Perspective Special Focus: Neurodegenerative Diseases

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An alternative approach to drug discovery for Alzheimers disease dementia

There are no drugs that halt the progression of any age-associated neurodegenerative disease. This may be due to the failure of drug developers to recognize that while there are mutations that predispose individuals to disease as they get older, the vast majority of neurodegenerative diseases arise from a confluence of multiple toxic insults. Thus, it is unlikely that the current single-target approach is going to yield useful drugs for these conditions. The identification of multi-target lead compounds is needed and their selection should be based upon a requirement for their efficacy in phenotypic screening assays that reflect the biology of the aging brain. This approach to neurodegenerative disease drug discovery is likely to produce safe and effective drugs.
to their dementia. While the b amyloid (Ab)producing pathway is clearly involved in AD and its choice as an initial therapeutic target was very logical, to date, drugs or immunotherapies directed solely towards its components have proven toxic or otherwise failed in clinical trials. This may be because the drug targets associated with this pathway are required for normal brain function, thereby resulting in toxicity when inhibited and/or because the incorrect molecular targets were selected. Since age is by far the major risk factor for AD and synapse loss and nerve cell death in AD are likely a consequence of many cumulative toxic events, there is a strong rationale for a more clinically relevant approach to drug discovery based upon the biology of aging. Phenotypic screening assays that reflect multiple, ageassociated neuro toxicity pathways rather than single molecular targets could be used, and compounds that have therapeutic efficacy in target pathways relevant to aging and cognition, as well as AD pathology, may be required. It is argued here that the employment of cell-based screening assays for AD drug discovery focused upon neuro protection from age-associated CNS stress pathways reduces the chance of cellular drug toxicity and increases the probability of finding an effective drug for age-associated cognitive impairment and AD, in large part because of the heterogeneity of aging and dementia itself. Background The majority of our current drugs are based upon the chemical scaffolds of compounds isolated from plants. The isolation and pharmaco logical characterization of pure compounds with
Future Med. Chem. (2012) 4(13), 16811688
ISSN 1756-8919

Alzheimers disease dementia (AD) is the most common age-associated disease where there are no treatments that can prevent, delay, slow or stop its progression. There are many reasons for the absence of an effective therapy, including the complexity of the disease, and until recently the paucity of efficient biomarkers for clinical trials. Indeed, it may be an illusion to consider AD a single disease at all, for while the current clinical criteria are primarily based upon alterations in behavior and cognition [1] , similar changes can result from multiple pathologies besides the plaques and tangles associated with AD. These primarily include various types of vascular disease [2,3] , but also behavioral variant fronto temporal dementia [4,5] and the numerous toxicities associated with normal aging [4] , including cumulative oxidative damage [5] . In fact, less than one-third of all people with dementia have pure AD pathology, and there is no threshold for any of these alterations that predicts dementia [3,6] . Therefore, both the mode of action of drugs and the relevant biomarkers for clinical trials for age-associated dementia should reflect the heterogeneity of the condition. In addition, a successful drug for dementia must combat the common end point of the majority of the above pathologies, which include synapse loss and nerve cell degeneration. Despite these realities, the focus of the pharma c eutical industry has been on single molecular targets associated with the rare form of familial AD (FAD). Although FAD accounts for only a few percent of all AD cases, essentially all clinical trials are done with patients that have the sporadic form of the disease in which a heterogeneous range of pathologies contribute
10.4155/FMC.12.109 2012 Future Science Ltd

David Schubert* & Pamela Maher

The Salk Institute for Biological Studies, Cellular Neurobiology Laboratory, 10010 North Torrey Pines Road, La Jolla, CA 92037-1002, USA *Author for correspondence: Tel.: +1 858 453 4100 (ext. 1528) Fax: +1 858 535 9062 E-mail: schubert@salk.edu

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medicinal properties started with digoxin from foxglove in 1785, morphine from poppies in 1806, and aspirin from salicylic acid in 1897 (for review see [7]). Not only did these molecules make tremendous contributions to medicine, they also were the major factors in the identification of the molecular pathways involving the Na + -K+ -ATP pump, opiate receptors and the cyclooxygenase enzymes, respectively, leading to a succession of derivative drugs that are in the clinic today. Following these initial screening assays based upon therapeutic efficacy in people, the standard drug-discovery paradigm for the pharmaceutical industry up until the last two decades was based upon biological or phenotypic screens using living organisms such as rodents, bacteria or fungi. More recently, there has been a dramatic shift away from phenotypic screening. The advent of molecular cloning and protein expression technologies, combinatorial chemistry to generate millions of novel molecules, and highthroughput screening (HTS) based upon a single protein target, has essentially eliminated biology-based drug discovery [8] . Despite the enormous investment in these high-tech drugdiscovery programs, no effective drugs for chronic neuro logical diseases such as AD and amyotrophic lateral sclerosis, or for ischemic stroke, have been developed, and the overall rate of drug discovery has greatly decreased over the last 10years [9,10] . The reason for this may be endemic to the approach itself. The selection & screening of drug targets While it is very frequently argued, if not assumed, that the single-target approach to drug discovery is the only relevant one [10,11] , there are surprisingly few targets for US FDA-approved drugs. Of approximately 1000 FDA-approved small-molecule drugs, there are only slightly more than 200 defined molecular targets, and many of these targets were discovered well after the therapeutic introduction of the drug [12] . The single target approach to drug discovery is based upon the following steps: pick a (protein) target with a presumptive cell or animal model validation; identify the best binder by HTS screening or rational design based upon the 3D structure of the binding pocket; carry out proof-ofprinciple experiments in animals; and devise a clinical development platform. While the last two steps are necessary for the development of any drug, the first two are unlikely to succeed
Future Med. Chem. (2012) 4(13)

Key Terms Phenotypic screening:

Drug screening based upon activity in living organisms or cells.

Neurodegeneration: Loss of
structure and function within the nervous system.

for neurodegenerative diseases because of the reasons outlined below. Due to the innate complexity of the brain and the multiple changes that occur with aging, it can be argued that the rationale for the selection of single putative therapeutic molecular targets for neurodegenerative diseases may be very weak, and that it is unwise to assume that we can choose the correct ones. In the case of microbial diseases, essentially all of the firstgeneration therapeutics were developed with no understanding of the molecular target. For many CNS drugs currently in the market for psychiatric illnesses, the presumptive target was identified and a specific drug made against this target, but it was later shown that the drug functions via unrelated targets; the antipsychotic drugs are perhaps the best examples of this [13] . For AD, the major presumptive drug target, the Ab pathway, was identified 20years ago. Dozens of drug and other therapeutic candidates against this pathway have been tested in the clinic, and all have failed to prevent disease progression [14] . Why is this and what are the alternatives to the single-target approach? Target selection in the case of AD has been primarily focused on a few percent of the FAD examples that result from dominant mutations in either the presenilins or the amyloid precursor protein. Since both sets of mutations lead to the increased production of A b and elevated plaque load, the study of this pathway for target selection was certainly justified. However, it has been known for many years that some AD patients have no plaques or tangles and that many cognitively normal old people have severe AD-like pathology [3,6,15,16] . Therefore, some AD patients in clinical trials do not have a significant plaque load (but are still cognitively impaired), and suffer from a combination of CNS pathologies unrelated to amyloid deposition. In addition, essentially all preclinical drug development is based upon FAD-transgenic mice, a model that has not successfully translated into the clinic [17] . Perhaps rodent models more relevant to the human disease would be very old animals or the senescence-accelerated SAMP8 mice, which acquire most of the pathology and behavioral deficits associated with AD, including amyloid accumulation [18] . Conversely, it would be a mistake to assume that if an anti-amyloid-based therapeutic is effective in FAD that it will help in the majority of sporadic cases, for FAD is essentially the same as transgenic AD mice where many drugs
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are effective but fail in clinical trials against sporadic AD. Age is by far the greatest risk factor for all forms of AD. Age-associated damage to the brain results from a combination of multiple accumulated stresses, lifestyle, environment and genetic risk factors with varying degrees of penetrance. The ApoE4 isoform is the only significant genetic risk factor so far identified, and while it is not predictive of AD, it is the only gene associated with an increased rate of age-related cognitive decline in humans [19] . Sufficient genetic information is currently available to make it likely that additional genetic risk factors for AD will be minor and combinatorial, making them unproductive therapeutic targets [20] . This leaves lifestyle and age-associated damage as potential therapeutic targets. It can be argued that lifestyle components such as exercise [21] and diet [22,23] affect the rate of physiological aging, and therefore the development of AD. Since lifestyle changes can be made in the absence of medication, the pathological components of the aging brain are the remaining therapeutic drug targets. These pathways include the loss of trophic factors such as BDNF [24] , increased oxidative stress and glutathione (GSH) depletion [25] , reduced energy metabolism [26] and the accumulation of misfolded, aggregated proteins [27,28] . An alternative approach to AD drug discovery is to identify neuroprotective compounds that reduce the toxic aspects of several or all of these pathways and will therefore be broadly effective against multiple causes of neurodegeneration and cognitive decline, not just one. In addition to the higher probability that such drugs can prevent disease progression compared with drugs directed against the amyloid pathway, there is a much greater chance of such neuroprotective drugs being successful in clinical trials where the patient base is heterogeneous and frequently a significant fraction has minimal amyloid burden [15] . An additional benefit is that compounds that elevate endogenous BDNF expression or directly activate its signaling pathways enhance learning and memory. This fact is significant for clinical trials because current measures of outcome for the FDA approval of AD drugs require a significant improvement in cognition. The second assumption of the single-target approach is that once the target is identified, the most potent, high-affinity ligand possible that activates or inactivates the target is needed. It is
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frequently stated that modifying the activity of a single target with a high-affinity drug is the most desirable approach because it minimizes undesirable side effects [11] . This assumption is, with rare exceptions, not true. The simple reason is that most proteins have a required normal function in the body so that their near complete inactivation in order to achieve a therapeutic effect often results in toxic outcomes. In addition, very few targets or target pathways are unique, resulting in modification of multiple targets or activities (polypharmacology) [29] . A recent example of this is the inhibition of g-secretase by the AD drug candidate semagacestat that resulted in both the worsening of clinical measures and an increased risk of skin cancer [30] . The reason for the CNS toxicity is unknown, but the skin cancer was predictable since blocking the cleavage of another g-secretase substrate, Notch 1, in mice leads to skin cancer [31] . An additional case in point is the repeated failure of glutamate-receptor antagonists for the treatment of ischemic stroke with the idea that they will inhibit ischemia-induced excitotoxicity, with insufficient appreciation of the fact that glutamate receptors are necessary for normal CNS function [32] . Phenotypic screening Due to the innate difficulties with the singletarget approach outlined above, what are the alternative approaches to drug screening that are likely to yield therapeutically relevant compounds for neurodegenerative diseases? Since there are rodent models of most of the neurodegenerative diseases, it could be argued that screening directly in animals is the best approach. However, it is now widely recognized that efficacy in animal models does not directly translate into success in clinical trials. In fact, over 400 compounds reportedly improve behavior or pathology in transgenic AD mice [17] , but no disease-altering AD therapeutics have emerged from these results. Therefore, before testing drug candidates in animals, a more rigorous invitro drug-candidate selection process is required. This can be achieved by creating cell-based assays that define molecular toxicity pathways relevant to age-associated neurodegeneration, rather than preselected molecular targets, and requiring that drug candidates work in multiple assays, not just one. These screening paradigms require disease relevance, reproducibility and reasonable throughput. Although alternatives clearly exist, an example will be used from our laboratories.
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Key Terms Multi-target drugs:
Discovery and development of drug candidates that alter more than one molecular target or target pathway within cells.

Natural products: Chemical

substance produced by a living organism.

We have described seven assays that represent distinct neurotoxicity pathways related to aging and neurodegenerative diseases (Table1) [33,34] . The levels of neurotrophic factors such as BDNF and NGF decline with age, and do so to a greater extent in AD and related conditions. In an assay of trophic factor withdrawal, primary embryonic cortical cells are plated at low density in serum-free medium. Under these conditions, the cells die within 2days, but can be rescued by combinations of neurotrophic growth factors, but not by one alone [35] . In contrast, cell death is prevented by compounds that activate neurotrophic cell signaling pathways. In a related assay, neurotrophic compounds are able to rescue a clone of the hippocampal nerve cell line HT22 expressing the BDNF receptor, TrkB, from serum starvation under conditions where the cells can be protected by BDNF [36] . BDNF is a molecule that is also involved in promoting memory and is reduced in the brain with age and in AD, as well as in other neurological disorders [3739] . The BDNF pathway has long been considered a drug target for AD [24] . Cells lacking TrkB are used to determine if compounds that rescue the cells from serum starvation activate the receptor or act on a signaling pathway downstream of the receptor. The oxytosis assay tests the ability of compounds to rescue cells from oxidative stressinduced programmed cell death caused by GSH depletion [40] . A reduction in GSH is a common denominator of old age and is accelerated in essentially all chronic CNS diseases [25] . The depletion of GSH leads to lipoxygenase activation, reactive oxygen species production and

calcium influx [40] . Because of the generality of the toxicity pathway in oxytosis and its mechanistic association with aging and essentially all age-associated neurodegenerative diseases, it has been used as the primary screen in most of our work. Recently, this pathway was rediscovered in the context of cancer and given another name[41] . Additional toxicity assays include protection from glucose starvation [33] , prevention of the loss of mitochondrial energy metabolism and ATP in an invitro ischemia model [42,43] , the ability to block extracellular amyloid toxicity using rat hippocampal neurons [44] , inhibition of intracellular Ab toxicity [45] and excitotoxicity [46] . Excitotoxicity can potentiate any condition where cell death is involved, in which nearby nerve cells have ionotropic glutamate receptors because dead cells release glutamate and glutamine. Glutamine is rapidly converted to glutamate by the abundant glutaminase enzyme[46] . This drug-discovery scheme has been effective at identifying several highly potent families of neuroprotective molecules. These include the curcumin derivative CNB-001 [33] that is effective in animal models of memory enhancement [47] , stroke [48] and traumatic brain injury [4951,
Valera Martin E, Chen Q, Dargusch R etal. Reduction of proteotoxicity by the limited activation of the unfolded protein response (2012), Manuscript in preparation] ,

Table1. Phenotypic screening assays listed in order of potential relevance to age-associated neurodegenerative disease.
Assays
Oxytosis Trophic factor withdrawal BDNF-like Invitro ischemia

Cell type
HT22 or primary cortical neurons Primary cortical neurons HT22 HT22

Protection target
Glutathione depletion and oxidative stress Loss of trophic support

Ref.
[25,40] [33,35]

Intracellular amyloid MC65 cells toxicity Extracellular amyloid Hippocampal neurons toxicity Excitotoxicity Primary cortical neurons

[33,36] Loss of BDNF-dependent neuroprotection [42,43] Reduced energy metabolism, loss of ATP [27,33,45] Proteotoxicity

Toxicity of b amyloid polymers Ionotropic glutamate receptor calcium flux

[33,44] [46]

and the highly potent neuroprotective compound J147, which is effective in memory enhancement in both normal and AD transgenic mice, and neuroprotective in AD animals [34] . Both compounds have neurotrophic and BDNFlike activities that are independent of the BDNF receptor, but also cause the accumulation of BDNF protein. Therefore, it may be possible to use BDNF levels in the cerebrospinal fluid as a biomarker for drug function in clinical trials. In addition, these assays were used to discover the rare flavone, fisetin, which has therapeutic efficacy in multiple animal disease models including memory enhancement [52] , stroke [43,53] , Huntingtons disease [54] and diabetic complications [55] , and a subset of these assays guided the synthesis of more potent fisetin derivatives [56] . In summary, a drug-discovery paradigm based upon multiple, age-related pathologies can yield compounds that get into cells, are likely not toxic, and target multiple disease-related toxicity pathways, not specific molecular targets. Lead compounds identified by these assays can then be improved by medicinal chemistry with
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An alternative approach to drug discovery for Alzheimers disease dementia

the maintenance of multiple activities, put into animal models and advanced toward the clinic. Furthermore, while the molecular targets may not be immediately identified, current technologies can be used to rapidly screen new drug candidates against essentially all known CNS receptors, transporters, and related molecules that are known to cause off-target toxicities. An example is the Ricerca lead profile screen. This fact negates the often used claim against multitarget drugs that a single target is required to limit off-target effects. Given this approach, what is the best source of the lead compounds that target multiple neuroprotective pathways? The relevance of natural products as lead compounds As outlined above, the single-target, highaffinity drug approach is unlikely to be effective for neurodegenerative diseases. To identify better lead compounds for diseases that require a long duration of drug exposure, a basic concept from immunology may be relevant. The efficacy of an immune response is determined by a concept called avidity, which dictates that the strength of the interaction of antigen molecules with several epitopes is a multiple of the affinities of the interactants and is not simply the sum of their affinities. Therefore, in the CNS, a drug that modestly modulates multiple relevant target pathways may be more potent in terms of therapeutic outcome than one that completely shuts down or activates a single activity. The most promising compounds of this class are derived from the original pharmacopoeia, plant-based polyphenolics. Natural products have a wide range of molecular targets because they resemble biosynthetic intermediates to a greater extent than synthetic drugs and are therefore able to compete with substrates for multiple enzymes [57] . The evolutionary selection for this type of small molecule is based upon the fact that the enormous number of plant secondary metabolites are made by a very limited repertoire of enzymes. Despite the fact that the majority of all FDA-approved drugs are derived from chemical scaffolds related to natural products, natural product lead compounds have fallen out of favor with the pharmaceutical world for a number of reasons, most of which are unjustified or reflect the failure of the industrys technology, not the compounds themselves. Drug developers frequently argue that polyphenolic compounds are poor lead compounds because they nonspecifically bind and modify
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the activity of many proteins, they lack a SAR, they simply function as general antioxidants, and they sometimes work in animals, but not in clinical trials. We believe that only one of these claims may be correct for technical reasons in the context of the most frequently used drugdiscovery paradigm, HTS against purified protein targets. But they are clearly not valid when cell-based phenotypic screening procedures are used. The protein-based, high-throughput screens generate false-positive hits with many hydrophobic small molecules, but in whole cells a certain level of stickiness may be an advantage for a drug because it localizes the drug to the cell surface where it can interact with a cell surface receptor or enter the cell by a number of mechanisms. This may, in fact, be the reason plants make these sticky polyphenolics. It should be pointed out that most, if not all, protein growth factors are quite basic and nonspecifically adhere to cell surfaces via heparin binding in order to more readily associate with their receptors. When the cells heparin-binding site is removed, the receptor binding and growth factor potency decrease significantly [58] . Conversely, drug candidates for CNS diseases may be missed because they need to have a high cLogP (hydrophobicity) to get through the bloodbrain barrier, and because of this they nonspecifically stick to the extensive plastic hardware associated with robotic screening. With respect to the SARs of polyphenolics, we have recently shown that their biological activities are exquisitely sensitive to ring substitutions, but that it is possible to improve their medicinal chemical properties and potency at the same time [34,56] . Synthetic compounds that have been made based upon curcumin and fisetin are many fold more potent than the parent compounds, maintain their biological activities and have much better medicinal chemical properties [34,56] . Therefore, it is indeed possible to chemically modify plant polyphenolics and maintain multiple biological activities at the same time. Furthermore, many polyphenolics are not direct antioxidants, and their biological activity is unrelated to antioxidant status [56] . In fact, it is impossible for the direct antioxidant properties of polyphenolics to have any relevance in animals because of the overwhelming antioxidant capabilities of endogenous antioxidant molecules in blood and tissues [59] . Finally, among the clinical trials for AD drugs published on the NIH website [101] , there have been no large-scale trials for any single
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polyphenolic compound, only two trials using mixtures of compounds derived from plants (tea and ginkgo), and two very small, short and underpowered trials with curcumin. It follows that SAR-driven medicinal chemistry based upon the multiple cell culture selection assays described above is a viable alternative to the single-target HTS screening paradigm that has dramatically failed for patients with AD, stroke and other neurodegenerative diseases. The current distaste of the pharmaceutical industry for polyphenolic lead compounds is not based upon their lack of therapeutic potential, and should not rule out the very great value that they may have for AD therapy. Nor should a drug-discovery program based upon cell biology rather than a single selected target molecule be frowned upon. Penicillin and many other drugs saved vast numbers of lives before their molecular targets were identified. Future perspective There is now something of a consensus that therapeutics directed solely against the amyloid pathway are unlikely to be effective in the treatment of most cases of AD [14,60,61] , and we have discussed why the single target approach will very likely not work. Therefore, either combination therapies or multi-target drugs will be required for the treatment of AD. Combinations are becoming much more common, for example in AIDS therapy [62] and in blood pressure management [63] . In the case of AD, perhaps combinations of nontoxic, antiamyloid compounds such as curcumin [64] and potent neuroprotective compounds such as J147
Executive summary

may be effective. If there is a real desire to find an AD therapeutic in an expedient manner, natural products such as fisetin or combinations of natural products are likely to be as effective as any synthetic drug, but due to the inability to defend patents on natural products and the profit imperative of the pharma ceutical world, these compounds are unlikely to ever get into clinical trials. Alternatively, an approach to AD drug discovery focused on synthetic derivatives of compounds such as fisetin that affect multiple, age-associated toxicity pathways rather than individual molecular targets may yield the best clinical candidates. In either case, the mindset of the pharmaceutical industry must change from a set of fixed ideas about the drug-discovery process to procedures based upon a greater fundamental understanding of the pathways involved in the disease itself and the use of drug selection criteria that reflect this understanding.
[34]

Acknowledgements
The authors thank E Villafranca and J Lewerenz for critical reading of the manuscript.

Financial & competing interests disclosure

This article was supported by the Alzheimers Drug Discovery Foundation and the Fritz Burns Foundation. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

There are no drugs that stop the progression of any age-associated neurodegenerative disease. The pharmaceutical industry has focused almost exclusively on the single-target, high-affinity, drug-discovery approach to these diseases, but have yet to make an effective drug. Normal aging and age-associated neurodegenerative disease is caused by a confluence of many toxicities, not just one. Therefore, an alternative approach is to use a drug-discovery paradigm based upon screening procedures that reflect the biology of aging and the disease pathology without preselecting a molecular target. Because of the complexity of the diseases, multi-target lead compounds should be chosen. Plant secondary metabolites (natural products) are the ideal source of lead compounds because they have evolved to interact at low affinity with multiple enzymes. The chemical scaffolds of the majority of drugs in the clinic are based upon those of natural products, and our laboratories have recently completed a series of proof-of-principle experiments demonstrating that some polyphenolic natural products are exceptionally effective in halting disease progression in a wide variety of animal models of age-associated neurodegenerative disease and ischemia, and that the potency and medicinal chemical properties of these compounds can be dramatically improved without loosing their multi-target activities. It is concluded that the single-target, high-affinity drug approach to neurodegenerative disease is unlikely to identify compounds that halt disease progression, and that drug-discovery paradigms based upon phenotypic screening are more likely to succeed.

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