Deuteration-Driven Photopharmacology: Deuterium-Labeled AzoCholine for Controlling Alpha 7 Nicotinic Acetylcholine Receptors.

Photopharmacology is a powerful approach to investigate biological processes and overcomes the common therapeutic challenges in drug development. Enhancing the photopharmacology properties of photoswitches contributes to extend their applications. Deuteration, a tiny structural modification, makes it possible to improve the photopharmacology and photophysical properties of prototype compounds, avoiding extra complex chemical changes or constructing multicomponent systems. In this work, we developed a series of D-labeled azobenzenes to expand the azobenzene photoswitchable library and introduced the D-labeled azobenzene unit into the photoagonist of α7 nicotinic acetylcholine receptors (α7 nAChRs) to investigate the effects of deuteration in photopharmacology. Spectral data indicated that deuteration maintained most of the photophysical properties of azobenzenes. The D-labeled photoagonist exhibited good control of the activity of α7 nAChRs than the prototype photoagonist. These results confirmed that deuteration is a promising strategy to improve the photopharmacological properties.

Novel BRD4-p53 Inhibitor SDU-071 Suppresses Proliferation and Migration of MDA-MB-231 Triple-Negative Breast Cancer Cells.

A promising alternative for cancer treatment involves targeted inhibition of the epigenetic regulator bromodomain-containing protein 4 (BRD4); however, available BRD4 inhibitors are constrained by their potency, oral bioavailability, and cytotoxicity. Herein, to overcome the drawback of the translational BRD4 inhibitors, we describe a novel BRD4-p53 inhibitor, SDU-071, which suppresses BRD4 interaction with the p53 tumor suppressor and its biological activity in MDA-MB-231 triple-negative breast cancer (TNBC) cells in vitro and in vivo. This novel small-molecule BRD4-p53 inhibitor suppresses cell proliferation, migration, and invasion by downregulating the expression of BRD4-targeted genes, such as c-Myc and Mucin 5AC, and inducing cell cycle arrest and apoptosis, as demonstrated in cultured MDA-MB-231 TNBC cells. Its antitumor activity is illustrated in an orthotopic mouse xenograft mammary tumor model. Overall, our results show that SDU-071 is a viable option for potentially treating TNBC as a new BRD4-p53 inhibitor.

Degradation of extracellular and membrane proteins in targeted therapy: Status quo and quo vadis.

Targeted protein degradation (TPD) strategies, such as proteolysis-targeting chimeras (PROTACs) only work for intracellular protein degradation because they involve the intracellular protein degradation machinery. Several new technologies have emerged in recent years for TPD of extracellular and membrane proteins. Even though some progress has been demonstrated in the extracellular and membrane protein degradation field, the application of these technologies is still in its infancy. In this review, we survey the therapeutic potential of existing technologies by summarizing and reviewing discoveries and hurdles in extracellular and membrane protein-of-interest (POI) degradation.

Fluorescent and theranostic probes for imaging nicotinamide phosphoribosyl transferase (NAMPT).

Nicotinamide phosphoribosyl transferase (NAMPT) has been regarded as an attractive target for cancer therapy. However, there is a lack of chemical tools for real-time visualization and detection of NAMPT. Herein, the first fluorescent and theranostic probes were designed for imaging NAMPT, which had dual functions of diagnosis and treatment. The designed probes possessed good affinity and environmental sensitivity to NAMPT with a turn-on mechanism and were successfully applied in fluorescence detecting and imaging of NAMPT at the level of living cells and tissue sections. They also effectively inhibited tumor cell proliferation and arrested cell cycle at the G2 phase. These fluorescent probes enabled detection and visualization of NAMPT, representing effective chemical tools for the pathological diagnosis and treatment of cancer.

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.

Targeted Protein Degradation Induced by HEMTACs Based on HSP90.

Targeted protein degradation (TPD) strategies open up new avenues for therapeutics and provide powerful tools for biological inquiry. Herein, we present a brand-new approach, termed heat shock protein 90 (HSP90)-mediated targeting chimeras (HEMTACs), to induce intracellular protein degradation by bridging a target protein to HSP90 to drive the downregulation of proteins. We successfully showcase HEMTACs for cyclin-dependent kinase 4 and 6 (CDK4/6) by using a flexible linker to connect the targeting warhead of CDK4/6 with the HSP90 ligand. Overall, our study delivers a series of evidence that HEMTACs can serve as a valuable addition to TPD strategies, most prominently proteolysis-targeting chimera technology.

Multiple rapid-responsive probes for hypochlorite detection based on dioxetane luminophore derivatives.

In recent years, various methods for detecting exogenous and endogenous hypochlorite have been studied, considering its essential role as a biomolecule. However, the existing technologies still pose obstacles such as their invasiveness, high costs, and complicated operation. In the current study, we developed a glow-type chemiluminescent probe, hypochlorite chemiluminescence probe (HCCL)-1, based on the scaffold of Schaap's 1,2-dioxetane luminophores. To better explore the physiological and pathological functions of hypochlorite, we modified the luminophore scaffold of HCCL-1 to develop several probes, including HCCL-2, HCCL-3, and HCCL-4, which amplify the response signal of hypochlorite. By comparing the luminescent intensities of the four probes using the IVIS® system, we determined that HCCL-2 with a limit of detection of 0.166 µM has enhanced sensitivity and selectivity for tracking hypochlorite both in vitro and in vivo.

Identification of anthelmintic parbendazole as a therapeutic molecule for HNSCC through connectivity map-based drug repositioning.

Head and neck squamous cell carcinoma (HNSCC) is one of the most common human cancers; however, its outcome of pharmacotherapy is always very limited. Herein, we performed a batch query in the connectivity map (cMap) based on bioinformatics, queried out 35 compounds with therapeutic potential, and screened out parbendazole as a most promising compound, which had an excellent inhibitory effect on the proliferation of HNSCC cell lines. In addition, tubulin was identified as a primary target of parbendazole, and the direct binding between them was further verified. Parbendazole was further proved as an effective tubulin polymerization inhibitor, which can block the cell cycle, cause apoptosis and prevent cell migration, and it exhibited reasonable therapeutic effect and low toxicity in the in vivo and in vitro anti-tumor evaluation. Our study repositioned an anthelmintic parbendazole to treat HNSCC, which revealed a therapeutic utility and provided a new treatment option for human cancers.

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.

Design, synthesis and biological evaluation of new parbendazole derivatives for the treatment of HNSCC.

Head and neck squamous cell carcinoma (HNSCC) is a lethal disease with a terrible prognosis, accounting for more than 900,000 new cases and 500,000 deaths each year, nevertheless, its pharmacotherapy is rather limited. Parbendazole was previously identified as a potential HNSCC therapy candidate in our research. Herein, we report the discovery of two series of parbendazole derivatives as tubulin inhibitors. Structure-activity relationship (SAR) analyses led to the discovery of compound 9q with the best pharmacological activities and pharmacokinetic properties. This compound exhibited reasonable inhibition activity on cell proliferation (HN6, CAL-27, Fadu) and tubulin polymerization, induced cell apoptosis, blocked cell cycle and suppressed cell migration and invasion. Compound 9q also displayed low toxicity and a favorable therapeutic effect on a xenograft tumor, indicating that it is a promising starting point for further research.

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.

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.

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.

Fluorescent Ligand-Based Discovery of Small-Molecule Sulfonamide Agonists for GPR120.

As a critical member of G protein-coupled receptors (GPCRs), G protein-coupled receptor 120 (GPR120) is a potential target for many physiological diseases, such as type 2 diabetes mellitus, inflammation, and obesity. Considering that small-molecule fluorescent ligands can combine the advantages of visualization, high sensitivity and selectivity, we initially undertook an effort to develop a series of fluorescent ligands to track GPR120 and establish a method to screen GPR120 agonists. The representative fluorescent ligand N1 possesses suitable optical property, equitable biological activity, and high fluorescence imaging feasibility, therefore, based on compound N1, we subsequently founded a bioluminescence resonance energy transfer (BRET) competition binding assay to screen three series of sulfonamide GPR120 agonists we developed herein. The activity evaluation results revealed that compound D5 was a potent GPR120 agonist with high activity and selectivity. Moreover, compound D5 exhibited a significant glucose-lowering effect in db/db mice, which indicates its potential application in the treatment of type 2 diabetes mellitus in vivo. It is anticipated that our fluorescent ligand-based method is a useful toolbox and will find broad applications in the discovery of small-molecule agonists for GPR120.

Discovery of small-molecule fluorescent probes for C-Met.

C-mesenchymal-epithelia transition factor (c-Met) is highly expressed in various solid tumors such as gastric cancer, liver cancer, and lung cancer, playing a pivotal role in the growth, maintenance, and development of different tumor cells. In this study, three small-molecule fluorescent probes (5, 11, 16) targeting c-Met were developed, and their design strategies were also initially explored. In general, the fluorescence properties of the probes themselves could meet the imaging requirements, and they have shown sufficient inhibitory activities against c-Met, especially probe 16, reflecting the targeting and acceptance. Also, fluorescence polarization assays and flow cytometry analysis verified the binding between the probes and c-Met. Cell imaging confirmed that these probes could be used to label c-Met on living cells. It is of positive significance for the development of c-Met kinase inhibitors and tumor pathology research.

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.

Multiple rapid-responsive probes for hypochlorite detection based on dioxetane luminophore derivatives / 药物分析学报

In recent years,various methods for detecting exogenous and endogenous hypochlorite have been studied,considering its essential role as a biomolecule.However,the existing technologies still pose obstacles such as their invasiveness,high costs,and complicated operation.In the current study,we developed a glow-type chemiluminescent probe,hypochlorite chemiluminescence probe(HCCL)-1,based on the scaffold of Schaap's 1,2-dioxetane luminophores.To better explore the physiological and pathological functions of hypochlorite,we modified the luminophore scaffold of HCCL-1 to develop several probes,including HCCL-2,HCCL-3,and HCCL-4,which amplify the response signal of hypo-chlorite.By comparing the luminescent intensities of the four probes using the IVIS? system,we determined that HCCL-2 with a limit of detection of 0.166 μM has enhanced sensitivity and selectivity for tracking hypochlorite both in vitro and in vivo.

Zebrafish Behavioral Phenomics Links Artificial Sweetener Aspartame to Behavioral Toxicity and Neurotransmitter Homeostasis.

Artificial sweeteners (ASs) are extensively used as food additives in drinks and beverages to lower calorie intake and prevent lifestyle diseases such as obesity. Although clinical and epidemiological data revealed the link between the chronic overconsumption of ASs and adverse health effects, there still exist controversies over the potential adverse neural toxic effect of ASs such as aspartame (APM), with acceptable daily intake (ADI) for a long time, on human health. In addition, whether APM and its metabolites are neurotoxic remains debatable due to a lack of data from an animal experiment or clinical investigation. Herein, to fully describe the potential neurological effect of APM, adult zebrafish served as the animal model to assess neurophysiological alteration induced by APM exposure within the range of the ADI (1, 10, and 100 mg/L) for 2 months. A cohort of standardized neurobehavioral phenotyping assays was conducted, including light/dark preference tests (LDP), novel tank diving tests, novel object recognition tests, social interaction tests, and color preference tests. For instance, in the LDP test, saccharin remarkably decreased the swimming time of zebrafish in the DARK part from 111 ± 10.8 (control group) to 72.2 ± 11.4 (100 mg/L groups). Besides, brain chemistry involved in the alteration of total neurotransmitters was determined by LC-MS/MS to confirm the behavioral results. Overall, current research studies revealed that APM within the range of the ADI altered the total behavioral profiles of zebrafish and disturbed the homeostasis of neurotransmitters in the brain. The present study has established a set of experimental paradigms, revealing the standardized procedure of using adult zebrafish to determine the neural activity or toxicity of AS molecules phenotypically. Zebrafish behavioral phenotyping methods, which were characterized by a cohort of behavioral fingerprints, can link the phenotypical alteration to changes in neurotransmitters in the brain, so as to provide a predictive reference for the further exploration of the molecular mechanism of phenotypic changes induced by ASs.

Polarity-based fluorescence probes: properties and applications.

Local polarity can affect the physical or chemical behaviors of surrounding molecules, especially in organisms. Cell polarity is the ultimate feedback of cellular status and regulation mechanisms. Hence, the abnormal alteration of polarity in organisms is closely linked with functional disorders and many diseases. It is incredibly significant to monitor and detect local polarity to explain the biological processes and diagnoses of some diseases. Because of their in vivo safe and real-time monitoring, several polarity-sensitive fluorophores and fluorescent probes have gradually emerged and been used in modern research. This review summarizes the fluorescence properties and applications of several representative polarity-sensitive fluorescent probes.