Hailin Zheng

Novel multifunctional neuroprotective iron chelator-monoamine oxidase inhibitor drugs for neurodegenerative diseases: in vitro studies on antioxidant activity, prevention of lipid peroxide formation and monoamine oxidase inhibition

Iron‐dependent oxidative stress, elevated levels of iron and of monoamine oxidase (MAO)‐B activity, and depletion of antioxidants in the brain may be major pathogenic factors in Parkinsons disease, Alzheimers disease and related neurodegenerative diseases. Accordingly, iron chelators, antioxidants and MAO‐B inhibitors have shown efficacy in a variety of cellular and animal models of CNS injury. In searching for novel antioxidant iron chelators with potential MAO‐B inhibitory activity, a series of new iron chelators has been designed, synthesized and investigated. In this study, the novel chelators were further examined for their activity as antioxidants, MAO‐B inhibitors and neuroprotective agents in vitro. Three of the selected chelators (M30, HLA20 and M32) were the most effective in inhibiting iron‐dependent lipid peroxidation in rat brain homogenates with IC50 values (12–16 µm), which is comparable with that of desferal, a prototype iron chelator that is not has orally active. Their antioxidant activities were further confirmed using electron paramagnetic resonance spectroscopy. In PC12 cell culture, the three novel chelators at 0.1 µm were able to attenuate cell death induced by serum deprivation and by 6‐hydroxydopamine. M30 possessing propargyl, the MAO inhibitory moiety of the anti‐Parkinson drug rasagiline, displayed greater neuroprotective potency than that of rasagiline. In addition, in vitro, M30 was a highly potent non‐selective MAO‐A and MAO‐B inhibitor (IC50 < 0.1 µm). However, HLA20 was more selective for MAO‐B but had poor MAO inhibition, with an IC50 value of 64.2 µm. The data suggest that M30 and HLA20 might serve as leads in developing drugs with multifunctional activities for the treatment of various neurodegenerative disorders.

Novel multifunctional neuroprotective iron chelator-monoamine oxidase inhibitor drugs for neurodegenerative diseases. In vivo selective brain monoamine oxidase inhibition and prevention of MPTP-induced striatal dopamine depletion

Several multifunctional iron chelators have been synthesized from hydroxyquinoline pharmacophore of the iron chelator, VK‐28, possessing the monoamine oxidase (MAO) and neuroprotective N‐propargylamine moiety. They have iron chelating potency similar to desferal. M30 is a potent irreversible rat brain mitochondrial MAO‐A and ‐B inhibitor in vitro (IC50, MAO‐A, 0.037 ± 0.02; MAO‐B, 0.057 ± 0.01). Acute (1–5 mg/kg) and chronic [5–10 mg/kg intraperitoneally (i.p.) or orally (p.o.) once daily for 14 days]in vivo studies have shown M30 to be a potent brain selective (striatum, hippocampus and cerebellum) MAO‐A and ‐B inhibitor. It has little effects on the enzyme activities of the liver and small intestine. Its N‐desmethylated derivative, M30A is significantly less active. Acute and chronic treatment with M30 results in increased levels of dopamine (DA), serotonin(5‐HT), noradrenaline (NA) and decreases in DOPAC (dihydroxyphenylacetic acid), HVA (homovanillic acid) and 5‐HIAA (5‐hydroxyindole acetic acid) as determined in striatum and hypothalamus. In the mouse MPTP (N‐methy‐4‐phenyl‐1,2,3,6‐tetrahydropyridine) model of Parkinsons disease (PD) it attenuates the DA depleting action of the neurotoxin and increases striatal levels of DA, 5‐HT and NA, while decreasing their metabolites. As DA is equally well metabolized by MAO‐A and ‐B, it is expected that M30 would have a greater DA neurotransmission potentiation in PD than selective MAO‐B inhibitors, for which it is being developed, as MAO‐B inhibitors do not alter brain dopamine.

Reduction of iron‐regulated amyloid precursor protein and β‐amyloid peptide by (–)‐epigallocatechin‐3‐gallate in cell cultures: implications for iron chelation in Alzheimer's disease

Brain iron dysregulation and its association with amyloid precursor protein (APP) plaque formation are implicated in Alzheimers disease (AD) pathology and so iron chelation could be considered a rational therapeutic strategy for AD. Here we analyzed the effect of the main polyphenol constituent of green tea, (–)‐epigallocatechin‐3‐gallate (EGCG), which possesses metal‐chelating and radical‐scavenging properties, on the regulation of the iron metabolism‐related proteins APP and transferrin receptor (TfR). EGCG exhibited potent iron‐chelating activity comparable to that of the prototype iron chelator desferrioxamine, and dose dependently (1–10 µm) increased TfR protein and mRNA levels in human SH‐SY5Y neuroblastoma cells. Both the immature and full‐length cellular holo‐APP were significantly reduced by EGCG, as shown by two‐dimensional gel electrophoresis, without altering APP mRNA levels, suggesting a post‐transcriptional action. Indeed, EGCG suppressed the translation of a luciferase reporter gene fused to the APP mRNA 5′‐untranslated region, encompassing the APP iron‐responsive element. The finding that Fe2SO4 reversed the action of EGCG on APP and TfR proteins reinforces the likelihood that these effects are mediated through modulation of the intracellular iron pool. Furthermore, EGCG reduced toxic β‐amyloid peptide generation in Chinese hamster ovary cells overexpressing the APP ‘Swedish’ mutation. Thus, the natural non‐toxic brain‐permeable EGCG may provide a potential therapeutic approach for AD and other iron‐associated disorders.

Therapeutic targets and potential of the novel brain- permeable multifunctional iron chelator-monoamine oxidase inhibitor drug, M-30, for the treatment of Alzheimer's disease.

Novel therapeutic approaches for the treatment of neurodegenerative disorders comprise drug candidates designed specifically to act on multiple CNS targets. We have synthesized a multifunctional non‐toxic, brain permeable iron chelator drug, M‐30, possessing propargyl monoamine oxidase (MAO) inhibitory neuroprotective and iron‐chelating moieties, from our prototype iron chelator VK‐28. In the present study M‐30 was shown to possess a wide range of pharmacological activities, including pro‐survival neurorescue effects, induction of neuronal differentiation and regulation of amyloid precursor protein (APP) and β‐amyloid (Aβ) levels. M‐30 was found to decrease apoptosis of SH‐SY5Y neuroblastoma cells in a neurorescue, serum deprivation model, via reduction of the pro‐apoptotic proteins Bad and Bax, and inhibition of the apoptosis‐associated phosphorylated H2A.X protein (Ser 139) and caspase 3 activation. In addition, M‐30 induced the outgrowth of neurites, triggered cell cycle arrest in G0/G1 phase and enhanced the expression of growth associated protein‐43. Furthermore, M‐30 markedly reduced the levels of cellular APP and β‐C‐terminal fragment (β‐CTF) and the levels of the amyloidogenic Aβ peptide in the medium of SH‐SY5Y cells and Chinese hamster ovary cells stably transfected with the APP ‘Swedish’ mutation. Levels of the non‐amyloidogenic soluble APPα and α‐CTF in the medium and cell lysate respectively were coordinately increased. These properties, together with its brain selective MAO inhibitory and propargylamine‐ dependent neuroprotective effects, suggest that M‐30 might serve as an ideal drug for neurodegenerative disorders, such as Parkinsons and Alzheimers diseases, in which oxidative stress and iron dysregulation have been implicated.

Prevention and restoration of lactacystin-induced nigrostriatal dopamine neuron degeneration by novel brain-permeable iron chelators

Dysfunction of the ubiquitin‐protea‐ some system (UPS) and accumulation of iron in substantia nigra (SN) are implicated in the pathogenesis of Parkinsons disease (PD). UPS dysfunction and iron misregulation may reinforce each others contribution to the degeneration of dopamine (DA) neurons. In the present study, we use a new brain‐permeable iron chelator, VK‐28 [5‐(4‐(2‐hydroxyethyl) piperazin‐1‐yl (methyl)‐8‐hydroxyquinoline], and its derivative M30 [5‐(N‐methyl‐N‐propargyaminomethyl)‐8‐hydroxyquino‐ line] in vivo to test their neuroprotective and neurorestorative properties against proteasome inhibitor (lactacystin) ‐induced nigrostriatal degeneration. Bilateral microinjections of lactacystin (1.25 μg/side) into the mouse medial forebrain bundle were performed. Administration of VK‐28 (5 mg/kg, once a day) or M30 (5 mg/kg, once a day) was applied intraperitoneally 7 days before or after the lactacystin microinjection until the mice were sacrificed 28 days after microinjection. We found that VK‐28 and M30 both significantly improved behavioral performances and attenuated lacta‐ cystin‐induced DA neuron loss, proteasomal inhibition, iron accumulation, and microglial activation in SN. In addition, M30 restored the Bcl‐2 level, which was suppressed after lactacystin injection. These findings suggest that brain‐permeable iron chelators can improve DA neuron survival under UPS impairment. Furthermore, M30, a derivative of VK‐28 and neuropro‐ tective agent rasagiline, may serve as a better neuropro‐ tective therapy for PD.— Zhu, W., Xie, W., Pan, T., Xu, P., Fridkin, M., Zheng, H., Jankovic, J., Youdim, M. B. H., Le, W. Prevention and restoration of lacta‐ cystin‐induced nigrostriatal dopamine neuron degeneration by novel brain‐permeable iron chelators. FASEB J. 21, 3835–3844 (2007)

Novel bifunctional drugs targeting monoamine oxidase inhibition and iron chelation as an approach to neuroprotection in Parkinson’s disease and other neurodegenerative diseases

Summary.Iron has been shown to accumulates at site where neurons degenerate in neurodegenerative diseases of Parkinson’s disease, Alzheimer’s disease, Huntington disease, amyotrophic lateral sclerosis and Friedreich ataxia. Iron is thought to participate or initiate oxidative stress via generation of reactive oxygen species (ROS), such as hydroxyl radical. Iron chelators are neuroprotective and prevent 6-hydroxydoapmine and MPTP dopaminergic neurotoxicity in rats and mice. However, their action on monoamine oxidase (MAO) A and B have not been determined previously since MAO-B inhibitors have been shown to be neuroprotective in cellular and animal models of Parkinson’s disease. The chelators 8-hydroxyquinoline, O-phenanthroline, 2,2′-dipyridyl, U74500A and U74600F showed a preference for inhibition of rat brain mitochondrial MAO-A over MAO-B. Their IC50 ranged from 10−3 M to 10−6 M, with 21-amino steroids (U74500A and U74006F) showing a greater selectivity and potency for MAO-A. Desferrioxamine (desferal), a prototype potent iron chelator, exhibited relatively poor MAO inhibitory. The inhibitions of MAO-A and B by 21-amino steroids (Lazaroids) were time dependent and irreversible. Those initiated by 8-hydroxyquinoline, 2,2′-dipyridyl and O-phenanthroline were fully reversible by enzyme dilution experiments. Both Fe2+ and Fe3+ reverse the MAO-A and B inhibition induced by the latter chelators, but not those initiated by 21-amino steroids. The data infer that either the inhibition of MAO by 21-amino steroids is either the resultant of their conversion to an irreversible covalently bound ligand or that the iron chelation moiety and MAO inhibitory activity in these compounds are not mutually shared. The results suggest that bifunctional brain penetrable drugs with iron chelating property and MAO inhibitory activity in could be the most feasible approach for neuroprotection in neurodegenerative diseases. Such drug would prevent participation of elevated iron in oxidative stress and formation of reactive hydroxyl radical, via its interaction with H2O2 (Fenton chemistry), generated as a consequence MAO and other oxidative enzyme reactions to generative cytotoxic reactive hydroxyl radical. We have now developed several of these compounds with neuroprotective, MAO inhibitory and iron chelating properties from our prototype iron chelators, VK-28 possessing propargylamine moiety of our anti-parkinson drug, rasagiline.

Site-Activated Multifunctional Chelator with Acetylcholinesterase and Neuroprotective— Neurorestorative Moieties for Alzheimer's Therapy

A novel strategy to develop site-activated multifunctional chelators for targeting multiple etiologies of Alzheimers disease is reported. The novel prochelator HLA20A with improved cytotoxicity shows little affinity for metal ions until it is activated by binding and inhibiting acetylcholinesterase (AChE), releasing an active chelator HLA20 that modulates amyloid precursor protein (APP) regulation and beta-amyloid (Abeta) reduction, suppresses oxidative stress, and passivates excess metal ions (Fe, Cu, and Zn) in the brain.

Site-activated chelators targeting acetylcholinesterase and monoamine oxidase for Alzheimer's therapy.

Chelators have the potential to treat the underlying cause of Alzheimers disease (AD), but their therapeutic use is hampered by their poor targeting and poor permeability to the brain and/or toxic effects. Here, we report a new strategy for designing site-activated chelators targeting both acetylcholinesterase (AChE) and monoamine oxidase (MAO). We demonstrated that our lead 2 inhibited both AChE and MAO in vitro, but with little affinity for metal (Fe, Cu, and Zn) ions. Compound 2 can be activated by inhibition of AChE to release an active chelator M30. M30 has been shown to be able to modulate amyloid precursor protein regulation and beta-amyloid reduction, suppress oxidative stress, and passivate excess metal ions (Fe, Cu, and Zn). Compound 2 was less cytotoxic and more lipophilic than the brain-permeable chelator M30. Our new strategy is relatively simple and generally produces small and simple molecules with drug-like properties; it thus holds a potential use in designing site-activated multifunctional chelators with safer and more efficacious properties for treating other metal-related diseases such as Parkinsons disease and cancer where specific elimination of metals in cancer cells is required.

From single target to multitarget/network therapeutics in Alzheimer's therapy.

Brain network dysfunction in Alzheimer’s disease (AD) involves many proteins (enzymes), processes and pathways, which overlap and influence one another in AD pathogenesis. This complexity challenges the dominant paradigm in drug discovery or a single-target drug for a single mechanism. Although this paradigm has achieved considerable success in some particular diseases, it has failed to provide effective approaches to AD therapy. Network medicines may offer alternative hope for effective treatment of AD and other complex diseases. In contrast to the single-target drug approach, network medicines employ a holistic approach to restore network dysfunction by simultaneously targeting key components in disease networks. In this paper, we explore several drugs either in the clinic or under development for AD therapy in term of their design strategies, diverse mechanisms of action and disease-modifying potential. These drugs act as multi-target ligands and may serve as leads for further development as network medicines.

M30, a novel multifunctional neuroprotective drug with potent iron chelating and brain selective monoamine oxidase-ab inhibitory activity for Parkinson’s disease

Iron and monoamine oxidase activity are increased in brain of Parkinsons disease (PD). They are associated with autoxidation and oxidative deamination of dopamine by MAO resulting in the generation of reactive oxygen species and the onset of oxidative stress to induce neurodegeneration. Iron chelators (desferal, Vk-28 and clioquinol) but not copper chelators have been shown to be neuroprotective in the 6-hydroxydoapmine and MPTP models of Parkinsons disease (PD), as are monoamine oxidase B inhibitors such as selegiline and rasagiline. These findings prompted the development of multifunctional anti PD drugs possessing iron chelating phamacophore of VK-28 and the propargylamine MAO inhibitory activity of rasagiline. M30 is a potent iron chelator, radical scavenger and brain selective irreversible MAO-A and B inhibitor, with little inhibition of peripheral MAO. It has neuroprotective activity in in vitro and in vivo models of PD and unlike selective MAO-B inhibitors it increases brain dopamine, serotonin and noradrenaline. These findings indicate beside its anti PD action, it may also possess antidepressant activity, similar to selective MAO-A and nonselective MAO inhibitors. These properties make it an ideal anti PD drug for which it is being developed.