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To investigate the effect of vidian trunk neurectomy and selective vidian branch neurectomy on treating moderate-to-severe persistent allergic rhinitis. Sixty patients with moderate-to-severe persistent allergic rhinitis treated at Zhejiang Hospital of Integrative Medicine participated in this study from June 2018 to June 2020. The patients in the observation group (n = 40) underwent a vidian trunk neurectomy. The patients in the control group (n = 20) underwent a vidian branch neurectomy. The patients in both groups were followed up after 6 months, 1 year, and 2 years of surgery. The efficacy was evaluated based on the AR diagnostic and the efficacy assessment criteria. The 4 symptoms of sneezing, runny nose, nasal congestion, and nasal itch were scored as efficacy indices before and 6 months, 1 year, and 2 years after surgery. Nasal endoscopy or sinus CT was performed to assess the postoperative inferior turbinate swelling. The postoperative tear secretion was followed up in both groups, and patients with dry eyes were counted in combination with ocular symptoms. The data recorded before and after surgery and between the 2 groups were analyzed statistically. The observation group had signs of 2.73 ± 0.452 before surgery, 1.20 ± 0.405 6 months after surgery, 1.25 ± 0.494 1 year after surgery, and 1.30 ± 0.564 2 years after surgery. The control group had signs of 2.75 ± 0.444 before surgery, 1.45 ± 0.686 6 months after surgery, 1.75 ± 0.716 1 year after surgery, and 1.90 ± 0.852 2 years after surgery. The between-subjects effect test between the groups showed an overall significant difference (P .05). The overall effective rate 2 years postoperatively was 38/40 (95.0%) in the observation group and 10/20 (50%) in the control group. Fisher's exact test showed a significant difference between the groups. No patient in either group had dry eyes 1.5 years after surgery. Both vidian trunk neurectomy and selective vidian branch neurectomy have good immediate therapeutic effects, and vidian trunk neurectomy has higher long-term efficacy than selective vidian branch neurectomy.

In previous work, we described 6–6’-bisindole compounds targeting a hydrophobic pocket on the N-heptad repeat region of viral glycoprotein-41 as effective inhibitors of HIV-1 fusion. Two promising compounds with sub-micromolar IC(50)’s contained a benzoic acid group and a benzoic acid ester attached at the two indole nitrogens. Here we have conducted a thorough SAR study evaluating the contribution of each of the ring systems and various substituents to compound potency. Hydrophobicity, polarity and charge were varied to produce 35 new compounds that were evaluated in binding, cell-cell fusion and viral infectivity assays. We found that (a) activity based solely on increasing hydrophobic content plateaued at ~ 200 nM; (b) the bisindole scaffold surpassed other heterocyclic ring systems in efficacy; (c) a polar interaction possibly involving Gln575 in the pocket could supplant less specific hydrophobic interactions; and (d) the benzoic acid ester moiety did not appear to form specific contacts with the pocket. The importance of this hydrophobic group to compound potency suggests a mechanism whereby it might interact with a tertiary component during fusion, such as membrane. A promising small molecule 10b with sub-μM activity was discovered with molecular weight < 500 da and reduced logP compared to earlier compounds. The work provides insight into requirements for small molecule inhibition of HIV-1 fusion.

Even being equipped with advanced surgical navigation systems, thorough analysis of patient's imaging diagnosis before procedures is still of great necessity for the operators. This study aimed to explore the morphological changes of sphenoid sinus lesions as observed on computed tomography (CT), and further carry out the fenestration of sphenoid sinus under guidance of the CT. A retrospective analysis of the characteristics of CT images was performed. The outcomes of three kinds of nasal endoscopic surgeries on benign lesions of sphenoid sinuses were investigated. For the 128 patients included, nasal-cavity-olfactory cleft pathway was performed in 64 patients, the Messerklinger pathway was adopted in 54 patients, while Wigand pathway was performed in the rest 10 patients. After follow-up visit, the drainage at the fenestration site of sphenoid sinus was found to be smooth, the symptoms were completely controlled, and no severe complication was reported during or after the surgery. No significant difference was observed in the curative effects among the 3 groups. CT of the paranasal sinus can guide the procedures of sphenoid sinus fenestration. No statistically significant differences were exhibited in terms of the amount of bleeding, surgical time and days of hospitalization among the 3 groups.

Small molecule inhibitors of glycoprotein-41 (gp41) are able to prevent HIV infection by binding to a hydrophobic pocket (HP) contained within the gp41 ectodomain, and preventing progression of fusion. There is little structural information on gp41 – ligand complexes, owing to hydrophobicity of the ligands, occlusion of the HP in folded gp41 ectodomain, and failure to form crystals of complexes. Here we used an engineered gp41 ectodomain protein containing an exposed HP and a small molecule designed to bind with weak affinity to the HP. We evaluated NMR methods, including WaterLOGSY, Saturation Transfer Difference spectroscopy (STD-NMR) and 1H relaxation rate difference spectroscopy with and without target irradiation (DIRECTION) for their ability to probe complex formation and structure. WaterLOGSY was the most sensitive technique for monitoring formation of the complex. STD-NMR and DIRECTION experiments gave similar pharmacophore mapping profiles, although the low dynamic range of the DIRECTION experiment limited its discrimination and sensitivity. A unique binding pose was identified from the STD data and provided clues for future optimization. Advantages and disadvantages of the techniques are discussed. This is the first example of the use of STD for structural analysis of a gp41- small molecule complex.

Background The hydrophobic pocket (HP) of HIV-1 glycoprotein-41 ectodomain is defined by two chains of the N-heptad repeat trimer, within the protein-protein interface that mediates 6HB formation. It is a potential target for inhibitors of viral fusion, but its hydrophobic nature and proximity to membrane in situ has precluded ready analysis of inhibitor interactions. Methods We evaluated the sensitivity of 19F NMR and fluorescence for detecting peptide and small molecule binding to the HP and explored the effect of non-denaturing detergent or phospholipid as cosolvents and potential mimics of the membrane environment surrounding gp41. Results Chemical shifts of aromatic fluorines were found to be sensitive to changes in the hydrogen bonding network that occurred when inhibitors transitioned from solvent into the HP or into ordered detergent micelles. Fluorescence intensities and emission maxima of autofluorescent compounds responded to changes in the local environment. Conclusions Gp41 – ligand binding occurred under all conditions, but was diminished in the presence of detergents. NMR and fluorescence studies revealed that dodecylphosphocholine (DPC) was a poor substitute for membrane in this system, while liposomes could mimic the membrane surroundings. General significance Our findings suggest that development of high potency small molecule binders to the HP may be frustrated by competition between binding to the HP and binding to the bilayer membrane.

Four new swapped-domain constructs of the ectodomain of human immunodeficiency virus type 1 glycoprotein-41 (gp41) were prepared. The gp41 ectodomain consists of 50-residue N-heptad repeat (NHR), 36-residue disulfide-bonded loop and 39-residue C-heptad repeat (CHR). It folds into a hairpin structure that forms a trimer along the NHR axis. The swapped-domain proteins feature CHR domains of length 39, 28 or 21 residues preceding a 4-residue loop and a 49- or 50-residue NHR domain. The effect of CHR truncation was to expose increasing lengths of the NHR groove, including the conserved hydrophobic pocket, an important drug target. A novel method for preparing proteins with extended exposed hydrophobic surfaces was demonstrated. Biophysical measurements, including analytical ultracentrifugation and ligand-detected Water-Ligand Observed via Gradient Spectroscopy and (1)H-(15)N-HSQC NMR experiments, were used to confirm that the proteins formed stable trimers in solution with exposed binding surfaces. These proteins could play an important role as receptors in structure-based drug discovery.

The conformational rearrangement of N- and C-heptad repeats (NHR, CHR) of the HIV-1 glycoprotein-41 (gp41) ectodomain into a trimer of hairpins triggers virus-cell fusion by bringing together membrane-spanning N- and C-terminal domains. Peptides derived from the NHR and CHR inhibit fusion by targeting a prehairpin intermediate state of gp41. Typically, peptides derived from the CHR are low nanomolar inhibitors, whereas peptides derived from the NHR are low micromolar inhibitors. Here, we describe the inhibitory activity of swapped-domain gp41 mimics of the form CHR-loop-NHR, which were designed to form reverse hairpin trimers exposing NHR grooves. We observed low nanomolar inhibition of HIV fusion in constructs that possessed the following properties: an extended NHR C-terminus, an exposed conserved hydrophobic pocket on the NHR, high helical content, and trimer stability. Low nanomolar activity was independent of CHR length. CD studies in membrane mimetic dodecylphosphocholine micelles suggested that bioactivity could be related to the ability of the inhibitors to interact with a membrane-associated prehairpin intermediate. The swapped-domain design resolves the problem of unstable and weakly active NHR peptides and suggests a different mechanism of action from that of CHR peptides in inhibition of HIV-1 fusion.

Gp41 is regarded as an attractive target for development of HIV-1 entry inhibitors since it mediates the fusion process of HIV- 1 entry into the target cell through the six-helix bundle (6-HB) formation between its N-heptad repeat (NHR) and C-heptad repeat (CHR). Any chemical entity that disrupts the six-helix bundle formation may inhibit the fusion process, thereby blocking HIV-1 entry into the target cells. A brief review of discovering small molecule inhibitors targeting gp41 is presented here, including the development of assay methods, current known small molecule inhibitors and their binding mode studies. Lessons learned and challenges remained in view of blocking protein-protein interaction between NHR and CHR are also discussed.

Polyphenols are bioactive natural products that appear to act against a wide range of pathologies. Mechanisms of activity have not been established, but recent studies have suggested that some polyphenols bind to membranes. This study examined the interaction between lipid bilayers and three structurally diverse polyphenols. It was hypothesized that features of the polyphenols such as polarity, molecular size, molecular geometry, and number and arrangement of phenol hydroxyl groups would determine the tendency to interact with the bilayer. The examined compounds included a mixed polyphenol, (-)-epigallocatechin gallate (EGCg); a proanthocyanidin trimer comprising catechin-(4→8)-catechin-(4→8)-catechin (cat₃; and a hydrolyzable tannin, 1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose (PGG). These polyphenols were incorporated at different levels into ²H-labeled 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) multilamellar vesicles (MLVs). ³¹P and ²H solid-state NMR experiments were performed to determine the dynamics of the headgroup region and the hydrophobic acyl chain region of the lipid bilayer upon addition of polyphenols. The chemical shift anisotropy (CSA) width of the ³¹P NMR spectra decreased upon addition of polyphenols. Addition of PGG induces a dramatic reduction on the CSA width compared with the control lipid bilayer sample, whereas addition of cat₃ barely reduces the CSA width. The ²H quadupolar splitting of the lipids also decreased upon addition of polyphenols. At the same concentration, PGG substantially reduced the quadrupolar splitting, whereas cat₃ barely reduced it when compared with the control sample. From a calculation of the order parameters of the acyl chain region of the lipid bilayer, it was concluded that the hydrophobic part of the lipid bilayer was perturbed by PGG, whereas cat₃ did not cause large perturbations. The data suggest that the polarity of the polyphenols affects the interaction between tannins and membranes. The interactions may relate to the biological activities of polyphenols.

A new approach for determining the membrane immersion depth of a spin-labeled probe has been developed using paramagnetic relaxation enhancement (PRE) in solid-state NMR spectroscopy. A DOXYL spin label was placed at different sites of 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC) phospholipid bilayers as paramagnetic moieties and the resulting enhancements of the longitudinal relaxation (T₁) times of ³¹P nuclei on the surface of the bilayers were measured by a standard inversion recovery pulse sequence. The ³¹P NMR spin-lattice relaxation times decrease steadily as the DOXYL spin label moves closer to the surface as well as the concentration of the spin-labeled lipids increase. The enhanced relaxation vs. the position and concentration of spin-labels indicate that PRE induced by the DOXYL spin label are significant to determine longer distances over the whole range of the membrane depths. When these data were combined with estimated correlation times τ(c), the r⁻⁶-weighted, time-averaged distances between the spin-labels and the ³¹P nuclei on the membrane surface were estimated. The application of using this solid-state NMR PRE approach coupled with site-directed spin labeling (SDSL) may be a powerful method for measuring membrane protein immersion depth.

Structurally well defined di-, tri- and tetra-valent cluster galactosides were synthesized in a convenient way. Oligo-glutamic acids were assembled as scaffolds. The presence of amine groups in these three ligands is expected to couple with drugs or genes for delivery. The binding affinities of these cluster galactoses to liver cells were determined by in vitro binding studies. Among them. the tetravalent cluster galactose (19) showed the highest affinity to Liver cell. It is therefore a promising targeting device for the specific delivery of drugs or genes to parenchymal liver cells.

Phospholamban (PLB) is an integral membrane protein regulating Ca(2+) transport through inhibitory interaction with sarco(endo)plasmic reticulum calcium ATPase (SERCA). The Asn27 to Ala (N27A) mutation of PLB has been shown to function as a superinhibitor of the affinity of SERCA for Ca(2+) and of cardiac contractility in vivo. The effects of this N27A mutation on the side-chain and backbone dynamics of PLB were investigated with (2)H and (15)N solid-state NMR spectroscopy in phospholipid multilamellar vesicles (MLVs). (2)H and (15)N NMR spectra indicate that the N27A mutation does not significantly change the side-chain or backbone dynamics of the transmembrane and cytoplasmic domains when compared to wild-type PLB. However, dynamic changes are observed for the hinge region, in which greater mobility is observed for the CD(3)-labeled Ala24 N27A-PLB. The increased dynamics in the hinge region of PLB upon N27A mutation may allow the cytoplasmic helix to more easily interact with the Ca(2+)-ATPase; thus, showing increased inhibition of Ca(2+)-ATPase.

2H, 31P, and 1H-magic-angle-spinning (MAS) solid-state NMR spectroscopic methods were used to elucidate the interaction between sorbic acid, a widely used weak acid food preservative, and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers under both acidic and neutral pH conditions. The linewidth broadening observed in the 31P NMR powder pattern spectra and the changes in the 31P longitudinal relaxation time (T1) indicate interaction with the phospholipid headgroup upon titration of sorbic acid or decanoic acid into DMPC bilayers over the pH range from 3.0 to 7.4. The peak intensities of sorbic acid decrease upon addition of paramagnetic Mn2+ ions in DMPC bilayers as recorded in the 1H MAS NMR spectra, suggesting that sorbic acid molecules are in close proximity with the membrane/aqueous surface. No significant 2H quadrupolar splitting (Δ γQ) changes are observed in the 2H NMR spectra of DMPC-d54 upon titration of sorbic acid, and the change of pH has a slight effect on Δ γQ, indicating that sorbic acid has weak influence on the orientation order of the DMPC acyl chains in the fluid phase over the pH range from 3.0 to 7.4. This finding is in contrast to the results of the decanoic acid/DMPC-d54 systems, where Δ γQ increases as the concentration of decanoic acid increases. Thus, in the membrane association process, sorbic acids are most likely interacting with the headgroups and shallowly embedded near the top of the phospholipid headgroups, rather than inserting deep into the acyl chains. Thus, antimicrobial mode of action for sorbic acid may be different from that of long-chain fatty acids.

Phospholamban (PLB)1 is a 52-amino acid transmembrane protein that interacts with the Ca-ATPase pump and lowers its affinity for Ca2+ (1-3). PLB plays a major role in the regulation process of the cardiac cycle (contraction and relaxation), which controls the heartbeat (3-5). Unphosphorylated PLB inhibits sarcoplasmic reticulum ATPase activity and stops the flow of Ca2+ ions, and this inhibition can be relieved by the cyclic AMP- and calmodulin-dependent phosphorylation of PLB (3-5). Since PLB is biologically significant and it is relatively small, many theoretical and biophysical experimental studies have aimed to investigate its structure in a membrane (6-22). On the basis of spectroscopic techniques and molecular modeling studies on pentameric WT-PLB, early structural reports on WT-PLB disagreed about whether the pentameric protein is composed of continuous α-helical subunits or composed of subunits that have two α-helices connected by an unstructured/β-sheet region (8, 21). Recently, the Chou group revealed an unusual bellflower-like assembly for pentameric WT-PLB that indicates an α-helical cytoplasmic domain of the pentamer that on average points away from the membrane surface (Figure 1A) (23). Alternatively, recent results from the Middleton group suggest that the cytoplasmic domain of PLB (residues 1-23) is stabilized only through its association with the phospholipid bilayer surface (24). In addition, new solution and solid-state NMR spectroscopic studies on the AFA-PLB mutant monomer (where C36, C41, and C46 have been mutated to A36, F41, and A46, respectively) have been reported (25, 26). The Veglia and Baldus groups debate whether the transmembrane domain of the monomeric AFA-PLB mutant structure is connected to an α-helical cytosolic segment that lies on and interacts with the membrane surface (L-shaped) (25) or a non-α-helical disordered cytosolic domain that has minimal interaction with the membrane surface (26). Figure 1 High-resolution solution NMR structures as well as the corresponding sequences for (A) pentameric (bellflower-like assembly) and (B) monomeric (L-shaped) phospholamban, by the Chou (23) and Veglia (26) groups, respectively. The two structures embedded ... Interestingly, the Veglia group resolved the solution NMR structure not only for the AFA-PLB monomer mutant (Figure 1B) but also for its phosphorylated form (27). The results of Veglia’s new phosphorylation study indicate that phosphorylation of serine 16 (located within the cytoplasmic domain) induces an order-to-disorder transition that disrupts the “L-shaped” monomer and causes a reduction in the extent of α-helical secondary structure around the phosphorylation site (27). However, the Chou group has not determined the phosphorylated form (P-PLB) of WT-PLB (23). They proposed that introducing a negative charge upon phosphorylation of WT-PLB could alter the average orientation of the cytoplasmic domain and decrease its accessibility to the calcium pump (23). Also, this latest model of WT-PLB interacting with SERCA introduced by the Chou group suggests that tightness of the PLB pentamer (supercoiled Leu/Ile zipper) could allow its cytoplasmic domain to fit into and interact with the groove of the SERCA cytoplasmic domain without the energetic cost of removing a subunit from the pentamer (23). Conversely, an earlier model suggests that phosphorylation of WT-PLB at Ser16 or Thr17 reduces its level of electrostatic binding with the pump and favors the association of the PLB monomers interacting with SERCA into PLB pentamers that do not interact with the pump (1, 3). Another recent model proposes that an allosteric interaction between the PLB monomer and SERCA takes place (28). Therefore, understanding the dynamic motion of WT-PLB with respect to the membrane is absolutely needed to completely describe the mechanism of binding of WT-PLB to Ca-ATPase. Complete knowledge of how different segments of phosphorylated and unphosphorylated PLB are moving within the membrane is essential for describing the mechanistic functions of the protein. EPR spectroscopic studies on the cytoplasmic domain of monomeric AFA-PLB have indicated large-scale dynamic changes (29). Also, Oxenoid and Chou hypothesized that this cytoplasmic domain motion is needed for WT-PLB/Ca-ATPase to function properly (23). It is clear that the cytoplasmic segment secondary structure, orientation, dynamics, and interaction with the membrane and SERCA of WT-PLB and P-PLB are under serious debate. However, it is important to emphasize that the high-resolution NMR solution structures of the monomeric PLB mutant (AFA-PLB) and pentameric WT-PLB in micelles reported by the Veglia (26) and Chou (23) groups, respectively, represent two of the best and widely accepted membrane protein structures available in the literature (see Figure 1). In addition, although the effect of phosphorylation on the cytoplasmic domain of WT-PLB has not been completely addressed in the literature, the Chou group suggested that their structure of WT-PLB in micelles sets the stage for a series of future experiments aimed at characterizing the effect of phosphorylation on the orientation and dynamics of its cytoplasmic helix (23). Our previous study indicated that phosphorylation does not significantly change the α-helical secondary structure of WT-PLB, and this finding agrees with the results previously reported by Arkin and co-workers (8, 30). In addition, that same report implies that P-PLB has less direct contact with the membranes than WT-PLB does (30). To extend and complement previous studies and to reveal new information about the dynamic properties of WT-PLB and its phosphorylated form (P-PLB), we have conducted NMR studies with PLB. These new studies will provide important physiological and mechanistic information regarding the regulatory role of WT-PLB and it phosphorylated form in biological systems (3, 23, 28). This dynamic information is crucial for understanding its binding to Ca-ATPase and inhibition. In this article, 2H and 15N solid-state NMR spectroscopic studies are utilized to ascertain pertinent information about the backbone and side chain dynamics of WT-PLB in phospholipid bilayers. 2H solid-state NMR spectroscopy is a powerful well-developed technique for studying the structural and dynamic properties of membrane proteins in phospholipid bilayers (31-34). The corresponding quadrupolar splitting and line shapes of the 2H solid-state NMR spectra can be used to probe the molecular dynamics of the side chain of selectively labeled residues in site-specific 2H-labeled integral membrane proteins (35-40). The primary amino acid sequence of full-length PLB has few alanines (mainly in the cytoplasmic domain, residues 1-15) and several leucines (the transmembrane domain, residues 23-52) along the length of the protein (23). In previous studies, methyl group motions have been well-characterized utilizing 2H NMR studies of CD3-labeled sites of alanines, valines, and leucines (35, 40-45). For the isotopically labeled alanines (short aliphatic side chains), the deuterated methyl group (CD3) rotates around the Cα-Cβ bond (see Figure 2A) and allows the deuterons to make jumps between three sites described by a tetrahedral geometry (46). However, for Leu, the long aliphatic side chain can be isotopically labeled at the δ- and/or ∊-CD3 sites, and the deuterium NMR powder pattern line shapes will be strongly influenced by the motions about the Cγ-Cδ bond axis as well as by additional librational motion about the Cα-Cβ and Cβ-Cγ bond axes (see Figure 2B) at various temperatures (40, 47). If the CD3-methyl probe of the protein undergoes no motion other than those associated with the axial rotation about the C-CD3 bond in a randomly dispersed sample, the resultant spectra will consist of a Pake pattern with a 40 kHz quadrupolar splitting (48). However, residues located outside the membrane are expected to be more motionally averaged and yield an isotropic peak (49). Figure 2 Chemical structure of the (A) alanine and (B) leucine amino acids. Additionally, 15N solid-state NMR spectroscopy has been used to ascertain direct information regarding backbone dynamics of membrane proteins (45, 50). Generally, the immobile (without large amplitude motions) amide sites of specific 15N-labeled proteins yield a broad static 15N powder pattern, whereas motionally averaged amide sites reveal isotropic peaks (50).

Octyl 2,3-di-O-sulfo-alpha-L-fucopyranosyl-(1-->3)-2-O-sulfo-alpha-L-fucopyranosyl-(1-->4)-2,3-di-O-sulfo-alpha-L-fucopyranosyl-(1-->3)-2-O-sulfo-alpha-L-fucopyranosyl-(1-->4)-2,3-di-O-sulfo-beta-L-fucopyranoside, a fucosyl pentasaccharide with a regular structure resembling the repeating unit of a natural sulfated fucan, was chemically synthesized using a convergent '2+3' strategy. Regioselective 3-O-silylation of beta-thiofucopyranoside and AgOTf-catalyzed glycosylation of the protected glycosyl trichloroacetimidate facilitated a one-pot trisaccharide synthesis. The synthesized target compound showed good antitumor activity in vivo, and promising anticoagulant activity in vitro.

PM3 calculations were performed on the inclusion complexation of α-cyclodextrin with acetophenone from a complete and unrestricted geometry optimization and conformational analysis. The complexation orientation in which the acetyl group is located near the secondary hydroxyl rim of the cyclodextrin cavity was found to be preferable, in contrast to the usual anti-parallel arrangement of host and guest dipoles in the molecular recognition of cyclodextrins. This abnormality was caused by the steric hindrance of the acetyl group in the guest, which led us to considerable van der Waals repulsion between the host and guest molecules upon complexation. It is suggested that steric effects play an important role in the energy and geometry of host–guest complexation.

The HIV-1 envelope glycoprotein gp41 fusion intermediate is a promising drug target for inhibiting viral entry. However, drug development has been impeded by challenges inherent in mediating the underlying protein-protein interaction. Here we report on the identification of fragments that bind to a C-terminal sub-pocket adjacent to the well-known hydrophobic pocket on the NHR coiled coil. Using a specifically designed assay and ligand-based NMR screening of a fragment library, we identified a thioenylaminopyrazole compound with a dissociation constant of ~500 μM. Interaction with the C-terminal sub-pocket was confirmed by paramagnetic relaxation enhancement NMR experiments, which also yielded the binding mode. Shape-based similarity searching detected additional phenylpyrazole and phenyltriazole fragments within the library, enriching the hit rate over random screening, and revealing molecular features required for activity. Discovery of the novel scaffolds and binding mechanism suggests avenues for extending the interaction surface and improving the potency of a hydrophobic pocket binding inhibitor.

2H, 31P, and 1H-magic-angle-spinning (MAS) solid-state NMR spectroscopic methods were used to elucidate the interaction between sorbic acid, a widely used weak acid food preservative, and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers under both acidic and neutral pH conditions. The linewidth broadening observed in the 31P NMR powder pattern spectra and the changes in the 31P longitudinal relaxation time (T1) indicate interaction with the phospholipid headgroup upon titration of sorbic acid or decanoic acid into DMPC bilayers over the pH range from 3.0 to 7.4. The peak intensities of sorbic acid decrease upon addition of paramagnetic Mn2+ ions in DMPC bilayers as recorded in the 1H MAS NMR spectra, suggesting that sorbic acid molecules are in close proximity with the membrane/aqueous surface. No significant 2H quadrupolar splitting (DeltanuQ) changes are observed in the 2H NMR spectra of DMPC-d54 upon titration of sorbic acid, and the change of pH has a slight effect on DeltanuQ, indicating that sorbic acid has weak influence on the orientation order of the DMPC acyl chains in the fluid phase over the pH range from 3.0 to 7.4. This finding is in contrast to the results of the decanoic acid/DMPC-d54 systems, where DeltanuQ increases as the concentration of decanoic acid increases. Thus, in the membrane association process, sorbic acids are most likely interacting with the headgroups and shallowly embedded near the top of the phospholipid headgroups, rather than inserting deep into the acyl chains. Thus, antimicrobial mode of action for sorbic acid may be different from that of long-chain fatty acids.

Wild-type phospholamban (WT-PLB) is a pentameric transmembrane protein that regulates the cardiac cycle (contraction and relaxation). From a physiological prospective, unphosphorylated WT-PLB inhibits sarcoplasmic reticulum ATPase activity; whereas, its phosphorylated form relieves the inhibition in a mechanism that is not completely understood. In this study, site-specifically (15)N-Ala-11- and (15)N-Leu-7-labeled WT-PLB and the corresponding phosphorylated forms (P-PLB) were incorporated into 1,2-dioleoyl-sn-glycero-3-phosphocholine/2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPC/DOPE) mechanically oriented lipid bilayers. The aligned (15)N-labeled Ala-11 and Leu-7 WT-PLB samples show (15)N resonance peaks at approximately 71ppm and 75ppm, respectively, while the corresponding phosphorylated forms P-PLB show (15)N peaks at 92ppm and 99ppm, respectively. These (15)N chemical shift changes upon phosphorylation are significant and in agreement with previous reports, which indicate that phosphorylation of WT-PLB at Ser-16 alters the structural properties of the cytoplasmic domain with respect to the lipid bilayers.

For the first time, 15N solid-state NMR experiments were conducted on wild-type phospholamban (WT-PLB) embedded inside mechanically oriented phospholipid bilayers to investigate the topology of its cytoplasmic and transmembrane domains. 15N solid-state NMR spectra of site-specific 15N-labeled WT-PLB indicate that the transmembrane domain has a tilt angle of 13 degrees+/-6 degrees with respect to the POPC (1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine) bilayer normal and that the cytoplasmic domain of WT-PLB lies on the surface of the phospholipid bilayers. Comparable results were obtained from site-specific 15N-labeled WT-PLB embedded inside DOPC/DOPE (1,2-dioleoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) mechanically oriented phospholipids' bilayers. The new NMR data support a pinwheel geometry of WT-PLB, but disagree with a bellflower structure in micelles, and indicate that the orientation of the cytoplasmic domain of the WT-PLB is similar to that reported for the monomeric AFA-PLB mutant.

Aligning lipid bilayers in nanoporous anodized aluminum oxide (AAO) is a new method to help study membrane proteins by electron paramagnetic resonance (EPR) and solid-state nuclear magnetic resonance (NMR) spectroscopic methods. The ability to maintain hydration, sample stability, and compartmentalization over long periods of time, and to easily change solvent composition are major advantages of this new method. To date, 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) has been the only phospholipid used for membrane protein studies with AAO substrates. The different properties of lipids with varying chain lengths require modified sample preparation procedures to achieve well formed bilayers within the lining of the AAO substrates. For the first time, the current study presents a simple methodology to incorporate large quantities of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), DMPC, and 1,2-dipalmitoyl-3-sn-phosphatidylcholine (DPPC) phospholipids inside AAO substrate nanopores of varying sizes. (2)H and (31)P solid-state NMR were used to confirm the alignment of each lipid and compare the efficiency of alignment. This study is the first step in standardizing the use of AAO substrates as a tool in NMR and EPR and will be useful for future structural studies of membrane proteins. Additionally, the solid-state NMR data suggest possible applications of nanoporous aluminum oxide in future vesicle fusion studies.

Cannabinoid receptors are G-protein-coupled receptors comprised of seven transmembrane helices. We hypothesized that the extended helix of the receptor interacts differently with POPC bilayers due to the differing distribution of charged amino acid residues. To test this, hCB1(T377-E416) and hCB2(K278-H316) peptides were studied with 31P and 2H solid-state NMR spectroscopy by incorporating them into 1-palmitoyl-2-oleoyl-sn-glycerophosphocholine bilayers. Lipid affinities of the 40- and 39-residue peptides were analyzed on the basis of 31P and 2H spectral line shapes, order parameters, and T1 relaxation measurements of the POPC bilayers. Lipid headgroup perturbations were noticed in the 31P NMR spectra in the lipid/peptide mixtures when compared with the pure lipids. 2H order parameters were calculated from the quadrupolar splitting of the de-Paked 2H NMR spectra. At the top of the acyl chain, pure lipids had an average S(CD) approximately = 0.20, whereas S(CD) approximately = 0.16 and S(CD) approximately = 0.18 were found in the presence of hCB1(T377-E416) and hCB2(K278-H316), respectively. S(CD) values decreased in the central part of the acyl chains when compared to the pure POPC lipids, indicating a change in the dynamic properties of the lipid membrane in the presence of the cannabinoid peptides. R(1Z) vs S2(CD) plots exhibited a linear dependency with and without the peptides, with an increase in slope upon addition of the peptides to the POPC, indicating that the dynamics of the lipid bilayer is dominated by fast axially symmetric motion. This study provides insights into the interaction of cannabinoid peptides with the membrane bilayer by investigating the headgroup and acyl chain dynamics.

The Ugi four-component reaction (U-4CR) was utilized to prepare divalent and trivalent cluster mannosides with different scaffolds. The glycoclusters obtained were tested for their relative inhibitory potency against the binding of yeast mannan to concanavalin A by solid-phase enzyme-linked lectin assays (ELLA) using methyl α- d -mannopyranoside as a standard. Among them, a divalent mannoside containing aromatic groups showed the strongest binding affinity to concanavalin A.