Binding Experiments Research Articles

Small Molecule Fluorescent Ligands for the Atypical Chemokine Receptor 3 (ACKR3).

The atypical chemokine receptor 3 (ACKR3) is a receptor that induces cancer progression and metastasis in multiple cell types. Therefore, new chemical tools are required to study the role of ACKR3 in cancer and other diseases. In this study, fluorescent probes, based on a series of small molecule ACKR3 agonists, were synthesized. Three fluorescent probes, which showed specific binding to ACKR3 through a luminescence-based NanoBRET binding assay (pKd ranging from 6.8 to 7.8) are disclosed. Due to their high affinity at the ACKR3, we have shown their application in both competition binding experiments and confocal microscopy studies showing the cellular distribution of this receptor.

Coordination Chemistry of Sulfur-Containing Bifunctional Chelators: Toward in Vivo Stabilization of 64Cu PET Imaging Agents for Alzheimer's Disease.

The broader utilization of 64Cu positron emission tomography (PET) imaging agents has been hindered by the unproductive demetalation induced by bioreductants. To advance the development of 64Cu-based PET imaging tracers for Alzheimer's Disease (AD), there is a need for novel ligand design strategies. In this study, we developed sulfur-containing dithiapyridinophane (N2S2) bifunctional chelators (BFCs) as well as all nitrogen-based diazapyridinophane (N4) BFCs to compare their abilities to chelate Cu and target Aβ aggregates. Through spectrophotometric titrations and electrochemical measurements, we have demonstrated that the N2S2-based BFCs exhibit >10 orders of magnitude higher binding affinity toward Cu(I) compared to their N4-based counterparts, while both types of BFCs exhibit high stability constants toward Cu(II). Notably, solid state structures for both Cu(II) and Cu(I) complexes supported by the two ligand frameworks were obtained, providing molecular insights into their copper chelating abilities. Aβ binding experiments were conducted to study the structure-affinity relationship, and fluorescence microscopy imaging studies confirmed the selective labeling of the BFCs and their copper complexes. Furthermore, we investigated the potential of these ligands for the 64Cu-based PET imaging of AD through radiolabeling and autoradiography studies. We believe our findings provide molecular insights into the design of bifunctional Cu chelators that can effectively stabilize both Cu(II) and Cu(I) and, thus, can have significant implications for the development of 64Cu PET imaging as a diagnostic tool for AD.

Umbrella-like Helical Structure of α-Synuclein at the Air-Water Interface Observed with Experimental and Theoretical Sum Frequency Generation Spectroscopy.

The misfolding of α-synuclein (αS) into amyloid aggregates is catalyzed by hydrophobic surfaces and associated with severe brain disorders, such as Parkinson's disease. Despite the important role of interfaces, the three-dimensional structure of αS at the interfaces is still not clear. We report interface-specific sum frequency generation (SFG) experiments of monomeric αS binding to the air-water interface, a model system for the important hydrophobic surfaces. We combine the SFG spectra with calculations of theoretical spectra based on molecular dynamics simulations to show that αS, which is an intrinsically disordered protein in solution, folds into a defined, mostly helical secondary structure at the air-water interface. The binding pose resembles an umbrella shape, where the C-terminus protrudes into the water phase, while the N-terminus and the NAC region span the canopy at the interface. In this binding pose, αS is prone to aggregate, which could explain the catalytic effect of hydrophobic interfaces and air bubbles on αS fibrillation.

Surface interaction of vancomycin with polystyrene microplastics and its effect on human serum albumin

Microplastics have a well-documented ability to adsorb various chemicals and contaminants found in the environment. By similar mechanisms, when medicines are stored in plastic packaging, the leaching of plastics into the contents poses the risk of possible toxicity and decreased drug efficacy. The work thus examines the presence of two categories of anthropogenic materials – microplastics (MPs) and medications - with their possible combined effects and fate in biological systems. A study on the kinetics and isotherm of the adsorption of vancomycin hydrochloride on the surface of polystyrene microspheres is performed, and the best-fitting models are obtained respectively as the pseudo-second-order model and the Temkin isotherm. Further, the interaction of each of, the drug, MPs and drug-adsorbed MPs with human serum albumin (HSA), the model protein chosen to validate the potential toxicity in humans, is determined by fluorescence spectroscopy. A thermodynamic analysis of this protein-ligand interaction shows that the process is spontaneous, endothermic and entropically favoured, and that hydrophobic forces operate between the interacting species. An unfolding of HSA is observed, disrupting its functions like the esterase activity. Competitive binding experiments with Warfarin and Ibuprofen as specific site markers on HSA reveal that all the studied ligands bind non-specifically to HSA.

Development of a neutralization monoclonal antibody with a broad neutralizing effect against SARS-CoV-2 variants

BackgroundThe emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has challenged the effectiveness of current therapeutic regimens. Here, we aimed to develop a potent SARS-CoV-2 antibody with broad neutralizing effect by screening a scFv library with the spike protein receptor-binding domain (RBD) via phage display.MethodsSKAI-DS84 was identified through phage display, and we performed pseudovirus neutralization assays, authentic virus neutralization assays, and in vivo neutralization efficacy evaluations. Furthermore, surface plasmon resonance (SPR) analysis was conducted to assess the physical characteristics of the antibody, including binding kinetics and measure its affinity for variant RBDs.ResultsThe selected clones were converted to human IgG, and among them, SKAI-DS84 was selected for further analyses based on its binding affinity with the variant RBDs. Using pseudoviruses, we confirmed that SKAI-DS84 was strongly neutralizing against wild-type, B.1.617.2, B.1.1.529, and subvariants of SARS-CoV-2. We also tested the neutralizing effect of SKAI-DS84 on authentic viruses, in vivo and observed a reduction in viral replication and improved lung pathology. We performed binding and epitope mapping experiments to understand the mechanisms underlying neutralization and identified quaternary epitopes formed by the interaction between RBDs as the target of SKAI-DS84.ConclusionsWe identified, produced, and tested the neutralizing effect of SKAI-DS84 antibody. Our results highlight that SKAI-DS84 could be a potential neutralizing antibody against SARS-CoV-2 and its variants.

Recombinant expression and immune function analysis of C-reactive protein (CRP) from Hexagrammos otakii

C-reactive protein (CRP) belongs to the short-chain pentraxin family and functions as a soluble pattern recognition molecule (PRM) aiding in host defense against pathogens. In the present study, a CRP gene, designated HoCRP, was cloned from Hexagrammos otakii for the first time. The full length of the HoCRP cDNA sequence is 821 bp, which contains an open reading frame (ORF) of 675 bp encoding a 224 amino acid protein. The deduced protein is predicted to have a theoretical isoelectric point (pI) of 5.30 and a molecular weight of 25.4 kDa. The recombinant HoCRP protein (rHoCRP) was expressed in E. coli to further characterize the functions of HoCRP. Saccharide binding experiments demonstrated that rHoCRP exhibited a high affinity for various pathogen-associated molecular patterns (PAMPs). Furthermore, bacterial binding and agglutination assays indicated that rHoCRP had the capability to recognize a wide spectrum of microorganisms. These findings suggest that HoCRP functions not only as a PRM for binding PAMPs but also as an immune effector molecule. Considering the role CRP plays in the classical complement pathway, the interaction between rHoCRP and rHoC1q was assessed and proven by a Pull-down and Elisa assay, which implied that rHoCRP may be able to activate complement. In addition, phagocytosis enhancement by rHoCRP in the presence or absence of complement components was analysed by flow cytometry. The results showed that rHoCRP could synergistically enhance the phagocytosis of RAW264.7 cells with complement, providing further evidence of complement activation by rHoCRP through the opsonization of specific complement components. In summary, our findings suggest that rHoCRP may play a crucial role in host antibacterial defense by recognizing pathogens, activating the complement system, and enhancing macrophage function.

Biophysical Characterization of the Thermodynamic Basis and Binding Orientation for Factor VIII Membrane Association

After vesicular damage occurs, factor VIII (fVIII) is proteolytically activated by thrombin and binds to activated factor IX (fIX) on the surface of activated platelets during the blood coagulation cascade to form the intrinsic “tenase” complex. Once activated, fVIII dissociates from von Willebrand factor and C1 and C2 domains bind to activated platelet surfaces. Previous studies have identified that the C1 and C2 domains bind to negatively charged membranes through both surface exposed hydrophobic residues located in the beta hairpin loops and adjacent positively charged residues. The thermodynamics of fVIII lipid binding are currently uncharacterized and our current understanding of the binding orientation of the C1 and C2 domains of fVIII on lipid membranes is incomplete. In this study, we investigate the thermodynamics and binding orientation of the C domains via mutagenesis to both surface exposed hydrophobic residues and adjacent charged residues. Binding measurements between phosphatidylserine (PS) containing lipid nanodiscs and fVIII C domains have revealed that the C1 domain lipid binding is driven by enthalpic interactions, whereas the C2 domain binding is driven by entropic interactions. Binding experiments on fVIII C2 domain mutants for both surface exposed hydrophobic and positively charged residues have revealed differential disruption to lipid binding. Lastly, we have utilized molecular dynamics to perform simulations on the C1 and C2 domains binding to lipid nanodiscs to elucidate the binding orientation of the C domains and identify novel residues that interact with the membrane. Our results from these simulations agree with previous work, showing that C domain lipid binding centers on R2163 (C1) and R2320 (C2).

Impact of Recombinant Factor VIII and Platelet Interaction on Platelet Functionality and Hemophilia a Treatment

Background: Platelets play an important role throughout the various stages of hemostasis. After vascular injury, platelets become activated in a pro-aggregatory state, and subsequently a subpopulation undergoes a phenotype shift to a pro-coagulant state. Pro-coagulant platelets bind to factor VIII (FVIII) and ensure efficient localization of FVIII at the site of injury. It is unclear whether modifications in recombinant FVIII (rFVIII) products impact FVIII-platelet binding and subsequent platelet signaling. Aims: To examine binding of different rFVIII products to platelets in vitro, and the impact of rFVIII on platelet phenotype and intracellular signaling. Methods: Platelet activation: Platelets isolated from healthy donors were activated with thrombin and cross-linked collagen-related peptide (CRP-XL). Platelet-FVIII binding: Activated platelets were incubated with simoctocog alfa, efmoroctocog alfa, rurioctocog alfa pegol or damoctocog alfa pegol. Binding was quantified by flow cytometry either by immunostaining with an anti-FVIII antibody conjugated to Alexa fluor (AF)647 or direct measurement of rFVIII concentrates labelled with AF647. Phenotype shift: During the shift to a pro-coagulant state, phosphatidylserine (PS) becomes exposed on the platelet membranes, with the extent of PS exposure correlating with the level of FVIII binding. To assess PS exposure, activated platelets were incubated with Annexin V-BV421, a molecular probe with high affinity for PS. Inhibition of integrin αIIbβ3 signaling: Activated platelets were incubated with the integrin αIIbβ3 inhibitor antibody 10E5. Results: Binding to pro-coagulant platelets was significantly higher with simoctocog alfa than with efmoroctocog alfa, rurioctocog alfa pegol or damoctocog alfa pegol irrespective of immunostaining method (p<0.05; Figure 1 shows detection with anti-FVIII antibody conjugated with AF647).To validate the variability in rFVIII-platelet interactions observed, we performed a dynamic platelet-binding assay with the two FVIII products exhibiting the strongest binding (simoctocog alfa and efmoroctocog alfa). The results of the dynamic assay confirmed the results of the static experiments (Figure 1). Exposure of activated platelets to simoctocog alfa and efmoroctocog alfa resulted in increased PS exposure, with a greater effect observed with simoctocog alfa, consistent with the results of the binding experiments. Immunostaining experiments revealed co-localization of simoctocog alfa and clusters of integrin αIIbβ3 in pro-aggregatory platelets. Inhibition of integrin αIIbβ3 by 10E5 decreased binding of simoctocog alfa to platelets and subsequent PS exposure in a dose-dependent manner (Figure 2). Analysis of platelet binding and phenotype shift in patients with hemophilia A are ongoing. Conclusion: Simoctocog alfa demonstrated higher binding to activated platelets in vitro compared with efmoroctocog alfa, rurioctocog alfa pegol or damoctocog alfa pegol, resulting in an increased phenotype shift of platelets from the pro-aggregatory to the pro-coagulant state. The increased binding of simoctocog alfa was associated with a phenotypic shift in platelets as evidenced by increased exposure of PS on the platelet membranes. The binding of simoctocog alfa to platelets was disrupted when integrin αIIbβ3 activation was inhibited, suggesting a role of integrin αIIbβ3 signaling following binding of FVIII to platelets. Variations in platelet binding and signaling between different rFVIIIs might impact their efficacy for the prevention of bleeds. Further experiments are ongoing to assess the impact of FVIII on the release of von Willebrand factor and thrombin generation by activated platelets, and to repeat the FVIII-binding experiments using platelets from healthy donors vs hemophilia A patients.

Synthesis and biochemical evaluation of 17-N-beta-aminoalkyl-4,5α-epoxynormorphinans

Opiate alkaloids and their synthetic derivatives are still widely used in pain management, drug addiction, and abuse. To avoid serious side effects, compounds with properly designed pharmacological profiles at the opioid receptor subtypes are long needed. Here a series of 17-N-substituted derivatives of normorphine and noroxymorphone analogues with five- and six-membered ring substituents have been synthesized for structure–activity study. Some compounds showed nanomolar affinity to MOR, DOR and KOR in in vitro competition binding experiments with selective agonists [3H]DAMGO, [3H]Ile5,6-deltorphin II and [3H]HS665, respectively. Pharmacological characterization of the compounds in G-protein signaling was determined by [35S]GTPγS binding assays. The normorphine analogues showed higher affinity to KOR compared to MOR and DOR, while most of the noroxymorphone derivatives did not bind to KOR. The presence of 14-OH substituent resulted in a shift in the pharmacological profiles in the agonist > partial agonist > antagonist direction compared to the parent compounds. A molecular docking-based in silico method was also applied to estimate the pharmacological profile of the compounds. Docking energies and the patterns of the interacting receptor atoms, obtained with experimentally determined active and inactive states of MOR, were used to explain the observed pharmacological features of the compounds.

Functional SNPs in SYISL promoter significantly affect muscle fiber density and muscle traits in pigs.

Our previous studies showed that SYISL is a negative regulator of muscle growth and regeneration in mice, pigs and humans. SYISL knockout resulted in an increase in the density of muscle fibers and muscle growth. However, it is unclear whether there are natural mutations in pig SYNPO2 intron sense-overlapping lncRNA (pSYISL) that affect the expression of pSYISL and muscle growth traits. In this study, three SNPs in exons and six SNPs within the promoter of pSYISL were identified. Association analysis showed that the two SNPs in exons are significantly associated with loin muscle area (p < 0.05); the six SNPs in the promoter that show complete linkage are significantly associated with live backfat thickness and live loin muscle area in American Large White pigs. Bioinformatics and luciferase reporter assays as well as in vitro binding experiments indicated that the mutation of SNP rs702045770 (g.539G>A) leads to the loss of YY1 binding to the promoter, thus affecting the expression level of pSYISL, and we found that Jiangshan Black pigs with genotype GG have a higher expression level of pSYISL than genotype AA individuals, but the muscle fiber density was significantly lower than in genotype AA individuals. Furthermore, the association analysis showed that the carcass backfat thickness of genotype GG of SNP rs702045770 was significantly higher than that of other genotypes in (Pietrain × Duroc) × (Landrace × Yorkshire) crossbred pigs (p < 0.05). The glycolytic potential of genotype GG was significantly higher than that of other genotypes (p < 0.05). These results provide novel insight into the identification of functional SNPs in non-coding genomic regions.

O2 adsorption on Fe3O4 (110) surface and effect of gangue element Al doping: combined study of binding experiment and ab initio molecular dynamics

O2 adsorption on Fe3O4 (110) surface and effect of gangue element Al doping: combined study of binding experiment and ab initio molecular dynamics

Exploiting the Catenane Mechanical Bond Effect for Selective Halide Anion Transmembrane Transport.

The first examples of [2]catenanes capable of selective anion transport across a lipid bilayer are reported. The neutral halogen bonding (XB) [2]catenanes were prepared via a chloride template-directed strategy in an unprecedented demonstration of using XB⋅⋅⋅anion interactions to direct catenane assembly from all-neutral components. Anion binding experiments in aqueous-organic solvent media revealed strong halide over oxoanion selectivity, and a marked enhancement in the chloride and bromide affinities of the catenanes relative to their constituent macrocycles. The catenanes additionally displayed an anti-Hofmeister binding preference for bromide over the larger iodide anion, illustrating the efficacy of employing sigma-hole interactions in conjunction with the mechanical bond effect to tune receptor selectivity. Transmembrane anion transport studies conducted in POPC LUVs revealed that the catenanes were more effective anion transporters than the constituent macrocycles, with high chloride over hydroxide selectivity, which is critical to potential therapeutic applications of anionophores. Remarkably these outperform existing acyclic halogen bonding anionophores with regards to this selectivity. Record chloride over nitrate anion transport selectivity was also observed. This represents a rare example of the direct translation of intrinsic anion binding affinities to anion transport behaviour, and demonstrates the key role of the catenane mechanical bond effect for enhanced anion transport selectivity.

Exploiting the Catenane Mechanical Bond Effect for Selective Halide Anion Transmembrane Transport

AbstractThe first examples of [2]catenanes capable of selective anion transport across a lipid bilayer are reported. The neutral halogen bonding (XB) [2]catenanes were prepared via a chloride template‐directed strategy in an unprecedented demonstration of using XB⋅⋅⋅anion interactions to direct catenane assembly from all‐neutral components. Anion binding experiments in aqueous‐organic solvent media revealed strong halide over oxoanion selectivity, and a marked enhancement in the chloride and bromide affinities of the catenanes relative to their constituent macrocycles. The catenanes additionally displayed an anti‐Hofmeister binding preference for bromide over the larger iodide anion, illustrating the efficacy of employing sigma‐hole interactions in conjunction with the mechanical bond effect to tune receptor selectivity. Transmembrane anion transport studies conducted in POPC LUVs revealed that the catenanes were more effective anion transporters than the constituent macrocycles, with high chloride over hydroxide selectivity, which is critical to potential therapeutic applications of anionophores. Remarkably these outperform existing acyclic halogen bonding anionophores with regards to this selectivity. Record chloride over nitrate anion transport selectivity was also observed. This represents a rare example of the direct translation of intrinsic anion binding affinities to anion transport behaviour, and demonstrates the key role of the catenane mechanical bond effect for enhanced anion transport selectivity.

Discovery of a Tritiated Radioligand with High Affinity and Selectivity for the Histamine H3 Receptor.

Radioligands used previously for histamine H3 receptor (H3R) are accompanied by a number of disadvantages. In this study, we report the synthesis of the new H3R radioligand [3H]UR-MN259 ([3H]11) with high (radio)chemical purity and stability. The radioligand exhibits sub-nanomolar affinity for the target receptor (pKi (H3R) = 9.56) and displays an outstanding selectivity profile within the histamine receptor family (>100,000-fold selective). [3H]UR-MN259 is ideally suitable for the characterization of H3R ligands in competition binding and shows one-site binding to the H3R in saturation binding experiments. The radiotracer shows fast association to the receptor (τassoc = 6.11 min), as well as full dissociation from the receptor (τdissoc = 14.48 min) in kinetic binding studies. The distinguished profile of [3H]UR-MN259 makes it a highly promising pharmacological tool to further investigate the role of the H3R in the CNS.

Design of an Anti-HMGB1 Synthetic Antibody for In Vivo Ischemic/Reperfusion Injury Therapy.

High-mobility group box 1 (HMGB1) is a multifunctional protein. Upon injury or infection, HMGB1 is passively released from necrotic and activated dendritic cells and macrophages, where it functions as a cytokine, acting as a ligand for RAGE, a major receptor of innate immunity stimulating inflammation responses including the pathogenesis of cerebral ischemia/reperfusion (I/R) injury. Blocking the HMGB1/RAGE axis offers a therapeutic approach to treating these inflammatory conditions. Here, we describe a synthetic antibody (SA), a copolymer nanoparticle (NP) that binds HMGB1. A lightly cross-linked N-isopropylacrylamide (NIPAm) hydrogel copolymer with nanomolar affinity for HMGB1 was selected from a small library containing trisulfated 3,4,6S-GlcNAc and hydrophobic N-tert-butylacrylamide (TBAm) monomers. Competition binding experiments with heparin established that the dominant interaction between SA and HMGB1 occurs at the heparin-binding domain. In vitro studies established that anti-HMGB1-SA inhibits HMGB1-dependent ICAM-1 expression and ERK phosphorylation of HUVECs, confirming that SA binding to HMGB1 inhibits the proteins' interaction with the RAGE receptor. Using temporary middle cerebral artery occlusion (t-MCAO) model rats, anti-HMGB1-SA was found to accumulate in the ischemic brain by crossing the blood-brain barrier. Significantly, administration of anti-HMGB1-SA to t-MCAO rats dramatically reduced brain damage caused by cerebral ischemia/reperfusion. These results establish that a statistical copolymer, selected from a small library of candidates synthesized using an "informed" selection of functional monomers, can yield a functional synthetic antibody. The knowledge gained from these experiments can facilitate the discovery, design, and development of a new category of drug.

Tailoring stimuli-responsive PVDF-based copolymer membrane with engineered pore structure for efficient antibody purification

Membrane chromatography is considered as a sustainable alternative for downstream purification to overcome the production bottleneck of biopharmaceuticals. However, two of the major factors impeding its industrial adoption are the lack of controllable functionalization and competitive binding capacity. This work aimed to develop a one-pot synthesis strategy for fabricating a new class of pH-responsive poly(vinylidene fluoride)-graft-poly(dimethylaminoethyl methacrylate) (PVDF-g-PDMAEMA) membranes with anion exchangers, to achieve fast and accurate removal of albumin impurities from antibody streams. By utilizing a pre-functionalized copolymer with controlled ligand density, PVDF-g-PDMAEMA membranes with advantageous bicontinuous pore network structure were obtained via an unconventional nonsolvent thermally induced phase separation method. The membrane showed a competitive water permeance of > 6000 L m-2h−1 bar−1. Combining with dedicated static binding and isotherm studies, the membranes demonstrated distinct pH-responsive binding behaviour to model impurity bovine serum albumin (BSA) with maximal binding of 318.0 mg/g at pH 5.5. The dynamic flow-through binding experiments indicated that the membrane could effectively capture BSA from immunoglobulin G (IgG) (model antibody)/BSA mixed solution with a competitive binding capacity of 210.5 mg/g, producing IgG with purity > 90 % and nearly full recovery. Also, comprehensive analysis demonstrated the robustness of the membrane performance through five consecutive operation cycles, with or without harsh alkaline regeneration. The biocompatibility analysis further proved the membranes’ suitability in biopharmaceutical applications. This study provided a new route for designing future chromatographic membranes to promote its industrial uptake, pushing for energy-saving and effective downstream bioprocessing.

IdeS, a secreted proteinase of Streptococcus pyogenes, is bound to a nuclease at the bacterial surface where it inactivates opsonizing IgG antibodies

The important bacterial pathogen Streptococcus pyogenes secretes IdeS (immunoglobulin G-degrading enzyme of S.pyogenes), a proteinase that cleaves human immunoglobulin G (IgG) antibodies in the hinge region resulting in Fc (fragment crystallizable) and F(ab')2 (fragment antigen-binding) fragments and protects the bacteria against phagocytic killing. Experiments with radiolabeled IdeS and flow cytometry demonstrated that IdeS binds to the surface of S.pyogenes, and the interaction was most prominent in conditions resembling those in the pharynx (acidic pH and low salt), the habitat for S.pyogenes. SpnA (S. pyogenes nuclease A) is a cell wall-anchored DNase. A dose-dependent interaction between purified SpnA and IdeS was demonstrated in slot binding and surface plasmon resonance spectroscopy experiments. Gel filtration showed that IdeS forms proteolytically active complexes with SpnA in solution, and super-resolution fluorescence microscopy revealed the presence of SpnA-IdeS complexes at the surface of S.pyogenes. Finally, specific IgG antibodies binding to S.pyogenes surface antigens were efficiently cleaved by surface-associated IdeS. IdeS is secreted by all S.pyogenes isolates and cleaves IgG antibodies with a unique degree of specificity and efficiency. These properties and the finding here that the proteinase is present and fully active at the bacterial surface in complex with SpnA implicate an important role for IdeS in S.pyogenes biology and pathogenesis.

A human monoclonal antibody binds within the poliovirus receptor-binding site to neutralize all three serotypes

Global eradication of poliovirus remains elusive, and it is critical to develop next generation vaccines and antivirals. In support of this goal, we map the epitope of human monoclonal antibody 9H2 which is able to neutralize the three serotypes of poliovirus. Using cryo-EM we solve the near-atomic structures of 9H2 fragments (Fab) bound to capsids of poliovirus serotypes 1, 2, and 3. The Fab-virus complexes show that Fab interacts with the same binding mode for each serotype and at the same angle of interaction relative to the capsid surface. For each of the Fab-virus complexes, we find that the binding site overlaps with the poliovirus receptor (PVR) binding site and maps across and into a depression in the capsid called the canyon. No conformational changes to the capsid are induced by Fab binding for any complex. Competition binding experiments between 9H2 and PVR reveal that 9H2 impedes receptor binding. Thus, 9H2 outcompetes the receptor to neutralize poliovirus. The ability to neutralize all three serotypes, coupled with the critical importance of the conserved receptor binding site make 9H2 an attractive antiviral candidate for future development.

Enhancing the Treatment of Uncontrolled Inflammation through the Targeted Delivery of TPCA-1-Loaded Nanoparticles.

Uncontrolled inflammation is a pathological state that underlies many diseases. Despite the development of numerous anti-inflammatory agents, the treatment of uncontrolled inflammation remains a challenging task. We developed a targeted delivery system for [5-(p-fluorophenyl)-2-ureido]thiophene-3-carboxamide (TPCA-1), a potent inhibitor of the NF-κB signaling pathway. The system comprises TPCA-1-loaded nanoparticles (NPs) functionalized with a monoclonal antibody (mAb) that specifically binds to the break point of the IgD6 region of the platelet/endothelial cell adhesion molecule-1 (PECAM-1) extracellular segment that is overexposed on the injured endothelium and activated macrophages during the pathogenesis of inflammation. In vitro binding and cellular uptake experiments revealed that the mAb modification on NPs could significantly enhance uptake by both Raw264.7 and HUVEC compared with unmodified NPs. In studies conducted at the cellular level focusing on anti-inflammatory and antioxidant effects, this formulation was found to effectively inhibit M1 polarization of macrophages, downregulate the secretion of pro-inflammatory cytokines, and reduce the production of reactive oxygen species (ROS) and nitric oxide (NO). In an animal model of vascular endothelial injury with acute inflammation, these NPs were capable of delivering TPCA-1 to inflammatory lesions in a targeted manner. Compared with the free agent-treated group, the NP-treated group exhibited reduced infiltration of inflammatory cells. In conclusion, our study demonstrates that this targeted delivery of TPCA-1-loaded NPs represents a promising strategy for improved mitigation of uncontrolled inflammation.

A Closed Cavity Strategy for Selective Dipeptide Binding by a Polyaromatic Receptor in Water.

Precise recognition of peptides is a daunting task owing to the substantial number of available amino acids and their combination into various oligo/polymeric structures in addition to the high hydration of their flexible frameworks. Here, we report the selective recognition of a dipeptide through a closed cavity strategy, in contrast to previous synthetic receptors with open cavities. A polyaromatic receptor with a virtually isolated, hydrophobic cavity exclusively binds one molecule of phenylalanine dipeptide from a mixture with its amino acid and tripeptide in water via multiple CH-π and hydrogen-bonding interactions in the complementary cavity. The binding selectivity persists even in the presence of other dipeptides, such as leucine-leucine, leucine-phenylalanine, tyrosine-phenylalanine, tryptophan-tryptophan, and aspartame, revealed by NMR/MS-based competitive binding experiments. ITC studies reveal that the selective binding of the phenylalanine dipeptide is relatively strong (Ka = 1.1 × 105 M-1) and an enthalpically and entropically favorable process (ΔH = -11.7 kJ mol-1 and TΔS = 17.0 kJ mol-1). In addition, the present receptor can be used for the emission detection of the dipeptide through a combination with a fluorescent dye in water.