Introduction. My PhD research project focused on development and application of analytical methodologies in the field of Alzheimer’s Disease (AD) drug discovery. AD is a neurodegenerative disease clinically characterized by progressive memory loss, cognitive dysfunction and behavioural change, and is the most common type of dementia. The major neuropathological hallmarks of AD are amyloid plaques, consisting of β-amyloid peptide, and neurofibrillary tangles (NFTs) mostly composed of paired helical filaments of hyperphosphorylated tau. Nowadays, AD is considered a global health concern for two main reasons: firstly, due to the gradual aging of the population, its incidence is estimated to increase; secondly, the approved drugs only alleviate the symptoms and delay the cognitive decline. The drugs currently used in the treatment are an antagonist of glutamate NMDA receptor (Memantine) and inhibitors of the enzyme acetylcholinesterase (Galantamine, Rivastigmine and Donepezil). Recently a controversial drug (Aducanumab) which is a human IgG1 anti-Aβ monoclonal antibody specific for β-amyloid oligomers and fibrils, passed successfully all the clinical trials. Until recently, AD has had no treatment that can be considered disease-modifying, because the therapies in use do not affect its incidence or the known AD altered pathways. Besides, late-stage clinical trials of new drugs failed due to the multifactorial nature of AD. Thus, a promising strategy might be designing multi-target directed ligands (MTDLs) which could interact simultaneously on different pathways.
In this context, the main goals of my Ph.D. thesis were to develop advanced analytical methodologies suitable for characterizing new potential MTDLs. This characterization would occur both in the first discovery stage, i.e. at the high-throughput screening level on isolated targets, and in the development phase in order to evaluate bioavailability and pharmacokinetics in vivo. The main targets involved in the AD pathogenesis taken into consideration were two enzymes, glycogen-synthase kinase 3 β (GSK-3β) and histone deacetylases (HDAC). GSK-3β is responsible for the hyperphosphorylated tau proteins involved in the intraneuronal neurofibrillary tangles, while HDAC for the histone deacetylation. Both GSK-3β and class I–II HDAC inhibitors are able to shift microglia from the neurotoxic activation (proinflammatory) to the neuroprotective (anti-inflammatory) phenotype. Moreover, the combined inhibition of GSK-3β and HDACs induces synergistic neuroprotective effects, potentially resulting in an improved therapeutic selectivity. My study also focused on another target, i.e. the β-amyloid (Aβ) aggregation process. This aggregation starts incidentally with the formation of oligomers species that are reorganized into protofibrils and fibrils, the latter found in amyloid plaques. Oligomers accumulated in the brain of AD patients are suggested to be the most toxic species for the cells. By permeabilizing cellular membranes, they can initiate a series of events leading to cell dysfunction and apoptosis. Due to the critical role of Aβ aggregation process in the development and progression of AD, many studies have been carried out on the characterization of molecules capable of contrasting this process. Furthermore, the Aβ cascade is strictly connected to other AD pathological events, including the formation of neurofibrillary tangles (NFTs), inflammatory reactions, increased oxidative stress and mitochondrial dysfunction, which are all factors of cell death and dementia.
Characterization of new potential active molecules in AD drug discovery.In view of developing methods to select HIT compounds towards main targets involved in the first stage of AD pathogenesis, the project focused on the optimization and application of an in vitro high-throughput luminescent assay for screening GSK-3β inhibitors. This is a homogeneous, non-radioactive kinase assay that proved to be solid, reproducible, sensitive and suitable for testing several compounds (ATP-competitive and ATP-non competitive inhibitors).
The method was applied for the characterization of the First-in-Class GSK-3β/ HDAC dual Inhibitors designed by the medicinal chemistry group supervised by Professor Andrea Milelli, Department for Life Quality Studies, University of Bologna. The in vitro high-throughput luminescent assay was able to select among the new series of molecules a promising hit compound with an inhibitory activity towards GSK-3β in the low micromolar range (3.19 ± 0.08 μM ). This compound induces an increase in histone acetylation and a reduction of tau phosphorylation. From its biochemical characterization it was demonstrated that it is non-toxic in SH-SY5Y neuroblastoma cell line. Moreover, it promotes neurogenesis and shows immunomodulatory effects. For concentrations ‹100 μM, it proved no lethality in a wt-zebrafish model and it has a high water solubility. For all these reasons this GSK-3β/HDAC dual inhibitor could be considered interesting to develop an innovative disease-modifying agent.
The luminescent kinase assay was also suitable to screen natural compounds. Notably, different alkaloids isolated at the Department of Pharmaceutical Botany, Charles University in Hradec Králové (Czech Republic) from various Amaryllidaceae plant species were characterized towards GSK-3β. This class of natural molecules proved to be interesting in the research of MTDL for being active on various targets given its many properties (antibacterial, antimalarial, antitumor and AChE inhibitors). In particular one of the drugs currently approved for AD (Galantamine) is an amaryllidaceae alkaloid. After a preliminary screening at single test concentration (50 µM), the most promising compounds were the alkaloids with a homolycorine and lycorine structure, whose inhibitory potency was then evaluated with IC50 values in the micromolar range. It has already been possible to determine some SARs (hydroxyl substitution in position 2 and tetrahydrofuran ring opening) but it will be necessary to characterize a wider range of natural and semi-synthetic compounds for further investigation.
Aggregation of the Aβ peptide, in particular Aβ42, is another crucial process in AD pathogenesis. Recent studies have proved the ability of carbon monoxide-releasing molecules (CORMs) to protect neurons and astrocytes from Aβ peptide toxicity. In fact, CORMs are able to carry and release controlled levels of CO that exert anti-inflammatory and anti-apoptotic activities at physiologically relevant concentrations. In order to investigate the reactivity of CORM-2 and CORM-3 with Aβ42 in vitro and the potential inhibition of its aggregation a multi-methodological approach has been applied. The ESI-MS analysis allowed the detection of stable Aβ42/CORMs adducts, involving the addition of the Ru(CO)2 portion of CORMs at histidine residues on the Aβ42 skeleton.
Furthermore, thioflavin T fluorescence assay showed that CORMs through formation of stable adducts with Aβ42 are able to inhibit its aggregation. The inhibitory effect was confirmed also by MS analysis. Moreover, CD studies of Aβ42 spectra recorded in the absence and in the presence of CORM-3 at a 1:1 molar ratio showed the ability of CORM-3 to stabilize the peptide in its soluble, unordered conformation. This stabilization delayed its misfolding and aggregation. The multi-methodological study developed allowed to deeply investigate the neuroprotective mechanism of CORMs, that can be considered promising active molecules towards amyloid aggregation and its toxicity.
A lipidomic approach to investigate Aß42 peptide toxicity on neuronal cells in view of Alzheimer’s Disease drug discovery. Given the central role of the amyloid pathway in the multifactorial nature of AD, I dedicated large part of my research to further explore the amyloid aggregation process and its mechanism of toxicity. Different studies highlighted connections between amyloid aggregation and AD extensive oxidative stress. Interestingly, β- amyloid aggregation and lipid peroxidation are strictly related. A possible explanation for this connection could be ascribed to the hydrophobic nature of the oligomeric peptide which makes it able to intercalate into the neuronal lipid bilayer.
We therefore aimed at clarifying the mechanism of Aß42 aggregation species toxicity through a lipidomic study on the most important classes of lipids, which we identified for the first time in differentiated SH-SY5Y neuroblastoma cells: Phosphatidylcholines, Lyso-Phosphatidylcholines, Ether-Phosphatidylcholines, Phosphatidylethanolamines, Sphingomyelins and Triacylglycerols. In fact, many mechanisms of toxicity have been proposed as a consequence of the effects of oxidative stress on poly unsaturated fatty acids (PUFA), included in lipids structures. Part of the work was carried out during the period I spent at the Institute of Pharmaceutical Science in Tuebingen (Germany) in the research group of Professor Laemmerhofer, whose field of expertise is grounded in lipidomic analysis. For this purpose, we optimised an LC-MS method to determine the lipid profile of differentiated human neuroblastoma derived SH-SY5Y cells. Cells were treated with increasing concentrations of Aß42 (2-50 µM) at different times of incubation, then the lipid extraction was carried out with IPA: H2O (90:10 v/v). LC-MS analyses of extracts were performed by RP-UHPLC system coupled to a high-resolution quadrupole TOF mass spectrometer in comprehensive data-independent SWATH acquisition mode. Data processing was achieved via MS-DIAL (version 4.24). Each lipid class profiling in SHSY5Y cells treated with Aβ42, was compared to those obtained for the untreated cells to identify some distinctive alterations in their lipidomic pattern. This approach was found suitable to underline some peculiar lipid alterations due to Aβ42 different aggregation species and to explore deeply the cellular response mechanisms to the toxic stimulus. Further studies will be conducted to identify some specific PC and PE oxidized species, which are useful as biomarkers. Thus, the present method can also be applied to underline the mechanism of action of amyloid aggregation inhibitors, able not only to inhibit the aggregation process, but also to reduce lipid alterations, in view of AD new drugs discovery.
Development and validation of analytical methods for the pharmacokinetic profile of nutraceutical formulations. In view of taking into consideration prevention aids of AD, a correlation between antioxidants and polyunsaturated fatty acids intake from diet have been demonstrated to play a key role in the protection of cognitive performance. In light of these premises, polyphenols are explored for their properties as interesting active agents able to prevent the oxidative stress as risk factor in the early altered pathways of the pathogenesis of AD. Therefore, in order to clarify their pharmacokinetics, a new chromatographic method by Ultra High Performance liquid chromatographic (UHPLC) technology, has been developed and validated for the determination of polydatin and resveratrol, as potential metabolite, in human plasma.
Polydatin is the natural precursor of resveratrol combined with glucose. Their antioxidant properties are known. Moreover, Polydatin demonstrated to have higher scavenging activity against hydroxyl radicals than resveratrol both in vitro and in vivo. This can be ascribed to its higher oral bioavailability. Thus, the metabolism of polydatin in vivo has been investigated through many studies. In rats it was showed that polydatin undergoes extensive first-pass deglycosylation and glucoronidation. Therefore, polydatin is primarily metabolized to resveratrol in the small intestine and liver. Then, resveratrol is metabolized into glucoronidated form. Resveratrol and glucoronidated resveratrol are mainly excreted by the kidneys. In this view, dietary supplements containing polydatin are gaining great interest. Since no studies were performed in humans, it was necessary to develop analytical methods for the characterization of polydatin pharmacokinetic. This could determine the right posology and elucidate the polydatin metabolism in humans.
For these reasons, my study focused on developing an Ultra High Performance liquid chromatographic (UHPLC) method for the determination of polydatin and resveratrol, as potential metabolite, in human plasma. After the optimization of the chromatographic conditions, the method has been validated on spiked human plasma samples. The optimized extraction allowed to obtain analytes recovery up to 98,48 ± 4,03%. Then, the isocratic elution in reversed phase mode provides a fast separation of polydatin and resveratrol (less than 10.0 min). Chromatographic analysis was performed on a C18, 10 cm x 3.0 mm, 2.7 µm stationary phase, by using triethanolamine phosphate solution (0.1 M, pH= 3.7) and ACN 85:15 (v/v) as mobile phase at a flow rate of 0.5mL/min. The UV detector was set at 306 nm for the analysis of both polydatin and resveratrol. The method resulted sensitive with a limit of detection (LoD) and the limit of quantification (LoQ) for polydatin in plasma samples found to be 7,82 ± 0,38 nM and 26,06 ± 1,28 nM respectively. The method was found to be accurate and precise with a coefficient for intra- and inter-day variation below 5%. The method was then applied to the analysis of plasma samples from orally treated volunteers with nutritional supplements containing polydatin. The polydatin distribution after dietary supplements (containing 40 or 75 mg of polydatin) administrated to 6 healthy volunteers was analysed in blood samples from all volunteers for one month. One urine sample was also analysed by employing the same UHPLC method. The results suggested that a dose higher than 75 mg/day has to be administered in order to determine a concentration in the blood suitable to exert its properties.
Conclusions. The analytical methodologies investigated in my PhD thesis resulted able to support AD drug discovery. In fact, they reliably and quickly identify true positive hit compounds, and perform structure activity relationship in view of the development and optimization of a lead structure with an improved activity and bioavailability. The new multimethodological approaches described give more innovative insights about the mechanism of toxicity Aβ aggregation that is crucial in AD pathogenesis and the combination of advance analytical methodology characterize new potential aggregation inhibitors in a comprehensive way. The identification and the relative quantification of some lipid altered species due to Aβ toxicity could be useful in the characterization of new biomarkers for the early detection of AD and in the therapeutic monitoring. Moreover, the methods developed showed to be suitable for the pharmacokinetic study of nutraceuticals formulation useful in AD prevention.
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