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We recently characterized the potent antiplasmodial activity of the aggregated protein dye YAT2150, whose presumed mode of action is the inhibition of protein aggregation in the malaria parasite. Using single-dose and ramping methods, assays were done to select Plasmodium falciparum parasites resistant to YAT2150 concentrations ranging from 3× to 0.25× the in vitro IC 50 of the compound (in the two-digit nM range) and performed a cross-resistance assessment in P. falciparum lines harboring mutations that make them resistant to a variety of antimalarial drugs. Resistant parasites did not emerge in vitro after 60 days of incubation, which postulates YAT2150 as an 'irresistible' antimalarial. The lyophilized compound is stable for at least one year stored at 25 °C. Tests performed in clinical isolates indicated that YAT2150 had also strong activity against Plasmodium vivax (IC 50 between 4 and 36 nM) and Leishmania infantum (1.27 and 1.11 µM), placing it as a unique compound with perspectives of becoming the first drug to be used against both malaria and leishmaniasis., Competing Interests: Declarations. Competing interests: A patent application (priority number: EP21382949.2; application number: PCT/EP2022/079438; application date: 21/10/2022) has been filed to protect some of the results presented in the paper, which includes as inventors IB-A, EMA, DM-T and XF-B. All other authors declare they have no competing interests., (© 2025. The Author(s).)

Background: Malaria during pregnancy implies a high risk for the mother and the developing child. However, the therapeutic options for pregnant women have historically been very limited, especially during the first trimester of pregnancy due to potential adverse effects on embryo-fetal development. Recently, there has been great controversy regarding these potential embryo-fetal adverse effects because the results of rodent studies were not in accordance with the clinical data available, and finally the WHO has changed the recommendations for pregnant women with uncomplicated P. falciparum malaria to treatment with artemether-lumefantrine during the first trimester. The discrepancy between pre-clinical and clinical studies has been attributed to species-differences in the duration of the window of susceptibility of circulating primitive erythroblasts., Methods: Here we provide a tool based on an alternative method to animal experimentation that accelerates the research of novel drugs for pregnant women. We have adapted the zebrafish embryo developmental toxicity assay to include hemoglobin staining in the embryos and two time-points of lethality and dysmorphogenesis evaluation. These two time-points were selected to include one when the development is independent of and one when the development is dependent of erythrocytes function. The method was used to test four marketed antimalarial drugs and three new antimalarial drug candidates., Results: Our combination of tests can correctly predict the teratogenic and non-teratogenic effects of several antimalarial marketed drugs (artemisinin, quinine, chloroquine, and dihydroartemisinin + desbutyl-lumefantrine). Furthermore, we have tested three new drug candidates (GS-GUAN, DONE3TCl, and YAT2150) with novel mechanisms of action, and different from those of the marketed antimalarial drugs., Conclusions: We propose a decision tree combining the results of the two time-points of evaluation together with the information on significant erythrocyte depletion. The aim of this decision tree is to identify compounds with no or lower hazard on teratogenicity or erythrocyte depletion at an early phase of the drug development process., Competing Interests: Declaration of competing interest Marta Barenys is a Guest Editor for Reproductive Toxicology and was not involved in the editorial review or the decision to publish this article. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: A patent application (WO, 2023/067170 A1; filing date: October 21, 2022) has been filed to protect some of the results presented in the paper, which includes as inventors DM-T and XF-B. All other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background/Objectives : YAT2150 is a first-in-class antiplasmodial compound that has been recently proposed as a new interesting drug for malaria therapy. Methods/Results : The fluorescence of YAT2150 rapidly increases upon its entry into Plasmodium , a property that can be of use for the design of highly sensitive diagnostic approaches. YAT2150 blocks the in vitro development of the ookinete stage of Plasmodium and, when added to an infected blood meal, inhibits oocyst formation in the mosquito. Thus, the compound could possibly contribute to future transmission-blocking antimalarial strategies. Cell influx/efflux studies in Caco-2 cells suggest that YAT2150 is internalized by endocytosis and also through the OATP2B1 transporter, whereas its main export route would be via OSTα. YAT2150 has an overall favorable drug metabolism and pharmacokinetics profile, and its moderate cytotoxicity can be significantly reduced upon encapsulation in immunoliposomes, which leads to a dramatic increase in the drug selectivity index to values close to 1000. Although YAT2150 binds amyloid-forming peptides, its in vitro fluorescence emission is stronger upon association with peptides that form amorphous aggregates, suggesting that regions enriched in unstructured proteins are the preferential binding sites of the drug inside Plasmodium cells. The reduction of protein aggregation in the parasite after YAT2150 treatment, which has been suggested to be directly related to the drug's mode of action, is also observed following treatment with quinoline antimalarials like chloroquine and primaquine. Conclusions : Altogether, the data presented here indicate that YAT2150 can represent the spearhead of a new family of compounds for malaria diagnosis and therapy due to its presumed novel mode of action based on the interaction with functional protein aggregates in the pathogen.

Multitarget compounds have emerged as promising drug candidates to cope with complex multifactorial diseases, like Alzheimer's disease (AD). Most multitarget compounds are designed by linking two pharmacophores through a tether chain (linked hybrids), which results in rather large molecules that are particularly useful to hit targets with large binding cavities, but at the expense of suffering from suboptimal physicochemical/pharmacokinetic properties. Molecular size reduction by removal of superfluous structural elements while retaining the key pharmacophoric motifs may represent a compromise solution to achieve both multitargeting and favorable physicochemical/PK properties. Here, we report the stepwise structural simplification of the dihydroxyanthraquinone moiety of a rhein-huprine hybrid lead by hydroxy group removal-ring contraction-ring opening-ring removal, which has led to new analogs that retain or surpass the potency of the lead on its multiple AD targets while exhibiting more favorable drug metabolism and pharmacokinetic (DMPK) properties and safety profile. In particular, the most simplified acetophenone analog displays dual nanomolar inhibition of human acetylcholinesterase and butyrylcholinesterase (IC 50 = 6 nM and 13 nM, respectively), moderately potent inhibition of human BACE-1 (48% inhibition at 15 µM) and Aβ42 and tau aggregation (73% and 68% inhibition, respectively, at 10 µM), favorable in vitro brain permeation, higher aqueous solubility (18 µM) and plasma stability (100/96/86% remaining in human/mouse/rat plasma after 6 h incubation), and lower acute toxicity in a model organism (zebrafish embryos; LC 50 >> 100 µM) than the initial lead, thereby confirming the successful lead optimization by structural simplification.

The problems associated with the drugs currently used to treat leishmaniasis, including resistance, toxicity, and the high cost of some formulations, call for the urgent identification of new therapeutic agents with novel modes of action. The aggregated protein dye YAT2150 has been found to be a potent antileishmanial compound, with a half-maximal inhibitory concentration (IC 50 ) of approximately 0.5 µM against promastigote and amastigote stages of Leishmania infantum . The encapsulation in liposomes of YAT2150 significantly improved its in vitro IC 50 to 0.37 and 0.19 µM in promastigotes and amastigotes, respectively, and increased the half-maximal cytotoxic concentration in human umbilical vein endothelial cells to >50 µM. YAT2150 became strongly fluorescent when binding intracellular protein deposits in Leishmania cells. This fluorescence pattern aligns with the proposed mode of action of this drug in the malaria parasite Plasmodium falciparum , the inhibition of protein aggregation. In Leishmania major , YAT2150 rapidly reduced ATP levels, suggesting an alternative antileishmanial mechanism. To the best of our knowledge, this first-in-class compound is the only one described so far having significant activity against both Plasmodium and Leishmania , thus being a potential drug for the treatment of co-infections of both parasites., Competing Interests: A patent application (priority number: EP21382949.2; application number: PCT/EP2022/079438; application date: 21/10/2022) has been filed to protect some of the results presented in the paper, which includes as inventors IB-A, EMA, DM-T and XF-B. All other authors declare they have no competing interests.

The so-called solid electrolyte interphase (SEI), a nanolayer formed on the negative electrode of lithium-ion batteries during the first cycles, largely influences some key performance indicators such as cycle life and specific power. The reason is due to the fact that the SEI prevents continuous electrolyte decomposition, making this protecting character extremely important. Herein, a specifically designed scanning droplet cell system (SDCS) is developed to study the protecting character of the SEI on lithium-ion battery (LIB) electrode materials. SDCS allows for automatized electrochemical measurements with improved reproducibility and time-saving experimentation. Besides the necessary adaptations for its implementation for non-aqueous batteries, a new operating mode, the so-called redox mediated-scanning droplet cell system (RM-SDCS), is established to investigate the SEI properties. By adding a redox mediator ( e.g. a viologen derivative) to the electrolyte, evaluation of the protecting character of the SEI becomes accessible. Validation of the proposed methodology was performed using a model sample (Cu surface). Afterwards, RM-SDCS was employed on Si-graphite electrodes as a case study. On the one hand, the RM-SDCS shed light on the degradation mechanisms providing direct electrochemical evidence of the rupture of the SEI upon lithiation. On the other hand, the RM-SDCS was presented as an accelerated method capable of searching for electrolyte additives. The results indicate an enhancement in the protecting character of the SEI when 4 wt% of both vinyl carbonate and fluoroethylene carbonate were used simultaneously., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Background: Alzheimer's disease (AD) is characterized by a polyetiological origin. Despite the global burden of AD and the advances made in AD drug research and development, the cure of the disease remains elusive, since any developed drug has demonstrated effectiveness to cure AD. Strikingly, an increasing number of studies indicate a linkage between AD and type 2 diabetes mellitus (T2DM), as both diseases share some common pathophysiological features. In fact, β-secretase (BACE1) and acetylcholinesterase (AChE), two enzymes involved in both conditions, have been considered promising targets for both pathologies. In this regard, due to the multifactorial origin of these diseases, current research efforts are focusing on the development of multi-target drugs as a very promising option to derive effective treatments for both conditions. In the present study, we evaluated the effect of rhein-huprine hybrid (RHE-HUP), a synthesized BACE1 and AChE inhibitor, both considered key factors not only in AD but also in metabolic pathologies. Thus, the aim of this study is to evaluate the effects of this compound in APP/PS1 female mice, a well-established familial AD mouse model, challenged by high-fat diet (HFD) consumption to concomitantly simulate a T2DM-like condition., Results: Intraperitoneal treatment with RHE-HUP in APP/PS1 mice for 4 weeks reduced the main hallmarks of AD, including Tau hyperphosphorylation, Aβ 42 peptide levels and plaque formation. Moreover, we found a decreased inflammatory response together with an increase in different synaptic proteins, such as drebrin 1 (DBN1) or synaptophysin, and in neurotrophic factors, especially in BDNF levels, correlated with a recovery in the number of dendritic spines, which resulted in memory improvement. Notably, the improvement observed in this model can be attributed directly to a protein regulation at central level, since no peripheral modification of those alterations induced by HFD consumption was observed., Conclusions: Our results suggest that RHE-HUP could be a new candidate for the treatment of AD, even for individuals with high risk due to peripheral metabolic disturbances, given its multi-target profile which allows for the improvement of some of the most important hallmarks of the disease., (© 2023. The Author(s).)

Background: By 2016, signs of emergence of Plasmodium falciparum resistance to artemisinin and partner drugs were detected in the Greater Mekong Subregion. Recently, the independent evolution of artemisinin resistance has also been reported in Africa and South America. This alarming scenario calls for the urgent development of new antimalarials with novel modes of action. We investigated the interference with protein aggregation, which is potentially toxic for the cell and occurs abundantly in all Plasmodium stages, as a hitherto unexplored drug target in the pathogen., Results: Attempts to exacerbate the P. falciparum proteome's propensity to aggregation by delivering endogenous aggregative peptides to in vitro cultures of this parasite did not significantly affect their growth. In contrast, protein aggregation inhibitors clearly reduced the pathogen's viability. One such compound, the bis(styrylpyridinium) salt YAT2150, exhibited potent antiplasmodial activity with an in vitro IC 50 of 90 nM for chloroquine- and artemisinin-resistant lines, arresting asexual blood parasites at the trophozoite stage, as well as interfering with the development of both sexual and hepatic forms of Plasmodium. At its IC 50 , this compound is a powerful inhibitor of the aggregation of the model amyloid β peptide fragment 1-40, and it reduces the amount of aggregated proteins in P. falciparum cultures, suggesting that the underlying antimalarial mechanism consists in a generalized impairment of proteostasis in the pathogen. YAT2150 has an easy, rapid, and inexpensive synthesis, and because it fluoresces when it accumulates in its main localization in the Plasmodium cytosol, it is a theranostic agent., Conclusions: Inhibiting protein aggregation in Plasmodium significantly reduces the parasite's viability in vitro. Since YAT2150 belongs to a novel structural class of antiplasmodials with a mode of action that potentially targets multiple gene products, rapid evolution of resistance to this drug is unlikely to occur, making it a promising compound for the post-artemisinin era., (© 2022. The Author(s).)

Alzheimer's disease (AD) is a progressive neurological disorder with multifactorial and heterogeneous causes. AD involves several etiopathogenic mechanisms such as aberrant protein accumulation, neurotransmitter deficits, synaptic dysfunction and neuroinflammation, which lead to cognitive decline. Unfortunately, the currently available anti-AD drugs only alleviate the symptoms temporarily and provide a limited therapeutic effect. Thus, new therapeutic strategies, including multitarget approaches, are urgently needed. It has been demonstrated that a co-treatment of acetylcholinesterase (AChE) inhibitor with other neuroprotective agents has beneficial effects on cognition. Here, we have assessed the neuroprotective effects of chronic dual treatment with a soluble epoxide hydrolase (sEH) inhibitor (TPPU) and an AChE inhibitor (6-chlorotacrine or rivastigmine) in in vivo studies. Interestingly, we have found beneficial effects after chronic low-dose co-treatment with TPPU and 6-chlorotacrine in the senescence-accelerated mouse prone 8 (SAMP8) mouse model as well as with TPPU and rivastigmine co-treatment in the 5XFAD mouse model, in comparison with the corresponding monotherapy treatments. In the SAMP8 model, no substantial improvements in synaptic plasticity markers were found, but the co-treatment of TPPU and 6-chlorotacrine led to a significantly reduced gene expression of neuroinflammatory markers, such as interleukin 6 ( Il-6 ), triggering receptor expressed on myeloid cell 2 ( Trem2 ) and glial fibrillary acidic protein ( Gfap ). In 5XFAD mice, chronic low-dose co-treatment of TPPU and rivastigmine led to enhanced protein levels of synaptic plasticity markers, such as the phospho-cAMP response element-binding protein (p-CREB) ratio, brain-derived neurotrophic factor (BDNF) and postsynaptic density protein 95 (PSD95), and also to a reduction in neuroinflammatory gene expression. Collectively, these results support the neuroprotectant role of chronic low-dose co-treatment strategy with sEH and AChE inhibitors in AD mouse models, opening new avenues for effective AD treatment., Competing Interests: The authors claim no financial conflict of interests.

Multitarget anti-Alzheimer agents are the focus of very intensive research. Through a comprehensive bibliometric analysis of the publications in the period 1990-2020, we have identified trends and potential gaps that might guide future directions. We found that: (i) the number of publications boomed by 2011 and continued ascending in 2020; (ii) the linked-pharmacophore strategy was preferred over design approaches based on fusing or merging pharmacophores or privileged structures; (iii) a significant number of in vivo studies, mainly using the scopolamine-induced amnesia mouse model, have been performed, especially since 2017; (iv) China, Italy and Spain are the countries with the largest total number of publications on this topic, whereas Portugal, Spain and Italy are the countries in whose scientific communities this topic has generated greatest interest; (v) acetylcholinesterase, β-amyloid aggregation, oxidative stress, butyrylcholinesterase, and biometal chelation and the binary combinations thereof have been the most commonly pursued, while combinations based on other key targets, such as tau aggregation, glycogen synthase kinase-3β, NMDA receptors, and more than 70 other targets have been only marginally considered. These results might allow us to spot new design opportunities based on innovative target combinations to expand and diversify the repertoire of multitarget drug candidates and increase the likelihood of finding effective therapies for this devastating disease.

Aβ(1-42) peptide is a neurotoxic agent strongly associated with the etiology of Alzheimer's disease (AD). Current treatments are still of very low effectiveness, and deaths from AD are increasing worldwide. Huprine-derived molecules have a high affinity towards the enzyme acetylcholinesterase (AChE), act as potent Aβ(1-42) peptide aggregation inhibitors, and improve the behavior of experimental animals. AVCRI104P4 is a multitarget donepezil-huprine hybrid that improves short-term memory in a mouse model of AD and exerts protective effects in transgenic Caenorhabditis elegans that express Aβ(1-42) peptide. At present, there is no information about the effects of this compound on human erythrocytes. Thus, we considered it important to study its effects on the cell membrane and erythrocyte models, and to examine its protective effect against the toxic insult induced by Aβ(1-42) peptide in this cell and models. This research was developed using X-ray diffraction and differential scanning calorimetry (DSC) on molecular models of the human erythrocyte membrane constituted by lipid bilayers built of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE). They correspond to phospholipids representative of those present in the external and internal monolayers, respectively, of most plasma and neuronal membranes. The effect of AVCRI104P4 on human erythrocyte morphology was studied by scanning electron microscopy (SEM). The experimental results showed a protective effect of AVCRI104P4 against the toxicity induced by Aβ(1-42) peptide in human erythrocytes and molecular models.

A new hypothesis highlights sleep-dependent learning/memory consolidation and regards the sleep-wake cycle as a modulator of β-amyloid and tau Alzheimer's disease (AD) pathologies. Sundowning behavior is a common neuropsychiatric symptom (NPS) associated with dementia. Sleep fragmentation resulting from disturbances in sleep and circadian rhythms in AD may have important consequences on memory processes and exacerbate the other AD-NPS. The present work studied the effect of training time schedules on 12-month-old male 3xTg-AD mice modeling advanced disease stages. Their performance in two paradigms of the Morris water maze for spatial-reference and visual-perceptual learning and memory were found impaired at midday, after 4 h of non-active phase. In contrast, early-morning trained littermates, slowing down from their active phase, exhibited better performance and used goal-directed strategies and non-search navigation described for normal aging. The novel multitarget anticholinesterasic compound AVCRI104P3 (0.6 µmol·kg -1 , 21 days i.p.) exerted stronger cognitive benefits than its in vitro equipotent dose of AChEI huprine X (0.12 μmol·kg -1 , 21 days i.p.). Both compounds showed streamlined drug effectiveness, independently of the schedule. Their effects on anxiety-like behaviors were moderate. The results open a question of how time schedules modulate the capacity to respond to task demands and to assess/elucidate new drug effectiveness.

Molecular factors involved in neuroprotection are key in the design of novel multitarget drugs in aging and neurodegeneration. AVCRI104P3 is a huprine derivative that exhibits potent inhibitory effects on human AChE, BuChE, and BACE-1 activities, as well as on AChE-induced and self-induced Aβ aggregation. More recently, cognitive protection and anxiolytic-like effects have also been reported in mice treated with this compound. Now, we have assessed the ability of AVCRI104P3 (0.43 mg/kg, 21 days) to modulate the levels of some proteins involved in the anti-apoptotic/apoptotic processes (pAkt1, Bcl2, pGSK3β, p25/p35), inflammation (GFAP and Iba1) and neurogenesis in C57BL/6 mice. The effects of AVCRI104P3 on AChE-R/AChE-S isoforms have been also determined. We have observed that chronic treatment of C57BL/6 male mice with AVCRI104P3 results in neuroprotective effects, increasing significantly the levels of pAkt1 and pGSK3β in the hippocampus and Bcl2 in both hippocampus and cortex, but slightly decreasing synaptophysin levels. Astrogliosis and neurogenic markers GFAP and DCX remained unchanged after AVCRI104P3 treatment, whereas microgliosis was found to be significantly decreased pointing out the involvement of this compound in inflammatory processes. These results suggest that the neuroprotective mechanisms that are behind the cognitive and anxiolytic effects of AVCRI104P3 could be partly related to the potentiation of some anti-apoptotic and anti-inflammatory proteins and support the potential of AVCRI104P3 for the treatment of brain dysfunction associated with aging and/or dementia.

Symptomatic treatment of myasthenia gravis is based on the use of peripherally-acting acetylcholinesterase (AChE) inhibitors that, in some cases, must be discontinued due to the occurrence of a number of side-effects. Thus, new AChE inhibitors are being developed and investigated for their potential use against this disease. Here, we have explored two alternative approaches to get access to peripherally-acting AChE inhibitors as new agents against myasthenia gravis, by structural modification of the brain permeable anti-Alzheimer AChE inhibitors tacrine, 6-chlorotacrine, and huprine Y. Both quaternization upon methylation of the quinoline nitrogen atom, and tethering of a triazole ring, with, in some cases, the additional incorporation of a polyphenol-like moiety, result in more polar compounds with higher inhibitory activity against human AChE (up to 190-fold) and butyrylcholinesterase (up to 40-fold) than pyridostigmine, the standard drug for symptomatic treatment of myasthenia gravis. The novel compounds are furthermore devoid of brain permeability, thereby emerging as interesting leads against myasthenia gravis., Competing Interests: The authors declare no conflict of interest.

Imidazolium salts are privileged compounds in organic chemistry, and have valuable biological properties. Recent studies show that symmetric imidazolium salts with bulky moieties can display antiparasitic activity against T. cruzi . After developing a facile methodology for the synthesis of tetrasubstituted imidazolium salts from propargylamines and isocyanides, we screened a small library of these adducts against the causative agents of African and American trypanosomiases. These compounds display nanomolar activity against T. brucei and low (or sub) micromolar activity against T. cruzi , with excellent selectivity indexes and favorable molecular properties, thereby emerging as promising hits for the treatment of Chagas disease and sleeping sickness., Competing Interests: The authors declare no conflict of interest.

[This corrects the article DOI: 10.1371/journal.pone.0177683.].

Aim: Simultaneous modulation of several key targets of the pathological network of Alzheimer's disease (AD) is being increasingly pursued as a promising option to fill the critical gap of efficacious drugs against this condition., Materials & Methods: A short series of compounds purported to hit multiple targets of relevance in AD has been designed, on the basis of their distinct basicities estimated from high-level quantum mechanical computations, synthesized, and subjected to assays of inhibition of cholinesterases, BACE-1, and Aβ42 and tau aggregation, of antioxidant activity, and of brain permeation., Results: Using, as a template, a lead rhein-huprine hybrid with an interesting multitarget profile, we have developed second-generation compounds, designed by the modification of the huprine aromatic ring. Replacement by [1,8]-naphthyridine or thieno[3,2-e]pyridine systems resulted in decreased, although still potent, acetylcholinesterase or BACE-1 inhibitory activities, which are more balanced relative to their Aβ42 and tau antiaggregating and antioxidant activities., Conclusion: Second-generation naphthyridine- and thienopyridine-based rhein-huprine hybrids emerge as interesting brain permeable compounds that hit several crucial pathogenic factors of AD.

The critical role of BACE-1 in the formation of neurotoxic ß-amyloid peptides in the brain makes it an attractive target for an efficacious treatment of Alzheimer's disease. However, the development of clinically useful BACE-1 inhibitors has proven to be extremely challenging. In this study we examine the binding mode of a novel potent inhibitor (compound 1, with IC50 80 nM) designed by synergistic combination of two fragments-huprine and rhein-that individually are endowed with very low activity against BACE-1. Examination of crystal structures reveals no appropriate binding site large enough to accommodate 1. Therefore we have examined the conformational flexibility of BACE-1 through extended molecular dynamics simulations, paying attention to the highly flexible region shaped by loops 8-14, 154-169 and 307-318. The analysis of the protein dynamics, together with studies of pocket druggability, has allowed us to detect the transient formation of a secondary binding site, which contains Arg307 as a key residue for the interaction with small molecules, at the edge of the catalytic cleft. The formation of this druggable "floppy" pocket would enable the binding of multisite inhibitors targeting both catalytic and secondary sites. Molecular dynamics simulations of BACE-1 bound to huprine-rhein hybrid compounds support the feasibility of this hypothesis. The results provide a basis to explain the high inhibitory potency of the two enantiomeric forms of 1, together with the large dependence on the length of the oligomethylenic linker. Furthermore, the multisite hypothesis has allowed us to rationalize the inhibitory potency of a series of tacrine-chromene hybrid compounds, specifically regarding the apparent lack of sensitivity of the inhibition constant to the chemical modifications introduced in the chromene unit. Overall, these findings pave the way for the exploration of novel functionalities in the design of optimized BACE-1 multisite inhibitors.

More than 46 million people worldwide suffer from Alzheimer's disease. A large number of potential treatments have been proposed; among these, the inhibition of the aggregation of amyloid β-peptide (Aβ), considered one of the main culprits in Alzheimer's disease. Limitations in monitoring the aggregation of Aβ in cells and tissues restrict the screening of anti-amyloid drugs to in vitro studies in most cases. We have developed a simple but powerful method to track Aβ aggregation in vivo in real-time, using bacteria as in vivo amyloid reservoir. We use the specific amyloid dye Thioflavin-S (Th-S) to stain bacterial inclusion bodies (IBs), in this case mainly formed of Aβ in amyloid conformation. Th-S binding to amyloids leads to an increment of fluorescence that can be monitored. The quantification of the Th-S fluorescence along the time allows tracking Aβ aggregation and the effect of potential anti-aggregating agents.

Introduction: The prion protein (PrP) binds to various molecular partners, but little is known about their potential impact on the pathogenesis of prion diseases, Results: Here, we show that PrP can interact in vitro with acetylcholinesterase (AChE), a key protein of the cholinergic system in neural and non-neural tissues. This heterologous association induced aggregation of monomeric PrP and modified the structural properties of PrP amyloid fibrils. Following its recruitment into PrP fibrils, AChE loses its enzymatic activity and enhances PrP-mediated cytotoxicity. Using several truncated PrP variants and specific tight-binding AChE inhibitors (AChEis), we then demonstrate that the PrP-AChE interaction requires two mutually exclusive sub-sites in PrP N-terminal domain and an aromatic-rich region at the entrance of AChE active center gorge. We show that AChEis that target this site impair PrP-AChE complex formation and also limit the accumulation of pathological prion protein (PrPSc) in prion-infected cell cultures. Furthermore, reduction of AChE levels in prion-infected heterozygous AChE knock-out mice leads to slightly but significantly prolonged incubation time. Finally, we found that AChE levels were altered in prion-infected cells and tissues, suggesting that AChE might be directly associated with abnormal PrP., Conclusion: Our results indicate that AChE deserves consideration as a new actor in expanding pathologically relevant PrP morphotypes and as a therapeutic target.

We describe the multigram synthesis and in vivo efficacy studies of a donepezil‒huprine hybrid that has been found to display a promising in vitro multitarget profile of interest for the treatment of Alzheimer's disease (AD). Its synthesis features as the key step a novel multigram preparative chromatographic resolution of intermediate racemic huprine Y by chiral HPLC. Administration of this compound to transgenic CL4176 and CL2006 Caenorhabditis elegans strains expressing human Aβ42, here used as simplified animal models of AD, led to a significant protection from the toxicity induced by Aβ42. However, this protective effect was not accompanied, in CL2006 worms, by a reduction of amyloid deposits. Oral administration for 3 months to transgenic APPSL mice, a well-established animal model of AD, improved short-term memory, but did not alter brain levels of Aβ peptides nor cortical and hippocampal amyloid plaque load. Despite the clear protective and cognitive effects of AVCRI104P4, the lack of Aβ lowering effect in vivo might be related to its lower in vitro potency toward Aβ aggregation and formation as compared with its higher anticholinesterase activities. Further lead optimization in this series should thus focus on improving the anti-amyloid/anticholinesterase activity ratio.

Oxidative stress is implicated in the pathogenesis of neurodegenerative disorders and hydrogen peroxide (H2O2) plays a central role in the stress. Huprines, a group of potent acetylcholinesterase inhibitors (AChEIs), have shown a broad cholinergic pharmacological profile. Recently, it has been observed that huprine X (HX) improves cognition in non transgenic middle aged mice and shows a neuroprotective activity (increased synaptophysin expression) in 3xTg-AD mice. Consequently, in the present experiments the potential neuroprotective effect of huprines (HX, HY, HZ) has been analyzed in two different in vitro conditions: undifferentiated and NGF-differentiated PC12 cells. Cells were subjected to oxidative insult (H2O2, 200 µM) and the protective effects of HX, HY and HZ (0.01 µM-1 µM) were analyzed after a pre-incubation period of 24 and 48 hours. All huprines showed protective effects in both undifferentiated and NGF-differentiated cells, however only in differentiated cells the effect was dependent on cholinergic receptors as atropine (muscarinic antagonist, 0.1 µM) and mecamylamine (nicotinic antagonist, 100 µM) reverted the neuroprotection action of huprines. The decrease in SOD activity observed after oxidative insult was overcome in the presence of huprines and this effect was not mediated by muscarinic or nicotinic receptors. In conclusion, huprines displayed neuroprotective properties as previously observed in in vivo studies. In addition, these effects were mediated by cholinergic receptors only in differentiated cells. However, a non-cholinergic mechanism, probably through an increase in SOD activity, seems to be also involved in the neuroprotective effects of huprines.

Two novel families of dual binding site acetylcholinesterase (AChE) inhibitors have been developed, consisting of a tacrine or 6-chlorotacrine unit as the active site interacting moiety, either the 5,6-dimethoxy-2-[(4-piperidinyl)methyl]-1-indanone fragment of donepezil (or the indane derivative thereof) or a 5-phenylpyrano[3,2-c]quinoline system, reminiscent to the tryciclic core of propidium, as the peripheral site interacting unit, and a linker of suitable length as to allow the simultaneous binding at both sites. These hybrid compounds are all potent and selective inhibitors of human AChE, and more interestingly, are able to interfere in vitro both formation and aggregation of the beta-amyloid peptide, the latter effects endowing these compounds with the potential to modify Alzheimer's disease progression., (Copyright (c) 2010 Elsevier Ireland Ltd. All rights reserved.)

A series of novel N,O-psiconucleosides has been prepared in both enantiomeric forms by resolution of an advanced racemic synthetic intermediate using (R)-N-phenylpantolactam as a chiral resolution agent. The absolute configuration of all of these compounds has been unequivocally established by chemical correlation with the novel (R)- or (S)-1-methyl-5-phenylpyrrolidine-2,3-dione, prepared from the known (R)- and (S)-1-methyl-5-phenylpyrrolidin-2-one, respectively.

Treatment of 1-indanones with aromatic aldehydes and NaOEt in THF affords complex spiropolycyclic compounds through a four-component reaction in which two molecules of each starting compound are combined with formation of four new carbon-carbon bonds, leading to the elaboration of a new five-membered ring that bears five contiguous stereogenic centers with a well-defined relative configuration. Different amounts of a minor epimer of the main product are also formed. The presence of methoxy substituents in the indanone component and the use of aldehydes derived from pi-excedent heterocycles make the dimerization step a slower transformation. In these cases, better yields of spirodimers are obtained starting from the preformed enones. The reaction seems to take place by cross-aldol condensation, dehydration, and dimerization of the thus formed enones. The molecular mechanism of the dimerization reaction of enone 5g has been studied using DFT methods at the B3LYP/6-31G level. The dimerization takes place through a process involving a Michael addition of a carbanion, obtained by deprotonation of 5g at the 3-position, to a second molecule of 5g, followed by an intramolecular Michael addition in the corresponding intermediate. The final protonation of the resulting anion accounts for the formation of the cis-fused pentacyclic system.