BDNF- and VEGF-Responsive Stimulus to an NGF Mimic Cyclic Peptide with Copper Ionophore Capability and Ctr1/CCS-Driven Signaling.

Neurotrophins are a family of growth factors that play a key role in the development and regulation of the functioning of the central nervous system. Their use as drugs is made difficult by their poor stability, cellular permeability, and side effects. Continuing our effort to use peptides that mimic the neurotrophic growth factor (NGF), the family model protein, and specifically the N-terminus of the protein, here we report on the spectroscopic characterization and resistance to hydrolysis of the 14-membered cyclic peptide reproducing the N-terminus sequence (SSSHPIFHRGEFSV (c-NGF(1-14)). Far-UV CD spectra and a computational study show that this peptide has a rigid conformation and left-handed chirality typical of polyproline II that favors its interaction with the D5 domain of the NGF receptor TrkA. c-NGF(1-14) is able to bind Cu2+ with good affinity; the resulting complexes have been characterized by potentiometric and spectroscopic measurements. Experiments on PC12 cells show that c-NGF(1-14) acts as an ionophore, influencing the degree and the localization of both the membrane transporter (Ctr1) and the copper intracellular transporter (CCS). c-NGF(1-14) induces PC12 differentiation, mimics the protein in TrkA phosphorylation, and activates the kinase cascade, inducing Erk1/2 phosphorylation. c-NGF(1-14) biological activities are enhanced when the peptide interacts with Cu2+ even with the submicromolar quantities present in the culture media as demonstrated by ICP-OES measurements. Finally, c-NGF(1-14) and Cu2+ concur to activate the cAMP response element-binding protein CREB that, in turn, induces the brain-derived neurotrophic factor (BDNF) and the vascular endothelial growth factor (VEGF) release.

Polymeric Nanozyme with SOD Activity Capable of Inhibiting Self- and Metal-Induced α-Synuclein Aggregation.

Despite decades of research, Parkinson's disease is still an idiopathic pathology for which no cure has yet been found. This is partly explained by the multifactorial character of most neurodegenerative syndromes, whose generation involves multiple pathogenic factors. In Parkinson's disease, two of the most important ones are the aggregation of α-synuclein and oxidative stress. In this work, we address both issues by synthesizing a multifunctional nanozyme based on grafting a pyridinophane ligand that can strongly coordinate CuII, onto biodegradable PEGylated polyester nanoparticles. The resulting nanozyme exhibits remarkable superoxide dismutase activity together with the ability to inhibit the self-induced aggregation of α-synuclein into amyloid-type fibrils. Furthermore, the combination of the chelator and the polymer produces a cooperative effect whereby the resulting nanozyme can also halve CuII-induced α-synuclein aggregation.

The Insulin-Degrading Enzyme from Structure to Allosteric Modulation: New Perspectives for Drug Design.

The insulin-degrading enzyme (IDE) is a Zn2+ peptidase originally discovered as the main enzyme involved in the degradation of insulin and other amyloidogenic peptides, such as the ß-amyloid (Aß) peptide. Therefore, a role for the IDE in the cure of diabetes and Alzheimer's disease (AD) has been long envisaged. Anyway, its role in degrading amyloidogenic proteins remains not clearly defined and, more recently, novel non-proteolytic functions of the IDE have been proposed. From a structural point of view, the IDE presents an atypical clamshell structure, underscoring unique enigmatic enzymological properties. A better understanding of the structure-function relationship may contribute to solving some existing paradoxes of IDE biology and, in light of its multifunctional activity, might lead to novel therapeutic approaches.

New Biotinylated GHK and Related Copper(II) Complex: Antioxidant and Antiglycant Properties In Vitro against Neurodegenerative Disorders.

Neurodegenerative diseases affect millions of people worldwide. The failure of the enzymatic degradation, the oxidative stress, the dyshomeostasis of metal ions, among many other biochemical events, might trigger the pathological route, but the onset of these pathologies is unknown. Multi-target and multifunctional molecules could address several biomolecular issues of the pathologies. The tripeptide GHK, a bioactive fragment of several proteins, and the related copper(II) complex have been largely used for many purposes, from cosmetic to therapeutic applications. GHK derivatives were synthesized to increase the peptide stability and improve the target delivery. Herein we report the synthesis of a new biotin-GHK conjugate (BioGHK) through orthogonal reactions. BioGHK is still capable of coordinating copper(II), as observed by spectroscopic and spectrometric measurements. The spectroscopic monitoring of the copper-induced ascorbate oxidation was used to measure the antioxidant activity Cu(II)-BioGHK complex, whereas antiglycant activity of the ligand towards harmful reactive species was investigated using MALDI-TOF. The affinity of BioGHK for streptavidin was evaluated using a spectrophotometric assay and compared to that of biotin. Finally, the antiaggregant activity towards amyloid-ß was evaluated using a turn-on fluorescent dye. BioGHK could treat and/or prevent several adverse biochemical reactions that characterize neurodegenerative disorders, such as Alzheimer's disease.

Copper(II) Complexes with Carnosine Conjugates of Hyaluronic Acids at Different Dipeptide Loading Percentages Behave as Multiple SOD Mimics and Stimulate Nrf2 Translocation and Antioxidant Response in In Vitro Inflammatory Model.

A series of copper(II) complexes with the formula [Cu2+Hy(x)Car%] varying the molecular weight (MW) of Hyaluronic acid (Hy, x = 200 or 700 kDa) conjugated with carnosine (Car) present at different loading were synthesized and characterized via different spectroscopic techniques. The metal complexes behaved as Cu, Zn-superoxide dismutase (SOD1) mimics and showed some of the most efficient reaction rate values produced using a synthetic and water-soluble copper(II)-based SOD mimic reported to date. The increase in the percentage of Car moieties parallels the enhancement of the I50 value determined via the indirect method of Fridovich. The presence of the non-functionalized Hy OH groups favors the scavenger activity of the copper(II) complexes with HyCar, recalling similar behavior previously found for the copper(II) complexes with Car conjugated using ß-cyclodextrin or trehalose. In keeping with the new abilities of SOD1 to activate protective agents against oxidative stress in rheumatoid arthritis and osteoarthritis diseases, Cu2+ interaction with HyCar promotes the nuclear translocation of erythroid 2-related factor that regulates the expressions of target genes, including Heme-Oxigenase-1, thus stimulating an antioxidant response in osteoblasts subjected to an inflammatory/oxidative insult.

Cyclodextrin Polymers Functionalized with Histidine and Carcinine as Chelating Therapeutics for Copper Dyshomeostasis.

In recent years, cyclodextrin polymeric nanoparticles have been designed to introduce new properties and extend their medical applications. Based on the features of cyclodextrins, we derivatized cross-linked cyclodextrin polymers with histidine or carcinine moieties. We found that amylases do not hydrolyze cyclodextrin polymers. The new polymers can form copper(II) complexes and may act as nanochelators to counteract copper(II) dyshomeostasis-related diseases. Furthermore, the copper(II) complexes show superoxide dismutase activity, similar to free carcinine and histidine complexes. The antioxidant biological activity of the copper(II) complex formed in situ may protect cells from oxidative damage related to copper dyshomeostasis.

Orobanche crenata Forssk. Extract Affects Human Breast Cancer Cell MCF-7 Survival and Viral Replication.

BACKGROUND: Breast cancer (BC) is the leading cause of death worldwide. The severity of BC strictly depends on the molecular subtype. The less aggressive hormone-positive subtype is treated with adjuvant endocrine therapy (AET), which causes both physical and psychological side effects. This condition strongly impacts the adherence and persistence of AET among oncologic patients. Moreover, viral infections also constitute a serious problem for public health. Despite their efficacy, antiviral agents present several therapeutic limits. Accordingly, in the present work, we investigated the antitumor and antiviral activities of Orobanche crenata Forssk. (O. crenata), a parasitic plant, endemic to the Mediterranean basin, traditionally known for its beneficial properties for human health. METHODS: The MTT assay was carried out to evaluate the cytotoxic effect of O. crenata leaf extract (OCLE) on human breast cancer cells (MCF-7 and MDA-MB-231) and the primary HFF-1 cell line. The lactic dehydrogenase (LDH) assay was performed on MCF-7 cells to analyze necrotic cell death. The antioxidant effect of OCLE was evaluated by intracellular determination of the reactive oxygen species and thiol groups, by DPPH and ABTS assays. The antiviral activity of OCLE was determined against Poliovirus 1, Echovirus 9, Human respiratory syncytial virus, Adenovirus type 2 and type 5, Coxsackievirus B1 (CoxB1) and B3 (CoxB3), Herpes simplex type 1 (HSV-1) and type 2 (HSV-2), and ß-Coronavirus by the plaque reduction assay. RESULTS: The extract, after 24 h of incubation, did not affect MDA-MB-231 and HFF-1 cell viability. However, at the same time point, it showed a dose-dependent inhibitory effect on MCF-7 cells, with an increase in LDH release. OCLE exhibited free radical scavenging activity and significantly increased non-protein thiol levels in MCF-7 cells. OCLE effectively inhibited HSV-1, HSV-2, CoxB1, and CoxB3 replication. CONCLUSIONS: The overall results showed an interesting inhibitory effect of OCLE on both MCF-7 cell survival and viral replication.

Synergistic Effect of L-Carnosine and Hyaluronic Acid in Their Covalent Conjugates on the Antioxidant Abilities and the Mutual Defense against Enzymatic Degradation.

Hyaluronic acid (Hy) is a natural linear polymer that is widely distributed in different organisms, especially in the articular cartilage and the synovial fluid. During tissue injury due to oxidative stress, Hy plays an important protective role. All the beneficial properties of Hy make the polymer attractive for many biomedical uses; however, the low stability and short biological half-life limit Hy application. To overcome these problems, the addition of small antioxidant molecules to Hy solution has been employed to protect the molecular integrity of Hy or delay its degradation. Carnosine (ß-alanyl-L-histidine, Car) protects cells from the damage due to the reactive species derived from oxygen (ROS), nitrogen (RNS) or carbonyl groups (RCS). Car inhibits the degradation of hyaluronan induced by free radical processes in vitro but, like Hy, the potential protective action of Car is drastically hampered by the enzymatic hydrolysis in vivo. Recently, we conjugated Hy to Car and the derivatives (HyCar) showed protective effects in experimental models of osteoarthritis and rheumatoid arthritis in vivo. Here we report the antioxidant activity exerted by HyCar against ROS, RNS and RCS. Moreover, we tested if the covalent conjugation between Hy and Car inhibits the enzymatic hydrolysis of the polymer and the dipeptide backbone. We found that the antioxidant properties and the resistance to the enzymatic hydrolysis of Hy and Car are greatly improved by the conjugation.

Neuroprotective Effect of Carnosine Is Mediated by Insulin-Degrading Enzyme.

l-Carnosine is an endogenous dipeptide that has high potential for therapeutic purposes, being an antioxidant with metal chelating, anti-aggregating, anti-inflammatory, and neuroprotective properties. Despite its potential therapeutic values, the biomolecular mechanisms involved in neuroprotection are not fully understood. Here, we demonstrate, at chemical and biochemical levels, that insulin-degrading enzyme plays a pivotal role in carnosine neuroprotection.

Terpyridine functionalized cyclodextrin nanoparticles: metal coordination for tuning anticancer activity.

Multi-metal and multi-cavity systems based on the coordination properties of terpyridine functionalized cyclodextrin polymers were synthesized and characterized. Nanoparticles decorated with terpyridine derivatives via metal coordination showed high antiproliferative activity in tumor cells.

Insulin-Degrading Enzyme Is a Non Proteasomal Target of Carfilzomib and Affects the 20S Proteasome Inhibition by the Drug.

Carfilzomib is a last generation proteasome inhibitor (PI) with proven clinical efficacy in the treatment of relapsed/refractory multiple myeloma. This drug is considered to be extremely specific in inhibiting the chymotrypsin-like activity of the 20S proteasome, encoded by the ß5 subunit, overcoming some bortezomib limitations, the first PI approved for multiple myeloma therapy which is however burdened by a significant toxicity profile, due also to its off-target effects. Here, molecular approaches coupled with molecular docking studies have been used to unveil that the Insulin-Degrading Enzyme, a ubiquitous and highly conserved Zn2+ peptidase, often found to associate with proteasome in cell-based models, is targeted by carfilzomib in vitro. The drug behaves as a modulator of IDE activity, displaying an inhibitory effect over 10-fold lower than for the 20S. Notably, the interaction of IDE with the 20S enhances in vitro the inhibitory power of carfilzomib on proteasome, so that the IDE-20S complex is an even better target of carfilzomib than the 20S alone. Furthermore, IDE gene silencing after delivery of antisense oligonucleotides (siRNA) significantly reduced carfilzomib cytotoxicity in rMC1 cells, a validated model of Muller glia, suggesting that, in cells, the inhibitory activity of this drug on cell proliferation is somewhat linked to IDE and, possibly, also to its interaction with proteasome.

Correction: Genovese et al. In Vitro Antibacterial, Anti-Adhesive and Anti-Biofilm Activities of Krameria lappacea (Dombey) Burdet & B.B. Simpson Root Extract against Methicillin-Resistant Staphylococcus aureus Strains. Antibiotics 2021, 10, 428.

The authors would like to make the following corrections to the published paper [...].

In Vitro Antibacterial, Anti-Adhesive and Anti-Biofilm Activities of Krameria lappacea (Dombey) Burdet & B.B. Simpson Root Extract against Methicillin-Resistant Staphylococcus aureus Strains.

Methicillin-resistant Staphylococcus aureus (MRSA) represents a serious threat to public health, due to its large variety of pathogenetic mechanisms. Accordingly, the present study aimed to investigate the anti-MRSA activities of Krameria lappacea, a medicinal plant native to South America. Through Ultra-High-Performance Liquid Chromatography coupled with High-Resolution Mass spectrometry, we analyzed the chemical composition of Krameria lappacea root extract (KLRE). The antibacterial activity of KLRE was determined by the broth microdilution method, also including the minimum biofilm inhibitory concentration and minimum biofilm eradication concentration. Besides, we evaluated the effect on adhesion and invasion of human lung carcinoma A549 cell line by MRSA strains. The obtained results revealed an interesting antimicrobial action of this extract, which efficiently inhibit the growth, biofilm formation, adhesion and invasion of MRSA strains. Furthermore, the chemical analysis revealed the presence in the extract of several flavonoid compounds and type-A and type-B proanthocyanidins, which are known for their anti-adhesive effects. Taken together, our findings showed an interesting antimicrobial activity of KLRE, giving an important contribution to the current knowledge on the biological activities of this plant.

Exploring Charged Polymeric Cyclodextrins for Biomedical Applications.

Over the years, cyclodextrin uses have been widely reviewed and their proprieties provide a very attractive approach in different biomedical applications. Cyclodextrins, due to their characteristics, are used to transport drugs and have also been studied as molecular chaperones with potential application in protein misfolding diseases. In this study, we designed cyclodextrin polymers containing different contents of ß- or γ-cyclodextrin, and a different number of guanidinium positive charges. This allowed exploration of the influence of the charge in delivering a drug and the effect in the protein anti-aggregant ability. The polymers inhibit Amiloid ß peptide aggregation; such an ability is modulated by both the type of CyD cavity and the number of charges. We also explored the effect of the new polymers as drug carriers. We tested the Doxorubicin toxicity in different cell lines, A2780, A549, MDA-MB-231 in the presence of the polymers. Data show that the polymers based on γ-cyclodextrin modified the cytotoxicity of doxorubicin in the A2780 cell line.

Antimicrobial, Antioxidant, and Cytotoxic Activities of Juglans regia L. Pellicle Extract.

The difficulty to treat resistant strains-related hospital-acquired infections (HAIs) promoted the study of phytoextracts, known sources of bioactive molecules. Accordingly, in the present study, the pharmacological activities of Juglans regia (L.) pellicle extract (WPE) were investigated. The antiviral effect was tested against Herpes simplex virus type 1 and 2, Poliovirus 1, Adenovirus 2, Echovirus 9, Coxsackievirus B1 through the plaque reduction assay. The antibacterial and antifungal activities were evaluated against medically important strains, by the microdilution method. DPPH and superoxide dismutase (SOD)s-like activity assays were used to determine the antioxidant effect. Besides, the extract was screened for cytotoxicity on Caco-2, MCF-7, and HFF1 cell lines by the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. The total phenolic and flavonoid contents were also evaluated. Interestingly, WPE inhibited Herpes simplex viruses (HSVs) replication, bacterial and fungal growth. WPE showed free radical scavenging capacity and inhibited superoxide anion formation in a dose-dependent manner. These effects could be attributed to the high content of phenols and flavonoids, which were 0.377 ± 0.01 mg GE/g and 0.292 ± 0.08 mg CE/g, respectively. Moreover, WPE was able to reduce Caco-2 cell viability, at both 48 h and 72 h. The promising results encourage further studies aimed to better elucidate the role of WPE in the prevention of human infectious diseases.

Hyaluronan-carnosine conjugates inhibit Aß aggregation and toxicity.

Alzheimer's disease is the most common neurodegenerative disorder. Finding a pharmacological approach that cures and/or prevents the onset of this devastating disease represents an important challenge for researchers. According to the amyloid cascade hypothesis, increases in extracellular amyloid-ß (Aß) levels give rise to different aggregated species, such as protofibrils, fibrils and oligomers, with oligomers being the more toxic species for cells. Many efforts have recently been focused on multi-target ligands to address the multiple events that occur concurrently with toxic aggregation at the onset of the disease. Moreover, investigating the effect of endogenous compounds or a combination thereof is a promising approach to prevent the side effects of entirely synthetic drugs. In this work, we report the synthesis, structural characterization and Aß antiaggregant ability of new derivatives of hyaluronic acid (Hy, 200 and 700 kDa) functionalized with carnosine (Car), a multi-functional natural dipeptide. The bioactive substances (HyCar) inhibit the formation of amyloid-type aggregates of Aß42 more than the parent compounds; this effect is proportional to Car loading. Furthermore, the HyCar derivatives are able to dissolve the amyloid fibrils and to reduce Aß-induced toxicity in vitro. The enzymatic degradation of Aß is also affected by the interaction with HyCar.

Carnoquinolines Target Copper Dyshomeostasis, Aberrant Protein-Protein Interactions, and Oxidative Stress.

Metal dysregulation, oxidative stress, protein modification, and aggregation are factors strictly interrelated and associated with neurodegenerative pathologies. As such, all of these aspects represent valid targets to counteract neurodegeneration and, therefore, the development of metal-binding compounds with other properties to combat multifactorial disorders is definitely on the rise. Herein, the synthesis and in-depth analysis of the first hybrids of carnosine and 8-hydroxyquinoline, carnoquinolines (CarHQs), which combine the properties of the dipeptide with those of 8-hydroxyquinoline, are reported. CarHQs and their copper complexes were characterized through several techniques, such as ESI-MS and NMR, UV/Vis, and circular dichroism spectroscopy. CarHQs can modulate self- and copper-induced amyloid-ß aggregation. These hybrids combine the antioxidant activity of their parent compounds. Therefore, they can simultaneously scavenge free radicals and reactive carbonyl species, thanks to the phenolic group and imidazole ring. These results indicate that CarHQs are promising multifunctional candidates for neurodegenerative disorders and they are worthy of further studies.

Structural and functional evidence for citicoline binding and modulation of 20S proteasome activity: Novel insights into its pro-proteostatic effect.

Citicoline or CDP-choline is a drug, made up by a cytidine 5'-diphosphate moiety and choline, which upon adsorption is rapidly hydrolyzed into cytidine 5'-diphosphate and choline, easily bypassing the blood-brain barrier. Once in the brain, these metabolites are used to re-synthesize citicoline in neurons and in the other cell histo-types which uptake them. Citicoline administration finds broad therapeutic application in the treatment of glaucoma as well as other retinal disorders by virtue of its safety profile and neuro-protective and neuroenhancer activity, which significantly improves the visual function. Further, though supported by limited clinical studies, this molecule finds therapeutic application in neurodegenerative disease, delaying the cognitive decline in Alzheimer's Disease (AD) and Parkinson's Disease (PD) subjects. In this work we show that citicoline greatly affects the proteolytic activity of the 20S proteasome on synthetic and natural substrates, functioning as a bimodal allosteric modulator, likely binding at multiple sites. In silico binding simulations identify several potential binding sites for citicoline on 20S proteasome, and their topology envisages the possibility that, by occupying some of these pockets, citicoline may induce a conformational shift of the 20S proteasome, allowing to sketch a working hypothesis for the structural basis of its function as allosteric modulator. In addition, we show that over the same concentration range citicoline affects the distribution of assembled proteasome populations and turn-over of ubiquitinated proteins in SH-SY5Y and SK-N-BE human neuroblastoma cells, suggesting its potential role as a regulator of proteostasis in nervous cells.

Acrolein and Copper as Competitive Effectors of α-Synuclein.

Mounting evidence supports the role of amyloidogenesis, oxidative stress, and metal dyshomeostasis in the development of neurodegenerative disorders. Parkinson's Disease is characterized by α-synuclein (αSyn) accumulation and aggregation in brain regions, also promoted by Cu2+ . αSyn is modified by reactive carbonyl species, including acrolein (ACR). Notwithstanding these findings, the interplay between ACR, copper, and αSyn has never been investigated. Therefore, we explored more thoroughly the effects of ACR on αSyn using an approach based on LC-MS/MS analysis. We also evaluated the influence of Cu2+ on the protein carbonylation and how the ACR modification impacts the Cu2+ binding and the production of Reactive Oxygen Species (ROS). Finally, we investigated the effects of ACR and Cu2+ ions on the αSyn aggregation by dynamic light scattering and fluorescence assays. Cu2+ regioselectively inhibits the modification of His50 by ACR, the carbonylation lowers the affinity of His50 for Cu2+ and ACR inhibits αSyn aggregation both in the presence and in the absence of Cu2+ .