SR-17018 Stimulates Atypical µ-Opioid Receptor Phosphorylation and Dephosphorylation

Opioid-associated overdoses and deaths due to respiratory depression are a major public health problem in the US and other Western countries. In the past decade, much research effort has been directed towards the development of G-protein-biased µ-opioid receptor (MOP) agonists as a possible means to circumvent this problem. The bias hypothesis proposes that G-protein signaling mediates analgesia, whereas ß-arrestin signaling mediates respiratory depression. SR-17018 was initially reported as a highly biased µ-opioid with an extremely wide therapeutic window. It was later shown that SR-17018 can also reverse morphine tolerance and prevent withdrawal via a hitherto unknown mechanism of action. Here, we examined the temporal dynamics of SR-17018-induced MOP phosphorylation and dephosphorylation. Exposure of MOP to saturating concentrations of SR-17018 for extended periods of time stimulated a MOP phosphorylation pattern that was indistinguishable from that induced by the full agonist DAMGO. Unlike DAMGO-induced MOP phosphorylation, which is reversible within minutes after agonist washout, SR-17018-induced MOP phosphorylation persisted for hours under otherwise identical conditions. Such delayed MOP dephosphorylation kinetics were also found for the partial agonist buprenorphine. However, buprenorphine, SR-17018-induced MOP phosphorylation was fully reversible when naloxone was included in the washout solution. SR-17018 exhibits a qualitative and temporal MOP phosphorylation profile that is strikingly different from any other known biased, partial, or full MOP agonist. We conclude that detailed analysis of receptor phosphorylation may provide novel insights into previously unappreciated pharmacological properties of newly synthesized MOP ligands.     

Synthesis of [18F]F-γ-T-3, a Redox-Silent γ-Tocotrienol (γ-T-3) Vitamin E Analogue for Image-Based In Vivo Studies of Vitamin E Biodistribution and Dynamics

Vitamin E, a natural antioxidant, is of interest to scientists, health care pundits and faddists; its nutritional and biomedical attributes may be validated, anecdotal or fantasy. Vitamin E is a mixture of tocopherols (TPs) and tocotrienols (T-3s), each class having four substitutional isomers (α-, β-, γ-, δ-). Vitamin E analogues attain only low concentrations in most tissues, necessitating exacting invasive techniques for analytical research. Quantitative positron emission tomography (PET) with an F-18-labeled molecular probe would expedite access to Vitamin E’s biodistributions and pharmacokinetics via non-invasive temporal imaging. (R)-6-(3-[¹⁸F]Fluoropropoxy)-2,7,8-trimethyl-2-(4,8,12-trimethyltrideca-3,7,11-trien-1-yl)-chromane ([¹⁸F]F-γ-T-3) was prepared for this purpose. [¹⁸F]F-γ-T-3 was synthesized from γ-T-3 in two steps: (i) 1,3-di-O-tosylpropane was introduced at C6-O to form TsO-γ-T-3, and (ii) reaction of this tosylate with [¹⁸F]fluoride in DMF/K₂₂₂. Non-radioactive F-γ-T-3 was synthesized by reaction of γ-T-3 with 3-fluoropropyl methanesulfonate. [¹⁸F]F-γ-T-3 biodistribution in a murine tumor model was imaged using a small-animal PET scanner. F-γ-T-3 was prepared in 61% chemical yield. [¹⁸F]F-γ-T-3 was synthesized in acceptable radiochemical yield (RCY 12%) with high radiochemical purity (>99% RCP) in 45 min. Preliminary F-18 PET images in mice showed upper abdominal accumulation with evidence of renal clearance, only low concentrations in the thorax (lung/heart) and head, and rapid clearance from blood. [¹⁸F]F-γ-T-3 shows promise as an F-18 PET tracer for detailed in vivo studies of Vitamin E. The labeling procedure provides acceptable RCY, high RCP and pertinence to all eight Vitamin E analogues.     

In Vitro Analyses of Spinach-Derived Opioid Peptides,Rubiscolins: Receptor Selectivity and Intracellular Activities through G Protein- and β-Arrestin-Mediated Pathways

Activated opioid receptors transmit internal signals through two major pathways: the G-protein-mediated pathway, which exerts analgesia, and the β-arrestin-mediated pathway, which leads to unfavorable side effects. Hence, G-protein-biased opioid agonists are preferable as opioid analgesics. Rubiscolins, the spinach-derived naturally occurring opioid peptides, are selective δ opioid receptor agonists, and their p.o. administration exhibits antinociceptive effects. Although the potency and effect of rubiscolins as G-protein-biased molecules are partially confirmed, their in vitro profiles remain unclear. We, therefore, evaluated the properties of rubiscolins, in detail, through several analyses, including the CellKey^TM assay, cADDis^® cAMP assay, and PathHunter^® β-arrestin recruitment assay, using cells stably expressing µ, δ, κ, or µ/δ heteromer opioid receptors. In the CellKey^TM assay, rubiscolins showed selective agonistic effects for δ opioid receptor and little agonistic or antagonistic effects for µ and κ opioid receptors. Furthermore, rubiscolins were found to be G-protein-biased δ opioid receptor agonists based on the results obtained in cADDis^® cAMP and PathHunter^® β-arrestin recruitment assays. Finally, we found, for the first time, that they are also partially agonistic for the µ/δ dimers. In conclusion, rubiscolins could serve as attractive seeds, as δ opioid receptor-specific agonists, for the development of novel opioid analgesics with reduced side effects.     

Development of Radiogallium-Labeled Peptides for Platelet-Derived Growth Factor Receptor β (PDGFRβ) Imaging: Influence of Different Linkers

The purpose of this study is to develop peptide-based platelet-derived growth factor receptor β (PDGFRβ) imaging probes and examine the effects of several linkers, namely un-natural amino acids (D-alanine and β-alanine) and ethylene-glycol (EG), on the properties of Ga-DOTA-(linker)-IPLPPPRRPFFK peptides. Seven radiotracers, ⁶⁷Ga-DOTA-(linker)-IPLPPPRRPFFK peptides, were designed, synthesized, and evaluated. The stability and cell uptake in PDGFRβ positive peptide cells were evaluated in vitro. The biodistribution of [⁶⁷Ga]Ga-DOTA-EG₂-IPLPPPRRPFFK ([⁶⁷Ga]27) and [⁶⁷Ga]Ga-DOTA-EG₄-IPLPPPRRPFFK ([⁶⁷Ga]28), which were selected based on in vitro stability in murine plasma and cell uptake rates, were determined in BxPC3-luc-bearing nu/nu mice. Seven ⁶⁷Ga-labeled peptides were successfully synthesized with high radiochemical yields (>85%) and purities (>99%). All evaluated radiotracers were stable in PBS (pH 7.4) at 37 °C. However, only [⁶⁷Ga]27 and [⁶⁷Ga]28 remained more than 75% after incubation in murine plasma at 37 °C for 1 h. [⁶⁷Ga]27 exhibited the highest BxPC3-luc cell uptake among the prepared radiolabeled peptides. As regards the results of the biodistribution experiments, the tumor-to-blood ratios of [⁶⁷Ga]27 and [⁶⁷Ga]28 at 1 h post-injection were 2.61 ± 0.75 and 2.05 ± 0.77, respectively. Co-injection of [⁶⁷Ga]27 and an excess amount of IPLPPPRRPFFK peptide as a blocking agent can significantly decrease this ratio. However, tumor accumulation was not considered sufficient. Therefore, further probe modification is required to assess tumor accumulation for in vivo imaging.     

Discovery of Amide-Functionalized Benzimidazolium Salts as Potent α-Glucosidase Inhibitors

α-Glucosidase inhibitors (AGIs) are used as medicines for the treatment of diabetes mellitus. The α-Glucosidase enzyme is present in the small intestine and is responsible for the breakdown of carbohydrates into sugars. The process results in an increase in blood sugar levels. AGIs slow down the digestion of carbohydrates that is helpful in controlling the sugar levels in the blood after meals. Among heterocyclic compounds, benzimidazole moiety is recognized as a potent bioactive scaffold for its wide range of biologically active derivatives. The aim of this study is to explore the α-glucosidase inhibition ability of benzimidazolium salts. In this study, two novel series of benzimidazolium salts, i.e., 1-benzyl-3-{2-(substituted) amino-2-oxoethyl}-1H-benzo[d]imidazol-3-ium bromide 9a–m and 1-benzyl-3-{2-substituted) amino-2-oxoethyl}-2-methyl-1H-benzo[d] imidazol-3-ium bromide 10a–m were screened for their in vitro α-glucosidase inhibitory potential. These compounds were synthesized through a multistep procedure and were characterized by ¹H-NMR, ¹³C-NMR, and EI-MS techniques. Compound 10d was identified as the potent α-glucosidase inhibitor among the series with an IC₅₀ value of 14 ± 0.013 μM, which is 4-fold higher than the standard drug, acarbose. In addition, compounds 10a, 10e, 10h, 10g, 10k, 10l, and 10m also exhibited pronounced potential for α-glucosidase inhibition with IC₅₀ value ranging from 15 ± 0.037 to 32.27 ± 0.050 µM when compared with the reference drug acarbose (IC₅₀ = 58.8 ± 0.12 μM). A molecular docking study was performed to rationalize the binding interactions of potent inhibitors with the active site of the α-glucosidase enzyme.     

Self-assembly of cyclic hexamers of γ-cyclodextrin in a metallosupramolecular framework with d-penicillamine

Cyclodextrins are widely used cyclic oligosaccharides of d-glucose whose hydrophilic exterior is covered by hydroxyl groups and whose hydrophobic interior is surrounded by lipophilic moieties. Because of this structural feature, cyclodextrin molecules commonly aggregate into dimensional structures via intermolecular hydrogen bonds, and their aggregation into closed oligomeric architectures has been achieved only via the attachment of functional substituent groups to the cyclodextrin rings. Here, we report the first structurally characterized self-assembly of non-substituted γ-cyclodextrin molecules into cyclic hexamers, which was realized in a chiral coordination framework composed of complex-anions with d-penicillamine rather than l- or dl-penicillamine. The self-assembly is accompanied by the 3D-to-2D structural transformation of porous coordination frameworks to form helical hexagonal cavities that accommodate helical γ-cyclodextrin hexamers. This finding provides new insight into the development of cyclodextrin chemistry and host–guest chemistry based on chiral recognition and crystal engineering processes.

The complex anions with d-penicillamine are organized into a 3D porous framework that allows the inclusion of γ-CD. The inclusion is accompanied by the 3D-to-2D transformation of porous frameworks so as to accept cyclic hexamers of γ-CD.     

The Amyloid Fibril-Forming β-Sheet Regions of Amyloid β and α-Synuclein Preferentially Interact with the Molecular Chaperone 14-3-3ζ

14-3-3 proteins are abundant, intramolecular proteins that play a pivotal role in cellular signal transduction by interacting with phosphorylated ligands. In addition, they are molecular chaperones that prevent protein unfolding and aggregation under cellular stress conditions in a similar manner to the unrelated small heat-shock proteins. In vivo, amyloid β (Aβ) and α-synuclein (α-syn) form amyloid fibrils in Alzheimer’s and Parkinson’s diseases, respectively, a process that is intimately linked to the diseases’ progression. The 14-3-3ζ isoform potently inhibited in vitro fibril formation of the 40-amino acid form of Aβ (Aβ₄₀) but had little effect on α-syn aggregation. Solution-phase NMR spectroscopy of ¹⁵N-labeled Aβ₄₀ and A53T α-syn determined that unlabeled 14-3-3ζ interacted preferentially with hydrophobic regions of Aβ₄₀ (L11-H21 and G29-V40) and α-syn (V3-K10 and V40-K60). In both proteins, these regions adopt β-strands within the core of the amyloid fibrils prepared in vitro as well as those isolated from the inclusions of diseased individuals. The interaction with 14-3-3ζ is transient and occurs at the early stages of the fibrillar aggregation pathway to maintain the native, monomeric, and unfolded structure of Aβ₄₀ and α-syn. The N-terminal regions of α-syn interacting with 14-3-3ζ correspond with those that interact with other molecular chaperones as monitored by in-cell NMR spectroscopy.     

Synthesis,Pharmacological Evaluation,and Computational Studies of Cyclic Opioid Peptidomimetics Containing β3-Lysine

Our formerly described pentapeptide opioid analog Tyr-c[D-Lys-Phe-Phe-Asp]NH₂ (designated RP-170), showing high affinity for the mu (MOR) and kappa (KOR) opioid receptors, was much more stable than endomorphine-2 (EM-2) in the rat brain homogenate and displayed remarkable antinociceptive activity after central (intracerebroventricular) and peripheral (intravenous ) administration. In this report, we describe the further modification of this analog, which includes the incorporation of a β³-amino acid, (R)- and (S)-β³-Lys, instead of D-Lys in position 2. The influence of such replacement on the biological properties of the obtained analogs, Tyr-c[(R)-β³-Lys-Phe-Phe-Asp]NH₂ (RP-171) and Tyr-c[(S)-β³-Lys-Phe-Phe-Asp]NH₂, (RP-172), was investigated in vitro. Receptor radiolabeled displacement and functional calcium mobilization assays were performed to measure binding affinity and receptor activation of the new analogs. The obtained data revealed that only one of the diastereoisomeric peptides, RP-171, was able to selectively bind and activate MOR. Molecular modeling (docking and molecular dynamics (MD) simulations) suggests that both compounds should be accommodated in the MOR binding site. However, in the case of the inactive isomer RP-172, fewer hydrogen bonds, as well as instability of the canonical ionic interaction to Asp¹⁴⁷, could explain its very low MOR affinity.     

Hydrogenation of β-Keto Sulfones to β-Hydroxy Sulfones with Alkyl Aluminum Compounds: Structure of Intermediate Hydroalumination Products

β-Hydroxy sulfones are important in organic synthesis. The simplest method of β-hydroxy sulfones synthesis is the hydrogenation of β-keto sulfones. Herein, we report the reducing properties of alkyl aluminum compounds R₃Al (R = Et, i-Bu, n-Bu, t-Bu and n-Hex); i-Bu₂AlH; Et₂AlCl and EtAlCl₂ in the hydrogenation of β-keto sulfones. The compounds i-Bu₂AlH, i-Bu₃Al and Et₃Al are the at best reducing agents of β-keto sulfones to β-hydroxy sulfones. In reactions of β-keto sulfones with aluminum trialkyls, hydroalumination products with β-hydroxy sulfone ligands [R₂AlOC(C₆H₅)CH₂S(O)₂(p-R¹C₆H₄]_n [where n = 1,2; 2aa: R = i-Bu, R¹ = CH₃; 2ab: R = i-Bu, R¹ = Cl; 2ba: R = Et, R¹ = CH₃; 2bb: R = Et, R¹ = Cl] and {[Et₂AlOC(C₆H₅)CH₂S(O)₂(p-ClC₆H₄]∙Et₃Al}_n 3bb were obtained. These complexes in the solid state have a dimeric structure, while in solutions, they appear as equilibrium monomer–dimer mixtures. The hydrolysis of both the isolated 2aa, 2ab, 2ba, 2bb and 3bb and the postreaction mixtures quantitatively leads to pure racemic β-hydroxy sulfones. Hydroalumination reaction of β-keto sulfones with alkyl aluminum compounds and subsequent hydrolysis of the complexes is a simple and very efficient method of β-hydroxy sulfones synthesis.     

Synthesis of Mono-Amino Substituted γ-CD: Host–Guest Complexation and In Vitro Cytotoxicity Investigation

Cyclodextrins (CDs) are cyclic oligosaccharides which can trap hydrophobic molecules and improve their chemical, physical, and biological properties. γ-CD showed the highest aqueous solubility with the largest cavity diameter among other CD types. The current study describes a direct and easy method for nucleophilic mono-aminos to be substituted with γ-CD and tested for their ability to host the guest curcumin (CUR) as a hydrophobic drug model. The mass spectrometry and NMR analyses showed the successful synthesis of three amino-modified γ-CDs: mono-6-amino-6-deoxy-cyclodextrine (γ-CD-NH₂), mono-6-deoxy-6-ethanolamine-γ-cyclodextrine (γ-CD-NHCH₂CH₂OH), and mono-6-deoxy-6-aminoethylamino)-γ-cyclodextrin (γ-CD-NHCH₂CH₂NH₂). These three amino-modified γ-CDs were proven to be able to host CUR as native γ-CDs with formation constants equal to 6.70 ± 1.02, 5.85 ± 0.80, and 8.98 ± 0.90 mM⁻¹, respectively. Moreover, these amino-modified γ-CDs showed no significant toxicity against human dermal fibroblast cells. In conclusion, the current work describes a mono-substitution of amino-modified γ-CDs that can still host guests and showed low toxicity in human dermal fibroblasts cells. Therefore, the amino-modified γ-CDs can be used as a carrier host and be conjugated with a wide range of molecules for different biomedical applications, especially for active loading methods.     

Mechanistic insights into dopaminergic and serotonergic neurotransmission – concerted interactions with helices 5 and 6 drive the functional outcome

Brain functions rely on neurotransmitters that mediate communication between billions of neurons. Disruption of this communication can result in a plethora of psychiatric and neurological disorders. In this work, we combine molecular dynamics simulations, live-cell biosensor and electrophysiological assays to investigate the action of the neurotransmitter dopamine at the dopaminergic D₂ receptor (D₂R). The study of dopamine and closely related chemical probes reveals how neurotransmitter binding translates into the activation of distinct subsets of D₂R effectors (i.e.: G_i2, G_oB, G_z and β-arrestin 2). Ligand interactions with key residues in TM5 (S5.42) and TM6 (H6.55) in the D₂R binding pocket yield a dopamine-like coupling signature, whereas exclusive TM5 interaction is typically linked to preferential G protein coupling (in particular G_oB) over β-arrestin. Further experiments for serotonin receptors indicate that the reported molecular mechanism is shared by other monoaminergic neurotransmitter receptors. Ultimately, our study highlights how sequence variation in position 6.55 is used by nature to fine-tune β-arrestin recruitment and in turn receptor signaling and internalization of neurotransmitter receptors.

Neurotransmitter contacts within the receptor binding site differentially contribute to the overall functional response: transmembrane helix (TM) 5 contacts promote G protein coupling whereas concerted TM5–TM6 contacts enhance β-arrestin recruitment.     

Specific Recognition of β-Galactofuranose-Containing Glycans of Synthetic Neoglycoproteins by Sera of Chronic Chagas Disease Patients

Chagas disease (CD) can be accurately diagnosed by detecting Trypanosoma cruzi in patients’ blood using polymerase chain reaction (PCR). However, parasite-derived biomarkers are of great interest for the serological diagnosis and early evaluation of chemotherapeutic efficacy when PCR may fail, owing to a blood parasite load below the method’s limit of detection. Previously, we focused on the detection of specific anti-α-galactopyranosyl (α-Gal) antibodies in chronic CD (CCD) patients elicited by α-Gal glycotopes copiously expressed on insect-derived and mammal-dwelling infective parasite stages. Nevertheless, these stages also abundantly express cell surface glycosylphosphatidylinositol (GPI)-anchored glycoproteins and glycoinositolphospholipids (GIPLs) bearing nonreducing terminal β-galactofuranosyl (β-Galf) residues, which are equally foreign to humans and, therefore, highly immunogenic. Here we report that CCD patients’ sera react specifically with synthetic β-Galf-containing glycans. We took a reversed immunoglycomics approach that entailed: (a) Synthesis of T. cruzi GIPL-derived Galfβ1,3Manpα-(CH₂)₃SH (glycan G29_SH) and Galfβ1,3Manpα1,2-[Galfβ1,3]Manpα-(CH₂)₃SH (glycan G32_SH); and (b) preparation of neoglycoproteins NGP29b and NGP32b, and their evaluation in a chemiluminescent immunoassay. Receiver-operating characteristic analysis revealed that NGP32b can distinguish CCD sera from sera of healthy individuals with 85.3% sensitivity and 100% specificity. This suggests that Galfβ1,3Manpα1,2-[Galfβ1,3]Manpα is an immunodominant glycotope and that NGP32b could potentially be used as a novel CCD biomarker.     

Involvement of the γ Isoform of cPLA2 in the Biosynthesis of Bioactive N-Acylethanolamines

Arachidonylethanolamide (anandamide) acts as an endogenous ligand of cannabinoid receptors, while other N-acylethanolamines (NAEs), such as palmitylethanolamide and oleylethanolamide, show analgesic, anti-inflammatory, and appetite-suppressing effects through other receptors. In mammalian tissues, NAEs, including anandamide, are produced from glycerophospholipid via N-acyl-phosphatidylethanolamine (NAPE). The ɛ isoform of cytosolic phospholipase A₂ (cPLA₂) functions as an N-acyltransferase to form NAPE. Since the cPLA₂ family consists of six isoforms (α, β, γ, δ, ɛ, and ζ), the present study investigated a possible involvement of isoforms other than ɛ in the NAE biosynthesis. Firstly, when the cells overexpressing one of the cPLA₂ isoforms were labeled with [¹⁴C]ethanolamine, the increase in the production of [¹⁴C]NAPE was observed only with the ɛ-expressing cells. Secondly, when the cells co-expressing ɛ and one of the other isoforms were analyzed, the increase in [¹⁴C]N-acyl-lysophosphatidylethanolamine (lysoNAPE) and [¹⁴C]NAE was seen with the combination of ɛ and γ isoforms. Furthermore, the purified cPLA₂γ hydrolyzed not only NAPE to lysoNAPE, but also lysoNAPE to glycerophospho-N-acylethanolamine (GP-NAE). Thus, the produced GP-NAE was further hydrolyzed to NAE by glycerophosphodiesterase 1. These results suggested that cPLA₂γ is involved in the biosynthesis of NAE by its phospholipase A₁/A₂ and lysophospholipase activities.     

Preparation of α-amino acids via Ni-catalyzed reductive vinylation and arylation of α-pivaloyloxy glycine

This work emphasizes easy access to α-vinyl and aryl amino acids via Ni-catalyzed cross-electrophile coupling of bench-stable N-carbonyl-protected α-pivaloyloxy glycine with vinyl/aryl halides and triflates. The protocol permits the synthesis of α-amino acids bearing hindered branched vinyl groups, which remains a challenge using the current methods. On the basis of experimental and DFT studies, simultaneous addition of glycine α-carbon (Gly) radicals to Ni(0) and Ar–Ni(ii) may occur, with the former being more favored where oxidative addition of a C(sp²) electrophile to the resultant Gly–Ni(i) intermediate gives a key Gly–Ni(iii)–Ar intermediate. The auxiliary chelation of the N-carbonyl oxygen to the Ni center appears to be crucial to stabilize the Gly–Ni(i) intermediate.

We have developed Ni-catalyzed reductive coupling of N-carbonyl protected α-pivaloyloxy glycine with Csp²-electrophiles that enabled facile preparation of α-amino acids, including those bearing hindered branched vinyl groups.     

Complexation of Cyclodextrins with Benzoic Acid in Water-Organic Solvents: A Solvation-Thermodynamic Approach

The aim of this research is to obtain new data about the complexation between β-cyclodextrin (β-CD) and benzoic acid (BA) as a model reaction of the complex formation of hydrophobic molecules with cyclodextrins (CDs) in various media. This research may help developing cyclodextrin-based pharmaceutical formulations through the choice of the appropriate solvent mixture that may be employed in the industrial application aiming to control the reactions/processes in liquid phase. In this paper, NMR results for the molecular complex formation between BA and β-CD ([BA⊂β-CD]) in D₂O-DMSO-d₆ and in D₂O-EtOH have shown that the stability of the complex in the H₂O-DMSO-d₆ varies within the experimental error, while decreases in H₂O-EtOH. Changes in the Gibbs energy of BA resolvation in water and water–dimethylsulfoxide mixtures have been obtained and have been used in the analysis of the reagent solvation contributions into the Gibbs energy changes of the [BA⊂β-CD] molecular complex formation. Quantum chemical calculations of the interaction energy between β-CD and BA as well as the structure of the [BA⊂β-CD] complex and the energy of β-CD and BA interaction in vacuum and in the medium of water, methanol and dimethylsulfoxide solvents are carried out. The stability of [BA⊂β-CD] complex in H₂O-EtOH and H₂O-DMSO solvents, obtained by different methods, are compared. The thermodynamic parameters of the [BA⊂β-CD] molecular complexation as well as the reagent solvation contributions in H₂O-EtOH and H₂O-DMSO mixtures were analyzed by the solvation-thermodynamic approach.     

New 4-Aminoproline-Based Small Molecule Cyclopeptidomimetics as Potential Modulators of α4β1 Integrin

Integrin α₄β₁ belongs to the leukocyte integrin family and represents a therapeutic target of relevant interest given its primary role in mediating inflammation, autoimmune pathologies and cancer-related diseases. The focus of the present work is the design, synthesis and characterization of new peptidomimetic compounds that are potentially able to recognize α₄β₁ integrin and interfere with its function. To this aim, a collection of seven new cyclic peptidomimetics possessing both a 4-aminoproline (Amp) core scaffold grafted onto key α₄β₁-recognizing sequences and the (2-methylphenyl)ureido-phenylacetyl (MPUPA) appendage, was designed, with the support of molecular modeling studies. The new compounds were synthesized through SPPS procedures followed by in-solution cyclization maneuvers. The biological evaluation of the new cyclic ligands in cell adhesion assays on Jurkat cells revealed promising submicromolar agonist activity in one compound, namely, the c[Amp(MPUPA)Val-Asp-Leu] cyclopeptide. Further investigations will be necessary to complete the characterization of this class of compounds.     

α-Amyrin and β-Amyrin Isolated from Celastrus hindsii Leaves and Their Antioxidant,Anti-Xanthine Oxidase,and Anti-Tyrosinase Potentials

Celastrus hindsii is a popular medicinal plant in Vietnam and Southeast Asian countries as well as in South America. In this study, an amount of 12.05 g of an α-amyrin and β-amyrin mixture was isolated from C. hindsii (10.75 g/kg dry weight) by column chromatography applying different solvent systems to obtain maximum efficiency. α-Amyrin and β-amyrin were then confirmed by gas chromatography-mass spectrometry (GC-MS), electrospray ionization-mass spectrometry (ESI-MS), and nuclear magnetic resonance (NMR). The antioxidant activities of the α-amyrin and β-amyrin mixture were determined via 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,20-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays with IC₅₀ of 125.55 and 155.28 µg/mL, respectively. The mixture exhibited a high potential for preventing gout by inhibiting a relevant key enzyme, xanthine oxidase (XO) (IC₅₀ = 258.22 µg/mL). Additionally, an important enzyme in skin hyperpigmentation, tyrosinase, was suppressed by the α-amyrin and β-amyrin mixture (IC₅₀ = 178.85 µg/mL). This study showed that C. hindsii is an abundant source for the isolation of α-amyrin and β-amyrin. Furthermore, this was the first study indicating that α-amyrin and β-amyrin mixture are promising in future therapies for gout and skin hyperpigmentation.     

Two Key Amino Acids Variant of α-l-arabinofuranosidase from Bacillus subtilis Str. 168 with Altered Activity for Selective Conversion Ginsenoside Rc to Rd

α-l-arabinofuranosidase is a subfamily of glycosidases involved in the hydrolysis of l-arabinofuranosidic bonds, especially in those of the terminal non-reducing arabinofuranosyl residues of glycosides, from which efficient glycoside hydrolases can be screened for the transformation of ginsenosides. In this study, the ginsenoside Rc-hydrolyzing α-l-arabinofuranosidase gene, BsAbfA, was cloned from Bacilus subtilis, and its codons were optimized for efficient expression in E. coli BL21 (DE3). The recombinant protein BsAbfA fused with an N-terminal His-tag was overexpressed and purified, and then subjected to enzymatic characterization. Site-directed mutagenesis of BsAbfA was performed to verify the catalytic site, and the molecular mechanism of BsAbfA catalyzing ginsenoside Rc was analyzed by molecular docking, using the homology model of sequence alignment with other β-glycosidases. The results show that the purified BsAbfA had a specific activity of 32.6 U/mg. Under optimal conditions (pH 5, 40 °C), the kinetic parameters K_m of BsAbfA for pNP-α-Araf and ginsenoside Rc were 0.6 mM and 0.4 mM, while the K_cat/K_m were 181.5 s⁻¹ mM⁻¹ and 197.8 s⁻¹ mM⁻¹, respectively. More than 90% of ginsenoside Rc could be transformed by 12 U/mL purified BsAbfA at 40 °C and pH 5 in 24 h. The results of molecular docking and site-directed mutagenesis suggested that the E173 and E292 variants for BsAbfA are important in recognizing ginsenoside Rc effectively, and to make it enter the active pocket to hydrolyze the outer arabinofuranosyl moieties at C₂₀ position. These remarkable properties and the catalytic mechanism of BsAbfA provide a good alternative for the effective biotransformation of the major ginsenoside Rc into Rd.     

[18F]Nifene PET/CT Imaging in Mice: Improved Methods and Preliminary Studies of α4β2* Nicotinic Acetylcholinergic Receptors in Transgenic A53T Mouse Model of α-Synucleinopathy and Post-Mortem Human Parkinson’s Disease

We report [¹⁸F]nifene binding to α4β2* nicotinic acetylcholinergic receptors (nAChRs) in Parkinson’s disease (PD). The study used transgenic Hualpha-Syn(A53T) PD mouse model of α-synucleinopathy for PET/CT studies in vivo and autoradiography in vitro. Additionally, postmortem human PD brain sections comprising of anterior cingulate were used in vitro to assess translation to human studies. Because the small size of mice brain poses challenges for PET imaging, improved methods for radiosynthesis of [¹⁸F]nifene and simplified PET/CT procedures in mice were developed by comparing intravenous (IV) and intraperitoneal (IP) administered [¹⁸F]nifene. An optimal PET/CT imaging time of 30–60 min post injection of [¹⁸F]nifene was established to provide thalamus to cerebellum ratio of 2.5 (with IV) and 2 (with IP). Transgenic Hualpha-Syn(A53T) mice brain slices exhibited 20–35% decrease while in vivo a 20–30% decrease of [¹⁸F]nifene was observed. Lewy bodies and α-synuclein aggregates were confirmed in human PD brain sections which lowered the [¹⁸F]nifene binding by more than 50% in anterior cingulate. Thus [¹⁸F]nifene offers a valuable tool for PET imaging studies of PD.     

Structural Insights into the Host–Guest Complexation between β-Cyclodextrin and Bio-Conjugatable Adamantane Derivatives

Understanding the host–guest chemistry of α-/β-/γ- cyclodextrins (CDs) and a wide range of organic species are fundamentally attractive, and are finding broad contemporary applications toward developing efficient drug delivery systems. With the widely used β-CD as the host, we herein demonstrate that its inclusion behaviors toward an array of six simple and bio-conjugatable adamantane derivatives, namely, 1-adamantanol (adm-1-OH), 2-adamantanol (adm-2-OH), adamantan-1-amine (adm-1-NH₂), 1-adamantanecarboxylic acid (adm-1-COOH), 1,3-adamantanedicarboxylic acid (adm-1,3-diCOOH), and 2-[3-(carboxymethyl)-1-adamantyl]acetic acid (adm-1,3-diCH₂COOH), offer inclusion adducts with diverse adamantane-to-CD ratios and spatial guest locations. In all six cases, β-CD crystallizes as a pair supported by face-to-face hydrogen bonding between hydroxyl groups on C2 and C3 and their adjacent equivalents, giving rise to a truncated-cone-shaped cavity to accommodate one, two, or three adamantane derivatives. These inclusion complexes can be terminated as (adm-1-OH)₂⊂CD₂ (1, 2:2), (adm-2-OH)₃⊂CD₂ (2, 3:2), (adm-1-NH₂)₃⊂CD₂ (3, 3:2), (adm-1-COOH)₂⊂CD₂ (4, 2:2), (adm-1,3-diCOOH)⊂CD₂ (5, 1:2), and (adm-1,3-diCH₂COOH)⊂CD₂ (6, 1:2). This work may shed light on the design of nanomedicine with hierarchical structures, mediated by delicate cyclodextrin-based hosts and adamantane-appended drugs as the guests.