Chromeno[3,4-b]xanthones as First-in-Class AChE and Aβ Aggregation Dual-Inhibitors

Alzheimer’s disease (AD) is a complex multifactorial disorder, mainly characterized by the progressive loss of memory and cognitive, motor, and functional capacity. The absence of effective therapies available for AD alongside the consecutive failures in the central nervous system (CNS) drug development has been motivating the search for new disease-modifying therapeutic strategies for this disease. To address this issue, the multitarget directed ligands (MTDLs) are emerging as a therapeutic alternative to target the multiple AD-related factors. Following this concept, herein we describe the design, synthesis, and biological evaluation of a family of chromeno[3,4-b]xanthones as well as their (E)-2-[2-(propargyloxy)styryl]chromone precursors, as first-in-class acetylcholinesterase (AChE) and β-amyloid (Aβ) aggregation dual-inhibitors. Compounds 4b and 10 emerged as well-balanced dual-target inhibitors, with IC₅₀ values of 3.9 and 2.9 μM for AChE and inhibitory percentages of 70 and 66% for Aβ aggregation, respectively. The molecular docking showed that most of the compounds bound to AChE through hydrogen bonds with residues of the catalytic triad and π-stacking interactions between the main scaffold and the aromatic residues present in the binding pocket. The interesting well-balanced activities of these compounds makes them interesting templates for the development of new multitarget compounds for AD.     

Structural Insights into the Ligand-Binding and -Releasing Mechanism of Helicoverpa armigera Pheromone-Binding Protein PBP1

Cotton bollworm (Helicoverpa armigera) is a worldwide agricultural pest in which the transport of pheromones is indispensable and perceived by pheromone-binding proteins (PBPs). However, three-dimensional structure, pheromone binding, and releasing mechanisms of PBPs are not completely illustrated. Here, we solved three structures of the cotton bollworm HarmPBP1 at different pH values and its complex with ligand, Z-9-hexadecenal. Although apo-HarmPBP1 adopts a common PBP scaffold of six α-helices surrounding a predominantly hydrophobic central pocket, the conformation is greatly distinct from other apo-PBPs. The Z-9-hexadecenal is bound mainly by hydrophobic interaction. The pheromone can enter this cavity through an opening between the helices α5 and α6, as well as the loop between α3 and α4. Structural analysis suggests that ligand entry into the pocket is followed by a shift of Lys94 and Lys138, which may act as a lid at the opening of the pocket. Acidic pH will cause a subtle structural change of the lid, which in turn affects its ligand-binding ability, differently from other family proteins. Taken together, this study provides structural bases for the interactions between pheromones and PBPs, the pH-induced conformational switch, and the design of small inhibitors to control cotton bollworms by disrupting male–female chemosensory communication.     

Hepatoprotective Effect of Oyster Peptide on Alcohol-Induced Liver Disease in Mice

Alcohol-induced liver disease (ALD) has become one of the major global health problems, and the aim of this study was to investigate the characterization of the structure as well as the hepatoprotective effect and mechanism of oyster peptide (OP, MW < 3500 Da) on ALD in a mouse model. The results demonstrate that ethanol administration could increase the activities of aspartate aminotransferase (AST), alanine transaminase (ALT), γ-Glutamyl transferase (GGT), reactive oxygen species (ROS), malondialdehyde (MDA), and triglycerides (TG), as well as increase the interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor (TNF-α) levels (p < 0.01), and reduce the activity of superoxide dismutase (SOD) and the concentration of glutathione (GSH). Those changes were significantly reversed by the application of different doses of OP. Furthermore, the mRNA expressions of nuclear factor elythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), and quinone oxidoreductase1 (NQO1) were significantly up-regulated in OP groups, and the mRNA expressions of nuclear factor kappa-light chain enhancer of B cells (NF-κB), TNF-α, and IL-6 were markedly reduced in OP groups compared to that of the model group. Thus, OP had a significant protective effect on ALD through the enhancement of the in vivo antioxidant ability and the inhibition of the inflammatory response as possible mechanisms of action, which therefore suggests that OP might be useful as a natural source to protect the liver from alcohol damage.     

Oxidase Reactivity of CuII Bound to N-Truncated Aβ Peptides Promoted by Dopamine

The redox chemistry of copper(II) is strongly modulated by the coordination to amyloid-β peptides and by the stability of the resulting complexes. Amino-terminal copper and nickel binding motifs (ATCUN) identified in truncated Aβ sequences starting with Phe4 show very high affinity for copper(II) ions. Herein, we study the oxidase activity of [Cu–Aβ_4−x] and [Cu–Aβ_1−x] complexes toward dopamine and other catechols. The results show that the Cu^II–ATCUN site is not redox-inert; the reduction of the metal is induced by coordination of catechol to the metal and occurs through an inner sphere reaction. The generation of a ternary [Cu^II–Aβ–catechol] species determines the efficiency of the oxidation, although the reaction rate is ruled by reoxidation of the Cu^I complex. In addition to the N-terminal coordination site, the two vicinal histidines, His13 and His14, provide a second Cu-binding motif. Catechol oxidation studies together with structural insight from the mixed dinuclear complexes Ni/Cu–Aβ_4−x reveal that the His-tandem is able to bind Cu^II ions independently of the ATCUN site, but the N-terminal metal complexation reduces the conformational mobility of the peptide chain, preventing the binding and oxidative reactivity toward catechol of Cu^II bound to the secondary site.     

Systematic Approach to Find the Global Minimum of Relaxation Dispersion Data for Protein-Induced B–Z Transition of DNA

Carr–Purcell–Meiboom–Gill (CPMG) relaxation dispersion spectroscopy is commonly used for quantifying conformational changes of protein in μs-to-ms timescale transitions. To elucidate the dynamics and mechanism of protein binding, parameters implementing CPMG relaxation dispersion results must be appropriately determined. Building an analytical model for multi-state transitions is particularly complex. In this study, we developed a new global search algorithm that incorporates a random search approach combined with a field-dependent global parameterization method. The robust inter-dependence of the parameters carrying out the global search for individual residues (GSIR) or the global search for total residues (GSTR) provides information on the global minimum of the conformational transition process of the Zα domain of human ADAR1 (hZα_ADAR1)–DNA complex. The global search results indicated that a α-helical segment of hZα_ADAR1 provided the main contribution to the three-state conformational changes of a hZα_ADAR1—DNA complex with a slow B–Z exchange process. The two global exchange rate constants, k_ex and k_ZB, were found to be 844 and 9.8 s⁻¹, respectively, in agreement with two regimes of residue-dependent chemical shift differences—the “dominant oscillatory regime” and “semi-oscillatory regime”. We anticipate that our global search approach will lead to the development of quantification methods for conformational changes not only in Z-DNA binding protein (ZBP) binding interactions but also in various protein binding processes.     

Structural Basis for Design of New Purine-Based Inhibitors Targeting the Hydrophobic Binding Pocket of Hsp90

Inhibition of the molecular chaperone heat shock protein 90 (Hsp90) represents a promising approach for cancer treatment. BIIB021 is a highly potent Hsp90 inhibitor with remarkable anticancer activity; however, its clinical application is limited by lack of potency and response. In this study, we aimed to investigate the impact of replacing the hydrophobic moiety of BIIB021, 4-methoxy-3,5-dimethylpyridine, with various five-membered ring structures on the binding to Hsp90. A focused array of N⁷/N⁹-substituted purines, featuring aromatic and non-aromatic rings, was designed, considering the size of hydrophobic pocket B in Hsp90 to obtain insights into their binding modes within the ATP binding site of Hsp90 in terms of π–π stacking interactions in pocket B as well as outer α-helix 4 configurations. The target molecules were synthesized and evaluated for their Hsp90α inhibitory activity in cell-free assays. Among the tested compounds, the isoxazole derivatives 6b and 6c, and the sole six-membered derivative 14 showed favorable Hsp90α inhibitory activity, with IC₅₀ values of 1.76 µM, 0.203 µM, and 1.00 µM, respectively. Furthermore, compound 14 elicited promising anticancer activity against MCF-7, SK-BR-3, and HCT116 cell lines. The X-ray structures of compounds 4b, 6b, 6c, 8, and 14 bound to the N-terminal domain of Hsp90 were determined in order to understand the obtained results and to acquire additional structural insights, which might enable further optimization of BIIB021.     

A Comparative Study of the Effects of Platinum (II) Complexes on β-Amyloid Aggregation: Potential Neurodrug Applications

Herein the effects of three platinum complexes, namely (SP-4-2)-(2,2′-bipyridine)dichloridoplatinum(II), Pt-bpy, (SP-4-2)-dichlorido(1,10-phenanthroline) platinum(II), Pt-phen, and (SP-4-2)-chlorido(2,2′:6′,2′′-terpyridine)platinum(II) chloride, Pt-terpy, on the aggregation of an amyloid model system derived from the C-terminal domain of Aβ peptide (Aβ_21–40) were investigated. Thioflavin T (ThT) binding assays revealed the ability of Pt(II) compounds to repress amyloid aggregation in a dose-dependent way, whereas the ability of Aβ_21–40 peptide to interfere with ligand field of metal complexes was analyzed through UV-Vis absorption spectroscopy and electrospray ionization mass spectrometry. Spectroscopic data provided micromolar EC₅₀ values and allowed to assess that the observed inhibition of amyloid aggregation is due to the formation of adducts between Aβ_21–40 peptide and complexes upon the release of labile ligands as chloride and that they can explore different modes of coordination toward Aβ_21–40 with respect to the entire Aβ_1–40 polypeptide. In addition, conformational studies through circular dichroism (CD) spectroscopy suggested that Pt-terpy induces soluble β-structures of monomeric Aβ_21–40, thus limiting self-recognition. Noticeably, Pt-terpy demonstrated the ability to reduce the cytotoxicity of amyloid peptide in human SH-SY5Y neuroblastoma cells. Presented data corroborate the hypothesis to enlarge the application field of already known metal-based agents to neurodegenerative diseases, as potential neurodrugs.     

In Silico and In Vitro Screening of 50 Curcumin Compounds as EGFR and NF-κB Inhibitors

The improvement of cancer chemotherapy remains a major challenge, and thus new drugs are urgently required to develop new treatment regimes. Curcumin, a polyphenolic antioxidant derived from the rhizome of turmeric (Curcuma longa L.), has undergone extensive preclinical investigations and, thereby, displayed remarkable efficacy in vitro and in vivo against cancer and other disorders. However, pharmacological limitations of curcumin stimulated the synthesis of numerous novel curcumin analogs, which need to be evaluated for their therapeutic potential. In the present study, we calculated the binding affinities of 50 curcumin derivatives to known cancer-related target proteins of curcumin, i.e., epidermal growth factor receptor (EGFR) and nuclear factor κB (NF-κB) by using a molecular docking approach. The binding energies for EGFR were in a range of −12.12 (±0.21) to −7.34 (±0.07) kcal/mol and those for NF-κB ranged from −12.97 (±0.47) to −6.24 (±0.06) kcal/mol, indicating similar binding affinities of the curcumin compounds for both target proteins. The predicted receptor-ligand binding constants for EGFR and curcumin derivatives were in a range of 0.00013 (±0.00006) to 3.45 (±0.10) µM and for NF-κB in a range of 0.0004 (±0.0003) to 10.05 (±4.03) µM, indicating that the receptor-ligand binding was more stable for EGFR than for NF-κB. Twenty out of 50 curcumin compounds showed binding energies to NF-κB smaller than −10 kcal/mol, while curcumin as a lead compound revealed free binding energies of >−10 kcal/mol. Comparable data were obtained for EGFR: 15 out of 50 curcumin compounds were bound to EGFR with free binding energies of <−10 kcal/mol, while the binding affinity of curcumin itself was >−10 kcal/mol. This indicates that the derivatization of curcumin may indeed be a promising strategy to improve targe specificity and to obtain more effective anticancer drug candidates. The in silico results have been exemplarily validated using microscale thermophoresis. The bioactivity has been further investigated by using resazurin cell viability assay, lactate dehydrogenase assay, flow cytometric measurement of reactive oxygen species, and annexin V/propidium iodide assay. In conclusion, molecular docking represents a valuable approach to facilitate and speed up the identification of novel targeted curcumin-based drugs to treat cancer.     

Co-Incubation with PPARβ/δ Agonists and Antagonists Modeled Using Computational Chemistry: Effect on LPS Induced Inflammatory Markers in Pulmonary Artery

Peroxisome proliferator activated receptor beta/delta (PPARβ/δ) is a nuclear receptor ubiquitously expressed in cells, whose signaling controls inflammation. There are large discrepancies in understanding the complex role of PPARβ/δ in disease, having both anti- and pro-effects on inflammation. After ligand activation, PPARβ/δ regulates genes by two different mechanisms; induction and transrepression, the effects of which are difficult to differentiate directly. We studied the PPARβ/δ-regulation of lipopolysaccharide (LPS) induced inflammation (indicated by release of nitrite and IL-6) of rat pulmonary artery, using different combinations of agonists (GW0742 or L−165402) and antagonists (GSK3787 or GSK0660). LPS induced release of NO and IL-6 is not significantly reduced by incubation with PPARβ/δ ligands (either agonist or antagonist), however, co-incubation with an agonist and antagonist significantly reduces LPS-induced nitrite production and Nos2 mRNA expression. In contrast, incubation with LPS and PPARβ/δ agonists leads to a significant increase in Pdk−4 and Angptl−4 mRNA expression, which is significantly decreased in the presence of PPARβ/δ antagonists. Docking using computational chemistry methods indicates that PPARβ/δ agonists form polar bonds with His287, His413 and Tyr437, while antagonists are more promiscuous about which amino acids they bind to, although they are very prone to bind Thr252 and Asn307. Dual binding in the PPARβ/δ binding pocket indicates the ligands retain similar binding energies, which suggests that co-incubation with both agonist and antagonist does not prevent the specific binding of each other to the large PPARβ/δ binding pocket. To our knowledge, this is the first time that the possibility of binding two ligands simultaneously into the PPARβ/δ binding pocket has been explored. Agonist binding followed by antagonist simultaneously switches the PPARβ/δ mode of action from induction to transrepression, which is linked with an increase in Nos2 mRNA expression and nitrite production.     

The Folding of de Novo Designed Protein DS119 via Molecular Dynamics Simulations

As they are not subjected to natural selection process, de novo designed proteins usually fold in a manner different from natural proteins. Recently, a de novo designed mini-protein DS119, with a βαβ motif and 36 amino acids, has folded unusually slowly in experiments, and transient dimers have been detected in the folding process. Here, by means of all-atom replica exchange molecular dynamics (REMD) simulations, several comparably stable intermediate states were observed on the folding free-energy landscape of DS119. Conventional molecular dynamics (CMD) simulations showed that when two unfolded DS119 proteins bound together, most binding sites of dimeric aggregates were located at the N-terminal segment, especially residues 5–10, which were supposed to form β-sheet with its own C-terminal segment. Furthermore, a large percentage of individual proteins in the dimeric aggregates adopted conformations similar to those in the intermediate states observed in REMD simulations. These results indicate that, during the folding process, DS119 can easily become trapped in intermediate states. Then, with diffusion, a transient dimer would be formed and stabilized with the binding interface located at N-terminals. This means that it could not quickly fold to the native structure. The complicated folding manner of DS119 implies the important influence of natural selection on protein-folding kinetics, and more improvement should be achieved in rational protein design.     

Design,Synthesis and Evaluation of Novel Derivatives of Curcuminoids with Cytotoxicity

Curcumin and curcuminoids have been discussed frequently due to their promising functional groups (such as scaffolds of α,β-unsaturated β-diketone, α,β-unsaturated ketone and β′-hydroxy-α,β-unsaturated ketone connected with aromatic rings on both sides) that play an important role in various bioactivities, including antioxidant, anti-inflammatory, anti-proliferation and anticancer activity. A series of novel curcuminoid derivatives (a total of 55 new compounds) and three reference compounds were synthesized with good yields using three-step organic synthesis. The anti-proliferative activities of curcumin derivatives were examined for six human cancer cell lines: HeLaS3, KBvin, MCF-7, HepG2, NCI-H460 and NCI-H460/MX20. Compared to the IC₅₀ values of all the synthesized derivatives, most α,β-unsaturated ketones displayed potent anti-proliferative effects against all six human cancer cell lines, whereas β′-hydroxy-α,β-unsaturated ketones and α,β-unsaturated β-diketones presented moderate anti-proliferative effects. Two potent curcuminoid derivatives were found among all the novel derivatives and reference compounds: (E)-5-hydroxy-7-phenyl-1-(3,4,5-trimethoxyphenyl)hept-1-en-3-one (compound 3) and (1E,4E)-1,7-bis(3,4,5-trimethoxyphenyl)hepta-1,4-dien-3-one (compound MD12a). These were selected for further analysis after the evaluation of their anti-proliferative effects against all human cancer cell lines. The results of apoptosis assays revealed that the number of dead cells was increased in early apoptosis and late apoptosis, while cell proliferation was also decreased after applying various concentrations of (E)-5-hydroxy-7-phenyl-1-(3,4,5-trimethoxyphenyl)hept-1-en-3-one (compound 3) and (1E,4E)-1,7-bis(3,4,5-trimethoxyphenyl)hepta-1,4-dien-3-one (compound MD12a) to MCF-7 and HpeG2 cancer cells. Analysis of the gene expression arrays showed that three genes (GADD45B, SESN2 and BBC3) were correlated with the p53 pathway. From the quantitative PCR analysis, it was seen that (1E,4E)-1,7-bis(3,4,5-trimethoxyphenyl)hepta-1,4-dien-3-one (compound MD12a) effectively induced the up-regulated expression of GADD45B, leading to the suppression of MCF-7 cancer cell formation and cell death. Molecular docking analysis was used to predict and sketch the interactions of the GADD45B-α,β-unsaturated ketone complex for help in drug design.     

Molecular Dynamics Simulation on the Conformational Transition of the Mad2 Protein from the Open to the Closed State

The Mad2 protein, with two distinct conformations of open- and closed-states, is a key player in the spindle checkpoint. The closed Mad2 state is more active than the open one. We carried out conventional and targeted molecular dynamics simulations for the two stable Mad2 states and their conformational transition to address the dynamical transition mechanism from the open to the closed state. The intermediate structure in the transition process shows exposure of the β6 strand and an increase of space around the binding sites of β6 strand due to the unfolding of the β7/8 sheet and movement of the β6/4/5 sheet close to the αC helix. Therefore, Mad2 binding to the Cdc20 protein in the spindle checkpoint is made possible. The interconversion between these two states might facilitate the functional activity of the Mad2 protein. Motion correlation analysis revealed the allosteric network between the β1 strand and β7/8 sheet via communication of the β5-αC loop and the β6/4/5 sheet in this transition process.     

Screening of β1- and β2-Adrenergic Receptor Modulators through Advanced Pharmacoinformatics and Machine Learning Approaches

Cardiovascular diseases (CDs) are a major concern in the human race and one of the leading causes of death worldwide. β-Adrenergic receptors (β1-AR and β2-AR) play a crucial role in the overall regulation of cardiac function. In the present study, structure-based virtual screening, machine learning (ML), and a ligand-based similarity search were conducted for the PubChem database against both β1- and β2-AR. Initially, all docked molecules were screened using the threshold binding energy value. Molecules with a better binding affinity were further used for segregation as active and inactive through ML. The pharmacokinetic assessment was carried out on molecules retained in the above step. Further, similarity searching of the ChEMBL and DrugBank databases was performed. From detailed analysis of the above data, four compounds for each of β1- and β2-AR were found to be promising in nature. A number of critical ligand-binding amino acids formed potential hydrogen bonds and hydrophobic interactions. Finally, a molecular dynamics (MD) simulation study of each molecule bound with the respective target was performed. A number of parameters obtained from the MD simulation trajectories were calculated and substantiated the stability between the protein-ligand complex. Hence, it can be postulated that the final molecules might be crucial for CDs subjected to experimental validation.     

Experimental and Theoretical Investigations on the Supermolecular Structure of Isoliquiritigenin and 6-O-α-d-Maltosyl-β-cyclodextrin Inclusion Complex

Isoliquiritigenin (ILTG) possesses many pharmacological properties. However, its poor solubility and stability in water hinders its wide applications. The solubility of bioactive compounds can often be enhanced through preparation and delivery of various cyclodextrin (CD) inclusion complexes. The 6-O-α-d-maltosyl-β-CD (G₂-β-CD), as one of the newest developments of CDs, has high aqueous solubility and low toxicity, especially stable inclusion characteristics with bioactive compounds. In this work, we for the first time construct and characterize the supermolecular structure of ILTG/G₂-β-CD by scanning electron microscopy (SEM), ultraviolet-visible spectroscopy (UV), Fourier transform infrared spectroscopy (FT-IR), and X-ray diffractometry (XRD). The solubility of ILTG in water at 25 °C rises from 0.003 to 0.717 mg/mL by the encapsulation with G₂-β-CD. Our experimental observations on the presence of the ILTG/G₂-β-CD inclusion complex are further supported by the ONIOM(our Own N-layer Integrated Orbital molecular Mechanics)-based QM/MM (Quantum Mechanics/Molecular Mechanics) calculations, typically substantiating these supermolecular characteristics, such as detailed structural assignments, preferred binding orientations, selectivity, solvent effects, interaction energies and forces of the ILTG/G₂-β-CD inclusion complex. Our results have elucidated how ILTG interacts with G₂-β-CD, demonstrating the primary host-guest interactions between ILTG and G₂-β-CD, characterized by hydrogen bonds, hydrophobic interactions, electrostatic forces, and conformational effects, are favored for the formation of the ILTG/G₂-β-CD inclusion.     

Urolithin and Reduced Urolithin Derivatives as Potent Inhibitors of Tyrosinase and Melanogenesis: Importance of the 4-Substituted Resorcinol Moiety

We previously reported (E)-β-phenyl-α,β-unsaturated carbonyl scaffold ((E)-PUSC) played an important role in showing high tyrosinase inhibitory activity and that derivatives with a 4-substituted resorcinol moiety as the β-phenyl group of the scaffold resulted in the greatest tyrosinase inhibitory activity. To examine whether the 4-substituted resorcinol moiety could impart tyrosinase inhibitory activity in the absence of the α,β-unsaturated carbonyl moiety of the (E)-PUSC scaffold, 10 urolithin derivatives were synthesized. To obtain more candidate samples, the lactone ring in synthesized urolithins was reduced to produce nine reduced urolithins. Compounds 1c (IC₅₀ = 18.09 ± 0.25 μM), 1h (IC₅₀ = 4.14 ± 0.10 μM), and 2a (IC₅₀ = 15.69 ± 0.40 μM) had greater mushroom tyrosinase-inhibitory activities than kojic acid (KA) (IC₅₀ = 48.62 ± 3.38 μM). The SAR results suggest that the 4-substituted resorcinol motif makes an important contribution to tyrosinase inhibition. To investigate whether these compounds bind to human tyrosinase, a human tyrosinase homology model was developed. Docking simulations with mushroom and human tyrosinases showed that 1c, 1h, and 2a bind to the active site of both tyrosinases with higher binding affinities than KA. Pharmacophore analyses showed that two hydroxyl groups of the 4-substituted resorcinol entity act as hydrogen bond donors in both mushroom and human tyrosinases. Kinetic analyses indicated that these compounds were all competitive inhibitors. Compound 2a inhibited cellular tyrosinase activity and melanogenesis in α-MSH plus IBMX-stimulated B16F10 melanoma cells more strongly than KA. These results suggest that 2a is a promising candidate for the treatment of skin pigment disorders, and show the 4-substituted resorcinol entity importantly contributes to tyrosinase inhibition.     

Zoledronic Acid-Loaded β-TCP Inhibits Tumor Proliferation and Osteoclast Activation: Development of a Functional Bone Substitute for an Efficient Osteosarcoma Treatment

Osteosarcoma has a poor survival rate due to relapse and metastasis. Zoledronic acid (ZOL), an anti-resorptive and anti-tumor agent, is used for treating osteosarcoma. Delivery of ZOL to the target region is difficult due to its high binding affinity to bone minerals. This study developed a novel treatment for osteosarcoma by delivering ZOL to the target region locally and sustainably. In this study, we fabricated a novel bone substitute by loading ZOL on β-tricalcium phosphate (β-TCP). The ZOL-loaded β-TCP (ZOL/β-TCP) would be expected to express the inhibitory effects via both bound-ZOL (bound to β-TCP) and free-ZOL (release from ZOL/β-TCP). To explore the ability to release ZOL from the ZOL/β-TCP, the amount of released ZOL was measured. The released profile indicates that a small amount of ZOL was released, and most of it remained on the β-TCP. Our data showed that ZOL/β-TCP could successfully express the effects of ZOL via both bound-ZOL and free-ZOL. In addition, we examined the biological effects of bound/free-ZOL using osteosarcoma and osteoclasts (target cells). The results showed that two states of ZOL (bound/free) inhibit target cell activities. As a result, ZOL/β-TCP is a promising candidate for application as a novel bone substitute.     

Development of a Highly Sensitive β-Glucan Detection System Using Scanning Single-Molecule Counting Method

To overcome the limitations of the Limulus amebocyte lysate (LAL) assay method for the diagnosis of invasive fungal infection, we applied a reaction system combining recombinant β-glucan binding proteins and a scanning single-molecule counting (SSMC) method. A novel (1→3)-β-D-glucan recognition protein (S-BGRP) and a (1→6)-β-glucanase mutant protein were prepared and tested for the binding of (1→6)-branched (1→3)-β-D-glucan from fungi. S-BGRP and (1→6)-β-glucanase mutant proteins reacted with β-glucan from Candida and Aspergillus spp. Although LAL cross-reacted with plant-derived β-glucans, the new detection system using the SSMC method showed low sensitivity to plant (1→3)-β-D-glucan, which significantly improved the appearance of false positives, a recognized problem with the LAL method. Measurement of β-glucan levels by the SSMC method using recombinant β-glucan-binding proteins may be useful for the diagnosis of fungal infections. This study shows that this detection system could be a new alternative diagnostic method to the LAL method.     

GABAA Receptor Modulators with a Pyrazolo[1,5-a]quinazoline Core: Synthesis,Molecular Modelling Studies and Electrophysiological Assays

As a continuation of our study in the GABA_A receptor modulators field, we report the design and synthesis of new 8-chloropyrazolo[1,5-a]quinazoline derivatives. Molecular docking studies and the evaluation of the ‘Proximity Frequencies’ (exploiting our reported model) were performed on all the final compounds (3, 4, 6a–c, 7a,b, 8, 9, 12a–c, 13a,b, 14–19) to predict their profile on the α1β2γ2-GABA_AR subtype. Furthermore, to verify whether the information coming from this virtual model was valid and, at the same time, to complete the study on this series, we evaluated the effects of compounds (1–100 µM) on the modulation of GABA_A receptor function through electrophysiological techniques on recombinant α1β2γ2L-GABA_A receptors expressed in Xenopus laevis oocytes. The matching between the virtual prediction and the electrophysiological tests makes our model a useful tool for the study of GABA_A receptor modulators.     

Integration of Adenylate Kinase 1 with Its Peptide Conformational Imprint

In the present study, molecularly imprinted polymers (MIPs) were used as a tool to grasp a targeted α-helix or β-sheet of protein. During the fabrication of the hinge-mediated MIPs, elegant cavities took shape in a special solvent on quartz crystal microbalance (QCM) chips. The cavities, which were complementary to the protein secondary structure, acted as a peptide conformational imprint (PCI) for adenylate kinase 1 (AK1). We established a promising strategy to examine the binding affinities of human AK1 in conformational dynamics using the peptide-imprinting method. Moreover, when bound to AK1, PCIs are able to gain stability and tend to maintain higher catalytic activities than free AK1. Such designed fixations not only act on hinges as accelerators; some are also inhibitors. One example of PCI inhibition of AK1 catalytic activity takes place when PCI integrates with an AK1_9-23 β-sheet. In addition, conformation ties, a general MIP method derived from random-coil AK1_133-144 in buffer/acetonitrile, are also inhibitors. The inhibition may be due to the need for this peptide to execute conformational transition during catalysis.