Fluorescence Energy Transfer Research Articles

Fluorescent "turn-on" aptamer sensor for sensitive and reliable detection of Golgi glycoprotein 73 based on nitrogen-doped graphene quantum dots and molybdenum disulfide nanosheets

The sensitivity and specificity of Golgi glycoprotein 73 (GP73) are very important for early diagnosis of hepatocellular carcinoma. Herein, we constructed a new-fashioned fluorescent aptamer sensor for GP73 determination based on nitrogen-doped graphene quantum dots (N-GQDS) and molybdenum disulfide (MoS2) nanosheets. N-GQDs with high fluorescence intensity and good stability were screened out, and GP73 aptamer (GP73Apt) is labeled with N-GQDs to form the N-GQDs-GP73Apt fluorescence probe. MoS2 nanosheets can quench the fluorescence of N-GQDs-GP73Apt owing to fluorescence resonance energy transfer mechanisms. After introducing GP73 into the biosensing system, the N-GQDs-GP73Apt specifically bound with GP73 to form the deployable structures, making N-GQDs-GP73Apt far away from MoS2, blocking the fluorescence energy transfer process, and restoring the fluorescence of N-GQDs-GP73Apt. When the GP73 concentration was in the extent of 2.5 ng/mL∼100 ng/mL, the relative fluorescence recovery is linearly relevant to the concentration of GP73, and the limit of detection (LOD) was 1.29 ng/mL (S/N = 3). Moreover in the application of actual serum sample detection, the recovery was range 98.85∼100.55 %. The fluorescent aptamer sensor can rapidly detect and analyze the serum marker GP73 with the characteristics of low-cost, high sensitivity, good specificity and recovery.

Single Molecule FRET Analysis of CRISPR Cas9 Single Guide RNA Folding Dynamics.

CRISPR Cas9 is an RNA guided endonuclease that is part of a bacterial adaptive immune system. Single guide RNA (sgRNA) can be designed to target genomic DNA, making Cas9 a programmable DNA binding/cutting enzyme and allowing applications such as epigenome editing, controlling transcription, and targeted DNA insertion. Some of the main hurdles against an even wider adoption are off-target effects and variability in Cas9 editing outcomes. Most studies that aim to understand the mechanisms that underlie these two areas have focused on Cas9 DNA binding, DNA unwinding, and target cleavage. The assembly of Cas9 RNA ribonucleoprotein complex (RNP) precedes all these steps and includes sgRNA folding and Cas9 binding to sgRNA. We know from the crystal structure of the Cas9 RNP what the final sgRNA conformation is. However, the assembly dynamics has not been studied in detail and a better understanding of RNP assembly could lead to better-designed sgRNAs and better editing outcomes. To study this process, we developed a single molecule FRET assay to monitor the conformation of the sgRNA and the binding of Cas9 to sgRNA. We labeled the sgRNA with a donor fluorophore and an acceptor fluorophore such that when the sgRNA folds, there are changes in FRET efficiency. We measured sgRNA folding dynamics under different ion conditions, under various methods of folding (refolding vs vectorial), and with or without Cas9. sgRNA that closely mimics the sgRNA construct used for high resolution structural analysis of the Cas9-gRNA complex showed two main FRET states without Cas9, and Cas9 addition shifted the distribution toward the higher FRET state attributed to the properly assembled complex. Even in the absence of Cas9, folding the sgRNA vectorially using a superhelicase-dependent release of the sgRNA in the direction of transcription resulted in almost exclusively high FRET state. An addition of Cas9 during vectorial folding greatly reduced a slow-folding fraction. Our studies shed light on the heterogeneous folding dynamics of sgRNA and the impact of co-transcriptional folding and Cas9 binding in sgRNA folding. Further studies of sequence dependence may inform rational design of sgRNAs for optimal function.

Macroscopic quantum electrodynamics approach to multichromophoric excitation energy transfer. II. Polariton-mediated population dynamics in a dimer system.

In this study, based on the theory developed in Paper I, we explore the combined effects of molecular fluorescence and excitation energy transfer in a minimal model-a pair of single-vibration-mode chromophores coupled to surface plasmon polaritons. For the chromophores with zero Huang-Rhys factors and strong couplings to surface plasmon polaritons, we find that the frequencies of Rabi oscillations (the strengths of strong light-matter couplings) are associated with the initial excitation conditions. On the other hand, for the chromophores weakly coupled to surface plasmon polaritons, our numerical calculations together with analytical analysis elaborate on the conditions for the superradiant and subradiant decay behaviors. Moreover, we show that the modified decay rate constants can be explicitly expressed in terms of generalized spectral densities (or dyadic Green's functions), revealing a relationship between photonic environments and the collective effects such as superradiance and subradiance. For the chromophores with nonzero Huang-Rhys factors and strong coupling to surface plasmon polaritons, the effects of molecular vibrations emerge. We demonstrate that the low-frequency vibrational modes do not affect the excited state population dynamics, while the high-frequency vibrational modes can modify either the period of Rabi oscillation (Franck-Condon Rabi oscillation) or the amplitude of excited state population. Our study shows that the collective effects, including superradiance and subradiance, can be controlled via dielectric environments and initial excitation conditions, providing new insights into polariton chemistry and the design of quantum optical devices.

Micrometer‐Scale Optical Web Made of Spider Dragline Fibers with Optical Gate Operations

AbstractDragline silk fibers produced by spiders are a masterpiece structural protein that is durable even in the wild conditions. Dragline silk fibers are known to have excellent mechanical toughness and flexibility and further possess optical transparency, which are highly promising as textiles, sensors, and optical devices. Here, the authors show that the dragline silk microfibers act as good optical waveguides with the optical loss coefficient as small as 0.03 decibel per micrometer. The light transportation in the dragline silk fiber is performed by fluorescence energy transfer between two microspheres located on a suspended dragline silk microfiber. By utilizing a micromanipulation equipment, micrometer‐scale spider web‐like structures are fabricated. The experimentally observed optical waveguiding properties of the weaves match well with simulation results using simple mathematical models. Furthermore, optical logic gate operations are demonstrated using photoswitchable microsphere resonators attached on the dragline fibers.

Non-fitting FLIM-FRET facilitates analysis of protein interactions in live zebrafish embryos.

Molecular interactions are key to all cellular processes, and particularly interesting to investigate in the context of gene regulation. Protein-protein interactions are challenging to examine in vivo as they are dynamic, and require spatially and temporally resolved studies to interrogate them. Foerster Resonance Energy Transfer (FRET) is a highly sensitive imaging method, which can interrogate molecular interactions. FRET can be detected by Fluorescence Lifetime Imaging Microscopy (FLIM-FRET), which is more robust to concentration variations and photobleaching than intensity-based FRET but typically needs long acquisition times to achieve high photon counts. New variants of non-fitting lifetime-based FRET perform well in samples with lower signal and require less intensive instrument calibration and analysis, making these methods ideal for probing protein-protein interactions in more complex live 3D samples. Here we show that a non-fitting FLIM-FRET variant, based on the Average Arrival Time of photons per pixel (AAT- FRET), is a sensitive and simple way to detect and measure protein-protein interactions in live early stage zebrafish embryos.

Potential matrix metalloproteinase-9 inhibitor of aurone compound isolated from Sterculia quadrifida leaves: In-vitro and in-silico studies

In our previous study, aurone compound were found in the n-butanol fraction of Sterculia quadrifida leaves. The aurone compound (compound 1) had high cytotoxic activity in cell lines with IC50 values of 4.05, 12.53, 15.38, and 25.91 μg/mL in 4T1, MCF7, MDA-MB-435, T47D, respectively. In this study, we performed the MMP-9 enzyme inhibition test with FRET assay, and to deepen the mechanism of action of the compounds use molecular docking and molecular dynamics. The results showed that compound 1 had the MMP-9 enzyme inhibition of 91.23% and IC50 48.27%. Furthermore, molecular docking and molecular dynamic simulation suggest compound 1 interacts firmly and stably at the enzyme catalytic site. In conclusion, this study can provide important information on the development of herbal medicines that are potentially cancer drugs.

Discovering cell-active BCL6 inhibitors: effectively combining biochemical HTS with multiple biophysical techniques, X-ray crystallography and cell-based assays

By suppressing gene transcription through the recruitment of corepressor proteins, B-cell lymphoma 6 (BCL6) protein controls a transcriptional network required for the formation and maintenance of B-cell germinal centres. As BCL6 deregulation is implicated in the development of Diffuse Large B-Cell Lymphoma, we sought to discover novel small molecule inhibitors that disrupt the BCL6-corepressor protein–protein interaction (PPI). Here we report our hit finding and compound optimisation strategies, which provide insight into the multi-faceted orthogonal approaches that are needed to tackle this challenging PPI with small molecule inhibitors. Using a 1536-well plate fluorescence polarisation high throughput screen we identified multiple hit series, which were followed up by hit confirmation using a thermal shift assay, surface plasmon resonance and ligand-observed NMR. We determined X-ray structures of BCL6 bound to compounds from nine different series, enabling a structure-based drug design approach to improve their weak biochemical potency. We developed a time-resolved fluorescence energy transfer biochemical assay and a nano bioluminescence resonance energy transfer cellular assay to monitor cellular activity during compound optimisation. This workflow led to the discovery of novel inhibitors with respective biochemical and cellular potencies (IC50s) in the sub-micromolar and low micromolar range.

Structure and inhibition of SARS-CoV-1 and SARS-CoV-2 main proteases by oral antiviral compound AG7404

Severe acute respiratory syndrome coronaviruses 1 and 2 (SARS-CoV-1 and SARS-CoV-2) pose a threat to global public health. The 3C-like main protease (Mpro), which presents structural similarity with the active site domain of enterovirus 3C protease, is one of the best-characterized drug targets of these viruses. Here we studied the antiviral activity of the orally bioavailable enterovirus protease inhibitor AG7404 against SARS-CoV-1 and SARS-CoV-2 from a structural, biochemical, and cellular perspective, comparing it with the related molecule rupintrivir (AG7800). Crystallographic structures of AG7404 in complex with SARS-CoV-1 Mpro and SARS-CoV-2 Mpro and of rupintrivir in complex with SARS-CoV-2 Mpro were solved, revealing that all protein residues interacting with the inhibitors are conserved between the two proteins. A detailed analysis of protein-inhibitor interactions indicates that AG7404 has a better fit to the active site of the target protease than rupintrivir. This observation was further confirmed by biochemical FRET assays showing IC50 values of 47 μM and 101 μM for AG7404 and rupintrivir, respectively, in the case of SARS-CoV-2 Mpro. Equivalent IC50 values for SARS-CoV-1 also revealed greater inhibitory capacity of AG7404, with a value of 29 μM vs. 66 μM for rupintrivir. Finally, the antiviral activity of the two inhibitors against SARS-CoV-2 was confirmed in a human cell culture model of SARS-CoV-2 infection, although rupintrivir showed a higher potency and selectivity index in this assay.

Rational design of the zonulin inhibitor AT1001 derivatives as potential anti SARS-CoV-2

Although vaccines are greatly mitigating the worldwide pandemic diffusion of SARS-Cov-2, therapeutics should provide many distinct advantages as complementary approach to control the viral spreading. Here, we report the development of new tripeptide derivatives of AT1001 against SARS-CoV-2 Mpro. By molecular modeling, a small compound library was rationally designed and filtered for enzymatic inhibition through FRET assay, leading to the identification of compound 4. X-ray crystallography studies provide insights into its binding mode and confirm the formation of a covalent bond with Mpro C145. In vitro antiviral tests indicate the improvement of biological activity of 4 respect to AT1001. In silico and X-ray crystallography analysis led to 58, showing a promising activity against three SARS-CoV-2 variants and a valuable safety in Vero cells and human embryonic lung fibroblasts. The drug tolerance was also confirmed by in vivo studies, along with pharmacokinetics evaluation. In summary, 58 could pave the way to develop a clinical candidate for intranasal administration.

Structural basis for the allosteric inhibition of hypoxia-inducible factor (HIF)-2 by belzutifan.

Hypoxia-inducible factor (HIF)-2α and its obligate heterodimerization partner aryl hydrocarbon receptor nuclear translocator (ARNT), are both members of the basic helix-loop-helix-PER-ARNT-SIM (bHLH-PAS) transcription factor family. Previous studies have identified HIF-2α as a key oncogenic driver in clear cell renal cell carcinoma (ccRCC), rendering it a promising drug target for this type of kidney cancer. Belzutifan is the first HIF-2α inhibitor approved for treating ccRCC and other cancers associated with the von Hippel-Lindau (VHL) disease. However, the detailed inhibitory mechanism of belzutifan at molecular level is still unclear. Here we obtained the crystal structure of HIF-2α-ARNT heterodimer in complex with belzutifan at 2.75 Å resolution. The complex structure shows that belzutifan binds into the PAS-B pocket of HIF-2α, and it destabilizes the dimerization of HIF-2α and ARNT through allosteric effects mainly mediated by the key residue M252 of HIF-2α near the dimer interface. We further explored the inhibitory effects of belzutifan using biochemical and functional assays. The time-resolved fluorescence energy transfer (TR-FRET)-based binding assay showed that belzutifan disrupts the dimerization of HIF-2α and ARNT with a Ki value of 20 nM. The luciferase reporter assay indicated that belzutifan can efficiently inhibit the transcriptional activity of HIF-2α with an IC50 value of 17 nM. Besides, the real-time PCR assay illustrated that belzutifan can reduce the expression of HIF-2α downstream genes in 786-O kidney cancer cells in a dose-dependent manner. Our work reveals the molecular mechanism by which belzutifan allosterically inhibits HIF-2α and provides valuable information for the subsequent drug development targeting HIF-2α. Significance Statement The bHLH-PAS family of transcription factors are an emerging group of small-molecule drug targets. Belzutifan, originally developed by Peloton Therapeutics, is the first FDA-approved drug directly binding to a bHLH-PAS protein, the hypoxia-inducible factor (HIF)-2α. Based on the protein-drug complex structure, biochemical binding assays, and functional profiling of downstream gene expression, this study reveals the regulatory mechanism of how belzutifan allosterically destabilizes HIF-2α's heterodimerization with its obligate partner protein, thus reducing their transcriptional activity that links to tumor progression.

Biological Evaluation and Reverse Pharmacophore Mapping of Innovative Bis-Triazoles as Promising Anticancer Agents

Here, we describe further cytotoxic studies and reverse pharmacophore mapping (pharmacophore profiling) for bis-triazoles MS44-53, which were designed and synthesized previously to stabilize the G-quadruplex nucleic acids capable of being formed at the telomeric region and promoter sequences of genes involved in cellular proliferation and oncogenes. Pharmacophore-based activity profiling screen demonstrated some biological targets that MS44-53 may modulate their biological response, and thus can be considered as potential drugs to treat different kinds of diseases, such as carcinoma, diabetes type II, bacterial infection and cardiovascular diseases. Potent cell growth inhibitory properties were shown by ligands MS47 and MS49 against human melanoma MDA-MB-435, colon cancer HCT-116 and COLO 205, and pancreatic cancer MIA PaCa-2 cell lines, as evidenced by MTT assay. Both ligands were more potent against cancer cells than in skin normal CCD-1064Sk fibroblasts. Aim: The aim of this study is to identify the molecular target and mechanism of action of our promising anticancer bis-triazoles MS44-53, focusing specifically on the G-quadruplex stabilizers MS47 and MS49. Background: In molecular biology, G-quadruplexes (also known as G4-DNA), one of the higher-order structures of polynucleotides, are four stranded structures formed by nucleic acid sequences which are rich in guanine. They are formed mainly at the single-stranded G-overhang of telomeric DNA and within promoter sequences of genes involved in cellular proliferation and oncogenes such as c-myc, c-kit, and Hsp90. Stabilization of DNA G-quadruplexes is one of the anticancer strategies that has the potential to treat all cancers regardless of the type. A new series of bis-triazoles MS44-53 were developed to stabilize G-quadruplex structures selectively, as G4 ligands and experimental antitumour agents. FRET assay showed that MS47 and MS49 were only the best binders towards the Hsp90 promoter G-guadruplexes. While all bis-triazoles MS44-53 exhibited potent cell growth inhibitory activity against human carcinoma cell lines, suggesting that the ligands perturb molecular targets and mechanisms of action, other than stabilizing G-quadruplexes, contributing to antitumor activity. Therefore, the molecular targets and mechanisms of action of bis-triazoles MS44-53 in different types of human cancer cell lines should be determined by performing further computational studies to MS44-53 and in vitro evaluations for the G-quadruplex stabilizers MS47 and MS49. Objectives: 1- Determining the exact IC50 for bis-triazoles MS47 & MS49 against four different types of human cancer cell lines; melanoma MDA-MB-435, pancreatic cancer MIA PaCa-2, and colon cancer HCT-116 and COLO 205 cell lines. 2- Predicting the biological targets that bis-triazoles MS44-53 may interact with to trigger or block their biological response. Methods: 1- MTT assay was used for in vitro evaluation of the antiproliferative activities of MS47 and MS49, and determination of IC50 values. 2- Reverse pharmacophore mapping (pharmacophore profiling) was used for predicting the biological targets of bis-triazoles MS44-53, and determining the % binding probabilities. Results: MS49 exhibited more potent proliferation inhibitory activity than MS47 and higher IC50 value against skin normal fibroblasts. Pharmacophore profiling demonstrated FGFR1, PDGFR2, FLT3, mTOR, PPAR-gamma, MUR-F and CETP as biological targets for bis-triazoles MS44-53. Conclusion: Bis-triazoles MS47 and MS49 are promising selective innovative compounds with wide spectrum cytotoxic activities against distinct cancer types. Bis-triazoles MS44-53 can be considered as potential drugs to treat different types of carcinoma, in addition to diabetes type II, bacterial infection and cardiovascular diseases. Other: Further in vitro evaluations will be performed for bis-triazoles MS44-53 in order to identify their molecular targets and mechanisms of action in different types of human cancer cell lines.

Novel benzylidene benzofuranone analogues as potential anticancer agents: design, synthesis and in vitro evaluation based on CDK2 inhibition assays

The classical anticancer agents do not have their efficacy on inhibiting the G2 phase of the cell cycle. There are a very few reports available on drugs that work at G2 phase. Flavopiridol is one such drug candidate. In the current study, we sought to make analogues of flavopiridol. Still, the conditions used during their synthesis were unfavourable for the formation of flavopiridol and led to the generation of benzofuranones. In the present work, a new series of benzylidene benzofuranones were designed, synthesized and evaluated for their antioxidant, anti-colorectal cancer activity. Molecular docking, MMGBSA and molecular dynamics studies were conducted to assess their binding affinity at the active site of CDK2. Based on the cytotoxicity exhibited by test compounds, the compound NISOA4 (from isopropyl series) was further selected for mechanistic anticancer studies on HCT 116 cell lines. The compound selected was evaluated by comet assay, DNA fragmentation assay, cell cycle analysis, apoptosis detection by annexin FITC, semi-quantitative RTPCR based gene expression studies and FRET assay on the target CDK2/Cyclin A. Compound NISOA4 exhibited marked olive moments in comet assay studies. The apoptotic DNA fragmentation for compound NISOA4 demonstrated a marked change in the DNA fragmentation. The compound exhibited cell cycle arrest at G2/M phase at both the test concentrations. Apoptosis induction was observed at both the test concentrations and the compound was found to be a potent proapoptotic agent. It exhibited marked inhibition for the CDK2 gene expression and did not show any effect on CyclinA gene expression. However, the compound NISOA4 along with other analogues showed appreciable inhibition for the CDK2/Cyclin A target enzyme.

Deciphering Regulatory Proteins of Prenylated Protein via the FRET Technique Using Nitroso-Based Ene-Ligation and Sequential Azidation and Click Reaction.

We report here the selective incorporations of nitroso species into a wide range of proteins targeting lysine residue(s). The corresponding azo functionalities were formed in a highly selective manner with excellent yields, displaying rather good stability under physiological conditions. Furthermore, the azodation proceeded smoothly in high yields on targeted peptides. Fluorescent and/or dual fluorescent labeling of varied proteins following this protocol have been determined efficiently and selectively. With this established protocol, we aim to determine its usage in the evaluation of the interaction of prenylated proteins with their interacted enzyme(s) via FRET assays. Delightedly, chemically modified proteins with a 1-pyrenyl fluorophore through 254 nm UV irradiation and the sequential azodation and click reaction of protein prenyl functionality, which enable the incorporation of naphthene, indeed increase the fluorescence energy transferred since we observed significantly enhanced absorption located at 218 nm in lysed HEK293T cells and a clearly strengthened greenish fluorescence in living HEK293T cells.

Dual Inhibition of HIV-1 and Cathepsin L Proteases by Sarcandra glabra.

The COVID-19 pandemic continues to impose a huge threat on human health due to rapid viral mutations. Thus, it is imperative to develop more potent antivirals with both prophylactic and treatment functions. In this study, we screened for potential antiviral compounds from Sarcandra glabra (SG) against Cathepsin L and HIV-1 proteases. A FRET assay was applied to investigate the inhibitory effects and UPLC-HRMS was employed to identify and quantify the bioactive components. Furthermore, molecular docking was carried out to get a glimpse of the binding of active compounds to the proteases. Our results showed that the SG extracts (SGW, SG30, SG60, and SG85) inhibited HIV-1 protease with an IC50 of 0.003~0.07 mg/mL and Cathepsin L protease with an IC50 of 0.11~0.26 mg/mL. Fourteen compounds were identified along with eight quantified from the SG extracts. Chlorogenic acid, which presented in high content in the extracts (12.7~15.76 µg/mg), possessed the most potent inhibitory activity against HIV-1 protease (IC50 = 0.026 mg/mL) and Cathepsin L protease (inhibition: 40.8% at 0.01 mg/mL). Thus, SG extracts and the active ingredients could potentially be used to prevent/treat viral infections, including SARS-CoV-2, due to their dual-inhibition functions against viral proteases.

Small-molecule screening of ribonuclease L binders for RNA degradation

Small molecules targeting the ubiquitous latent ribonuclease (RNase L), which has limited sequence specificity toward single-stranded RNA substrates, hold great potential to be developed as broad-spectrum antiviral drugs by modulating the RNase L-mediated innate immune responses. The recent development of proximity-inducing bifunctional molecules, as described in the strategy of ribonuclease targeting chimeras, demonstrated that small-molecule RNase L activators can function as the essential RNase L-recruiting component to design bifunctional molecules for targeted RNA degradation. However, only a single screening study on small-molecule RNase L activators with poor potency has been reported to date. Herein, we established a FRET assay and conducted a screening of 240,000 small molecules to identify new RNase L activators with improved potency. The extremely low hit rate of less than 0.03% demonstrated the challenging nature of RNase L activation by small molecules available from current screening collections. A few hit compounds induced enhanced thermal stability of RNase L upon binding, although validation assays did not lead to the identification of compounds with significantly improved RNase L activating potency. The sulfonamide compound 17 induced a thermal shift of ~ 0.9 °C upon binding to RNase L, induced significant apoptosis in cancer cells, and showed single-digit micromolar inhibitory activity against cancer cell proliferation. This study paves the way for future structural optimization for the development of small-molecule RNase L binders.

Fluorescence energy transfer biosensing platform based on hyperbranched rolling circle amplification and multi-site strand displacement for ultrasensitive detection of miRNA

A novel fluorescence biosensor based on hyperbranched rolling circle amplication (HRCA) and multi-site strand displacement amplification (SDA) strategy was constructed to realize super-sensitive detection of miRNA-21. The target miRNA-21 was specifically used to trigger HRCA reaction and generated abundant DNA sequences with different lengths. These sequences could initiate the SDA reaction with hairpin HP1 and HP2 to form the fluorescence signal double-stranded bodies (FSDB), and produced the fluorescence resonance energy transfer (FRET) for target assay. The circle template, reverse primer, hairpin HP1 and HP2 were skillfully designed to much improve the assay selectivity. This FRET signal ratio-based strategy not only avoided the false positive signal produced by traditional detection methods, but also minimized the influence of system fluctuation. So the detection limit of the target could reach fM level. In addition, this method could also be applied to the detection of other miRNAs, proteins and biomolecules, and had great potential in biomedical research, environmental detection and clinical diagnostic applications.

Fragment Screening Yields a Small-Molecule Stabilizer of 14-3-3 Dimers That Modulates Client Protein Interactions.

The development of protein‐protein interaction (PPI) inhibitors has been a successful strategy in drug development. However, the identification of PPI stabilizers has proven much more challenging. Here we report a fragment‐based drug screening approach using the regulatory hub‐protein 14‐3‐3 as a platform for identifying PPI stabilizers. A homogenous time‐resolved FRET assay was used to monitor stabilization of 14‐3‐3/peptide binding using the known interaction partner estrogen receptor alpha. Screening of an in‐house fragment library identified fragment 2 (VUF15640) as a putative PPI stabilizer capable of cooperatively stabilizing 14‐3‐3 PPIs in a cooperative fashion with Fusicoccin‐A. Mechanistically, fragment 2 appears to enhance 14‐3‐3 dimerization leading to increased client‐protein binding. Functionally, fragment 2 enhanced potency of 14‐3‐3 in a cell‐free system inhibiting the enzyme activity of the nitrate reductase. In conclusion, we identified a general PPI stabilizer targeting 14‐3‐3, which could be used as a tool compound for investigating 14‐3‐3 client protein interactions.

Whole-cell FRET monitoring of transcription factor activities enables functional annotation of signal transduction systems in living bacteria

Bacteria adapt to their constantly changing environments largely by transcriptional regulation through the activities of various transcription factors (TFs). However, techniques that monitor TF–promoter interactions in situ in living bacteria are lacking. Herein, we developed a whole-cell TF–promoter binding assay based on the intermolecular FRET between an unnatural amino acid, l-(7-hydroxycoumarin-4-yl) ethylglycine, which labels TFs with bright fluorescence through genetic encoding (donor fluorophore) and the live cell nucleic acid stain SYTO 9 (acceptor fluorophore). We show that this new FRET pair monitors the intricate TF–promoter interactions elicited by various types of signal transduction systems, including one-component (CueR) and two-component systems (BasSR and PhoPQ), in bacteria with high specificity and sensitivity. We demonstrate that robust CouA incorporation and FRET occurrence is achieved in all these regulatory systems based on either the crystal structures of TFs or their simulated structures, if 3D structures of the TFs were unavailable. Furthermore, using CueR and PhoPQ systems as models, we demonstrate that the whole-cell FRET assay is applicable for the identification and validation of complex regulatory circuit and novel modulators of regulatory systems of interest. Finally, we show that the FRET system is applicable for single-cell analysis and monitoring TF activities in Escherichia coli colonizing a Caenorhabditis elegans host. In conclusion, we established a tractable and sensitive TF–promoter binding assay, which not only complements currently available approaches for DNA–protein interactions but also provides novel opportunities for functional annotation of bacterial signal transduction systems and studies of the bacteria–host interface.

CDI Exerts Anti-Tumor Effects by Blocking the FoxM1-DNA Interaction.

The Forkhead box protein M1 (FoxM1) is an appealing target for anti-cancer therapeutics as this cell proliferation-associated transcription factor is overexpressed in most human cancers. FoxM1 is involved in tumor invasion, angiogenesis, and metastasis. To discover novel inhibitors that disrupt the FoxM1-DNA interaction, we identified CDI, a small molecule that inhibits the FoxM1–DNA interaction. CDI was identified through an assay based on the time-resolved fluorescence energy transfer response of a labeled consensus oligonucleotide that was bound to a recombinant FoxM1-dsDNA binding domain (FoxM1-DBD) protein and exhibited potent inhibitory activity against FoxM1-DNA interaction. CDI suppressed cell proliferation and induced apoptosis in MDA-MB-231 cells obtained from a breast cancer patient. Furthermore, it decreased not only the mRNA and protein expression of FoxM1 but also that of downstream targets such as CDC25b. Additionally, global transcript profiling of MDA-MB-231 cells by RNA-Seq showed that CDI decreases the expression of FoxM1-regulated genes. The docking and MD simulation results indicated that CDI likely binds to the DNA interaction site of FoxM1-DBD and inhibits the function of FoxM1-DBD. These results of CDI being a possible effective inhibitor of FoxM1-DNA interaction will encourage its usage in pharmaceutical applications.

Structure and luminescence properties of color-tunable phosphor Sr2La3(SiO4)3F:Tb3+,Sm3+

A series of single-phase and color-tunable phosphors Sr2La3(SiO4)3F:0.15Tb3+,xSm3+ (SLSOF:0.15Tb3+,xSm3+) was prepared using solid-state route. The X-ray diffraction (XRD) was used to characterize the phase of the as-prepared samples. The synthesized phosphors have apatite-type structure without other impurities. Sm3+ and Tb3+ ions substitute La3+ into the lattice and form a single-phase solid solution. The elements are evenly distributed in the sample according to the scanning electron microscopy (SEM) results. The crystal structure of the host phosphor was refined by the Rietveld method. The optical properties were studied in detail by investigation of the luminescence and diffuse spectra, fluorescence decay curves and energy transfer efficiency. The phosphor color can be changed from green (0.29, 0.48) to yellow/orange and red (0.57, 0.42) via adjusting the doping ratio of Sm3+. The SLSOF:0.15Tb3+,xSm3+ phosphors can emit typical multicolor light such as green, yellow, orange and red with gradually increasing Sm3+ doping concentration. All results indicate the occurrence of the energy transfer which results in the color-tunable properties of the phosphors.