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Targeted protein degradation (TPD) is emerging as one of the most innovative strategies to tackle infectious diseases. Particularly, proteolysis-targeting chimera (PROTAC)-mediated protein degradation may offer several benefits over classical anti-infective small-molecule drugs. Because of their peculiar and catalytic mechanism of action, anti-infective PROTACs might be advantageous in terms of efficacy, toxicity, and selectivity. Importantly, PROTACs may also overcome the emergence of antimicrobial resistance. Furthermore, anti-infective PROTACs might have the potential to (i) modulate "undruggable"targets, (ii) "recycle"inhibitors from classical drug discovery approaches, and (iii) open new scenarios for combination therapies. Here, we try to address these points by discussing selected case studies of antiviral PROTACs and the first-in-class antibacterial PROTACs. Finally, we discuss how the field of PROTAC-mediated TPD might be exploited in parasitic diseases. Since no antiparasitic PROTAC has been reported yet, we also describe the parasite proteasome system. While in its infancy and with many challenges ahead, we hope that PROTAC-mediated protein degradation for infectious diseases may lead to the development of next-generation anti-infective drugs.

Majority of drugs act by interacting with chiral counterparts, e.g., proteins, and we are, unfortunately, well-aware of how chirality can negatively impact the outcome of a therapeutic regime. The number of chiral, non-racemic drugs on the market is increasing, and it is becoming ever more important to prepare these compounds in a safe, economic, and environmentally sustainable fashion. Asymmetric organocatalysis has a long history, but it began its renaissance era only during the first years of the millennium. Since then, this field has reached an extraordinary level, as confirmed by the awarding of the 2021 Chemistry Nobel Prize. In the present review, we wish to highlight the application of organocatalysis in the synthesis of enantio-enriched molecules that may be of interest to the pharmaceutical industry and the medicinal chemistry community. We aim to discuss the different activation modes observed for organocatalysts, examining, for each of them, the generally accepted mechanisms and the most important and developed reactions, that may be useful to medicinal chemists. For each of these types of organocatalytic activations, select examples from academic and industrial applications will be disclosed during the synthesis of drugs and natural products. The authors acknowledge the Spanish Agencia Estatal de Investigación (FEDER-PID2020-118422-GB-I00), the Basque Government (Grupos IT1558-22), and the University of Bologna for financial support.

Proteolysis targeting chimera (PROTAC)-mediated protein degradation has prompted a radical rethink and is at a crucial stage in driving a drug discovery transition. To fully harness the potential of this technology, a growing paradigm toward enriching PROTACs with other therapeutic modalities has been proposed. Could researchers successfully combine two modalities to yield multifunctional PROTACs with an expanded profile? In this Perspective, we try to answer this question. We discuss how this possibility encompasses different approaches, leading to multitarget PROTACs, light-controllable PROTACs, PROTAC conjugates, and macrocycle-and oligonucleotide-based PROTACs. This possibility promises to further enhance PROTAC efficacy and selectivity, minimize side effects, and hit undruggable targets. While PROTACs have reached the clinical investigation stage, additional steps must be taken toward the translational development of multifunctional PROTACs. A deeper and detailed understanding of the most critical challenges is required to fully exploit these opportunities and decisively enrich the PROTAC toolbox.

Small molecules able to bind non-canonical G-quadruplex DNA structures (G4) have been recently tested as novel potential agents for the treatment of prostate cancer thanks to their repression of aberrant androgen receptor gene. However, metastatic castration-resistant prostate cancer (mCRPC), a letal form of prostate cancer, is still incurable. Here we tested two naphthalenediimide derivatives, previously reported as multitarget agents, on a couple of relevant mCRPC cell models (DU145 and PC-3). We showed that these compounds interfere with the RAS/MEK/ERK and PI3K/AKT pathways. Interestingly, both these two biological processes depend upon Epidermal Growth Factor Receptor (EGFR) activation. By means of biological and analytical tools we showed that our compounds are efficient inducers of the structural transition of the EGFR promoter towards a G-quadruplex conformation, ultimately leading to a reduction of the receptor production. The overall result is an interesting cytotoxic profile for these two derivatives. Thanks to their activity at different steps, these compounds can open the way to novel therapeutic approaches for mCRPC that could contribute to escape resistance to selective treatments.

The self-assembly of amyloid peptides (A beta), in particular A beta(1-42), into oligomers and fibrils is one of the main pathological events related to Alzheimer's disease. Recent studies have demonstrated the ability of carbon monoxide-releasing molecules (CORMs) to protect neurons and astrocytes from A beta(1-42) toxicity. In fact, CORMs are able to carry and release controlled levels of CO and are known to exert a wide range of anti-inflammatory and anti-apoptotic activities at physiologically relevant concentrations. In order to investigate the direct effects of CORMs on A beta(1-42), we studied the reactivity of CORM-2 and CORM-3 with A beta(1-42) in vitro and the potential inhibition of its aggregation by mass spectrometry (MS), as well as fluorescence and circular dichroism spectroscopies. The application of an electrospray ionization-MS (ESI-MS) method allowed the detection of stable A beta(1-42)/CORMs adducts, involving the addition of the Ru(CO)(2) portion of CORMs at histidine residues on the A beta(1-42) skeleton. Moreover, CORMs showed anti-aggregating properties through formation of stable adducts with A beta(1-42) as demonstrated by a thioflavin T fluorescence assay and MS analysis. As further proof, comparison of the CD spectra of A beta(1-42) recorded in the absence and in the presence of CORM-3 at a 1: 1 molar ratio showed the ability of CORM-3 to stabilize the peptide in its soluble, unordered conformation, thereby preventing its misfolding and aggregation. This multi-methodological investigation revealed novel interactions between A beta(1-42) and CORMs, contributing new insights into the proposed neuroprotective mechanisms mediated by CORMs and disclosing a new strategy to divert amyloid aggregation and toxicity.

Histone deacetylase inhibitors (HDACIs) are responsible for controlling gene expression by modulating the acetylation status of histone proteins. Furthermore, they modulate the activity of cytoplasmic non-histone proteins. Due to the involvement of HDACs in neurodevelopment, memory formation, and cognitive processes, HDACIs have been suggested as innovative agents for the treatment of neurodegenerative disorders such as Alzheimer's disease (AD). Given their mechanisms of action and the complex nature of AD, HDACIs have been proposed for the design of novel multitarget ligands (MTLs). To this aim, the fragment responsible for HDAC inhibition has been coupled with other structures that are able to provide additional biological actions, such as antioxidant activity or the inhibition of phosphodiesterase 5, transglutaminase 2, and glycogen synthase kinase 3β. Herein we discuss recent efforts to design HDACI-based MTLs as potential disease-modifying entities.

[Image: see text] Several evidence pointed out the role of epigenetics in Alzheimer’s disease (AD) revealing strictly relationships between epigenetic and “classical” AD targets. Based on the reported connection among histone deacetylases (HDACs) and glycogen synthase kinase 3β (GSK-3β), herein we present the discovery and the biochemical characterization of the first-in-class hit compound able to exert promising anti-AD effects by modulating the targeted proteins in the low micromolar range of concentration. Compound 11 induces an increase in histone acetylation and a reduction of tau phosphorylation. It is nontoxic and protective against H(2)O(2) and 6-OHDA stimuli in SH-SY5Y and in CGN cell lines, respectively. Moreover, it promotes neurogenesis and displays immunomodulatory effects. Compound 11 shows no lethality in a wt-zebrafish model (

Mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene cause a rare neurodevelopmental disorder characterized by early-onset seizures and severe cognitive, motor, and visual impairments. To date there are no therapies for CDKL5 deficiency disorder (CDD). In view of the severity of the neurological phenotype of CDD patients it is widely assumed that CDKL5 may influence the activity of a variety of cellular pathways, suggesting that an approach aimed at targeting multiple cellular pathways simultaneously might be more effective for CDD. Previous findings showed that a single-target therapy aimed at normalizing impaired GSK-3β or histone deacetylase (HDAC) activity improved neurodevelopmental and cognitive alterations in a mouse model of CDD. Here we tested the ability of a first-in-class GSK-3β/HDAC dual inhibitor, Compound 11 (C11), to rescue CDD-related phenotypes. We found that C11, through inhibition of GSK-3β and HDAC6 activity, not only restored maturation, but also significantly improved survival of both human CDKL5-deficient cells and hippocampal neurons from Cdkl5 KO mice. Importantly, in vivo treatment with C11 restored synapse development, neuronal survival, and microglia over-activation, and improved motor and cognitive abilities of Cdkl5 KO mice, suggesting that dual GSK-3β/HDAC6 inhibitor therapy may have a wider therapeutic benefit in CDD patients.

A series of hybrid compounds was designed to target histone deacetylases and ds-/G-quadruplex DNAs by merging structural features deriving from Scriptaid and compound 1. Compound 6 binds different DNA arrangements, inhibits HDACs both in vitro and in cells, and is able to induce a reduction of cell proliferation. Moreover, compound 6 displays cell phenotype-reprogramming properties since it prevents the epithelial to mesenchymal transition in cancer cells, inducing a less aggressive and migratory phenotype, which is one of the goals of present innovative strategies in cancer therapies.

Drugs targeting human topoisomerase II (topoII) are used in clinical practice since decades. Nevertheless, there is an urgent need for new and safer topoII inhibitors due to the emergence of secondary malignancies and the appearance of resistance mechanisms upon treatment with topoII-targeted anticancer drugs. In the present investigation, we report the discovery of a new topoII inhibitor, whose design was based on the structure of the natural product trypthantrin, a natural alkaloid containing a basic indoloquinazoline moiety. This new topoII inhibitor, here numbered compound 5, is found to inhibit topoII with an IC50 of 26.6 ± 4.7 μM. Notably, compound 5 is more potent than the template compound trypthantrin, and even than the widely used topoII-targeted clinical drug etoposide. In addition, compound 5 also exhibits high water solubility, and a promising antiproliferative activity on different tumor cell lines such as acute leukemia, colon, and breast cancer. In light of these results, compound 5 represents a promising lead for developing new topoII inhibitors as anti-cancer therapeutic agents.

Benextramine is a well kown tetraamine disulfide acting as irreversible α-adrenergic antagonist and able to hit additional targets involved in neurodegeneration, such as nicotinic and muscarinic receptors and acetylcholinesterase enzyme. The disulfide group of benextramine can react with Cys residues of receptors and enzymes leading to irreversible alterations of their biological activity. On this basis, the potential inhibitory action of ten benextramine analogues was investigated on other well-known targets involved in neurodegenerative diseases, monoamine oxidases (MAOs), whose molecular structures contain various Cys residues. Most of the tested compounds were found able to inactivate both MAO isoforms, with inactivation rate constant values dependent on the polyamine structure as well as on MAO isoforms. Benextramine showed the highest kinact values (0.98 min-1) for MAO A and the “short” CM367 was found the most potent MAO B inactivator (kinact/KIE =1x105 M-1min-1). CM331, with a cathecol moiety at the two terminal ends, showed the best selectivity vs MAO B. Docking experiments highlight the role of Cys323 (in MAO A) and Cys 172 (in MAO B) as targets of the reactive disulfide moiety of benextramine derivatives, suggesting thatthe sterical hindrace of the bound inhibitor may hinder the entrance of substrate towards the FAD cofactor of the active site. The effectiveness of benextramine in inactivating monoamine oxidases in cells was confirmed using human neuroblastoma cell line SH SY5Y. Optimization studies are now in progress with the aim to modify the structure of the described compounds in order to improve their affinity and selectivity for potential application in neurodegenerative diseases.

Epigenetic modulators Histone deacetylases (HDACs) and Lysine demethylase (LSD1) are validated targets for anticancer therapy. Both HDAC1/2 and LSD1 are found in association with the repressor protein CoREST in a transcriptional co-repressor complex, which is responsible for gene silencing. Combined modulation of both targets results in a synergistic antiproliferative activity. In the present investigation, we report about the design and synthesis of a series of polyamine-based HDACs-LSD1 dual binding inhibitors obtained by coupling Vorinostat and Tranylcypromine. Compound 4 emerged as the most promising of the synthesized series, showing good inhibitory activity towards HDAC1 and LSD1 either in vitro and in cell-based assay (Ki = 42.52 ± 8.94 nM and IC50 = 3.85 μM, respectively). Furthermore, at 70.0 µM compound 4 induced a more pronounced cytotoxic effect than Vorinostat (68.6% vs 56.6% of dead cells) in MCF7 cancer cell line.

Purpose of review Traumatic brain injury (TBI) is one of the leading causes of death in the developed world. Despite advances at the bedside, pharmacological interventions have yet to be successful likely because of the need for a better understanding of disease mechanisms as potential targets for intervention. Recent evidence implicates a family of enzymes, namely transglutaminases, in the pathological mechanisms of TBI. Recent findings Transglutaminases are multifunctional, calcium-dependent enzymes that are significantly upregulated in TBI. They are known for their transamidase activity that consists of the covalent crosslinking of glutamines and lysines. Recent data support their ability to aminylate proteins with primary amines such as polyamines or monoamines like serotonin and histamine and to regulate gene transcription. Summary In this review, we will discuss data that support a role for transglutaminases, in particular transglutaminase 2, in mitochondrial damage, excitotoxicity and inflammation and their relationship to the pathobiology of TBI. We will review past evidence and outline the need for new experiments that could clarify the role of these enzymes in cell injury and death associated with traumatic brain injury.

Several evidence pointed out the role of epigenetics in Alzheimer's disease (AD) revealing strictly relationships between epigenetic and "classical" AD targets. Based on the reported connection among histone deacetylases (HDACs) and glycogen synthase kinase 3β (GSK-3β), herein we present the discovery and the biochemical characterization of the first-in-class hit compound able to exert promising anti-AD effects by modulating the targeted proteins in the low micromolar range of concentration. Compound

Fourteen polyamine analogues, asymmetric or symmetric substituted spermine (1–9) or methoctramine (10–14) analogues, were evaluated as potential inhibitors or substrates of two enzymes of the polyamine catabolic pathway, spermine oxidase (SMOX) and acetylpolyamine oxidase (PAOX). Compound 2 turned out to be the best substrate for PAOX, having the highest affinity and catalytic efficiency with respect to its physiological substrates. Methoctramine (10), a well-known muscarinic M2 receptor antagonist, emerged as the most potent competitive PAOX inhibitor known so far (Ki = 10 nM), endowed with very good selectivity compared with SMOX (Ki=1.2 μM vs SMOX). The efficacy of methoctramine in inhibiting PAOX activity was confirmed in the HT22 cell line. Methoctramine is a very promising tool in the design of drugs targeting the polyamine catabolism pathway, both to understand the physio-pathological role of PAOX vs SMOX and for pharmacological applications, being the polyamine pathway involved in various pathologies.

The two human monoamine oxidase isoforms (namely MAO A and MAO B) are enzymes involved in the catabolism of monoamines, including neurotransmitters, and for this reason are well-known and attractive pharmacological targets in neuropsychiatric and neurodegenerative diseases, for which novel pharmacological approaches are necessary. Benextramine is a tetraamine disulfide mainly known as irreversible alpha-adrenergic antagonist, but able to hit additional targets involved in neurodegeneration. As the molecular structures of monoamine oxidases contain nine cysteine residues, the aim of this study was to evaluate benextramine and eleven structurally related polyamine disulfides as potential MAO inhibitors. Most of the compounds were found to induce irreversible inactivation of MAOs with inactivation potency depending on both the polyamine structure and the enzyme isoform. The more effective compounds generally showed preference for MAO B. Structure-activity relationships studies revealed the key role played by the disulfide core of these molecules in the inactivation mechanism. Docking experiments pointed to Cys323, in MAO A, and Cys172, in MAO B, as target of this type of inhibitors thus suggesting that their covalent binding inside the MAO active site sterically impedes the entrance of substrate towards the FAD cofactor. The effectiveness of benextramine in inactivating MAOs was demonstrated in SH-SY5Y neuroblastoma cell line. These results demonstrated for the first time that benextramine and its derivatives can inactivate human MAOs exploiting a mechanism different from that of the classical MAO inhibitors and could be a starting point for the development of pharmacological tools in neurodegenerative diseases.

In the cold environments of the interstellar medium, a variety of molecules in which a hydrogen (H) atom has been replaced by its heavier isotope deuterium (D) can be found. From its emergence, life had to counteract the toxic action of many agents, which posed a constant threat to its development and propagation. Oxygen-reactive species are archaic toxicants that lead to protein damage and genomic instability. Most of the oxidative lesions involve cleavage of C-H bonds and H abstraction. According to free radical chemistry principles, the substitution of D for H in oxidation-sensitive positions of cellular components should confer protection against the oxidative attack without compromising the chemical identity of the compounds. Here, we show that deuterated nucleosides and proteins protect from oxidative damage. Our data suggest a new, subtle but likely role of D in terrestrial life’s evolution in that its inclusion in critical biomolecules might have facilitated their resistance during the infinite generations of life entities, cells, and organisms.

Preclinical Research A novel and promising approach to overcome the limits of single-target therapy is represented by the multitarget approach. This strategy aims to simultaneously modulate several targets involved in the pathophysiology of a multifactorial disease, with the potential to enhance therapeutic effectiveness and improve drug safety. Although there has been a marked growth in the design of multitarget drugs (MTDs) in the last years in the context of anti-Alzheimer and anti-cancer drug discovery, a parallel expansion was not observed in antipsychotic drugs, even that for psychiatric disorders there is a cogent medical need for new treatments. The discovery of new MTDs is a challenging task and we will describe the main strategies that have been developed over the years for the design of multifunctional molecules in antipsychotic drug discovery. In particular, we will focus on the few available MTDs based on the design of selective serotonin re-uptake inhibitors, used as antidepressants and in the treatment of schizophrenia. Drug Dev Res 77 : 437-443, 2016. © 2016 Wiley Periodicals, Inc.

Epidermal growth factor receptor inhibitors (EGFR-TKIs) represent a class of compounds widely used in anticancer therapy. An increasing number of studies reports on combination therapies in which the block of the EGFR-TK activity is associated with inhibition of its downstream pathways, as PI3K-Akt. Sulforaphane targets the PI3K-Akt pathway whose dysregulation is implicated in many functions of cancer cells. According to these considerations, a series of multitarget molecules have been designed by combining key structural features derived from an EGFR-TKI, PD168393, and the isothiocyanate sulforaphane. Among the obtained molecules 1-6, compound 6 emerges as a promising lead compound able to exert antiproliferative and proapoptotic effects in A431 epithelial cancer cell line by covalently binding to EGFR-TK, and reducing the phosphorylation of Akt without affecting the total Akt levels.

Introduction: Chemoprevention has emerged as a promising strategy to reduce the risk and to control cancer. In this context, isothiocyanates (ITCs), found in abundance in the form of glucosinolates in cruciferous vegetables, have gained increasing consideration for their chemopreventive activity. ITCs exert their effects mainly by inducing carcinogen metabolism or by inhibiting tumor cell proliferation. Areas covered: In recent years, novel combination treatments, by coupling chemopreventive agents and typical chemotherapeutics, have been exploited to increase the antitumor activities. The aim of this article is to examine the foremost studies carried out, so far, on the effects of dietary and synthetic ITCs on different signaling pathways involved in the pharmacokinetics and pharmacodynamics of chemotherapeutic agents, in order to enhance their effectiveness. Expert opinion: Undoubtedly, the beneficial anticarcinogenic potential of ITCs, both singly and in combination, has emerged in in vitro and in vivo studies. However, only a few clinical trials have been carried out so far with ITCs, which try to better define both the pharmacokinetic and pharmacodynamic impacts in humans. More toxicological evaluations after long-term administration of ITCs in different species are required for the clinical development of ITCs as anticarcinogenic agents.

The biological activities of six symmetrically substituted 2-methoxy-benzyl polymethylene tetraamines (1–4) and diphenylethyl polymethylene tetraamines (5 and 6) as N-methyl- d -aspartate (NMDA) receptor channel blockers, were evaluated in vitro and in vivo. Although all compounds exhibited stronger channel block activities in comparison to memantine in Xenopus oocytes voltage clamped at −70 mV, only compound 2 (0.4 mg/kg intravenous injection) decreased the size of brain infarction in a photochemically induced thrombosis model mice at the same extent of memantine (10 mg/kg intravenous injection). Other compounds (1, 3, 4, 5 and 6) did not decrease the size of brain infarction significantly due to the limited injection doses. The present study suggests that compound 2 could represent a valuable lead compound to design low toxicity polyamines for clinical use against stroke.

One important strategy to develop effective anticancer agents is based on natural products. Many active phytochemicals are in human clinical trials and have been used for a long time, alone and in association with conventional anticancer drugs, for the treatment of various types of cancers. A great number of in vitro, in vivo and clinical reports document the multi-target anticancer activities of isothiocyanates and of compounds characterized by a naphthalenetetracarboxylic diimide scaffold. In order to search for new anticancer agents with a better pharmaco-toxicological profile, we investigated hybrid compounds obtained by inserting isothiocyanate group(s) on a naphthalenetetracarboxylic diimide scaffold. Moreover, since water-soluble fullerene derivatives can cross cell membranes thus favoring the delivery of anticancer therapeutics, we explored the cytostatic and cytotoxic activity of hybrid compounds conjugated with fullerene. We studied their cytostatic and cytotoxic effects on a human T-lymphoblastoid cell line by using different flow cytometric assays. In order to better understand their pharmaco-toxicological potential, we also analyzed their genotoxicity. Our global results show that the synthesized compounds reduced significantly the viability of leukemia cells. However, the conjugation with a non-toxic vector did not increase their anticancer potential. This opens an interesting research pattern for certain fullerene properties.

The design of multifunctional agents represents one of the most active research field in medicinal chemistry. In particular, tacrine, a well known Acetylcholinesterase inhibitor, is one of the most used starting point to develop multifunctional ligands and hundreds of papers report about these new agents. This is the third review of a series concerning tacrinebased multifunctional ligands; in particular, in the present, we will summarize and discuss the most intriguing examples of tacrine-based multifunctional agents published since 2013 until 2016.We analyzed the bibliographic databases for peer-reviewed publications concerning tacrine-based multifunctional agents possessing biological actions that go beyond the simple "cholinergic" blockage. These papers have been subdivided according to their biological activities. Since this is the third review of a series, we took into considerations only the papers appeared since 2013 until 2016.In this review, we have analyzed more than 33 papers. All the reported compounds retain good inhibitory activity towards acetyl- and butyryl-cholinesterase. The other biological activities concern mostly inhibition of a) β-amyloid aggregation, b) β-secretase, c) monoamino oxidases, modulation of τ and ROS and metal chelation.The analysis of the current literature reported in this review confirm the importance of tacrine as scaffold to develop multifunctional agents potentially usefull to contrast Alzheimer's disease. Furthermore, the compounds herein reported showed very intriguing biological activities that could be used as starting point to develop new compounds even more interesting and, hopefully, clinically usefull to contrast Alzheimer's Disease.

Drug consumption in our modern society has reached unequaled proportions and it is still increasing. Recent data on the revenue of the worldwide pharmaceutical market pointed out a potent increase in the global drug market starting from the 390.1 billion US dollars for 2001 up to 1057.2 billion US dollars of revenue for 2014 (data taken from statistical website). We consider this data only for their general significance, and we cannot examine the market of the different classes of drugs, but we report these data only to realize the massive diffusion of drugs in the world and the dimensions of this phenomenon. On the other hand, pollution represents another reliable problem due to its global dimensions. These two issues are not distinct, but they overlap one to another and they can influence heavily the human health. In fact, drugs and pollutants are metabolized by the same enzymatic systems, which will no longer be able to process these massive doses of substrates. In this chapter, we will consider the interactions between drugs and environmental pollutants based on scientific evidence which appeared recently on the literature.

none 7 no The crucial role of Glycogen Synthase Kinase 3 (GSK-3β) as a pivotal player in Alzheimer's Disease (AD) has recently inspired significant attempts to design and synthesize potent kinase inhibitors. In fact GSK-3β is considered the main kinase which catalyzes the microtubule-associated protein tau hyper-phosphorylation and the neurofibrillary tangles (NFT) in vitro and in vivo, The first classes of GSK-3β inhibitors were classified as ATP-competitive and, therefore, they lack of an efficient degree of selectivity over other kinases. In light of this consideration, many efforts are devoted to characterize new non ATP-competitive GSK-3β inhibitors, endowed with high selectivity. In parallel, there is an urgent need to develop new analytical methodologies for the hit selection (highthroughput screening) and ligand binding characterization in terms of potency, affinity and mechanism of action. The new methodology for GSK-3β enzymatic activity determination can be adopted as a realistic alternative to the currently used radioactive, luminescence and fluorescence detection methods, each showing limitations in terms of safety and interferences. Herein, we propose an alternative and selective electrospray ionization quadrupole time-of-flight (ESI-QTOF) method, based on the direct quantification of phosphorylated substrate muscle glycogen synthase GSM, a peptide resembling the high affinity sequence of natural substrate muscle glycogen synthase 1, for the detailed characterization of GSK-3β inhibitors. The method was validated in terms of accuracy and reproducibility of GSM signal intensity with a relative standard deviation RSD% value of 3.55%; Limit of Detection (LOD): 0.006μM; Lower Limit of Quantification (LLOQ): 0.02μM; linearity r 2 0.9951. The kinetic constants (K M and v max) of the GSK-3β catalyzed kinase reaction and the inhibitory potency of known ligands (IC50), were determined. All the obtained results were in agreement with those reported in literature or obtained in house by the standard reference luminometric approach. The proposed method was applied to the elucidation of well known inhibitors mechanism of action by the construction of a Lineweaver-Burk plot and the K i determination. Furthermore, the potency, affinity and mechanism of action of a new non ATP-competitive compound were established. We demonstrated the ESI-QTOF method to be more feasible than the classic kinase assays since it avoids drawbacks inherently connected with radioisotope labeling or the indirect detection of kinase activity, so far. It is also scalable to the screening of large library collections and suitable for pharmaceutical industries purposes. none De Simone, Angela; Fiori, Jessica; Naldi, Marina; D'Urzo, Annalisa; Tumiatti, Vincenzo; Milelli, Andrea; Andrisano, Vincenza De Simone, Angela; Fiori, Jessica; Naldi, Marina; D'Urzo, Annalisa; Tumiatti, Vincenzo; Milelli, Andrea; Andrisano, Vincenza

none 14 si Alzheimer's disease (AD) is a multifactorial pathology that requires multifaceted agents able to address its peculiar nature. In recent years, a plethora of proteins and biochemical pathways has been proposed as possible targets to counteract neurotoxicity. Although the complex scenario is not completely elucidated, close relationships are emerging among some of these actors. In particular, increasing evidence has shown that aggregation of amyloid beta (Aβ), glycogen synthase kinase 3β (GSK-3β) and oxidative stress are strictly interconnected and their concomitant modulation may have a positive and synergic effect in contrasting AD-related impairments. We designed compound 3 which demonstrated the ability to inhibit both GSK-3β (IC50 = 24.36 ± 0.01 μM) and Aβ42 self-aggregation (IC50 = 9.0 ± 1.4 μM), to chelate copper (II) and to act as exceptionally strong radical scavenger (kinh = 6.8 ± 0.5 · 105 M−1s−1) even in phosphate buffer at pH 7.4 (kinh = 3.2 ± 0.5 · 105 M−1s−1). Importantly, compound 3 showed high-predicted blood-brain barrier permeability, did not exert any significant cytotoxic effects in immature cortical neurons up to 50 μM and showed neuroprotective properties at micromolar concentration against toxic insult induced by glutamate. De Simone, Angela; Bartolini, Manuela; Baschieri, Andrea; Apperley, Kim Y.P.; Chen, Huan Huan; Guardigni, Melissa; Montanari, Serena; Kobrlova, Tereza; Soukup, Ondrej; Valgimigli, Luca; Andrisano, Vincenza; Keillor, Jeffrey W.; Basso, Manuela; Milelli, Andrea De Simone, Angela; Bartolini, Manuela; Baschieri, Andrea; Apperley, Kim Y.P.; Chen, Huan Huan; Guardigni, Melissa; Montanari, Serena; Kobrlova, Tereza; Soukup, Ondrej; Valgimigli, Luca; Andrisano, Vincenza; Keillor, Jeffrey W.; Basso, Manuela; Milelli, Andrea

Naphthalene diimides (NDIs) have been widely used as scaffold to design DNA-directed agents able to target peculiar DNA secondary arrangements endowed with relevant biochemical roles. Recently, we have reported disubstituted linear- and macrocyclic-NDIs that bind telomeric and non-telomeric G-quadruplex with high degree of affinity and selectivity. Herein, the synthesis, biological evaluation and molecular modelling studies of a series of asymmetrically substituted NDIs are reported. Among these, compound 9 emerges as the most interesting of the series being able to bind telomeric G-quadruplex (ΔTm = 29 °C at 2.5 μM), to inhibit the activity of DNA processing enzymes, such as topoisomerase II and TAQ-polymerase, and to exert antiproliferative effects in the NCI panel of cancer cell lines with GI50 values in the micro-to nanomolar concentration range (i.e. SR cell line, GI50 = 76 nM). Molecular mechanisms of cell death have been investigated and molecular modelling studies have been performed in order to shed light on the antiproliferative and DNA-recognition processes.

The aim of this study was to incorporate a new naphthalenediimide derivative (AN169) with a promising anticancer activity into pegylated liposomes to an extent that allows its in vitro and in vivo testing without use of toxic solvent. AN169-loaded liposomes were prepared using the thin-film hydration method and characterized for size, polydispersity index, drug content and drug release. We examined their lyophilization ability in the presence of cryoprotectants (trehalose, sucrose and lysine) and the long-term stability of the lyophilized products stored at 4 °C for 3 and 6 months by particle size changes and drug leakage. AN169 was successfully loaded into liposomes with an entrapment efficiency of 87.3 ± 2.5%. The hydrodynamic diameter of these liposomes after sonication was ∼145 nm with a high degree of monodispersity. Trehalose was found to be superior to the other lyoprotectants. In particular, trehalose 1:10 lipid:cryoprotectant molar ratio may provide stable lyophilized liposomes with the conservation of physicochemical properties upon freeze-drying and long-term storage conditions. We also assessed their in vitro antitumor activity in human cancer cell lines (HTLA-230 neuroblastoma, Mel 3.0 melanoma, OVCAR-3 ovarian carcinoma and SV620 prostate cancer cells). However, only after 72 h incubation, loaded liposomes showed almost the same IC50 as free AN169. In conclusion, we developed a stable lyophilized liposomal formulation for intravenous administration of AN169 as anticancer drug, with the advantage of avoiding the use of potentially toxic solubilizing agents for future in vivo experiments

Redox impairment is a prominent feature of Alzheimer’s disease (AD). It has led to the “oxidative stress hypothesis”, which proposes antioxidants as beneficial therapeutic tools in AD treatment. To date, a wide variety of antioxidants have been examined as neuroprotectants. However, success has been elusive in clinical trials. Several factors have contributed to this failure, including the complexity of the redox system in vivo. Potentially critical aspects include the fine-tuned equilibrium between antioxidant defenses and free radical production, the lack of specific antioxidant target(s), and the inherent difficulty in delivering antioxidants where they are needed. Herein, we highlight significant progress in the field. Future directions of antioxidant research are also presented.

Tacrine (1) was the first acetylcholinesterase inhibitor (AChEI) introduced in therapy for the treatment of Alzheimer's disease (AD), but similarly to the most recent approved AChEIs and memantine, an N-methyl-D-aspartate receptor (NMDAR) antagonist, it does not represent an effective drug in halting the progression of AD. The continuous research in this field has contributed to delineate AD as a multifactorial syndrome with several biological targets involved in its etiology. On these bases, the development of new effective therapeutics becomes crucial and the design of molecules that address more than one specific AD target should represent thus a succeeded strategy for AD treatment. This review will focus on and summarize multifunctional 1 derivatives starting from our last paper published on the same topic in 2010. In the last three years, the design and synthesis of 1 homo- and heterodimers, as well as of 1-hybrid structures for AD therapy, was aimed mainly to discover safer drugs, with decreased hepatotoxicity in comparison to 1, taking also into account the multifactorial pathogenesis of the disease. Most of these new hetero/homo-dimers and/or hybrids of 1, although addressed mainly to acetylcholinesterase (AChE) and Aβ aggregation inhibition, are able to hit additional targets relevant to AD, among which, β-secretase (BACE1), reactive oxygen species (ROS), calcium channels, NMDAR and M1- muscarinic receptors.

Redox impairment is a prominent feature of Alzheimer's disease (AD). It has led to the "oxidative stress hypothesis", which proposes antioxidants as beneficial therapeutic tools in AD treatment. To date, a wide variety of antioxidants have been examined as neuroprotectants. However, success has been elusive in clinical trials. Several factors have contributed to this failure, including the complexity of the redox system in vivo. Potentially critical aspects include the fine-tuned equilibrium between antioxidant defenses and free radical production, the lack of specific antioxidant target(s), and the inherent difficulty in delivering antioxidants where they are needed. Herein, we highlight significant progress in the field. Future directions of antioxidant research are also presented.

Lipoic acid (LA) is a natural antioxidant. Its structure was previously combined with that of the acetylcholinesterase inhibitor tacrine to give lipocrine (1), a lead compound multitargeted against Alzheimer's disease (AD). Herein, we further explore LA as a privileged structure for developing multimodal compounds to investigate AD. First, we studied the effect of LA chirality by evaluating the cholinesterase profile of 1's enantiomers. Then, a new series of LA hybrids was designed and synthesized by combining racemic LA with motifs of other known anticholinesterase agents (rivastigmine and memoquin). This afforded 4, which represents a step forward in the search for balanced anticholinesterase and antioxidant capacities.

To date, the pharmacotherapy of Alzheimer's disease (AD) has relied on acetylcholinesterase (AChE) inhibitors (AChEIs) and, more recently, an N-methyl-D-aspartate receptor (NMDAR) antagonist. AD is a multifactorial syndrome with several target proteins contributing to its etiology. “Multi-target-directed ligands” (MTDLs) have great potential for treating complex diseases such as AD because they can interact with multiple targets. The design of compounds that can hit more than one specific AD target thus represents an innovative strategy for AD treatment. Tacrine was the first AChEI introduced in therapy. Recent studies have demonstrated its ability to interact with different AD targets. Furthermore, numerous tacrine homo- and heterodimers have been developed with the aim of improving and enlarging its biological profile beyond its ability to act as an AChEI. Several tacrine hybrid derivatives have been designed and synthesized with the same goal. This review will focus on and summarize the last two years of research into the development of tacrine derivatives able to hit AD targets beyond simple AChE inhibition.

It is shown that racemic oxazolones are excellent reagents for the synthesis of chiral quaternary amino acids and its derivatives by the diastereo- and enantioselective nucleophilic addition to alpha,beta-unsaturated aldehydes catalyzed by diarylprolinol silyl ethers. The scope of this new organocatalytic reaction is demonstrated for different oxazolones having aromatic and alkyl groups at the reactive carbon atom and different aromatic and aliphatic substituted alpha,beta-unsaturated aldehydes, for which the stereoselective reaction proceeds with good yield, moderate to good to very high diastereoselectivity, and very high enantioselectivity. The potential of the reaction is shown for the synthesis of optically active alpha,alpha-disubstituted alpha-amino acids, alpha-quaternary proline derivatives, amino alcohols, lactams, and tetrahydropyranes. Furthermore, we have calculated by DFT-methods the transition-state structures that account for both the diastereo- and enantioselectivity observed for the addition of oxazolones to the alpha,beta-unsaturated aldehydes. For one class of compounds, the stereoselectivity is controlled by a hydrogen-bonding interaction of the enolate-form of the oxazolone with an ortho-hydroxy-phenyl substituent of the alpha,beta-unsaturated aldehyde, whereas the benzhydryl-protecting group in the oxazolone determines the diastereo- and enantioselectivity in a more general manner for both aromatic and aliphatic alpha,beta-unsaturated aldehydes.

The aim of the present paper was to investigate the role of the octamethylene spacer of methoctramine ( 1 ) on the biological profile. Thus, this spacer was incorporated into a dianiline or dipiperidine moiety to determine whether flexibility and the basicity of the inner nitrogen atoms are important determinants of potency with respect to muscarinic receptors. The most potent compound was 4 , which displayed, in the functional assays, a comparable potency at muscarinic M 2 receptors with respect to 1 , and, in the binding assays, a loss of potency and selectivity toward muscarinic M 1 and M 3 receptor subtypes. Both compounds were endowed with antinociceptive activity. Furthermore, in microdialysis tests in rat parietal cortex, they enhanced acetylcholine release, most likely by antagonizing presynaptic muscarinic receptor subtypes.

none 6 no Altered GSK-3β activity can contribute to a number of pathological processes including Alzheimer's disease (AD). Indeed, GSK-3β catalyzes the hyperphosphorylation of tau protein by transferring a phosphate moiety from ATP to the protein substrate serine residue causing the formation of the toxic insoluble neurofibrillary tangles; for this reason it represents a key target for the development of new therapeutic agents for AD treatment. Herein we describe a new selective UHPLC methodology developed for the direct characterization of GSK-3β kinase activity and for the determination of its inhibition, which could be crucial in AD drug discovery. The UHPLC-UV (DAD) based method was validated for the very fast determination of ATP as reactant and ADP as product, and applied for the analysis of the enzymatic reaction between a phosphate primed peptide substrate (GSM), resembling tau protein sequence, ATP and GSK-3β, with/without inhibitors. Analysis time was ten times improved, when compared with previously published chromatographic methods. The method was also validated by determining enzyme reaction kinetic constants (KM and vmax) for GSM and ATP and by analyzing well known GSK-3β inhibitors. Inhibition potency (IC50) values for SB-415286 (81 ± 6 nM) and for Tideglusib (251 ± 17 nM), found by the newly developed UHPLC method, were in good agreement with the luminescence method taken as independent reference method. Further on, the UHPLC method was applied to the elucidation of Tideglusib mechanism of action by determining its inhibition constants (Ki). In agreement with literature data, Tideglusib resulted a GSM competitive inhibitor, whereas SB-415286 was found inhibiting GSK-3β in an ATP competitive manner. This method was applied to the determination of the potency of a new lead compound and was found potentially scalable to inhibitor screening of large compounds collections. D'Urzo, Annalisa; DE SIMONE, Angela; Fiori, Jessica; Naldi, Marina; Milelli, Andrea; Andrisano, Vincenza D'Urzo, Annalisa; DE SIMONE, Angela; Fiori, Jessica; Naldi, Marina; Milelli, Andrea; Andrisano, Vincenza

none 18 no Neuroblastoma is a childhood cancer with poor long-term prognosis in advanced stages. A major aim in neuroblastoma therapy is to develop targeted drug delivery systems to ameliorate drug therapeutic index and efficacy. In this study, a novel bortezomib (BTZ) liposomal formulation was set-up and characterized. Since BTZ is freely permeable across the lipidic bilayer, an amino-lactose (LM) was synthesized as complexing agent to entrap BTZ inside the internal aqueous compartment of stealth liposomes. High encapsulation efficiency was achieved by a loading method based on the formation of boronic esters between the boronic acid moiety of BTZ and the hydroxyl groups of LM. Next, NGR peptides were linked to the liposome surface as a targeting-ligand for the tumor endothelial cell marker, aminopeptidase N. Liposomes were characterized for size, Z-potential, polydispersity index, drug content, and release. Lyophilization in the presence of cryoprotectants (trehalose, sucrose) was also examined in terms of particle size changes and drug leakage. BTZ was successfully loaded into non-targeted (SL[LM-BTZ]) and targeted (NGR-SL[LM-BTZ]) liposomes with an entrapment efficiency of about 68% and 57%, respectively. These nanoparticles were suitable for intravenous administration, presenting an average diameter of 170nm and narrow polydispersity. Therefore, orthotopic NB-bearing mice were treated with 1.0 or 1.5mg/kg of BTZ, either in free form or encapsulated into liposomes. BTZ loaded liposomes showed a significant reduction of drug systemic adverse effects with respect to free drug, even at the highest dose tested. Moreover, mice treated with 1.5mg/kg of NGR-SL[LM-BTZ] lived statistically longer than untreated mice (P=0.0018) and SL[LM-BTZ]-treated mice (P=0.0256). Our results demonstrate that the novel vascular targeted BTZ formulation is endowed with high therapeutic index and low toxicity, providing a new tool for future applications in neuroblastoma clinical studies. Zuccari, G.; Milelli, A.; Pastorino, F.; Loi, M.; Petretto, A.; Parise, A.; Marchetti, C.; Minarini, A.; Cilli, M.; Emionite, L.; Di Paolo, D.; Brignole, C.; Piaggio, F.; Perri, P.; Tumiatti, V.; Pistoia, V.; Pagnan, G.; Ponzoni, M. Zuccari, G.; Milelli, A.; Pastorino, F.; Loi, M.; Petretto, A.; Parise, A.; Marchetti, C.; Minarini, A.; Cilli, M.; Emionite, L.; Di Paolo, D.; Brignole, C.; Piaggio, F.; Perri, P.; Tumiatti, V.; Pistoia, V.; Pagnan, G.; Ponzoni, M.

The synthesis, biological and molecular modeling evaluation of a series of macrocyclic naphthalene diimides is reported. The present investigation expands on the study of structure–activity relationships of prototype compound 2 by constraining the molecule into a macrocyclic structure with the aim of improving its G-quadruplex binding activity and selectivity. The new derivatives, compounds 4 – 7 carry spermidine- and spermine-like linkers while in compound 8 the inner basic nitrogen atoms of spermine have been replaced with oxygen atoms. The design strategy has led to potent compounds stabilizing both human telomeric (F21T) and c-KIT2 quadruplex sequences, and high selectivity for quadruplex in comparison to duplex DNA. Antiproliferative effects of the new derivatives 4 – 8 have been evaluated in a panel of cancer cell lines and all the tested compounds showed activity in the low micromolar or sub-micromolar range of concentrations. In order to rationalize the molecular basis of the DNA G-quadruplex versus duplex recognition preference, docking and molecular dynamics studies have been performed. The computational results support the observation that the main driving force in the recognition is due to electrostatic factors.

New polyamine derivatives 1-8, related to the previously reported N(1),N(12)-dibenzyldodecane-1,12-diamine (Bis-Bza-Diado) and N(1)-benzyl-spermine (BD6), have been synthesized and used as "probes" (potential substrates or inhibitors) of the human monoamine oxidases (MAO A and MAO B) and Vascular-Adhesion-protein -1 (VAP-1). Compound 8, the most effective inhibitor of the series, is characterized by a 12-methylene carbon chain ending with an isothiocyanate (ITC) group. Interestingly, it behaves as competitive inhibitor of MAO B and as irreversible inhibitor of MAO A. Compound 3, an asymmetric spermine analogue bearing a thiophene ring, acts as a reversible mixed inhibitor, selective for MAO B (K(IE) = 23 μM). Docking studies performed using the available Protein Data Bank (PDB) structures of MAO A and MAO B, suggested that the different mode of inhibition of 8 may be explained by the different binding poses of 8 into the active site cavities of the two MAO isoforms. The ε-amino group of Lys 305 of MAO A is proposed as possible target of the ITC group of the inhibitor. Further studies are in progress to confirm this hypothesis. These results indicate a potential use of the polyamine scaffold for the development of new MAO inhibitors for application in human pathologies involving these enzymes.

The ability of symmetrically substituted long chain polymethylene tetramines, methoctramine (1) and its analogs 2-4 to kill cancer cells was studied. We found that an elevated cytotoxicity was correlated with a 12 methylene chain length separating the inner amine functions (6-12-6 carbon backbone), together with the introduction of diphenylethyl moieties on the terminal nitrogen atoms (compound 4) of a tetramine backbone. Compound 4 triggered dissipation of mitochondrial transmembrane potential and increased intracellular peroxide levels, leading to a caspase-independent HeLa cell death associated with a rapid activation of autophagy. The antioxidant N-acetylcysteine inhibited cell death and activation of autophagy, indicating a link between oxidative stress and autophagy. Autophagy was rapidly triggered even by tetramines 2 and 3, indicating that is related to their polyamine structure. Autophagy did not protect HeLa cells against cytotoxicity elicited by compound 4. The present study shows that, by modifications of the methoctramine structure, it is possible to design polyamine derivatives highly cytotoxic against tumor cells and that the appropriate design of molecules bearing polyamine-like structures leads to powerful inducers of autophagy.

Anticancer chemotherapy is strongly hampered by the low therapeutic index of most anticancer drugs and the development of chemoresistance. Therefore, there is a continued need for the identification of new molecular targets in order to selectively hit cancer cells. RNA has been recently validated as a cancer target by the use of different specific ligands and/or by different agents able to destroy its diverse forms. The ability of synthetic polyamines to interact and to alter the RNA structure has been already reported. In the present paper the interaction and the ability to damage RNA structure by several synthetic polyamines were evaluated and quantified by microfluid capillary electrophoresis. This technique allowed us to visualize both the RNA impairment through different electropherograms and to assess the RNA integrity number. Finally, the ability to discriminate between RNA and DNA by these synthetic polyamines was also evaluated.

The multimodal effective compound (I) inhibits the anticholinesterase (AChE) activity in a submicromolar range and shows a significant reduction of AChE-induced anti-aggregating efficacy without a cytotoxicity in neuronal-like cells SH-SY5Y up to the highest concentration of 50 μM.

In the last few decades, medicinal chemists have carried out extensive research on synthetic polyamines for use as anticancer drugs and multitarget-directed ligands in neurodegenerative diseases. The aim of this study was to evaluate the effect of some synthetic polyamines as inhibitors of two new potential targets, human semicarbazide-sensitive amine oxidase/vascular adhesion protein-1 (SSAO/VAP-1) and monoamine oxidases B (MAO B), enzymes involved in various multi-factorial diseases such as Alzheimer's disease. N,N'-Dibenzyl-dodecane-1,12-diamine (Bis-Bza-Diado), a newly synthesised compound, and ELP 12, a muscarinic cholinergic M(2) receptor antagonist, were found to behave as reversible and mixed non-competitive inhibitors of both amine oxidases (dissociation constants of about 100 microM). ELP 12 was found to be more selective for SSAO/VAP-1. Combining kinetic and structural approaches, the binding mode of ELP 12 to SSAO/VAP-1 was investigated. ELP 12 may bind at the entrance of the active site channel by ionic interactions with ASP446 and/or ASP180; one end of the polyamine may be accommodated inside the channel, reaching the TPQ cofactor area. The binding of ELP 12 induces rearrangement of the secondary structure of the enzyme and impedes substrate entry and/or product release and catalysis. These structural data reveal that the entrance and the first part of the SSAO/VAP-1 channel may be considered as a new target area, or a "secondary binding site", for modulators of human SSAO/VAP-1 activity. These results indicate ELP 12 and Bis-Bza-Diado as new "skeletons" for the development of novel SSAO/VAP-1 and MAO B inhibitors.