Evaluation of the effects of acetylcholinesterase inhibitors in the zebrafish touch-evoked response: quantitative vs. qualitative assessment

Background: The difficulty of finding new treatments for neurological diseases with great impact in our society like Alzheimer's disease can be ascribed in part to the complexity of the nervous system and the lack of quick and costeffective screening tools. Such tools could not only help to identify potential novel treatments, but could also be used to test environmental contaminants for their potential to cause neurotoxicity. It has been estimated that 5-10% of the anthropogenic chemicals are developmental neurotoxic (DNT) and exposure to DNT compounds has been linked to several neurological diseases. Within this study we were testing the applicability of a quick and cost-effective behavioural test using zebrafish embryos: the touch-evoked response assay, in this case, an assay evaluating the swimming response to a tap in the tail. Two acetylcholinesterase (AChE) inhibitors positive controls (paraoxon and huprine Y), as well as 10 huprine-derivative compounds were tested and the results were evaluated using 2 different methods, a quantitative and a qualitative one. Results: We could show that the methodology presented is able to detect behavioural effects of AChE inhibitors. A good correlation between the results obtained with the quantitative and the qualitative method was obtained (R2 = 0.84). Conclusions: Our proposed method enables combination of screening for new drugs with toxicity screening in a whole embryo model alternative to animal experimentation, thereby merging 2 drug development steps into one

A novel rhein-huprine hybrid ameliorates disease-modifying properties in preclinical mice model of Alzheimer's disease exacerbated with high fat diet

Background: Alzheimer's disease (AD) is characterized by a polyetiological origin. Despite the global burden of AD and the advances made in AD drug research and development, the cure of the disease remains elusive, since any developed drug has demonstrated effectiveness to cure AD. Strikingly, an increasing number of studies indicate a linkage between AD and type 2 diabetes mellitus (T2DM), as both diseases share some common pathophysiological features. In fact, β-secretase (BACE1) and acetylcholinesterase (AChE), two enzymes involved in both conditions, have been considered promising targets for both pathologies. In this regard, due to the multifactorial origin of these diseases, current research efforts are focusing on the development of multi-target drugs as a very promising option to derive effective treatments for both conditions. In the present study, we evaluated the effect of rhein-huprine hybrid (RHE-HUP), a synthesized BACE1 and AChE inhibitor, both considered key factors not only in AD but also in metabolic pathologies. Thus, the aim of this study is to evaluate the effects of this compound in APP/PS1 female mice, a well-established familial AD mouse model, challenged by high-fat diet (HFD) consumption to concomitantly simulate a T2DM-like condition. Results: Intraperitoneal treatment with RHE-HUP in APP/PS1 mice for 4 weeks reduced the main hallmarks of AD, including Tau hyperphosphorylation, Aβ42 peptide levels and plaque formation. Moreover, we found a decreased inflammatory response together with an increase in different synaptic proteins, such as drebrin 1 (DBN1) or synaptophysin, and in neurotrophic factors, especially in BDNF levels, correlated with a recovery in the number of dendritic spines, which resulted in memory improvement. Notably, the improvement observed in this model can be attributed directly to a protein regulation at central level, since no peripheral modification of those alterations induced by HFD consumption was observed. Conclusions: Our results suggest that RHE-HUP could be a new candidate for the treatment of AD, even for individuals with high risk due to peripheral metabolic disturbances, given its multi-target profile which allows for the improvement of some of the most important hallmarks of the disease

Discovery and In Vivo Proof of Concept of a Highly Potent Dual Inhibitor of Soluble Epoxide Hydrolase and Acetylcholinesterase for the Treatment of Alzheimer's Disease

With innumerable clinical failures of target-specific drug candidates for multifactorial diseases, such as Alzheimer's disease (AD), which remains inefficiently treated, the advent of multitarget drug discovery has brought a new breath of hope. Here, we disclose a class of 6-chlorotacrine (huprine)‒TPPU hybrids as dual inhibitors of the enzymes soluble epoxide hydrolase (sEH) and acetylcholinesterase (AChE), a multitarget profile to provide cumulative effects against neuroinflammation and memory impairment. Computational studies confirmed the gorge-wide occupancy of both enzymes, from the main site to a secondary site, including a so far non-described AChE cryptic pocket. The lead compound displayed in vitro dual nanomolar potencies, adequate brain permeability, aqueous solubility, and human microsomal stability and lack of neurotoxicity, and rescued memory, synaptic plasticity and neuroinflammation in an AD mouse model, after low dose chronic oral administration

A novel class of multitarget anti-Alzheimer benzohomoadamantane‒chlorotacrine hybrids modulating cholinesterases and glutamate NMDA receptors

The development of multitarget compounds against multifactorial diseases, such as Alzheimer's disease, is an area of very intensive research, due to the expected superior therapeutic efficacy that should arise from the simultaneous modulation of several key targets of the complex pathological network. Here we describe the synthesis and multitarget biological profiling of a new class of compounds designed by molecular hybridization of an NMDA receptor antagonist fluorobenzohomoadamantanamine with the potent acetylcholinesterase (AChE) inhibitor 6-chlorotacrine, using two different linker lengths and linkage positions, to preserve or not the memantine-like polycyclic unsubstituted primary amine. The best hybrids exhibit greater potencies than parent compounds against AChE (IC50 0.33 nM in the best case, 44-fold increased potency over 6-chlorotacrine), butyrylcholinesterase (IC50 21 nM in the best case, 24-fold increased potency over 6-chlorotacrine), and NMDA receptors (IC50 0.89 µM in the best case, 2-fold increased potency over the parent benzohomoadamantanamine and memantine), which suggests an additive effect of both pharmacophoric moieties in the interaction with the primary targets. Moreover, most of these compounds have been predicted to be brain permeable. This set of biological properties makes them promising leads for further anti-Alzheimer drug development

From virtual screening hits targeting a cryptic pocket in BACE-1 to a nontoxic brain permeable multitarget anti-Alzheimer lead with disease-modifying and cognition-enhancing effects

Starting from six potential hits identified in a virtual screening campaign directed to a cryptic pocket of BACE-1, at the edge of the catalytic cleft, we have synthesized and evaluated six hybrid compounds, designed to simultaneously reach BACE-1 secondary and catalytic sites and to exert additional activities of interest for Alzheimer's disease (AD). We have identified a lead compound with potent in vitro activity towards human BACE-1 and cholinesterases, moderate Ab42 and tau antiaggregating activity, and brain permeability, which is nontoxic in neuronal cells and zebrafish embryos at concentrations above those required for the in vitro activities. This compound completely restored short- and long-term memory in a mouse model of AD (SAMP8) relative to healthy control strain SAMR1, shifted APP processing towards the non-amyloidogenic pathway, reduced tau phosphorylation, and increased the levels of synaptic proteins PSD95 and synaptophysin, thereby emerging as a promising disease-modifying, cognitionenhancing anti-AD lead

Multitarget strategies in search of novel drug candidates against Alzheimer’s disease

[eng] Alzheimer’s disease (AD) is the most common form of dementia and one of the most important health-care problems in the world, due to its high prevalence and unaffordable personal and economic impact. Moreover, current commercialised treatments are only symptomatic, but are not capable of preventing, curing or even delaying the disease progression. Because AD arises from a complex network of pathological events, such as dysfunction in neurotransmitter systems (mainly cholinergic and glutamatergic), β-amyloid and tau proteins disorders, oxidative stress or neuroinflammation, amongst others, the traditional medicinal chemistry paradigm of “one molecule-one target” is increasingly regarded as clearly ineffective. On the contrary, it becomes evident that a more comprehensive, complex pharmacological approach is needed to tackle AD. As a consequence, the use of multitarget directed ligands, where one single molecule is able to interact simultaneously with multiple targets of the pathological network, is emerging as a promising and more realistic way to confront this disease. In this context, the purpose of the present Thesis was the design, synthesis and biological evaluation of three novel families of compounds, endowed with multitarget biological profile, in order to find novel treatments for AD: 1) firstly, a new series of compounds designed by substitution of the rhein subunit of a rhein–huprine hybrid lead, previously developed in our group, by more simplified scaffolds, with the aim of finding optimized hybrids with reduced lipophilicity and better drug-like properties, while maintaining favourable activities against cholinesterases, BACE1, β- amyloid and tau aggregation, and antioxidant properties; 2) secondly, a novel family of huprine- derived hybrids, designed to perform a dual binding site interaction within BACE1 through the linkage of a huprine moiety to new scaffolds, selected by their predicted binding affinities towards a secondary transient pocket in BACE1, which were expected to combine cholinesterases and BACE1 inhibitory activities, as well as activity against β-amyloid and tau aggregation, and antioxidant properties; 3) finally, a family of huprine–TPPU and tacrine–TPPU hybrids, which were designed to be dual inhibitors of acetylcholinesterase (AChE) and soluble epoxide hydrolase (sEH). The blood–brain barrier permeability was also assessed for all these compounds, as it is a crucial factor for drugs acting in the central nervous system, while other important physicochemical and pharmacokinetic parameters, such as solubility and microsomal stability were determined for the latter series of compounds. Also, the toxicity of some compounds was evaluated. Finally, using the same assay that was employed for the determination of the β-amyloid and tau antiaggregating activity of the first two families and other compounds synthesised by our group, we demonstrated that a single compound can be able of inhibiting the aggregation of different types of amyloid-prone proteins, with these results supporting the notion that common mechanisms exist for the aggregation of different amyloidogenic proteins and that a generic treatment of conformational diseases is possible

Multitarget strategies in search of novel drug candidates against Alzheimer’s disease

Alzheimer’s disease (AD) is the most common form of dementia and one of the most important health-care problems in the world, due to its high prevalence and unaffordable personal and economic impact. Moreover, current commercialised treatments are only symptomatic, but are not capable of preventing, curing or even delaying the disease progression. Because AD arises from a complex network of pathological events, such as dysfunction in neurotransmitter systems (mainly cholinergic and glutamatergic), β-amyloid and tau proteins disorders, oxidative stress or neuroinflammation, amongst others, the traditional medicinal chemistry paradigm of “one molecule-one target” is increasingly regarded as clearly ineffective. On the contrary, it becomes evident that a more comprehensive, complex pharmacological approach is needed to tackle AD. As a consequence, the use of multitarget directed ligands, where one single molecule is able to interact simultaneously with multiple targets of the pathological network, is emerging as a promising and more realistic way to confront this disease. In this context, the purpose of the present Thesis was the design, synthesis and biological evaluation of three novel families of compounds, endowed with multitarget biological profile, in order to find novel treatments for AD: 1) firstly, a new series of compounds designed by substitution of the rhein subunit of a rhein–huprine hybrid lead, previously developed in our group, by more simplified scaffolds, with the aim of finding optimized hybrids with reduced lipophilicity and better drug-like properties, while maintaining favourable activities against cholinesterases, BACE1, β- amyloid and tau aggregation, and antioxidant properties; 2) secondly, a novel family of huprine- derived hybrids, designed to perform a dual binding site interaction within BACE1 through the linkage of a huprine moiety to new scaffolds, selected by their predicted binding affinities towards a secondary transient pocket in BACE1, which were expected to combine cholinesterases and BACE1 inhibitory activities, as well as activity against β-amyloid and tau aggregation, and antioxidant properties; 3) finally, a family of huprine–TPPU and tacrine–TPPU hybrids, which were designed to be dual inhibitors of acetylcholinesterase (AChE) and soluble epoxide hydrolase (sEH). The blood–brain barrier permeability was also assessed for all these compounds, as it is a crucial factor for drugs acting in the central nervous system, while other important physicochemical and pharmacokinetic parameters, such as solubility and microsomal stability were determined for the latter series of compounds. Also, the toxicity of some compounds was evaluated. Finally, using the same assay that was employed for the determination of the β-amyloid and tau antiaggregating activity of the first two families and other compounds synthesised by our group, we demonstrated that a single compound can be able of inhibiting the aggregation of different types of amyloid-prone proteins, with these results supporting the notion that common mechanisms exist for the aggregation of different amyloidogenic proteins and that a generic treatment of conformational diseases is possible

Multitarget strategies in search of novel drug candidates against Alzheimer’s disease

Alzheimer’s disease (AD) is the most common form of dementia and one of the most important health-care problems in the world, due to its high prevalence and unaffordable personal and economic impact. Moreover, current commercialised treatments are only symptomatic, but are not capable of preventing, curing or even delaying the disease progression. Because AD arises from a complex network of pathological events, such as dysfunction in neurotransmitter systems (mainly cholinergic and glutamatergic), β-amyloid and tau proteins disorders, oxidative stress or neuroinflammation, amongst others, the traditional medicinal chemistry paradigm of “one molecule-one target” is increasingly regarded as clearly ineffective. On the contrary, it becomes evident that a more comprehensive, complex pharmacological approach is needed to tackle AD. As a consequence, the use of multitarget directed ligands, where one single molecule is able to interact simultaneously with multiple targets of the pathological network, is emerging as a promising and more realistic way to confront this disease. In this context, the purpose of the present Thesis was the design, synthesis and biological evaluation of three novel families of compounds, endowed with multitarget biological profile, in order to find novel treatments for AD: 1) firstly, a new series of compounds designed by substitution of the rhein subunit of a rhein–huprine hybrid lead, previously developed in our group, by more simplified scaffolds, with the aim of finding optimized hybrids with reduced lipophilicity and better drug-like properties, while maintaining favourable activities against cholinesterases, BACE1, β- amyloid and tau aggregation, and antioxidant properties; 2) secondly, a novel family of huprine- derived hybrids, designed to perform a dual binding site interaction within BACE1 through the linkage of a huprine moiety to new scaffolds, selected by their predicted binding affinities towards a secondary transient pocket in BACE1, which were expected to combine cholinesterases and BACE1 inhibitory activities, as well as activity against β-amyloid and tau aggregation, and antioxidant properties; 3) finally, a family of huprine–TPPU and tacrine–TPPU hybrids, which were designed to be dual inhibitors of acetylcholinesterase (AChE) and soluble epoxide hydrolase (sEH). The blood–brain barrier permeability was also assessed for all these compounds, as it is a crucial factor for drugs acting in the central nervous system, while other important physicochemical and pharmacokinetic parameters, such as solubility and microsomal stability were determined for the latter series of compounds. Also, the toxicity of some compounds was evaluated. Finally, using the same assay that was employed for the determination of the β-amyloid and tau antiaggregating activity of the first two families and other compounds synthesised by our group, we demonstrated that a single compound can be able of inhibiting the aggregation of different types of amyloid-prone proteins, with these results supporting the notion that common mechanisms exist for the aggregation of different amyloidogenic proteins and that a generic treatment of conformational diseases is possible