Fluorescence Polarization Research Articles

Advances in the Development of Non-Structural Protein 1 (NsP1) Inhibitors for the Treatment of Chikungunya Virus Infection.

Chikungunya is a re-emerging viral infection of worldwide concern, and new antiviral therapeutics are necessary to combat this disease. Inhibitors of the non-structural protein 1 (NsP1), which shows Methyltransferase (MTase) activity and plays a crucial in the Chikungunya virus (ChikV) replication, are exhibiting promising results. This review aimed to describe recent advances in the development of NsP1 inhibitors for the treatment of Chikungunya disease. High-throughput screening of novel ChikV NsP1 inhibitors has been widely performed for the identification of new molecule hits through fluorescence polarization, Western blotting, ELISA-based assay, and capillary electrophoresis assays. Additionally, cell-based assays confirmed that the inhibition of ChikV NsP1 abolishes viral replication. In summary, pyrimidine and pyrimidin-7(6H)-one derivatives, GTP and nucleoside analogs have been demonstrated to show inhibitory activity and are considered promising scaffolds that provide useful knowledge for the research and development of new NsP1 inhibitors as potential treatment of Chikungunya re-emerging disease.

Discovery of KPT-6566 as STAG1/2 Inhibitor sensitizing PARP and NHEJ Inhibitors to suppress tumor cells growth in vitro

Stromal antigen 1 and 2 (STAG1 and STAG2) are two mutually exclusive components of the cohesin complex that is crucial for centromeric and telomeric cohesion. Beyond its structural role, STAG2 also plays a pivotal role in homologous recombination (HR) repair and has emerged as a promising therapeutic target in cancer treatment. Here, we employed a fluorescence polarization (FP)-based high-throughput screening and identified KPT-6566 as a dual inhibitor of STAG1 and STAG2. Biochemical and biophysical analyses demonstrated that KPT-6566 directly binds to STAG1 and STAG2, disrupting their interactions with SCC1 and double-stranded DNA. A metaphase chromosome spread assay showed that KPT-6566 causes premature chromosome separation and induces chromosome damages in HeLa cells. Furthermore, KPT-6566 also impairs DNA damage repair, leading to the accumulation of double-strand breaks and cell apoptosis. Finally, KPT-6566 can sensitize HeLa and HepG2 cells to PARP inhibitor Olaparib and the NHEJ inhibitor UMI-77, exhibiting a synergistic effect in suppressing cell proliferation. Our findings highlight the potential of STAG1/2 as promising therapeutic targets in cancer treatment, particularly when they are targeted in combination with other DNA damage response inhibitors.

PROTAC Beyond Cancer- Exploring the New Therapeutic Potential of Proteolysis Targeting Chimeras.

In the realm of oncology, the transformative impact of PROTAC (PROteolysis TAgeting Chimeras) technology has been particularly pronounced since its introduction in the 21st century. Initially conceived for cancer treatment, PROTACs have evolved beyond their primary scope, attracting increasing interest in addressing a diverse array of medical conditions. This expanded focus includes not only oncological disorders but also viral infections, bacterial ailments, immune dysregulation, neurodegenerative conditions, and metabolic disorders. This comprehensive review explores the broadening landscape of PROTAC application, highlighting ongoing developments and innovations aimed at deploying these molecules across a spectrum of diseases. Careful consideration of the design challenges associated with PROTACs reveals that, when appropriately addressed, these compounds present significant advantages over traditional therapeutic approaches, positioning them as promising alternatives. To evaluate the efficacy of PROTAC molecules, a diverse array of assays is employed, ranging from High-Throughput Imaging (HTI) assays to Cell Painting assays, CRBN engagement assays, Fluorescence Polarization assays, amplified luminescent proximity homogeneous assays, Timeresolved fluorescence energy transfer assays, and Isothermal Titration Calorimetry assays. These assessments collectively contribute to a nuanced understanding of PROTAC performance. Looking ahead, the trajectory of PROTAC technology suggests its potential recognition as a versatile therapeutic strategy for an expansive range of medical conditions. Ongoing progress in this field sets the stage for PROTACs to emerge as valuable tools in the multifaceted landscape of medical treatments.

Anisotropic Fluorescence Surface Quenching of Quantum Dot-in-Rods on Silver Nanopatterned Film

Semiconductor quantum dot-in-rods (QDIRs) have served in the broad applications such as luminescent applications, sensor technology, quantum information processing, due to their polarized emission and polarized absorption characteristics. The localized surface plasmon resonance (LSPR) characteristics of the noble metal nanostructures can regulate the fluorescence intensity by controlling the radiation and non-radiation channels of fluorescent substances. However, the reports on the fluorescence surface quenching of QDIRs by plasmonic nanoparticles (NPs) are very rare. Herein, we propose a new method to realize QDIRs orientation, as well as the fluorescence intensity can be regulated. The effect of Ag nanopatterned film on the fluorescence surface quenching of QDIRs is discussed from both theoretical and experimental perspectives. Generally, the direct combination of Ag nanopatterned film and QDIRs results in 75% fluorescence surface quenching. Through FDTD simulation, we further analyzed the modulation of fluorescence intensity of the dipole source by the Ag nanopatterned film. This work will be useful for the development of QDIRs in the future.

Studying Exoribonuclease Activity Using Fluorescence Anisotropy Assay.

Fluorescence anisotropy is a powerful technique, widely used for investigating ligand-macromolecule binding and high-throughput screens for drugs. Here, we employ fluorescence anisotropy to quantitatively study the activity of exoribonucleases exemplified by the Xrn2 enzyme. Recording changes in the fluorescence anisotropy over time allows real-time detection of enzymatic activity and provides a framework that can be tailored to particular questions. We discuss the experimental setup, the potential substrate RNAs and highlight data analysis. We envision that this assay can be applied to study other nucleic acid-degrading enzymes and further expanded to include competition and inhibitor screens.

Computational Model-Assisted Development of a Nonenzymatic Fluorescent Glucose-Sensing Assay.

Deep-red fluorescence was implemented in this fully injectable, nonenzymatic glucose biosensor design to allow for better light penetration through the skin, particularly for darker skin tones. In this work, a novel method was developed to synthesize Cy5.5 labeled mannose conjugates (Cy5.5-mannobiose, Cy5.5-mannotriose, and Cy5.5-mannotetraose) to act as the fluorescent competing ligand in a competitive binding assay with the protein Concanavalin A acting as the recognition molecule. Using fluorescence anisotropy (FA) data, a computational model was developed to determine optimal concentration ratios of the assay components to allow for sensitive glucose measurements within the physiological range. The model was experimentally validated by measuring the glucose response via FA of the three Cy5.5-labeled mannose conjugates synthesized with Cy5.5-mannotetraose demonstrating the most sensitive response to glucose across the physiological range. The developed method may be broadly applied to a vast range of commercially available fluorescent dyes and opens up opportunities for glucose measurements using nonenzymatic assays.

Effect of Hyaluronic Acid on the Activity of Methylene Blue in Photogeneration of 1O2

The effect of a natural polysaccharide (hyaluronic acid (HA)) on the photocatalytic activity of methylene blue (MB) was studied both under model conditions (a tryptophan photooxidation reaction in water) and with in vitro experiments on P. aeruginosa and S. aureus bacterial cultures. It was shown spectrophotometrically that, in the presence of HA, an increase in the optical density of the absorption bands λ = 665 nm and 620 nm—which correspond to the monomeric and dimeric forms of the dye, respectively—was observed in the EAS of the dye, while the ratio of the optical density of these bands remained practically unchanged. When adding HA to MB, the intensity of singlet oxygen 1O2 photoluminescence and the degree of fluorescence polarization of MB increase. The observed effects are associated with the disaggregation of molecular associates of the dye in the presence of HA. The maximum increase in the photocatalytic activity of MB (by 1.6 times) was observed in the presence of HA, with concentrations in a range between 0.0015 wt.% and 0.005 wt.%.

Growth and spectral properties of Er3+/Yb3+/Ce3+:YPO4 crystal

Er3+/Yb3+/Ce3+ tir-doped YPO4 crystals were successfully synthesized by high temperature solution method with Pb2P2O7 as flux. The growth, structures and spectral characterizations, including polarization absorption spectra, polarization fluorescence spectra and lifetime, were systematically discussed. It has broad absorption band around 973 nm with a full width at half maximum of 20.76 nm and 14.71 nm for π- and σ-polarizations, respectively, which is suitable for efficient pumping of InGaAs laser diodes. Based on J-O theory, spectral parameters such as oscillator strength, line strength, J-O intensity, fluorescence branching ratio, spontaneous transition rate and radiation lifetime were calculated systematically. In the polarization fluorescence spectra, four strong emission peaks were observed at 1505, 1537, 1561 and 1586 nm. The results show that Er3+/Yb3+/Ce3+:YPO4 crystal may be a promising laser crystal suitable for 1.5–1.6 μm.

Glycocalyx Interactions Modulate the Cellular Uptake of Albumin-Coated Nanoparticles.

Albumin-based nanoparticles (ABNPs) represent promising drug carriers in nanomedicine due to their versatility and biocompatibility, but optimizing their effectiveness in drug delivery requires understanding their interactions with and uptake by cells. Notably, albumin interacts with the cellular glycocalyx, a phenomenon particularly studied in endothelial cells. This observation suggests that the glycocalyx could modulate ABNP uptake and therapeutic efficacy, although this possibility remains unrecognized. In this study, we elucidate the critical role of the glycocalyx in the cellular uptake of a model ABNP system consisting of silica nanoparticles (NPs) coated with native, cationic, and anionic albumin variants (BSA, BSA+, and BSA-). Using various methodologies-including fluorescence anisotropy, dynamic light scattering, microscale thermophoresis, surface plasmon resonance spectroscopy, and computer simulations─we found that both BSA and BSA+, but not BSA-, interact with heparin, a model glycosaminoglycan (GAG). To explore the influence of albumin-GAG interactions on NP uptake, we performed comparative uptake studies in wild-type and GAG-mutated Chinese hamster ovary cells (CHO), along with complementary approaches such as enzymatic GAG cleavage in wild-type cells, chemical inhibition, and competition assays with exogenous heparin. We found that the glycocalyx enhances the cell uptake of NPs coated with BSA and BSA+, while serving as a barrier to the uptake of NPs coated with BSA-. Furthermore, we showed that harnessing albumin-GAG interactions increases cancer cell death induced by paclitaxel-loaded albumin-coated NPs. These findings underscore the importance of albumin-glycocalyx interactions in the rational design and optimization of albumin-based drug delivery systems.

Novel antimicrobial coating for hernia meshes.

Antibiotic coating for several medical devices has been carried out; however, there are only few studies about coating hernia meshes with antimicrobial substances. In this study we checked the capacity of different commercially available hernia meshes to act as drug carrier. The meshes were coated with gentamicin palmitate, chlorhexidine palmitic acid and chlorhexidine palmitate. The coating mass and subsequent in vitro delivery rate were evaluated for gentamicin palmitate by fluorescence polarization. For Chlorhexidine coated devices the coating mass was determined by weighing. The in vitro delivery rate was determined by UV absorption (255 nm). The interaction of each mesh to the different coating substances was observed by scanning electron microscopy. 1. Certain uniformity was observed on the quantity of chlorhexidine coating the surface of each mesh used when compared with gentamicin palmitate coating. 2.We did not detect significant difference between the amounts of gentamicin palmitate released from each mesh. 3. The release of chlorhexidine palmitate and chlorhexidine palmitic acid from UltraPro™ and Mersilene™ were significantly higher (p<0.05) in comparison with the other two meshes. 4. The coating substances covered the surface of the fibers without damaging its structure. 5. The coating substances were distributed all along the fibers in all samples. We suggest the use of chlorhexidine palmitate and chlorhexidine palmitic acid, as well as gentamicin palmitate, for coating of hernia meshes aiming prevention of infections. Further investigation of the bactericidal effect of coated hernia meshes against biofilm form of S. aureus and other device-related infections is suggested.

Fluorescence spatial anisotropy of emission dipoles in an orthogonal imaging system

In this study, we explore the relatively unexplored spatially anisotropic fluorescence emission induced by rotationally polarized excitation light in orthogonal imaging systems, a phenomenon that is particularly pronounced in orthogonal setups compared to coaxial configurations. Despite its significance, this aspect has been largely neglected, given the prevalent use of coaxial Fluorescence Polarization Microscope (FPM) setups. Our research endeavors to bridge this gap by formulating a physical model to investigate the polarization-dependent emission of dipoles and the corresponding fluorescence signal within an orthogonal imaging configuration. Additionally, we introduce the Fluorescence Spatial Anisotropy Index (FSAI) to quantify fluorescence spatial anisotropy.

Fluorescence scattering under the Stokes–Mueller formalism determines the orientational distribution of fluorophores and the nature of optically active biological proteins

Abstract In the current paper, we represent intrinsic fluorescence anisotropies as four-dimensional normalized Stokes vectors defined by the maximum excitation and emission in the fluorescence process with respect to linear, linear-45 and circular polarizations of light. Depending upon the transition moments for absorption/excitation and emission of fluorophores, eight types of these Stokes vectors can be realized from the Mueller fluorescence matrix of the system. These Stokes vectors probe the orientational distribution of fluorophores and predict the nature of optically active biological proteins, whether laevorotatory or dextrorotatory. The orthogonality relation between the Stokes vectors corresponding to the excitation and emission processes of fluorescence connects the molecular ground and excited states of biological and non-biological systems.

Anisotropic Laser Performance in an Optically Isotropic Crystal by Phonon Engineering

Symmetry is an eternal motif in understanding the characteristics of nearly all the laws and courses of science. The symmetry‐breaking in the optical crystal will generate a rich variety of unprecedented physical phenomena and create new functional properties. Herein, the symmetry‐breaking in a photon–phonon collaboratively pumped laser, dominated by the anisotropic lattice vibrations is investigated. Using an optically isotropic Nd:YAG crystal as an example, the phonon‐assisted electronic transitions accompanied by anisotropic fluorescence emission are observed, beyond the intrinsic structural symmetry ruled by Neumann's principle. For the first time, the phonon‐assisted new‐wavelength lasers at 1151 and 1166 nm are achieved in Nd:YAG with divergent light polarization, determined by the vibrational direction of involved phonons. These results provide a flexible degree of freedom for photonics by phonon engineering and pave the way to new frontiers in the field of laser generation and manipulation.

Two conserved arginine residues facilitate C-S bond cleavage and persulfide transfer in Suf family cysteine desulfurases.

Under conditions of oxidative stress or iron starvation, iron-sulfur cluster biogenesis in E. coli is initiated by the cysteine desulfurase, SufS, via the SUF pathway. SufS is a type II cysteine desulfurase that catalyzes the PLP-dependent breakage of an L-cysteine C-S bond to generate L-alanine and a covalent active site persulfide as products. The persulfide is transferred from SufS to SufE and then to the SufBC2D complex, which utilizes it in iron-sulfur cluster biogenesis. Several lines of evidence suggest two conserved arginine residues that line the solvent side of the SufS active site could be important for function. To investigate the mechanistic roles of R56 and R359, the residues were substituted using site-directed mutagenesis to obtain R56A/K and R359A/K SufS variants. Steady state kinetics indicated R56 and R359 have moderate defects in the desulfurase half reaction but major defects in the transpersulfurase step. Fluorescence polarization binding assays showed that the loss of activity was not due to a defect in forming the SufS/SufE complex. Structural characterization of R56A SufS shows loss of electron density for the α3-α4 loop at the R56/G57 positions, consistent with a requirement of R56 for proper loop conformation. The structure of R359A SufS exhibits a conformational change in the α3-α4 loop allowing R56 to enter the active site and mimics the residue's position in the PLP-cysteine aldimine structure. Taken together, the kinetic, binding, and structural data support a mechanism where R359 plays a role in linking SufS catalysis with modulation of the α3-α4 loop to promote a close-approach interaction of SufS and SufE conducive to persulfide transfer.

Illuminating cellular architecture and dynamics with fluorescence polarization microscopy.

Ever since Robert Hooke's 17th century discovery of the cell using a humble compound microscope, light-matter interactions have continuously redefined our understanding of cell biology. Fluorescence microscopy has been particularly transformative and remains an indispensable tool for many cell biologists. The subcellular localization of biomolecules is now routinely visualized simply by manipulating the wavelength of light. Fluorescence polarization microscopy (FPM) extends these capabilities by exploiting another optical property - polarization - allowing researchers to measure not only the location of molecules, but also their organization or alignment within larger cellular structures. With only minor modifications to an existing fluorescence microscope, FPM can reveal the nanoscale architecture, orientational dynamics, conformational changes and interactions of fluorescently labeled molecules in their native cellular environments. Importantly, FPM excels at imaging systems that are challenging to study through traditional structural approaches, such as membranes, membrane proteins, cytoskeletal networks and large macromolecular complexes. In this Review, we discuss key discoveries enabled by FPM, compare and contrast the most common optical setups for FPM, and provide a theoretical and practical framework for researchers to apply this technique to their own research questions.

Recurrent Neurodevelopmentally Associated Variants of the Pre-mRNA Splicing Factor U2AF2 Alter RNA Binding Affinities and Interactions.

De novo mutations affecting the pre-mRNA splicing factor U2AF2 are associated with developmental delays and intellectual disabilities, yet the molecular basis is unknown. Here, we demonstrated by fluorescence anisotropy RNA binding assays that recurrent missense mutants (Arg149Trp, Arg150His, or Arg150Cys) decreased the binding affinity of U2AF2 for a consensus splice site RNA. Crystal structures at 1.4 Å resolutions showed that Arg149Trp or Arg150His disrupted hydrogen bonds between U2AF2 and the terminal nucleotides of the RNA site. Reanalysis of publicly available RNaseq data confirmed that U2AF2 depletion altered splicing of transcripts encoding RNA binding proteins (RBPs). These results confirmed that the impaired RNA interactions of Arg149Trp and Arg150His U2AF2 variants could contribute to dysregulating an RBP-governed neurodevelopmental program of alternative splicing.

Fluorescence Polarization Assay for Infection Diagnostics: A Review

Rapid and specific diagnosis is necessary for both the treatment and prevention of infectious diseases. Bacteria and viruses that enter the bloodstream can trigger a strong immune response in infected animals and humans. The fluorescence polarization assay (FPA) is a rapid and accurate method for detecting specific antibodies in the blood that are produced in response to infection. One of the first examples of FPA is the non-competitive test for detecting brucellosis in animals, which was followed by the development of other protocols for detecting various infections. Fluorescently labeled polysaccharides (in the case of brucellosis and salmonellosis) or specific peptides (in the case of tuberculosis and salmonellosis, etc.) can be used as biorecognition elements for detecting infections. The availability of new laboratory equipment and mobile devices for fluorescence polarization measurements outside the laboratory has stimulated the development of new fluorescence polarization assays (FPAs) and the emergence of commercial kits on the market for the detection of brucellosis, tuberculosis, and equine infectious anemia viruses. It has been shown that, in addition to antibodies, the FPA method can detect both viruses and nucleic acids. The development of more specific and sensitive biomarkers is essential for the diagnosis of infections and therapy monitoring. This review summarizes studies published between 2003 and 2023 that focus on the detection of infections using FPA. Furthermore, it demonstrates the potential for using new biorecognition elements (e.g., aptamers, proteins, peptides) and the combined use of FPA with new technologies, such as PCR and CRISPR/Cas12a systems, for detecting various infectious agents.

Fluorescent Labeling Can Significantly Perturb Measured Binding Affinity and Selectivity of Peptide-Protein Interactions.

Peptide-based drugs are powerful inhibitors of therapeutically relevant protein-protein interactions. Their affinity and selectivity for target proteins are commonly assessed using fluorescence-based assays such as anisotropy/polarization or quantitative microarrays. This study reveals that labeling can perturb peptide/protein binding by more than 1 order of magnitude. We have recently developed inhibitors targeted to the N-terminal Src homology 2 (SH2) domain of oncogenic phosphatase SHP2. Despite their high activity and selectivity, these molecules demonstrated an undesired interaction with the SH2 domain of another protein, known as APS, in a fluorescence microarray assay. Fluorescence anisotropy measurement in solution showed that the dissociation constant was significantly influenced by labeling (∼10 times), and the effect depended on the specific fluorophore and SH2 domain. Notably, displacement assays performed with unlabeled peptides were successfully used to eliminate these artifacts, demonstrating that the inhibitors' affinity for their target is over 1,000 times higher than for APS.

Probing the Closed Association of Oligoquinoline Foldamers by Time-Resolved Fluorescence Anisotropy.

The metal-mediated dimerization of oligoquinoline foldamers terminated at one end with an oligo(phenylenevinylene) and at the other with a carboxylic acid (OPV-QnA, where n = 4, 8, 17, and 33), and the complexation of OPV-Q8A and Q16A was promoted in chloroform by the addition of a concentrated 16 M aqueous sodium hydroxide solution. UV-vis absorption and time-resolved fluorescence anisotropy (TRFA) experiments were conducted to determine, respectively, the concentration and the average rotational time ⟨ϕ⟩ of the mixture of unassociated and associated foldamers across a range of foldamer concentrations spanning 5 orders of magnitude. Plots of ⟨ϕ⟩ as a function of acid group concentration revealed that ⟨ϕ⟩ increased with increasing foldamer concentration only when the foldamer solution in chloroform was vigorously mixed with the 16 M sodium hydroxide aqueous solution. Furthermore, all plots showed that ⟨ϕ⟩ reached a plateau at high foldamer concentration. The increase in ⟨ϕ⟩ reflected the association of foldamers into larger objects through metal ion coordination with the carboxylate anions generated by deprotonation of the carboxylic acid of OPV-QnA with NaOH, while the plateau obtained at high foldamer concentration indicated that these interactions led to the dimerization of the foldamers via a closed association mechanism. Analysis of the ⟨ϕ⟩ trends yielded the equilibrium constants (K) describing the foldamer dimerization, whose value equaled 1.0 (±0.2) × 106 M-1 for the three longer OPV-QnA foldamers, but was about 10 times smaller for the shortest one (n = 4). Association of OPV-Q8A and Q16A yielded a complex with a ⟨ϕ⟩ matching that of OPV-Q24A, and K for this complexation was similar to that for dimerization. These experiments illustrate the robust nature of TRFA as an experimental method to probe the size of rigid, self-assembled foldamers in solution.

High Affinity Inhibitors of the Macrophage Infectivity Potentiator Protein from Trypanosoma cruzi, Burkholderia pseudomallei, and Legionella pneumophila─A Comparison.

Since Chagas disease, melioidosis, and Legionnaires' disease are all potentially life-threatening infections, there is an urgent need for new treatment strategies. All causative agents, Trypanosoma cruzi, Burkholderia pseudomallei, and Legionella pneumophila, express a virulence factor, the macrophage infectivity potentiator (MIP) protein, emerging as a promising new therapeutic target. Inhibition of MIP proteins having a peptidyl-prolyl isomerase activity leads to reduced viability, proliferation, and cell invasion. The affinity of a series of pipecolic acid-type MIP inhibitors was evaluated against all MIPs using a fluorescence polarization assay. The analysis of structure-activity relationships led to highly active inhibitors of MIPs of all pathogens, characterized by a one-digit nanomolar affinity for the MIPs and a very effective inhibition of their peptidyl-prolyl isomerase activity. Docking studies, molecular dynamics simulations, and quantum mechanical calculations suggest an extended σ-hole of the meta-halogenated phenyl sulfonamide to be responsible for the high affinity.