Fluorescence Resonance Energy Transfer Assay Research Articles

Analysis of IFITM-IFITM Interactions by a Flow Cytometry-Based FRET Assay.

The interferon-induced transmembrane proteins 1–3 (IFITM1–3) inhibit host cell entry of several viruses. However, it is incompletely understood how IFITM1–3 exert antiviral activity. Two phenylalanine residues, F75 and F78, within the intramembrane domain 1 (IM1) were previously shown to be required for IFITM3/IFITM3 interactions and for inhibition of viral entry, suggesting that IFITM/IFITM interactions might be pivotal to antiviral activity. Here, we employed a fluorescence resonance energy transfer (FRET) assay to analyze IFITM/IFITM interactions. For assay calibration, we equipped two cytosolic, non-interacting proteins, super yellow fluorescent protein (SYFP) and super cyan fluorescent protein (SCFP), with signals that target proteins to membrane rafts and also analyzed a SCFP-SYFP fusion protein. This strategy allowed us to discriminate background signals resulting from colocalization of proteins at membrane subdomains from signals elicited by protein–protein interactions. Coexpression of IFITM1–3 and IFITM5 fused to fluorescent proteins elicited strong FRET signals, and mutation of F75 and F78 in IFITM3 (mutant IFITM3-FF) abrogated antiviral activity, as expected, but did not alter cellular localization and FRET signals. Moreover, IFITM3-FF co-immunoprecipitated efficiently with wild type (wt) IFITM3, lending further support to the finding that lack of antiviral activity of IFITM3-FF was not due to altered membrane targeting or abrogated IFITM3-IFITM3 interactions. Collectively, we report an assay that allows quantifying IFITM/IFITM interactions. Moreover, we confirm residues F75 and F78 as critical for antiviral activity but also show that these residues are dispensable for IFITM3 membrane localization and IFITM3/IFITM3 interactions.

Potential anti-Alzheimer effects of selected Lamiaceae plants through polypharmacology on glycogen synthase kinase-3β, β-secretase, and casein kinase 1δ

The aerial parts of Lamiaceae plants have been used since ancient times all over the world, especially in Mediterranean cultures, for their medicinal and culinary properties. Although some of them have a historical role in the improvement of cognition and memory, the precise mechanism of these plants remains unclear.This study evaluated the potential anti-Alzheimer effects of some popular cultivated Lamiaceae plants via their inhibitory potential towards glycogen synthase kinase (GSK-3β), β-secretase (BACE-1), and casein kinase 1δ (CK-1δ), enzymes related to AD.Luminometric-based assays were used for the evaluation of GSK-3β and CK-1δ inhibitory activities in which a fluorescence resonance energy transfer (FRET) assay was used for determining BACE-1 inhibitory activity. The major compounds were identified by high pressure liquid chromatography with diode array detection coupled with electrospray ionization tandem mass spectrometry (LC-DAD-ESI-MS/MS).Among the extracts tested, Origanum onites L., O. majorana L., and Mentha piperita L. were the most active ones for GSK-3β inhibitory activity, with IC50 values under 15 μg/mL, while O. majorana and S. officinalis L. were the most active for BACE-1 and O. onites, O. majorana, and Melissa officinalis L. for CK-1δ, with half maximal inhibitory concentration (IC50) values under 250 μg/mL. On the basis of the composition of these active extracts it is apparent that flavonoids, i.e. apigenin, luteolin, and their glycoside derivatives, along with phenolic acids, i.e. rosmarinic acid and salvianolic acids, are the major principles of these active extracts.The outcomes of this study supported the traditional use of Lamiaceae plants for cognitive disorders and highlighted their underlying mechanisms, targeting GSK-3β/BACE-1/CK-1δ together for the first time.

Fluorescence resonance energy transfer combined with asymmetric PCR for broad and sensitive detection of porcine reproductive and respiratory syndrome virus 2

With its ever-increasing viral genetic diversity, accurate diagnosis of porcine reproductive and respiratory syndrome virus (PRRSV) infection is indispensable for PRRSV control. Here, a sensitive graphene oxide (GO)-based FRET method was developed to detect PRRSV-2 based on the ability of GO to quench fluorophore by fluorescence resonance energy transfer (FRET). Using primers and a fluorophore-labeled ssDNA probe targeting a conserved region between the PRRSV M gene and 3′UTR, asymmetric PCR specifically amplified viral ssDNA that could anneal with probe to generate dsDNA only in the presence of virus. Upon exonuclease III treatment to release the probe fluorophore, which degrades dsDNA with blunt ends or recessed 3´-termini, the ssDNA annealed with other probe to generate enhanced fluorescence. This GO-based FRET assay specifically detected both classical and highly pathogenic PRRSV, with analytical sensitivity approaching 10 copies/μL, similar to that of real-time PCR but greater than that of conventional reverse transcription PCR (RT-PCR). Consistent with real-time RT-PCR detection, the assay developed here exhibited high diagnostic sensitivity for virus detection of sera from experimentally and naturally infected pigs. Thus, this novel GO-based FRET assay combined with asymmetric PCR detection is sensitive and specific and will be valuable for future PRRSV diagnosis.

A General TR-FRET Assay Platform for High-Throughput Screening and Characterizing Inhibitors of Methyl-Lysine Reader Proteins

Chromatin regulatory complexes localize to specific sites via recognition of posttranslational modifications (PTMs) on N-terminal tails of histone proteins (e.g., methylation, acetylation, and phosphorylation). Molecular recognition of modified histones is mediated by “reader” protein subunits. The recruited complexes govern processes such as gene transcription, DNA replication, and chromatin remodeling. Dysregulation of histone modifications and consequent downstream effects have been associated with a variety of disease states, leading to an interest in developing small-molecule inhibitors of reader proteins. Herein, we describe a generalized time-resolved fluorescence resonance energy transfer (TR-FRET) assay for a panel of methyl-lysine (Kme) reader proteins. These assays are facile, robust, and reproducible. Importantly, this plug-and-play assay can be used for high-throughput screening (HTS) campaigns, generation of structure–activity relationships (SARs), and evaluation of inhibitor selectivity. Successful demonstration of this assay format for compound screening is highlighted with a pilot screen of a focused compound set with CBX2. This assay platform enables the discovery and characterization of chemical probes that can potently and selectively inhibit Kme reader proteins to ultimately accelerate studies of chromatin reader proteins in normal biology and disease states.

Abstract 374: Discovery and targeting MKK3-MYC protein-protein interaction for therapeutic interrogation in cancer

Abstract Protein-protein interactions (PPIs) dictate the signal transduction and regulate diverse physiological programs, including cell death and survival. Recently we have developed a PPI high-throughput screening platform to detect the PPIs between cancer-associated proteins in the context of cancer cells. Characterization of ~3,500 PPIs tested for a set of lung-cancer related proteins resulted in a network of high-confidence direct PPIs, termed OncoPPi. To enable streamlined and integrated analysis of PPI datasets, we have developed the OncoPPi Portal, a web-based resource that integrates the network of experimentally detected cancer-associated PPIs with cancer genomics, pharmacological, and protein structural data to support cancer research. The analysis of the OncoPPi network revealed a new connectivity between transcription factor MYC and Mitogen-Activated Protein Kinase Kinase 3 (MKK3) known an activator of p38 signaling pathway. MYC is frequently amplified across the vast majority of cancer types, and extensive clinical and biological studies have established MYC as a highly attractive therapeutic target in cancer. However, currently, there are no FDA-approved MYC inhibitors, and novel therapeutic strategies to control MYC-dependency are urgently needed. We found that in contrast to other MYC-regulating kinases, such as ERK, MKK3 enhances MYC protein stability and transcriptional activity in a phosphorylation-independent manner. The biochemical studies guided by computational molecular modeling have revealed specific domains of MKK3 and MYC that are responsible for their interaction. Furthermore, we found that MKK3-MYC PPI can be disrupted by a short 27 amino acid residue fragment derived from MYC helix-loop-helix domain. Overexpression of this peptide was correlated with the cell growth inhibition and induction of apoptosis in multiple cancer cell lines. To identify small molecule chemical probes to test MKK3-MYC dependency in cancer, we have developed a time-resolved fluorescence resonance energy transfer (TR-FRET) assay to monitor the interaction of MKK3 and MYC in mammalian cells. We have further optimized the assay for ultra-high-throughput screening (uHTS) in 1536-well plate format. A pilot screening with a library of 3,280 compounds has identified several positive hits that were further validated in orthogonal affinity pull-down PPI inhibition assays. In summary, the integration of experimentally determined OncoPPi network with bioinformatics approaches has established a new biological role for MKK3 as a direct regulator of MYC-driven cell growth and proliferation. Our HTS PPI inhibitor platform has revealed first small molecule inhibitors for MKK3-MYC PPI. Together, this data defines the MKK3/MYC interface as a new potential therapeutic target to control MYC-driven tumorigenesis. Citation Format: Xuan Yang, Yuhong Du, Haian Fu, Andrey A. Ivanov. Discovery and targeting MKK3-MYC protein-protein interaction for therapeutic interrogation in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 374.

Strand displacement dual amplification miRNAs strategy with FRET between NaYF4:Yb,Tm/Er upconversion nanoparticles and Ti3C2 nanosheets

Due to the low abundance of miRNAs and the coordinated regulation of multiple miRNAs toward some biological processes, a method that can sensitively and simultaneously detect multiple miRNAs is needed. Here, a strand displacement dual amplification (SDDA) strategy was developed for multiple miRNAs analyses using a unique single strand-double strand-single strand DNA (sdsDNA), which generated by the target recognition probe hybridizing with the site region probe. During the process of SDDA, miRNA hybridized to the target recognition domain of sdsDNA, thereby releasing the site, which triggered the first strand displacement reaction. This strand displacement event resulted in the release of the other site, triggering the second strand displacement reaction. Both two strand displacement reactions in each assembly circle were integrated for amplified signals, which were tested by a fluorescence resonance energy transfer (FRET) assay. NaYF4:Yb,Tm/Er UCNPs and Ti3C2 nanosheets were used as the energy donor-acceptor pair. By employing 1:2 ratio of donor-acceptor, the energy transfer efficiency between NaYF4:Yb,Tm/Er UCNPs and Ti3C2 nanosheets could reach 90%, which enhanced signal-to-background ratio. Combining the benefits of dual amplification and efficient energy transfer on miRNAs detection, this assay exhibited a linear range from 5 fM to 100 pM for miRNA-21 and miRNA-10b. Moreover, this assay was applied to estimate the expression of miRNA-21 and miRNA-10b in human hepatoma and hepatocyte cell lines. Therefore, this FRET-based SDDA strategy has been used for highly sensitive detection of multiple miRNAs in cell lysate and facilitates the development of efficient biosensor platform.

An optical and non-invasive method to detect the accumulation of ubiquitin chains.

The accumulations of excess amounts of polyubiquitinated proteins are cytotoxic and frequently observed in pathologic tissue from patients of neurodegenerative diseases. Therefore, optical and non-invasive methods to detect the increase of the amounts of polyubiquitinated proteins in living cells is a promising strategy to find out symptoms and environmental cause of neurodegenerative diseases, also for identifying compounds that could inhibit gathering of polyubiquitinated proteins. Therefore, we generated a pair of fluorescent protein [Azamigreen (Azg) and Kusabiraorange (Kuo)] tagged ubiquitin on its N-terminus (Azg-Ub and Kuo-Ub) and developed an Azg/Kuo-based Fluorescence Resonance Energy Transfer (FRET) assay to estimate the amount of polyubiquitin chains in vitro and in vivo. The FRET intensity was attenuated in the presence of ubiquitin-activating enzyme inhibitor, PYR-41, indicating that both fluorescent ubiquitin is incorporated into ubiquitin chains likewise normal ubiquitin. The FRET intensity was enhanced by the addition of the proteasome inhibitor, MG-132, and was reduced in the presence of the autophagy activator Rapamycin, designating that ubiquitin chains with fluorescent ubiquitin act as the degradation signal equally with normal ubiquitin chains. In summary, the above optical methods provide powerful research tools to estimate the amounts of polyubiquitin chains in vitro and in vivo, especially non-invasively in living cells.

Toxoplasma gondii ROP18 inhibits human glioblastoma cell apoptosis through a mitochondrial pathway by targeting host cell P2X1

BackgroundApoptosis plays a critical role in the embryonic development, homeostasis of immune system and host defense against intracellular microbial pathogens. Infection by the obligate intracellular pathogen Toxoplasma gondii can both inhibit and induce host cell apoptosis; however, the parasitic factors involved remain unclear. The T. gondii virulence factor ROP18 (TgROP18) has been reported to regulate host cell apoptosis; nevertheless, results for this regulation have been rarely reported or have provided contradictory findings. Human purinergic receptor 1 (P2X1) is an ATP-gated ion channel that responds to ATP stimulation and functions in cell apoptosis mediation. The precise roles of TgROP18 in T. gondii pathogenesis, and the relationship between TgROP18 and host P2X1 in host cell apoptosis are yet to be revealed.MethodsApoptosis rates were determined by flow cytometry (FCM) and TUNEL assay. The interaction between TgROP18 and the host P2X1 was measured by fluorescence resonance energy transfer (FRET) and co-immunoprecipitation (co-IP) assay. Calcium influx and mitochondrial membrane depolarization were determined by FCM after JC-1 staining. The translocation of cytochrome C (Cyt C), Bax and Bcl2 proteins, expression of the apoptotic proteins PARP and caspase activation were detected by western blotting.ResultsThe apoptosis rates of glial or immune cells (human SF268, mouse RAW264.7 and human THP-1 cells) infected by any T. gondii strain (RH-type I, ME49-type II and VEG-type III) were significantly inhibited compared with their uninfected controls. TgROP18 inhibited ATP-induced apoptosis of SF268 with P2X1 expression, but had no effect on RAW264.7 or THP-1 cells without detectable P2X1 expression. It was further identified that TgROP18 interacted with P2X1, and overexpression of ROP18 in COS7 cells significantly inhibited cell apoptosis mediated by P2X1. Moreover, TgROP18 also inhibited P2X1-mediated Ca2+ influx, translocation of cytochrome C from the mitochondria to the cytosol, and ATP-triggered caspase activation.ConclusionsToxoplasma gondii infection inhibits ATP-induced host cell apoptosis, regardless of strain virulence and host cell lines. TgROP18 targets the purinergic receptor P2X1 of the SF268 human neural cells and inhibits ATP-induced apoptosis through the mitochondrial pathway, suggesting a sensor role for the host proapoptotic protein P2X1 in this process.

A high-throughput screen of pharmacologically active compounds for inhibitors of UHRF1 reveals epigenetic activity of anthracycline derivative chemotherapeutic drugs.

DNA methylation can mediate epigenetic silencing of tumor suppressor and cancer protective genes. The protein ubiquitin-like containing PHD and ring finger domains 1 (UHRF1) is an essential component in cells for DNA methylation maintenance. The SET- and RING-associated (SRA) domain of UHRF1 can bind hemimethylated DNA, and mediate recruitment of DNA methyltransferases to copy the methylation pattern to the newly synthesized daughter strand. Loss of UHRF1 function can lead to demethylation and re-expression of epigenetically silenced tumor suppressor genes and can reduce cancer cell growth and survival. We created a high-throughput time-resolved fluorescence resonance energy transfer (TR-FRET) assay to screen for inhibitors capable of disrupting the interaction between the UHRF1-SRA domain and hemimethylated DNA. Using this assay (Z’ factor of 0.74 in 384-well format) we screened the Library of Pharmacologically Active Compounds (LOPAC) for UHRF1-SRA inhibitors, and validated 7 hit compounds. These compounds included the anthracycline derivatives idarubicin and mitoxantrone, which are commonly used chemotherapeutic drugs known to mediate cytotoxicity by acting as class II topoisomerase (TOP2) poisons. In a panel of additional known topoisomerase poisons, only the anthracycline derivatives showed dose responsive inhibition of UHRF1-SRA. Additionally, mitoxantrone and doxorubicin showed dose-responsive global DNA demethylation and demonstrated a synergistic growth inhibition of multiple cancer cell lines when combined with the DNA methyltransferase (DNMT) inhibitor decitabine. These data validate a novel TR-FRET assay for identification of UHRF1 inhibitors, and revealed unexpected epigenetic properties of commonly used chemotherapeutic drugs that showed synergistic cytotoxicity of cancer cells when combined with a demethylating agent.

Chicken immunoglobulin Y based FRET assay for TSST-1 detection and its validation onto clinical isolates

Staphylococcus aureus (S. aureus) is commensal bacteria implicated in various opportunistic infections worldwide. Their numerous virulence factors along with mounting antibiotic resistance is an ominous problem associated with its clinical management. Among the numerous S. aureus virulence factors, toxic shock syndrome toxin 1 (TSST-1) is a superantigen that is pivotal in life-threatening multisystem disorder. The present study reports the development of a fluorescence resonance energy transfer (FRET) assay with hybrid gold nanoparticles (GNP) conjugated chicken antibodies (IgY) and Cadmium Telluride (CdTe) quantum dots labeled rabbit antibodies for TSST-1 detection. The TSST-1 gene cloned into pET22b (+) vector for recombinant protein production and employed for anti-TSST-1 antibody generation from chicken and rabbit systems. Thereafter, CdTe-QDs tagged anti-TSST-1 IgG was employed as the fluoroprobe and subsequently, GNP conjugated anti-TSST-1 IgY served as its quencher pair. Further analysis results showed that the assay was simple, sensitive and reliable with a limit of detection (LOD) of 0.0005 μgmL-1 and shows no cross-reactivity towards closely related toxins. The artificially spiked clinical sample analysis revealed insignificant matrix interference as well as considerable toxin recovery range (81.21%–99.3%) for the assay. Furthermore, its co-evaluation with commercially available TSST-1 detection kit demonstrated their feasibility for toxin screening with substantially less assay duration. Therefore, the assay may aid in better toxic shock management, apparently reducing empirical treatment, medical costs, and antimicrobial resistance.

DNA Aptamers Targeting BACE1 Reduce Amyloid Levels and Rescue Neuronal Deficiency in Cultured Cells

β-amyloid (Aβ) plays an essential role in the pathogenesis of Alzheimer’s disease (AD). Beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) is indispensable for Aβ production, and knockout of BACE1 has no overt phenotypes in mouse. Thus, fine modulation of BACE1 may be a safe and effective treatment for AD patients. However, the large active site of BACE1 makes it challenging to target BACE1 with classical small-molecule inhibitors. DNA aptamer can have high affinity and specificity against diverse targets, and it provides an alternative strategy to target BACE1. In this study, we used a novel cell-systematic evolution of ligands by exponential enrichment (SELEX) strategy to select specific DNA aptamers optimized to target BACE1 under physiological status. After 17 rounds of selection, we identified two DNA aptamers against BACE1: BI1 and BI2. The identified aptamers interacted with BACE1 in pull-down assay, inhibited BACE1 activity in in vitro fluorescence resonance energy transfer (FRET) assay and HEK293-APP stable cell line, reduced Aβ in the culture medium of HEK293-amyloid protein precursor (APP) stable cell line and APP-PS1 primary cultured neurons, and rescued Aβ-induced neuronal deficiency in APP-PS1 primary cultured neurons. In contrast, the identified aptamers had no effect on α- or γ-secretase. In addition, cholesteryl tetraetylene glycol (TEG) modification further improved the potency of the identified aptamers. Our study suggests that it is feasible and effective to target BACE1 with DNA aptamers, and the therapeutic potential of the identified aptamers deserves further investigation.

Full anti-apoptotic function of Bcl-XL complexed with Beclin-1 verified by live-cell FRET assays

Binding of Bcl-XL to Beclin-1 reduces Beclin-1’s capacity to induce autophagy. This report aims to explore whether this interaction affects Bcl-XL’s anti-apoptotic function. Using fluorescence resonance energy transfer (FRET) two-hybrid assay to quantify the stoichiometry of Bcl-XL-Beclin-1 complex in living cells coexpressing Bcl-XL-CFP and Beclin-1-YFP, we showed that Bcl-XL bond to Beclin-1 to form hetero-oligomers whose stoichiometry increases from 1:1 to 2:1 or higher with the increasing relative expression level of Bcl-XL, indicating the multiple binding sites of Beclin-1 with Bcl-XL. Co-expression of Bcl-XL and Beclin-1 exhibited consistent anti-apoptotic ability against staurosporine (STS)-induced apoptosis with expression of Bcl-XL alone irrespective of the relative expression level between Beclin-1 and Bcl-XL. Collectively, Bcl-XL complexed with Beclin-1 maintains full anti-apoptotic ability independent of the stoichiometry of Bcl-XL-Beclin-1 complex.

Imaging-based assays for investigating functions of the RNA polymerase II elongation factor Elongin and the Elongin ubiquitin ligase.

Elongin A binds to Elongins B and C to form the RNA polymerase II transcription elongation factor Elongin. It also functions as the substrate recognition subunit of a ubiquitin ligase that is formed by binding of Elongin to Cullin protein CUL5 and RING finger protein RBX2 and that targets RNA polymerase II for ubiquitination. In this article, we describe use of acceptor photobleaching fluorescence resonance energy transfer (AP-FRET) and laser microirradiation-based assays to study regulated assembly of the Elongin ubiquitin ligase and its recruitment to regions of localized DNA damage.

Design, synthesis, and biological evaluation of 4-aminopyrimidine or 4,6-diaminopyrimidine derivatives as beta amyloid cleaving enzyme-1 inhibitors.

A series of novel aminopyrimidine and diaminopyrimidine derivatives were designed and optimized to improve their potency and permeability relative to lead compound 1 (IC50 =37.4μM), which was discovered in a previous virtual screening. The potency of the optimized compound, 13g (IC50 =1.4μM), was 26-fold greater than that of 1 based on a fluorescence resonance energy transfer assay, and a parallel artificial membrane permeability assay suggested that it could pass through the blood-brain barrier. Additionally, several compounds containing selenium showed good potencies and deserve further investigation as anti-Alzheimer's agents.

Discovery of N-indanyl benzamides as potent RORγt inverse agonists

The retinoic acid receptor-related orphan receptor-gamma-t (RORγt) is a promising therapeutic target for treatment of Th17 cell-mediated autoimmune diseases. Based on a scaffold hopping/conformational restriction strategy, a series of N-indanyl benzamides as novel RORγt inverse agonists was discovered. Exploration of structure-activity relationship on the piperazine ring, benzoyl moiety and cyclopentyl moiety of N-indanyl benzamides 2a and 2d led to identification of potent RORγt inverse agonists. Compound 5c with (S)-enantiomer was found having an IC50 of 153.7 nM in Fluorescence Resonance Energy Transfer (FRET) assay, and an IC50 of 47.1 nM in mouse Th17 cell differentiation assay, which represents a promising starting point for developing potent small molecule RORγt inverse agonists. Binding modes of the two enantiomers 5c and 5d in RORγt ligand binding domain were also discussed.

Abstract WP151: Ischemic Injury in Diabetes Augments Cerebral Microvascular Matrix Metalloproteinase Activation in Female Rats

Introduction: Diabetic female patients are at greater risk of stroke and worsened recovery. We have previously shown that matrix metalloprotease 3 (MMP3) exacerbates hemorrhagic transformation (HT) in hyperglycemic stroke in male rats while high glucose and hypoxia increases MMP3 activity in brain microvascular endothelial cells (BMVECs). Furthermore, diabetic female rats develop greater HT. Current study was designed to test the hypothesis that ischemic stroke increases the cerebral microvascular MMP3 activity in diabetic female animals. Methods: Female rats after 7-8 weeks of diabetes (high fat diet and low dose of streptozotocin) were subjected to 60 min MCAO. After 3 days, adhesive removal time (ART) and behavioral composite score were measured and cerebral microvessels were isolated. Human female BMVECs were grown under normal (5.5mM glucose) or diabetic condition (25mM glucose plus 100μM sodium palmitate) for 48 hours, followed by hypoxia insult for 12 hours (200μM cobalt chloride). Cell lysate, media and microvascular homogenates were analyzed for MMP profiling by immunoblotting, zymography and MMP3 enzyme activity by fluorescence resonance energy transfer (FRET) assay. Results: MMP3 protein expression and activity were significantly increased in the ischemic microvasculature and this was accompanied by a worse neurobehavioral score (Table 1). In vitro, expression of MMP3, 2 and 9 proteins in cell lysate were not different between the groups. However, MMP3 and MMP9 enzyme activities were significantly increased in the diabetes condition while, there was no additional increase in enzyme activity with hypoxia. MMP2 activity was significantly increased with hypoxia in diabetes condition (Table 1). Conclusions: In female rats, diabetes and hypoxia increase MMP activity in the microvasculature and endothelial cells. These observations provide initial evidence to further explore the role of MMPs in the increased HT seen in females with diabetes.

Development of a High-Throughput Cul3-Keap1 Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay for Identifying Nrf2 Activators

Nrf2, a master regulator of the phase II gene response to stress, is kept at low concentrations in the cell through binding to Keap1, an adaptor protein for the Cul3 ubiquitin ligase complex. To identify Nrf2 activators, two separate time-resolved fluorescence resonance energy transfer (TR-FRET) assays were developed to monitor the binding of Nrf2-Keap1 and Cul3-Keap1, respectively. The triterpenoid, 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl] imidazole (CDDO-Im) and its analogs, exhibited approximately 100-fold better potency in the Cul3-Keap1 assay than in the Nrf2-Keap1 assay, and this difference was more profound at 37 °C than at room temperature in the Nrf2-Keap1 assay, but this phenomenon was not observed in the Cul3-Keap1 assay. A full diversity screen of approximately 2,200,000 GSK compounds was run with the Cul3-Keap1 TR-FRET assay and multiple chemical series were identified and characterized.

Twin-FRET: A New Molecular Ruler for Biomolecules

Conformational changes in biomolecules underpin all biological processes and being able to quantify these structural changes in solution is crucial to understand biological function. By carefully positioning two fluorophores within the biomolecule, it is possible to use fluorescence resonance energy transfer (FRET) as a molecular ruler to measure the desired distance. In current FRET assays, the biomolecule needs to be labelled with two different chemical fluorophores acting as a donor-acceptor FRET pair. Incorporation of these two chemically different species at specific positions within the biomolecule is challenging due to limited chemical labelling strategies. Here, we present a radically different strategy for measuring distances in biomolecules. We have developed the concept of TWIN-FRET which removes the need for two different fluorophores attached to the biomolecule by chemically encoding the FRET pair within the structure of the fluorophore itself. We have designed and synthesised a fluorescent molecule to prove this concept. The fluorophore (FH) has an acid-base equilibrium with a ground state pKa∼8.9. We have derivatized the fluorophore to its succinimide ester, and used this derivative, to label a duplex DNA with two molecules of the same fluorophore at specific positions. Our results demonstrate the transfer of non-radiative energy from the neutral (FH) to the anionic (F−) state of the fluorescent molecule. We further demonstrate the use of TWIN-FRET to measure nanometer-size distances within the DNA duplex, and we obtained distance values similar to those obtained using a conventional FRET pair (Alexa488-Cy3). By removing the need to introduce two different chemical structures within the biomolecule, we greatly simplified the methodology to measure nanometer-size distances in biomolecules. We expect this technique to be widely used in structural and biophysical studies of nucleic acids, proteins and interactions between them.

Discovery of alkoxy benzamide derivatives as novel BPTF bromodomain inhibitors via structure-based virtual screening

Bromodomain PHD finger transcription factor (BPTF), a bromodomain-containing protein, plays a crucial role in the regulation of downstream gene expression through the specific recognition of lysine acetylation on bulk histones. The dysfunction of BPTF is closely involved with the development and progression of many human diseases, especially cancer. Therefore, BPTF bromodomain has become a promising drug target for epigenetic cancer therapy. However, unlike BET family inhibitors, few BPTF bromodomain inhibitors have been reported. In this study, by integrating docking-based virtual screening with biochemical analysis, we identified a novel selective BPTF bromodomain inhibitor DCB29 with the IC50 value of 13.2 ± 1.6 μM by homogenous time-resolved fluorescence resonance energy transfer (HTRF) assays. The binding between DCB29 and BPTF was confirmed by NMR and SPR. Molecular docking disclosed that DCB29 occupied the pocket of acetylated H4 peptide substrate and provided detailed SAR explanations for its derivatives. Collectively, DCB29 presented great potential as a powerful tool for BPTF-related biological research and further medicinal chemistry optimization.

Prevalence of herpes simplex virus-1 in hospitalized adult patients with clinical diagnosis of meningoencephalitis using real-time polymerase chain reaction: A single-center, cross-sectional study.

Background:Early diagnosis of herpes simplex virus-1 (HSV-1) meningoencephalitis is very important because antiviral therapy significantly decreases mortality and morbidity. Polymerase chain reaction (PCR) is a reliable method with high sensitivity and specificity in detection of HSV-1. The aim of this study is to determine the prevalence of HSV-1 in patients with diagnosis of meningoencephalitis using real-time PCR.Materials and Methods:The cerebrospinal fluid samples were collected from 126 patients with clinical diagnosis of HSV-1 meningoencephalitis in Alzahra Referral Hospital in Isfahan, Iran. After deoxyribonucleic acid (DNA) extraction, real-time PCR was performed by fluorescence resonance energy transfer assay and participants underwent brain magnetic resonance imaging, as well.Results:Among 126 patients, 68.3% were male and 31.7% were female. The mean age of the participants was 41.96 ± 22.36 years. Most of the participants were in the age group of 20–29 years. Three patients (2.4%) had positive and 123 patients (97.6%) had negative HSV-1 DNA test. Among three positive cases, two were in the age group of 20–29 years and one in the age group of ≥80 years. No HSV-2 DNA was detected.Conclusion:According to the estimated prevalence of HSV-1 meningoencephalitis in the current study, it seems that the prevalence of HSV-1 meningoencephalitis is not too high in our community; therefore, initial empiric acyclovir therapy is frequently overused.