Alzheimer's disease, enzyme targets and drug discovery struggles: from natural products to drug prototypes.

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Contents lists available at ScienceDirect

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Alzheimer’s disease, enzyme targets and drug discovery struggles: From natural products to drug prototypes

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Tiago Silva, Joana Reis, José Teixeira, Fernanda Borges ∗ Department of Chemistry and Biochemistry, Faculty of Sciences of Porto, Porto, Portugal

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Article history: Received 20 November 2013 Received in revised form 26 March 2014 Accepted 31 March 2014 Available online xxx

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Keywords: Alzheimer’s disease Acetylcholinesterase and inhibitors ␤- and ␥-secretases and inhibitors Sirtuins and inhibitors Caspases and inhibitors Glycogen synthase kinase-3 and inhibitors Autophagy enhancers Synaptogenesis enhancers

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Alzheimer’s disease (AD) is an incapacitating neurodegenerative disease that slowly destroys brain cells. This disease progressively compromises both memory and cognition, culminating in a state of full dependence and dementia. Currently, AD is the main cause of dementia in the elderly and its prevalence in the developed world is increasing rapidly. Classic drugs, such as acetylcholinesterase inhibitors (AChEIs), fail to decline disease progression and display several side effects that reduce patient’s adhesion to pharmacotherapy. The past decade has witnessed an increasing focus on the search for novel AChEIs and new putative enzymatic targets for AD, like ␤- and ␥-secretases, sirtuins, caspase proteins and glycogen synthase kinase-3 (GSK-3). In addition, new mechanistic rationales for drug discovery in AD that include autophagy and synaptogenesis have been revised. Herein, we describe the state-of-the-art of the development of recent enzymatic inhibitors and enhancers with therapeutic potential on the treatment of AD. © 2014 Published by Elsevier B.V.

Alzheimer’s disease: an overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Classic enzymatic targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Acetylcholinesterase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: 2AADPR, 2 -O-acetylADP-ribose; 3AADPR, 3 -O-acetylADP-ribose; 6-BIO, 6-bromo-3 -oxime; Å, Ångström; ACh, acetylcholine; AChE, acetylcholinesterase; AChEIs, acetylcholinesterase inhibitors; AD, Alzheimer’s disease; ADAM, a desintegrin and metalloprotease; ADME, absorption, distribution, metabolism and excretion; APH1, anterior pharynx-defective phenotype-1; APOE4, ␧4 allele of the apolipoprotein E gene; APP, amyloid precursor protein; APPsw -Tauvlw mice, transgenic mice expressing human APP and Tau; ATP, adenosine triphosphate; A␤, ␤-amyloid protein; BACE1, ␤-site APP cleaving enzyme-1; BACEIs, ␤-site APP cleaving enzyme-1 inhibitors; BBB, blood–brain barrier; BChE, butyrylcholinesterase; BDNF, brain derived neurotrophic factors; cAMP, cyclic adenosine monophosphate; CAS, catalytic anionic site; CAT, choline acetyltransferase; CDK, cyclin dependant kinases; Chol, choline; CK1, casein kinase 1; CMGC, cyclin-dependant kinases, mitogen-activated protein kinases, glycogen synthases kinases and cyclin-dependant kinase-like kinases; CNS, central nervous system; CSF, cerebrospinal fluid; CYP, cytochrome P450; DARP-32, dopamine and cAMP regulated phosphoprotein, Mr. 32 kDa; EC50 , half maximal effective concentration; EeAChE, Electrophorus electricus acetylcholinesterase; EOAD, early-onset alzheimer’s disease; EqBChE, equine butyrylcholinesterase; ES, esteratic site; FDA, Food and Drug Administration; GS, glycogen synthase; GSK-3, glycogen synthase kinase-3; H2 O2 , hydrogen peroxyde; hAChE, human acetylcholinesterase; hBChE, human butyrylcholinesterase; HDAC, histone deacetylase; hSIRT1, human sirtuin-1; hSIRT2, human sirtuin-2; HupA, huperzine A; IC50 , half maximal inhibitory concentration; IP3 , inositol triphosphate; kDa, Kylo Dalton; LOAD, late-onset alzheimer’s disease; mAChR, muscarinic receptors; MEF, mouse embryonic fibroblasts; mESCs, mouse embryonic stem cells; MPP(+), 1-methyl-4-phenylpyridinium; mTOR, mammalian target of rapamycin; nAChR, nicotinic receptors; NAD+ , nicotinamide adenine dinucleotide; NAM, nicotinamide; NaNO2 , sodium nitrite; NFT, neurofibrilary tangles; NF-␬B, nuclear factor kappa-light-chain-enhancer of activated B cells; NLGs, neuroligins; NMDA, n-methyl-D-aspartate; nNOS, neuronal nitric oxide synthase; NPC, human neural progenitor cells; NRXs, neurexins; OKA, okadaic acid; PAF, platelet activating factor; PAS, peripheral anionic site; PD, Parkinson’s disease; PEN-2, presenilin enhancer-2; PIK, phosphoinositide kinase; PKA, protein kinase A; PKC, protein kinase C; PKPD, pharmacokinetic and pharmacodynamics; PPAR␥, peroxisome proliferator-activated receptor ␥; PrP, prion peptide; PSEN-1, presenilin-1; PSEN-2, presenilin-2; PSP, progressive supranuclear palsy; QSAR, quantitative structure–activity relationship; rAChE, rat acetylcholinesterase; RAR␣, retinoic acid receptor ␣; rSIRT1, rat sirtuin-1; rSIRT2, rat sirtuin-2; sAPP␣, soluble APP␣ peptide; sAPP␤, soluble APP␤ peptide; SIRT, sirtuins; SMERs, small molecule enhancers of rapamycin; SNP, senile neuritic plaques; SOD, superoxide dismutase; Tau, tau protein; tau-P, hyperphosphorylated tau protein; TcAChE, Torpedo californica acetylcholinesterase; TSP, thrombospondin. ∗ Corresponding author. E-mail addresses: [email protected], [email protected] (F. Borges). http://dx.doi.org/10.1016/j.arr.2014.03.008 1568-1637/© 2014 Published by Elsevier B.V.

Please cite this article in press as: Silva, T., et al., Alzheimer’s disease, enzyme targets and drug discovery struggles: From natural products to drug prototypes. Ageing Res. Rev. (2014), http://dx.doi.org/10.1016/j.arr.2014.03.008

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2.1.1. Acetylcholinesterase inhibitors in therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.2. Discovery and development of novel acetylcholinesterase inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Non-classic targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Secretases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.1. ␤-Secretase (BACE1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.2. ␥-Secretase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.3. ␣-Secretase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Sirtuins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.1. Sirtuin-2 (SIRT2) inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Caspases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.1. Caspases inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Glycogen synthase kinase-3 (GSK-3) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.1. GSK-3 inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. New mechanistic rationales for drug discovery in AD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.1. Autophagy enhancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.2. Small molecule enhancers of synaptogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Final remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Uncited reference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Alzheimer’s disease: an overview

Alzheimer’s disease (AD), a multifactorial neurodegenerative 50 disease, is single-handedly the main factor behind dysfunction 51 among persons over age 85, and the major cause of dementia in 52 old age (Larson et al., 1992). According to epidemiological surveys, 53 an estimated 7–10% of individuals over 65 and 50–60% over 85 suf54 fer from AD (Evans et al., 1989; McKhann et al., 1984), reaching 4 55Q3 approximately 35 million people worldwide. In Europe, 7.3 mil56 lions of citizens suffer from dementia. Owing to the increased life 57 expectancy this number will increase dramatically in the future, 58 escalating up to 114 million cases by 2050 (Ferri et al., 2005; 59 Brodaty et al., 2011; Berr et al., 2005; Minati et al., 2009). 60 AD patients develop a gradual and insidious cognitive deficit 61 that becomes incapacitating in the advanced stages of the disease. These devastating symptoms significantly compromise the 62 patients’ quality of life, leading to absolute dependence, hospital63 ization and, unavoidably, death (Hughes et al., 1982). The most 64 common form of Alzheimer’s is the late-onset AD (LOAD) form, 65 which accounts for approximately 95% of AD cases (Monczor, 2005). 66 Although the specific cause of AD is unknown, analyzing the risk 67 factors, age and family history in a first-degree are arguably the 68 most important for developing dementia (Aliev et al., 2008). How69 ever, the mentioned risk factors alone cannot be responsible for 70 all documented cases of Alzheimer disease. Some detected cases, 71 particularly with early onset (EOAD), are familial and inherited as 72 autosomal dominant disorder. Familial AD risk is markedly genetic 73 and, so far, four genes have been associated with AD pathology: the 74 APP (amyloid protein precursor), preselinin 1 (PSEN-1), preselinin 75 2 (PSEN-2) and the ␧4 allele of the apolipoprotein E gene (APOE4) 76 (Wollmer, 2010; Frank and Gupta, 2005). 77 Classic features found in the brains of AD patients include 78 neuronal loss in regions associated with memory and cognition, 79 particularly of cholinergic neurons, neurotransmitter depletion 80 (mainly acetylcholine, ACh) and synaptic dysfunction (Monczor, 81 2005; St George-Hyslop, 2000). Microscopically, the most common 82 findings are abnormal protein deposits, including senile neu83 ritic plaques (SNP) and neurofibrillary tangles (NFT) (Goedert and 84 Spillantini, 2006). Senile plaques are the result of the extracellular 85 accumulation of insoluble aggregates of ␤-amyloid protein (A␤) 86 while NFT occur intracellularly and are composed of paired heli87 cal filaments of hyperphosphorylated tau protein (tau-P). These 88 abnormalities lead to the activation of neurotoxic cascades and 89 to cytoskeletal changes that eventually cause synaptic dysfunc90 tion and neuronal death (Goedert and Spillantini, 2006). Protein 91 misfolding and abnormal aggregation both play a critical role in 92 49

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AD pathology, leading to the formation of insoluble pathological conformers that cause neuronal degeneration and cellular death (Sadqi et al., 2002). The main feature of AD etiology is the multiplicity of pathological stimuli associated with increased risk, disease development and progression, commonly referred to as hypothesis. Indeed, several of these hypotheses have been proposed to date, including the amyloid hypothesis (Goedert and Spillantini), cholinergic hypothesis (Craig et al., 2011), glutamatergic hypothesis (Bezprozvanny and Mattson, 2008), oxidative stress hypothesis (Pratico, 2008), metal hypothesis (Bonda et al., 2011) and the inflammatory hypothesis (Trepanier and Milgram, 2010). Current therapies with acetylcholinesterase inhibitors (AChEIs) and N-methyl-d-aspartate (NMDA) receptor antagonists are based on the cholinergic and glutamatergic hypothesis, respectively (Schmidt et al., 2008). Though active at ameliorating AD symptoms, none of the current drugs are able to modify disease progression, a fact that has provided the driving force behind the ongoing research for new and potent anti-Alzheimer compounds (Schmitt et al., 2004; Schmidt et al., 2008). Hence, the wide range of pathologic features in AD is continuously broadened and gives rise to a growing set of promising therapeutic targets. Mainly, these non-classic macromolecular targets are enzymes involved in key physiological and pathological processes that have been connected with neurotoxicity and neurodegeneration, such as secretases, sirtuins, and caspases. In this review, we provide a thorough insight on the state-ofthe-art of the development of enzymatic inhibitors and enhancers of classic and non-classic targets with therapeutic potential for AD. These new approaches drive the drug discovery in neurodegeneration toward the development of novel potent disease-modifying agents that aim at improving the quality of life of AD patients and paving the way to solving the complex and yet unresolved puzzle of AD.

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2. Classic enzymatic targets

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2.1. Acetylcholinesterase

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Cholinergic transmission has a vital role in cerebral cortical development and activity, cerebral blood flow, sleep-wake cycle, learning, memory and cognition (Rees and Brimijoin, 2003; Brimijoin, 1983). Acetylcholine is responsible for stimulating contractions of smooth muscle in the gastrointestinal tract, urinary tract and eye, as well as decreasing heart rate and relaxing the smooth muscle of blood vessels, causing vasodilation. The biological response to cholinergic stimuli is dependent on the type of


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Fig. 1. Catalytic activity of acetylcholinesterase (AChE).

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post-synaptic receptor that is activated, which can be either muscarinic receptors (mAChR) or nicotinic receptors (nAChR) (Rees and Brimijoin, 2003; Brimijoin, 1983). Acetylcholinesterase (AChE, E.C. 3.1.1.7) is a serine-protease that hydrolyses the carboxylic ester of neurotransmitter acetylcholine (ACh) to afford choline (Chol) and acetic acid (Fig. 1), thus playing a key role in cholinergic neurotransmission within the autonomic and somatic nervous system. AChE is mainly expressed in nervous tissue, neuromuscular junctions, plasma and red blood cells (Brimijoin, 1983; Heller and Hanahan, 1972; de Almeida and Saldanha, 2010). Structural insight on AChE was driven by studies on Torpedo californica (Sussman et al., 1991), which reported that the active site of the enzyme (the catalytic anionic site, CAS) is located in the bottom of a deep narrow gorge (Fig. 2), characterized by several subsites: the anionic site, where the interaction with ACh occurs, the esteratic site (ES), that contains three residues of the catalytic triad, the oxyanion hole, and the acyl pocket, which confers substrate selectivity. Another important subunit known as the peripheral anionic site (PAS) is located approximately 15 A˚ from the CAS (Dvir et al., 2010; Silman and Sussman, 2008). The hydrolytic activity of AChE inactivates ACh and serves as an essential physiological mechanism for terminating its effects.

The cholinergic neurons have been described to undergo extensive degenerative changes in AD, resulting in ACh depletion and cholinergic hypofunction, which contribute to the progressive memory deficit and cognitive decline in AD (Schliebs and Arendt, 2006, 2011). Therefore, the use of AChEIs to restore the synaptic levels of ACh and to treat symptoms caused by cholinergic imbalance represents a rational approach in AD pharmacotherapy. Additionally, it has been shown that AChE is one of the proteins that colocalizes with A␤ deposits and directly promotes A␤ assembly and aggregation into insoluble plaques, a classic biochemical hallmark of AD pathology (Inestrosa et al., 1996; Rees and Brimijoin, 2003; Lane et al., 2004). These secondary noncholinergic functions of AChE are attributed to the PAS of the enzyme’s active site. Furthermore, the neuronal and nonneuronal cholinergic systems have important roles in the modulation of regional cerebral blood flow. These findings may modify the overly simplistic cholinergic hypothesis in AD that is limited to symptomatic treatment and disregards the potential of cholinergic therapies as disease-modifying agents (Lane et al., 2004). While AChE predominates in the healthy brain, butyrylcholinesterase (BChE) is considered to play a minor role in the regulation of synaptic ACh levels. This scenario is modified in the context of AD, as the activity of AChE remains unchanged and

Fig. 2. Schematic view of the active site gorge of Torpedo californica acetylcholinesterase (Tc AChE). CAS, catalytic anionic site; PAS, peripheral anionic site.


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Fig. 3. FDA approved AChEIs for the treatment of Alzheimer’s disease.

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BChE activity progressively increases (Greig et al., 2002). The two enzymes differ in substrate specificity, kinetics and activity in different brain regions. While BChE inhibition may also be considered a valid approach to restore cholinergic function in AD (Lane et al., 2004, 2006), its role in the regulation of cholinergic transmission in humans is not yet fully understood, and AChE remains the main target within this hypothesis. Nevertheless, BChE inhibition assays are carried out in parallel with the AChE inhibition studies, to attain selectivity parameters of the compounds under study. 2.1.1. Acetylcholinesterase inhibitors in therapy Acetylcholinesterase inhibitors are used in the treatment of several neuromuscular disorders and have provided the first generation of pharmaceuticals for the treatment of AD. To date, four classic drugs that act as AChEIs have been approved by the U.S. Food and Drug Administration (FDA) to treat AD symptoms: donepezil (Aricept® ), galantamine (Razadyne® ), rivastigmine (Exelon® ) and tacrine (Cognex® ) (compounds 1–4, Fig. 3). These drugs are prescribed to treat symptoms related to memory, thinking, language, judgment and other cognitive processes. However, due to poor tolerability and unfavorable pharmacological profile, tacrine is currently no longer marketed (Francis et al., 1999). In summary, these drugs offered a significant advance in the pharmacological management of dementia, yet the search for novel and potent AChEIs to treat AD is still an ongoing endeavor and AChE remains a highly viable target for the symptomatic improvement in AD (Wilkinson et al., 2004). 2.1.2. Discovery and development of novel acetylcholinesterase inhibitors Currently, the pursuit of new chemical entities as AChEIs with increased affinity and selectivity is mainly oriented by following the lead of natural compounds with cholinesterase inhibiting properties and/or by developing dual-binding site inhibitors, able to interact with both the central and peripheral sites of the enzyme. Actually, some dual or multi-target directed compounds, blocking more than one feature of the AD pathological cascade with a single chemical entity, have been developed. Our query for new AChEIs is divided in two main categories, related to the origin of the compounds – naturally inspired or of synthetic origin. 2.1.2.1. Naturally based acetylcholinesterase inhibitors. The main compounds in this category are alkaloids present in medicinal herbs, some of which are used as alternative medicines in China and India. These compounds include huperzine A, huperzine B, berberine and their semi-synthetic derivatives. 2.1.2.1.1. Huperzine A and derivatives. Huperzine A (Hup A) (Table 1, compound 5) is a highly selective, reversible and potent AChEI found in the Chinese medicinal herb Huperzia serrata. Hup A has a higher bioavailability and potency when compared to tacrine and donepezil, while being less active toward BChE (Bai et al., 2000). It is considered the drug choice in China for the treatment

of memory disorders (Mehta et al., 2012). HupA is associated with Q4 an enhancement in cognitive, clinical, behavioral and functional status, with no significant adverse effects in AD subjects. Nevertheless, there is no irrefutable clinical support concerning its use as a drug for AD treatment. Rigorous design, randomized, multicenter, large-sample trials of Huperzine A for AD are needed to further assess the effects (Li et al., 2008). Currently, Hup A is extensively used as a scaffold for the development of new AChEIs. The AChE inhibitory activity of Hup A can be improved by the formation of imine derivatives with an additional aromatic ring, preferably with small substituents, such as methoxy or ciano groups (Table 1, compounds 6 and 7). Results show effective inhibition of hAChE within nanomolar range and no inhibitory effect on hBChE was detected. The phenyl ring enables additional interactions at the CAS, making derivates 6 and 7 more active than the parent Hup A (Yan et al., 2009). Huprines (Table 1, compounds 8–10), a series of innovative tacrine–huperzine A hybrids, have also emerged as promising cholinesterase inhibitors with potential interest for treating AD. These compounds were originally designed in an empirical way by combination of the pharmacophores of huperzine A (carbobicycle substructure) and tacrine (4-aminoquinoline substructure) (Camps et al., 2000). The introduction of a fluorine substituent at position 3 was found to be advantageous, leading to compounds 15 times more active than tacrine (e.g. Huprine Z, Table 1, compound 8). The replacement of fluorine by chlorine was found to improve the inhibitory activity, which means an affinity around 1200-fold higher than that of tacrine. Huprines Y and X (Fig. 4, compounds 9 and 10, respectively) are potent and reversible AChEIs bearing a chlorine at C3. The inhibition was found quite selective, with low activity toward hBChE a condition that reduced the potential of peripheral side effects. Both are potent and reversible inhibitors able to cross the BBB (Camps et al., 2000). 2.1.2.1.2. Huperzine B and derivatives. Huperzine B (Hup B) (Table 1, compound 11) is also a compound present in Huperzia serrata. This compound is a reversible and effective AChEI, but it is less potent than Hup A. Nevertheless8, Hup B is also used as a natural template for the development of more potent AChEIs (Mehta et al., 2012). X-ray crystallography data has shown that the secondary amine of Hup B is needed for proper interaction with the enzyme. Based on this discovery novel Hup B 16-substituted derivatives were rationally designed (Table 1, compounds 12 and 13) (Shi et al., 2009). The Hup B moiety from the 16 position is connected through a tether chain with a terminal aromatic ring favoring ␲–␲ interactions with the PAS. Compounds 12 and 13 reveal to be reversible inhibitors 481- and 1430-fold more potent, respectively, than the parent compound. Additionally, they also possess neuroprotective properties against H2 O2 -induced insults (Shi et al., 2009). Bis-huperzine B derivatives consisting of two Hup B molecules linked together by a tether carbon–nitrogen chain through the secondary amine group were also reported (Feng et al., 2005). The most potent bis-huperzine B (Table 1, compound 14) exhibited 3900fold increase in AChE inhibition and 930-fold greater selectivity for


232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285



5

Table 1 Naturally based acetylcholinesterase inhibitors: Huperzines A, B and their derivatives. Structure

Compound

IC50

5, Huperzine A

Reference(s)

hAChE (nM)

hBChE (nM)

rAChE (nM)

rBChE (nM)

260

>10,000 n.d.

n.d.

Bai et al. (2000), Li et al. (2008) and Camps et al. (2000)

n.d.

n.d.

n.d.

Yan et al. (2009)

n.d.

n.d.

6

R1 = OCH3 R2 = OH

16

7

R1 = H R2 = CN

22

8, Huprine Z

R1 = CH3 R2 = F R1 = CH3 R2 = Cl R1 = CH2 CH3 R2 = Cl

4.58

197

0.78

236

0.75

15.8

14,300

214,000 n.d.

n.d.

Shi et al. (2009)

n.d.

n.d.

29.7

572

Shi et al. (2009)

10.5

137

4.93

54,300

9, Huprine Y 10, Huprine X

11, Huperzine B

12 13

R=F X = CH R=H X= N

14, Bis-Huperzine B

n.d.

n.d.

Camps et al. (2000)

Feng et al. (2005)

hAChE, human AChE; hBChE, human BChE; rAChE, rat AChE; rBChE, ratBChE.

286 287 288 289 290 291 292 293 294 295 296 297 298

AChE vs. BChE than its parent compound. The concurrent interactions via hydrogen bonds and hydrophobic interactions of 14 with the central site, gorge and peripheral site of the enzyme are behind the increase in AChE inhibitory potency (Feng et al., 2005). 2.1.2.1.3. Berberine and derivatives. Berberine (Table 2, compound 15) is an isoquinoline alkaloid present in the roots, rhizomes and stem bark of a vast number of plants (e.g. Hydrastis canadensis, Coptis chinensis, Berberis vulgaris, Berberis aquifolium, Berberis aristata) (Kulkarni and Dhir, 2010). Berberine has multiple biological activities, ranging from immunomodulation, antimicrobial and antidiabetic to anticancer. On systemic administration, berberine can cross the blood–brain barrier and reach the central nervous system (CNS), where it not only function as an AChEI

(Kulkarni and Dhir, 2010; Imanshahidi and Hosseinzadeh, 2008; Jung et al., 2009) but also as a neuroprotective agent, by decreasing NMDA-induced excitotoxicity, central A␤ load and inflammation (Kulkarni and Dhir, 2010). This compound binds primarily to the PAS of AChE, which explains both its poor interaction with BChE (since BChE does not bear a peripheral domain) and its A␤-lowering properties. Berberine chloride improved the spatial memory in rat models of AD (Zhu and Qian, 2006) but to date no clinical evidence on human patients is available. Derivatives of berberine containing triazole-containing type substituents were designed to improve the interaction of berberin with the CAS (Shi et al., 2011). Compound 16 (Table 2) features a diisopropylamino substitution at the 4-position of the triazole ring


299 300 301 302 303 304 305 306 307 308 309 310 311



6

Fig. 4. Enzymatic processing of transmembrane APP: the non-amyloidogenic (A) and amyloidogenic pathways (B). ␣: ␣-secretase, ␤: ␤-secretase (BACE1) and ␥: ␥-secretase.

312 313 314 315 316 317 318 319 320

and was found to be both a potent AChEI and a fair inhibitor of A␤ aggregation. The berberine moiety adopts an orientation that allows ␲–␲ stacking with the residues of the PAS and the triazole moiety positioned at the CAS, displaying a face-to-face ␲–␲ stacking conformation, while the diisopropylamino moiety formed hydrophobic interactions with residues outside the CAS (Shi et al., 2011). The presence of a flexible bulk of branch-chain secondary amine favors AChE inhibition. Compound 17 (Table 2) is also a 9-substituted berberine derivative, bearing a cyclohexylamino group linked to berberin by a three

carbon spacer and 18-fold more potent than the parent compound (Huang et al., 2010). Similarly to the previously described triazole derivatives, compound 17 interacts with both the PAS, via berberine moiety, the enzyme gorge, via cation-␲ stacking with the quaternary nitrogen of berberin, and the CAS, via cation-␲ interactions with the protonated nitrogen from the cyclohexylamino group (Huang et al., 2010). Data relative to the inhibition of A␤ aggregation is not yet available, but given the compounds strong interaction with the enzyme’s PAS it is expected that 17 will be able to decrease A␤ aggregation.

Table 2 Naturally based acetylcholinesterase inhibitors: Berberin and berberin derivatives. Structure

Compound

IC50

Reference(s)

hAChE (nM)

hBChE (nM)

TcAChE (nM)

eqBChE (nM)

15, Berberin

n.d.

n.d.

374

18,200

16

n.d.

n.d.

44

6210

Shi et al. (2011)

17

n.d.

n.d.

20

4710

Huang et al. (2010)

Kulkarni and Dhir (2010), Imanshahidi and Hosseinzadeh (2008), Jung et al. (2009), Zhu et al. (2006) and Shi et al. (2011)

hAChE, human AChE; hBChE, human BChE; TcAChE, Torpedo californica AChE; eqBChE, equine BChE.


321 322 323 324 325 326 327 328 329 330



331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396

2.1.2.2. Synthetic acetylcholinesterase inhibitors. The majority of compounds in this category are tacrine-based dimers and hybrids, built to interact with the central and peripheral sites of the enzyme. Since the development of the first tacrine dimers, the search for AChEIs able to simultaneously bind to its peripheral and catalytic site has become an area of very active research. This endeavor has also been extended to other scaffolds, yielding a vast diversity of new compounds not only with AChE inhibiting properties but also with other biological activities relevant in the context of AD. The synergism of these activities provides an effective therapeutic strategy to prevent and slow down neurodegeneration, while improving cognitive function. 2.1.2.2.1. Tacrine-based dual-binding inhibitors. Bis(7)tacrine (Table 3, compound 18), the first reported dual binding site AChEI, consists of two tacrine units linked by a heptylene chain. The two ring nitrogens lay up to 18 A˚ apart, allowing each of the tacrine subunits to interact with the central and peripheral sites, with additional hydrophobic interactions between the heptylene chain and the enzyme’s gorge. Accordingly, bis(7)-tacrine proved 1000-fold more potent and 10,000-fold more selective than tacrine in inhibiting rat brain AChE (Pang et al., 1996). In pre-clinical settings, bis(7)-tacrine showed low toxicity and excellent efficacy for improving cognitive deficits in several animal models (Li et al., 2009a,b). Additionally, this compound displays remarkable neuroprotective activities in vitro and in vivo: it prevents glutamate-induced neurotoxicity by moderately blocking NMDA receptor and selectively inhibition of neuronal nitric oxide synthase (nNOS), and attenuates ␤-amyloid induced neuronal apoptosis (Li et al., 2009a,b). Tacrine-donepezil hybrids (Table 3, compounds 19 and 20) were designed to simultaneously interact with the active, peripheral and midgorge binding sites of AChE (Camps et al., 2008). An ethylene (19) or propylene (20) tether connects the 5,6-dimethoxy2-[(4-piperidinyl)methyl]-1-indanone moiety of donepezil to the 6-chlorotacrine unit. The latter unit is firmly bound to the CAS, by aromatic stacking, hydrogen bonding via quinoline nitrogen (which becomes protonated at physiological pH) and the interaction of 6-chlorine with a hydrophobic pocket of the CAS (Camps et al., 2008). As observed with huprines Y (9) and X (10), 6-chlorine greatly enhances inhibition potency when compared to the parent and 6-unsubstituted compounds. The linker is aligned along the gorge and features relevant interactions with the enzyme formed by the piperidine ring, which are enhanced with the longer propylene tether of 20. Finally, the indanone ring is stacked onto the aromatic residues of the peripheral site and the carbonyl group of the indanone forms water-mediated contacts with other peripheral residues (Camps et al., 2008). Combined, this network of interactions provides highly potent inhibitors of human AChE, exhibiting IC50 values in the subnanomolar range. Additionally, it was found that compounds 19 and 20 exhibit significant A␤ anti-aggregating activity, making them promising therapeutic agents for AD. Other tacrine-based inhibitors were developed linking a 6chlorotacrine unit with an indole moiety connected by a tether with appropriate length bearing a carbamate group (Munoz-Ruiz et al., 2005). These tacrine-indole hybrids (Table 3, compounds 21 and 22) have a great potency displaying IC50 values within the picomolar range toward human AChE. Similarly to what was observed with other tacrine-based hybrids, the 6-chlorotacrine moiety is firmly stacked to the CAS, and the indole moiety interacts with the peripheral residues; however, the introduction of an amide group in the tether defines a network of interactions that couple several residues of the enzyme gorge and different structural units of the inhibitor, thus explaining the noteworthy reliance of the drug potency on the position of this group (Munoz-Ruiz et al., 2005). Outstandingly, compounds 21 and 22 inhibit A␤ aggregation in the presence and absence of AChE. Combined, these properties render interesting

7

pharmacological profiles of promising disease-modifying agents for AD. Similar encouraging results have also been obtained by developing a family of tacrine–huprine Y hybrids (Table 3, compounds 23 and 24), yielding very potent inhibitors of hAChE with IC50 values in the subnanomolar range (Galdeano et al., 2012). Compound 23 is roughly equipotent to huprine Y and around 400-fold more potent than tacrine, while compound 24 approximately 2- and 1000-fold more potent than parents huprine Y and tacrine, respectively. In these compounds the 6-chlorotacrine unit is binding with the peripheral site rather than the central site of the enzyme, which is filled by the huprine Y moiety, while the tether chain interacts with the enzyme’s midgorge. Both compounds are able to cross the BBB and display anti A␤ aggregation properties. Furthermore, at 5 ␮M compound 23 is able to inhibit ␤-secretase, the enzyme responsible for A␤ synthesis. Additionally, compounds 23 and 24 inhibit prion peptide (PrP) aggregation, which is also associated with CNS pathology. These compounds are promising lead with potential disease-modifying properties (Galdeano et al., 2012). Other research teams designed hybrid drugs with distinct pharmacological activities. This is the case of tacripyrines (Table 3, compound 25), which are tacrine–dihydropyridine hybrids developed as multi-target directed drugs for AD (Marco-Contelles et al., 2009). Compound 25 combines the anti-AChE properties of tacrine with the l-type Ca2+ channel antagonism of nimodipine. Calcium antagonism is relevant since Ca2+ overload is the main factor that triggers the processes that lead to cell death. Furthermore, calcium dysfunction increases A␤ formation and tau hyperphosphorylation. Compound 25 is a selective and potent non-competitive AChE inhibitor with an IC50 within the nanomolar range, binding mainly to the PAS via ␲-stacking interactions, hydrogen bonds with the NH group and stacking of the 4-methoxyphenyl group. Moreover, compound 25 is able to cross the BBB and displays a remarkable neuroprotective effect against both Ca2+ overload and oxidative stress. These findings indicate that tacripyrines are interesting new chemical identities with potential therapeutic interest (MarcoContelles et al., 2009). Another example of multi-target directed drugs are compounds 26 and 27 (Table 3), hybrids of tacrine and naturally occurring antioxidants ferulic acid (Pi et al., 2012) and caffeic acid (Chao et al., 2012), respectively. The AChE (Torpedo californica – TcAChE) inhibitory data allow concluding that compound 26 present an IC50 in the low nanomolar range and that compound 27 is significantly less potent. Besides inhibiting A␤ aggregation, compound 26 significantly improves cognition while increasing choline acetyltransferase (CAT) and superoxide dismutase (SOD) activity (Pi et al., 2012), gathering the requisites to be classified as a multi-target directed drug with potential interest in AD. 2.1.2.2.2. Miscellaneous acetylcholinesterase inhibitors. PMS777 (Table 4, compound 28) is a tetrahydrofuran derivative AChEI with an additional anti platelet activating factor (anti-PAF) antagonist activity (Li et al., 2007). PAF is potent pro-inflammatory mediator that can induce chemotaxis of microglia and up-regulate pro-inflammatory citokyne expression, hence exacerbating inflammatory brain damage and further contributing to AD pathology (Li et al., 2007, 2009a,b). A single compound which both inhibits AChE activity and blocks PAF-induced inflammatory response may provide therapeutic advantages over single-acting agents. The trimethoxyphenyl group is responsible for the anti-PAF activity, while the quaternary ammonium ion enables the interaction with AChE and its inhibition. Additionally, PMS777 inhibits A␤-induced neuroinflammation and apoptosis (Li et al., 2007, 2009a,b) while modulating amyloid metabolism toward a lower central A␤ load (Yang et al., 2009). BZYX (Table 4, compound 29) is a donepezil-rivastigmine hybrid designed as a dual-binding site inhibitor, characterized


397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462



8

Table 3 Tacrine-based dual binding-site acetylcholinesterase inhibitors. Structure

Compound

IC50

A␤ aggr. inhibition (%)

Reference(s)

hAChE (nM)

hBChE (nM)

EeAChE (nM)

eqBChE (nM)

0.81

5.66

0.4

390

68

Pang et al. (1996), Li et al. (2009a,b) and Bolognesi et al. (2010)

R = Cl n=2 R = Cl n=3

0.67

1.36

n.d.

n.d.

46.1

0.27

66.3

Camps et al. (2008)

21

X = (CH2 )6

0.07

2.9

n.d.

n.d.

49

22

X = (CH2 )7

0.02

0.1

MunozRuiz et al. (2005)

23

X = CH2

0.74

51.3

24

X = N-CH3

0.31

51.3

105

>10,000 n.d.

n.d.

34.9

MarcoContelles et al. (2009)

n.d.

4.4

6.7

50.27

Pi et al. (2012)

300

29,300

18, Bis(7)-tacrine

19 20

25

26

27

R1 = H R2 = OCH3 n=6 R1 = Cl R2 = H n=3

65

n.d.

n.d.

30.9 38.6

Galdeano et al. (2012)

Chao et al. (2012)

hAChE, human AChE; hBChE, human BChE; EeAChE, Electrophorus eletricus AChE; eqBChE, equine BChE.




9

Table 4 Other synthetic acetylcholinesterase inhibitors. Structure

Compound

IC50

Reference(s)

hAChE (nM)

hBChE (nM)

rAChE (nM)

rBChE (nM)

28, PMS777

n.d.

n.d.

2420

4730

29, BZYX

n.d.

n.d.

58

235,000

Li et al. (2007, 2009a,b) and Yang et al. (2009)

Zhang et al. (2009a,b)

hAChE, human AChE; hBChE, human BChE; rAChE, rat AChE; rBChE, rat BChE.

463 464 465 466 467 468 469 470 471 472

by combining the 5,6-dimethoxyindan-1-one from donepezil and the dialkylbenzylamine from rivastigmine (Zhang et al., 2009a,b). BZYX exhibits high and selective anti-AChE activity and shows a comparable in vivo effect to donepezil and rivastigmine on cognitive function tests. Furthermore, it remarkably improves memory impairment induced by ethanol, NaNO2 , scopolamine and H2 O2 in rodents, presenting significant potential as a neuroprotective and cognitive enhancing agent (Zhang et al., 2009a,b). Combined, BZYX sums up a pharmacological profile of a potential candidate for AD therapy.

473

3. Non-classic targets

474

3.1. Secretases

475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504

An abnormality in proteolytic processing of APP, which results in increased production and accumulation in the brain of neurotoxic forms of A␤, plays a pivotal role in dysfunction and death of neurons in Alzheimer disease (Mattson, 2004). According to the amyloid hypothesis, the oligomeric form of A␤ peptide is the main cause of neuronal death in AD (Imbimbo, 2008). A␤ is a natively unfolded protein and can misfold and assemble into a variety of aggregate forms, including several oligomeric species, some of which very neurotoxic. A␤ is generated by proteolytic processing of the transmembrane APP (Boddapati et al., 2011). APP is an integral membrane protein with a single membrane-spanning domain, a large extracellular glycosylated N-terminus and a shorter cytoplasmic C-terminus, which is ubiquitously expressed, being the amount produced influenced by the developmental and physiological state of the cells (Mattson, 2004). The three main enzymes that affect processing of APP are called secretases (␣-, ␤-, and ␥-secretases). APP can be processed through a non-amyloidogenic pathway (Fig. 7A) wherein the enzyme ␣secretase first cleaves APP, releasing the soluble APP␣ peptide (sAPP␣) fragment, and the shorter membrane-bound C-terminal fragment (C83) (Boddapati et al., 2011). C83 is further cleaved by ␥-secretase, producing the non toxic p3 and C59 fragments (Willem et al., 2009). In the amyloidogenic processing pathway (Fig. 7B), APP is first cleaved by the enzyme ␤-secretase (or ␤-site APP cleaving enzyme 1, BACE1), releasing a large soluble fragment called sAPP␤ (Boddapati et al., 2011). The remaining 99 aminoacid C-terminal fragment of APP (C99) is further processed by ␥-secretase to produce A␤40/42 in endocytic compartments (Mattson, 2004). A␤40 is the more frequent but A␤42 has higher propensity to aggregate and is greatly enriched in amyloid plaques (Mangialasche et al., 2010).

Current drug development in AD is mainly driven by the amyloid hypothesis. As secretases are responsible for the production of amyloid-␤ peptide, limiting amyloidogenic processing of APP by inhibiting BACE1 or promoting non-amyloidogenic processing by increasing ␣-secretase activity is a promising therapeutic strategy for treating AD. Heterogeneous proteolysis by ␥-secretase, which leads to the formation of A␤ peptides prone to aggregation, is also an interesting disease-modifying approach for AD treatment. 3.1.1. ˇ-Secretase (BACE1) BACE1 (E.C. 3.4.23.46) is a 501-amino-acid protein, member of the pepsin-like family of aspartyl proteases and ubiquitously expressed, particularly in the brain and pancreas. High BACE1 enzymatic activity was found in human brains extracts, evidence that is consistent with the highest levels of A␤ found in neurons (Willem et al., 2009). In neurons, it is transported to axonal membrane surfaces and it is mainly localized in pre-synaptic terminals. BACE1 is a type I membrane protein that contains the characteristic dual active site motif (D-T/S-G-T/S) of aspartic proteases in its ectodomain (Willem et al., 2009). The crystal structures (Ghosh et al., 2008) (Fig. 5) allow verifying that the active site is a long cleft for substrate recognition, with two catalytic aspartic residues positioned at the site of bond hydrolysis. Along the active-site cleft are side-chain pockets that interact with amino acid residues of the substrates to correctly position the substrates for hydrolysis (Ghosh et al., 2008). BACE1 has maximal activity at acid pH, namely, in the acidic subcellular compartments of the secretary pathway, including Golgi apparatus and endosomes (Vassar, 2002). As BACE1 proteolysis is the rate limiting activity in A␤ production, this enzyme is a potential target for antiamyloidogenic drugs (Stockley et al., 2006). The development of BACE1 inhibitors (BACEIs) is challenging as this enzyme has many substrates (e.g. Notch receptor) and a wide substrate-binding domain. Thus, complete blockage of BACE1 activity by BACEIs may cause unpredicted side effects (De Strooper et al., 2010). Furthermore, drugs targeting BACE1 must cross the BBB and achieve therapeutic levels within the CNS (Willem et al., 2009; Mangialasche et al., 2010). AsBACE1-mediated proteolysis is an intracellular process, within endosomes or endoplasmatic reticulum and Golgi apparatus, the inhibitors must have good drug-like absorption, distribution, metabolism and excretion (ADME) properties to effectively penetrate subcellular membranes and reach the sites where A␤ is produced (Ghosh et al., 2008, 2012). 3.1.1.1. ˇ-Secretase inhibitors. Several approaches have been undertaken to find effective human BACE1 inhibitors (hBACEIs) (Turner et al., 2004). One potent inhibitor, OM99-2 (Table 5,


505 506 507 508 509 510 511 512

513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545

546 547 548

G Model ARR 513 1–30 10


Fig. 5. 3D crystal structure of BACE1 as reported by Hong et al. (2000).

549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579

580 581 582 583 584 585 586

compound 30) is a peptide (Glu–Val–Asn–Leu–Ala–Ala–Glu–Phe) in which the bond between Leu and Ala was replaced by an isostere hydroxyethylenic transition-state (Ghosh et al., 2008). The cocrystallized inhibitor/secretase structure shows strong hydrogen bonding, namely between the OH of a hydroxyethylene transition state analog and the catalytic aspartic acids (Sathya et al., 2012). An isosteric replacement by a hydroxymethylcarbonyl function as a transition-state mimic lead to the development of a potent BACE1 inhibitor KMI-429 (in vitro IC50 = 3.9 nM) (Table 5, compound 31), which reduces brain A␤ production in both wild-type and APP transgenic mice (Asai et al., 2006). GSK 188909 (Table 5, compound 32) is a small non-peptidic compound developed from substrate-based design that displays a cellular IC50 of 5 nM. It presents excellent selectivity over other aspartyl proteases and can reduce brain A␤ levels in mice (Luo and Yan, 2010; Hussain et al., 2007). A new orally effective 4phenoxypyrrolidine-based BACEI (Table 5, compound 33) was developed having good selectivity toward BACE1 (Ki = 0.7 nM) and appropriate PKPD properties (Luo and Yan, 2010; Iserloh et al., 2008). Incorporation of a 3-methoxybenzyl derivative as the P2 -ligand provided a very potent inhibitor, GRL-8234, which exhibited a Ki of 1.8 nM (Table 5, compound 34). GRL-8234 presents a remarkable cellular BACE inhibitory activity with an IC50 of 1 nM and promising effects on cerebrospinal fluid (CSF) and plasma A␤ levels in transgenic mice models (Chang et al., 2011). CTS-21166 (Table 5, compound 35), which reduces brain A␤ levels by over 35% and plaque load by 40%, is the only ␤-secretase inhibitor that has passed phase I clinical trials. It was well tolerated and reduces effectively the plasma A␤ concentrations in healthy volunteers (Mangialasche et al., 2010). 3.1.2. -Secretase ␥-Secretase is a multi-subunit aspartyl protease that cleaves APP in the last metabolic step and generates A␤ peptide, among other products (De Strooper et al., 2010). ␥-Secretase is a protein complex composed by PSEN-1 and -2, nicastrin, anterior pharynx-defective phenotype-1(APH-1) and presenilin enchancer 2 (PEN-2) (Fig. 10). It seems consensual that the PSEN-1 and -2 are intrinsically

related with amyloidogenic processing of APP, forming the catalytic subunit of the enzyme (Turner et al., 2004). On the other hand, nicastrin, APH-1 and PEN-2 seem to be associated in the maturation and stabilization of the complex (De Strooper et al., 2010). The catalytic mechanism is similar to that of classical aspartic proteases Q5 (Turner et al., 2004) (see Fig. 6). Development of ␥-secretase inhibitors presents challenges similar to those named for BACEIs due to the interference with the metabolization non-amyloid substrates, which results in undesired side effects (e.g. hematological and gastrointestinal toxicity, skin reactions and changes in hair color) (Mangialasche et al., 2010). As observed with BACEIs, involvement with the Notch signaling pathway is a major source of collateral damage (Mangialasche et al., 2010). The diverse composition of the subunits of the ␥-secretase complex indicates that they can be differentially inhibited, potentially leading to efficacy against APP cleavage but also an acceptable adverse effect profile by sparing other functions of the complex (De Strooper et al., 2010; Woodward, 2012). 3.1.2.1. -Secretase inhibitors. ␥-Secretase is a valid target in AD therapy due its involvement in the regulation of the A␤ formation. Although bioavailable and brain-penetrant, the toxicity associated with ␥-secretase inhibition remains a major hindrance in this pharmacological approach (De Strooper et al., 2010). DAPT was the first dipeptidic compound with ␥-secretase inhibitory activity tested in vivo (Table 6, compound 36) (Wolfe, 2008; Dovey et al., 2001). This compound inhibits A␤ production in cells (IC50 = 115 nM), but high dosage has been found to be required to lower A␤ in the brains of young APP transgenic mice (Lanz et al., 2003). DAPT has also been found to be an inhibitor of Notch signaling pathway (Wolfe, 2008). Furthermore, the PK profiling revealed potential acid instability of the t-butyl ester group. To overcome the pharmacokinetic constrains a series of amino imidazoles based on DAPT were synthesized: compound 37 (Table 6) emerged as a promising candidate exhibiting a robust cellular potency as well as significant reduction of brain, plasma and CSF A␤ peptides (Brodney et al., 2011). LY-450139 or semagacestat (Table 6, compound 38) is a nonselective ␥-secretase inhibitor that can inhibit APP and Notch


587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604

605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624



11

Table 5 BACE1 inhibitors. Structure

Structure not available

625 626 627 628 629 630 631 632 633 634 635

cleavage (APP IC50 = 15 nM and Notch EC50 = 49 nM) (Imbimbo, 2008). In phase III trials, the drug showed no considerable effect on brain plaque deposition in chronic studies in transgenic mice expressing mutated human APPV717F (PDAAPP mice). In humans, any major reduction was seen in CSF A␤ levels and also no differences were seen in cognitive or functional measures between placebo- and LY-450139-treated patients (Imbimbo, 2008; Mangialasche et al., 2010). The major concern regarding this nonselective ␥-secretase inhibitor is its Notch-related adverse effects (De Strooper et al., 2010), including atrophy of the thymus and decrease in lymphocytes.

Compound

hBACE1

Reference(s)

Ki (nM)

Cell free IC50 (nM)

Cellular IC50 (nM)

30, OM99-2

1.60

n.d.

n.d.

Ghosh et al. and Sathya et al. (2012)

31, KMI-429

n.d.

3.90

n.d.

Asai et al. (2006) and Luo and Yan (2010)

32, GSK 188909

n.d.

n.d.

5.00

Luo and Yan (2010) and Hussain et al. (2007)

33

0.70

n.d.

21

Luo and Yan (2010) and Iserloh et al. (2008)

34, GRL-8234

1.80

n.d.

1.00

Chang et al. (2011)

35, CTS-21166

n.d.

n.d.

1.20–3.60

Mangialasche et al. (2010)

To overcome the above-mentioned toxicity issues, a second generation of ␥-secretase inhibitors was developed, the Notch-sparing ␥-secretase inhibitors (Portelius et al., 2010). BMS-708163 (Table 7, compound 39) is a potent Notch-sparing ␥-secretase inhibitor that shows a 193-fold selectivity for A␤40 vs. Notch cleavage (A␤40 IC50 = 0.3 nM and Notch EC50 = 58 nM) (Imbimbo, 2008). Consequently BMS-708163 is able to decrease brain and CSF A␤40 levels without causing Notch-related adverse effects. In healthy young subjects it is well tolerated and decreases, dose-dependently, A␤40 levels in CSF with a peak inhibition of approximately 55% (Mangialasche et al., 2010; De Strooper et al., 2010). PF-3084014


636 637 638 639 640 641 642 643 644 645 646

G Model ARR 513 1–30 12


3.1.3.1. ˛-Secretase enhancers.

Fig. 6. Representation of the ␥-secretase complex. ␥-Secretase is composed by four different integral proteins: presenilin (PS), presenilin enhancer 2 (PEN-2), anterior pharynx-defective phenotype-1 (APH-1) and nicastrin.

647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663

664 665

(Table 7, compound 40) is a Notch-sparing ␥-secretase inhibitor with high selectivity for APP that appears to be a potent, noncompetitive but reversible inhibitor of human ␥-secretase activity (IC50 = 6.2 nM) (Imbimbo, 2008). In young Tg2576 transgenic mice PF-3084014 promoted a dose-dependent reduction in plasma and brain A␤ concentrations (Mangialasche et al., 2010). PF-3084014 has been tested in human but no clinical data has been released. GSI-953 (begacestat) (Table 7, compound 41) is a potent ␥secretase inhibitor, 15-fold more selective toward inhibiting APP cleavage vs. Notch cleavage. GSI-953 inhibits A␤ production in both cellular (A␤42 IC50 = 15 nM) and cell-free (IC50 = 8 nM) assays. In Tg2576 mice, high doses of GSI-953 reduced A␤41 levels in brain, CSF and plasma. In addition, GSI-953 was found to be able to reverse contextual memory deficits (Imbimbo, 2008). Begacestat has shown promising results in phase I clinical trials and is currently under clinical evaluation for AD treatment (De Strooper et al., 2010; Hopkins, 2012). 3.1.3. ˛-Secretase ␣-Secretase mediated proteolysis of APP is non-amyloidogenic and prevents A␤ formation (Woodward, 2012). Its activity is

mediated by a series of membrane-bound proteases, members of the ADAM (a desintegrin and metalloprotease) family (De Strooper et al., 2010). ADAM-10, ADAM-17 and ADAM-9 have been proposed as ␣-secretases. All proteases are type I integral membrane proteins and have multi-domain structure (Endres and Fahrenholz, 2012; Hooper and Turner, 2002). The precise identity and involvement of each ␣-secretase to the catalytic activity in the brain has not been entirely unveiled. The development of this field has been hindered by the lack of selective ␣-secretase inhibitors and the frequently observed lethality upon ablation of ␣-secretase encoding genes (De Strooper et al., 2010). Thus, an alternative and indirect method of promoting ␣-secretase activity may be the stimulation of one or more of the signal pathways involved in its regulation. Upregulation of ␣-secretase activity can decrease A␤ formation and increase the production of sAPP␣, which is potentially neuroprotective (Mangialasche et al., 2010). On the other hand, the outcome of chronically upregulating ␣secretase-mediated cleavage of other substrates is still undefined (De Strooper et al., 2010). In consequence, the development of ␣-secretase enhancers remains an innovative yet undisclosed alternative.

Table 6 ␥-Secretase inhibitors. Structure

Compound

␥-Secretase

Reference(s)

Ki (nM)

Cell free IC50 (nM)

Cellular IC50 (nM)

36, DAPT

1.60

n.d.

115

Wolfe (2008), Dovey et al. (2001) and Lanz et al. (2003)

37

n.d.

1.10

0.40

Brodney et al. (2011)

38, LY-450139 or Semagacestat

n.d.

15

n.d.

Imbimbo (2008), Mangialasche et al. (2010) and De Strooper et al. (2010)


666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686



13

Table 7 Notch-sparing ␥-secretase inhibitors. Structure

687 688 689 690 691 692 693 694 695 696 697 698 699 700

701

702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719

␥-Secretase

Notch pathway

Ki (nM)

Cell free IC50 (nM)

EC50 (nM)

39, BMS-708163

n.d.

n.d.

58

Imbimbo (2008), Mangialasche et al. (2010) and De Strooper et al. (2010)

40

n.d.

6.20

n.d.

Imbimbo (2008)

41, GSI-953 or Begacestat

15

8

n.d.

Imbimbo (2008)

Compound

Etazolate or EHT-0202 (Table 8, compound 42) is a compound that stimulates neuronal ␣-secretase and increases sAPP␣ production (Marcade et al., 2008). This compound has been recently tested in a phase II study in patients with mild-to-moderate Alzheimer disease, displaying precognitive activity (Desire et al., 2009). Bryostatin-1 (Table 8, compound 43) is a macrocyclic lactone that can stimulate ␣-secretase activity by activating protein kinase C (PKC) and promoting sAPP␣ secretion (Etcheberrigaray et al., 2004). Bryostatin-1 is undergoing phase II clinical studies to evaluate its safety in mild-to-moderate AD patients. Exebryl-1 (Table 8, compound 44) modulates ␣- and ␤secretase activity, which cause a significant reduction in A␤ peptide production and accumulation in the mouse brain, with consequent memory improvements (Snow et al., 2009).

3.2. Sirtuins Sirtuins (SIRT) are a family of nicotinamide adenine dinucleotide (NAD+ )-dependant protein deacetylases that function as key regulators of multiple pathways associated with the life span and the overhealth of several organisms, from simple bacteria to complex mammal organisms. Seven sirtuins (SIRT1-7), which are categorized by their highly conserved central NAD+ -binding and catalytic domain, have been described in humans (Haigis and Sinclair, 2010). Each enzyme presents a distinct biological activity profile, due to their substrate specificities, subcellular localization and expression pattern (Haigis and Sinclair, 2010). With the exception of SIRT4, all the sirtuins catalyze a unique deacetylation in which NAD+ is consumed as a co-substrate, yielding the deacetylated substrate protein and NAD+ -cleavage products nicotinamide (NAM) and 2 O-acetylADP-ribose (2AADPR) (Fig. 7A) (Haigis and Sinclair, 2010; Lavu et al., 2008). The latter can undergo a non-enzymatical acetyltransfer reaction originating the 3 -O-acetyl isomer (3AADPR) (Lavu et al., 2008). Additionally, SIRT4 and SIRT6 can act as ADP-ribosyl transferases, linking the ADP-ribosyl moiety of NAD+ to its protein

Reference(s)

substrate and yielding the ADP-ribosyl product and NAM (Fig. 7B) (Lavu et al., 2008). Since the original studies in yeast, significant advance has been made. There is increasing data on the physiological role of sirtuins and their involvement in disease-relevant pathways, such as cell-cycle regulation, oxidative stress resistance, insulin secretion, mitochondrial energetic and inflammation. Accordingly, sirtuins have been associated with age-related disorders, namely type II diabetes mellitus, cardiovascular disease, cancer and neurodegenerative disorders (Haigis and Sinclair, 2010; Lavu et al., 2008; Donmez, 2012). The biological implications of sirtuins are mainly related to the type of substrates they act upon, which can be categorized in three main groups: transcriptional regulating, apoptosis regulating and metabolic regulating (Sauve et al., 2006). In particular, SIRT2 deacetylates histone proteins in the nucleus acting also upon non-histone cytolosic substrates like ␣-tubulin, FOXO-1 and p63 (Haigis and Sinclair, 2010; Lavu et al., 2008; Donmez, 2012). Studies in mice show that SIRT2 inhibition increased acetylated ␣-tubulin, reduced hyperphosphorylated tau protein and restored cognition in AD transgenic mice. Additionally, SIRT2 inhibition displayed in vitro and in vivo neuroprotective effects in models of Parkinson’s and Huntington’s disease (Suzuki et al., 2012). Furthermore, SIRT2 has been shown to accumulate in the aging brain (Maxwell et al., 2011). The crystal structure of the catalytic core of human SIRT2 (hSIRT2) (Fig. 8) is composed by 323 aminoacids and exhibits a NAD-binding domain, a smaller domain composed of a helical module and a zinc-binding module and a conserved large groove, at the interface of the previously described domains; intersecting the large groove there is a pocket lined by hydrophobic residues (Finnin et al., 2001).

3.2.1. Sirtuin-2 (SIRT2) inhibitors Among human sirtuins, SIRT1 and SIRT2 remain the most studied to date. Though significant, the study of sirtuin biology within


720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750

751 752 753

G Model ARR 513 1–30 14


Table 8 ␣-Secretase enhancers. Structure

Structure not available

754 755 756 757 758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791

Compound

Reference(s)

42, Etazolate or EHT-0202

Marcade et al. (2008) and Desire et al. (2009)

43, Bryostatin-1

Etcheberrigaray et al. (2004)

44, Exebryl-1

Snow et al. (2009)

the complexity of the human organism is still in its infancy and ongoing research is yet to determine the specific pathways that intertwine sirtuin-mediated activity and several disease mechanisms. Moreover, the lack of substrate-bound crystal structures of SIRT2 is a drawback in the drug design of new candidates for these targets (Huhtiniemi et al., 2011). Hence, the development of sirtuin activators or inhibitors will provide additional insight on sirtuin biology in health and disease state and will help to elucidate how SIRTs interact with diverse type of ligands. There is solid evidence that associate SIRT2 to neurodegeneration and neurodegenerative diseases, therefore the art of human SIRT2 inhibitors will be reviewed. The most widely known inhibitor of SIRT2, AGK2 (Table 9, compound 45), has an estimated IC50 for SIRT2 of 3.5 ␮M (Outeiro et al., 2007), representing a significant increase over the low selective SIRT2 inhibitors that had been previously described, like salermide (Lara et al., 2009) and EX-527 (Napper et al., 2005) (Table 9, compounds 46 and 47, respectively). AGK2 displays slight inhibition of SIRT1 and SIRT3 at concentrations over 40 ␮M, being more than 15-fold more selective for SIRT2. AGK-mediated SIRT2 inhibition reversed ␣-synuclein toxicity and altered inclusion morphology in a cellular model of Parkinson’s disease (PD) (Outeiro et al., 2007). Compounds 48 and 49 (Table 9) are bis(indolyl)maleimides developed as adenosine mimetic PKC-␤ inhibitors. Given the overlap of the structures of adenosine triphosphate (ATP, the substrate of PKC-␤) and NAD+ (SIRT co-substrate), due to the fact they both bear an adenosine moiety, it was hypothesized that PKC-␤ inhibitors might have anti-SIRT activity. Hence, these compounds were screened for their SIRT inhibition potential (Trapp et al., 2006). Indeed, they were potent inhibitors of SIRT1 and SIRT2. Although compound 49 is a more potent SIRT2 inhibitor, compound 48 displays better selectivity toward SIRT2 vs. SIRT1. Additionally, compound 49 inhibits SIRT2 in vivo and induces hyperacetylation of tubulin. Cambinol derivates are a class of SIRT inhibitors that also display antitumor activity in preclinical models. New N1-alkyl substituted derivates (Table 9, compound 50) led to increased potency and selectivity (Medda et al., 2009). The N-butyl moiety of 50 forms

hydrophobic interactions with a narrow lipophilic channel in the SIRT2 active site, an explanation that can justify the compound’s selectivity (SIRT2 vs. SIRT1). Cell-based assays are consistent with the in vitro data, showing that compound 50 increased levels of acetylated ␣-tubulin (Medda et al., 2009). 2-Anilinobenzamide analogs, such as compounds 51 and 52 (Table 9), are potent and selective SIRT2 inhibitors, displaying IC50 values in the low (51) and submicromolar range (52) (Suzuki et al., 2012). Preliminary SAR studies and molecular modeling indicated that the phenethyl group of 51 and 52 is critical for the SIRT2 selectivity and that the ethylene tether is important for the potency. Furthermore, compound 51 promoted a selective increase of ␣tubulin acetylation, proving itself as a cell-active SIRT2-selective inhibitor (Suzuki et al., 2012). AK-7 (Table 9, compound 53) is a halogenated benzamide derivate that exhibit in vitro SIRT2 selective inhibitory activity (Taylor et al., 2011). AK-7 is a potent brain permeable SIRT2 inhibitor that downregulates cholesterol biosynthesis and reduces total cholesterol levels in primary striatal neurons. This effect was also observed in cultured naïve neuronal cells and brain slices of wild-type mice. Although compound 53 presents several drawbacks related to metabolism and stability, it is now regarded as a lead compound to the development of drugs for the treatment of human illnesses that feature cholesterol homeostasis deregulation, which is the case of ND and, particularly, AD (Fonseca et al., 2010). Another rational design strategy for the discovery of SIRT2 inhibitors pursued by Pesnot et al. (2011) led to the development of a series of novel 8-aryl substituted NAD derivatives with activity against human SIRT2. The most active of the synthetic nucleotides was compound 54 (Table 9), due to the presence of a 4-chlorine substituent. Although in vivo data is not yet available, it is already known that these synthetic nucleotides seem to adopt an anticonformation when bound to the enzyme, similarly to what is observed to the NAD+ co-substrate (Pesnot et al., 2011). This conformational preference appears to interfere with cofactor binding at SIRT2, which may underpin their isoform selectivity and provides a starting point for the rational development of new synthetic NAD analogs.


792 793 794 795 796 797 798 799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825 826 827 828 829



15

Fig. 7. (A) SIRT-catalyzed deacetylation of acetylysine protein residues; (B) ADP-rybosyl tranferase activity of SIRT4 and SIRT6.

830 831 832 833 834 835 836 837 838 839 840 841 842 843 844

The chromone and chromane scaffold provides a valid support for the rational design and development of novel SIRT2 inhibitors (Friden-Saxin et al., 2012). A series of chromone/chromane derivatives display SIRT2 inhibitory activity within the low micromolar range. SAR studies disclosed important features for chromone/chromane-based SIRT2 inhibitors: an alkyl chain with three to five carbons in the 2-position, large electron-withdrawing groups in the 6- and 8-position and an intact carbonyl group (essential for hydrogen bonding interactions with SIRT2 catalytic site) (Friden-Saxin et al., 2012). Compound 55 (Table 9) stands up as the lead compound: the (−)-55 enantiomer was a more potent inhibitor compared to the (+)-55 enantiomer and racemic mixture. Splitomicin (Table 9, compound 56) is a lactone inhibitor active against yeast SIRT2 but inactive against the human sirtuin isoforms, namely hSIRT2. As the lactone is prone to hydrolytic ring opening,

yielding an inactive hydrolyzed form, other type of derivatives have been synthesized (Freitag et al., 2011). The development of splitomicin ␤-aryl derivatives led to the identification of SIRT2 inhibitors that are active within the low micromolar range, particularly the derivatives with a 4-methylphenyl group (Table 9, compounds 57 and 58) (Neugebauer et al., 2008). However, it should be noted that the in vitro screening was carried out with recombinant SIRT2. Virtual screening and docking studies confirmed that the ␤-phenyl group and its orientation are important to anti-SIRT2 activity and point out toward a non-competitive mechanism of inhibition (Neugebauer et al., 2008). AC 93253 (Table 9, compound 59) is a selective and potent SIRT2 inhibitor, active in the low micromolar range and inhibiting SIRT2 7.5- and 4-fold more potently than SIRT1 and SIRT3, respectively (Zhang et al., 2009a,b). AC 93253 significantly enhances the


845 846 847 848 849 850 851 852 853 854 855 856 857 858 859

G Model ARR 513 1–30 16


Fig. 8. Structure of human SIRT2, as reported by Finnin et al. (2001).

860 861 862 863 864 865

866

867 868 869 870 871 872 873 874 875 876 877 878 879 880 881 882 883 884 885 886 887 888 889 890 891 892 893 894 895 896 897

acetylation of ␣-tubulin, p53 and histone H4. This compound was previously described as a potent retinoic acid receptor ␣ (RAR␣), though its structure does not resemble any known RAR␣ agonist. Hence, this RAR␣ agonism may be due to the effects on gene expression downstream from the interaction of AC 93253 with its molecular target, SIRT2 (Zhang et al., 2009a,b). 3.3. Caspases In addition to the neurodegenerative pathways associated with the toxicity of A␤ and P-tau, the involvement of caspases in AD and its apoptic cascade is a major accelerator of cell death (Cavallucci and D’Amelio, 2011). Apoptosis is a programmed cell death pathway characterized by distinct morphological features and biochemical mechanisms enhanced by the slow accumulation of the A␤ peptide within plaques of the brain of patients with AD. Inappropriate apoptosis is associated with neurodegenerative diseases, ischemic damage, autoimmune disorders and many types of cancer (Elmore, 2007). Caspases are a family of 14 mammalian cysteine-containing proteases which can be classified into initiator caspases and executioner caspases (Cavallucci and D’Amelio, 2011). The apoptic cascade consists in the activation of initiator caspases (caspase-8, -9, -10) by autocatalytic cleavage, which in turn cleave and activate the executioner caspases (caspase-3, -6, -7) which leads the production of characteristic cellular changes related to apoptosis (Fig. 9) (Cavallucci and D’Amelio, 2011). Recent studies have indeed shown that caspases are not exclusively associated with end-stage events in AD (Rohn and Head, 2009). In fact, it has reported that caspase activation and cleavage of tau colocalizes with the formation of NFTs (Rohn et al., 2002; Guo et al., 2004; Gamblin et al., 2003; Yin and Kuret, 2006; Ding et al., 2006; Gastard et al., 2003; Guillozet-Bongaarts et al., 2005). Moreover studies involving animal models and postmortem AD patients have given a deeper length to hypothesis that associates executioner caspases to AD development. As the studies performed so far suggest a critical involvement of caspases and the apoptotic regulator Bcl-2 in the etiology associated with AD it has been thought that the development of caspase inhibitors or compounds that increase expression of Bcl-2 in the

brain can be looked as effective therapeutic targets. Although Bcl2 agonists are innovative antiapoptotic approaches no selective agonist has yet been reported (Rohn and Head, 2009).

3.3.1. Caspases inhibitors The inhibition of apoptosis can be attained through the use of caspase inhibitors, such as carbobenzoxy-valyl-alanyl-aspartyl-[Omethyl] fluoromethylketone (Z-VAD-FMK) (Table 10, compound 60). Li et al. (2000) reported that Z-VAD-FMK treatment in transgenic mice treated delayed both disease onset and mortality. Nonetheless, even knowing that Z-VAD-FMK has shown good results in animal models for apoptosis, the associate problems with its bioavailability and especially its selectivity toward specific caspases is described to be a major disadvantage. In this context, quinolyl-valyl-O-methylaspartyl-[-2,6difluorophenoxy]-methyl ketone (Q-VD-OPh) (Table 10, compound 61) was developed. Indeed, Q-VD-OPh inhibits recombinant caspases with an IC50 within the nanomolar range (Mischak, 2002). In addition, in animal studies in TgCRND8, Q-VD-OPh displayed inhibition of caspase-7 and decreased pathological changes in tau protein (Rohn et al., 2009). Nevertheless, no variations in extracellular A␤ deposition have been detected. Furthermore, Q-VD-OPh represents an improvement over Z-VAD-FMK showing higher potency, stability, cell permeability and lower toxicity (Rohn and Head, 2009). Minocycline (Table 10, compound 62) is a high lipophilic semisynthetic tetracycline analog that effectively crosses the BBB (Kim and Suh, 2009). Minocycline inhibits the release of cytochrome c and prevents the activation of caspase-3, displaying net neuroprotective activity (Zhu et al., 2002). Additionally, in vitro studies have shown that minocycline prevents apoptosis in kidney epithelial cells by selectively increasing the expression of Bcl-2 mRNA and protein (Wang et al., 2004). Moreover, minocycline was shown to slow neuronal cell death in Tg2576 mice as well as in A␤42 infused rats (Choi et al., 2007). Furthermore, minocycline displays oral bioavailability and its safety has been proven both in animal models of AD and in human subjects. Additionally, its dual function as anti-apoptotic and anti-inflammatory agent may be useful in AD therapy (Rohn and Head, 2009).


898 899 900

901 902 903 904 905 906 907 908 909 910 911 912 913 914 915 916 917 918 919 920 921 922 923 924 925 926 927 928 929 930 931 932 933 934 935



17

Table 9 Sirtuin-2 inhibitors. Structure

Compound

IC50

Reference(s)

hSIRT2 (␮M)

hSIRT1 (␮M)

rSIRT2 (␮M)

rSIRT1 (␮M)

45, AGK2

3.5

>40.

n.d.

n.d.

Outeiro et al. (2007)

46, Salermide

25a

100a

n.d.

n.d.

Lara et al. (2009)

47, EX-527

19.6

0.098

n.d.

n.d.

Napper et al. (2005)

48, Ro 31-8220

0.8

3.5

n.d.

n.d.

Trapp et al. (2006)

49, GF 109203X

7.3

>50

n.d.

n.d.

50

1.0

>200.

n.d

n.d.

Medda et al. (2009)


G Model ARR 513 1–30 18


Table 9 (Continued) Structure

Compound

IC50

938 939 940 941 942 943 944

hSIRT1 (␮M)

rSIRT2 (␮M)

rSIRT1 (␮M)

n.d.

n.d.

Suzuki et al. (2012)

R=H

1.0

>100

52

R=F

0.57

>100

53, AK-7

15.5

n.d.

n.d.

n.d.

Taylor et al. (2011)

54

35

>50

n.d.

n.d.

Pesnot et al. (2011)

(−)-55

1.5

n.d.

n.d.

n.d.

Neugebauer et al. (2008)

1.5

n.a.

1.5

n.a.

n.d.

n.d.

56, Splitomicin

R1 = R2= H

57

R1 = 4-MePh R2 = Br R1 = 4-MePh R2 = CH3

59, AC 93253

937

hSIRT2 (␮M)

51

58

936

Reference(s)

3.4. Glycogen synthase kinase-3 (GSK-3) Glycogen synthase kinase 3 (GSK-3, E.C. 2.7.11.1), a serine/threonine kinase of the CMGC kinase group, has been originally described as a key regulator of glycogen synthesis and glucose metabolism (Roach et al., 2012) and, later on, as a participant in the Wnt signaling pathway (Clevers and Nusse, 2012). GSK-3 targets a wide range of substrates, namely metabolic proteins (e.g. cyclin D1, APP and presenilin) (Takahashi-Yanaga and Sasaguri, 2008; Phiel et al., 2003; Koo and Kopan, 2004), structural proteins (e.g. tau

Not active

6

45.3

Freitag et al. (2011) and Neugebauer et al. (2008)

Zhang et al. (2009a,b)

and other microtubule associated proteins) (Hernandez et al., 2010; Hanger et al., 1998; Mandelkow et al., 1995) and transcription factors (e.g. NF-␬B, p53 and Notch) (Sanchez et al., 2003; Charvet et al., 2011; Espinosa et al., 2003; Foltz et al., 2011). This plethora of biological substrates places GSK-3 as an important feature in cell cycle and development, bioenergetics and apoptosis, unveiling its role as a master regulator of key physiological processes (Medina and Wandosell, 2011). Mammals display two isoforms of the enzyme, GSK-3␣ (51 kDa, 483 a.a. in humans) and GSK-3␤ (47 kDa, 433 a.a. in humans),


945 946 947 948 949 950 951 952 953 954



19

Fig. 9. Caspase activation and apoptotic cascade.

Table 10 Caspase inhibitors. Structure

955 956 957 958 959 960 961 962 963 964 965 966 967

Compound

IC50 (nM)

Reference(s)

60, Z-VAD-FMK

–

Li et al. (2000)

61, Q-VD-OPh

25–400

Mischak (2002)

62, Minocycline

–

Kim and Suh (2009), Zhu et al. (2002), Wang et al. (2004) and Choi et al. (2007)

encoded by the homologue genes gsk-3␣ and gsk-3␤, respectively. The ATP binding pocket of both isoforms presents a high level of homology (98%), but significant differences are observed within the N- an C-terminal domains (Medina and Wandosell, 2011). GSK-3␣ and -␤ are expressed ubiquitously, although some tissues show preferential accumulation of a particular isoform, which is the case of GSK-3␤ in neuronal tissue (Woodgett, 1990). Additionally, neurons feature a specific alternative splicing isoform with a 13 a.a. insertion in the catalytic domain, named GSK-3␤2 (Mukai et al., 2002). The crystal structure of GSK-3␤ yields the characteristic double-domain kinase fold with a ␤-strand domain (residues 25–138) at the N-terminal end and an ␣-helix domain at the C-terminal end (residues 139–343), displaying the ATP

binding pocket within the interface of the mentioned sites (Fig. 10) (ter Haar et al., 2001). Other key structural features of the kinase fold include a hinge, a glycine-rich loop and an activation loop. The orientation of the domains and the loops supports the unique primed substrate mechanism observed for GSK-3, in which the substrates require a previous phosphorylation by a priming kinase (such as CDK-5, casein kinase I, PKC or PKA) on a Ser or Thr residue located four aminoacids C-terminal to the Ser or Thr residue to be catalyzed by GSK-3 (Fig. 11) (Medina and Wandosell, 2011; ter Haar et al., 2001). Hence, most substrate proteins are phosphorylated by a priming kinase at the P + 4 Ser/Thr in the S/TXXXS/T sequence before GSK-3 phosphorylates the Ser/Thr residue in the P position. ter Haar et al. (2001) estimate that the prime phosphate


968 969 970 971 972 973 974 975 976 977 978 979 980

G Model ARR 513 1–30 20


Fig. 10. Structure of GSK-3␤ as reported by Haat et al. (2001) (PDB ID: 1I09).

981 982 983 984 985 986 987 988 989 990 991 992 993 994 995 996 997 998 999 1000 1001 1002 1003 1004 1005 1006 1007 1008 1009 1010 1011 1012 1013 1014 1015 1016 1017 1018 1019 1020 1021 1022 1023 1024 1025

occupies a positively charged binding pocket near the activation loop (containing Arg96, Arg180 and Lys205). This feature facilitates substrate recognition and enhances the overall efficiency of the catalytic process. Notwithstanding, not all substrates undergo prime phosphorylation prior to GSK-3 catalysis (e.g. axin, ␤-catenin and Tau), involving other phosphorylation mechanisms. Besides the priming/substrate specificity mechanism, the regulation of GSK-3 activity relies on a combination of complex mechanisms, namely by hetero or autophosphorylation of specific Ser or Thr residues, association with protein complexes, subcellular localization and proteolytic cleavage (Medina and Wandosell, 2011). These mechanisms form a fine network of complex regulatory pathways that can yield up- or down-regulated GSK-3 which, in turn, can have a significant impact on both physiological and pathological processes, including cancer, renal diseases, neurodegenerative diseases and human immunodeficiency virus (HIV)-associated dementia (Medina and Wandosell, 2011). Given its involvement in a wide range of relevant human diseases, GSK-3 has gathered a spotlight status as a potential drug target and is currently one of the most extensively studied macromolecules for drug development (Amar et al., 2011; Martinez et al., 2002a,b; Gao et al., 2012; Rayasam et al., 2009). Proper GSK-3 signaling is particularly crucial for neuronal development and control of multiple aspects such as neurogenesis, neuronal migration and differentiation, synaptogenesis and cell survival (Medina and Wandosell, 2011; Kim and Snider, 2011; Kim et al., 2009; Hur and Zhou, 2010). Deregulation of GSK-3 associated signaling pathways and GSK-3 activity is related with the pathogenesis of several neurological and psychiatric disorders, particularly neurodegenerative diseases such as AD (Kaidanovich-Beilin and Woodgett, 2011; Takashima, 2006; Valvezan and Klein, 2012). GSK3 knock-out mice are not viable and die in utero (Hoeflich et al., 2000) while GSK-3 over-expressing mice display cognitive deficit and neurodegenerative alterations (Sirerol-Piquer et al., 2011). Accordingly, administration of GSK-3 inhibitors improves subjects’ performance in the water maze test, preserves the dendritic structure in the frontal cortex and hippocampus and decreases Tau phosphorylation (Rockenstein et al., 2007). Indeed, GSK-3 is central to AD physiopathology. GSK-3 is significantly involved in the hyper-phosphorylation of Tau (Hernandez et al., 2010; Hanger et al., 1998; Mandelkow et al., 1995; Hooper et al., 2008) but also in memory impairment, amyloidogenesis, inflammatory response and ACh deficit (Hooper et al., 2008). Furthermore, it has been shown to co-localize with dystrophic neurites

Fig. 11. Schematics of the canonical phosphorylation sequence recognized by GSK-3.

and NFTs, appearing in its hyper-active form in the frontal cortex of AD patients and in neurons with pre-tangle alterations. Additionally, it is up-regulated in the hippocampus of AD brains. Up-regulated GSK-3 can also be found in peripheral lymphocytes in both AD and mild cognitively impaired subjects. These findings support the rationale for the GKS-3 hypothesis for AD (Hooper et al., 2008) making this kinase a particularly interesting and promising target for the development novel disease modifying therapies for AD. 3.4.1. GSK-3 inhibitors GSK-3 inhibitors are valuable candidates for new therapeutic approaches toward innovative disease-modifying drugs. Several GSK-3 inhibitors have been described in literature, including the monovalent cation lithium and derivatives of structurally diverse chemical families, such as thiadiazolidinediones (TDZDs), paullones, maleimides, indirubines and diverse natural alkaloids derived from marine sponges (Martinez et al., 2002a,b; Avila and Hernandez, 2007; Bhat et al., 2004). Lithium (Li+ , compound 63, Table 11) is a classic mood stabilizer widely used as a first-line treatment to bipolar disorder (Burgess et al., 2001). Later on, lithium was reported as a direct inhibitor of GSK-3 via competition with magnesium, which supported the significant effects observed on the morphogenesis on the early development of numerous organisms (Klein and Melton, 1996; Stambolic et al., 1996). Preliminary in vitro data suggested a rather high Ki value (approximately 1–2 mM) (Klein and Melton, 1996; Stambolic et al., 1996), but there is strong in vivo evidence that Li+ produces a physiologically significant inhibition of brain GSK-3 at plasmatic therapeutic concentrations (Gould et al., 2004; Mendes et al., 2009). Interestingly, it does not display inhibitory activity on other kinases (Klein and Melton, 1996; Stambolic et al., 1996). Furthermore, Li+ induces the phosphorylation of Ser9 and Ser21, which results in the formation of down-regulated forms of GSK-3␤ and -3␣, respectively (Gould and Manji, 2005; ChaleckaFranaszek and Chuang, 1999). The significant pre-clinical data supported the design of clinical trials to evaluate the effects of Li+ on AD patients. Accordingly, Li+ treatment was associated with a significant increase in brain derived neurotrophic factor (BDNF) and an improvement in the ADAS-Cog score of AD patients in a single-blinded, placebo-controlled, parallel group multicenter 10 week study (Leyhe et al., 2009), pointing toward a possible neuroprotective effect of Li+ . Similarly, Li+ administration was associated with a significant decrease in CSF levels of phosphorylated Tau in amnestic mild cognitive impairment patients in a randomized, double-blinded, placebo-controlled 12 months trial (Forlenza et al.,


1026 1027 1028 1029 1030 1031 1032 1033 1034

1035 1036 1037 1038 1039 1040 1041 1042 1043 1044 1045 1046 1047 1048 1049 1050 1051 1052 1053 1054 1055 1056 1057 1058 1059 1060 1061 1062 1063 1064 1065 1066 1067 1068 1069 1070

G Model

ARTICLE IN PRESS

ARR 513 1–30


21

Table 11 hGSK-3␤ inhibitors. Structure

Li+

Compound

IC50 (nM)

Reference(s)

hGSK-3␤

Other kinases

63, Lithium

Ki = 1–2 mM

–

Klein and Melton (1996) and Stambolic et al. (1996)

64, Tideglusib, NP-12 or NP031112

73.74% inhibition at 10 ␮M

29.50% inhibition of hGSK-3␣ at 10 ␮M

Sereno et al. (2009)

Leost et al. (2000)

65, Alsterpaullone

R = NO2

4

CDK1/cyclin B, 35 CDK2/cyclin A, 15 CDK5/p25, 40 CK1, >100,000

66, Kenpaullone

R = Br

23

67, 9-Cyanopaullone

R = CN

10

68, 9-chloropaullone

R = Cl

24

69, 9-trifluoromethylpaullone

R = CF3

30

CDK1/cyclin B, 400 CDK2/cyclin A, 680 CDK5/p25, 850 CDK1/cyclin B, 35 CDK2/cyclin A, 15 CDK5/p25, 40 CDK1/cyclin B, 600 CDK5/p25, 800 CDK1/cyclin B, 400 CDK5/p25, 600

70, SB-415268

R1 = 2-NO2 R2 = 3-Cl-4-OH

78

71

R1 = 3-NO2 R2 = 3,5-diCl-4-OH

20

72, SB-216763

R1 = 2,4-diCl R2 = Me

73, Indirubin

No significant inhibition

Smith et al. (2001) and Martin et al. (2013)

34

No significant inhibition

Martin et al. (2013)

R=O

600

CDK1/cyclin B, 10,000 CDK5/p25, 5500

Leclerc et al. (2001)

74, Indirubin-3 -oxime

R = NOH

22

CDK1/cyclin B, 180 CDK5/p25, 100

75, 6-BIO

R=H

5

CDK1, 320 CDK5, 10,000

Meijer et al. (2003)

30

CDK1, >10,000 CDK5, 10,000

Vougogiannopoulou et al. (2008)

76, 6-BIDECO


G Model

ARTICLE IN PRESS

ARR 513 1–30


22 Table 11 (Continued) Structure

1071 1072 1073 1074 1075 1076 1077 1078 1079 1080 1081 1082 1083 1084 1085 1086

Compound

IC50 (nM)

Reference(s)

hGSK-3␤

Other kinases

77, 6-BIMYEO

110

CDK1, 200 CK5, 900

78, Hymenialdisine

100

CDK1/cyclin B, 220 CDK5/p25, 280 CK1, 350

Leost et al. (2000)

79, Dibromocantharelline

3000

No inhibition

Zhang et al. (2011)

80, Manzamine A

10,200

CDK-5, 1500

Hamann et al. (2007)

Hamann et al. (2007)

81

R=H

5.4

n.a.

82

R = OCOMe

4.8

n.a.

2011), suggesting a possible disease-modifying mechanism with clinical relevance. This is consistent with the findings of a casecontrol study (Nunes et al., 2007), in which lithium treatment reduces the prevalence of AD in patients with bipolar disorder to the levels in the general population. The same outcome was reported in a larger observational cohort (Kessing et al., 2008) and in a follow-up study (Kessing et al., 2010). Notwithstanding, Li+ treatment was not correlated with clinical benefit in mild AD patients in a single-blinded, randomized, placebo-controlled, multicenter 10 week study (Hampel et al., 2009). Additional epidemiological and clinical data is required to fully attain the therapeutic potential of Li+ treatment in AD. Tideglusib (NP-12/NP031112, compound 64, Table 11) is a TDZD derivative that acts as an ATP-noncompetitive irreversible inhibitor of GSK-3␤ (Dominguez et al., 2012). The carbonyl groups of the TDZD motif interact with Arg96 and Lys205 via hydrogen

bonding and the N-aryl moieties form ␲–␲ stacking interactions mainly with Tyr 216 (Martinez et al., 2002a,b). Pre-clinical reports show that tideglusib (per os and i.v.) displays significant neuroprotection of hippocampal neurons after excytotoxic injury in adult rats, possibly via nuclear peroxisomal proliferator-activator receptor ␥ (PPAR␥) activation (Luna-Medina et al., 2007). Moreover, tideglusib administration in transgenic APPsw -tauvlw mice decreased phosphorylated Tau levels and amyloid deposition, induced neuroprotection of cortical and hippocampal neurons and improved spatial memory (Sereno et al., 2009). The encouraging pre-clinical data gave a solid rationale for clinical studies of tideglusib, reaching phase IIb clinical trials for the treatment of AD and progressive supranuclear palsy (PSP), a rare tauopathy. In these clinical settings tideglusib was well tolerated in mild-to-moderate AD patients, producing positive alterations in cognitive scores but without statistical significance due to the small size of the sample


1087 1088 1089 1090 1091 1092 1093 1094 1095 1096 1097 1098 1099 1100 1101 1102



(Del Ser et al., 2013). The clinical efficacy of tideglusib in AD patients needs to be attained in a trial with a larger patient enrollment, to obtain statistically significant results. 1105 Paullones (compounds 65–69, Table 11) are family of ben1106 zazepinones that have been described as potent ATP-competitive 1107 inhibitors of GSK-3␤, with IC50 values within the low nanomolar 1108 range. Alsterpaullone (compound 65, Table 11) inhibits in vivo 1109 phosphorylation of both Tau at AD-specific sites by GSK-3␤ and 1110 dopamine and cAMP regulated phosphoprotein Mr 32 kDa (DARPP1111 32, a striatum protein acting downstream of dopamine) in isolated 1112 striatum slices by CDK5 (Leost et al., 2000). Kenpaullone (com1113 pound 66, Table 11) enhances neurogenesis in human neural 1114 progenitor cells (NPC) without changing cell cycle exit or cell 1115 survival, and may therefore be used to direct the differentia1116 tion of neural stem and progenitor cells (Lange et al., 2011). 1117 Kenpaullone prevents 1-methyl-4-phenylpyridinium (MPP(+))1118 induced cell death by blocking mitochondrial dysfunction and 1119 subsequent caspase-9 and -3 activation, displaying neuroprotec1120 tive effects against GSK-3␤-mediated mitochondrial dysfunction 1121 and stress-induced cell death (Petit-Paitel et al., 2009). The intro1122 duction of a 9-halogen in the indolobenzazepinone scaffold favors 1123 not only the formation of a hydrogen bond with a water molecule, 1124 as observed in the crystal structure of CDK2 bound with ATP, 1125 as well as the interaction with the Leu83 residue, via hydro1126 gen bond (Martinez et al., 2002a,b). Indeed, paullones with a 1127 9-halogen substitution pattern display improved inhibitory activity 1128 toward GSK-3␤ (compounds 65–69, Table 11). This class of GSK1129 3 inhibitors interacts more extensively with the Val135 residue 1130 of GSK-3 when compared to the Cys85 residue of CDK-5 (Chen 1131 et al., 2011), which justifies the enhanced selectivity for GSK-3 1132 and further validates paullones as a promising template for the 1133 development of selective and potent GSK-3␤ inhibitors. 1134 Maleimides (compounds 70–72, Table 11) were identified 1135 as potent ATP-competitive GSK-3 inhibitors in high throughput 1136 screening assays. Relevant derivatives include anilinomaleimides 1137 (compounds 70 and 71, Table 11) (Smith et al., 2001), particu1138 Q6 larly SB-415286 (compound 70, table 11). SB-415286 is a highly 1139 selective, potent and cell permeable GSK-3 inhibitor that stim1140 ulates hepatic glycogen synthesis and induces the expression of 1141 ␤-catenin (Coghlan et al., 2000), which is consistent with Wnt 1142 signaling activation. Furthermore, SB-415286 protects both cen1143 tral and peripheral neurons in culture from phosphoinositide (PI) 1144 kinase-induced apoptosis and decreases Tau hyperphosphoryla1145 tion (Cross et al., 2001). Interestingly, SB-415286 (40 ␮M) was 1146 shown to induce cell growth inhibition and apoptosis in leukemic 1147 cell lines KG1a, K562 and CMK (Mirlashari et al., 2012), a differ1148 ent outcome to that observed in neurodegeneration models, but 1149 with potential therapeutic interest in the field of oncology. The 1150 potency of anilinomaleimides is enhanced by introducing addi1151 tional chlorine substituents (compound 71, Table 11), suggesting 1152 that the greater acidity of the phenol may result in a stronger 1153 hydrogen bonding interaction with the kinase active site or that 1154 the potency is enhanced with increased lipophilicity (Smith et al., 1155 2001). Quantitative structure–activity relationship (QSAR) (Akhtar 1156 and Bharatam, 2012) suggests that an improvement in GSK-3␤ 1157 binding affinity can be achieved through conformational restric1158 tion in the aniline ring and the introduction of electronegative 1159 and/or hydrophobic substituents in the amino-aryl ring. Another 1160 example of maleimide derivatives with anti-GSK-3 activity are 1161 arylindolmaleimides, like SB-216763 (compound 72, Table 11). 1162 This compound is also a potent and selective cell permeable GSK-3 1163 inhibitor and exhibits similar glycogen and ␤-catenin enhancing 1164 behavior as that reported for the anilinomaleimide SB-415286 1165 (Coghlan et al., 2000). Intravenous SB-216763 in mice previously 1166 treated with oligomeric A␤ reversed the majority of the deleterious 1167 effects associated with A␤ neurotoxicity, although SB-216763 itself 1168 1103 1104

23

induced mild behavioral deficits (Hu et al., 2009). Notwithstanding, SB-216763 exhibits neuroprotective effects in isolated neurons in culture, possibly through activation of neuroprotective signaling pathways (Cross et al., 2001). Furthermore, SB-216763 was the first GSK-3 inhibitor reported to promote self-renewal in mouse embryonic stem cells (mESCs) co-cultured with mouse embryonic fibroblasts (MEFs) (Kirby et al., 2012). Taken together, the gathered data places the maleimide nucleus as a valid and promising scaffold for the development of new potent and selective drug candidates Indirubines (compounds 73–77, Table 11) are bis-indole compounds that can be found in a variety of natural sources. The most well-known compound of this family is indirubin (compound 73, Table 11), which was identified as the active ingredient of a Chinese medicine (Danggui Longhui Wan) used in the treatment of cronic diseases, such as leukemia. Indirubin displays not only a significant antiproliferative effect (Han, 1988), but also acts as a potent inhibitor of CDKs (50–100 nM) and GSK-3␤ (5–50 nM) (Leclerc et al., 2001), through an ATP-competitive mechanism. However, indirubin is scarcely soluble, shows poor absorption and exerts gastrointestinal toxicity (Martinez et al., 2002a,b). Several indirubin derivatives with improved solubility and anti-GSK-3␤ activity have been reported, namely indirrubine-3 -monoxime (compound 74, Table 11). This compound displays increased potency as a GSK-3␤ inhibitor while effectively preventing in vivo and in vitro Tau phosphorylation at AD-specific sites and phosphorylation of DARPP-32 by CDK5 (Leclerc et al., 2001). The introduction of a bromide in C6 on compound 74 yields 6-bromoindirubin-3 -oxime (6-BIO, compound 75, Table 11), a synthetic cell-permeable derivative that displays remarkable inhibition of GSK-3␤ within the low nanomolar range (Meijer et al., 2003). This 6-bromide derivative binds in a planar conformation into a narrow hydrophobic pocket within the ATP-binding site of the enzyme. 6-BIO inhibits the phosphorylation of GSK-3␣/␤ at Tyr279 and Tyr216, respectively, blocking the formation of the hyperactive forms of GSK-3. Additionally, 6BIO reduces the phosphorylation of ␤-catenin and Tau in COS-1 and SH-SY5Y cells and mimics Wnt signaling in Xenopus leavis embryos (Meijer et al., 2003), but displays high cellular toxicity (LD50 = 5 ␮M in SH-SY5Y cells) (Martin et al., 2013). Nevertheless, 6-BIO is a valuable scaffold for the development of selective and potent GSK-3␤ inhibitors, like 6-BIDECO and 6-BIMYEO (compounds 76 and 77, respectively, Table 11). These compounds retain the pharmacophore structure, consisting in the lactam nitrogen and carbonyl and the heterocyclic nitrogen, which forms key hydrogen bonds with the kinase active site, and the 6-bromide, which is a selectivity determinant for GSK-3␤ (Vougogiannopoulou et al., 2008). 6-BIDECO and 6-BIMYEO are non-toxic water-soluble GSK-3␤ selective derivatives of 6-BIO able to reverse Tau phosphorylation and reduce OKA (okadaic acid)-induced apoptosis in SH-SY5Y cells (Vougogiannopoulou et al., 2008) and primary neuronal cultures (Martin et al., 2011), displaying a neuroprotective effect that may be therapeutically significant in the context of AD and other neurodegenerative disorders. Information regarding in vivo safety and efficacy of 76 and 77 in animal models remains unavailable. Marine sponges (Porifera sp.) are a highly valuable resource in the search for bioactive chemical compounds with potential pharmacological interest, being a natural source of kinase inhibitors (De Zoysa, 2012; Johnson et al., 2011). Hymenialdisine (compound 78, Table 11), a bromopyrrole alkaloid isolated from marine sponges (e.g. Axinellidæ, Agelasidæ and Halichondriidæ), is a natural compound that acts as an ATP competitive inhibitor of several kinases, including GSK-3␤ (Meijer et al., 2000). Docking studies show that compound 78 is well located within the ATP-binding pocket of the kinase, via hydrophobic interactions of the pyrrole and azepinone rings with Ile62, Val110, Leu188 and Leu132. The nitrogen atoms of the pyrrole


1169 1170 1171 1172 1173 1174 1175 1176 1177 1178 1179 1180 1181 1182 1183 1184 1185 1186 1187 1188 1189 1190 1191 1192 1193 1194 1195 1196 1197 1198 1199 1200 1201 1202 1203 1204 1205 1206 1207 1208 1209 1210 1211 1212 1213 1214 1215 1216 1217 1218 1219 1220 1221 1222 1223 1224 1225 1226 1227 1228 1229 1230 1231 1232 1233 1234

G Model ARR 513 1–30 24


1268

and azepinone rings and the carbonyl group also establish important hydrogen bonds with residues of the kinase hinge region (Asp133 and Val 135), a common feature of ATP-competitive kinase inhibitors. Furthermore, the oxygen and nitrogen atoms of the imidazolone ring form electrostatic interactions with polar residues Lys85, Asn186 and Asp200 (Zhang et al., 2011). Moreover, hymenialdisine strongly inhibits other important kinases like CDKs and CK1. Furthermore, compound 78 inhibits in vivo phosphorylation of Tau and displays anti-inflammatory activity (Meijer et al., 2000). Another sponge metabolite, dibromocantharelline (compound 79, Table 11), displays moderate inhibition of GSK-3␤ (IC50 = 3 ␮M) compared to compound 78, but does not show significant inhibition of related kinases, such as CDK1 and CDK5 (Zhang et al., 2011). Thus, compound 79 is a less potent yet selective GSK-3␤ inhibitor, yielding a valid natural template for the development of semi-synthetic selective and potent GSK-3␤ inhibitors. The guanidine ring of 79 is oriented 90◦ relative to the other three co-planar rings of the molecule, being more deeply inserted in the GSK-3␤ binding cavity. This specific structural feature is behind the lack of affinity toward CDKs (Zhang et al., 2011). Manzamine alkaloids (compounds 80–82, Table 11) are another class of marine derived compounds with potential therapeutic interest in CNS disorders. Manzamines are complex ␤-carboline polycyclic alkaloids isolated from Indo-Pacific sponges. Manzamine A (compound 80, Table 11) is a non-competitive GSK-3␤ inhibitor with an IC50 within the micromolar range, also inhibiting CDK-5 within the low micromolar range (Hamann et al., 2007). Treatment with manzamine A decreases tau hyperphosphorylation on human neuroblastoma cell lines (SH-SY5Y cells) (Hamann et al., 2007). Synthetic derivatives of manzamine A with increased conformational restrictions (compounds 81 and 82, Table 11) in the aliphatic chains exhibit increased inhibitory activity toward GSK-3␤ (Hamann et al., 2007).

1269

3.5. New mechanistic rationales for drug discovery in AD

1235 1236 1237 1238 1239 1240 1241 1242 1243 1244 1245 1246 1247 1248 1249 1250 1251 1252 1253 1254 1255 1256 1257 1258 1259 1260 1261 1262 1263 1264 1265 1266 1267

1270 1271 1272 1273 1274 1275 1276

1277 1278 1279 1280 1281 1282 1283 1284 1285 1286 1287 1288 1289 1290 1291 1292 1293 1294 1295 1296 1297

The search for NCE for AD has long evolved from the early stages of the cholinergic and amyloid hypothesis. The discovery of new mechanisms of neuronal regulation has paved the way for the discovery of new targets which may lead to more effective drugs. The cascade of molecular events leading to mechanisms like synaptogenesis and autophagy may provide new mechanistic rationales for the discovery of innovative drugs with disease-modifying ability. 3.5.1. Autophagy enhancers Autophagy is a mechanism for the self-degradation of cellular components mediated by acid hydrolases within the lysosomes. Originally described as an adaptive response to nutrient deprivation in mitotic cells, this cellular mechanism was soon discovered to play an important role in the balance of cell biosynthesis and turnover while providing means to cellular component recycling (He and Klionsky, 2009). Moreover, along with the ubiquitinproteosome system, autophagy is a major route for the clearance of protein aggregates and damaged organelles, preventing the accumulation of toxic fragments. Although this process has been first described in 1963, only more recently has it been suggested that impaired autophagy can play a significant role in human disease. Neurons are particularly susceptible to deregulated autophagy, mainly due to the accumulation of toxic debris that triggers neuronal dysfunction, death and neurodegeneration (Banerjee et al., 2010; Son et al., 2012; Nixon, 2013; Tan and Yu, 2014). Autophagy is regulated by various signaling factors including beclin-1, an autophagy effector involved in the formation of autophagosomes (Kang et al., 2011) and mammalian target of rapamycin (mTOR), a negative regulator of autophagy (Kim et al., 2011). Reports in APP

transgenic mice have shown that beclin-1 deficiency leads to accumulation of A␤, which is attenuated by beclin-1 overexpression. Moreover, post-mortem analysis of AD brains showed reduced levels of beclin-1 (Pickford and Masliah, 2008). Furthermore, levels of mTOR and its downstream molecular targets were found to be dramatically increased in AD (Li et al., 2005) and positively correlated with the accumulation of A␤ and Tau (Caccamo and Majumder, 2010) and cognitive decline (Paccalin and Pain-Barc, 2006). Conversely, inhibition of mTOR slowed AD progression in a transgenic mouse model of the disease (Spilman and Podlutskaya, 2010). These data suggests that the acceleration of the removal of damaged cell components and proteins by enhancing autophagy may prevent neuronal death, which can have a favorable clinical outcome in AD. In this context, the use of autophagy enhancers may be a valid therapeutic approach toward AD. This premise drove the search for modulators of autophagy, and several small molecules have been identified as autophagy enhancers (Sarkar and Rubinsztein, 2008). Rapamycin (Sirolimus, compound 83, Table 12) is a macrolide antibiotic isolated from Streptomyces hygroscopicus used as an immunosuppressant in post transplantation subjects. Rapamycin acts as an autophagy enhancer via mTOR inhibition, by stabilizing the functional complexes of mTOR and inhibiting its kinase activity (Kim et al., 2002). Several lines of evidence show that rapamycin enhances autophagic degradation of aggregate proteins including tau and A␤ and promotes neuroprotection (Berger et al., 2006; Spilman and Podlutskaya, 2010; Viscomi and D’Amelio, 2012; Yang and Chen, 2014). However, due to its strong immunosuppressant effect, several concerns arise regarding the long-term use of this drug, given the significant potential for dangerous side effects, particularly in the elderly. Temsirolimus (CCI-779, compound 84, Table 12), a rapamycin analog and mTOR inhibitor approved in 2007 for the treatment of renal carcinoma (Kwitkowski et al., 2010), was also found to enhance the autophagic clearance of A␤ and promote neuroprotection in APP/PS1 mice after intraperitonial administration (2 mg/kg) (Jiang et al., 2014). Previous studies in Drosophila models of tauopathy showed the same protective outcome (Berger et al., 2006). Recent screening programs for small molecule enhancers of rapamycin (SMERs) have effectively identified enhancers of mammalian autophagy (compounds 85–87, Table 12) (Sarkar et al., 2007). These SMERs include different chemical scaffolds such as an aminopyrimidone (SMER10, compound 85), vinylogous amide (SMER28, compound 86) and a bromo-substituted quinazoline (SMER28, compound 87). These compounds induced mTORindependent autophagy and reduced mutant aggregates in HD cell and animal models. Preliminary SAR showed that the pyrimidine scaffold of compound 85 and the hydroxyl group of compound 86 are essential for autophagy inducing activity (Sarkar et al., 2007). Other miscallenoeous autophagy inducers include latrepirdine (Dimebon® , compound 88, Table 12) and carbamazepine (compound 89, Table 12). Latrepirnide, originally marketed as a non-selective anti-histamine in Russia, has been shown to induce autophagy, reduce A␤ deposition and prevent behavioral decline in 3 months old TgCRND8 mice (Steele and Gandy, 2013). These observations are in accordance with previous results in Saccharomyces cerevisae (Bharadwaj and Verdile, 2012). The effects of latrepirnide on A␤ are thought to be the result of direct activation of intracellular catabolic pathways. Notwithstanding, this compound did not show efficacy in phase III clinical trials for AD and HD (Bharadwaj and Bates, 2013). However, a recent meta-analysis has reported that in spite of the failure to demonstrate efficacy, latrepirnide tended to improve cognitive scores (Cano-Cuenca et al., 2014). Carbamazepine (compound 89, Table 12), along with other anticonvulsant drugs, has been shown to enhance autophagy, relief the A␤ load and improve memory deficit in mouse models of AD (Li et al., 2013). These effects are mTOR-independent and occur via


1298 1299 1300 1301 1302 1303 1304 1305 1306 1307 1308 1309 1310 1311 1312 1313 1314 1315 1316 1317 1318 1319 1320 1321 1322 1323 1324 1325 1326 1327 1328 1329 1330 1331 1332 1333 1334 1335 1336 1337 1338 1339 1340 1341 1342 1343 1344 1345 1346 1347 1348 1349 1350 1351 1352 1353 1354 1355 1356 1357 1358 1359 1360 1361 1362 1363



25

Table 12 Autophagy enhancers. Structure

Compound

Reference

83, Rapamycin or Sirolimus

Berger et al. (2006), Spilman and Podlutskaya (2010), Viscomi and D’Amelio (2012) and Yang and Chen (2014)

84, Temsirolimus or CCI-779

Berger et al. (2006) and Jiang et al. (2014)

85, SMER10

Sarkar et al. (2007)

86, SMER18

87, SMER28

88, Latrepirdine or Dimebon®

Bharadwaj and Verdile (2012) and Steele (2013)

89, Carbamazepine

Li et al. (2013)


G Model ARR 513 1–30 26


Table 13 Small molecule enhancers of synaptogenesis. Structure

1364 1365

1366 1367 1368 1369 1370 1371 1372 1373 1374 1375 1376 1377 1378 1379 1380 1381 1382 1383 1384 1385 1386 1387 1388 1389 1390 1391 1392 1393 1394 1395 1396 1397 1398

Compound

IC 50 (nM)

Reference

Class-I HDACs

Class-II HDACs

HDAC1 < 2 HDAC2 < 2 HDAC3 = 4.4 HDAC1 < 2 HDAC2 < 2 HDAC3

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