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REV7 is a HORMA (H op1, R ev7, M ad2) family adaptor protein best known as an accessory subunit of the translesion synthesis (TLS) DNA polymerase ζ (Polζ). In this role, REV7 binds REV3, the catalytic subunit of Polζ, by locking REV7-binding motifs (RBMs) in REV3 underneath the REV7 safety-belt loop. The same mechanism is used by REV7 to interact with RBMs from other proteins in DNA damage response (DDR) and mitosis. Because of the importance of REV7 for TLS and other DDR pathways, targeting REV7:RBM protein-protein interactions (PPIs) with small molecules has emerged as a strategy to enhance cancer response to genotoxic chemotherapy. To identify druggable pockets at the REV7:RBM interface, we performed computational analyses of REV7 complexed with several RBM partners. The contributions of different interface regions to REV7:RBM stabilization were corroborated experimentally. These studies provide insights into key intermolecular interactions and establish targetable regions of REV7 for the design of REV7:RBM PPI inhibitors. [Display omitted] • REV7 is a key adaptor protein in translesion synthesis, DNA repair, and cell-cycle control • REV7 binds partners by locking the REV7-binding motif underneath its safety belt region • REV7:RBM interaction hot-spots characterized that contribute most of the binding energy • Binding hot-spot analysis will aid targeting REV7 interactions for anti-cancer drug design Dash et al. utilized computational and biochemical studies to identify key amino acids in REV7 that govern binding of its partner proteins. The studies provide guidance for developing small molecules that can disrupt REV7 protein-protein interactions. [ABSTRACT FROM AUTHOR]

Transthyretin (TTR) and thyroxine-binding globulin (TBG) are two major thyroid hormone (TH) distributor proteins in human plasma, playing important roles in stabilizing the TH levels in plasma, delivery of TH to target tissues, and trans-barrier transport. Binding of xenobiotics to these distributor proteins can potentially affect all these three important roles of distributor proteins. Therefore, fast and cost-effective experimental methods are required for both TTR and TBG to screen both existing and new chemicals for their potential binding. In the present study, the TTR-binding assay was therefore simplified, optimized and pre-validated, while a new TBG-binding assay was developed based on fluorescence polarization as a readout. Seven model compounds (including positive and negative controls) were tested in the pre-validation study of the optimized TTR-binding assay and in the newly developed TBG-binding assay. The dissociation constants of the natural ligand (thyroxine, T4) and potential competitors were determined and compared between two distributor proteins, showing striking differences for perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA). [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

ABSTRACTDesmosomes are intercellular junctions that regulate mechanical integrity in epithelia and cardiac muscle. Dynamic desmosome remodeling is essential for wound healing and development, yet the mechanisms governing junction assembly remain elusive. While we and others have shown that cadherin ectodomains are highly organized, how this ordered architecture emerges during assembly is unknown. Using fluorescence polarization microscopy, we show that desmoglein 2 (Dsg2) ectodomain order gradually increases during 8 h of assembly, coinciding with increasing adhesive strength. In a scratch wound assay, we observed a similar increase in order in desmosomes assembling at the leading edge of migratory cells. Together, our findings indicate that cadherin organization is a hallmark of desmosome maturity and may play a role in conferring adhesive strength.

Abstract Fluorescence polarization (Fpol) imaging of methylene blue (MB) is a promising quantitative approach to thyroid cancer detection. Clinical translation of MB Fpol technology requires reduction of the data analysis time that can be achieved via deep learning-based automated cell segmentation with a 2D U-Net convolutional neural network. The model was trained and tested using images of pathologically diverse human thyroid cells and evaluated by comparing the number of cells selected, segmented areas, and Fpol values obtained using automated (AU) and manual (MA) data processing methods. Overall, the model segmented 15.8% more cells than the human operator. Differences in AU and MA segmented cell areas varied between − 55.2 and + 31.0%, whereas differences in Fpol values varied from − 20.7 and + 10.7%. No statistically significant differences between AU and MA derived Fpol data were observed. The largest differences in Fpol values correlated with greatest discrepancies in AU versus MA segmented cell areas. Time required for auto-processing was reduced to 10 s versus one hour required for MA data processing. Implementation of the automated cell analysis makes quantitative fluorescence polarization-based diagnosis clinically feasible.

Molecular targets for a promising antitumor agent based on trans-stilbene containing a pyridoxine fragment were identified. The lead compound, (E)-6-(3,4-dimethoxystyryl)-2,2,5,8-tetramethyl-4H-[1,3] dioxino[4,5-c]pyridine, was found to selectively induce apoptosis in MCF-7 breast adenocarcinoma cells overexpressing estrogen receptor, but not in MDA-MB-231 cells negative for estrogen receptor. The mechanism by which the novel trans-stilbene derivative acts as a selective estrogen receptor modulator was analyzed, and the affinity for human estrogen receptor α (ERα) was assessed by fluorescence polarization. Unlike its structural analogs—tamoxifen and raloxifene, the lead compound showed no affinity for ERα and did not form complexes with it. Therefore, it was concluded that the selective action of the pyridoxine-containing derivative of trans-stilbene on estrogen-positive breast cancer cells occurs through an alternative mechanism. The EC50 values for the displacement of the fluorescent ligand from the ERα active site were 22, 120, and 595 nM for estradiol, raloxifene, and tamoxifen, respectively.

The adenosine A2A receptor (A2AR) has been identified as a therapeutic target for treating neurodegenerative diseases and cancer. In recent years, we have highlighted the 2-aminoquinazoline heterocycle as an promising scaffold for designing new A2AR antagonists, exemplified by 6-bromo-4-(furan-2-yl)quinazolin-2-amine 1 (Ki (hA2AR) = 20 nM). Here, we report the synthesis of new 2-aminoquinazoline derivatives with substitutions at the C6- and C7-positions, and the introduction of aminoalkyl chains containing tertiary amines at the C2-position to enhance antagonist activity and solubility properties. Compound 5m showed a high affinity for hA2AR with a Ki value of 5 nM and demonstrated antagonist activity with an IC50 of 6 µM in a cyclic AMP assay. Introducing aminopentylpiperidine and 4-[(piperidin-1-yl)methyl]aniline substituents maintained the binding affinities (9x, Ki = 21 nM; 10d, Ki = 15 nM) and functional antagonist activities (9x, IC50 = 9 µM; 10d, IC50 = 5 µM) of the synthesized compounds while improving solubility. This study provides insights into the future development of A2AR antagonists for therapeutic applications. [ABSTRACT FROM AUTHOR]

Fluorescence polarization (Fpol) imaging of methylene blue (MB) is a promising quantitative approach to thyroid cancer detection. Clinical translation of MB Fpol technology requires reduction of the data analysis time that can be achieved via deep learning-based automated cell segmentation with a 2D U-Net convolutional neural network. The model was trained and tested using images of pathologically diverse human thyroid cells and evaluated by comparing the number of cells selected, segmented areas, and Fpol values obtained using automated (AU) and manual (MA) data processing methods. Overall, the model segmented 15.8% more cells than the human operator. Differences in AU and MA segmented cell areas varied between − 55.2 and + 31.0%, whereas differences in Fpol values varied from − 20.7 and + 10.7%. No statistically significant differences between AU and MA derived Fpol data were observed. The largest differences in Fpol values correlated with greatest discrepancies in AU versus MA segmented cell areas. Time required for auto-processing was reduced to 10 s versus one hour required for MA data processing. Implementation of the automated cell analysis makes quantitative fluorescence polarization-based diagnosis clinically feasible. [ABSTRACT FROM AUTHOR]

The beta-galactoside-binding mammalian lectin galectin-1 can bind, via its carbohydrate recognition domain (CRD), to various cell surface glycoproteins and has been implicated in a range of cancers. As a consequence of binding to sugar residues on cell surface receptors, it has been shown to have a pleiotropic effect across many cell types and mechanisms, resulting in immune system modulation and cancer progression. As a result, it has started to become a therapeutic target for both small and large molecules. In previous studies, we used fluorescence polarization (FP) assays to determine KD values to screen and triage small molecule glycomimetics that bind to the galectin-1 CRD. In this study, surface plasmon resonance (SPR) was used to compare human and mouse galectin-1 affinity measures with FP, as SPR has not been applied for compound screening against this galectin. Binding affinities for a selection of mono- and di-saccharides covering a 1000-fold range correlated well between FP and SPR assay formats for both human and mouse galectin-1. It was shown that slower dissociation drove the increased affinity at human galectin-1, whilst faster association was responsible for the effects in mouse galectin-1. This study demonstrates that SPR is a sound alternative to FP for early drug discovery screening and determining affinity estimates. Consequently, it also allows association and dissociation constants to be measured in a high-throughput manner for small molecule galectin-1 inhibitors. [ABSTRACT FROM AUTHOR]

Recently, geraniin has been identified as a potent antiviral agent targeting SARS-CoV-2 main protease (Mpro). Considering the potential of geraniin in COVID-19 treatment, a stringent validation for its Mpro inhibition is necessary. Herein, we rigorously evaluated the in vitro inhibitory effect of geraniin on Mpro using the fluorescence resonance energy transfer (FRET), fluorescence polarization (FP), and dimerization-dependent red fluorescent protein (ddRFP) assays. Our data indicate that geraniin is not a potential inhibitor against Mpro based on the results from a set of in vitro assays. These results suggest a stringent in vitro validation with diverse biochemical assays is essential for the discovery of Mpro inhibitors, and the fluorescence quenching effect caused by natural products should be considered when evaluating Mpro inhibitors. [ABSTRACT FROM AUTHOR]

Fluorescence induced by the excitation of a fluorophore with plane-polarized light has a different polarization depending on the size of the fluorophore-containing reagent and the rate of its rotation. Based on this effect, many analytical systems have been implemented in which an analyte contained in a sample and labeled with a fluorophore (usually fluorescein) competes to bind to antibodies. Replacing antibodies in such assays with aptamers, low-cost and stable oligonucleotide receptors, is complicated because binding a fluorophore to them causes a less significant change in the polarization of emissions. This work proposes and characterizes the compounds of the reaction medium that improve analyte binding and reduce the mobility of the aptamer–fluorophore complex, providing a higher analytical signal and a lower detection limit. This study was conducted on aflatoxin B1 (AFB1), a ubiquitous toxicant contaminating foods of plant origins. Eight aptamers specific to AFB1 with the same binding site and different regions stabilizing their structures were compared for affinity, based on which the aptamer with 38 nucleotides in length was selected. The polymers that interact reversibly with oligonucleotides, such as poly-L-lysine and polyethylene glycol, were tested. It was found that they provide the desired reduction in the depolarization of emitted light as well as high concentrations of magnesium cations. In the selected optimal medium, AFB1 detection reached a limit of 1 ng/mL, which was 12 times lower than in the tris buffer commonly used for anti-AFB1 aptamers. The assay time was 30 min. This method is suitable for controlling almond samples according to the maximum permissible levels of their contamination by AFB1. The proposed approach could be applied to improve other aptamer-based analytical systems. [ABSTRACT FROM AUTHOR]

The heightened transmissibility and capacity of African swine fever virus (ASFV) induce fatal diseases in domestic pigs and wild boars, posing significant economic repercussions and global threats. Despite extensive research efforts, the development of potent vaccines or treatments for ASFV remains a persistent challenge. Recently, inhibiting the AsfvPolX, a key DNA repair enzyme, emerges as a feasible strategy to disrupt viral replication and control ASFV infections. In this study, a comprehensive approach involving pharmacophore-based inhibitor screening, coupled with biochemical and biophysical analyses, were implemented to identify, characterize, and validate potential inhibitors targeting AsfvPolX. The constructed pharmacophore model, Phar-PolX-S, demonstrated efficacy in identifying a potent inhibitor, D-132 (IC50 = 2.8 ± 0.2 µM), disrupting the formation of the AsfvPolX-DNA complex. Notably, D-132 exhibited strong binding to AsfvPolX (KD = 6.9 ± 2.2 µM) through a slow-on-fast-off binding mechanism. Employing molecular modeling, it was elucidated that D-132 predominantly binds in-between the palm and finger domains of AsfvPolX, with crucial residues (R42, N48, Q98, E100, F102, and F116) identified as hotspots for structure-based inhibitor optimization. Distinctively characterized by a 1,2,5,6-tetrathiocane with modifications at the 3 and 8 positions involving ethanesulfonates, D-132 holds considerable promise as a lead compound for the development of innovative agents to combat ASFV infections.

Lysozyme is a well-known enzyme found in many biological fluids which plays an important role in the antibacterial protection of humans and animals. Lysozyme assays are used for the diagnosis of a number of diseases and utilized in immunohistochemistry, genetic and cellular engineering studies. The assaying methods are divided into two categories measuring either the concentration of lysozyme as a protein or its activity as an enzyme. While the first category of methods traditionally uses an enzyme-linked immunosorbent assay (ELISA), the methods for the determination of the enzymatic activity of lysozyme use either live bacteria, which is rather inconvenient, or natural peptidoglycans of high heterogeneity and variability, which leads to the low reproducibility of the assay results. In this work, we propose the use of a chemically synthesized substrate of a strictly defined structure to measure in a single experiment both the concentration of lysozyme as a protein and its enzymatic activity by means of the fluorescence polarization (FP) method. Chito-oligosaccharides of different chain lengths were fluorescently labeled and tested leading to the selection of the pentasaccharide as the optimal size tracer and the further optimization of the assay conditions for the accurate (detection limit 0.3 μM) and rapid (<30 min) determination of human lysozyme. The proposed protocol was applied to assay human lysozyme in tear samples and resulted in good correlation with the reference assay. The use of synthetic fluorescently labeled tracer, in contrast to natural peptidoglycan, in FP analysis allows for the development of a reproducible method for the determination of lysozyme activity. [ABSTRACT FROM AUTHOR]

A fluorescence polarization immunoassay method has been developed for the determination of antibiotic tylosin in honey. For this purpose, rabbits were immunized with a conjugate of tylosin and bovine serum albumin, and antisera were obtained. The conditions for the interaction of antisera in a competitive format with tylosin in the sample and with tylosin labeled with fluorescein were optimized. The developed method is characterized by a limit of detection for tylosin of 34.7 ng/mL and an analytical range of 65.2 to 564.0 ng/mL. High reproducibility of measurements using a portable detector was demonstrated, with relative standard deviation (RSD) values of 1.5–2.5%. The method was validated using honey samples, showing recovery ranging from 98 to 103.8%. An analysis time, excluding sample preparation, is 10 min. [ABSTRACT FROM AUTHOR]

CRISPR/Cas12a is a potent biosensing tool known for its high specificity in DNA analysis. Cas12a recognizes the target DNA and acquires nuclease activity toward single-stranded DNA (ssDNA) probes. We present a straightforward and versatile approach to transforming common Cas12a-cleavable DNA probes into enhancing tools for fluorescence anisotropy (FA) measurements. Our study involved investigating 13 ssDNA probes with linear and hairpin structures, each featuring fluorescein at one end and a rotation-slowing tool (anchor) at the other. All anchors induced FA changes compared to fluorescein, ranging from 24 to 110 mr. Significant FA increases (up to 180 mr) were obtained by adding divalent metal salts (Mg2+, Ca2+, Ba2+), which influenced the rigidity and compactness of the DNA probes. The specific Cas12a-based recognition of double-stranded DNA (dsDNA) fragments of the bacterial phytopathogen Erwinia amylovora allowed us to determine the optimal set (probe structure, anchor, concentration of divalent ion) for FA-based detection. The best sensitivity was obtained using a hairpin structure with dC10 in the loop and streptavidin located near the fluorescein at the stem in the presence of 100 mM Mg2+. The detection limit of the dsDNA target was equal to 0.8 pM, which was eight times more sensitive compared to the common fluorescence-based method. The enhancing set ensured detection of single cells of E. amylovora per reaction in an analysis based on CRISPR/Cas12a with recombinase polymerase amplification. Our approach is universal and easy to implement. Combining FA with Cas12a offers enhanced sensitivity and signal reliability and could be applied to different DNA and RNA analytes. [ABSTRACT FROM AUTHOR]

Cathepsin L (CTSL), a lysosomal cysteine proteinase, is primarily dedicated to the metabolic turnover of intracellular proteins. It is involved in various physiological processes and contributes to pathological conditions such as viral infection, tumor invasion and metastasis, inflammatory status, atherosclerosis, renal disease, diabetes, bone diseases, and other ailments. The coronavirus disease 2019 (COVID-19), with its rapid global spread and significant mortality, has been a worldwide epidemic since the late 2019s. Notably, CTSL plays a role in the processing of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein, providing a potential avenue to block coronavirus host cell entry and thereby inhibit SARS-CoV-2 infection in humans. In this study, we have developed a novel method using fluorescence polarization (FP) for screening CTSL inhibitors in a high-throughput format. The optimized assay demonstrated its appropriateness for high-throughput screening (HTS) with a Z-factor of 0.9 in a 96-well format. Additionally, the IC50 of the known inhibitor, Z-Phe-Tyr-CHO, was determined to be 188.50 ± 46.88 nM. Upon screening over 2000 small molecules, we identified, for the first time, the anti-CTSL properties of a benzothiazoles derivative named IMB 8015. This work presents a novel high-throughput approach and its application in discovering and evaluating CTSL inhibitors.

Chaperones of the heat shock protein 70 (Hsp70) family engage in protein-protein interactions with many cochaperones. One "hotspot" for cochaperone binding is the EEVD motif, found at the extreme C terminus of cytoplasmic Hsp70s. This motif is known to bind tetratricopeptide repeat domain cochaperones, such as the E3 ubiquitin ligase CHIP. In addition, the EEVD motif also interacts with a structurally distinct domain that is present in class B J-domain proteins, such as DnaJB4. These observations suggest that CHIP and DnaJB4 might compete for binding to Hsp70's EEVD motif; however, the molecular determinants of such competition are not clear. Using a collection of EEVD-derived peptides, including mutations and truncations, we explored which residues are critical for binding to both CHIP and DnaJB4. These results revealed that some features, such as the C-terminal carboxylate, are important for both interactions. However, CHIP and DnaJB4 also had unique preferences, especially at the isoleucine position immediately adjacent to the EEVD. Finally, we show that competition between these cochaperones is important in vitro, as DnaJB4 limits the ubiquitination activity of the Hsp70-CHIP complex, whereas CHIP suppresses the client refolding activity of the Hsp70-DnaJB4 complex. Together, these data suggest that the EEVD motif has evolved to support diverse protein-protein interactions, such that competition between cochaperones may help guide whether Hsp70-bound proteins are folded or degraded.

The adenosine A2A receptor (A2AR) has been identified as a therapeutic target for treating neurodegenerative diseases and cancer. In recent years, we have highlighted the 2-aminoquinazoline heterocycle as an promising scaffold for designing new A2AR antagonists, exemplified by 6-bromo-4-(furan-2-yl)quinazolin-2-amine 1 (Ki (hA2AR) = 20 nM). Here, we report the synthesis of new 2-aminoquinazoline derivatives with substitutions at the C6- and C7-positions, and the introduction of aminoalkyl chains containing tertiary amines at the C2-position to enhance antagonist activity and solubility properties. Compound 5m showed a high affinity for hA2AR with a Ki value of 5 nM and demonstrated antagonist activity with an IC50 of 6 µM in a cyclic AMP assay. Introducing aminopentylpiperidine and 4-[(piperidin-1-yl)methyl]aniline substituents maintained the binding affinities (9x, Ki = 21 nM; 10d, Ki = 15 nM) and functional antagonist activities (9x, IC50 = 9 µM; 10d, IC50 = 5 µM) of the synthesized compounds while improving solubility. This study provides insights into the future development of A2AR antagonists for therapeutic applications.

The beta-galactoside-binding mammalian lectin galectin-1 can bind, via its carbohydrate recognition domain (CRD), to various cell surface glycoproteins and has been implicated in a range of cancers. As a consequence of binding to sugar residues on cell surface receptors, it has been shown to have a pleiotropic effect across many cell types and mechanisms, resulting in immune system modulation and cancer progression. As a result, it has started to become a therapeutic target for both small and large molecules. In previous studies, we used fluorescence polarization (FP) assays to determine KD values to screen and triage small molecule glycomimetics that bind to the galectin-1 CRD. In this study, surface plasmon resonance (SPR) was used to compare human and mouse galectin-1 affinity measures with FP, as SPR has not been applied for compound screening against this galectin. Binding affinities for a selection of mono- and di-saccharides covering a 1000-fold range correlated well between FP and SPR assay formats for both human and mouse galectin-1. It was shown that slower dissociation drove the increased affinity at human galectin-1, whilst faster association was responsible for the effects in mouse galectin-1. This study demonstrates that SPR is a sound alternative to FP for early drug discovery screening and determining affinity estimates. Consequently, it also allows association and dissociation constants to be measured in a high-throughput manner for small molecule galectin-1 inhibitors.

Target-based discovery of first-in-class therapeutics demands an in-depth understanding of the molecular mechanisms underlying human diseases. Precise measurements of cellular and biochemical activities are critical to gain mechanistic knowledge of biomolecules and their altered function in disease conditions. Such measurements enable the development of intervention strategies for preventing or treating diseases by modulation of desired molecular processes. Fluorescence-based techniques are routinely employed for accurate and robust measurements of in-vitro activity of molecular targets and for discovering novel chemical molecules that modulate the activity of molecular targets. In the current review, the authors focus on the applications of fluorescence-based high throughput screening (HTS) and fragment-based ligand discovery (FBLD) techniques such as fluorescence polarization (FP), Förster resonance energy transfer (FRET), fluorescence thermal shift assay (FTSA) and microscale thermophoresis (MST) for the discovery of chemical probe to exploring target’s role in disease biology and ultimately, serve as a foundation for drug discovery. Some recent advancements in these techniques for compound library screening against important classes of drug targets, such as G-protein-coupled receptors (GPCRs) and GTPases, as well as phosphorylation- and acetylation-mediated protein-protein interactions, are discussed. Overall, this review presents a landscape of how these techniques paved the way for the discovery of small-molecule modulators and biologics against these targets for therapeutic benefits.

The high expression of BLM (Bloom syndrome) DNA helicase in tumors involves its strong association with cell expansion. Bisbenzylisoquinoline alkaloids own an antitumor property and have developed as candidates for anticancer drugs. This paper aimed to study the antitumor effect of fangchinoline derivative HY-2 by targeting BLM642–1290 DNA helicase, and then explore its inhibitory mechanism on proliferation of MDA-MB-435 breast cancer cells. We confirmed that the mRNA and protein levels of BLM DNA helicase in breast cancer were higher than those in normal tissues. HY-2 could inhibit the DNA binding, ATPase and DNA unwinding of BLM642–1290 DNA helicase with enzymatic assay. HY-2 could also inhibit the DNA unwinding of DNA helicase in cells. In addition, HY-2 showed an inhibiting the MDA-MB-435, MDA-MB-231, MDA-MB-436 breast cancer cells expansion. The mRNA and protein levels of BLM DNA helicase in MDA-MB-435 cells increased after HY-2 treatment, which might contribute to HY-2 inhibiting the DNA binding, ATPase and DNA unwinding of BLM DNA helicase. The mechanism of HY-2 inhibition on BLM DNA helicase was further confirmed with the effect of HY-2 on the ultraviolet spectrogram of BLM642–1290 DNA helicase and Molecular dynamics simulation of the interacting between HY-2 and BLM640–1291 DNA helicase. Our study provided some valuable clues for the exploration of HY-2 in the living body and developing it as an anticancer drug.

In eukaryotic organisms, genomic DNA associates with histone proteins to form nucleosomes. Nucleosomes provide a basis for genome compaction, epigenetic markup, and mediate interactions of nuclear proteins with their target DNA loci. A negatively charged (acidic) patch located on the H2A-H2B histone dimer is a characteristic feature of the nucleosomal surface. The acidic patch is a common site in the attachment of various chromatin proteins, including viral ones. Acidic patch-binding peptides present perspective compounds that can be used to modulate chromatin functioning by disrupting interactions of nucleosomes with natural proteins or alternatively targeting artificial moieties to the nucleosomes, which may be beneficial for the development of new therapeutics. In this work, we used several computational and experimental techniques to improve our understanding of how peptides may bind to the acidic patch and what are the consequences of their binding. Through extensive analysis of the PDB database, histone sequence analysis, and molecular dynamic simulations, we elucidated common binding patterns and key interactions that stabilize peptide–nucleosome complexes. Through MD simulations and FRET measurements, we characterized changes in nucleosome dynamics conferred by peptide binding. Using fluorescence polarization and gel electrophoresis, we evaluated the affinity and specificity of the LANA1-22 peptide to DNA and nucleosomes. Taken together, our study provides new insights into the different patterns of intermolecular interactions that can be employed by natural and designed peptides to bind to nucleosomes, and the effects of peptide binding on nucleosome dynamics and stability. [ABSTRACT FROM AUTHOR]

The fluidity and polar environment of ~100 nm hybrid vesicles combining dipalmitoylphosphatidylcholine (DPPC) and poly(1,2-butadiene)-block-polyethylene oxide (PBd-PEO, average molecular weight 950 g/mol) were studied upon vesicle heating using the fluorescence spectroscopy techniques of DPH anisotropy and laurdan generalized polarization (GP). These techniques indicated PBd-PEO membranes are less ordered than solid DPPC, but slightly more ordered than fluid DPPC or dioleoylphosphatidylcholine (DOPC) membranes. We find the DPH anisotropy values are less than expected from additivity of the components' anisotropies in the fluid phase mixture of DPPC and PBd-PEO, inferring that DPPC strongly fluidizes the PBd-PEO. We use transitions in DPH anisotropy and laurdan GP to create a temperature/composition phase diagram for DPPC/PBd-PEO which we find displays a significantly broader solid/fluid phase coexistence region than DPPC/DOPC, showing that DPPC partitions less readily into fluid PBd-PEO than into fluid DOPC. The existence of a broad solid/fluid phase coexistence region in DPPC/PBd-PEO vesicles is verified by Förster resonance energy transfer results and the visualization of phase separation in giant unilamellar vesicles containing up to 95% PBd-PEO and a single phase in 100% PBd-PEO vesicles at room temperature. These results add to the limited knowledge of phase behavior and phase diagrams of hybrid vesicles, and should be useful in understanding and tailoring membrane surface architecture toward biomedical applications such as drug delivery or membrane protein reconstitution.

Protein-protein interaction (PPI) networks are fundamental for cellular processes. Small-molecule PPI enhancers have been shown to be powerful tools to fundamentally study PPIs and as starting points for potential new therapeutics. Yet, systematic approaches for their discovery are not widely available, and the design prerequisites of "molecular glues" are poorly understood. Covalent fragment-based screening can identify chemical starting points for these enhancers at specific sites in PPI interfaces. We recently reported a mass spectrometry-based disulfide-trapping (tethering) approach for a cysteine residue in the hub protein 14-3-3, an important regulator of phosphorylated client proteins. Here, we invert the strategy and report the development of a functional read-out for systematic identification of PPI enhancers based on fluorescence anisotropy (FA-tethering) with the reactive handle now on a client-derived peptide. Using the DNA-binding domain of the nuclear receptor Estrogen Related Receptor gamma (ERRγ), we target a native cysteine positioned at the 14-3-3 PPI interface and identify several fragments that form a disulfide bond to ERRγ and stabilize the complex up to 5-fold. Crystallography indicates that fragments bind in a pocket comprised of 14-3-3 and the ERRγ phosphopeptide. FA-tethering presents a streamlined methodology to discover molecular glues for protein complexes.

Fluorescence induced by the excitation of a fluorophore with plane-polarized light has a different polarization depending on the size of the fluorophore-containing reagent and the rate of its rotation. Based on this effect, many analytical systems have been implemented in which an analyte contained in a sample and labeled with a fluorophore (usually fluorescein) competes to bind to antibodies. Replacing antibodies in such assays with aptamers, low-cost and stable oligonucleotide receptors, is complicated because binding a fluorophore to them causes a less significant change in the polarization of emissions. This work proposes and characterizes the compounds of the reaction medium that improve analyte binding and reduce the mobility of the aptamer–fluorophore complex, providing a higher analytical signal and a lower detection limit. This study was conducted on aflatoxin B1 (AFB1), a ubiquitous toxicant contaminating foods of plant origins. Eight aptamers specific to AFB1 with the same binding site and different regions stabilizing their structures were compared for affinity, based on which the aptamer with 38 nucleotides in length was selected. The polymers that interact reversibly with oligonucleotides, such as poly-L-lysine and polyethylene glycol, were tested. It was found that they provide the desired reduction in the depolarization of emitted light as well as high concentrations of magnesium cations. In the selected optimal medium, AFB1 detection reached a limit of 1 ng/mL, which was 12 times lower than in the tris buffer commonly used for anti-AFB1 aptamers. The assay time was 30 min. This method is suitable for controlling almond samples according to the maximum permissible levels of their contamination by AFB1. The proposed approach could be applied to improve other aptamer-based analytical systems.

The detailed study of the effect of dimethylsulfoxide (or diethylsulfoxide) on interaction between methylene blue (MB) and calf thymus DNA was performed at 293.15 K using steady-state fluorescence quenching and fluorescence polarization. The combination of steady-state fluorescence quenching and fluorescence polarization reveal the main binding modes at different concentrations of DNA and the effect of sulfoxides on binding mechanism. For comparison, the other polar solvents such as N,N-dimethyl formamide and acetonitrile were used. From Stern–Volmer equation, the values of binding constant and standard Gibbs free energy change were determined. At low concentrations of DNA with addition of various organic cosolvents, the binding constant of DNA–MB complex decreases indicating that hydrogen bonds are predominant, and thus, the main binding mode is groove binding. At high concentrations of DNA, the binding mechanism changes and intercalation becomes the main binding mode in all solutions. Addition of cosolvents distorts the groove binding and, as a result, weakens the intercalation between MB and base pairs of double-stranded DNA. [ABSTRACT FROM AUTHOR]

EPR imaging techniques are known to be successful tools for mapping living bodies, especially because of the high transparency of tissues in the microwave range. This technique assumes the presence of radicals whose in vivo transport is also controlled by serum albumins. Accordingly, in this study, the interactions between 3-hydroxymethyl-1-oxyl-4-(pyren-1-yl)-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrole radical and the human serum albumin molecules were investigated. To clarify the adsorption processes of this radical onto the surface of human serum albumin (HSA), the interaction of the OMe derivative of the radical was also examined parallel with the studies on the radical—HSA interactions. Considering the solubility issues and also to modulate the transport, inclusion complexes of the radical with a cavitand derivative were also studied. The latter interactions were observed through fluorescence spectroscopy, fluorescence polarization, and by EPR spectroscopy. As a double-sensor molecule, we found that the fluorophore nitroxide is a good candidate as it gave further information about host-guest interactions (fluorescence, fluorescence polarization, and EPR). We also found that in the presence of a cavitand, a complex with greater stability was formed between the sensor molecule and the human serum albumin. Based on these observations, we can conclude that applying this double-sensor (spin, fluorescent) molecule is useful in cases when different interactions can affect the EPR measurements. [ABSTRACT FROM AUTHOR]

Simple Summary: The evaluation of breast fine needle aspiration cytology specimens requires subjective, visual assessments of cytomorphology, resulting in suboptimal diagnostic accuracy. The fluorescence polarization of methylene blue demonstrated significant potential as a quantitative marker for cellular level breast cancer diagnosis in clinical aspirates. Results indicate the technology could be implemented as a standalone approach for breast cancer detection in singe cells or augment conventional approaches to reduce the incidence of indeterminate cytopathology. Breast cancer is the most common malignancy in women. The standard of care for diagnosis involves invasive core needle biopsy followed by time-consuming histopathological evaluation. A rapid, accurate, and minimally invasive method to diagnose breast cancer would be invaluable. Therefore, this clinical study investigated the fluorescence polarization (Fpol) of the cytological stain methylene blue (MB) for the quantitative detection of breast cancer in fine needle aspiration (FNA) specimens. Cancerous, benign, and normal cells were aspirated from excess breast tissues immediately following surgery. The cells were stained in aqueous MB solution (0.05 mg/mL) and imaged using multimodal confocal microscopy. The system provided MB Fpol and fluorescence emission images of the cells. Results from optical imaging were compared to clinical histopathology. In total, we imaged and analyzed 3808 cells from 44 breast FNAs. Fpol images displayed quantitative contrast between cancerous and noncancerous cells, whereas fluorescence emission images showed the morphological features comparable to cytology. Statistical analysis demonstrated that MB Fpol is significantly higher (p < 0.0001) in malignant vs. benign/normal cells. It also revealed a correlation between MB Fpol values and tumor grade. The results indicate that MB Fpol could provide a reliable, quantitative diagnostic marker for breast cancer at the cellular level. [ABSTRACT FROM AUTHOR]

Elevated concentrations of toxic organic compounds observed in food products pose serious dangers to human health. Both natural and artificial pollutants can cause food contamination. The stages of food production, packaging, transportation, and storage can also largely cause the appearance of undesirable substances in food products. The health consequences of ingesting food containing toxic contaminants range from mild gastroenteritis to deaths resulting from dysfunctional internal organs and neurological syndromes. The World Health Organization (WHO) sets recommendations for the content of such chemicals in food, including a minimum allowable concentration considered safe for human consumption. However, the control of food products from chemical pollutants is necessary. Moreover, fast, sensitive, and inexpensive methods are needed to detect them at the point of need. Currently, immune analysis methods are most widely used to determine pollutants in food. The development of fluorescence polarization immunoassay (FPIA) methods in a competitive format is a powerful and modern tool for detecting organic molecules in various matrices, thereby making FPIA methods useful for food safety applications. Due to the availability of portable devices for measuring the fluorescence polarization signal, FPIA methods can be used at the point of need. The variety of fluorescent labels and recognizing elements (receptors, monoclonal and polyclonal antibodies, and nanobodies) permits fluorescence polarization (FP) assays to detect significantly lower limits of organic substances. The FP assay is a homogeneous, fast, and quantitative method. The development of various formats of FP assays makes them promising in determining food pollutants. This review summarizes publications on FP analyses for detecting organic contaminants (pesticides, hormones, toxins, antibiotics, and other pharmaceuticals) in food products during 2018–2023. Further, it demonstrates the prospects for using this method to determine pollutants at the point of need and for detecting high molecular weight substances, fungi, and bacterial infections during food safety inspections.

Lysozyme is a well-known enzyme found in many biological fluids which plays an important role in the antibacterial protection of humans and animals. Lysozyme assays are used for the diagnosis of a number of diseases and utilized in immunohistochemistry, genetic and cellular engineering studies. The assaying methods are divided into two categories measuring either the concentration of lysozyme as a protein or its activity as an enzyme. While the first category of methods traditionally uses an enzyme-linked immunosorbent assay (ELISA), the methods for the determination of the enzymatic activity of lysozyme use either live bacteria, which is rather inconvenient, or natural peptidoglycans of high heterogeneity and variability, which leads to the low reproducibility of the assay results. In this work, we propose the use of a chemically synthesized substrate of a strictly defined structure to measure in a single experiment both the concentration of lysozyme as a protein and its enzymatic activity by means of the fluorescence polarization (FP) method. Chito-oligosaccharides of different chain lengths were fluorescently labeled and tested leading to the selection of the pentasaccharide as the optimal size tracer and the further optimization of the assay conditions for the accurate (detection limit 0.3 μM) and rapid (

A fluorescent magnetic surface molecular imprinting method was used to detect naringin by fluorescence polarization technology. By using SiO2-coated magnetic particles as substrate and methacrylic acid and acrylamide as monomers, a surface molecular imprinting polymer with both fluorescence and magnetic characteristics was prepared and loaded with fluorescein isothiocyanate. The binding ability of the prepared polymer was tested by fluorescence polarization and ultraviolet (UV) spectrophotometry. Compared with UV detection method, the fluorescence polarization method was more sensitive, and its limit of detection was 0.1 mg/L. The recovery of the fluorescence polarization method was higher than 81.3%. It was shown that the fluorescent magnetic surface molecular imprinting technique could be a new method to detect naringin quickly and efficiently in food. [ABSTRACT FROM AUTHOR]

Due to its unique structure and properties, human breast milk lactoferrin (hLF) has many nutritional and health-promoting functions in infants, including protection against inflammation and bacterial infections. The lack of LF in breastmilk or formula can result in the weakening of the infant's immune system. Noncompetitive polarization fluorescence immunoassay (FPIA) is a promising method for hLF quantification in milk and dairy products, which does not require the separation of the bound and free protein and allows to avoid time-consuming sample preparation. The use of fluorescently labeled single-domain camelid antibodies (nanobodies) for protein recognition in FPIA makes it possible to quantify relatively large antigens, in particular, hLF. In this work, we used previously obtained fluorescein isothiocyanate (FITC)-conjugated anti-hLF5 and anti-hLF16 nanobodies, which selectively recognized two different human lactoferrin epitopes, but did not bind to goat lactoferrin. The kinetics of hLF interaction with the FITC-labeled nanobodies was studied. The dissociation constant (KD) for the anti-LF5 and anti-LF16 nanobodies was 3.2 ± 0.3 and 4.9 ± 0.4 nM, respectively, indicating the high-affinity binding of these nanobodies to hLF. We developed the FPIA protocol and determined the concentration of FITC-labeled anti-hLF5 and anti-hLF16 nanobodies that provided the optimal fluorescence signal and stable fluorescence polarization value. We also studied the dependence of fluorescence polarization on the hLF concentration in the noncompetitive FPIA with FITC-anti-hLF5 nanobody. The detection limit for hLF was 2.1 ± 0.2 µg/ml and the linear range for determining the hLF concentration was 3-10 µg/ml. FPIA is commonly used to assay low-molecular-weight substances; however, the use of fluorescently labeled nanobodies allows quantification of high-molecular-weight proteins. Here, we demonstrated that FPIA with fluorescently labeled nanobodies can be used for hLF quantification in milk. [ABSTRACT FROM AUTHOR]

The signal transducer and activation of transcription (STAT) proteins are a family of Src homology 2 (SH2) domain-containing transcription factors. The family member STAT4 is a mediator of IL-12 signalling and has been implicated in the pathogenesis of multiple autoimmune diseases. The activity of STAT4 requires binding of phosphotyrosine-containing motifs to its SH2 domain. Selective inhibitors of the STAT4 SH2 domain have not been published to date. Here, we present a fluorescence polarization-based assay for the identification of inhibitors of the STAT4 SH2 domain. The assay is based on the interaction between the STAT4 SH2 domain and the fluorophore-labelled peptide 5-carboxyfluorescein-GpYLPQNID (Kd = 34 ± 4 nM). The assay is stable with respect to DMSO concentrations of up to 10% and incubation times of at least 8 h. The Z'-value of 0.85 ± 0.01 indicates that the assay is suited for use in high-throughput screening campaigns aimed at identifying new therapeutic modalities for the treatment of autoimmune diseases. [ABSTRACT FROM AUTHOR]

Most membrane protein molecules undergo conformational changes as they transition from one functional state to another one. An understanding of the mechanism underlying these changes requires the ability to resolve individual conformational states, whose changes often occur on millisecond and angstrom scales. Tracking such changes and acquiring a sufficiently large amount of data remain challenging. Here, we use the amino-acid transporter AdiC as an example to demonstrate the application of a high-resolution fluorescence-polarization-microscopy method in tracking multistate conformational changes of a membrane protein. We have successfully resolved four conformations of AdiC by monitoring the emission-polarization changes of a fluorophore label and quantified their probabilities in the presence of a series of concentrations of its substrate arginine. The acquired data are sufficient for determining all equilibrium constants that fully establish the energetic relations among the four states. The KD values determined for arginine in four individual conformations are statistically comparable to the previously reported overall KD determined using isothermal titration calorimetry. This demonstrated strong resolving power of the present polarization-microscopy method will enable an acquisition of the quantitative information required for understanding the expected complex conformational mechanism underlying the transporter’s function, as well as those of other membrane proteins.

Heat shock factor 1 (HSF1) is a stress-responsive transcription factor that regulates expression of protein chaperones and cell survival factors. In cancer, HSF1 plays a unique role, hijacking the normal stress response to drive a cancer-specific transcriptional program. These observations suggest that HSF1 inhibitors could be promising therapeutics. However, HSF1 is activated through a complex mechanism, which involves release of a negative regulatory domain, leucine zipper 4 (LZ4), from a masked oligomerization domain (LZ1-3), and subsequent binding of the oligomer to heat shock elements (HSEs) in HSF1-responsive genes. Recent crystal structures have suggested that HSF1 oligomers are held together by extensive, buried contact surfaces, making it unclear whether there are any possible binding sites for inhibitors. Here, we have rationally designed a series of peptide-based molecules based on the LZ4 and LZ1-3 motifs. Using a plate-based, fluorescence polarization (FP) assay, we identified a minimal region of LZ4 that suppresses binding of HSF1 to the HSE. Using this information, we converted this peptide into a tracer and used it to understand how binding of LZ4 to LZ1-3 suppresses HSF1 activation. Together, these results suggest a previously unexplored avenue in the development of HSF1 inhibitors. Furthermore, the findings highlight how native interactions can inspire the design of inhibitors for even the most challenging protein-protein interactions (PPIs).

The stoichiometry of a ligand binding reaction to a protein is given by a parameter (n). The value of this parameter may indicate the presence of protein monomer or dimers in the binding complex. Members of the lipocalin superfamily show variation in the stoichiometry of binding to ligands. In some cases the stoichiometry parameter (n) has been variously reported for the same protein as mono- and multimerization of the complex. Prime examples include retinol binding protein, β lactoglobulin and tear lipocalin, also called lipocalin-1(LCN1). Recent work demonstrated the stoichiometric ratio for ceramide:tear lipocalin varied (range n = 0.3-0.75) by several different methods. The structure of ceramide raises the intriguing possibility of a lipocalin dimer complex with each lipocalin molecule attached to one of the two alkyl chains of ceramide. The stoichiometry of the ceramide-tear lipocalin binding complex was explored in detail using size exclusion chromatography and time resolved fluorescence anisotropy. Both methods showed consistent results that tear lipocalin remains monomeric when bound to ceramide. Delipidation experiments suggest the most likely explanation is that the low 'n' values result from prior occupancy of the binding sites by native ligands. Lipocalins such as tear lipocalin that have numerous binding partners are particularly prone to an underestimated apparent stoichiometry parameter.

Abstract Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly transmissible and has caused a pandemic named coronavirus disease 2019 (COVID-19), which has quickly spread worldwide. Although several therapeutic agents have been evaluated or approved for the treatment of COVID-19 patients, efficacious antiviral agents are still lacking. An attractive therapeutic target for SARS-CoV-2 is the main protease (Mpro), as this highly conserved enzyme plays a key role in viral polyprotein processing and genomic RNA replication. Therefore, the identification of efficacious antiviral agents against SARS-CoV-2 Mpro using a rapid, miniaturized and economical high-throughput screening (HTS) assay is of the highest importance at the present. Results In this study, we first combined the fluorescence polarization (FP) technique with biotin-avidin system (BAS) to develop a novel and step-by-step sandwich-like FP screening assay to quickly identify SARS-CoV-2 Mpro inhibitors from a natural product library. Using this screening assay, dieckol, a natural phlorotannin component extracted from a Chinese traditional medicine Ecklonia cava, was identified as a novel competitive inhibitor against SARS-CoV-2 Mpro in vitro with an IC50 value of 4.5 ± 0.4 µM. Additionally, dieckol exhibited a high affinity with SARS-CoV-2 Mpro using surface plasmon resonance (SPR) analysis and could bind to the catalytic sites of Mpro through hydrogen-bond interactions in the predicted docking model. Conclusions This innovative sandwich-like FP screening assay enables the rapid discovery of antiviral agents targeting viral proteases, and dieckol will be an excellent lead compound for generating more potent and selective antiviral agents targeting SARS-CoV-2 Mpro.

Target‐directed dynamic combinatorial chemistry has emerged as a useful tool for hit identification, but has not been widely used, in part due to challenges associated with analyses involving complex mixtures. We describe an operationally simple alternative: in situ inhibitor synthesis and screening (ISISS), which links high‐throughput bioorthogonal synthesis with screening for target binding by fluorescence. We exemplify the ISISS method by showing how coupling screening for target binding by fluorescence polarization with the reaction of acyl‐hydrazides and aldehydes led to the efficient discovery of a potent and novel acylhydrazone‐based inhibitor of human prolyl hydroxylase 2 (PHD2), a target for anemia treatment, with equivalent in vivo potency to an approved medicine. [ABSTRACT FROM AUTHOR]

Rapid and sensitive detection of heavy metal cadmium ions (Cd2+) is of great significance to food safety and environmental monitoring, as Cd2+ contamination and exposure cause serious health risk. In this study we demonstrated an aptamer-based fluorescence anisotropy (FA) sensor for Cd2+ with a single tetramethylrhodamine (TMR)-labeled 15-mer Cd2+ binding aptamer (CBA15), integrating the strengths of aptamers as affinity recognition elements for preparation, stability, and modification, and the advantages of FA for signaling in terms of sensitivity, simplicity, reproducibility, and high throughput. In this sensor, the Cd2+-binding-induced aptamer structure change provoked significant alteration of FA responses. To acquire better sensing performance, we further introduced single phosphorothioate (PS) modification of CBA15 at a specific phosphate backbone position, to enhance aptamer affinity by possible strong interaction between sulfur and Cd2+. The aptamer with PS modification at the third guanine (G) nucleotide (CBA15-G3S) had four times higher affinity than CBA15. Using as an aptamer probe CBA15-G3S with a TMR label at the 12th T, we achieved sensitive selective FA detection of Cd2+, with a detection limit of 6.1 nM Cd2+. This aptamer-based FA sensor works in a direct format for detection without need for labeling Cd2+, overcoming the limitations of traditional competitive immuno-FA assay using antibodies and fluorescently labeled Cd2+. This FA method enabled the detection of Cd2+ in real water samples, showing broad application potential. [ABSTRACT FROM AUTHOR]

The global scourge of COVID-19 is a serious threat to public health, but effective therapies remain very limited for this disease. Therefore, the discovery of novel antiviral agents is urgently needed to fight against COVID-19. In the lifecycle of SARS-CoV-2, the causing pathogen of COVID-19, papain-like protease (PLpro) is responsible for the cleavage of polyprotein into functional units as well as immune evasion of vaccines. Hence, PLpro has been regarded as an attractive target to develop antiviral agents. Herein, we first developed a robust and simple sandwich-like fluorescence polarization (FP) screening assay for the discovery of PLpro inhibitors, and identified anacardic acid as a novel competitive inhibitor against PLpro in vitro with an IC 50 value of 24.26 ± 0.4 μM. This reliable FP screening assay could provide a prospective avenue for rapid discovery of antiviral agents targeting PLpro in a large-scale screening. [Display omitted] • Development of a simple, robust, and affordable sandwich-like fluorescence polarization (FP) screening assay for the discovery of SARS-CoV-2 papain-like protease (PLpro) inhibitors. • This FP screening assay identified anacardic acid as a competitive inhibitor against SARS-CoV-2 PLpro in vitro with an IC 50 value of 24.26 ± 0.4 μM. • This reliable FP screening assay will be vital for rapid screening of antiviral agents targeting SARS-CoV-2 PLpro in a large-scale screening. [ABSTRACT FROM AUTHOR]

The use of dendrimers as nanovectors for nucleic acids or drugs requires the understanding of their interaction with biological membranes. This study investigates the impact of 1st generation polyphenolic carbosilane dendrimers on biological and model lipid membranes using several biophysical methods. While the increase in the z-average size of DMPC/DPPG liposomes correlated with the number of caffeic acid residues included in the dendrimer structure, dendrimers that contained polyethylene glycol chains generated lower zeta potential when interacting with a liposomal membrane. The increase in the fluorescence anisotropy of DPH and TMA-DPH probes incorporated into erythrocyte membranes predicted the ability of dendrimers to affect membrane fluidity in the hydrophobic interior and hydrophilic/polar region of a lipid bilayer. The presence of caffeic acid and polyethylene glycol chains in the dendrimer structure affected the thermodynamical properties of the membrane lipid matrix., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

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.

The determination of ligand-receptor binding affinities plays a key role in the development process of pharmaceuticals. While the classical radiochemical binding assay uses radioligands, fluorescence-based binding assays require fluorescent probes. Usually, radio- and fluorescence-labeled ligands are dissimilar in terms of structure and bioactivity, and can be used in either radiochemical or fluorescence-based assays. Aiming for a close comparison of both assay types, we synthesized tritiated fluorescent neurotensin receptor ligands ([ 3 H] 13 , [ 3 H] 18 ) and their nontritiated analogues ( 13 , 18 ). The labeled probes were studied in radiochemical and fluorescence-based (high-content imaging, flow cytometry, fluorescence anisotropy) binding assays. Equilibrium saturation binding yielded well-comparable ligand-receptor affinities, indicating that all these setups can be used for the screening of new drugs. In contrast, discrepancies were found in the kinetic behavior of the probes, which can be attributed to technical differences of the methods and require further studies with respect to the elucidation of the underlying mechanisms.

The abundant nuclear protein hnRNP U interacts with a broad array of RNAs along with DNA and protein to regulate nuclear chromatin architecture. The RNA-binding activity is achieved via a disordered ∼100 residue C-terminal RNA-binding domain (RBD) containing two distinct RGG/RG motifs. Although the RNA-binding capabilities of RGG/RG motifs have been widely reported, less is known about hnRNP U's RNA-binding selectivity. Furthermore, while it is well established that hnRNP U binds numerous nuclear RNAs, it remains unknown whether it selectively recognizes sequence or structural motifs in target RNAs. To address this question, we performed equilibrium binding assays using fluorescence anisotropy (FA) and electrophoretic mobility shift assays (EMSAs) to quantitatively assess the ability of human hnRNP U RBD to interact with segments of cellular RNAs identified from eCLIP data. These RNAs often, but not exclusively, contain poly-uridine or 5'-AGGGAG sequence motifs. Detailed binding analysis of several target RNAs reveal that the hnRNP U RBD binds RNA in a promiscuous manner with high affinity for a broad range of structured RNAs, but with little preference for any distinct sequence motif. In contrast, the isolated RGG/RG of hnRNP U motif exhibits a strong preference for G-quadruplexes, similar to that observed for other RGG motif bearing peptides. These data reveal that the hnRNP U RBD attenuates the RNA binding selectivity of its core RGG motifs to achieve an extensive RNA interactome. We propose that a critical role of RGG/RG motifs in RNA biology is to alter binding affinity or selectivity of adjacent RNA-binding domains., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: “The authors declare the following competing financial interest(s): R.T.B. serves on the Scientific Advisory Boards of Expansion Therapeutics, SomaLogic and MeiraGTx.”, (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Fluorescence polarization (FP) has been applied in detecting chemicals and biomolecules for early-stage diagnosis, food safety analyses, and environmental monitoring. Compared to organic dyes, inorganic nanomaterials such as quantum dots have special fluorescence properties that can enhance the photostability of FP-based biosensing. In addition, nanomaterials, such as metallic nanoparticles, can be used as signal amplifiers to increase fluorescence polarization. In this review paper, different types of nanomaterials used in in FP-based biosensors have been reviewed. The role of each type of nanomaterial, acting as a fluorescent element and/or the signal amplifier, has been discussed. In addition, the advantages of FP-based biosensing systems have been discussed and compared with other fluorescence-based techniques. The integration of nanomaterials and FP techniques allows biosensors to quickly detect analytes in a sensitive and cost-effective manner and positively impact a variety of different fields including early-stage diagnoses. [ABSTRACT FROM AUTHOR]

Here we explore the click chemistry-based synthesis of high affinity multivalent aptamers and their application for virus detection. In contrast to attempts based on rigid constructs, such as aptamer-modified nanoparticles or hybridization-connected constructs, we propose highly flexible "star-shaped" multivalent aptamers to facilitate the cooperative binding to randomly distributed viral proteins on the surface of pleomorphic viruses such as the respiratory syncytial virus (RSV). Polyethylene glycols (PEGs) with 4 azide terminated flexible arms spanning from a pentaerythritol core were conjugated to aptamer strands with dibenzocyclooctyne and a fluorescent label at their, 3' and 5' terminus, respectively. By controlling the aptamer - PEG ratio and following electrophoretic separation all the different valency aptamer conjugates could be prepared. For proof of concept, we used an aptamer selected for the G protein of RSV. We show that significantly higher apparent affinities can be obtained by using multivalent aptamers for both the G protein and RSV virus than with the single stranded aptamer, i.e. with up to ca. 2 orders of magnitude. The inherent convenience of synthesizing fluorescently labelled multivalent aptamers leads to a versatile class of affinity ligands for sensing as demonstrated through the improved detection sensitivity of tetravalent aptamer tagged RSV by fluorescence nanoparticle tracking analysis. [Display omitted] • Star-shaped flexible PEG arm multivalent aptamers can be synthesized by click chemistry. • Different valency PEG core aptamers can be electrophoretically separated. • Flexible tetravalent aptamers showed increased affinity for the pleomorphic RSV. • Multivalent aptamers resist well to displacement from their RSV target. • Multivalent fluorescent aptamers improved the detectability of RSV in F-NTA. [ABSTRACT FROM AUTHOR]