Selectively Bind Research Articles

Characterization of insulin and bile acid complexes in liposome by different mass spectrometry techniques.

Insulin bound with ligand molecules can improve its bioavailability in oral formulations. In this work, the interactions between insulin and bile acids of taurocholic acid (TCA) and glycocholic acid (GCA) are characterized using different mass spectrometry (MS) methods. Electrospray (ESI)-MS analysis revealed that GCA and TCA could interact with insulin individually or together through non-covalent bonds, and the products included mGCA-insulin, nTCA-insulin, and mGCA-nTCA-insulin complexes. Their binding stoichiometry, relative intensity ratio (IRa), and binding affinity were determined. ESI-MS/MS data and the calculated association constants both suggest that TCA has stronger affinity to insulin than GCA. The mixtures of various insulin, GCA, andTCA complexes with different charge states were separated, and distinct trend lines were observed using ion mobility mass spectrometry (IMMS). Moreover, liposomes containing insulin and GCA and/or TCA were prepared, and directly characterized using ESI-MS, and the interaction products of insulin with GCA and TCA were found in the liposome formulation. AutoDock was used to simulate molecular binding and select binding sites between insulin and GCA or TCA to explore the interaction mechanisms. The findings in this work could help understand the mechanism of actionof insulin protection with bile acids in the body.

Motif distribution and DNA methylation underlie distinct Cdx2 binding during development and homeostasis

Transcription factors guide tissue development by binding to developmental stage-specific targets and establishing an appropriate enhancer landscape. In turn, DNA and chromatin modifications direct the genomic binding of transcription factors. However, how transcription factors navigate chromatin features to selectively bind a small subset of all the possible genomic target loci remains poorly understood. Here we show that Cdx2—a lineage defining transcription factor that binds distinct targets in developing versus adult intestinal epithelial cells—has a preferential affinity for a non-canonical CpG-containing motif in vivo. A higher frequency of this motif at embryonic Cdx2 targets and methylated state of the CpG during development enables selective Cdx2 binding and activation of developmental enhancers and genes. In adult cells, demethylation at these enhancers prevents ectopic Cdx2 binding, instead directing Cdx2 to its canonical motif without a CpG. This shift in Cdx2 binding facilitates Ctcf and Hnf4 recruitment, establishing super-enhancers during development and homeostatic enhancers in adult cells, respectively. Induced DNA methylation in adult mouse epithelium or cultured cells recruits Cdx2 to developmental targets, promoting corecruitment of partner transcription factors. Thus, Cdx2’s differential CpG motif preferences enable it to navigate distinct DNA methylation profiles, activating genes specific to appropriate developmental stages.

Feasibility of Ex Vivo Ligandomics.

We developed ligandomics for the in vivo profiling of vascular ligands in mice, discovering secretogranin III (Scg3) as a novel angiogenic factor that selectively binds to retinal vessels of diabetic but not healthy mice. This discovery led to the development of anti-Scg3 therapy for ocular vasculopathies. However, in vivo ligandomics requires intracardial perfusion to remove unbound phage clones, limiting its use to vascular endothelial cells (ECs). To extend ligandomics to non-vascular cells, we investigated ex vivo ligandomics. We isolated ECs and retinal ganglion cells (RGCs) from diabetic and healthy mouse retinas by immunopanning. We quantified the binding of clonal phages displaying Scg3 and vascular endothelial growth factor (VEGF), confirming that their binding patterns to isolated diabetic versus healthy ECs matched in vivo patterns. Additionally, Scg3 and VEGF binding to isolated RGCs reflected their in vivo activity. These results support the feasibility of ex vivo ligandomics. We further mapped ligands binding to immunopanned diabetic and healthy ECs and RGCs by ligandomics, confirming that Scg3 was enriched with selective binding to diabetic ECs but not healthy ECs or diabetic/healthy RGCs. These findings demonstrate the feasibility of ex vivo ligandomics, which can be broadly applied to various cell types, tissues, diseases, and species.

Green Tea Polyphenol Epigallocatechin Gallate Interactions with Copper-Serum Albumin.

Epigallocatechin gallate (EGCg), an abundant phytochemical in green tea, is an antioxidant that also binds proteins and complex metals. After gastrointestinal absorption, EGCg binds to serum albumin in the hydrophobic pocket between domains IIA and IIIA and overlaps with the Sudlow I site. Serum albumin also has two metal binding sites, a high-affinity N-terminal site (NTS) site that selectively binds Cu(II), and a low-affinity, less selective multi-metal binding site (MBS). We proposed to determine whether EGCg binds or reacts with Cu(II)-serum albumin using fluorescence, UV-Visible and electron paramagnetic resonance (EPR) spectroscopy. Our results suggest that when serum albumin is loaded with Cu(II) in both sites, EGCg binds to the MBS-Cu(II) and reduces the copper to Cu(I). EGCg does not bind to or react with Cu(II) in the high-affinity NTS site. Potential consequences include changes in copper homeostasis and damage from pro-oxidative Fenton reactions.

Discovery of pentacyclic triterpene conjugates as HBV polymerase/NTCP dual-targeting inhibitors with potent anti-HBV activities.

The inhibition of HBV DNA and elimination of HBsAg has already been established as an indicator for HBV clinic cure, and a novel dual-targeting inhibitors of HBV polymerase/entry are designed and synthesized in this study. Pentacyclic triterpenes (PTs) scaffold of exhibiting a high affinity to NTCP, including glycyrrhitinic acid (GA), oleanolic acid (OA), ursolic acid (UA), and betulinic acid (BA) were linked neatly with the nucleoside drug zidovudine (AZT) through a molecular hybrid strategy to synthesize twenty of PTs-AZT conjugates for targeting HBV polymerase as well as sodium taurocholate cotransporting polypeptide (NTCP). The conjugates showed significant inhibitory effects on the secretion of HBsAg and HBeAg in HepG2.2.15 cells, and the activity on HBsAg were better. Moreover, HBV DNA replication was also notably suppressed after incubated with the conjugates. The IC50 value of BA-AZT1 on HBsAg inhibition was 0.65±0.07μM, and it was 284.2 times and 442.2 times higher comparing to corresponding parent compound BA and AZT. Additionally, the therapeutic index (TI) was also improved by 87.8 times than AZT. And the IC50 value of BA-AZT1 on inhibition of HBV DNA replication was 0.70±0.02μM, 10.4 times higher than that of AZT besides conspicuous TI. Molecular docking suggested that AZT skeleton of conjugate BA-AZT1 interacted with B region of HBV Polymerase reverse transcription region, and BA structure simultaneously targeted to C region of polymerase via hydrophobic chain, establishing strong binding interactions with the HBV Pol protein. In addition, docked with NTCP, BA-AZT1 with flat pentacyclic structure inserted into the interface and also formed hydrogen bonds, hydrophobic and van der Waals forces with the amino residue 157-165 of NTCP. Further SPR analysis demonstrated the binding affinity of BA-AZT1 to C region of polymerase was 19.55μM, stronger than 53.21μM of BA and 31.82μM of AZT. BA-AZT1 selectively bound to the 157-165 epitopes of NTCP receptors in host cell but not PreS1 of virus. As a result, we deduced that the designed conjugates targeted NTCP and HBV polymerase, not only prevented HBV from entering host cells via selective binding NTCP, but also inhibited HBV DNA replication through obstructing the function of HBV polymerase, and it could potentially serve as a promising dual-functional and dual-target inhibitor with both replication and entry inhibition to exert anti-HBV activity.

Application of Tetrodotoxin for Anesthetic and Pain Management

Tetrodotoxin (TTX) is a potent neurotoxin that is predominately sourced from marine animals, which serves a dual role as both a fatal toxin and a potential therapeutic compound. TTX functions by selectively binding to voltage-gated sodium channels (VGSCs), preventing sodium ions from entering nerve cells and thereby blocking action potential formation. While its toxicity can lead to severe outcomes like whole-body paralysis and even death, TTX’s unique mechanism of action creates possibilities for anesthetic use and pain management. Unlike traditional local anesthetics, TTX offers long-lasting effects with a lower risk of cardiac toxicity due to its selective binding. TTX can effectively manage chronic and neuropathic pain, including cancer-related and chemotherapy-induced pain, with relatively mild side effects when used at controlled doses. Combining TTX with drugs like bupivacaine has shown a synergistic impact, significantly extending pain relief duration while reducing potential toxicity. The lacks of an antidote for TTX necessitates strict dosing controls and further research to ensure safety. This research explores the mechanisms and safety considerations of TTX, and most importantly, its emerging applications will be analyzed.

Binding of Bioactive Ammonium Ions in Water with a Cavity-Based Selectivity: Water Solubilization versus Micellar Incorporation.

Many bioactive molecules contain primary ammonium groups, generating significant interest in developing selective receptors for ammonium ions. A promising strategy involves the use of polyaromatic cavitands to achieve size and shape selectivity through their cavity. However, designing effective receptors for ammonium ions in aqueous media is challenging due to the competitive nature of water. Calix[5]arenes are known to selectively bind primary ammonium ions over secondary, tertiary, and quaternary ammonium ions in organic solvents. Here, we report on the binding properties of a calix[5]arene, which bears carboxyl groups on its small rim, in organic solvents and aqueous media. This receptor was transferred in water either through deprotonation of its carboxyl groups or by incorporation into dodecylphosphocholine micelles. 1H Nuclear Magnetic Resonance data confirmed the endo complexation of various primary ammonium ions in not only organic solvents but also both aqueous media. Cavity-based selectivity was also observed, validating the cavitand strategy for the selective binding of ammonium ions in water. Unique binding properties, driven by the calix[5]arene's intrinsic recognition ability and the hydrophobic effect, were observed in water. Notably, binding affinities for dopamine and lysine derivatives with log Ka values of >3.9 were determined. The direct solubilization of the receptor outperformed micellar incorporation due to the hydrophilic nature of the primary ammonium ions, which hinders their uptake into micelles. These results offer promising perspectives for the development of efficient chemosensors for the characterization of bioactive ammonium ions in water.

MuGger Toxins: Exploring the Selective Binding Mechanism of Clostridial Glucosyltransferase Toxin B and Host GTPases.

(a) Clostridioides difficile (C. difficile) bacterium can cause severe diarrhea and its over-colonization in the host's intestinal tract lead to the development of pseudomembranous colitis, generally due to antibiotic usage. The primary exotoxins involved are toxin A (TcdA) and toxin B (TcdB), the latter being more pathogenic. TcdB has glucosyltransferase activity and mediates monoglycosylation by targeting host cell enzymes (mainly Rho and Ras family of GTPases) with differential selectivity. Here, we aim to provide structural and dynamic insights into how TcdB impacts the host's intestinal epithelial cells focusing on the glycosylation mechanism of Rho GTPases, Cdc42, and Rac1, at the molecular level. To this aim, we modeled the unknown TcdB-host protein complex structures, based on the available experimental structures of TcdB, through protein-protein docking. Then, we elaborated on TcdB-Rho GTPase models as TcdB is known to selectively interact with GDP-bound inactive states of Rho GTPases, over the GTP-bound active ones, but the mechanism is unclear. Through a total of 6 μs-long molecular dynamics simulation of TcdB and GTP/GDP-bound Rac1 and Cdc42 complexes, TcdB's selective binding mechanism was revealed for Rac1. TcdB-Rac1 complexes were further analyzed with enhanced sampling techniques such as well-tempered metadynamics simulations and umbrella sampling to reveal selective binding mechanism between TcdB and GDP-bound Rac1. Our results show that TcdB selectively binds to GDP-bound Rac1, over the GTP-bound one, driven by its affinity for the Mg2+ ion. A destabilized Mg2+ ion incapable of coordinating GDP disrupts Rac1's GTPase function, shedding light on the molecular basis of TcdB's pathogenic effects.

3D‐Printable Polymer Filters for the Selective Complexation of Silver Ions

ABSTRACTThis study presents the fabrication of polymeric filters, which are able to selectively bind silver ions from aqueous solution. Those filters are fabricated within an easy approach via 3D‐digital light processing (DLP)‐printing enabling a tailor‐made design. In first order, an acrylamide containing a benzo‐trithiacrown‐ether (BTCE) functionality is synthesized. This monomer is further 3D‐printed via layer by layer photo‐polymerization together with commercially available comonomers, resulting in polymer networks containing the BTCE‐groups in their side chains as selective binding moiety for silver ions. Within isothermal‐titration calorimetry (ITC) measurements, the complexation abilities of the BTCE to bind the Ag+ ions are determined. Furthermore, the resulting 3D‐printed filters are investigated regarding their complexation behavior, in a detailed fashion applying inductively coupled plasma optical emission spectroscopy (ICP‐OES).

Multiplex Pulsed Current Sensors for Anion Detection

Low-cost and stable sensors are needed for long-duration monitoring of nitrate and phosphate in waterways to help improve farming practices and understand the negative environmental and economic impacts caused by the release of these ions. One established platform that shows promise for detecting these ions is the use of solid-contact ion selective electrodes (SC−ISEs). Conventional SC-ISE measurements monitor electrode potential under open circuit conditions to quantify the amount of one target analyte. These conventional SC-ISEs rely on highly engineered membrane materials that are formulated to selectively bind to the target ion. These engineered membrane materials can achieve high selectivity for nitrate and phosphate, but are vulnerable to certain interferents, require frequent calibration, and have poor long-term stability. In recent work, we demonstrated the use of pulsed current technique for anion detection in SC-ISEs, which allows for anion detection based on differences in anion diffusion through the membrane rather than selective anion binding – opening up a wide range of materials as candidates for ion sensing membranes. In this report, we employ pulsed current measurement to examine anion detection using three common polymers as membrane layers: polyvinyl acetate (PVA), polyvinylchloride (PVC), and polyacrylonitrile (PAN). We measure the potential vs. time response for these three membrane materials in solutions containing various anions such as nitrate, phosphate, chloride, and carbonate at varying pulse currents. Afterwards, we employ tree-based and neural-based machine learning algorithms to examine the feasibility of detecting multiple ions simultaneously using the combined response data from multiple electrodes. Our experiments illustrate that our method for anion detection, used with machine learning analysis, is useful for investigating the interaction mechanisms of anions with the polymer matrix.

Design and validation of a selective binding 3D printer for bio-based construction materials

We aim to address that home-made design and assembly can be effective to investigate several sizes and natures of construction materials. This study offers a new design that accepts different powder and bio-based materials to enhance the functionality of construction elements. We developed a system in which a numerical controlled machine is coupled with a 6 axis articulated-arm to selectively bind a mineral powder bed with liquid's droplets. A home-made frame has been designed and built to adapt the geometrical space that allows the displacement of the numerical controlled machine and the articulated arm. The powder deposition is carried out using a tank controlled by an Arduino board. In parallel, we propose a fluid deposition system using a pressurised tank and a valve jetting that allows the deposition of millimetric liquid droplet. 3D printing tests using cement particles, water retention agent, and a composite of cement with hemp aggregates were carried out to validate the prototype. We then highlight, as a proof of concept, that an innovative selective binding 3D printer could bring new composites or biobased materials in construction industry.

Copper-saturated chitosan beads (CuSCBs) for Enhanced phosphate removal

This study investigates the sequential use of non-cross-linked chitosan beads (CBs) for copper and phosphate adsorption, emphasizing copper site saturation and selective phosphate binding. Initially, CBs were loaded with copper until saturation was achieved, where all available sites were occupied by copper at an initial concentration of 100 mg/L. These copper-saturated chitosan beads (CuSCBs) were subsequently applied for phosphate adsorption, achieving a maximum adsorption capacity of 85.89 mg/g at pH 5. X-ray Photoelectron Spectroscopy (XPS) analysis revealed a shift in the Cu2O binding energy to lower values upon phosphate adsorption, indicating that phosphate ions interact primarily with the copper ions intercalated on the CBs rather than the chitosan matrix. Additionally, the nitrogen spectra showed no shift, confirming that the amine groups of chitosan do not participate in phosphate binding. The findings suggest a 1:1 interaction ratio between copper and phosphate ions, with each phosphate ion binding to one copper ion, creating a stable and selective adsorption site. The adsorption data fit the Langmuir isotherm and pseudo-second-order kinetic models, suggesting a homogeneous monolayer adsorption mechanism driven by chemisorption, where each copper site on the chitosan surface selectively binds one phosphate ion. Furthermore, the CuSCBs demonstrated minimal copper leaching and retained 90 % of their phosphate adsorption capacity over multiple adsorption–desorption cycles, highlighting their potential as effective and reusable adsorbents for water treatment applications targeting copper and phosphate removal.

Fully human monoclonal antibody targeting the cysteine-rich substrate-interacting region of ADAM17 on cancer cells

ADAM17 sheds EGFR/erbB ligands and triggers oncogenic pathways that lead to the progression of solid tumors. We targeted the ADAM17 disintegrin and cysteine rich domain region (D+C) to generate a panel of single-chain antibody fragments (scFvs) that selectively bind to the D or C domains of ADAM17, but not of ADAM10 or ADAM19. From the panel, we selected one scFv, referred to as C12, based on its high binding affinity towards the target, and re-formatted it to a full IgG for further studies. High-resolution cryo-electron microscopy studies documented that the mAb binds to the ADAM17 C-domain that in ADAM proteases, notably ADAM10 and ADAM17, is known to impart substrate-specificity. The C12 mAb significantly inhibited EGFR phosphorylation in cancer cell lines by hindering the cleavage of EGFR ligands tethered to the cell surface. This inhibition provides a mechanism for potential anti-tumor effects, and indeed C12 diminished the viability of a variety of EGFR-expressing cancer cell lines. Cell-based ELISA studies revealed that C12 preferentially bound to activated ADAM17 present on tumor cells, as compared to the autoinhibited ADAM17 that is the predominant form on HEK293 and other non-tumor cells. C12 also exhibited tumor growth inhibition in an ovarian cancer xenograft mouse model. Consistent with its selective tumor cell binding in vitro, radioimmuno PET (positron emission tomography) imaging with 89Zr-DFO-C12 in mouse xenograft models confirmed tumoral accumulation of the C12 mAb.

The Influence of Peptide Linkers on Functional Properties of Hybrid Structures with the Selective pH-Dependent Binding to Cancer Cells

Most of the modern cancer therapies are non-specific and have adverse side effects on the body. Nowadays, targeted cancer therapies are being developed, in particular using targeting peptides that selectively bind to cancer cells. The aim of the present work is to explore the prospects of using a peptide pHLIP that binds to cancer cells at decreased pH values, as a part of recombinant protein-peptide construct for cancer diagnosis and targeted therapy. Hybrid structures based on the fluorescent protein EGFP and a linker sequence connecting fluorescent protein to two different pHLIP variants were obtained. The effect of different linkers on the pH-dependent binding of the constructs to cells, as well as on the efficiency of EGFP chromophore synthesis within the hybrid construct was investigated.

Virus-Based Separation of Rare Earth Elements.

The utilization of biomaterials for the separation of rare earth elements (REEs) has attracted considerable interest due to their inherent advantages, including diverse molecular structures for selective binding and the use of eco-friendly materials for sustainable systems. We present a pioneering methodology for developing a safe virus to selectively bind REEs and facilitate their release through pH modulation. We engineered the major coat protein of M13 bacteriophage (phage) to incorporate a lanthanide-binding peptide. The engineered lanthanide-binding phage (LBPh), presenting ∼3300 copies of the peptide, serves as an effective biological template for REE separation. Our findings demonstrate the LBPh's preferential binding for heavy REEs over light REEs. Moreover, the LBPh exhibits remarkable robustness with excellent recyclability and stability across multiple cycles of separations. This study underscores the potential of genetically integrating virus templates with selective binding motifs for REE separation, offering a promising avenue for environmentally friendly and energy-efficient separation processes.

Structural and biophysical insights into targeting of claudin-4 by a synthetic antibody fragment

Claudins are a 27-member family of ~25 kDa membrane proteins that integrate into tight junctions to form molecular barriers at the paracellular spaces between endothelial and epithelial cells. As the backbone of tight junction structure and function, claudins are attractive targets for modulating tissue permeability to deliver drugs or treat disease. However, structures of claudins are limited due to their small sizes and physicochemical properties—these traits also make therapy development a challenge. Here we report the development of a synthetic antibody fragment (sFab) that binds human claudin-4 and the determination of a high-resolution structure of it bound to claudin-4/enterotoxin complexes using cryogenic electron microscopy. Structural and biophysical results reveal this sFabs mechanism of select binding to human claudin-4 over other homologous claudins and establish the ability of sFabs to bind hard-to-target claudins to probe tight junction structure and function. The findings provide a framework for tight junction modulation by sFabs for tissue-selective therapies.

Multicomponent, Multicavity Metallacages That Contain Different Binding Sites for Allosteric Recognition.

The encapsulation of different guest molecules by their different recognition domains of proteins leads to selective binding, catalysis, and transportation. Synthetic hosts capable of selectively binding different guests in their different cavities to mimic the function of proteins are highly desirable but challenging. Here, we report three ladder-shaped, triple-cavity metallacages prepared by multicomponent coordination-driven self-assembly. Interestingly, the porphyrin-based metallacage is capable of heteroleptic encapsulation of fullerenes (C60 or C70) and coronene using its different cavities, allowing distinct allosteric recognition of coronene upon the addition of C60 or C70. Owing to the different binding affinities of the cavities, the metallacage hosts one C60 molecule in the central cavity and two coronene units in the side cavities, while encapsulating two C70 molecules in the side cavities and one coronene molecule in the central cavity. The rational design of multicavity assemblies that enable heteroleptic encapsulation and allosteric recognition will guide the further design of advanced supramolecular constructs with tunable recognition properties.

Development of Benziodarone Analogues with Enhanced Potency for Selective Binding to Transthyretin in Human Plasma.

Transthyretin amyloidosis is a fatal disorder caused by transthyretin amyloid aggregation. Stabilizing the native structure of transthyretin is an effective approach to inhibit amyloid aggregation. To develop kinetic stabilizers of transthyretin, it is crucial to explore compounds that selectively bind to transthyretin in plasma. Our recent findings demonstrated that the uricosuric agent benziodarone selectively binds to transthyretin in plasma. Here, we report the development of benziodarone analogues with enhanced potency for selective binding to transthyretin in plasma compared to benziodarone. These analogues featured substituents of chlorine, bromine, iodine, a methyl group, or a trifluoromethyl group, at the 4-position of the benzofuran ring. X-ray crystal structure analysis revealed that CH···O hydrogen bonds and a halogen bond are important for the binding of the compounds to the thyroxine-binding sites. The bioavailability of benziodarone analogues with 4-Br, 4-Cl, or 4-CH3 was comparable to that of tafamidis, a current therapeutic agent for transthyretin amyloidosis.

A charge-neutral organic cage selectively binds strongly hydrated sulfate anions in water.

In biological systems, enzymes and transport proteins can bind anions in aqueous media solely by forming hydrogen bonds with charge-neutral motifs. Reproducing this functionality in synthetic systems presents challenges and incurs high costs, particularly when targeting strongly hydrated anions such as sulfate. Here we report a [2.2.2]urea cryptand (cage), synthesized in one pot, that selectively binds sulfate in a mixture of dimethyl sulfoxide and water and in water with affinities in the micromolar to millimolar range. The neutral cage bearing six urea groups donates 12 strong hydrogen bonds to encapsulate a sulfate anion, showing favourable enthalpy even in pure water. Sulfate binding can be further enhanced by using micelles to provide a low-polarity microenvironment. The cage finds utility in analysing divalent anions in water and beverage samples or in removing sulfate. The work demonstrates the achievability of robust and selective anion binding in water with minimal synthetic efforts, by using neutral NH hydrogen bonds akin to those found in biology.

Rna Buffering Fluorogenic Probe for Nucleolar Morphology Stable Imaging And Nucleolar Stress-Generating Agents Screening.

In the realm of cell research, membraneless organelles have become a subject of increasing interest. However, their ever-changing and amorphous morphological characteristics have long presented a formidable challenge when it comes to studying their structure and function. In this paper, a fluorescent probe Nu-AN is reported, which exhibits the remarkable capability to selectively bind to and visualize the nucleolus morphology, the largest membraneless organelle within the nucleus. Nu-AN demonstrates a significant enhancement in fluorescence upon its selective binding to nucleolar RNA, due to the inhibited twisted intramolecular charge-transfer (TICT) and reduced hydrogen bonding with water. What sets Nu-AN apart is its neutral charge and weak interaction with nucleolus RNA, enabling it to label the nucleolus selectively and reversibly. This not only reduces interference but also permits the replacement of photobleached probes with fresh ones outside the nucleolus, thereby preserving imaging photostability. By closely monitoring morphology-specific changes in the nucleolus with this buffering fluorogenic probe, screenings for agents are conducted that induce nucleolar stress within living cells.