Current Alzheimer Research 2013, 9, 797-808

Multipotent, Permeable Drug ASS234 Inhibits Aβ Aggregation, Possesses Antioxidant properties and Protects from Aβ-induced Apoptosis In vitro.
 

Bolea I, Gella A, Monjas l, Perez C, Rodriguez-Franco MI, Marco-Contelles J, Samadi A, and Unzeta M.

Departament de Bioquímica i Biologia Molecular, Facultat de Medicina, Institut de Neurociències, Universitat Autónoma de Barcelona, Spain.

 

Abstract

Amyloid beta (Aβ) aggregation and deposition is a key pathological hallmark of AD. Growing evidence suggests that neurotoxicity of this peptide is related to the formation of toxic oligomeric aggregates. Therefore, a deeply investigated therapeutic strategy comes at present from blocking the formation of these species to non-toxic aggregates. Among other considered strategies, the multi-target approach has been proposed as a more suitable potential therapy, precisely due to the multifactorial nature of AD. In this context, we recently identified ASS234, a novel compound that possesses a significant multipotent profile since it is able to inhibit cholinesterase and Monoamine oxidase enzymes as well as to interfere in Ab aggregation process. In this work, we investigated more in detail the effects of ASS234 on Ab aggregation and toxicity in vitro as well as we explored its ability to penetrate to the CNS. We report that ASS234 inhibited Ab1-42 self-aggregation more efficiently than that of Ab1-40, limiting the formation of fibrillar and oligomeric species. Additionally, ASS234 completely blocked the aggregation mediated by AChE of both Ab1-42 and Ab1-40, showing a dual binding site to AChE. Interestingly, ASS234 significantly reduced Ab1-42-mediated toxicity in SH-SY5Y human neuroblastoma cells through the prevention of the mitochondrial apoptosis pathway activation. Also importantly, we observed a significant ability of ASS234 to capture free-radical species in vitro as well as a potent effect in preventing the Ab1-42-induced depletion of antioxidant enzymes (catalase and SOD-1). Finally, we report the capability of ASS234 to cross the blood-brain barrier. Overall, our in vitro results show that ASS234 may have an impact on different processes involved in AD pathogenesis and provide evidences that it has encouraging attributes as a therapeutic lead compound.

 

Supplement:

Alzheimer´s disease (AD) is the fourth leading cause of death and the most common form of dementia in the elderly population. The predominant clinical manifestations are a progressive memory loss and cognitive decline as a result of the depletion of cholinergic neurons. Two neuropathological hallmarks characterize the disease: the presence of intraneuronal neurofibrillary tangles formed by tau protein and the deposition of extracellular amyloid plaques composed of Ab peptide. The pathogenesis is not yet fully understood but  there is a wide consensus in describing it as a multifactorial disease caused by  several factors,  including cholinergic dysfunction, excessive protein misfolding and aggregation, oxidative stress and free radical formation, metal dyshomeostasis, excitotoxic and neuroinflammatory processes, as well as disturbances in the monoaminergic and glutamatergic systems. There are at present 5 drugs approved by the Food and Drug Administartion (FDA) to treat the cognitive symptoms, namely Donepezil, Rivastigmine, Galantamine, Tacrine and Memantine. However, half of the patients do not respond to them and in the other half these drugs only modestly slow disease progression. It has been suggested that this lack of efficacy may be due to their “single-target” effect, as these drugs were designed only to inhibit the enzyme responsible for acetylcholine breakdown. By contrast, at present it is widely accepted that a more effective therapy for the multifactorial nature of AD would result from the development of the “one drug, multiple target” strategy, also called the multi target directed ligand (MTDL) approach, which suggests the use of compounds with multiple activities at different biological targets (Figure 1)

 

fig1Figure 1: Pathways leading to the discovery of new drugs: On the left, the “one target-one molecule” paradigm and on the right the “multitarget-directed ligand” (MTDL) approach

 

Based on the MTDL approach, we designed and synthesised  ASS234, a new compound based on the structure of Donepezil responsible for the anti-cholinergic activity, and the structure of a PF 9601N analogue, a potent Monoamine Oxidase (MAO) inhibitor with  neuroprotective properties [1](Figure 2).  MAO is the enzyme responsible for monoamine oxidation. Due to the monoaminergic system deficiencies observed in AD patients, MAO inhibition appears an important target to be considered when designing MTDLs against AD [4].

 

fig22

Figure 2. Design strategy leading to the multifunctional compound ASS234. The N- benzylpiperidine moiety of Donepezil was combined with the [1-methyl-1H-indol-2-yl]-N-methylprop-2yn-1-amine, moiety of an analogue of PF9601N.

 

One of the hallmarks of AD pathology is the presence of Ab plaques. It had been reported that reducing plaque content could be of value when treating AD, however it was later reported no correlation between the number of plaques and disease severity. By contrast, soluble oligomeric Ab species appear to be the responsible for neuronal loss.

Both Aβ 1-40 and Aβ 1-42, isoforms are present forming the senile plaques. The MTDL ASS234, is able to prevent the formation of toxic Aβ 1-42 oligomers This anti-Aβ aggregating effect was also observed in the presence of AChE, demonstrating the binding capacity of ASS234 to the peripheral anionic site (PAS) of AChE. These results confer to ASS234 a potential therapeutic value, (Figure 3).

 

fig3

Figure 3. Schematic representation of ASS234 targets involved in AD pathogenesis

 

The oxidative imbalance and the subsequent increased oxidative stress are key events in the pathogenesis of AD [3], thus targeting the source of ROS early in the disease progression is a matter of interest. ASS234 inhibits both MAO isoforms, preventing hydrogen peroxide formation. In addition, it is able to restore the expression of the antioxidant enzymes catalase and superoxide dismutase-1 (SOD-1) in vitro. ASS234 also possesses an anti-apoptotic effect in the mitochondrial pathway by preventing the cleavage and activation of caspase-9 as well as the downstream executioner caspase-3. As previously reported by the parent compound and other propargylamines, the propargyl group present in the molecule is the responsible for this protective effect .An important property that a molecule for AD treatment should possess is to cross the blood brain barrier (BBB) to reach its cerebral targets. However, almost the 98% of the developed compounds aimed to prevent Aβ oligomerization can not penetrate the BBB. The MTDL ASS234 gave an interesting profile in vitro and was able to cross the BBB in vivo [4,5].

Overall, our results show that ASS234 may have an impact on different process involved in AD pathogenesis and provide evidences that it has encouraging attributes as a theraoeutic lead compound for further optimisation of formulations capable of addressing the biological complexity of this disorder.

 

References

[1] Irene Bolea, Jordi Juarez-Jimenez, Cristobal de los Rios, Mourad Chioua, Ramón Pauplana, F.Javier Luque, Mercedes Unzeta, José Luis Marco-Contelles and Abdelouaid Samadi. Synthesis, biological evaluation and molecular modeling of Donepezil and N-[(5-(Benzyloxy)-1-m2thyl-1H-indol-2-yl)methyl]-N-methylprop-2-yn-1-amine Hybrids as new Multipotent Cholinesterase/Monoamine Oxidase inhibitors for the treatment of Alzheimer´s disease. Journal of Medicinal Chemistry 2011, 54, 8251-8270.

[2] Irene Bolea, Alejandro Gella, Leticia Monjas, Concepción Pérez, Maria Isabel Rodriguez-Franco, José Luis Marco-Contelles ,  Abdelouaid Samadi and  Mercedes Unzeta. Multipotent, Permeable Drug ASS234 inhibits Aβ aggregation, possesses antioxidant properties and portects from Aβ-induced apoposis in vitro. Current Alzheimer Research 2013, 9,797-808.

[3] Alejandro Gella & Irene Bolea. “Oxidative Stress in Alzheimer’s disease: Pathogenesis, Biomarkers & Therapy“. Alzheimer’s disease Pathogenesis-Core Concepts, Shifting Paradigms and Therapeutic Targets, Suzanne De La Monte (Ed.), ISBN: 978-953-307-690-4, InTech

[4] Bolea I, Gella A, Unzeta M. Propargylamine-derived multitarget-directed ligands: fighting Alzheimer’s disease with monoamine oxidase inhibitors. J Neural Transm 120: 893-902

[5] Stasiak A, Mussur M, Unzeta M, Samadi A, Marco-Contelles JL, Fogel WA. Effects of novel monoamine oxidases and cholinesterases targeting compounds on brain neurotransmitters and behavior in rat model of vascular dementia. Curr Pharm Des. 2014;20(2):161-71.

 

Acknowledgements

This work was realized in the framework of COST Actions D34/0003/05 (2005-2010) and CM1103 (2011-2015). J.M.-C. thanks MICINN (SAF2006-08764-C02-01; SAF2009-07271) for support, M.I.R.-F. acknowledges the financial support from the Spanish Ministry of Economy and Competitiveness (SAF2012-31035 and SAF2009-13015) and CSIC (PIE-201280E074).

fig4

Prof. Mercedes Unzeta Departament de Bioquímica i Biologia Molecular

Institut de Neurociències Edifici M, Lab M2-125/M2-104 Universitat Autònoma de Barcelona 08193, Cerdanyola del Vallès , Barcelona Spain

E-mail: mercedes.unzeta@uab.es Telf: (+34)935811523 FAX: (+34)935811573

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