK) cells, in addition to their cytotoxicity function, harbor prominent cytokine production capabilities and contribute to regulating autoimmune responses. T-cell immunoglobulin and mucin domain containing protein-3 (Tim-3) is one of the inhibitory receptors on NK cells and a promising immune checkpoint target. We recently found that phosphatidylserine (PS) binding to Tim-3 can suppress NK cell activation. Therefore, based on the therapeutic potential of Tim-3 in NK-cell-mediated diseases, we developed a photoswitchable ligand of Tim-3, termed photophosphatidylserine (phoPS), that mimics the effects of PS. Upon 365 or 455 nm light irradiation, the isomer of phoPS cyclically conversed the cis/trans configuration, resulting in an active/inactive Tim-3 ligand, thus modulating the function of NK cells in vitro and in vivo. We also demonstrated that reversible phoPS enabled optical control of acute hepatitis. Together, phoPS may be an appealing tool for autoimmune diseases and cytokine storms in the future.

Discovery of Environment-Sensitive Fluorescent Ligands of ß-Adrenergic Receptors for Cell Imaging and NanoBRET Assay.

By fusing several environment-sensitive fluorophores to the pharmacophore mirabegron, a series of new fluorescent ligands for ß-adrenergic receptors (ß-ARs) were produced with a turn-on mechanism and high binding affinity to ß-ARs efficiently. Compound L5 with the pyridinium moiety possessed the most favorable combination of properties after systematic comparison and optimization, including high affinity and acceptable cytotoxicity, remarkable fluorescence enhancement (up to 30-fold) upon binding with ß-ARs, and feasible visualizing ability of ß-ARs in living cells under no-wash conditions. Furthermore, a NanoLuc-based bioluminescence resonance energy transfer (NanoBRET) binding assay based on compound L5 was developed and may be used in high-throughput screening (HTS) in the drug discovery of ß-ARs due to its unique fluorescence spectroscopic features. Overall, as the first environment-sensitive fluorescent ligand, molecule L5 could be a useful tool for understanding the pharmacology of ß-ARs.

Au-24 as a potential thioredoxin reductase inhibitor in hepatocellular carcinoma cells.

A novel TrxR inhibitor Au-24 and its inhibitory ability to hepatocellular carcinoma in vitro and in vivo is reported herein. Au-24 can suppress HepG2 cells from proliferating by lowering mitochondrial membrane potential (MMP) and increasing reactive oxygen species (ROS) levels, resulting in oxidative stress, which causes DNA damage, autophagy, cell cycle arrest, and apoptosis. This compound can also affect the normal function of apoptosis, MAPK, PI3K/AKT/mTOR, NF-κB, STAT3 signaling pathways. In vivo experiments revealed that Au-24 inhibited HepG2 tumor growth more effectively than AA1 (chloro(triethylphosphine)gold(I)) by decreasing Ki67 and CD31 protein expression and promoting tumor cell apoptosis and necrosis lesions. As a result, Au-24 was found to be a promising candidate as a TrxR inhibitor for the treatment of hepatocellular carcinoma (HCC) in both in vivo and in vitro experiments.

Constructing firefly luciferin bioluminescence probes for in vivo imaging.

Bioluminescence imaging (BLI) is a widely applied visual approach for real-time detecting many physiological and pathological processes in a variety of biological systems. Based on the caging strategy, lots of bioluminescent probes have been well developed. While the targets react with recognizable groups, caged luciferins liberate luciferase substrates, which react with luciferase generating a bioluminescent response. Among the various bioluminescent systems, the most widely utilized bioluminescent system is the firefly luciferin system. The H and carboxylic acid of luciferin are critically caged sites. The introduced self-immolative linker extends the applications of probes. Firefly luciferin system probes have been successfully applied for analyzing physiological processes, monitoring the environment, diagnosing diseases, screening candidate drugs, and evaluating the therapeutic effect. Here, we systematically review the general design strategies of firefly luciferin bioluminescence probes and their applications. Bioluminescence probes provide a new approach for facilitating investigation in a diverse range of fields. It inspires us to explore more robust light emission luciferin and novel design strategies to develop bioluminescent probes.

Discovery of alkene-conjugated luciferins for redshifted and improved bioluminescence imaging in vitro and in vivo.

The firefly luciferase system is the most extensively utilized bioluminescence system in the field of life science at the moment. In this work, we designed and synthesized a series of alkene-conjugated luciferins to develop new firefly bioluminescence substrates, and further evaluated their activities in vitro and in vivo. It is worth noting that the maximum biological emission wavelength of novel luciferin analogue AL3 ((S,E)-2-(6-hydroxy-5-(3-methoxy-3-oxoprop-1-en-1-yl)benzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4-carboxylic acid) is 100 nm red-shifted compared with D-luciferin, while that of analogue AL4 ((S,E)-2-(5-(2-cyanovinyl)-6-hydroxybenzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4-carboxylic acid) is 75 nm red-shifted. The new substrate AL2 ((S,E)-2-(6-hydroxy-7-(3-methoxy-3-oxoprop-1-en-1-yl)benzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4-carboxylic acid) showed better bioluminescence performance in vivo.

Photoinduced Electron Transfer-Based Fluorescent Agonists for α1-Adrenergic Receptors Imaging.

The novel fluorescent agonists were discovered herein for α1-adrenergic receptors (α1-ARs) based on photoinduced electron transfer (PeT) off-on switch by conjugating the fluorophore 7-(diethylamino)coumarin-3-carboxylic acid with phenylephrine. After careful evaluation, these probes exhibited efficient binding affinity with α1-ARs and could be applied to selectively imaging α1-ARs or successfully tracing the dynamic process of α1-AR internalization in living cells. Meanwhile, a bioluminescence resonance energy transfer binding assay with these new probes has been well-established and applied. Therefore, these PeT-based on-off agonists may serve as powerful tools for the α1-AR-associated study during drug discovery.

Bright chemiluminescent dioxetane probes for the detection of gaseous transmitter H2S.

Hydrogen sulfide (H2S), the third gaseous transmitter after CO and NO, is a double-edged sword in the human body. A specific concentration of H2S can attenuate myocardial ischemia-reperfusion injury by preserving mitochondrial function, in contrast, cause illness, including inflammation and stroke. There are already some probes for the real-time monitoring of the level of H2S in the biological environment. However, they have some disadvantages, such as phototoxicity, low sensitivity, and low quantum yield. In this research, by linking 4-dinitrophenyl-ether (DNP), a specific recognition group for H2S, with a chemiluminophore 1,2-dioxetane, we designed and synthesized the probe SCL-1. To tackle the barrier that the traditional chemiluminescent group has a short emission wavelength and is not easy to penetrate deep tissues, an acrylonitrile electron-withdrawing substituent was installed to the ortho-position of the 1,2-dioxanol hydroxy group. According to the same design strategy as SCL-1, the probe SCL-2 was designed with the modified chemiluminescent group. Studies have shown that SCL-2 with electron-withdrawing acrylonitrile has higher luminescence quantum yield and high sensitivity than SCL-1, realizing real-time detection of H2S in vitro and in vivo. The LOD of SCL-2 was 0.185 µM, which was the best among the currently available luminescent probes for detecting H2S. We envisage that SCL-2 may be a practical toolbox for studying the biological functions of H2S and H2S-related diseases.

Novel furimazine derivatives for nanoluciferase bioluminescence with various C-6 and C-8 substituents.

Nanoluciferase (NLuc) is the emerging commercially available luciferase considering its small size and superior bioluminescence performance. Nevertheless, this bioluminescence system has some limitations, including narrow emission wavelength and single substrate. Herein, a series of novel furimazine derivatives at the C-6 and C-8 positions of the imidazopyrazinone core have been designed and synthesized for extension of the bioluminescence substrates. It should be noted that two compounds, molecules A2 (2-(furan-2-ylmethyl)-6-(4-(hydroxymethyl)phenyl)-8-(phenylthio)imidazo[1,2-a]pyrazin-3(7H)-one) and A3 (2-(furan-2-ylmethyl)-6-(4-amino-3-fluorophenyl)-8-(phenylthio)imidazo[1,2-a]pyrazin-3(7H)-one), display reasonable bioluminescence properties for in vitro and in vivo biological evaluations. In particular, compound A3 can broaden the application of NLuc bioluminescence techniques, especially for in vivo bioluminescent imaging.

Optical Control of CRAC Channels Using Photoswitchable Azopyrazoles.

The Ca2+ release-activated Ca2+ (CRAC) channels control many Ca2+-modulated physiological processes in mammals. Hyperactivating CRAC channels are known to cause several human diseases, including Stormorken syndrome. Here, we show the design of azopyrazole-derived photoswitchable CRAC channel inhibitors (designated piCRACs), which enable optical inhibition of store-operated Ca2+ influx and downstream signaling. Moreover, piCRAC-1 has been applied in vivo to alleviate thrombocytopenia and hemorrhage in a zebrafish model of Stormorken syndrome in a light-dependent manner.

Discovery of Turn-On Fluorescent Probes for Detecting PDEδ Protein in Living Cells and Tumor Slices.

The first small-molecule fluorescent turn-on probes for detecting PDEδ protein were rationally designed, showing reasonable fluorescent properties and the fluorescent ability has been applied for visualization of the PDEδ protein in living cells and at tissue levels. The qPCR results showed that the mRNA expression of KRAS, PDEδ, AKT1, MAPK1, MEK7, RAF1, and mTOR were downregulated by probes 1-3 through PI3K/AKT/mTOR and MAPK signal pathways. The probes also can downregulate the protein level of pErk and tErk. Therefore, these small-molecule fluorescent probes are expected to be used in the screening of antipancreatic cancer drugs targeting the PDEδ protein, as well as in obtaining a better understanding of the pathological and physiological roles of PDEδ protein.

Novel NanoLuc-type substrates with various C-6 substitutions.

NanoLuc (NLuc)-furimazine bioluminescence system offers several advantages over established systems, including improved stability, smaller size, and >150-fold enhancement in bioluminescence. Herein, we designed and synthesized a series of bioluminescent substrates with varying at the C-6 position of furimazine for NLuc-furimazine bioluminescence system. Among all derivatives, compounds A6 and A11 provided excellent bioluminescence characteristics compared with furimazine in vitro and in vivo. We believe that these new NLuc substrates can broaden the application of NLuc bioluminescence techniques, especially in vivo bioluminescent imaging.

Environment-sensitive fluorescent inhibitors of histone deacetylase.

Histone deacetylases (HDACs) are proteases that can catalyze the deacetylation of histones to inhibit gene transcription. Since mutations and/or aberrant expression of various HDACs are frequently associated with human diseases, particularly cancers, HDACs are important therapeutic targets for many human tumors. However, there are still relatively few studies on HDAC small molecule fluorescent probes. Herein, we designed and synthesized a class of environment-sensitive fluorescent inhibitors with a switch mechanism to study HDAC activity. In vitro, the enzyme inhibition activity of compound 6b was comparable to the positive control drug SAHA, and it presented suitable imaging in living cells and tumor-tissue slices. This environment-sensitive fluorescent inhibitor provides a new idea for the diagnosis and treatment of HDACs-related diseases.

Bioluminescent Probe for Monitoring Endogenous Fibroblast Activation Protein-Alpha.

Fibroblast activation protein-α (FAP), as a crucial member of cell surface glycoprotein, highly expresses in reactive fibroblasts of tumors and several fibrosis diseases. It is a potential target for drug design and also reported as a prodrug strategy to increase the therapeutic window of some anticancer agents. In this work, we developed the first bioluminogenic probe for FAP with a limit-of-detection of 0.254 ng/mL, which could be applied to evaluate the FAP inhibitors in vitro. The experiments of transgenic mice and tumor-bearing nude mice validated our probe 1 could reflect the endogenous FAP level in vivo. Furthermore, this probe was successfully used to reflect FAP up-regulation in the lung homogenates of the bleomycin-induced idiopathic pulmonary fibrosis mice.

Discovery of Environment-Sensitive Fluorescent Agonists for α1-Adrenergic Receptors.

A series of novel fluorescent agonists were well developed herein with turn-on switch for α1-adrenergic receptors (α1-ARs) by conjugating the environment-sensitive fluorophore 4-chloro-7-nitrobenzoxadiazole with phenylephrine. Overall, these probes exhibited efficient binding and apparent fluorescence intensity changes (up to 10-fold) upon binding with α1-ARs. Moreover, these probes have been successfully applied for selectively imaging α1-ARs in the living cells. The dynamic process of α1-ARs internalization was traced successfully with these newly designed fluorescent agonists. Fluorescence polarization assay demonstrated specific interactions between these probes and α1-ARs. With these new probes, a bioluminescence resonance energy transfer binding assay has been well established and applied to the high-throughput screening of unlabeled α1-ARs agonist and antagonist. It is expected that these environment-sensitive fluorescent turn-on agonists may provide useful new tools in studying pharmacology and physiology of α1-ARs during drug discovery.

Discovery of Turn-On Fluorescent Probes for Detecting Bcl-2 Protein.

B-cell-lymphoma-2-gene (Bcl-2) family proteins play a central role in regulating programmed cell death. In cancer, antiapoptotic Bcl-2 proteins, such as Bcl-2 and Mcl-1, are overexpressed. However, there are few developed labeling techniques for tracing the dynamic processes of Bcl-2. To study the physiological process of Bcl-2 protein, a novel series of small-molecule fluorescent probes (1-3) were designed and evaluated for their labeling properties. Our probes can be applied to the identification of tumor-tissue slices and the differentiation of tumor and normal tissues effectively, a feature that renders these probes compatible with future cancer diagnosis in clinical practice.

Discovery of Small-Molecule Sulfonamide Fluorescent Probes for GPR120.

GPR120 is a novel target for the treatment of metabolic disease and type 2 diabetes. The small-molecule fluorescent probe could help us locate GPR120 visually and guide in-depth study of GPR120. In this study, we synthesized six nonacidic sulfonamide fluorescent probes and tested their optical and biological properties. Compared to previous probes for GPR120, these probes, with sulfonamide structure, have high selectivity on GPR120. We used these probes to establish a BRET binding assay system to screen agonists and antagonists of GPR120. It is expected that these novel fluorescent probes may become useful tools in studying pharmacology and physiology of GPR120.

Bioluminescent Probe for Detection of Starvation-Induced Pantetheinase Upregulation.

Pantetheinase, a glycosylphosphatidylinositol (GPI) anchored enzyme, overexpresses in intestine, liver, and kidney with various biological functions such as its linkage to the inflammation and some metabolic diseases. It can hydrolyze pantetheine to cysteamine, an antioxidant, and pantothenic acid (Vitamin B5) that is an essential component of coenzyme A (CoA). Until now, very few analytic methods were developed for this enzyme, hampering the further investigation of its biological functions. In this work, we report the design, synthesis, and biological examination of a highly sensitive bioluminogenic probe for pantetheinase with a limit of detection of 1.14 ng/mL. Furthermore, animal experiments validated that our probe can be applied to detect the endogenous pantetheinase activity. To the best of our knowledge, this is the first bioluminogenic probe achieving the detection of pantetheinase level in vivo.

In Vivo Bioluminescence Imaging of Cobalt Accumulation in a Mouse Model.

As a trace element nutrient, cobalt is critical for both prokaryotes and eukaryotes. In the current study, a turn-on Cobalt Bioluminescent Probe 1 (CBP-1) for the detection of cobalt has been successfully developed based on oxidative C-O bond cleavage. This probe exhibited high selectivity and sensitivity toward cobalt over other analytes. By using CBP-1, the successful in vivo imaging of cobalt accumulation was carried out in a mouse model. Such an ability to determine cobalt in living animals provides a powerful technology for studying the system distribution, toxic potency, and biological effect of Co2+.