Abstract
Neurodegenerative diseases (ND) affect millions of people worldwide and even though treatments may help to partly relieve some of the associated physical or mental symptoms, currently none of them cure the diseases. Preclinical studies point out the therapeutic potential of neurotrophins, which have been shown to play a significant role in neurogenesis and neuroprotection through binding to their respective receptors. However, due to their poor pharmacokinetic properties, neurotrophins cannot be used as drugs. Neurosteroids play a central role in the control of survival, development and function of neurons while, their reduced levels in the brain have been associated with neurodegenerative diseases. Recent studies have demonstrated that the well-known neurosteroid dehydroepiandrosterone (DHEA) possesses notable neuroprotective effects and acts as a neurotrophic factor in the brain by interacting with the neurotrophin receptors TrkA and p75NTR. Considering that DHEA is metabolized in hu ...
Neurodegenerative diseases (ND) affect millions of people worldwide and even though treatments may help to partly relieve some of the associated physical or mental symptoms, currently none of them cure the diseases. Preclinical studies point out the therapeutic potential of neurotrophins, which have been shown to play a significant role in neurogenesis and neuroprotection through binding to their respective receptors. However, due to their poor pharmacokinetic properties, neurotrophins cannot be used as drugs. Neurosteroids play a central role in the control of survival, development and function of neurons while, their reduced levels in the brain have been associated with neurodegenerative diseases. Recent studies have demonstrated that the well-known neurosteroid dehydroepiandrosterone (DHEA) possesses notable neuroprotective effects and acts as a neurotrophic factor in the brain by interacting with the neurotrophin receptors TrkA and p75NTR. Considering that DHEA is metabolized in humans into estrogens and androgens and long-term administration could increase the risk of cancer, it cannot be used as a drug. Hence, there is a need to overcome this limitation by developing small molecules, neurotrophin mimetics, as potential therapeutics for neurodegenerative diseases. Therefore, we embarked on the synthesis of new DHEA analogues with the aim to improve the neuroprotective and antiapoptotic activity of the parent molecule, without the undesired hormonal side effects. The present work involves the design and synthesis of 41 C17-spirocyclopropyl DHEA derivatives aiming to evaluate, through structure-activity relationship (SAR) studies, their neurotrophic mimetic activity. The design of these analogues was based on previous findings which showed that the C17-spiroepoxy DHEA derivative BNN-27, synthesized by the group of Dr. Theodora Calogeropoulou, possesses neuroprotective activity by interacting with the TrkA and p75NTR receptors. The synthesized derivatives of the present thesis are divided into 1st and 2nd generation C17-spirocyclopropyl analogues, fluorescently labelled derivatives and multitarget compounds. Initially, eighteen 1st generation compounds were designed and synthesized in an effort to expand the toolbox of pharmacophores with neurotrophic, neuroprotective and anti-inflammatory activity. Upon the completion of the biological assessment, a number of 2nd generation compounds were synthesized focusing on targeted structural modifications of the most promising compound (ENT-A010). To this end, the identification of the structural features responsible for selective TrkA or TrkB agonistic activity was accomplished. Additionally, two fluorescently labelled derivatives of the dual TrkA/TrkB agonist ENT-A010 were synthesized to be used for mechanistic studies. Finally, five multitarget compounds were prepared combining a neurotrophin mimetic and an Amyotrophic Lateral Sclerosis (ALS) drug (edaravone or riluzole) in one molecular scaffold. In total, we were able to identify two TrkA agonists (ENT-A013 and ENT-A040), five TrkB agonists (ENT-A011, ENT- 2 A022, ENT-A044, ENT-A045 and ENT-A061) and one dual TrkA/TrkB agonist (ENT-A010). The compounds were evaluated for their ability to activate TrkA and/or TrkB receptors and the respective downstream kinases. Furthermore, the TrkA agonists were assessed for neuroprotection against serum-deprivation induced cell death using PC12 cells while, the TrkB agonists using NIH-3T3TrkB cells. Selected agonists were evaluated for their ability to promote survival of mature neurons, as well as proliferation and differentiation of neural stem cells. In addition, the protective effects of the compounds on neuronal or neural stem cells against toxic Aβ oligomers was studied. Furthermore, the compounds’ ability to attenuate the lipopolysaccharide (LPS)-induced inflammatory responses in primary murine microglia was also examined and ADMET studies were performed for the most promising compounds. Additionally, one of the most active compounds, ENT-A010, which acts as a dual activator, was also found to interact with the extracellular domain (EDC) of both TrkA and TrkB receptors and it is chemically stable in acidic, neutral and basic environments up to 24 hours. In the end, two selected compounds were synthesized in a large scale for in vivo experiments. More specifically, compound ENT-A010 was tested in the cuprizone mice model to evaluate its ability to promote remyelination and in LPS-induce inflammation in mice model to study the restoration of microglial homeostatic features in the hippocampus of LPS-treated mice. Additionally, the selective TrkB agonist, ENT-A061, was scaled-up for in vivo studies in the 5xFAD animal model of Alzheimer’s disease (AD).
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