Interactions of HIV‐1 Antibodies 2F5 and 4E10 with a gp41 Epitope Prebound to Host and Viral Membrane Model Systems

Point of recognition : Surface plasmon resonance was used to study the role of the viral membrane in HIV immunogen recognition by the monoclonal antibodies 2F5 and 4E10. The different behaviour of the antibodies towards membranes and immunogens embedded within membranes, provide insight into the emerging membrane‐based or membrane‐conformation strategies of immunogen design.

     

Structure-activity relationships and mechanism of action of Eph-ephrin antagonists: interaction of cholanic acid with the EphA2 receptor

The Eph-ephrin system, including the EphA2 receptor and the ephrinA1 ligand, plays a critical role in tumor and vascular functions during carcinogenesis. We previously identified (3α,5β)-3-hydroxycholan-24-oic acid (lithocholic acid) as an Eph-ephrin antagonist that is able to inhibit EphA2 receptor activation; it is therefore potentially useful as a novel EphA2 receptor-targeting agent. Herein we explore the structure-activity relationships of a focused set of lithocholic acid derivatives based on molecular modeling investigations and displacement binding assays. Our exploration shows that while the 3-α-hydroxy group of lithocholic acid has a negligible role in recognition of the EphA2 receptor, its carboxylate group is critical for disrupting the binding of ephrinA1 to EphA2. As a result of our investigation, we identified (5β)-cholan-24-oic acid (cholanic acid) as a novel compound that competitively inhibits the EphA2-ephrinA1 interaction with higher potency than lithocholic acid. Surface plasmon resonance analysis indicates that cholanic acid binds specifically and reversibly to the ligand binding domain of EphA2, with a steady-state dissociation constant (K(D) ) in the low micromolar range. Furthermore, cholanic acid blocks the phosphorylation of EphA2 as well as cell retraction and rounding in PC3 prostate cancer cells, two effects that depend on EphA2 activation by the ephrinA1 ligand. These findings suggest that cholanic acid can be used as a template structure for the design of effective EphA2 antagonists, and may have potential impact in the elucidation of the role played by this receptor in pathological conditions.     

The Calcium Phosphate Matrix of FGF-2-Apatite Composite Layers Contributes to Their Biological Effects

The purpose of the present study was to fabricate fibroblast growth factor (FGF)-2-apatite composite layers on titanium (Ti) pins in one step at 25 °C using a supersaturated calcium phosphate (CaP) solution, and to evaluate the physicochemical characteristics and biological effects of the coated Ti pins compared with coated Ti pins fabricated at 37 °C. Ti pins were immersed in a supersaturated CaP solution containing 0.5, 1.0, or 2.0 µg/mL FGF-2 at 25 °C for 24 h (25F0.5, 25F1.0, and 25F2.0) or containing 4.0 µg/mL FGF-2 at 37 °C for 48 h (37F4.0). Except for the 25F0.5, the chemical compositions and the mitogenic activity levels of FGF-2 of the composite layers formed by these two methods were similar, except for the Ca/P molar ratio, which was markedly smaller at 25 °C (1.55–1.56 ± 0.01–0.02, p = 0.0008–0.0045) than at 37 °C (1.67 ± 0.11). Thus, either the apatite was less mature or the amount of amorphous calcium phosphate was higher in the composite layer formed at 25 °C. In vivo, the pin tract infection rate by visual inspection for 37F4.0 (45%) was lower than that for 25F1.0 (80%, p = 0.0213), and the rate of osteomyelitis for 37F4.0 (35%) was lower than that for 25F0.5 (75%, p = 0.0341). The extraction torque for 37F4.0 (0.276 ± 0.117 Nm) was higher than that for 25F0.5 (0.192 ± 0.117 Nm, p = 0.0142) and that for 25F1.0 (0.176 ± 0.133 Nm, p = 0.0079). The invasion rate of S. aureus for 37F4.0 (35%) was lower than that for 25F0.5 (75%, p = 0.0110). On the whole, the FGF-2-apatite composite layer formed at 25 °C tended to be less effective at improving fixation strength in the bone-pin interface and resisting pin tract infections. These results suggest that the chemistry of the calcium phosphate matrix that embeds FGF-2, in addition to FGF-2 content and activity, has a significant impact on composite infection resistance and fixation strength.     

X‐ray Structural Analysis of Tau‐Tubulin Kinase 1 and Its Interactions with Small Molecular Inhibitors

Tau‐tubulin kinase 1 (TTBK1) is a serine/threonine/tyrosine kinase that putatively phosphorylates residues including S422 in tau protein. Hyperphosphorylation of tau protein is the primary cause of tau pathology and neuronal death associated with Alzheimer’s disease. A library of 12 truncation variants comprising the TTBK1 kinase domain was screened for expression in Escherichia coli and insect cells. One variant (residues 14–313) could be purified, but mass spectrometric analysis revealed extensive phosphorylation of the protein. Co‐expression with lambda phosphatase in E. coli resulted in production of homogeneous, nonphosphorylated TTBK1. Binding of ATP and several compounds to TTBK1 was characterized by surface plasmon resonance. Crystal structures of TTBK1 in the unliganded form and in complex with ATP, and two high‐affinity ATP‐competitive inhibitors, 3‐[(6,7‐dimethoxyquinazolin‐4‐yl)amino]phenol ( 1 ) and methyl 2‐bromo‐5‐(7H‐pyrrolo[2,3‐d]pyrimidin‐4‐ylamino)benzoate ( 2 ), were elucidated. The structure revealed two clear basic patches near the ATP pocket providing an explanation of TTBK1 for phosphorylation‐primed substrates. Interestingly, compound 2 displayed slow binding kinetics to TTBK1, the structure of TTBK1 in complex with this compound revealed a reorganization of the L199–D200 peptide backbone conformation together with altered hydrogen bonding with compound 2 . These conformational changes necessary for the binding of compound 2 are likely the basis of the slow kinetics. This first TTBK1 structure can assist the discovery of novel inhibitors for the treatment of Alzheimer’s disease.     

Synthesis of a heparan sulfate mimetic library targeting FGF and VEGF via click chemistry on a monosaccharide template

A disulfated methyl 6-azido-6-deoxy-α-D-mannopyranoside template was used as a core structure for binding to the angiogenic growth factors FGF-1, FGF-2, and VEGF. The core structure was diversified in a rapid, parallel manner by employing the Cu(I)-catalyzed Huisgen azide-alkyne cycloaddition ("click") reaction. The diversity was further extended by incorporating a Swern oxidation-Wittig reaction sequence on a click adduct of propargyl alcohol. Thus, the sulfated core was linked by various spacers to selected hydrophobic or polar motifs, which were designed to probe the protein surface surrounding the cationic heparan sulfate binding sites of the growth factors in order to improve affinity and selectivity. The affinities of the compounds for the growth factors were measured by surface plasmon resonance solution affinity assays. A lead compound was identified with micromolar binding affinity toward both FGF-1 and VEGF (K(d)=84 and 49 μM, respectively) and good selectivity over FGF-2 (29- and 51-fold, respectively).     

Growth Factors VEGF-A165 and FGF-2 as Multifunctional Biomolecules Governing Cell Adhesion and Proliferation

Vascular endothelial growth factor-A₁₆₅ (VEGF-A₁₆₅) and fibroblast growth factor-2 (FGF-2) are currently used for the functionalization of biomaterials designed for tissue engineering. We have developed a new simple method for heterologous expression and purification of VEGF-A₁₆₅ and FGF-2 in the yeast expression system of Pichia pastoris. The biological activity of the growth factors was assessed in cultures of human and porcine adipose tissue-derived stem cells (ADSCs) and human umbilical vein endothelial cells (HUVECs). When added into the culture medium, VEGF-A₁₆₅ stimulated proliferation only in HUVECs, while FGF-2 stimulated the proliferation of both cell types. A similar effect was achieved when the growth factors were pre-adsorbed to polystyrene wells. The effect of our recombinant growth factors was slightly lower than that of commercially available factors, which was attributed to the presence of some impurities. The stimulatory effect of the VEGF-A₁₆₅ on cell adhesion was rather weak, especially in ADSCs. FGF-2 was a potent stimulator of the adhesion of ADSCs but had no to negative effect on the adhesion of HUVECs. In sum, FGF-2 and VEGF-A₁₆₅ have diverse effects on the behavior of different cell types, which maybe utilized in tissue engineering.     

An amphiphilic conjugate approach toward the design and synthesis of betulinic acid-polyphenol conjugates as inhibitors of the HIV-1 gp41 fusion core formation

Exploration of potent inhibitors of the HIV-1 gp41 fusion core formation is a promising strategy to discover small-molecule HIV-1 entry inhibitors for the treatment of HIV-1 infection. In this paper, a series of novel betulinic acid-polyphenol conjugates was designed, guided by molecular modeling of the binding of betulinic acid (BA) and phenolic galloyl/caffeoyl groups in the groove on the gp41 N-terminal heptad repeat (NHR) trimeric coiled coil. These conjugates were synthesized via conjugation of galloyl and caffeoyl groups with BA at the C-28 position. Their inhibitory activities of HIV gp41 six-helix bundle (6-HB) formation between the NHR peptide N36 and the C-terminal heptad repeat (CHR) peptide C34 were evaluated with size-exclusion HPLC. Conjugates bearing a galloyl group were found to exhibit four to sixfold higher inhibitory activities than that of parent compound BA, suggesting that they may be exploitable as HIV-1 fusion/entry inhibitors targeting gp41. The docking study on BA and its derivatives suggests that hydrophobic and hydrogen-bonding pockets exist in the groove of the gp41 NHR trimeric coiled coil and that a potent inhibitor should have amphiphilic structures to cooperatively interact with both pockets. This possibility was explored by incorporating both lipophilic and hydrophilic groups into the conjugates in a well-defined orientation to bind with both pockets in the gp41 NHR-trimer.     

Characterisation of the binding of cationic amphiphilic drugs to phospholipid bilayers using surface plasmon resonance

The interactions of three cationic amphiphilic drugs (CPZ, AMI, PROP) with phospholipid vesicles comprising DOPC, DMPC, or DSPC were investigated using surface plasmon resonance (SPR). Responses for CAD concentrations in the range 15.625 to 1500 microM were measured. The greatest uptake by each phospholipid bilayer occurred with CPZ. Inclusion of CAD concentrations between 750 and 1500 microM provided evidence for a second nonsaturable binding process, which may arise from intercalation of the drugs within the lipid bilayer. CAD binding was additionally shown to be dependent on membrane fluidity. Responses were initially fitted over a concentration range of 15.625 to 500 microM using a model which incorporated terms for a saturable binding site. This yielded very poor values of K(D) and nonsensible values of saturation responses. Subsequently, responses were fit to the expression for a model which incorporated terms for both a saturable binding site and second nonsaturable site. Measurable binding affinities (K(D) values ranged from 170 to 814 microM) were obtained for DOPC and DMPC bilayers which are similar to values reported previously. This work demonstrates that SPR studies with synthetic phospholipid bilayers provide a potentially useful approach for characterising drug-membrane binding interactions and for providing insight into the processes that contribute to drug-membrane binding.     

Virtual Drug Repositioning as a Tool to Identify Natural Small Molecules That Synergize with Lumacaftor in F508del-CFTR Binding and Rescuing

Cystic fibrosis is a hereditary disease mainly caused by the deletion of the Phe 508 (F508del) of the cystic fibrosis transmembrane conductance regulator (CFTR) protein that is thus withheld in the endoplasmic reticulum and rapidly degraded by the ubiquitin/proteasome system. Cystic fibrosis remains a potentially fatal disease, but it has become treatable as a chronic condition due to some CFTR-rescuing drugs that, when used in combination, increase in their therapeutic effect due to a synergic action. Also, dietary supplementation of natural compounds in combination with approved drugs could represent a promising strategy to further alleviate cystic fibrosis symptoms. On these bases, we screened by in silico drug repositioning 846 small synthetic or natural compounds from the AIFA database to evaluate their capacity to interact with the highly druggable lumacaftor binding site of F508del-CFTR. Among the identified hits, nicotinamide (NAM) was predicted to accommodate into the lumacaftor binding region of F508del-CFTR without competing against the drug but rather stabilizing its binding. The effective capacity of NAM to bind F508del-CFTR in a lumacaftor-uncompetitive manner was then validated experimentally by surface plasmon resonance analysis. Finally, the capacity of NAM to synergize with lumacaftor increasing its CFTR-rescuing activity was demonstrated in cell-based assays. This study suggests the possible identification of natural small molecules devoid of side effects and endowed with the capacity to synergize with drugs currently employed for the treatment of cystic fibrosis, which hopefully will increase the therapeutic efficacy with lower doses.     

Glycosylation assists binding of HIV protein gp120 to human CD4 receptor

The role of glycosylation of proteins on its binding affinity is not well understood. Even a monosaccharide (magenta) placed at a glycosylation site can significantly enhance binding of peptides to their receptor. If glycosylated, an HIV protein binds stronger and faster to its primary receptors on human cells.     

The LSPR regulation of TiO2: W nanocrystals and its application in enhanced upconversion luminescence

The localized surface plasmon resonance (LSPR) absorption peaks of semiconductor nanocrystals are mainly concentrated in the infrared band, and the absorption characteristics can be controlled by the amount of element doping. The coupling of upconversion nanocrystals (UCNPs) and semiconductor nanocrystals can improve the upconversion luminescence (UCL) of rare-earth ions. Here, the LSPR absorption and morphology of the semiconductor nanocrystalline TiO₂: W were adjusted by using ammonium fluoride during synthesis. Significant absorption enhancement of TiO₂: W in the near-infrared region was obtained to enhance the UCL of NaYF₄: Yb³⁺, Er³⁺. The Glass/NaYF₄: Yb³⁺, Er³⁺/TiO₂: W@SiO₂ layered structure films were fabricated through spin coating. Compared with Glass/NaYF₄: Yb³⁺, Er³⁺, the green and red lights of the Glass/NaYF₄: Yb³⁺, Er³⁺/TiO₂: W@SiO₂ films were enhanced by 15.9 and 17.8 times, respectively. The UCL enhancement of Glass/NaYF₄: Yb³⁺, Er³⁺/TiO₂: W@SiO₂ was derived from the LSPR property of TiO₂: W through the enhancement of the excitation. The present work is important for possible applications of these layered structures as biomarkers, photocatalysts, flexible materials, and photoluminescence display panels.     

A new concept for glycosyltransferase inhibitors: nonionic mimics of the nucleotide donor of the human blood group B galactosyltransferase

Glycosyltransferases play an important role in the formation of oligosaccharides and glycoconjugates. To find suitable and selective inhibitors for this class of enzymes is still challenging. Here, we describe a novel concept that allows the design of inhibitors based on the structure of the donor substrate binding pocket. As a first step we describe the design, synthesis and analysis of inhibitors of the human blood group B galactosyltransferase (GTB). This enzyme served as a model system to study the concept, which can be used for easy access of glycosyltransferase inhibitors in general. In silico docking of bicyclic heteroaromatic ligands to GTB and experimental verification of binding affinities by saturation transfer difference NMR (STD NMR) spectroscopy gave 9-N-pentityl uric acid derivatives as non-ionic mimics of UDP. Two derivatives were synthesized and showed inhibitory activity for GTB as determined by competitive STD NMR experiments and by a radiolabeled enzyme assay.     

Preparation of exposed (001) facets TiO2/Ti3C2 MXene and performance of near infrared light photodegradation

The (001)TiO₂/Ti₃C₂ MXene heterojunction photocatalyst was prepared with the hydrothermal oxidation method by using the two-dimensional Ti₃C₂ MXene as the substrate. The formation of TiO₂ surface heterojunctions, Schottky junctions at the interface between (001) facets TiO₂ and Ti₃C₂ MXene, and the local surface plasmon resonance effect of plasma Ti₃C₂ MXene combine together to promote the effective separation of electrons and holes after photoexcitation. After 150 min of near infrared light irradiation and sunlight irradiation, the degradation rates of tetracycline by (001)TiO₂/Ti₃C₂ MXene photocatalyst are 28.63% and 52.74%, respectively, with near infrared light and full spectrum degradation characteristics. Our work is expected to provide a valuable reference for the design of MXene based full spectrum photocatalysts.     

Modulation of CD14 and TLR4⋅MD‐2 Activities by a Synthetic Lipid A Mimetic

Monosaccharide lipid A mimetics based on a glucosamine core linked to two fatty acid chains and bearing one or two phosphate groups have been synthesized. Compounds 1 and 2 , each with one phosphate group, were practically inactive in inhibiting LPS‐induced TLR4 signaling and cytokine production in HEK‐blue cells and murine macrophages, but compound 3 , with two phosphate groups, was found to be active in efficiently inhibiting TLR4 signal in both cell types. The direct interaction between compound 3 and the MD‐2 coreceptor was investigated by NMR spectroscopy and molecular modeling/docking analysis. This compound also interacts directly with the CD14 receptor, stimulating its internalization by endocytosis. Experiments on macrophages show that the effect on CD14 reinforces the activity on MD‐2 ? TLR4 because compound 3 's activity is higher when CD14 is important for TLR4 signaling (i.e., at low LPS concentration). The dual targeting of MD‐2 and CD14, accompanied by good solubility in water and lack of toxicity, suggests the use of monosaccharide 3 as a lead compound for the development of drugs directed against TLR4related syndromes.     

Peptide Fragments of Odin‐Sam1: Conformational Analysis and Interaction Studies with EphA2‐Sam

Odin is a protein belonging to the ANKS family, and has two tandem Sam domains. The first, Odin‐Sam1, binds to the Sam domain of the EphA2 receptor (EphA2‐Sam); this interaction could be crucial for the regulation of receptor endocytosis and might have an impact on cancer. Odin‐Sam1 associates with EphA2‐Sam by adopting a “mid‐loop/end‐helix” model. In this study three peptide sequences, encompassing the mid‐loop interacting portion of Odin‐Sam1 and its C‐terminal α5 helix, were designed. Their conformational properties were analyzed by CD and NMR. In addition, their abilities to interact with EphA2‐Sam were investigated by SPR studies. The peptides adopt a predominantly disordered state in aqueous buffer, but a higher helical content is evident in the presence of the cosolvent trifluoroethanol. Dissociation constants towards EphA2‐Sam were in the high micromolar range. The structural findings suggest further routes for the design of potential anti‐cancer therapeutics as inhibitors of EphA2‐Sam heterotypic interactions.     

An experimental and theoretical understanding of a UV photodetector based on Ag nanoparticles decorated Er-doped TiO2 thin film

Glancing angle deposition (GLAD) was employed to synthesise plasmonic Silver (Ag) nanoparticles (NPs) on the chemically prepared Erbium-doped Titanium dioxide (Er:TiO₂) thin films (TFs). The impact of using Ag NPs on the morphological, optical, and electrical aspects of Er:TiO₂ TFs were sequentially analysed. From the field emission scanning electron microscopy (FESEM) image, the Ag NPs appeared spherical and uniformly distributed on the Er:TiO₂ TFs. The size (diameter) of the maximum number of Ag NPs was ~15 nm (calculated from FESEM image). Energy dispersive X-ray (EDX) spectra assured the presence of Ag NPs on the TFs. X-ray diffraction (XRD) pattern for Ag NPs decorated Er:TiO₂ TFs closely resembled the face centred cubic crystal structure of Ag NPs and body centred tetragonal Ag–O compound. The optical spectroscopy (UV–visible diffuse reflectance and photoluminescence) elucidated that the absorption of light was significantly enhanced in the UV–visible spectral range for the TFs in which Ag NPs were sandwiched between Er:TiO₂ TF layers (Er:TiO₂/Ag NPs/Er:TiO₂). The Schottky contact-based Au/Er:TiO₂/Si photodetector (PD) and Au/Er:TiO₂/Ag NPs/Er:TiO₂/Si (plasmonic) PD were constructed. The plasmonic PD offered a better photo-responsivity of ~4.5 fold higher as compared to Er:TiO₂ TF-based PD upon 380 nm illumination under ?6 V bias. An increase in detectivity and a decrease in noise equivalent power was observed for the plasmonic device compared to Er:TiO₂ device in the UV region. A theoretical approach had been adopted to calculate the wavelength-dependent responsivity for both devices. Further, the important parameters like photoconductive gain, electron transit time and electron mobility were calculated by simulating the experimental responsivity curves of the devices. These parameters exhibited improvement in the UV regime for the plasmonic PD. The fast temporal response with short rise and decay time proves the excellent efficiency of the plasmonic UV PD.