Discovering Effective Chiral Dipeptides against Aß(1-42) Aggregation by the Computational Screening Strategy.

The ß-sheet-breaker (BSB) peptides inhibiting amyloidogenic aggregation have been extensively studied. However, the inhibition efficacy of ultrashort chiral dipeptides remains inadequately understood. In this study, we proposed a computational screening strategy to identify chiral dipeptides as BSB with optimal antiaggregation performance against Aß(1-42) aggregation. We constructed a complete dipeptide library encompassing all possible chiral sequence arrangements and then filtered the library by cascaded molecular docking-molecular dynamics (MD) simulation. Our screening strategy discovered dipeptide DWDP (superscript for chirality) that displayed strong interactions with Aß fibrils and inhibitory effects on Aß aggregation, validated by subsequent experiments. Mechanistic investigation by both MD and replica-exchange molecular dynamics (REMD) simulations revealed that DWDP interacts with Aß by hydrophobic contacts and hydrogen bonds and thus inhibits Aß intermolecular contacts and salt bridge formation, therefore inhibiting Aß aggregation and disrupting Aß aggregates. Totally, our strategy presents a viable approach to discover potential dipeptides with effective antiaggregation ability as potential therapeutic agents for Alzheimer's disease.

Exploiting the potential of rivastigmine-melatonin derivatives as multitarget metal-modulating drugs for neurodegenerative diseases.

The multifaceted nature of the neurodegenerative diseases, as Alzheimer's disease (AD) and Parkinson's disease (PD) with several interconnected etiologies, and the absence of effective drugs, led herein to the development and study of a series of multi-target directed ligands (MTDLs). The developed RIV-IND hybrids, derived from the conjugation of an approved anti-AD drug, rivastigmine (RIV), with melatonin analogues, namely indole (IND) derivatives, revealed multifunctional properties, by associating the cholinesterase inhibition of the RIV drug with antioxidant activity, biometal (Cu(II), Zn(II), Fe(III)) chelation properties, inhibition of amyloid-ß (Aß) aggregation (self- and Cu-induced) and of monoamine oxidases (MAOs), as well as neuroprotection capacity in cell models of AD and PD. In particular, two hybrids with hydroxyl-substituted indoles (5a2 and 5a3) could be promising multifunctional compounds that inspire further development of novel anti-neurodegenerative drugs.

Critical aggregation concentration and reversibility of amyloid-ß (1-40) oligomers.

Amyloid-beta (Aß) aggregation is a critical factor in the pathogenesis of Alzheimer's disease, with distinct aggregation behaviours observed between its isoforms Amyloid-ß 1-40 (Aß40) and 1-42 (Aß42). In this study, we investigated the aggregation properties of Aß40 using fluorescence correlation spectroscopy (FCS) and detailed data analysis. Our results reveal that Aß40 undergoes a two-step cooperative aggregation process. The first step, characterized by a critical aggregation concentration (cac) of 0.5 ± 0.3 µM, results in the formation of metastable oligomers of 5 to 25 monomers and stable oligomers of 50 to 100 monomers, with less than 10% of the amyloid aggregated. The second step, with a cac of 18.9 ± 2.2 µM, leads to the formation of much larger aggregates, consistent with protofibrils, and approximately 50% aggregated amyloid. Notably, the cac for Aß40 is significantly higher, and the fraction of aggregated amyloid is much lower compared to Aß42, indicating a lower propensity for aggregation. Additionally, our findings suggest that Aß40 early oligomers are reversible upon dilution, albeit with a kinetic barrier to disaggregation. These insights into the aggregation mechanisms of Aß40 enhance our understanding of its role in Alzheimer's disease and may inform therapeutic strategies targeting amyloid aggregation.

Computational insights into the aggregation mechanism and amyloidogenic core of aortic amyloid medin polypeptide.

Medin amyloid, prevalent in the vessel walls of 97 % of individuals over 50, contributes to arterial stiffening and cerebrovascular dysfunction, yet our understanding of its aggregation mechanism remains limited. Dividing the full-length 50-amino-acid medin peptide into five 10-residue segments, we conducted individual investigations on each segment's self-assembly dynamics via microsecond-timescale atomistic discrete molecular dynamics (DMD) simulations. Our findings showed that medin1-10 and medin11-20 segments predominantly existed as isolated unstructured monomers, unable to form stable oligomers. Medin31-40 exhibited moderate aggregation, forming dynamic ß-sheet oligomers with frequent association and dissociation. Conversely, medin21-30 and medin41-50 segments demonstrated significant self-assembly capability, readily forming stable ß-sheet-rich oligomers. Residue pairwise contact frequency analysis highlighted the critical roles of residues 22-26 and 43-49 in driving the self-assembly of medin21-30 and medin41-50, acting as the ß-sheet core and facilitating ß-strand formation in other regions within medin monomers, expecting to extend to oligomers and fibrils. Regions containing residues 22-26 and 43-49, with substantial self-assembly abilities and assistance in ß-sheet formation, represent crucial targets for amyloid inhibitor drug design against aortic medial amyloidosis (AMA). In summary, our study not only offers deep insights into the mechanism of medin amyloid formation but also provides crucial theoretical and practical guidance for future treatments of AMA.

Exploring the therapeutic potential of Potentilla fragarioides var. major (Rosaceae) extract in Alzheimer's disease using in vitro and in vivo models: A multi-faceted approach.

Alzheimer's disease (AD) is the most common cause of dementia and is caused by various factors including amyloid-beta (Aß) aggregation. We investigated the pharmacological effects of the ethanol extract of Potentilla fragarioides var. major (Rosaceae) (EEPF) on AD-related pathogenesis, which remain elusive. We observed the effects of EEPF on Aß disaggregation and free-radical scavenging activities for 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) using in vitro assays, evaluated the effects of EEPF on memory loss in two animal models, and examined the molecular regulatory mechanisms of EEPF using an antibody-protein microarray in EEPF-treated neuronal cell lines. EEPF inhibited Aß aggregation in a concentration-dependent manner and enhanced free-radical scavenging activities for ABTS and DPPH. EEPF significantly inhibited memory impairment in the passive avoidance task, Y-maze test, and Morris water maze test in scopolamine-induced short-term memory loss mice and Aß-injected AD-like mice. Nissl staining and immunohistochemistry for NeuN and Iba-1 confirmed the neuroprotective and anti-inflammatory effects of EEPF in both animal models. In H2O2-treated HT22 hippocampal cells, EEPF significantly prevented cell damage, enhanced CaMK2, and reduced ferric reductase. In lipopolysaccharide (LPS)-stimulated BV-2 microglia, EEPF significantly inhibited LPS-induced production of inflammatory factors, such as nitric oxide, prostaglandin E2, tumor necrosis factor-α, and interleukin-6, and decreased the phosphorylation of Smad3 and cyclin D3. High-performance liquid chromatography confirmed that EEPF has five major components: neochlorogenic acid, chlorogenic acid, polydatin, isochlorogenic acid A, and buddleoside, with amounts ranging across 1.91-9.41 mg/g. EEPF may be a promising drug for treatment of AD and AD-related brain disorders.

Guluronic acid disaccharide inhibits reactive oxygen species production and amyloid-ß oligomer formation.

In general, Cu(II) is the critical factor in catalyzing reactive oxygen species (ROS) production and accelerating amyloid-ß (Aß) oligomer formation in Alzheimer's disease (AD). Natural chelating agents with good biocompatibility and appropriate binding affinity with Cu(II) have emerged as potential candidates for AD therapy. Herein, we tested the capability of guluronic acid disaccharide (Di-GA), a natural chelating agent with the moderate association affinity to Cu(II), in inhibiting ROS production and Aß oligomer formation. The results showed that Di-GA was capable of chelating with Cu(II) and reducing ROS production, even in solutions containing Fe(II), Zn(II), and Aß. In addition, Di-GA can also capture Cu(II) from Cu-Aß complexes and decrease Aß oligomer formation. The cellular results confirmed that Di-GA prevented SH-SY5Y cells from ROS and Aß oligomer damage by reducing the injury of ROS and Aß oligomers on cell membrane and decreasing their intracellular damage on mitochondria. Notably, the slightly higher efficiency of Di-GA in chelating with Cu(I) than Cu(II) can be benefit for its in vivo applications, as Cu(I) is not only the most active but also the special intermediate specie during ROS production process. All of these results proved that Di-GA could be a promising marine drug candidate in reducing copper-related ROS damage and Aß oligomer toxicity associated with AD.

"Cyclophilin A" Enzymatic Effect on the Aggregation Behavior of 1N4R Tau Protein: An Overlooked Crucial Determinant that should be Re-considered in Alzheimer's Disease Pathogenesis.

BACKGROUND: Neurodegenerative disorders like Alzheimer's disease (AD) involve the abnormal aggregation of tau protein, which forms toxic oligomers and amyloid deposits. The structure of tau protein is influenced by the conformational states of distinct proline residues, which are regulated by peptidyl-prolyl isomerases (PPIases). However, there has been no research on the impact of human cyclophilin A (CypA) as a PPIase on (non-phosphorylated) tau protein aggregation. METHODS: On the basis of these explanations, we used various spectroscopic techniques to explore the effects of CypA on tau protein aggregation behavior. RESULTS: We demonstrated the role of the isomerization activity of CypA in promoting the formation of tau protein amyloid fibrils with well-defined and highly ordered cross-ß structures. According to the "cistauosis hypothesis," CypA's ability to enhance tau protein fibril formation in AD is attributed to the isomerization of specific proline residues from the trans to cis configuration. To corroborate this theory, we conducted refolding experiments using lysozyme as a model protein. The presence of CypA increased lysozyme aggregation and impeded its refolding process. It is known that proper refolding of lysozyme relies on the correct (trans) isomerization of two critical proline residues. CONCLUSION: Thus, our findings confirmed that CypA induces the trans-to-cis isomerization of specific proline residues, ultimately leading to increased aggregation. Overall, this study highlights the emerging role of isomerization in tau protein pathogenesis in AD.

Evaluation of the Effect of ß-Wrapin AS69 in a Mouse Model Based on Alpha-Synuclein Overexpression.

Aggregation of the protein α-Synuclein (αSyn) is a hallmark of Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple systems atrophy, and alleviating the extent of αSyn pathology is an attractive strategy against neurodegeneration. The engineered binding protein ß-wrapin AS69 binds monomeric αSyn. AS69 reduces primary and secondary nucleation as well as fibril elongation in vitro. It also mitigates aSyn pathology in a mouse model based on intrastriatal injection of aSyn pre-formed fibrils (PFFs). Since the PFF-based model does not represent all aspects of PD, we tested here whether AS69 can reduce neurodegeneration resulting from αSyn overexpression. Human A53T-αSyn was overexpressed in the mouse Substantia nigra (SN) by using recombinant adeno-associated viral vector (rAAV). AS69 was also expressed by rAAV transduction. Behavioral tests and immunofluorescence staining were used as outcomes. Transduction with rAAV-αSyn resulted in αSyn pathology as reported by phospho-αSyn staining and caused degeneration of dopaminergic neurons in the SN. The co-expression of rAAV-AS69 did not reduce αSyn pathology or the degeneration of dopaminergic neurons. We conclude that αSyn monomer binding by rAAV-AS69 was insufficient to protect from aSyn pathology resulting from αSyn overexpression.

Natural products targeting amyloid-ß oligomer neurotoxicity in Alzheimer's disease.

Alzheimer's disease (AD) constitutes a major global health issue, characterized by progressive neurodegeneration and cognitive impairment, for which no curative treatment is currently available. Current therapeutic approaches are focused on symptom management, highlighting the critical need for disease-modifying therapy. The hallmark pathology of AD involves the aggregation and accumulation of amyloid-ß (Aß) peptides in the brain. Consequently, drug discovery efforts in recent decades have centered on the Aß aggregation cascade, which includes the transition of monomeric Aß peptides into toxic oligomers and, ultimately, mature fibrils. Historically, anti-Aß strategies focused on the clearance of amyloid fibrils using monoclonal antibodies. However, substantial evidence has highlighted the critical role of Aß oligomers (AßOs) in AD pathogenesis. Soluble AßOs are now recognized as more toxic than fibrils, directly contributing to synaptic impairment, neuronal damage, and the onset of AD. Targeting AßOs has emerged as a promising therapeutic approach to mitigate cognitive decline in AD. Natural products (NPs) have demonstrated promise against AßO neurotoxicity through various mechanisms, including preventing AßO formation, enhancing clearance mechanisms, or converting AßOs into non-toxic species. Understanding the mechanisms by which anti-AßO NPs operate is useful for developing disease-modifying treatments for AD. In this review, we explore the role of NPs in mitigating AßO neurotoxicity for AD drug discovery, summarizing key evidence from biophysical methods, cellular assays, and animal models. By discussing how NPs modulate AßO neurotoxicity across various experimental systems, we aim to provide valuable insights into novel therapeutic strategies targeting AßOs in AD.

TDP-43 Promotes Amyloid-Beta Toxicity by Delaying Fibril Maturation via Direct Molecular Interaction.

Amyloid-ß (Aß) is a peptide that undergoes self-assembly into amyloid fibrils, which compose the hallmark plaques observed in Alzheimer's disease (AD). TAR DNA-binding protein 43 (TDP-43) is a protein with mislocalization and aggregation implicated in amyotrophic lateral sclerosis and other neurodegenerative diseases. Recent work suggests that TDP-43 may interact with Aß, inhibiting the formation of amyloid fibrils and worsening AD pathology, but the molecular details of their interaction remain unknown. Using all-atom discrete molecular dynamics simulations, we systematically investigated the direct molecular interaction between Aß and TDP-43. We found that Aß monomers were able to bind near the flexible nuclear localization sequence of the N-terminal domain (NTD) of TDP-43, adopting ß-sheet rich conformations that were promoted by the interaction. Furthermore, Aß associated with the nucleic acid binding interface of the tandem RNA recognition motifs of TDP-43 via electrostatic interactions. Using the computational peptide array method, we found the strongest C-terminal domain interaction with Aß to be within the amyloidogenic core region of TDP-43. With experimental evidence suggesting that the NTD is necessary for inhibiting Aß fibril growth, we also simulated the NTD with an Aß40 fibril seed. We found that the NTD was able to strongly bind the elongation surface of the fibril seed via extensive hydrogen bonding and could also diffuse along the lateral surface via electrostatic interactions. Our results suggest that TDP-43 binding to the elongation surface, thereby sterically blocking Aß monomer addition, is responsible for the experimentally observed inhibition of fibril growth. We conclude that TDP-43 may promote Aß toxicity by stabilizing the oligomeric state and kinetically delaying fibril maturation.

Naringenin mitigates nanoparticulate-aluminium induced neuronal degeneration in brain cortex and hippocampus through downregulation of oxidative stress and neuroinflammation.

Alumunium usage and toxicity has been a global concern especially an increased use of nanoparticulated aluminum (Al-NPs) products from the environment and the workplace. Al degrades in to nanoparticulate form in the environment due to the routine process of bioremediation in human body. Al-NPs toxicity plays key role in the pathophysiology of neurodegeneration which is characterised by the development of neurofibrillary tangles and neuritic plaques which correlates to the Alzheimer's disease. This study evaluated the Al-NPs induced neurodegeneration and causative behavioral alterations due to oxidative stress, inflammation, DNA damage, ß-amyloid aggregation, and histopathological changes in mice. Furthermore, the preventive effect of naringenin (NAR) as a potent neuroprotective flavonoid against Al-NPs induced neurodegeneration was assessed. Al-NPs were synthesized and examined using FTIR, XRD, TEM, and particle size analyzer. Mice were orally administered with Al-NPs (6 mg/kg b.w.) followed by NAR treatment (10 mg/kg b.w. per day) for 66 days. The spatial working memory was determined by novel object recognition, T-maze, Y-maze, and Morris Water Maze tests. We measured nitric oxide, advanced oxidation of protein products, protein carbonylation, lipid peroxidation, superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, reduced glutathione, oxidised glutathione, and acetylcholine esterase, as well as cytokines analysis, immunohistochemistry, and DNA damage. Al-NPs significantly reduced the learning memory power, increased oxidative stress, reduced antioxidant enzymatic activity, increased DNA damage, altered the levels of cytokines, and increased ß-amyloid aggregation in the cortex and hippocampus regions of the mice brain. These neurobehavioral impairments, neuronal oxidative stress, and histopathological alterations were significantly attenuated by NAR supplementation. In conclusion, Al-NPs may be potent neurotoxic upon exposure and that NAR could serve as a potential preventive measure in the treatment and management of neuronal degeneration.

Pro-inflammatory protein S100A9 targeted by a natural molecule to prevent neurodegeneration onset.

Accumulation of the pro-inflammatory protein S100A9 has been implicated in neuroinflammatory cascades in neurodegenerative diseases (NDs) such as Alzheimer's disease (AD) and Parkinson's disease (PD). S100A9 co-aggregates with other proteins such as α-synuclein in PD and Aß in AD, contributing to amyloid plaque formation and neurotoxicity. The amyloidogenic nature of this protein and its role in chronic neuroinflammation suggest that it may play a key role in the pathophysiology of these diseases. Research into molecules targeting S100A9 could be a potential therapeutic strategy to prevent its amyloidogenic self-assembly and to attenuate the neuroinflammatory response in affected brain tissue. This work suggests that bioactive natural molecules, such as those found in the Mediterranean diet, may have the potential to alleviate neuroinflammation associated with the accumulation of proteins such as S100A9 in neurodegenerative diseases. A major component of extra virgin olive oil (EVOO), hydroxytyrosol (HT), with its ability to interact with and modulate S100A9 amyloid self-assembly and expression, offers a compelling approach for the development of novel and effective interventions for the prevention and treatment of ND. The findings highlight the importance of exploring natural compounds, such as HT, as potential therapeutic options for these complex and challenging neurological conditions.

Power of Dopamine: Multifunctional Compound Assisted Conversion of the Most Risk Factor into Therapeutics of Alzheimer's Disease.

In Alzheimer's disease (AD), reactive oxygen species (ROS) plays a crucial role, which is produced from molecular oxygen with extracellular deposited amyloid-ß (Aß) aggregates through the reduction of a Cu2+ ion. In the presence of a small amount of redox-active Cu2+ ion, ROS is produced by the Aß-Cu2+ complex as Aß peptide alone is unable to generate excess ROS. Therefore, Cu2+ ion chelators are considered promising therapeutics against AD. Here, we have designed and synthesized a series of Schiff base derivatives (SB) based on 2-hydroxy aromatic aldehyde derivatives and dopamine. These SB compounds contain one copper chelating core, which captures the Cu2+ ions from the Aß-Cu2+ complex. Thereby, it inhibits copper-induced amyloid aggregation as well as amyloid self-aggregation. It also inhibits copper-catalyzed ROS production through sequestering of Cu2+ ions. The uniqueness of our designed ligands has the dual property of dopamine, which not only acts as a ROS scavenger but also chelates the copper ion. The crystallographic analysis proves the power of the dopamine unit. Therefore, dual exploration of dopamine core can be considered as potential therapeutics for future AD treatment.

Glyceraldehyde-3-phosphate dehydrogenase is involved in the pathogenesis of Alzheimer's disease.

One of important characteristics of Alzheimer's disease is a persistent oxidative/nitrosative stress caused by pro-oxidant properties of amyloid-beta peptide (Aß) and chronic inflammation in the brain. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is easily oxidized under oxidative stress. Numerous data indicate that oxidative modifications of GAPDH in vitro and in cell cultures stimulate GAPDH denaturation and aggregation, and the catalytic cysteine residue Cys152 is important for these processes. Both intracellular and extracellular GAPDH aggregates are toxic for the cells. Interaction of denatured GAPDH with soluble Aß results in mixed insoluble aggregates with increased toxicity. The above-described properties of GAPDH (sensitivity to oxidation and propensity to form aggregates, including mixed aggregates with Aß) determine its role in the pathogenesis of Alzheimer's disease.

Multifunctional Nanocarrier for Synergistic Treatment of Alzheimer's Disease by Inhibiting ß-Amyloid Aggregation and Scavenging Reactive Oxygen Species.

The excessive depositions of ß-amyloid (Aß) and abnormal level of reactive oxygen species (ROS) are considered as the important pathogenic factors of Alzheimer's disease (AD). Strategies targeting only one of them have no obvious effects in clinic. In this study, a multifunctional nanocarrier CICe@M-K that crosses the blood-brain barrier (BBB) efficiently was developed for inhibiting Aß aggregation and scavenging ROS synchronously. Antioxidant curcumin (Cur) and photosensitizer IR780 were loaded in mesoporous silica nanomaterials (MSNs). Their surfaces were grafted with cerium oxide nanoparticles (CeO2 NPs) and a short peptide K (CKLVFFAED). Living imaging showed that CICe@M-K was mainly distributed in the brain, liver, and kidneys, indicating CICe@M-K crossed BBB efficiently and accumulated in brain. After the irradiation of 808 nm laser, Cur was continuously released. Both of Cur and the peptide K can recognize and bind to Aß through multiple interaction including π-π stacking interaction, hydrophobic interaction, and hydrogen bond, inhibiting Aß aggregation. On the other hand, Cur and CeO2 NPs cooperate to relieve the oxidative stress in the brains by scavenging ROS. In vivo assays showed that the CICe@M-K could diminish Aß depositions, alleviate oxidative stress, and improve cognitive ability of the APP/PS1 AD mouse model, which demonstrated that CICe@M-K is a potential agent for AD treatment.

Molecular Dynamics Insights into the Aggregation Behavior of N-Terminal ß-Lactoglobulin Peptides.

ß-lactoglobulin (BLG) forms amyloid-like aggregates at high temperatures, low pH, and low ionic strengths. At a pH below 2, BLG undergoes hydrolysis into peptides, with N-terminal peptides 1-33 and 1-52 being prone to fibrillization, forming amyloid-like fibrils. Due to their good mechanical properties, BLG amyloids demonstrate great potential for diverse applications, including biosensors, nanocomposites, and catalysts. Consequently, further studies are essential to comprehensively understand the factors governing the formation of BLG amyloid-like morphologies. In this study, all-atom molecular dynamics simulations were employed to explore the aggregation of N-terminal 1-33 and 1-52 BLG peptides under conditions of pH 2 and at 10 mM NaCl concentration. The simulations revealed that the peptides spontaneously assembled into aggregates of varying sizes. The aggregation process was enabled by the low charge of peptides and the presence of hydrophobic residues within them. As the peptides associated into aggregates, there was a concurrent increase in ß-sheet structures and the establishment of hydrogen bonds, enhancing the stability of the aggregates. Notably, on average, 1-33 peptides formed larger aggregates compared to their 1-52 counterparts, while the latter exhibited a slightly higher content of ß-sheets and higher cluster orderliness. The applied approach facilitated insights into the early stages of amyloid-like aggregation and molecular-level insight into the formation of ß-sheets, which serve as nucleation points for further fibril growth.

The effects of histidine substitution of aromatic residues on the amyloidogenic properties of the fragment 264-277 of nucleophosmin 1.

Histidine (His) plays a key role in mediating protein interactions and its unique side chain determines pH responsive self-assembling processes and thus in the formation of nanostructures. In this study, To identify novel self-assembling bioinspired sequences, we analyzed a series of peptide sequences obtained through the point mutation of aromatic residues of 264-277 fragment of nucleophosmin 1 (NPM1) with single and double histidines. Through several orthogonal biophysical techniques and under different pH and ionic strength conditions we evaluated the effects of these substitutions in the amyloidogenic features of derived peptides. The results clearly indicate that both the type of aromatic mutated residue and its position can have different effect on amyloid-like behaviors. They corroborate the crucial role exerted by Tyr271 in the self-assembling process of CTD of NPM1 in AML mutated form and add novel insights in the accurate investigation of how side chain orientations can determine successful design of innovative bioinspired materials.

Redesigning the kinetics of lysozyme amyloid aggregation by cephalosporin molecules.

In lysozyme amyloidosis, fibrillar aggregates of lysozyme are associated with severe renal, hepatic, and gastrointestinal manifestations, with no definite therapy. Current drugs are now being tested in amyloidosis clinical trials as aggregation inhibitors to mitigate disease progression. The tetracycline group among antimicrobials in use is in phase II of clinical trials, whereas some macrolides and cephalosporins have shown neuroprotection. In the present study, two cephalosporins, ceftazidime (CZD) and cefotaxime (CXM), and a glycopeptide, vancomycin (VNC), are evaluated for inhibition of amyloid aggregation of hen egg white lysozyme (HEWL) under two conditions (i) 4 M guanidine hydrochloride (GuHCl) at pH 6.5 and 37° C, (ii) At pH 1.5 and 65 °C. Fluorescence quench titration and molecular docking methods report that CZD, CXM, and VNC interact more strongly with the partially folded intermediates (PFI) in comparison to the protein's natural state (N). However, only CZD and CXM proficiently inhibit the aggregation. Transmission electron microscopy, tinctorial assessments, and aggregation kinetics all support oligomer-level inhibition. Transition structures in CZD-HEWL and CXM-HEWL aggregation are shown by circular dichroism (CD). On the other hand, kinetic variables and soluble fraction assays point to a localized association of monomers. Intrinsic fluorescence (IF),1-Anilino 8-naphthalene sulphonic acid, and CD demonstrate structural and conformational modifications redesigning the PFI. GuHCl-induced unfolding and differential scanning fluorimetry suggested that the PFI monomers bound to CZD and CXM exhibited partial stability. Our results present two mechanisms that function in both solution conditions, creating a novel avenue for the screening of putative inhibitors for drug repurposing. We extend our proposed mechanisms in the designing of physical inhibitors of amyloid aggregation considering shorter time frames and foolproof methods.Communicated by Ramaswamy H. Sarma.

Drug repurposing has overcome failures in drug discovery and has reduced the overall time and cost of drug discovery and development.We examined the effect of screened antibiotics, ceftazidime (CZD), cefotaxime (CXM), and vancomycin (VNC) on lysozyme aggregation under two solution conditions.These antibiotics inhibit/modulate the aggregation reactions by strongly interacting with aggregation-prone intermediate and modulation of conformation and stability.Our study puts forward with caution two cephalosporins for aggregation inhibition studies.

Novel 6-alkyl-bridged 4-arylalkylpiperazin-1-yl derivatives of azepino[4,3-b]indol-1(2H)-one as potent BChE-selective inhibitors showing protective effects against neurodegenerative insults.

Due to the putative role of butyrylcholinesterase (BChE) in regulation of acetylcholine levels and functions in the late stages of the Alzheimer's disease (AD), the potential of selective inhibitors (BChEIs) has been envisaged as an alternative to administration of acetylcholinesterase inhibitors (AChEIs). Starting from our recent findings, herein the synthesis and in vitro evaluation of cholinesterase (ChE) inhibition of a novel series of some twenty 3,4,5,6-tetrahydroazepino[4,3-b]indol-1(2H)-one derivatives, bearing at the indole nitrogen diverse alkyl-bridged 4-arylalkylpiperazin-1-yl chains, are reported. The length of the spacers, as well as the type of arylalkyl group affected the enzyme inhibition potency and BChE/AChE selectivity. Two compounds, namely 14c (IC50 = 163 nM) and 14d (IC50 = 65 nM), bearing at the nitrogen atom in position 6 a n-pentyl- or n-heptyl-bridged 4-phenethylpiperazin-1-yl chains, respectively, proved to be highly potent mixed-type inhibitors of both equine and human BChE isoforms, showing more than two order magnitude of selectivity over AChE. The study of binding kinetics through surface plasmon resonance (SPR) highlighted differences in their BChE residence times (8 and 47 s for 14c and 14d, respectively). Moreover, 14c and 14d proved to hit other mechanisms known to trigger neurodegeneration underlying AD and other CNS disorders. Unlike 14c, compound 14d proved also capable of inhibiting by more than 60% the in vitro self-induced aggregation of neurotoxic amyloid-ß (Aß) peptide at 100 µM concentration. On the other hand, 14c was slightly better than 14d in counteracting, at 1 and 10 µM concentration, glutamate excitotoxicity, due to over-excitation of NMDA receptors, and hydrogen peroxide-induced oxidative stress assessed in neuroblastoma cell line SH-SY5Y. This paper is dedicated to Prof. Marcello Ferappi, former dean of the Faculty of Pharmacy of the University of Bari, in the occasion of his 90th birthday.