Revealing interaction between sulfobutylether‐β‐cyclodextrin and reserpine by chemiluminescence and site‐directed molecular docking

The host–guest interaction between sulfobutylether‐β‐cyclodextrin (SBE‐β‐CD) and reserpine (RSP) is described using flow injection‐chemiluminescence (FI‐CL) and site‐directed molecular docking methods. It was found that RSP could inhibit the CL intensity produced by a luminol/SBE‐β‐CD system. The decrease in CL intensity was logarithmic over an RSP concentration range of 0.03 to 700.0 nM, giving a regression equation of ?I = 107.1lgC_RES + 186.1 with a detection limit of 10 pM (3σ). The CL assay was successfully applied in the determination of RSP in injection, saliva and urine samples with recoveries in the range 93.5–106.1%. Using the proposed CL model, the binding constant (K_CD‐R) and the stoichiometric ratio of SBE‐β‐CD/RSP were calculated to be 7.4 × 10⁶ M^‐1 and 1 : 1, respectively. Using molecular docking, it was confirmed that luminol binds to the small cavity of SBE‐β‐CD with a nonpolar interaction, while RSP targeted the larger cavity of SBE‐β‐CD and formed a 1 : 1 complex with hydrogen bonds. The proposed new CL method has the potential to become a powerful tool for revealing the host–guest interaction between CDs and drugs, as well as monitoring drugs with high sensitivity. Copyright © 2013 John Wiley & Sons, Ltd.     

Structural insights into the inclusion complexes between clomiphene citrate and β‐cyclodextrin: The mechanism of preferential isomeric selection

A major challenge in pharmaceuticals for clinical applications is to alter the solubility, stability, and toxicity of drug molecules in living systems. Cyclodextrins (CDs) have the ability to form host–guest inclusion complexes with pharmaceuticals for further development of new drug formulations. The inclusion complex of clomiphene citrate (CL), a poorly water‐soluble drug, with native β‐cyclodextrin (β‐CD) was characterized by a one and two‐dimensional nuclear magnetic resonance (NMR) spectroscopic approach and also by molecular docking techniques. Here we report NMR and a computational approach in preferential isomeric selection of CL, which exists in two stereochemical isomers, enclomiphene citrate (ENC; E isomer) and zuclomiphene citrate (ZNC; Z isomer) with β‐CD. β‐CD cavity protons, namely, H‐3′ and H‐5′, experienced shielding in the presence of CL. The aromatic ring protons of the CL molecule were observed to be deshielded in the presence of β‐CD. The stoichiometric ratio of the β‐CD:CL inclusion complex was observed by NMR and found to be 1:1. The overall binding constant of β‐CD:CL inclusion complexes was based on NMR chemical shifts and was calculated to be 50.21 M⁻¹. The change in Gibb's free energy (∆G) was calculated to be −9.80 KJ mol⁻¹. The orientation and structure of the β‐CD:CL inclusion complexes are proposed on the basis of NMR and molecular docking studies. 2D ¹H‐¹H ROESY confirmed the involvement of all three aromatic rings of CL in the inclusion complexation with β‐CD in the solution, confirming the multiple equilibria between β‐CD and CL. Molecular docking and 2D ¹H‐¹H ROESY provide insight into the inclusion complexation of two isomers of CL into the β‐CD cavity. A molecular docking technique further provided the different binding affinities of the E and Z isomers of CL with β‐CD and confirmed the preference of the Z isomer binding for β‐CD:CL inclusion complexes. The study indicates that the formation of a hydrogen bond between –O– of CL and the hydrogen atom of the hydroxyl group of β‐CD was the main factor for noncovalent β‐CD:CL inclusion complex formation and stabilization in the aqueous phase.     

Enantioseparation of ketoconazole and miconazole by capillary electrophoresis and a study on their inclusion interactions with β‐cyclodextrin and derivatives

A chiral separation method coupled with capillary electrophoresis (CE) analysis for ketoconazole and miconazole enantiomers using chiral selectors such as β‐cyclodextrin (β‐CD) and hydroxypropyl‐β‐CD (HP‐β‐CD) was developed in this study, which included the optimisation, validation and application of the method on the antifungal cream samples. The formation of inclusion complex between the hosts (β‐CD and HP‐β‐CD) and guests (ketoconazole and miconazole) were compared and analysed using ultraviolet–visible spectrophotometry, nuclear magnetic resonance (NMR) spectroscopy and molecular docking methods. Results from the study showed that in a concentration that ranged between 0.25 and 50 mg L^?1, the linear calibration curves of each enantiomer had a high coefficient of regression (R² > 0.999), low limit of detection (0.075 mg L^?1) and low limit of quantification (0.25 mg L^?1). The relative standard deviation (RSD) of the intraday and interday analyses ranged from 0.79% to 8.01% and 3.30% to 11.43%, respectively, while the recoveries ranged from 82.0% to 105.7% (RSD < 7%, n = 3). The most probable structure of the inclusion complexes was proposed based on the findings from the molecular docking studies conducted using the PatchDock server.     

Supramolecular interactions between β‐lapachone with cyclodextrins studied using isothermal titration calorimetry and molecular modeling

Supramolecular interactions between β‐lapachone (β‐lap) and cyclodextrins (CDs) were investigated by isothermal titration calorimetry. The most favorable host: guest interaction was characterized using X‐ray powder diffraction (XRD), differential scanning calorimetry and thermogravimetry (DSC/TG), spectroscopy (FT‐IR), spectroscopy (2D ROESY) nuclear magnetic resonance (NMR), and molecular modeling. Phase solubility diagrams showed β‐, HP‐β‐, SBE‐β‐, γ‐, and HP‐γ‐CDs at 1.5% (w/w) allowed an increase in apparent solubility of β‐lap with enhancement factors of 12.0, 10.1, 11.8, 2.4, and 2.2, respectively. β‐lap has a weak interaction with γ‐ and HP‐γ‐CDs and tends to interact more favorably with β‐CD and its derivatives, especially SBE‐β‐CD (K = 4160 M⁻¹; ΔG = −20.66 kJ·mol⁻¹). Thermodynamic analysis suggests a hydrophobic interaction associated with the displacement of water from the cavity of the CD by the β‐lap. In addition, van der Waals forces and hydrogen bonds were responsible for the formation of complexes. Taken together, the results showed intermolecular interactions between β‐lap and SBE‐β‐CD, thereby confirming the formation of the inclusion complex. Molecular docking results showed 2 main orientations in which the interaction of benzene moiety at the wider rim of the SBE‐β‐CD is the most stable (average docking energy of −7.0 kcal/mol). In conclusion, β‐lap:SBE‐β‐CD is proposed as an approach for use in drug delivery systems in cancer research.     

β‐cyclodextrin encapsulated polyphenols as effective antioxidants

Formation of dityrosine (DT) cross‐linkages in proteins is one of the most widely used markers of oxidative stress. Ribonuclease A (RNase A) has 6 Tyr residues and shows a characteristic DT fluorescence peak upon oxidation in addition to major changes in its secondary structure. DT formation can be prevented by using polyphenols (GA, ECG, and EGCG) which are known to have strong antioxidant activity. However, it has been observed that ECG and EGCG initiate protein oligomerization due to protein‐polyphenol cross‐linkages. To prevent the formation of such cross‐linkages we have used β‐cyclodextrin (β‐CD) to encapsulate the polyphenols and studied its antioxidant properties along with that of free polyphenols. The polyphenol/β‐cyclodextrin (β‐CD) inclusion complexes not only prevent DT formation but also reduce protein oligomerization. This may be attributed to the fact that the quinone forming rings of ECG and EGCG become encapsulated in the cavity of β‐CD and are no longer available for protein cross‐linking.     

GC Separation of Enantiomers of Alkyl Esters of 2‐Bromo Substituted Carboxylic Acids Enantiomers on 6‐TBDMS‐2,3‐di‐alkyl‐ β‐ and γ‐Cyclodextrin Stationary Phases

The gas chromatographic separation of enantiomers of 2‐Br carboxylic acid derivatives was studied on four different 6‐TBDMS‐2,3‐di‐O‐alkyl‐ β‐ and ‐γ‐CD stationary phases. The differences in thermodynamic data {ΔH and –ΔS} for the 15 structurally related racemates were evaluated. The influence of structure differences in the alkyl substituents covalently attached to the stereogenic carbon atom, as well as in the ester group of the homologous analytes, and the selectivity of modified β‐ and γ‐ cyclodextrin derivatives was studied in detail. The cyclodextrin cavity size, as well as elongation of alkyl substituents in positions 2 and 3 of 6‐TBDMS‐β‐CD, also affected their selectivity. The quality of enantiomeric separations is influenced mainly by alkyl chains of the ester group of the molecule and this appears to be independent of the CD stationary phase used. In some cases the separations occur as the result of external adsorption rather than inclusion complexations with the chiral selector. It was found that the temperature dependencies of the selectivity factor were nonlinear. Chirality 26:279–285, 2014. © 2014 Wiley Periodicals, Inc.     

Cholesterol‐β1AR interaction versus cholesterol‐β2AR interaction

Two 8‐µs all‐atom molecular dynamics simulations have been performed on the two highly homologous G protein‐coupled receptor (GPCR) subtypes, β₁‐ and β₂‐adrenergic receptors, which were embedded in a lipid bilayer with randomly dispersed cholesterol molecules. During the simulations, cholesterol molecules accumulate to different surface regions of the two receptors, suggesting the subtype specificity of cholesterol–β‐adrenergic receptor interaction and providing some clues to the physiological difference of the two subtypes. Meanwhile, comparison between the two receptors in interacting with cholesterols shed some new light on general determinants of cholesterol binding to GPCRs. Our results indicate that although the concave surface, charged residues and aromatic residues are important, neither of these stabilizing factors is indispensable for a cholesterol interaction site. Different combinations of these factors lead to the diversified binding modes of cholesterol binding to the receptors. Our long‐time simulations, for the first time, revealed the pathway of a cholesterol molecule entering the consensus cholesterol motif (CCM) site, and the binding process of cholesterol to CCM is accompanied by a side chain flipping of the conserved Trp4.50. Moreover, the simulation results suggest that the I‐/V‐/L‐rich region on the extracellular parts of helix 6 might be an alternatively conserved cholesterol‐binding site for the class‐A GPCRs. Proteins 2014; 82:760–770. © 2013 Wiley Periodicals, Inc.     

Modeling interactions between a β‐O‐4 type lignin model compound and 1‐allyl‐3‐methylimidazolium chloride ionic liquid

Aiming at understanding the molecular mechanism of the lignin dissolution in imidazolium‐based ionic liquids (ILs), this work presents a combined quantum chemistry (QC) calculation and molecular dynamics (MD) simulation study on the interaction of the lignin model compound, veratrylglycerol‐β‐guaiacyl ether (VG) with 1‐allyl‐3‐methylimidazolium chloride ([Amim]Cl). The monomer of VG is shown to feature a strong intramolecular hydrogen bond, and its dimer is indicated to present important π‐π stacking and intermolecular hydrogen bonding interactions. The interactions of both the cation and anion of [Amim]Cl with VG are shown to be stronger than that between the two monomers, indicating that [Amim]Cl is capable of dissolving lignin. While Cl^– anion forms a hydrogen‐bonded complex with VG, the imidazolium cation interacts with VG via both the π‐π stacking and intermolecular hydrogen bonding. The calculated interaction energies between VG and the IL or its components (the cation, anion, and ion pair) indicate the anion plays a more important role than the cation for the dissolution of lignin in the IL. Theoretical results provide help for understanding the molecular mechanism of lignin dissolution in imidazolium‐based IL. The theoretical calculations on the interaction between the lignin model compound and [Amim]Cl ionic liquid indicate that the anion of [Amim]Cl plays a more important role for lignin dissolution although the cation also makes a substantial contribution.     

Enantiodifferentiation of α‐hydroxyalkanephosphonic acids in 31P NMR with application of α‐cyclodextrin as chiral discriminating agent

α‐Cyclodextrin was shown to be convenient chemical shift reagent for determination of the enantiomeric composition of α‐hydroxyphosphonic acids by means of ³¹P NMR. The developed methodology appeared to be reliable, repetitive, easy to perform and simple for interpretation. Enantiomeric discrimination in the ³¹P NMR spectra for 12 of 13 studied hydroxyphosphonates was achieved, with baseline separation of resonances obtained for eight compounds. In those cases, the chemical nonequivalence values ranged from 0.069 to 0.313 ppm. The studies showed that enantioselectivity is strongly influenced by the solution pD and the optimal condition was found at pD 2 or 10 depending on the guest structure. On the basis of the ROESY spectra the complexation modes of selected hydroxyphosphonates with α‐cyclodextrin was postulated. Chirality 2010. © 2009 Wiley‐Liss, Inc.     

Pre‐fibrillar α‐synuclein variants with impaired β‐structure increase neurotoxicity in Parkinson's disease models

The relation of α‐synuclein (αS) aggregation to Parkinson's disease (PD) has long been recognized, but the mechanism of toxicity, the pathogenic species and its molecular properties are yet to be identified. To obtain insight into the function different aggregated αS species have in neurotoxicity in vivo, we generated αS variants by a structure‐based rational design. Biophysical analysis revealed that the αS mutants have a reduced fibrillization propensity, but form increased amounts of soluble oligomers. To assess their biological response in vivo, we studied the effects of the biophysically defined pre‐fibrillar αS mutants after expression in tissue culture cells, in mammalian neurons and in PD model organisms, such as Caenorhabditis elegans and Drosophila melanogaster. The results show a striking correlation between αS aggregates with impaired β‐structure, neuronal toxicity and behavioural defects, and they establish a tight link between the biophysical properties of multimeric αS species and their in vivo function.     

Effect of 2‐hydroxypropyl‐β‐cyclodextrin on thermal stability and aggregation of glycogen phosphorylase b from rabbit skeletal muscle

The study of the kinetics of thermal aggregation of glycogen phosphorylase b (Phb) from rabbit skeletal muscles by dynamic light scattering at 48°C showed that 2‐hydroxypropyl‐β‐cyclodextrin (HP‐β‐CD) accelerated the aggregation process and induced the formation of the larger protein aggregates. The reason of the accelerating effect of HP‐β‐CD is destabilization of the protein molecule under action of HP‐β‐CD. This conclusion was supported by the data on differential scanning calorimetry and the kinetic data on thermal inactivation of Phb. It is assumed that destabilization of the Phb molecule is due to preferential binding of HP‐β‐CD to intermediates of protein unfolding in comparison with the original native state. The conclusion regarding the ability of the native Phb for binding of HP‐β‐CD was substantiated by the data on the enzyme inhibition by HP‐β‐CD. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 986–993, 2010.     

Optical resolution and mechanism using enantioselective cellulose,sodium alginate and hydroxypropyl‐β‐cyclodextrin membranes

Chiral solid membranes of cellulose, sodium alginate, and hydroxypropyl‐β‐cyclodextrin were prepared for chiral dialysis separations. After optimizing the membrane material concentrations, the membrane preparation conditions and the feed concentrations, enantiomeric excesses of 89.1%, 42.6%, and 59.1% were obtained for mandelic acid on the cellulose membrane, p ‐hydroxy phenylglycine on the sodium alginate membrane, and p ‐hydroxy phenylglycine on the hydroxypropyl‐β‐cyclodextrin membrane, respectively. To study the optical resolution mechanism, chiral discrimination by membrane adsorption, solid phase extraction, membrane chromatography, high‐pressure liquid chromatography ultrafiltration were performed. All of the experimental results showed that the first adsorbed enantiomer was not the enantiomer that first permeated the membrane. The crystal structures of mandelic acid and p ‐hydroxy phenylglycine are the racematic compounds. We suggest that the chiral separation mechanism of the solid membrane is “adsorption – association – diffusion,” which is able to explain the optical resolution of the enantioselective membrane. This is also the first report in which solid membranes of sodium alginate and hydroxypropyl‐β‐cyclodextrin were used in the chiral separation of p ‐hydroxy phenylglycine.     

Syntheses and anti‐tubercular activity of β‐substituted and α,β‐disubstituted peptidyl β‐aminoboronates and boronic acids

Tuberculosis is still affecting millions of people worldwide, and new resistant strains of Mycobacterium tuberculosis are being found. It is therefore necessary to find new compounds for treatment. In this paper, we report the synthesis and in vitro testing of peptidyl β‐aminoboronic acids and β‐aminoboronates with anti‐tubercular activity. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

5α‐Androst‐16‐en‐3α‐ol β‐D‐Glucuronide,Precursor of 5α‐Androst‐16‐en‐3α‐ol in Human Sweat

5α‐Androst‐16‐en‐3α‐ol (α‐androstenol) is an important contributor to human axilla sweat odor. It is assumed that α‐andostenol is excreted from the apocrine glands via a H₂O‐soluble conjugate, and this precursor was formally characterized in this study for the first time in human sweat. The possible H₂O‐soluble precursors, sulfate and glucuronide derivatives, were synthesized as analytical standards, i.e., α‐androstenol, β‐androstenol sulfates, 5α‐androsta‐5,16‐dien‐3β‐ol (β‐androstadienol) sulfate, α‐androstenol β‐glucuronide, α‐androstenol α‐glucuronide, β‐androstadienol β‐glucuronide, and α‐androstenol β‐glucuronide furanose. The occurrence of α‐androstenol β‐glucuronide was established by ultra performance liquid chromatography (UPLC)/MS (heated electrospray ionization (HESI)) in negative‐ion mode in pooled human sweat, containing eccrine and apocrine secretions and collected from 25 female and 24 male underarms. Its concentration was of 79 ng/ml in female secretions and 241 ng/ml in male secretions. The release of α‐androstenol was observed after incubation of the sterile human sweat or α‐androstenol β‐glucuronide with a commercial glucuronidase enzyme, the urine‐isolated bacteria Streptococcus agalactiae, and the skin bacteria Staphylococcus warneri DSM 20316, Staphylococcus haemolyticus DSM 20263, and Propionibacterium acnes ATCC 6919, reported to have β‐glucuronidase activities. We demonstrated that if α‐ and β‐androstenols and androstadienol sulfates were present in human sweat, their concentrations would be too low to be considered as potential precursors of malodors; therefore, the H₂O‐soluble precursor of α‐androstenol in apocrine secretion should be a β‐glucuronide.     

Comparative Enantioseparation of Ketoprofen with Trimethylated α‐, β‐, and γ‐Cyclodextrins in Capillary Electrophoresis and Study of Related Selector–Selectand Interactions Using Nuclear Magnetic Resonance Spectroscopy

The enantiomers of ketoprofen were separated by capillary electrophoresis using the (2,3,6‐tri‐O‐methyl)‐derivatives of α‐, β‐, and γ‐cyclodextrin (CyD) as chiral selectors. The affinity pattern of the ketoprofen enantiomers toward these CyDs changed depending on their cavity size. Thus, with hexakis (2,3,6‐tri‐O‐methyl)‐α‐CyD and heptakis (2,3,6‐tri‐O‐methyl)‐β‐CyD, the R enantiomer of the drug migrated first, whereas the enantiomer migration order was reversed in the presence of octakis(2,3,6‐tri‐O‐methyl)‐γ‐CyD. The change in the migration order was rationalized on the basis of changes in the structure of the complexes between the ketoprofen enantiomers and the chiral selectors as derived from nuclear magnetic resonance spectroscopy experiments. Chirality, 25:79–88, 2013.© 2012 Wiley Periodicals, Inc.     

The β‐sheet breakers and π‐stacking

Fibrillation of β‐amyloid is recognized as a key process leading to the development of Alzheimer's disease. Small peptides called β‐sheet breakers were found to inhibit the process of β‐amyloid fibrillation and to dissolve amyloid fibrils in vitro, in vivo, and in cell culture studies [1,2]. The mechanism by which peptide inhibition takes place remains elusive and a detailed model needs to be established. Here, we present new insights into the possible role of consecutive Phe residues, present in the structure of β‐sheet breakers, supported by the results obtained by means of MD simulations. We performed a 30‐ns MD of two β‐sheet breakers: iAβ5 (LPFFD) and iAβ6 (LPFFFD) which have two and three consecutive Phe residues, respectively. We have found that Phe rings in these peptides tend to form stacked conformations. For one of the peptides – iAβ6 – the calculated electrostatic contribution to free energy of one of the conformers with three rings stacked (c2) is significantly lower than that corresponding to the unstacked one (c1), two rings stacked (c0) and second conformer with three rings stacked (c3). This may favor the interaction of the c2 conformer with the target on amyloid fibril. We hypothesize that the mechanism of inhibition of amyloidogenesis by β‐sheet breaker involves competition among π‐stacked Phe residues of the inhibitor and π‐stacking within the β‐amyloid fibril. iAβ6 may be a promising candidate for a lead compound of amyloidogenesis inhibitors. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

β‐strand interactions at the domain interface critical for the stability of human lens γD‐crystallin

Human age‐onset cataracts are believed to be caused by the aggregation of partially unfolded or covalently damaged lens crystallin proteins; however, the exact molecular mechanism remains largely unknown. We have used microseconds of molecular dynamics simulations with explicit solvent to investigate the unfolding process of human lens γD‐crystallin protein and its isolated domains. A partially unfolded folding intermediate of γD‐crystallin is detected in simulations with its C‐terminal domain (C‐td) folded and N‐terminal domain (N‐td) unstructured, in excellent agreement with biochemical experiments. Our simulations strongly indicate that the stability and the folding mechanism of the N‐td are regulated by the interdomain interactions, consistent with experimental observations. A hydrophobic folding core was identified within the C‐td that is comprised of a and b strands from the Greek key motif 4, the one near the domain interface. Detailed analyses reveal a surprising non‐native surface salt‐bridge between Glu135 and Arg142 located at the end of the ab folded hairpin turn playing a critical role in stabilizing the folding core. On the other hand, an in silico single E135A substitution that disrupts this non‐native Glu135‐Arg142 salt‐bridge causes significant destabilization to the folding core of the isolated C‐td, which, in turn, induces unfolding of the N‐td interface. These findings indicate that certain highly conserved charged residues, that is, Glu135 and Arg142, of γD‐crystallin are crucial for stabilizing its hydrophobic domain interface in native conformation, and disruption of charges on the γD‐crystallin surface might lead to unfolding and subsequent aggregation.     

Mutations of key hydrophobic surface residues of 11β‐hydroxysteroid dehydrogenase type 1 increase solubility and monodispersity in a bacterial expression system

11β‐Hydroxysteroid dehydrogenase type 1 (11β‐HSD1) is a key enzyme in the conversion of cortisone to the functional glucocorticoid hormone cortisol. This activation has been implicated in several human disorders, notably the metabolic syndrome where 11β‐HSD1 has been identified as a novel target for potential therapeutic drugs. Recent crystal structures have revealed the presence of a pronounced hydrophobic surface patch lying on two helices at the C‐terminus. The physiological significance of this region has been attributed to facilitating substrate access by allowing interactions with the endoplasmic reticulum membrane. Here, we report that single mutations that alter the hydrophobicity of this patch (I275E, L266E, F278E, and L279E in the human enzyme and I275E, Y266E, F278E, and L279E in the guinea pig enzyme) result in greatly increased yields of soluble protein on expression in E. coli. Kinetic analyses of both reductase and dehydrogenase reactions indicate that the F278E mutant has unaltered K_m values for steroids and an unaltered or increased k_cat. Analytical ultracentrifugation shows that this mutation also decreases aggregation of both the human and guinea pig enzymes, resulting in greater monodispersity. One of the mutants (guinea pig F278E) has proven easy to crystallize and has been shown to have a virtually identical structure to that previously reported for the wild‐type enzyme. The human F278E enzyme is shown to be a suitable background for analyzing the effects of naturally occurring mutations (R137C, K187N) on enzyme activity and stability. Hence, the F278E mutants should be useful for many future biochemical and biophysical studies of the enzyme.