Natural Antioxidants in Hybrids for the Treatment of Neurodegenerative Diseases: A Successful Strategy?

Future Medicinal ChemistryVol. 9, No. 8 EditorialFree AccessNatural antioxidants in hybrids for the treatment of neurodegenerative diseases: a successful strategy?Sandra Gunesch, Simon Schramm & Michael DeckerSandra Gunesch Pharmazeutische & Medizinische Chemie, Institut für Pharmazie & Lebensmittelchemie, Julius-Maximilians-Universität Würzburg, Am Hubland, D-97074 Würzburg, Germany, Simon Schramm Pharmazeutische & Medizinische Chemie, Institut für Pharmazie & Lebensmittelchemie, Julius-Maximilians-Universität Würzburg, Am Hubland, D-97074 Würzburg, Germany & Michael Decker*Author for correspondence: E-mail Address: michael.decker@uni-wuerzburg.de Pharmazeutische & Medizinische Chemie, Institut für Pharmazie & Lebensmittelchemie, Julius-Maximilians-Universität Würzburg, Am Hubland, D-97074 Würzburg, GermanyPublished Online:9 May 2017https://doi.org/10.4155/fmc-2017-0073AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInReddit Keywords: drug designoxidative stressreactive oxygen speciesFirst draft submitted: 20 March 2017; Accepted for publication: 21 March 2017; Published online: 9 May 2017It is well established that oxidative stress is one of the characteristics of neurodegenerative diseases, such as Alzheimer's disease (AD) and Parkinson's disease [1]. Free radicals, that is, reactive oxygen species (ROS) and reactive nitrogen species, are damaging a variety of biomolecules like lipids, proteins and DNA. Such radicals are generated in the pathology of neurodegenerative diseases and eventually lead to neuronal cell death. They contribute to the overall pathophysiology and pathogenesis of these diseases, but are not their sole cause. In fact, it is unclear until now at which stage of the pathophysiological cascades oxidative stress occurs. Irrespective of the stage though, it is obvious that ROS enhance if not multiply the effects of the neuropathological cascades [1].In AD, for example, pathological mechanisms mainly include hyperphosphorylation of T proteins, reduction of acetylcholine receptor levels, mitochondrial dysfunction, neuronal DNA damage, cerebral glucose hypometabolism, aggregation of β-amyloid peptides (Aβ) and oxidative stress [1,2]. With several mechanisms contributing to a complex pathological network, development of therapeutically applicable drugs is extremely challenging. A drug addressing only one biological target is probably not going to yield any success in stopping neurodegenerative diseases. Therefore, multitarget or hybrid drugs – combining two or more pharmacologically active units – that are capable of modifying several biological targets, may overcome the shortcomings of a single-target drug. Many multitarget compounds have been developed in recent years [3]. A very significant number of those compounds contain – mostly natural – antioxidants to target oxidative stress, therefore counteract the formation of ROS and are claimed to hold the potential to interfere with the neuropathological cascades [3,4]. The term 'antioxidant' is actually kind of vague and unspecific in both the chemical and biological sense and more specific terms are desirable. The combination of natural antioxidants with other moieties can often lead to disadvantageous drug properties, such as high MW, and therefore low bioavailability, poor solubility, rapid metabolism and hampered blood–brain barrier penetration. Problems typical for natural antioxidants also include rapid CYP metabolism – often 3A4 – and CYP induction.We want to shortly discuss both the physicochemical as well as the cell-based in vitro assays that have been applied for assessing the 'antioxidant' properties of multitarget compounds containing antioxidant moieties and discuss what the outcomes of these assays actually mean, before we report on some very recent in vivo studies.In vitro cell-free assaysNatural antioxidants belong to very diverse chemical classes including carotenoids, vitamins C and E, stilbene derivatives, curcuminoids, phenolic acids (such as cinnamic, caffeic and ferulic acids) and flavonoids (such as quercetin, rutin and genistein). Due to this structural diversity it is assumed that they exhibit different modes of action [4]. The cell-free assays used to assess antioxidant activity in vitro are fairly similar in what properties they determine, namely their ability to scavenge radicals. These assays include the DPPH assay, an ABTS scavenging assay, the NBT assay and the oxygen radical absorbance capacity assay [5]. The oxygen radical absorbance capacity assay, for example, provides quantification of the test compounds' abilities to protect fluorescein from being oxidized by scavenging peroxyl radicals. Tacrine–ferulic acid hybrids, for example, show even higher values than the natural antioxidant ferulic acid by itself [6,7].The ability of molecules to scavenge radicals determined in these assays is a prerequisite and an indication for the occurrence of effects in cell-based assays or in vivo, but not more than that. For that reason, more advanced cell-based assays are indispensable for better prediction of 'antioxidant' and therapeutically desirable activities in vivo.In vitro cell-based assaysA variety of set-ups can be used in vitro to simulate disease-like conditions in cells, including oxidative stress, and to investigate the neuroprotective properties of test compounds. To quantify the protection against ROS-mediated apoptosis, cells are incubated with ROS-inducing or producing agents in the presence of the antioxidant compounds and the experimental read-out is the reduced cell viability determined in an MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) or a chemically related assay.Different cell lines are established that reflect the quite diverse origins of ROS. Mouse hippocampal HT-22 cells are used to study neuroprotective effects of hybrids toward ROS produced by glutamate-induced toxicity [8]. Presence of high glutamate concentrations in culture medium leads to glutathione depletion inside the cells. This causes ROS accumulation leading to cell death by apoptosis. Aβ exposure can also induce oxidative damage in cells and lead to apoptosis due to Aβ-induced mitochondrial dysfunction [9]. Protection against Aβ-induced oxidative stress through ferulic–tacrine acid hybrids was shown in PC-12 cells [10], a pheochromocytoma cell line of the rat adrenal medulla. MC-65 and SH-SY5Y are human neuroblastoma cell lines and are also applied in such assays since they derive from neuronal tissue and are therefore very suitable to investigate neurodegenerative diseases. For example, SH-SY5Y cells were used to investigate neuroprotection of tacrine–melatonin hybrids against Aβ-induced toxicity [11]. This cell line also served to investigate ROS production induced by application of hydrogen peroxide and rotenone. Rotenone specifically inhibits mitochondrial complex I leading to increased ROS formation and mitochondrial-induced apoptosis [12], whereas hydrogen peroxide causes DNA damage and lipid peroxidation, being a source of nonselective reactive oxygen species [13].Drawbacks & first in vivo dataCurcumin represents a prime example for a natural product for which numerous beneficial effects have been claimed and that has also been used in hybrid molecules. A remarkable review has been published recently [14], that discusses the positive results of numerous in vitro assays found for this compound and derivatives thereof and the article raises some questions: low solubility, instability due to the oxidation-prone phenolic and enolic hydroxyl groups, the Michael system and its fluorescent properties, respectively, are likely leading to false-positive results in vitro for curcumin and curcumin-derived compounds. Albeit to a lower extent, these false positives can also occur with other natural products that, for example, possess a Michael system.Numerous multitarget compounds containing antioxidant moieties have been claimed to be lead structures for AD therapy after their investigation in one or more of the above described in vitro assays (cell-free and/or cell-based). Nevertheless, only in vivo studies giving access to information about bioavailability, blood–brain barrier penetration and in vivo neuroprotection plus antineuroinflammation, are really answering the question, whether such compounds hold true therapeutic potential. Only very recently first in vivo data has become available [15]. A melatonin–curcumin hybrid was applied to 4-month-old transgenic mice and in vitro results could be confirmed in vivo with regard to reduction of markers of oxidative stress on the molecular level [16], even though no behavioral effects have been reported. However, a tacrine–ferulic acid hybrid described above showed reduction of oxidative stress markers as well as positive effects on learning and memory in transgenic AD mice [10].SummaryTaken together, multitarget (hybrid) compounds and other chemical compounds containing antioxidant moieties are prone to several disadvantages: metabolic instability, false-positive results in (some – nonsuitable) in vitro assays and unknown modes of action. There are also problems of transferring the relevance of in vitro data to an in vivo setup. Nevertheless, this does not mean that antioxidants are not versatile and promising components of hybrids, but only specific assays for assessing the compounds' activities are suitable. The methods deemed suitable have to be carefully selected on the basis of the chemical structure of the compounds under investigation. Positive results should not be overinterpreted. The often unknown modes of action of natural antioxidants with regard to neuroprotection in vitro and in vivo still hamper the development of effective hybrid molecules.Financial & competing interests disclosureFinancial support by the German Research Foundation (Deutsche Forschungsgemeinschaft) DFG DE 1546/6-1 and 6-3 is gratefully acknowledged. 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.References1 Thanan R, Oikawa S, Hiraku Y et al. Oxidative stress and its significant roles in neurodegenerative diseases and cancer. Int. J. Mol. Sci. 16(1), 193–217 (2015).Crossref, Google Scholar2 Karran E, De Strooper B. The amyloid cascade hypothesis: are we poised for success of failure? J. Neurochem. 139(Suppl. 2), 237–252 (2016).Crossref, Medline, CAS, Google Scholar3 Ismaili L, Romero A, do Carmo Carreiras M, Marco-Contelles J. Multi-target directed antioxidants as therapeutic agents: putting the focus on the oxidative stress. In: Design of Hybrid Molecules for Drug Development. Decker M (Ed.). Elsevier, Oxford, UK, 5–46 (2017).Crossref, Google Scholar4 Pisoschi AM, Pop A. The role of antioxidants in the chemistry of oxidative stress: a review. Eur. J. Med. Chem. 97, 55–74 (2015).Crossref, Medline, CAS, Google Scholar5 López-Alarcón C, Denicola A. Evaluating the antioxidant capacity of natural products: a review on chemical and cellular-based assays. Anal. Chim. Acta 763, 1–10 (2013).Crossref, Medline, CAS, Google Scholar6 Benchekroun M, Bartolini M, Egea J et al. Novel tacrine-grafted Ugi adducts as multipotent anti-Alzheimer drugs: a synthetic renewal in tacrine-ferulic acid hybrids. ChemMedChem 10(3), 523–539 (2015).Crossref, Medline, CAS, Google Scholar7 Fang L, Kraus B, Lehmann J, Heilmann J, Zhang Y, Decker M. Design and synthesis of tacrine-ferulic acid hybrids as multi-potent anti-Alzheimer drug candidates. Bioorg. Med. Chem. Lett. 18(9), 2905–2909 (2008).Crossref, Medline, CAS, Google Scholar8 Kling B, Bücherl D, Palatzky P et al. Flavonoids, flavonoid metabolites, and phenolic acids inhibit oxidative stress in the neuronal cell line HT-22 monitored by ECIS and MTT assay: a comparative study. J. Nat. Prod. 77(3), 446–454 (2014).Crossref, Medline, CAS, Google Scholar9 Lin MT, Beal MF. Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. Nature 443(7113), 787–795 (2006).Crossref, Medline, CAS, Google Scholar10 Pi R, Mao X, Chao X et al. Tacrine-6-ferulic acid, a novel multifunctional dimer, inhibits amyloid-β-mediated Alzheimer's disease-associated pathogenesis in vitro and in vivo. PLoS ONE 7(2), e31921 (2012).Crossref, Medline, CAS, Google Scholar11 Fernandez-Bachiller MI, Perez C, Campillo NE et al. Tacrine-melatonin hybrids as multifunctional agents for Alzheimer's disease, with cholinergic, antioxidant, and neuroprotective properties. ChemMedChem 4(5), 828–841 (2009).Crossref, Medline, CAS, Google Scholar12 Sipos I, Tretter L, Adam-Vizi V. Quantitative relationship between inhibition of respiratory complexes and formation of reactive oxygen species in isolated nerve terminals. J. Neurochem. 84(1), 112–118 (2003).Crossref, Medline, CAS, Google Scholar13 Halliwell B, Aruoma OI. DNA damage by oxygen-derived species. Its mechanism and measurement in mammalian systems. FEBS Lett. 281(1–2), 9–19 (1991).Crossref, Medline, CAS, Google Scholar14 Nelson KM, Dahlin JL, Bisson J, Graham J, Pauli GF, Walters MA. The essential medicinal chemistry of curcumin. J. Med. Chem. 60(5), 1620–1637 (2017).Crossref, Medline, CAS, Google Scholar15 Muñoz-Torrero D. Multi-target anti-Alzheimer hybrid compounds: do they work in vivo? In: Design of hybrid molecules fordrug development. Decker M (Ed.). Elsevier, Oxford, UK, 167–192 (2017).Crossref, Google Scholar16 Chojnacki JE, Liu K, Yan X et al. Discovery of 5-(4-hydroxyphenyl)-3-oxo-pentanoic acid [2-(5-methoxy-1H-indol-3-yl)-ethyl]-amide as a neuroprotectant for Alzheimer's disease by hybridization of curcumin and melatonin. ACS Chem. Neurosci. 5(8), 690–699 (2014).Crossref, Medline, CAS, Google ScholarFiguresReferencesRelatedDetailsCited ByOxidation degree of a cell membrane model and its response to structural changes, a coarse-grained molecular dynamics approach16 October 2020 | Journal of Biomolecular Structure and Dynamics, Vol. 40, No. 5Azobioisosteres of Curcumin with Pronounced Activity against Amyloid Aggregation, Intracellular Oxidative Stress, and Neuroinflammation5 March 2021 | Chemistry – A European Journal, Vol. 27, No. 19Sterubin: Enantioresolution and Configurational Stability, Enantiomeric Purity in Nature, and Neuroprotective Activity in Vitro and in Vivo15 May 2020 | Chemistry – A European Journal, Vol. 26, No. 327-O-Esters of taxifolin with pronounced and overadditive effects in neuroprotection, anti-neuroinflammation, and amelioration of short-term memory impairment in vivoRedox Biology, Vol. 29Novel multi target-directed ligands targeting 5-HT4 receptors with in cellulo antioxidant properties as promising leads in Alzheimer's diseaseEuropean Journal of Medicinal Chemistry, Vol. 182The Impact of Natural Compounds on the Treatment of Neurodegenerative DiseasesCurrent Organic Chemistry, Vol. 23, No. 3Regioselective synthesis of 7-O-esters of the flavonolignan silibinin and SARs lead to compounds with overadditive neuroprotective effectsEuropean Journal of Medicinal Chemistry, Vol. 146Neurodegenerative drug discovery: building on the past, looking to the futureMaria Laura Bolognesi15 May 2017 | Future Medicinal Chemistry, Vol. 9, No. 8 Vol. 9, No. 8 Follow us on social media for the latest updates Metrics History Published online 9 May 2017 Published in print May 2017 Information© Future Science LtdKeywordsdrug designoxidative stressreactive oxygen speciesFinancial & competing interests disclosureFinancial support by the German Research Foundation (Deutsche Forschungsgemeinschaft) DFG DE 1546/6-1 and 6-3 is gratefully acknowledged. 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.PDF download