Miriam Ratia

Huprine–Tacrine Heterodimers as Anti-Amyloidogenic Compounds of Potential Interest against Alzheimer’s and Prion Diseases

A family of huprine-tacrine heterodimers has been developed to simultaneously block the active and peripheral sites of acetylcholinesterase (AChE). Their dual site binding for AChE, supported by kinetic and molecular modeling studies, results in a highly potent inhibition of the catalytic activity of human AChE and, more importantly, in the in vitro neutralization of the pathological chaperoning effect of AChE toward the aggregation of both the β-amyloid peptide (Aβ) and a prion peptide with a key role in the aggregation of the prion protein. Huprine-tacrine heterodimers take on added value in that they display a potent in vitro inhibitory activity toward human butyrylcholinesterase, self-induced Aβ aggregation, and β-secretase. Finally, they are able to cross the blood-brain barrier, as predicted in an artificial membrane model assay and demonstrated in ex vivo experiments with OF1 mice, reaching their multiple biological targets in the central nervous system. Overall, these compounds are promising lead compounds for the treatment of Alzheimers and prion diseases.

Novel huprine derivatives with inhibitory activity toward β-amyloid aggregation and formation as disease-modifying anti-Alzheimer drug candidates.

A new family of dual binding site acetylcholinesterase (AChE) inhibitors has been designed, synthesized, and tested for their ability to inhibit AChE, butyrylcholinesterase (BChE), AChE‐induced and self‐induced β‐amyloid (Aβ) aggregation and β‐secretase (BACE‐1), and to cross the blood–brain barrier. The new heterodimers consist of a unit of racemic or enantiopure huprine Y or X and a donepezil‐related 5,6‐dimethoxy‐2‐[(4‐piperidinyl)methyl]indane moiety as the active site and peripheral site to mid‐gorge‐interacting moieties, respectively, connected through a short oligomethylene linker. Molecular dynamics simulations and kinetics studies support the dual site binding to AChE. The new heterodimers are potent inhibitors of human AChE and moderately potent inhibitors of human BChE, AChE‐induced and self‐induced Aβ aggregation, and BACE‐1, and are predicted to be able to enter the central nervous system (CNS), thus constituting promising multitarget anti‐Alzheimer drug candidates with the potential to modify the natural course of this disease.

Effect of Huprine X on β-Amyloid, Synaptophysin and α7 Neuronal Nicotinic Acetylcholine Receptors in the Brain of 3xTg-AD and APPswe Transgenic Mice

Background: Several studies implicate acetylcholinesterase (AChE) in the pathogenesis of Alzheimer’s disease (AD), raising the question of whether inhibitors of AChE also might act in a disease-modifying manner. Huprine X (HX), a reversible AChE inhibitor hybrid of tacrine and huperzine A, has shown to affect the amyloidogenic process in vitro. In this study, the aim was to investigate whether HX could affect the AD-related neuropathology in vivoin two mouse models. Methods:Tg2576 (K670M/N671L) (APPswe) and 3xTg-AD (K670M/N671L, PS1M146V, tauP301L) mice were treated with HX (0.12 µmol/kg, i.p., 21 days) or saline at 6–7 months. Human β-amyloid (Aβ) was measured by ELISA, synaptophysin by Western blot and α7 neuronal nicotinic acetylcholine receptors (nAChRs) were analyzed by [125I]α-bungarotoxin autoradiography. Results: Treatment with HX reduced insoluble Aβ1–40 (about 40%) in the hippocampus of 3xTg-AD mice, while showing no effect in APPswe mice. Additionally, HX markedly increased cortical synaptophysin levels (about 140%) and decreased (about 30%) the levels of α7 nAChRs in the caudate nucleus of 3xTg-AD mice, while increasing (about 10%) hippocampal α7 nAChRs in APPswe mice. Conclusion: The two mouse models react differently to HX treatment, possibly due to their differences in brain neuropathology. The modulation of Aβ and synaptophysin by HX in 3xTg-AD mice might be due to its suggested interaction with the peripheral anionic site on AChE, and/or via cholinergic mechanisms involving activation of cholinergic receptors. Our results provide further evidence that drugs targeting AChE affect some of the fundamental processes that contribute to neurodegeneration, but whether HX might act in a disease-modifying manner in AD patients remains to be proven.

Huprine X and huperzine A improve cognition and regulate some neurochemical processes related with Alzheimer's disease in triple transgenic mice (3xTg-AD).

Background: Different studies have established that cholinergic neurodegeneration could be a major pathological feature of Alzheimer’s disease (AD). Thus, enhancement of the central cholinergic neurotransmission has been regarded as one of the most promising strategies for the symptomatic treatment of AD, mainly by means of reversible acetylcholinesterase inhibitors (AChEIs). The cognitive-enhancing properties of both huprine X, a new AChEI, and the structurally related huperzine A, as well as their effects on the regulation of several neurochemical processes related to AD have been studied in triple transgenic mice (3xTg-AD). Methods: Seven-month-old homozygous 3xTg-AD male mice, which received chronic intraperitoneal treatment with either saline, huprine X (0.12 µmol·kg–1) or huperzine A (0.8 µmol·kg–1) were subjected to a battery of behavioural tests after 3 weeks of treatment and thereafter the brains were dissected to study the neurochemical effects induced by the two AChEIs. Results: Treatments with huprine X and huperzine A improved learning and memory in the Morris water maze and some indicators of emotionality without inducing important adverse effects. Moreover, huprine X and huperzine A activate protein kinase C/mitogen-activated protein kinase pathway signalling, α-secretases (ADAM 10 and TACE) and increase the fraction of phospho-glycogen synthase kinase 3-β. Conclusion: Results obtained herein using a sample of 3xTg-AD animals strongly suggest that the treatment with the two AChEIs not only improves the cognitive performance of the animals but also induces some neurochemical changes that could contribute to the beneficial effects observed.

Behavioural effects and regulation of PKCα and MAPK by huprine X in middle aged mice

The behavioural effects of huprine X, a new anticholinesterasic inhibitor, as well as its effects on the regulation of protein kinase C (PKC), mitogen activated protein kinase (MAPK) and alpha-secretase (ADAM10 and TACE/ADAM17) related to amyloid precursor protein (APP) processing remain to be established. In the present work, 12 month old 126/SvxC57b/6 male mice which received chronic i.p. treatment with either saline, huprine X (0.04 micromol kg(-1) or huprine X (0.12 micromol kg(-1), were submitted to a battery of behavioural tests and thereafter the brains were dissected to study the neurochemical effects induced by huprine X. The results show that, in a dose dependent manner, huprine X facilitates learning and memory in the Morris water maze and improves some indicators of emotionality without inducing adverse effects, affecting motor activity nor anxiety-like behaviours, as measured in the open-field and corner tests. Moreover activation of downstream PKC/MAPK signaling pathways may underly these behavioural effects as well as the stimulation of the non-amyloidogenic processing of APP. Results obtained herein using a sample of aged animals strongly suggest that huprine X constitutes a promising therapeutic agent for the treatment of cholinergic dysfunction underlying aging and/or dementias.

Effect of acetylcholinesterase inhibitors on AChE-induced PrP106-126 aggregation.

Transmissible spongiform encephalopaties are caused by an extracellular surface protein, the scrapie prion protein (PrPsc), which is an aberrant form of normal and functional cellular PrP (PrPc). The pathological hallmarks of these diseases are the accumulation and deposition of PrPsc in the form of amyloid fibrils in the central nervous system (Tateishi et al., 1988), similar to amyloid-beta (Abeta) protein in Alzheimers disease (AD). In some patients, Abeta and prion pathology can coexist (Hainfellner et al., 1998), and a common spatial pattern of protein deposition has been described (Armstrong et al., 2001). In addition, it is well-known that acetylcholinesterase (AChE) colocalizes with Abeta deposits of brains in AD patients and accelerates assembly of Abeta peptides through the peripheral site of the enzyme (Inestrosa et al., 1996). The aim of the present study was to analyze time course and concentration dependence of the AChE proaggregating effect on synthetic peptide-spanning residues 106-126 of human PrP (PrP106-126) and the reversion of this effect by different AChE inhibitors (AChEIs).

Expanding the multipotent profile of huprine-tacrine heterodimers as disease-modifying anti-Alzheimer agents.

Background: Multifactorial diseases such as Alzheimer’s disease (AD) should be more efficiently tackled by drugs which hit multiple biological targets involved in their pathogenesis. We have recently developed a new family of huprine-tacrine heterodimers, rationally designed to hit multiple targets involved upstream and downstream in the neurotoxic cascade of AD, namely β-amyloid aggregation and formation as well as acetylcholinesterase catalytic activity. Objective: In this study, the aim was to expand the pharmacological profiling of huprine-tacrine heterodimers investigating their effect on muscarinic M1 receptors as well as their neuroprotective effects against an oxidative insult. Methods: Sprague-Dawley rat hippocampus homogenates were used to assess the specific binding of two selected compounds in competition with 1 nM [3H]pirenzepine (for M1 receptors) or 0.8 nM [3H]quinuclidinyl benzilate (for M2 receptors). For neuroprotection studies, SHSY5Y cell cultures were subjected to 250 µM hydrogen peroxide insult with or without preincubation with some huprine-tacrine heterodimers. Results: A low nanomolar affinity and M1/M2 selectivity has been found for the selected compounds. Huprine-tacrine heterodimers are not neurotoxic to SHSY5Y cells at a range of concentrations from 1 to 0.001 µM, and some of them can protect cells from the oxidative damage produced by hydrogen peroxide at concentrations as low as 0.001 µM. Conclusion: Even though it remains to be determined if these compounds act as agonists at M1 receptors, as it is the case of the parent huprine Y, their low nanomolar M1 affinity and neuroprotective effects expand their multitarget profile and increase their interest as disease-modifying anti-Alzheimer agents.

Subject Index Vol. 7, 2010

Cell culture model 108 – cycle 108 Central nervous system 96 Cerebral hypoperfusion 116 Cerebrospinal fluid 42, 251, 373 Cerebrovascular lesions 112 Cholesterol 183 c-jun NH2-terminal kinase 68 Cognition 206 Cognitive activities 160 – impairment 163 Conformational change 24 – disease 272 Continuous dopaminergic stimulation 213 Corticobasal degeneration 300 Costs 365 CSF biomarkers 136 Cytokines 38 Czech psychiatry 6