Search Results (27)

Protein–protein interactions (PPIs) play a crucial role in various biological processes, including signal transduction, transcriptional regulation, and metabolic pathways. Over the years, many methods have been developed to study PPIs, such as yeast two-hybrid (Y2H), co-immunoprecipitation (Co-IP), pull-down assays, and surface plasmon resonance (SPR). However, each of these techniques has its own limitations, including false positives, a lack of specific binding partners, and restricted interaction zones. Fluorescence resonance energy transfer (FRET) has emerged as a powerful technique for investigating PPIs, offering several advantages over traditional methods. Recent advancements in fluorescence microscopy have further enhanced its application in PPI studies. In this review, we summarize recent developments in FRET-based approaches and their applications in PPIs research over the past five years, including conventional FRET, time-resolved FRET (TR-FRET), fluorescence lifetime imaging microscopy-FRET (FLIM-FRET), single-molecule FRET (smFRET), fluorescence cross-correlation spectroscopy FRET (FCCS-FRET), and provide guidance on selecting the most appropriate method for PPIs studies. Full article

The study’s aim was to evaluate electroretinographic (ERG) alterations in Fragile X syndrome (FXS), FMR1 premutation carriers, and controls, and to explore correlations with peripheral blood FMRP expression levels and behavioral outcomes. ERG recordings were obtained using a handheld device across three stimulus protocols in 43 premutation carriers, 39 individuals with FXS, and 23 controls. Peripheral blood FMRP expression levels were quantified using TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer). Correlations were assessed with cognitive and behavioral measures including IQ (Intelligence Quotient), ABC_FX (Aberrant Behavior Checklist for Fragile X Syndrome), SNAP-IV (Swanson, Nolan, and Pelham Teacher and Parent Rating Scale), SEQ (Sensory Experiences Questionnaire), ADAMS (Anxiety, Depression, and Mood Scale), and the Vineland III Adaptive Behavior Scale standard score. Significant group differences were observed in multiple ERG parameters, particularly in 2 Hz b-wave amplitude (p = 0.0081), 2 Hz b-wave time to peak (p = 0.0164), 28.3 Hz flash combined amplitude (p = 0.0139), 3.4 Hz red/blue flash b-wave amplitude (p = 0.0026), and PhNR amplitude (p = 0.0026), indicating both outer and inner retinal dysfunction in FXS and premutation groups. Despite high test–retest reliability for ERG (ICC range = 0.71–0.92) and FMRP (ICC = 0.70), no correlation was found between ERG metrics and FMRP or behavioral measures. However, FMRP levels strongly correlated with IQ (ρ = 0.69, p < 0.0001) and inversely with behavioral impairment [ABC_FX (ρ = −0.47, p = 0.0041), SNAP-IV (ρ = −0.48, p = 0.0039), SEQ (ρ = −0.43, p = 0.0146), and the Vineland III standard score (ρ = 0.56, p = 0.0019)]. ERG reveals distinct retinal functional abnormalities in FMR1-related conditions but does not correlate with peripheral FMRP expression levels, highlighting the need for multimodal biomarkers integrating radiological, physiological, behavioral, and molecular measures. Full article

Glycyrrhizic acid (GA) and its derivatives have been reported to have potent pharmacological effects against viral infections, including SARS-CoV and SARS-CoV-2. However, their antiviral mechanisms against coronaviruses are not fully understood. In this study, we found that diammonium glycyrrhizinate (DG) can effectively reduce infections of several human coronaviruses, including HCoV-OC43, HCoV-229E, and SARS-CoV-2, as well as newly emerged variants, with EC₅₀ values ranging from 115 to 391 μg/mL being recorded. Time-of-addition and pseudotype virus infection studies indicated that DG treatment dramatically inhibits the process of virus entry into cells. Furthermore, we demonstrated that DG broadly binds to the RBD of human coronaviruses, thereby blocking spike-mediated cellular entry, by using TR-FRET-based receptor-binding domain (RBD)-ACE2 interaction assay, capillary electrophoresis (CE), and surface plasmon resonance (SPR) assay. In support of this notion, studies of molecular docking and amino acid mutation showed that DG may directly bind to a conserved hydrophobic pocket of the RBD of coronaviruses. Importantly, intranasal administration of DG had a significant protective effect against viral infection in a HCoV-OC43 mouse model. Finally, we found that combinations of DG and other coronavirus inhibitors exhibited antiviral synergy. In summary, our studies strongly reveal that DG exerts broad-spectrum antiviral activity against human coronaviruses by interrupting spike-mediated cellular entry, demonstrating the pharmacological feasibility of using DG as a candidate for alternative treatment and prevention of coronavirus infection. Full article

Peroxisome proliferator-activated receptors (PPARs) are considered good drug targets for breast cancer because of their involvement in fatty acid metabolism that induces cell proliferation. In this study, we used the KAIMRC1 breast cancer cell line. We showed that the PPARE-Luciferase reporter gets highly activated without adding any exogenous ligand when PPAR alpha is co-transfected, and the antagonist GW6471 can inhibit the activity. Using this reporter system, we screened 240 compounds representing kinase inhibitors, epigenetic modulators, and stem cell differentiators and identified compounds that inhibit the PPARα-activated PPARE-Luciferase reporter in the KAIMRC1 cell. We selected 11 compounds (five epigenetic modulators, two stem cell differentiators, and four kinase inhibitors) that inhibited the reporter by at least 40% compared to the controls (DMSO-treated cells). We tested them in a dose-dependent manner and measured the KAIMRC1 cell viability after 48 h. All 11 compounds induced the cell killing at different IC50 values. We selected two compounds, PHA665752 and NSC3852, to dissect how they kill KAIMRC1 cells compared to the antagonist GW6741. First, molecular docking and a TR-FRET PPARα binding assay showed that compared to GW6471, these two compounds could not bind to PPARα. This means they inhibit the PPARα pathway independently rather than binding to the receptor. We further confirmed that PHA665752 and NSC3852 induce cell killing depending on the level of PPARα expression, and as such, their potency for killing the SW620 colon cancer cell line that expresses the lowest level of PPARα was less potent than for the KAIMRC1 and MDA-MB-231 cell lines. Further, using an apoptosis array and fatty acid gene expression panel, we found that both compounds regulate the PPARα pathway by controlling the genes involved in the fatty acid oxidation process. Our findings suggest that these two compounds have opposite effects involving fatty acid oxidation in the KAIMRC1 breast cancer cell line. Although we do not fully understand their mechanism of action, our data provide new insights into the potential role of these compounds in targeting breast cancer cells. Full article

Recent advances in drug discovery have established biosensors as indispensable tools, particularly valued for their precision, sensitivity, and real-time monitoring capabilities. The review begins with a brief overview of cancer drug discovery, underscoring the pivotal role of biosensors in advancing cancer research. Various types of biosensors employed in cancer drug discovery are then explored, with particular emphasis on fluorescence- and bioluminescence-based technologies such as FRET, TR-FRET, BRET, NanoBRET, and NanoBiT. These biosensors have enabled breakthrough discoveries, including the identification of Celastrol as a novel YAP-TEAD inhibitor through NanoBiT-based screening, and the development of TR-FRET assays that successfully identified Ro-31-8220 as a SMAD4R361H/SMAD3 interaction inducer. The integration of biosensors in high throughput screening and validation for cancer drug compounds is examined, highlighting successful applications such as the development of LATS biosensors that revealed VEGFR as an upstream regulator of the Hippo signaling pathway. Real-time monitoring of cellular responses through biosensors has yielded invaluable insights into cancer cell signaling pathways, as demonstrated by NanoBRET assays detecting RAF dimerization and HiBiT systems monitoring protein degradation dynamics. The review addresses challenges linked to biosensor applications, such as maintaining stability in complex tumor microenvironments and achieving consistent sensitivity in HTS applications. Emerging trends are discussed, including integrating artificial intelligence and advanced nanomaterials for enhanced biosensor performance. In conclusion, this review offers a comprehensive analysis of fluorescence- and bioluminescence-based biosensor applications in the dynamic cancer drug discovery field, presenting quantitative evidence of their impact and highlighting their potential to revolutionize targeted cancer treatments. Full article

Thiazolidinediones (TZDs) including rosiglitazone and pioglitazone function as peroxisome proliferator-activated receptor gamma (PPARγ) full agonists, which have been known as a class to be among the most effective drugs for the treatment of type 2 diabetes mellitus (T2DM). However, side effects of TZDs such as fluid retention and weight gain are associated with their full agonistic activities toward PPARγ induced by the AF-2 helix-involved “locked” mechanism. Thereby, this study aimed to obtain novel PPARγ partial agonists without direct interaction with the AF-2 helix. Through performing virtual screening of the Targetmol L6000 Natural Product Library and utilizing molecular dynamics (MD) simulation, as well as molecular mechanics Poisson–Boltzmann surface area (MM-PBSA) analysis, four compounds including tubuloside b, podophyllotoxone, endomorphin 1 and paliperidone were identified as potential PPARγ partial agonists. An in vitro TR-FRET competitive binding assay showed podophyllotoxone displayed the optimal binding affinity toward PPARγ among the screened compounds, exhibiting IC₅₀ and ki values of 27.43 µM and 9.86 µM, respectively. Further cell-based transcription assays were conducted and demonstrated podophyllotoxone’s weak agonistic activity against PPARγ compared to that of the PPARγ full agonist rosiglitazone. These results collectively demonstrated that podophyllotoxone could serve as a PPARγ partial agonist and might provide a novel candidate for the treatment of various diseases such as T2DM. Full article

Exosomal biomarker detection holds great importance in the field of in vitro diagnostics, offering a non-invasive and highly sensitive approach for early disease detection and personalized treatment. Here, we proposed an “APPROACH” strategy, combining aptamer-mediated proximity ligation assay (PLA) with rolling circle amplification (RCA) and time-resolved Förster resonance energy transfer (TR-FRET) for the sensitive and semi-homogenous detection of exosomal biomarkers. PLA probes consisted of a cholesterol-conjugated oligonucleotide, which anchored to the membrane of an exosome, and a specific aptamer oligonucleotide that recognized a target protein of the exosome; the proximal binding of pairs of PLA probes to the same exosome positioned the oligonucleotides in the vicinity of each other, guiding the hybridization and ligation of two subsequently added backbone and connector oligonucleotides to form a circular DNA molecule. Circular DNA formed from PLA underwent rolling circle amplification (RCA) for signal amplification, and the resulting RCA products were subsequently quantified by TR-FRET. The limits of detection provided by APPROACH for the exosomal biomarkers CD63, PD-L1, and HER2 were 0.46 ng∙μL⁻¹, 0.77 ng∙μL⁻¹, and 1.1 ng∙μL⁻¹, respectively, demonstrating excellent analytical performance with high sensitivity and quantification accuracy. Furthermore, the strategy afforded sensitive detection of exosomal CD63 with a LOD of 1.56 ng∙μL⁻¹ in complex biological matrices, which underscored its anti-interference capability and potential for in vitro detection. The proposed strategy demonstrates wide-ranging applicability in quantifying diverse exosomal biomarkers while exhibiting robust analytical characteristics, including high sensitivity and accuracy. Full article

Fragile X syndrome (FXS) is the most common heritable cause of intellectual disability and autism spectrum disorder. The syndrome is often caused by greatly reduced or absent protein expression from the fragile X messenger ribonucleoprotein 1 (FMR1) gene due to expansion of a 5′-non-coding trinucleotide (CGG) element beyond 200 repeats (full mutation). To better understand the complex relationships among FMR1 allelotype, methylation status, mRNA expression, and FMR1 protein (FMRP) levels, FMRP was quantified in peripheral blood mononuclear cells for a large cohort of FXS (n = 154) and control (n = 139) individuals using time-resolved fluorescence resonance energy transfer. Considerable size and methylation mosaicism were observed among individuals with FXS, with FMRP detected only in the presence of such mosaicism. No sample with a minimum allele size greater than 273 CGG repeats had significant levels of FMRP. Additionally, an association was observed between FMR1 mRNA and FMRP levels in FXS samples, predominantly driven by those with the lowest FMRP values. This study underscores the complexity of FMR1 allelotypes and FMRP expression and prompts a reevaluation of FXS therapies aimed at reactivating large full mutation alleles that are likely not capable of producing sufficient FMRP to improve cognitive function. Full article

To enhance the specificity and sensitivity, cut the cost, and realize joint detection of multiple indicators, an immunoassay system based on the technology of time-resolved fluorescence resonance energy transfer (TR-FRET) was studied. Due to the FRET of the reagent, the donor probe and acceptor probe emitted specific fluorescence to enhance specificity. Long-lifetime specific fluorescence from the acceptor probe was combined with time-resolved technology to enhance sensitivity. A xenon flash lamp and a photomultiplier tube (PMT) were selected as the light source and detector, respectively. A filter-switching mechanism was placed in the light path, so the fluorescence signal from the donor and acceptor was measured alternately. The instrument’s design is given, and some specificI parts are described in detail. Key technical specifications of the instrument and procalcitonin (PCT), C-reactive protein (CRP), and interleukin-6(IL-6) were tested, and the test results were presented subsequently. The CV value of the self-designed counting module is better than 0.01%, and the instrument noises for 620 nm and 665 nm are 41.44 and 10.59, respectively. When set at 37 °C, the temperature bias (B) is 0.06 °C, and the temperature fluctuation is 0.10 °C. The CV and bias are between ±3% and 5%, respectively, when pipetting volumes are between 10 μL and 100 μL. Within the concentration range of 0.01 nM to 10 nM, the luminescence values exhibit linear regression correlation coefficients greater than 0.999. For PCT detection, when the concentration ranges from 0.02 ng/mL to 50 ng/mL, the correlation coefficient of linear fitting exceeds 0.999, and the limit of quantification is 0.096 ng/mL. For CRP and IL-6, the detection concentration ranges from 0 ng/mL to 500 ng/mL and 0 ng/mL to 20 ng/mL, respectively, with limits of quantification of 2.70 ng/mL and 2.82 ng/mL, respectively. The experimental results confirm the feasibility of the technical and instrumental solutions. Full article

Edible berries such as the fruits of black chokeberry (Aronia melanocarpa (Michx.) Elliott) and bilberry (Vaccinium myrtillus L.) are considered to be rich in phenolic compounds, which are nowadays attracting great interest due to their promising health benefits. The main objective of our study was to investigate, for the first time, their inhibitory properties on Src tyrosine kinase activity, as this enzyme plays an important role in multiple cellular processes and is activated in both cancer and inflammatory cells. In hydroethanolic fruit extracts, 5.0–5.9% of total polyphenols were determined spectrophotometrically, including high amounts of hydroxycinnamic acid derivatives. HPLC analysis revealed that the black chokeberry and bilberry extracts contained 2.05 mg/g and 2.54 mg/g of chlorogenic acid, respectively. Using a time-resolved fluorescence resonance energy transfer (TR-FRET) assay, the extracts studied were found to have comparable inhibitory effects on Src tyrosine kinase, with IC₅₀ values of 366 µg/mL and 369 µg/mL, respectively. The results also indicated that chlorogenic acid contributes significantly to the observed effect. In addition, both fruit extracts exhibited antioxidant activity by scavenging DPPH and NO radicals with SC₅₀ values of 153–352 µg/mL. Our study suggested that black chokeberry and bilberry fruits may be beneficial in cancer and other inflammation-related diseases. Full article

Simufilam is a novel oral drug candidate in Phase 3 clinical trials for Alzheimer’s disease (AD) dementia. This small molecule binds an altered form of filamin A (FLNA) that occurs in AD. This drug action disrupts FLNA’s aberrant linkage to the α7 nicotinic acetylcholine receptor (α7nAChR), thereby blocking soluble amyloid beta_1–42 (Aβ₄₂)’s signaling via α7nAChR that hyperphosphorylates tau. Here, we aimed to clarify simufilam’s mechanism. We now show that simufilam reduced Aβ₄₂ binding to α7nAChR with a 10-picomolar IC₅₀ using time-resolved fluorescence resonance energy transfer (TR-FRET), a robust technology to detect highly sensitive molecular interactions. We also show that FLNA links to multiple inflammatory receptors in addition to Toll-like receptor 4 (TLR4) in postmortem human AD brains and in AD transgenic mice: TLR2, C-X-C chemokine receptor type 4 (CXCR4), C-C chemokine receptor type 5 (CCR5), and T-cell co-receptor cluster of differentiation 4 (CD4). These aberrant FLNA linkages, which can be induced in a healthy control brain by Aβ₄₂ incubation, were disrupted by simufilam. Simufilam reduced inflammatory cytokine release from Aβ₄₂-stimulated human astrocytes. In the AD transgenic mice, CCR5–G protein coupling was elevated, indicating persistent activation. Oral simufilam reduced both the FLNA–CCR5 linkage and the CCR5–G protein coupling in these mice, while restoring CCR5′s responsivity to C-C chemokine ligand 3 (CCL3). By disrupting aberrant FLNA–receptor interactions critical to AD pathogenic pathways, simufilam may promote brain health. Full article

SARS-CoV-2 exploits the homotrimer transmembrane Spike glycoproteins (S protein) during host cell invasion. The Omicron XBB subvariant, delta, and prototype SARS-CoV-2 receptor-binding domain show similar binding strength to hACE2 (human Angiotensin-Converting Enzyme 2). Here we utilized multiligand virtual screening to identify small molecule inhibitors for their efficacy against SARS-CoV-2 virus using QPLD, pseudovirus ACE2 Inhibition -Time Resolved Forster/Fluorescence energy transfer (TR-FRET) Assay Screening, and Molecular Dynamics simulations (MDS). Three hundred and fifty thousand compounds were screened against the macrodomain of the nonstructural protein 3 of SARS-CoV-2. Using TR-FRET Assay, we filtered out two of 10 compounds that had no reported activity in in vitro screen against Spike S1: ACE2 binding assay. The percentage inhibition at 30 µM was found to be 79% for “Compound F1877-0839” and 69% for “Compound F0470-0003”. This first of its kind study identified “FILLY” pocket in macrodomains. Our 200 ns MDS revealed stable binding poses of both leads. They can be used for further development of preclinical candidates. Full article

Human immunodeficiency virus-1 (HIV-1) transactivator (Tat)-mediated transcription is essential for HIV-1 replication. It is determined by the interaction between Tat and transactivation response (TAR) RNA, a highly conserved process representing a prominent therapeutic target against HIV-1 replication. However, owing to the limitations of current high-throughput screening (HTS) assays, no drug that disrupts the Tat-TAR RNA interaction has been uncovered yet. We designed a homogenous (mix-and-read) time-resolved fluorescence resonance energy transfer (TR-FRET) assay using europium cryptate as a fluorescence donor. It was optimized by evaluating different probing systems for Tat-derived peptides or TAR RNA. The specificity of the optimal assay was validated by mutants of the Tat-derived peptides and TAR RNA fragment, individually and by competitive inhibition with known TAR RNA-binding peptides. The assay generated a constant Tat-TAR RNA interaction signal, discriminating the compounds that disrupted the interaction. Combined with a functional assay, the TR-FRET assay identified two small molecules (460-G06 and 463-H08) capable of inhibiting Tat activity and HIV-1 infection from a large-scale compound library. The simplicity, ease of operation, and rapidity of our assay render it suitable for HTS to identify Tat-TAR RNA interaction inhibitors. The identified compounds may also act as potent molecular scaffolds for developing a new HIV-1 drug class. Full article

The main objective of this research was to develop novel compounds from readily accessed natural products especially eugenol with potential biological activity. Eugenol, the principal chemical constituent of clove (Eugenia caryophyllata) from the family Myrtaceae is renowned for its pharmacological properties, which include analgesic, antidiabetic, antioxidant, anticancer, and anti-inflammatory effects. According to reports, PPARγ regulates inflammatory reactions. The synthesized compounds were structurally analyzed using FT-IR, ¹HNMR, ¹³CNMR, and mass spectroscopy techniques. Molecular docking was performed to analyze binding free energy and important amino acids involved in the interaction between synthesized derivatives and the target protein. The development of the structure–activity relationship is based on computational studies. Additionally, the stability of the best-docked protein–ligand complexes was assessed using molecular dynamic modeling. The in-vitro PPARγ competitive binding Lanthascreen TR-FRET assay was used to confirm the affinity of compounds to the target protein. All the synthesized derivatives were evaluated for an in vitro anti-inflammatory activity using an albumin denaturation assay and HRBC membrane stabilization at varying concentrations from 6.25 to 400 µM. In this background, with the aid of computational research, we were able to design six novel derivatives of eugenol synthesized, analyzed, and utilized TR-FRET competitive binding assay to screen them for their ability to bind PPARγ. Anti-inflammatory activity evaluation through in vitro albumin denaturation and HRBC method revealed that 1f exhibits maximum inhibition of heat-induced albumin denaturation at 50% and 85% protection against HRBC lysis at 200 and 400 µM, respectively. Overall, we found novel derivatives of eugenol that could potentially reduce inflammation by PPARγ agonism. Full article

KcapTR488 is a dual-fluorophore peptide sensor for the real-time reporting of programmed cell death by fluorescence imaging. KcapTR488 contains a nuclear localization sequence (NLS) conjugated with Texas Red, a caspase-cleavable sequence (DEVD), and a C-terminus conjugated to Alexa Fluor 488 (AF488). The synthesis and preliminary evaluation in cellulo of KcapTR488 for monitoring cell death by fluorescence imaging has been previously reported, but its utility in vivo has yet to be tested or validated. Herein, in vitro solution experiments verified the intramolecular fluorescence resonance energy transfer (FRET) between the two fluorophores and enabled a quantitative analysis of enzyme rates and selectivity. The sensor delivery kinetics in live rat models were quantified by ex vivo fluorescence microscopy. Studies in healthy control retinas demonstrated that KcapTR488 concentrated in the nucleus of retinal ganglion cells (RGC), with a strong colocalization of red and green fluorescence signals producing robust FRET signals, indicating an intact reporter. By contrast, using an acute but mild NMDA-induced retinal injury model, dual-color confocal ex vivo microscopy of cleaved KcapTR488 identified sensor activation as early as 2 h after injection. Quantitative changes in fluorescence colocalization were superior to changes in FRET for monitoring injury progression. Longitudinal monitoring revealed that the NLS-Texas Red fragment of the cleaved sensor moved out of the cell body, down the axon, and exited the retina, consistent with anterograde axonal transport. Thus, KcapTR488 may be a powerful tool to study RGC death pathways in live preclinical models of glaucoma. Full article