Oxidative Stress in Alzheimer's Disease: Are We Connecting the Dots?

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Oxidative Stress in Alzheimer’s Disease: Are We Connecting the Dots? Miniperspective Michela Rosini,*,† Elena Simoni,† Andrea Milelli,§ Anna Minarini,† and Carlo Melchiorre*,† †

Department of Pharmacy and Biotechnology, Alma Mater StudiorumUniversity of Bologna, Via Belmeloro 6, 40126 Bologna, Italy Department for Life Quality Studies, Alma Mater StudiorumUniversity of Bologna, Corso d’Augusto 237, 47921 Rimini, Italy

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ABSTRACT: 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.

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INTRODUCTION Alzheimer’s disease (AD) is the most common cause of memory impairment and dementia, for which effective cures are urgently needed.1 Besides diffuse neuronal loss, AD brains exhibit accumulation of misfolded protein deposits as amyloid β (Aβ) plaques and tau-dependent neurofibrillary tangles. These pathological lesions have been interpreted as causative features, attracting considerable efforts in the search for mechanismbased therapies.2 However, with a deepening understanding of the mechanisms leading to neurodegeneration, numerous other pathogenic factors have emerged, including excitotoxicity, calcium impairment, mitochondrial dysfunction, neuroinflammation, and oxidative stress. There is a near consensus that these mechanisms coexist, affecting each other at multiple levels.3 In this respect, oxidative stress, which could be secondary to several other pathophysiological events, appears to be a major determinant of AD pathogenesis and progression. Experimental evidence indicates that a dysregulation of the redox state strongly participates in an early stage of AD, inducing and activating multiple cell signaling pathways that contribute to the initial progression of the neurodegenerative process.4 Indeed, constant evidence of reactive oxygen species (ROS) mediated injury and reactive nitrogen species (RNS) mediated injury is observed in AD brain.5 Increased levels of oxidative markers of biomolecules (proteins, lipids, carbohydrates, and nucleic acids) have been detected in a large number of studies in AD central and peripheral systems. 6−8 Furthermore, levels of antioxidant enzymes were found to be altered in brain regions of AD patients.9 All these data support the “oxidative stress hypothesis” of AD. Positing oxidative stress as a key event in AD onset and progression, this theory © XXXX American Chemical Society

proposes antioxidants as beneficial therapeutic tools in AD treatment.10 To date, a wide variety of antioxidants have been examined as neuroprotectants. This has afforded promising results in in vitro studies and animal models. However, success has been elusive in clinical trials.11 Much criticism has been directed toward the reliability of the negative trials, whose design has often been questioned.12 However, there has recently been a growing consensus that the oxidative stress hypothesis requires a critical rethink. Attention has first focused on the poor bioavailability and low permeability across the blood−brain barrier of most of the exogenous antioxidants. New delivery systems, such as those based on nanoparticles, are emerging as effective approaches for overcoming this issue.13 Although nanomedicine is of paramount importance in searching for effective antioxidant cures, the more we expand our knowledge of the role of oxidative stress in AD, the more the complexity of the redox system in vivo emerges as the nodal point. Oxidative stress is a widespread cellular process that lacks a specific treatment target such as a receptor or a single major metabolic pathway.14 The wide variety of sources and sites of production of oxidant species goes along with an even higher heterogeneity in the antioxidant response. Antioxidants can function by preventing the formation, detoxifying, or scavenging (of) oxidant species.5 They can inhibit pro-oxidant enzymes, neutralize radicals, or chelate transition-metal ions that catalyze radicals’ generation. In addition, some antioxidants Received: June 27, 2013

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dx.doi.org/10.1021/jm400970m | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Four of the five drugs marketed for AD are AChE inhibitors (AChEIs), designed to attenuate cholinergic deficit by reducing degradation of the neurotransmitter acetylcholine. As with all the available AD drugs, AChEIs offer limited and transient benefits to patients. This has prompted researchers to endow these molecules with antioxidant features to hopefully increase the likelihood of success. Although this approach is still in its infancy, the bifunctional drug ladostigil (1) has already provided the proof of concept.20 In a single molecule, 1 combines the neuroprotective effects of the selective monoamine oxidase (MAO) B inhibitor rasagiline with the AChE inhibitory activity of the anti-AD drug rivastigmine (Figure 2). Compound 1 has been licensed to Avraham Pharmaceuticals, and data from their phase II trial (NCT01354691) are expected very soon.21

exert their effects by raising endogenous antioxidant defenses, up-regulating the expression of redox-sensitive transcriptional factors. Hence, redox homeostasis in cells is derived from a finetuning of numerous factors. In AD, there are established interconnections between oxidative stress and other key AD events, which amplify the complexity of this issue (Figure 1). Nevertheless, oxidative

Figure 1. Prevailing interconnections between oxidative stress and other key players in AD.

stress has recently been read as a common key element capable of articulating the divergent nature of different pathogenic mechanisms of AD.3 This offers the chance to connect the dots of this intricate picture and possibly to create disease-modifying antioxidant approaches to confronting the disease. Extensive literature exists on single pharmacophore “pure antioxidants” as therapeutics for neurodegenerative disorders. The interested reader is directed to the excellent review by Trippier et al. and references cited therein.15 Herein, we highlight some relevant advances, focusing on medicinal chemists’ efforts to design antioxidant agents able to tackle AD on multiple fronts. We will discuss the intertwined relationship between oxidative stress and other active players of the neurotoxic cascade. We will examine how, by targeting the main sites of ROS production, researchers have seized an intriguing opportunity to seek more efficient antioxidant approaches to AD treatment. We will also present current progress and future directions in activating inducible antioxidant defense.

Figure 2. Design strategy and structures of rivastigmine, rasagiline, and ladostigil (1).

The discovery that AChE plays other roles, in addition to its “classical” function in terminating the cholinergic impulse,22 has created new interest in this target. In particular, the AChE peripheral anionic site (PAS) is thought to be associated with the neurotoxic cascade underlying AD through AChE-induced Aβ aggregation. The concomitant inhibition of the PAS may turn AChEIs into potential disease-modifying agents.23,24 Considering the nonclassical AChE functions, their relationships with Aβ processing and deposition, and the putative role of the PAS in these functions,25 dual-binding site AChEIs have acquired new importance for AD treatment.26 The rational modification of their structures to provide them with antioxidant properties has led us and others to discover a number of interesting multifunctional bivalent ligands. In 2005, racemic lipocrine (2) was at the forefront of this approach.27 Compound 2 combined, in the same molecule, the structure of the natural antioxidant lipoic acid (LA) with a derivative of tacrine, the first AChEI approved for AD treatment (Figure 3). Experimental evidence indicated that 2 could bind both catalytic and peripheral sites of AChE, thus acting as a mixed-type inhibitor. This bivalent interaction afforded one of the most potent AChEIs ever found (IC50 = 0.25 nM), together with the ability to reduce AChE-induced Aβ aggregation (IC50 = 45 μM). In addition, LA contributed to the multimodal profile of 2, protecting human SH-SY5Y cells from ROS formation evoked by oxidative stress (with 2 being more active than LA). In 2011, this study was expanded, exploiting the role of LA’s stereochemistry.28 In particular, it had been reported that stereochemistry is not significant for the protective effect of LA against oxidative cell damage.29 However, to verify whether it could affect AChE inhibition, the two enantiomers of 2, (S)-2 and (R)-2, were synthesized and studied. Their inhibitory

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MULTIFUNCTIONAL ANTIOXIDANTS Epidemiological studies and clinical trials have not convincingly ruled out the oxidative stress hypothesis. However, they have significantly contributed to suggesting that antioxidant efficacy is not the only thing needed to modify AD progression. Antioxidants presenting additional pharmacological effects are thought to offer a good possibility of combating this complex disease, in which free radicals are significant but not exclusive drivers.16 The rationale is that single molecules, endowed with antioxidant properties and able to act at different steps in the neurodegenerative process, can produce additional neuroprotective effects against AD.17,18 Research has thus gradually moved from an oxidative-stress-centered approach to one that considers other targets relevant to AD’s pathogenesis. In recent years, many multifunctional antioxidants have been rationally designed.19 Attention has focused on acetylcholinesterase (AChE), a target that remains crucial to AD therapy. B



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Figure 3. Design strategy and structures of tacrine, lipoic acid, and lipocrine (2) and its enantiomers.

Figure 4. Structures of the multifunctional antioxidants 3−5. Antioxidant features are highlighted with dashed boxes.38

anticholinesterase motif.35−37 Compounds 3−5 inhibited both catalytic activities of AChE and AChE-induced Aβ aggregation and exerted antioxidant and/or neuroprotective efficacy (Figure 4). Moreover, when endowed with antiaggregating efficacy, the antioxidant features also provided hybrid compounds with the ability to modulate Aβ self-induced aggregation. Interestingly, the detailed pharmacological characterization of the FA−tacrine adduct 5 in in vitro and in vivo studies correlates well with the established connection between oxidative stress and Aβ processing and deposition. Markers of oxidative stress in AD brains colocalize with Aβ plaques, a situation that is also observed in animal models of the disease.3 Oxidative stress is known to promote Aβ toxicity through the production of free radicals. It also triggers the amyloidogenic pathway in human cell lines.39 At the same time, several lines of evidence indicate that Aβ induces oxidative stress in AD,40 confirming the etiopathogenic loop generated by Aβ and ROS overproduction. In light of this, it is interesting that the FA− tacrine adduct 5 prevents cell death and reduces intracellular ROS accumulation induced by Aβ1−40 in PC12 cells. It also

potencies were slightly different on AChE. Enantiomer (R)-2 was only twice as potent as (S)-2 (IC50 = 0.23 nM and IC50 = 0.47 nM, respectively). The lack of a biologically significant difference between racemic 2 and the most active enantiomer (R)-2 rationalized the design of new anticholinesterasic LA adducts as racemic compounds. Compound 2 is currently marketed as a pharmacological tool for studying AD. Preliminary ADMET studies have investigated whether this lead compound could result in a suitable multifunctional drug for AD treatment. There has been a particular focus on the distribution pattern30 and in vivo studies.31 From 2005, the repertoire of AChE dual-binding antioxidants continued to grow.32−34 Herein, we focus on tacrine conjugates for which the ability to interact with the AChE PAS has been investigated through the study of AChE-induced Aβ aggregation. In particular, carbazole, cystamine, and ferulic acid (FA), which possess antioxidant features, have been exploited affording hybrid molecules with a significantly improved pharmacological profile with respect to the solely C



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Figure 5. Structure of curcumin (6) (keto and enol forms).

ameliorates the impairment of learning and memory37 on a validated AD mice model for anti-AD drug development.41 The effects of 5 on the cholinergic system were also measured in AD mice, together with its antioxidant efficacy. Compound 5 significantly increased choline acetyltranferase activity and reduced AChE efficacy, thus potentiating the cholinergic system after intracerebroventricular injection of Aβ1−40. In the same conditions, 5 could considerably reduce Aβ-induced oxidative stress as revealed by raised superoxide dismutase activity and decreased levels of malondialdehyde, which are two factors indicative of the oxidative status. Altogether, these results point to 5 as a promising multifunctional antioxidant for treating AD. Summing up, endowing anticholinesterase agents with antioxidant abilities is an intriguing strategy for broadening their efficacy. Notably, the observed improvement of the pharmacological profile generally runs parallel to a pronounced decrease in toxicity.27,28,36,37 In addition, the multifunctional compounds herein described are able to protect neuronal cells from oxidative or Aβ-induced injuries. This ability is ascribed to the antioxidant moieties. This suggests that antioxidant features may significantly contribute to contrasting the progressive neurodegeneration responsible for the decline in efficacy of AChEIs, thus lengthening the time in which anticholinesterase drugs can provide beneficial effects on memory and cognition.

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amyloid aggregation provided the rationale for its selective targeting to neuronal membranes. Several lines of evidence indicate that the initial events of Aβ oligomerization and deposition in AD involve the interaction of soluble oligomers with neuronal membranes.47 Indeed, it has been proposed that Aβ generated from cleavage of the transmembrane amyloid precursor protein (APP), once produced, could plausibly integrate within the lipid bilayer. This is because of its hydrophobic nature. In addition, evidence suggested that lipid rafts, which are ordered membrane microdomains enriched in cholesterol and sphingolipids, could accelerate the cell membrane binding of Aβ. APP, APP cleavage enzymes (βand γ-secretases), Aβ, and Aβ oligomers have all been identified in lipid rafts. Thus, they may be a critical platform for Aβ production and oligomerization.48 Once associated with the membranes, Aβ oligomers are thought to serve ROS formation and initiate lipid peroxidation, causing Aβ-induced oxidative stress.40 The products of oxidation are thought to further diffuse through the membrane, affecting other cellular compartments and significantly amplifying the effect of the original Aβcentered free radicals. On this basis, Zhang and co-workers49 sought to direct the antioxidant and antiaggregating efficacy of 6 to cell membrane/ lipid rafts (CM/LR), selecting cholesterol as the anchor motif. A series of multifunctional adducts containing the structure of both cholesterol and 6 were designed, synthesized, and biologically characterized as potential treatments for AD (Figure 6). Appropriate connection of the two pharmacophores, in terms of both spacer length and attaching position, emerged as an important structural determinant for their biological profile. In particular, compound 7, carrying a 21atom linker, was shown to localize to the CM/LR of human neuroblastoma MC65 cells, to inhibit the formation of Aβ oligomers, and to protect MC65 cells and differentiated SHSY5Y cells from Aβ-induced cytotoxicity. It retained the antioxidant property of 6 while exhibiting superior ability to reach Aβ oligomers by interacting with the CM/LR, thus showing better overall protective activities than 6 in these cells. Interestingly, in 2012, 7 was evaluated as a fluorescent probe for its ability to label and detect aggregated Aβ peptide.50 Compound 7 bound to different Aβ42 species with micromolar binding affinity while displaying the appropriate fluorescent properties for labeling and imaging Aβ plaques in situ. In in vivo studies, 7 crossed the blood−brain barrier. Furthermore, it specifically bound Aβ plaques in both AD human patients and transgenic mouse brain tissues, thus holding promise as a fluorescent probe for Aβ imaging. In order to develop alternatives to the cholesterol-anchored series of compounds, researchers have synthesized new 6 conjugates carrying cholesterylamine (CLA) as the vehicle tool.51 Notably, compound 8 (Figure 6), possessing a 17-atom linker, exerted a significantly increased neuroprotection in MC65 cells (EC50 = 0.083 μM) with respect to the cholesterolcontaining series (EC50 = 3 μM for compound 7). In particular, 8 inhibited production of Aβ oligomers in MC65 cells and

TARGETING OF ANTIOXIDANT FEATURES

Disappointing results from clinical trials have also raised the question of whether antioxidants could efficiently target oxidative stress sources. In recent years, a number of targeted antioxidants have been developed, with the aim of increasing their ability to counteract oxidative stress. The development of curcumin (6) (Figure 5) exemplifies medicinal chemistry’s efforts in this direction. Polyphenol 6 is a natural compound derived from turmeric Curcuma longa. It is a pleiotropic agent with several molecular targets and biological activities. In addition to anti-inflammatory, antiproliferative, and neuroprotective effects,42 it possesses direct primary and indirect secondary antioxidant activity.43 Over the past decade, 6 has been the object of intensive study, which demonstrated that it could modulate pathways involved in the pathophysiology of AD, such as the Aβ cascade, tau phosphorylation, neuroinflammation, and oxidative stress.44 As with many other antioxidants, the potential use of 6 as a therapeutic tool for AD treatment is severely affected by its low water solubility and poor bioavailability.45 This is despite its broad effects on the biological functions of cells. Enhancing 6’s solubility and stability following oral administration is a challenge that has recently been addressed by technological approaches44 and derivatization strategies.46 In parallel, its multifaceted mechanism of action has been interpreted as a versatile feature that, appropriately targeted to ROS sources, could produce a more specific therapeutic effect. Targeting Neuronal Membrane. In this respect, the ability of 6 to counteract oxidative species and interfere with D



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Figure 7. Representation of antioxidant delivery strategies. Driven by appropriate vehicle tools, antioxidant features preferentially localize into cell membranes or mitochondria.

impaired activity of the electron-transport chain found in AD patients and tissues from AD brain.60 Dysfunction of mitochondria is associated with increased ROS production, abnormal intracellular calcium levels, and reduced mitochondrial ATP.61 Notably, mitochondria are both a major site of production and a primary target of ROS. Mitochondrially derived ROS initiate toxic APP processing and thereby trigger Aβ production.62 At the same time, Aβ itself causes mitochondrial dysfunction. It localizes to mitochondria and interferes with their normal functioning, disrupting the respiratory chain, contributing to synaptic damage, and in a vicious manner, altering redox homeostasis.63 Given the central role that mitochondria and mitochondrial dysfunction play in AD, great interest has been devoted to protecting this subcellular compartment from ROS-mediated damage.61 Researchers have thus sought to direct the antioxidant therapy to this organelle specifically (Figure 7) by developing purposely designed mitochondria-targeted antioxidants.18 In the past decade, considerable progress has been made in this direction, based on two distinct mitochondrial features: the organelle protein import machinery and the high membrane potential across the inner membrane.64 In particular, the latter offers a unique chemical opportunity for selectively targeting the mitochondrion, as verified for several lipophilic cations, which efficiently accumulate into this subcellular compartment taking advantage of electrostatic forces. There are a wide range of cations that could in principle be used for drug-targeting purposes. To date, however, only one, triphenylphosphonium, has been extensively characterized in this context. It consists of a positively charged phosphorus atom surrounded by three hydrophobic phenyl functions. This has been used to drive the selective uptake of a variety of antioxidants into mitochondria. In particular, MitoQ (9) (Figure 8), which has a ubiquinone moiety, has emerged as a promising lead.65 Overall, numerous mitochondria-targeted antioxidant candidates have been developed. Their potential use in combating neurodegenerative diseases has been detailed elsewhere.66 In this context, researchers have pursued the idea of endowing the multifaceted curcumin with an additional action. Mitochondria are involved in such apparently different diseases as AD and cancer, for which 6’s efficacy is currently under clinical investigation.44,67 This has prompted researchers to

Figure 6. Multifunctional adducts 7 and 8, bearing the structures of both cholesterol and 6.

significantly reduced intracellular oxidative species in a dosedependent manner. The metal complexation of 8 and cholesterol derivative 7 was also investigated because of the known ability of 6 to chelate biometals.52 Indeed, metal ion dyshomeostasis is believed to underlie numerous neurodegenerative diseases, including AD. Experimental evidence indicates that metals might play a major role in aggregation/ precipitation of Aβ soluble oligomers.53 Numerous studies presented accelerated rates of Aβ aggregation in the presence of certain divalent metal cations such as Cu2+ and Zn2+. These findings are in agreement with the usually high metal content of the senile plaques.54 In addition, a large body of evidence suggests that metal ions might participate in Aβ-induced oxidative stress through their redox cycling while bound to an Aβ peptide.55 In particular, redox-active metals (e.g., copper, iron) associated with Aβ species have been implicated in Fenton-like chemistry, causing oxidative stress and neuronal damage.56 Intriguingly, compound 7 formed complexes with both Cu2+ and Fe2+, while no appreciable complexation between 7 and Zn2+ was detected. These findings are consistent with those reported for 6.57 Notably, under the same experimental conditions, 8 was able to complex Cu2+, Fe2+, and Zn2+. This suggests the involvement of more groups of the molecule (such as the NH group of the anchoring motif CLA) in complexation rather than just the 6 moiety. Collectively, these results strongly support the hypothesis that multifunctional antioxidants specifically directed to the neuronal membrane might offer new ways of developing effective AD treatments (Figure 7). Mitochondria-Targeted Antioxidants. Another central player in AD pathogenesis is the organelle mitochondrion.58 Mitochondria provide cellular energy and are crucial for neuronal activity and survival.59 Multiple lines of evidence suggest that mitochondrial dysfunction plays a crucial role in AD onset and progression.60 This evidence comes from E



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Prototypical polyamine conjugate 11 (Figure 9) significantly decreased ROS production, induced in both submitochondrial particles and a cellular context, with an efficacy similar to that of the reference compound 6.68 In addition, the constrained curcumin analogue 10 was more efficacious than 6 in activating HO-1. Including a spermine tail in the constrained polyphenolic nucleus only slightly decreased its efficacy. These promising results suggest that the synthesized compounds are, like 6, multimodal antioxidants able to raise cell defenses by means of direct and indirect mechanisms. Moreover, as verified with proliferation studies on a series of tumor and normal cell lines, the well-retained antioxidant activity observed for the polyamine conjugates was accompanied by a significant loss of cytotoxicity with respect to the polyphenolic core, suggesting that these compounds may be promising molecules for neuroprotectant lead discovery. This concept finds support in the increased blood−brain barrier penetration observed for previously reported CNS active therapeutics, following their conjugation with naturally occurring polyamines.72 The efficient intracellular uptake and mitochondria targeting of the new compounds were assessed by inverted fluorescence microscopy, using polyamine 12 as the fluorescent probe (Figure 9). Images of T67 glioma cells pretreated with 12 and incubated in the presence and absence of a mitochondrial uncoupler strongly propose the mitochondrial potential as the driving force.68 This suggests that polyamines might offer new perspectives in addressing the considerable challenge of targeted drug delivery. Overall, it is important to further explore subcellular targeting strategies. Most interestingly, this approach could lead to an increase in drug specificity.73 In particular, low concentrations of radical species serve physiological functions and are a part of numerous cell signaling pathways.74 It is therefore conceivable that selective targeting of antioxidants

Figure 8. Structure of MitoQ (9).

selectively direct the polyphenolic compound into this subcellular compartment.68 In particular, on the basis of the numerous modifications recently performed on 6’s structure, its 3,5-dibenzylidenepiperidin-4-one analogue 10 was selected instead (Figure 9). This is because of its higher antioxidant and antiproliferative efficacies.69 This moiety also offered the chance of an improved pharmacokinetic profile.70 To direct the bioactive functionality into mitochondria passing through cell and mitochondria membranes, polyamine chains were selected as the vehicle agents. Polyamines had already been used to improve the cellular import of drug molecules.71 Being protonated at physiological pH, they offered the possibility of serving mitochondria targeting in a gradient-dependent manner. Thus, a series of polyamine-based curcumin congeners was synthesized and biologically investigated. In this context, the antioxidant profile of 6 deserves comment. Antioxidants can show their effects through various mechanisms, which are not mutually exclusive. In particular, 6 not only exhibits free-radical-scavenging properties but also protects against oxidative injury by means of indirect antioxidant mechanisms such as heme oxygenase 1 (HO-1) modulation.43 As for other antioxidant enzymes, HO-1 induction is achieved through activation of the redox-sensitive Keap1−Nrf2 signaling system, which will be examined in the following section.

Figure 9. Structures of curcumin (6), its congener (10), 10−polyamine conjugate (11), and the fluorescent probe (12). F



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could help differentiate the physiological level of oxidative species from the pathological level, resulting in a better therapeutic outcome.

The interaction between Nrf2 and its repressor Keap1 is a nodal point for regulating Nrf2 activity. Most of the known Nrf2 activators, including endogenous metabolites and natural products, function as electrophiles.78 They indirectly inhibit Keap1−Nrf2 interaction through covalent S-alkylation of critical cysteine thiols of Keap1 (Figure 10, left side). The covalent nature of the interaction raises a wide discussion on the effective druggability of electrophilic compounds, which are often considered as toxicophores. However, most of the Nrf2 activators (Michael acceptors, for example) are highly reversible. This could significantly mitigate off-target issues relative to irreversible agents.75 In recent years, numerous Nrf2 activators have been developed, and some are currently undergoing clinical investigation.79 Notable examples include dimethyl fumarate (13) and the triterpenoid bardoxolone methyl ester (CDDOMe) (14) (Figure 11).

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THE ROAD AHEAD: MECHANISM-BASED APPROACHES Regulators of antioxidant defenses are assuming consolidated importance in pursuing the challenging issue of finely tuned ROS and RNS levels. One example is transcriptional activation of antioxidant signaling cascades. The nuclear E2-related factor 2 (Nrf2) plays a pivotal role in the inducible cell defense system. It activates the expression of phase II detoxification and antioxidant enzymes including HO-1, glutathione S-transferase, and NAD(P)H:quinone oxidoreductase. Under basal conditions, Nrf2 is associated with the repressor Kelch ECH associating protein 1 (Keap1) and retained in the cytoplasm. When exposed to oxidative insults, the redox sensitive Keap1 is covalently modified at specific cysteine residues. This disrupts Nrf2 binding and in turn promotes its nuclear translocation. Once in the nucleus, Nrf2 dimerizes with members of the small Maf (sMaf) family and binds to the antioxidant response element (ARE) to stimulate the transcription of cytoprotective genes (Figure 10, left side). For a detailed discussion of the

Figure 11. Structures of electrophilic Nrf2 activators dimethyl fumarate (13) and CDDO-Me (14).

Compound 13 has been shown to be both safe and highly efficacious in the treatment of multiple sclerosis (MS).80 At the later stage of the disease, it exerts neuroprotective effects dependent on Nrf2-mediated antioxidant pathways which are relevant in a variety of CNS pathological conditions.81 Its approval by the FDA (March 2013) for MS treatment is a significant success. It corroborates the efficacy of Nrf2 activators as therapeutic agents for diseases involving oxidative stress and inflammation, encouraging further developments of this approach in the AD field. In contrast, the promising CDDO-Me 14 reached a phase III clinical study (NCT01351675) for chronic kidney disease, but recently failed (October 2012) because of adverse events. Although the nature of these unexpected results has not been disclosed, the failure raises the question of target specificity, the most complicated issue for antioxidant therapy to date. Electrophilic compounds have beneficial or detrimental impacts depending on their respective cysteine thiol targets, which are responsible for these diverse effects.82 This has recently given rise to inspiring strategies. In particular, Lipton and co-workers83 advocate the use of proelectrophilic compounds that, functioning as prodrugs, convert to electrophiles by oxidation after reaching the intended target. These drugs are specifically activated by a redox pathological state (labeled “pathologically activated therapeutics” by the same authors). They act by providing neuroprotection where it is needed. Reported examples of proelectrophiles are tert-butylhydroquinone (TBHQ) and carnosic acid (CA), which require oxidative conversion from their hydroquinone form to the quinone one before they can activate the Keap1/Nrf2 pathway (Figure 12).84 As such, these chemical features may lead to drugs that do not produce serious clinical side effects caused by interferences

Figure 10. Proposed mechanisms of Nrf2 activation. The square shapes represent the bioactive molecules inducing the Keap1−Nrf2− ARE pathway. The model represents the direct inhibition of Keap1− Nrf2 protein−protein interaction on the right side and the binding of critical cysteines on Keap1 on the left side. Both mechanisms allow for Nrf2 translocation to the nucleus, where it joins other transcription factors (e.g., Maf in yellow) and binds ARE to initiate the transcription of cytoprotective genes.

molecular mechanisms of the Keap1−Nrf2 system, the interested reader is directed to the excellent review by Moody et al. and references cited therein.75 Regulating inducible antioxidant defense moves from classical antioxidant interventions (based on the administration of free radical scavengers) to a functional/mechanistic approach. This has stimulated substantial interest in identifying Nrf2 activators as therapeutic agents for many diseases, including AD.76,77 G



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sites of ROS production offers an intriguing opportunity to seek more efficient antioxidant approaches to AD treatment. In addition, the issue of target specificity, which is one of the most complex issues for antioxidant therapy to date, is directing considerable interest toward functional/mechanistic strategies. These new approaches may compensate for the poor target specificity that characterizes most antioxidant compounds. They may also fill the remaining gap in our understanding of the exact mechanisms underlying ROS’ contribution to AD pathogenesis. The design and assessment of antioxidant treatments remain a great challenge. However, medicinal chemists have laid the foundations for selectively directing therapeutic efforts to pathological oxidative events, creating new possibilities for disease-modifying antioxidant AD cures.

Figure 12. Structures of proelectrophiles TBHQ and CA.

with normal brain function. This farsighted concept can be extended to other types of chemical structures, helping medicinal chemists develop new, well-tolerated, and effective antioxidants for AD treatment. In the effort to limit the risk of “off-targets” toxic effects, an alternative strategy for regulating Nrf2−Keap1 interaction is now emerging. The Keap1−Nrf2 interface has recently evolved as a direct molecular target of ARE activation (Figure 10, right side), offering new opportunities for the design of reversible ARE inducers with high potency and specificity.79 High resolution structural data are available, which offer the opportunity to successfully address the binding of Nrf2 to its repressor Keap1.85 Compound (SRS)15 is a small molecule hit identified by high-throughput screening as a first-in-class inhibitor of the Keap1−Nrf2 protein−protein interaction (Figure 13).86

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AUTHOR INFORMATION

Corresponding Authors

*M.R.: e-mail, [email protected]; phone/fax, +39 051 2099722/34. *C.M.: e-mail, [email protected]; phone/fax, +39 051 2099706/34 Notes

The authors declare no competing financial interest. Biographies Michela Rosini obtained her degree in Pharmaceutical Chemistry and Technology in 1997 followed by a Ph.D. in Pharmaceutical Sciences in 2001 from the University of Bologna, Italy. In 1998 and 2000, she spent some months at The Royal Danish School of Pharmacy of Copenhagen, Denmark. At present, she is Researcher (Assistant Professor) in the Department of Pharmacy and Biotechnology at the University of Bologna. Her research focuses on the design and synthesis of small molecules as probes for the investigation of biological processes or as drug candidates for neurodegenerative diseases.

Figure 13. Structure of the Keap1−Nrf2 protein−protein interaction inhibitor (SRS)15.

Elena Simoni graduated in Pharmaceutical Chemistry and Technology from the University of Bologna, Italy, in 2006 and received her Ph.D. in Pharmaceutical Sciences in 2010 from the same institution. In 2008−2009, as a part of her Ph.D. program, she was a Visiting Scholar in Arun K. Ghosh’s laboratory at Purdue University (IN, U.S.). In 2011−2012, she joined the Italian Institute of Technology (Genoa, Italy) as a Postdoctoral Fellow. At the Department of Pharmacy and Biotechnology at the University of Bologna, she is currently Temporary Research Fellow. Her research focuses on the design and synthesis of small molecules with the aim of modulating specific targets in the context of neurodegeneration.

It has been demonstrated to bind the Keap1 Kelch domain (Kd = 1 μM) and promote nuclear translocation (EC50 = 12 μM) and ARE-induced activity (EC50 = 18 μM) in cell-based functional assays. Experimental evidence confirms that inhibition of Nrf2 binding to its repressor is not mediated by covalent interaction with cysteine residues, offering perspectives for avoiding possible toxic side effects due to nonspecific cysteine modification.86 Direct inhibition of the Keap1−Nrf2 protein−protein interaction has also been demonstrated by Xray crystallography for other small molecules,87 supporting the feasibility of this approach to rationally designing Nrf2 activators.

Andrea Milelli received his degree in Pharmaceutical Chemistry and Technology in 2005 from the University of Bologna, Italy, followed by a Ph.D. in Pharmaceutical Sciences in 2009. He spent a research period at Aarhus University, Denmark, working under the supervision of Prof. Karl Anker Jørgensen. At present, he is Researcher at the Department of Life Quality Sciences at the University of Bologna. His research focuses on the design and synthesis of new agents for the treatment of multifactorial diseases.

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CONCLUSIONS Most of the antioxidants studied so far have had limited success in AD clinical trials. However, there is a clear consensus that oxidative stress is a prominent feature of AD. Redox dysregulation is thought to be crucial to the complex nature of its pathogenesis. Yet growing evidence suggests that merely hitting oxidative stress will not be enough to undermine AD architecture. In recent years, therefore, research into antioxidant therapies has embraced alternative strategies. The close relationship between ROS overproduction and other key AD features has prompted the design of antioxidants endowed with other relevant anti-AD abilities, which could in tandem contribute to confronting the disease. Targeting the main

Anna Minarini graduated in Pharmaceutical Chemistry and Technology from the University of Bologna, Italy, in 1987 and received her Ph.D. in Pharmaceutical Sciences in 1992 from the same university. In 1990−1991, she was a Visiting Scientist at the University of Buffalo, NY. In 1998, she was appointed Associate Professor of Medicinal Chemistry at the Department of Pharmaceutical Sciences of the University of Bologna. Her current research interests include the H



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(13) Fazil, M.; Shadab; Baboota, S.; Sahni, J. K.; Ali, J. Nanotherapeutics for Alzheimer’s disease (AD): past, present and future. J. Drug Targeting 2012, 20, 97−113. (14) Galasko, D. R.; Peskind, E.; Clark, C. M.; Quinn, J. F.; Ringman, J. M.; Jicha, G. A.; Cotman, C.; Cottrell, B.; Montine, T. J.; Thomas, R. G.; Aisen, P. Antioxidants for Alzheimer disease: a randomized clinical trial with cerebrospinal fluid biomarker measures. Arch. Neurol. 2012, 69, 836−841. (15) Trippier, P. C.; Jansen Labby, K.; Hawker, D. D.; Mataka, J. J.; Silverman, R. B. Target- and mechanism-based therapeutics for neurodegenerative diseases: strength in numbers. J. Med. Chem. 2013, 56, 3121−3147. (16) Zhang, H. Y.; Yang, D. P.; Tang, G. Y. Multipotent antioxidants: from screening to design. Drug Discovery Today 2006, 11, 749−754. (17) Cavalli, A.; Bolognesi, M. L.; Minarini, A.; Rosini, M.; Tumiatti, V.; Recanatini, M.; Melchiorre, C. Multi-target-directed ligands to combat neurodegenerative diseases. J. Med. Chem. 2008, 51, 347−372. (18) Bolognesi, M. L.; Matera, R.; Minarini, A.; Rosini, M.; Melchiorre, C. Alzheimer’s disease: new approaches to drug discovery. Curr. Opin. Chem. Biol. 2009, 13, 303−308. (19) Tailor, N.; Sharma, M. Antioxidant hybrid compounds: a promising therapeutic intervention in oxidative stress induced diseases. Mini-Rev. Med. Chem. 2013, 13, 280−297. (20) Weinreb, O.; Amit, T.; Bar-Am, O.; Yogev-Falach, M.; Youdim, M. B. The neuroprotective mechanism of action of the multimodal drug ladostigil. Front. Biosci. 2008, 13, 5131−5137. (21) Weinreb, O.; Amit, T.; Bar-Am, O.; Youdim, M. B. Ladostigil: a novel multimodal neuroprotective drug with cholinesterase and brainselective monoamine oxidase inhibitory activities for Alzheimer’s disease treatment. Curr. Drug Targets 2012, 13, 483−494. (22) Silman, I.; Sussman, J. L. Acetylcholinesterase: “classical” and “non-classical” functions and pharmacology. Curr. Opin. Pharmacol. 2005, 5, 293−302. (23) Bolognesi, M. L.; Minarini, A.; Rosini, M.; Tumiatti, V.; Melchiorre, C. From dual binding site acetylcholinesterase inhibitors to multi-target-directed ligands (MTDLs): a step forward in the treatment of Alzheimer’s disease. Mini-Rev. Med. Chem. 2008, 8, 960− 967. (24) Munoz-Torrero, D. Acetylcholinesterase inhibitors as diseasemodifying therapies for Alzheimer’s disease. Curr. Med. Chem. 2008, 15, 2433−2455. (25) Inestrosa, N. C.; Dinamarca, M. C.; Alvarez, A. Amyloidcholinesterase interactions. Implications for Alzheimer’s disease. FEBS J. 2008, 275, 625−632. (26) Castro, A.; Martinez, A. Targeting beta-amyloid pathogenesis through acetylcholinesterase inhibitors. Curr. Pharm. Des. 2006, 12, 4377−4387. (27) Rosini, M.; Andrisano, V.; Bartolini, M.; Bolognesi, M. L.; Hrelia, P.; Minarini, A.; Tarozzi, A.; Melchiorre, C. Rational approach to discover multipotent anti-Alzheimer drugs. J. Med. Chem. 2005, 48, 360−363. (28) Rosini, M.; Simoni, E.; Bartolini, M.; Tarozzi, A.; Matera, R.; Milelli, A.; Hrelia, P.; Andrisano, V.; Bolognesi, M. L.; Melchiorre, C. Exploiting the lipoic acid structure in the search for novel multitarget ligands against Alzheimer’s disease. Eur. J. Med. Chem. 2011, 46, 5435−5442. (29) Tirosh, O.; Sen, C. K.; Roy, S.; Kobayashi, M. S.; Packer, L. Neuroprotective effects of alpha-lipoic acid and its positively charged amide analogue. Free Radical Biol. Med. 1999, 26, 1418−1426. (30) Bertucci, C.; De Simone, A.; Pistolozzi, M.; Rosini, M. Reversible human serum albumin binding of lipocrine: a circular dichroism study. Chirality 2011, 23, 827−832. (31) Rosini, M.; Andrisano, V.; Bartolini, M.; Melchiorre, C. Tetrahydro-acridine and Dithiolane Derivatives. US7307083B2, 2007. (32) Minarini, A.; Milelli, A.; Simoni, E.; Rosini, M.; Bolognesi, M. L.; Marchetti, C.; Tumiatti, V. Multifunctional tacrine derivatives in Alzheimer’s disease. Curr. Top. Med. Chem. 2013, 13, 1771−1786. (33) Cavalli, A.; Bolognesi, M. L.; Capsoni, S.; Andrisano, V.; Bartolini, M.; Margotti, E.; Cattaneo, A.; Recanatini, M.; Melchiorre,

design and synthesis of new chemical entities against neurodegenerative diseases and cancer. Carlo Melchiorre received his degree in Chemistry from Camerino University, Italy (1966), where he was appointed Full Professor (1980). In 1988, he joined the faculty of Pharmacy at the University of Bologna, Italy, where he is presently Alma Mater Professor. He has longstanding interests in neurotransmitter receptors. His current research focuses on identifying MTDLs for the treatment of neurodegenerative disorders.

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ACKNOWLEDGMENTS We are grateful to Grace Fox for invaluable assistance with the manuscript. This work was supported by a grant from MIUR (PRIN 2009ESXPT2_001) and the University of Bologna, Italy.

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ABBREVIATIONS USED AChE, acetylcholinesterase; AChEI, acetylcholinesterase inhibitor; AD, Alzheimer’s disease; Aβ, amyloid β; APP, amyloid precursor protein; ARE, antioxidant response element; CA, carnosic acid; CM/LR, cell membrane/lipid raft; CLA, cholesterylamine; FA, ferulic acid; Keap1, Kelch ECH associating protein 1; HO-1, heme oxygenase 1; LA, lipoic acid; MAO, monoamine oxidase; MS, multiple sclerosis; Nrf2, nuclear E2-related factor 2; PAS, peripheral anionic site; RNS, reactive nitrogen species; ROS, reactive oxygen species; TBHQ, tert-butylhydroquinone

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