Advances in Multi-Functional Ligands and the Need for Metal-Related Pharmacology for the Management of Alzheimer Disease

Alzheimer’s disease (AD) is the age linked neurodegenerative disorder with no disease modifying therapy currently available. The available therapy only offers short term symptomatic relief. Several hypotheses have been suggested for the pathogenesis of the disease while the molecules developed as possible therapeutic agent in the last decade, largely failed in the clinical trials. Several factors like tau protein hyperphosphorylation, amyloid-β (Aβ) peptide aggregation, decline in acetyl cholinesterase and oxidative stress might be contributing toward the pathogenesis of AD. Additionally, biometals dyshomeostasis (Iron, Copper, and Zinc) in the brain are also reported to be involved in the pathogenesis of AD. Thus, targeting these metal ions may be an effective strategy for the development of a drug to treat AD. Chelation therapy is currently employed for the metal intoxication but we lack a safe and effective chelating agents with additional biological properties for their possible use as multi target directed ligands for a complex disease like AD. Chelating agents possess the ability to disaggregate Aβ aggregation, dissolve amyloid plaques, and delay the cognitive impairment. Thus there is an urgent need to develop disease modifying therapeutic molecules with multiple beneficial features like targeting more than one factor responsible of the disease. These molecules, as disease modifying therapeutic agents for AD, should possess the potential to inhibit Aβ-metal interactions, the formation of toxic Aβ aggregates; and the capacity to reinstate metal homeostasis

Molecular scaffold and biological activities of anti- Alzheimer agents

Alzheimer’s disease (AD) is an age-associated and neurodegenerative illness which results in progressive dementia and severe cognitive malfunctions. The pathogenesis of AD is affected by some factors such as accumulation of β-amyloid, aggregation of tau protein, cholinergic insufficiency, neuroinflammation, oxidative stress and apoptosis. Factors such as gene mutation, as well as environmental, psychical and other co-existing diseases influence the pathogenesis of AD to varying extents. While there are no available drugs for arresting AD-associated neurodegeneration, the characteristics that result from AD treatment are considered as indexes of symptomatic cure. Several medications with varied scaffolds have been used for the treatment of many cognitive syndromes, including AD. These medications act as anti-inflammatory and antioxidant agents, and as inhibitors of cholinesterase and β-secretase. Moreover, these drugs suppress the accumulation of β-amyloid and its fibril. This review is an update and compilation of various scaffolds of anti-AD medications used to ameliorate the deleterious effects of the disease, based on their pharmacologic characteristics

Development of Small Molecules as Chemical Tools for Investigating the Role of Metal-Protein Interactions in Neurodegenerative Diseases.

Metals play an essential part in biological processes in humans. When these beneficial metal ions become misregulated, the resulting metal ion dyshomeostasis can be catastrophic. This occurs in several neurodegenerative diseases where the aberrant interactions of metal ions with proteins can lead to their abnormal aggregation, production of oxidative stress, and neuronal death (reactivity). To better understand the role of metal−protein complexes in the pathogenesis of these diseases, small molecules have been developed as chemical tools that target these complexes and mediate their reactivity. In this thesis, first, design considerations along with the approaches of developing and studying the activity of such molecules were discussed in the context of the most prevalent neurodegenerative diseases, Alzheimer’s disease (AD). Next, one such compound, L2-b, was demonstrated to target metal complexes of amyloid-beta (Abeta), an AD pathological feature, over metal-free Abeta, and reduce the reactivity of these species using biochemical and biophysical techniques. Upon application of L2-b to 5XFAD AD model mice, metal−Abeta was targeted and modulated in the brain; amyloid pathology was reduced; and AD-associated cognitive deficits were improved. These in vivo studies are the first time experimental evidence has directly linked metal−Abeta to AD pathogenesis. Subsequent investigations developed new small molecules that could target and mediate abnormal metal-free and metal-induced reactivity. Initial studies began with a small series of stilbene-based compounds that were found to have different activity toward controlling metal-free Abeta and metal−Abeta reactivity despite their structural similarity. In-depth (bio)chemical and DFT calculations were also performed to propose modes of action for these molecules. This knowledge was then used to create a library of chemical tools that have different abilities toward mediating abnormal metal-free and metal-induced Abeta reactivity. Additionally, two more frameworks were developed and their ability to control metal−Abeta reactivity was explored. Overall, the small molecules designed and analyzed here demonstrate that increased mechanistic understanding of their activity allows for the development of compounds with targeted abilities to control the reactivity of metal−protein complexes. Application of such compounds in vivo could lead to the elucidation of the pathogenesis of these devastating diseases, which could result in effective therapeutic discovery.PhDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/113567/1/beckmw_3.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/113567/2/beckmw_2.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/113567/3/beckmw_1.pd

Key targets for multi-target ligands designed to combat neurodegeneration

This article is based upon work from COST Action CM1103 “Structure-based drug design for diagnosis and treatment of neurological diseases: dissecting and modulating complex function in the monoaminergic systems of the brain”, supported by COST (European Cooperation in Science and Technology). The authors thank the participants in COST Action for productive collaborations. M. Majekova acknowledges the support of VEGA 2/0033/14, and M. Medina the support of MINECO, Spain (BIO2013-42978-P)Growing evidence supports the view that neurodegenerative diseases have multiple and common mechanisms in their aetiologies. These multifactorial aspects have changed the broadly common assumption that selective drugs are superior to ‘dirty drugs’ for use in therapy. This drives the research in studies of novel compounds that might have multiple action mechanisms. In neurodegeneration, loss of neuronal signaling is a major cause of the symptoms, so preservation of neurotransmitters by inhibiting the breakdown enzymes is a first approach. Acetylcholinesterase (AChE) inhibitors are the drugs preferentially used in AD and that one of these, rivastigmine, is licensed also for PD. Several studies have shown that monoamine oxidase (MAO) B, located mainly in glial cells, increases with age and is elevated in Alzheimer (AD) and Parkinson’s Disease’s (PD). Deprenyl, a MAO B inhibitor, significantly delays the initiation of levodopa treatment in PD patients. These indications underline that AChE and MAO are considered a necessary part of multi-target designed ligands (MTDL). However, both of these targets are simply symptomatic treatment so if new drugs are to prevent degeneration rather than compensate for loss of neurotransmitters, then oxidative stress and mitochondrial events must also be targeted. MAO inhibitors can protect neurons from apoptosis by mechanisms unrelated to enzyme inhibition. Understanding the involvement of MAO and other proteins in the induction and regulation of the apoptosis in mitochondria will aid progress towards strategies to prevent the loss of neurons. In general, the oxidative stress observed both in PD and AD indicate that antioxidant properties are a desirable part of MTDL molecules. After two or more properties are incorporated into one molecule, the passage from a lead compound to a therapeutic tool is strictly linked to its pharmacokinetic and toxicity. In this context the interaction of any new molecules with cytochrome P450 and other xenobiotic metabolic processes is a crucial point. The present review covers the biochemistry of enzymes targeted in the design of drugs against neurodegeneration and the cytochrome P450-dependent metabolism of MTDLs.Publisher PDFPeer reviewe

Towards a unifying, systems biology understanding of large-scale cellular death and destruction caused by poorly liganded iron: Parkinson’s, Huntington’s, Alzheimer’s, prions, bactericides, chemical toxicology and others as examples

Exposure to a variety of toxins and/or infectious agents leads to disease, degeneration and death, often characterised by circumstances in which cells or tissues do not merely die and cease to function but may be more or less entirely obliterated. It is then legitimate to ask the question as to whether, despite the many kinds of agent involved, there may be at least some unifying mechanisms of such cell death and destruction. I summarise the evidence that in a great many cases, one underlying mechanism, providing major stresses of this type, entails continuing and autocatalytic production (based on positive feedback mechanisms) of hydroxyl radicals via Fenton chemistry involving poorly liganded iron, leading to cell death via apoptosis (probably including via pathways induced by changes in the NF-κB system). While every pathway is in some sense connected to every other one, I highlight the literature evidence suggesting that the degenerative effects of many diseases and toxicological insults converge on iron dysregulation. This highlights specifically the role of iron metabolism, and the detailed speciation of iron, in chemical and other toxicology, and has significant implications for the use of iron chelating substances (probably in partnership with appropriate anti-oxidants) as nutritional or therapeutic agents in inhibiting both the progression of these mainly degenerative diseases and the sequelae of both chronic and acute toxin exposure. The complexity of biochemical networks, especially those involving autocatalytic behaviour and positive feedbacks, means that multiple interventions (e.g. of iron chelators plus antioxidants) are likely to prove most effective. A variety of systems biology approaches, that I summarise, can predict both the mechanisms involved in these cell death pathways and the optimal sites of action for nutritional or pharmacological interventions

An implantable nano-enabled bio-robotic intracranial device for targeted and prolonged drug delivery

A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, in fulfillment of the requirements for the degree of Doctor of PhilosophyAlzheimer’s disease (AD) is the most prevalent and progressive neurodegenerative disorder (ND). It is characterized by a progressive decline of cognitive function, complete loss of memory, deterioration of visual capacity and the inability to function independently. According to the World Health Organization (WHO) it is estimated that about 26 million people suffer with AD worldwide. Although the etiology of AD is not fully understood, the aggregation of β-amyloidal (A) peptides that are associated with the formation of extracellular neurotoxin senile plaques and neurofibrillary tangles comprising hyperphosphorylated tau proteins have been recognized as the primary constituents that play a crucial role in AD. Several potential neurotherapeutic agents that can improve the management of AD such as metal chelators and alkaloid drugs have been approved by the US Food and Drug Administration (FDA) and European Medicines Agency (EMA). Metal chelators [e.g. histidine, Ethylenediaminetetraacetic acid (EDTA) and zinc acetate (ZnAc)] are the main therapy used for modulating Aβ peptide aggregation with biological metals (such as zinc and copper ions) which is associated with promoting neurotoxicity in AD. While alkaloid drugs, such as donepezil, galantamine and rivastigmine, are used to inhibit the enzyme acetylcholinesterase (AChE); memantine is used to block the N-methyl-D-aspartate (NMDA) receptors associated with pathological activation. Despite the availability of these indispensable drugs, the clinical utility of these drugs is hampered by their poor retention and difficulty in bypassing the highly restrictive Blood Brain Barrier (BBB). Therefore this study aimed at developing novel nanoliposomes (NLPs) surface-engineered with chelating and synthetic peptides that are capable of crossing the BBB thus improving delivery efficacy and modulating the extracellular neurotoxicity associated with β-Amyloid aggregates of AD. Furthermore, since this system was designed for a chronic condition, a temporary depot-based polymeric system was integrated for further enhancement of the liposomal half-life, storage and prolonged drug delivery over a period of 50 days. The surface-engineered NLPs produced were spherical in shape, 100-149±28nm ~ size, with a zeta potential range of -9.59 to -37.3mV and a polydispersity index (PdI) of 0.02-0.2. A Box-Behnken experimental design was employed for maximizing the ligand coupling efficiency (40-78%) and drug entrapment efficiency (DEE) that ranged from 42-79%. The optimized peptide-based ligand NLP formulation showed sustained drug release (30% of drug released within 48 hours). Chelating ligands on the surface of NLPs showed 50-68% modulation of neurotoxicity on PC12 neuronal cells induced by ZnAβ (1-42) or CuAβ (1-42) aggregates. When drug-loaded functionalized NLPs were embedded within the temporal hydrophilic hydrogel network/scaffold as an implantable nano-enabled bio-robotic intracranial device (BICD), the physicomechanical and physicochemical dynamics showed improvement of liposomal structure such as the stability, and homogeneity in distribution of the liposomes within the internal core of the hydrogel networks and post-lyophilized scaffold. In vitro studies in simulated cerebrospinal fluid (CSF) showed prolonged release behavior of the drug-loaded functionalized NLPs from the BICD with 50-70% released over 50 days. Scanning Electron Microscopy (SEM) and confocal microscopy confirmed intact liposomal structures within the temporal polymeric scaffold/depot post-fixation and post-lyophilization. Ex vivo studies confirmed cell proliferation and a low level of lactate dehydrogenase (LDH), which is associated with cell membrane damage/injury, after PC12 neuronal cells were exposed to the BICD. In addition, when PC12 neuronal cells were exposed to the BICD high accumulation of galantamine (GAL) into these PC12 neuronal cells was observed post-cultivation. This outcome indicated that the released drug-loaded functionalized NLPs from the BICD were still in their intact form and capable of serving as bio-robotic markers for the delivery of GAL into the neuronal cells in response to AD. Furthermore, intracellular activity validated that the synthetic peptide has the potency for targeted delivery of the drug-loaded NLPs post-release of the BICD in ex vivo studies. Overall, results from this study revealed that the BICD device had superior cytocompatibility and may be suitable for application as a prolonged and targeted delivery system for GAL into neuronal cells to treat AD

Developing a cell-based fluorescent assay for screening Dicer-activating compounds

MicroRNAs are ~22 nucleotide long RNA strands which regulate gene expression by binding to the 3’UTRs of messenger RNAs. MicroRNAs are predicted to regulate about a half of all protein-coding genes in the human genome thus affecting many cellular processes. One crucial part of microRNA biogenesis is the cleaving of pre-miRNA strands into mature microRNAs by the type III RNase enzyme, Dicer. Dicer has been shown to be downregulated due to aging and in many disease states. Particularly central nervous system disorders are linked to dysregulated microRNA processing. According to the latest studies, Dicer is crucial to the survival of dopaminergic neurons and conditional Dicer knockout mice show severe nigrostriatal dopaminergic cell loss, which is a hallmark of Parkinson’s disease. By activating Dicer with a small-molecule drug, enoxacin, the survival of dopaminergic cells exposed to stress is significantly improved. However, enoxacin, which is a fluoroquinolone antibiotic, activates Dicer only at high concentrations (10-100 μM) and is polypharmacological, which may cause detrimental side effects. Therefore, enoxacin is not a suitable drug candidate for Dicer deficiencies and better Dicer-activating drug candidates are needed. The aim of this work was to develop a cell-based fluorescent assay to screen for Dicer-activating compounds. Assays which measure Dicer activity have already been developed, but they have some pitfalls which don’t make them optimal to use for high-throughput screening of Dicer-activating compounds. Some are cell-free enzyme-based assays and thus neglect Dicer in its native context. The RNA to be processed by Dicer does not represent a common mammalian RNA type. Most assays do not have internal normalizing factors, such as a second reporter protein to account for e.g. cell death, or the analysis method is not feasible for high-throughput screening data. Considering these disadvantages, the study started by designing a reporter plasmid in silico. The plasmid expresses two fluorescent proteins, mCherry (red) and EGFP (green), and a mCherry transcripttargeting siRNA implemented into a pre-miR155 backbone which is processed by Dicer. Thus, measuring the ratios of red and green fluorescence intensities will give an indication on Dicer activity. The plasmid also has additional regulatory elements for stabilizing expression levels. The plasmid was then produced by molecular cloning methods and its functionality was tested with Dicer-modulating compounds. The assay was optimised by testing it in different cell lines and varying assay parameters, and stable cell lines were created to make large-scale screening more convenient. Finally, a small-scale screen was done with ten pharmacologically active compounds. Transiently transfected, in Chinese hamster ovarian cells, mCherry silencing was too efficient for reliable detection of improvement in silencing efficiency due to floor effect. With an inducible, Tet-On, system in FLP-IN 293 T-Rex cells, the expression could be controlled by administering doxycycline and the improvement in silencing was quantifiable. The assay seemed to be functional after 72 hours and 120 hours of incubation using enoxacin (100 μM) as a positive control. However, the screening found no compounds to significantly reduce mCherry/EGFP fluorescence ratio and, additionally, the effect of enoxacin was abolished. Therefore, a more thorough analysis on the effects of enoxacin was done and, although statistically significant, enoxacin was only marginally effective in reducing mCherry/EGFP fluorescence ratio after 72 hours of treatment. It should be noted from the small-scale screening that metformin and BDNF, compounds previously shown to elevate Dicer levels, showed similar effects to enoxacin. The quality of the assay in terms of high-throughput screening was determined by calculating Zfactors and coefficients of variations for the experiments, which showed that the variability of the assay was acceptable, but the differences between controls was not large enough for reliable screening. In conclusion, the effects of metformin and BDNF should be further studied and regarding the assay, more optimisation is needed for large-scale, high-throughput, screening to be done with minimal resources.MikroRNA:t ovat noin 22 nukleotidiä pitkiä RNA-juosteita, jotka estävät geenien ilmentymistä sitoutumalla lähetti-RNA:n 3’UTR-alueille. MikroRNA:t osallistuvat laajalti moneen soluprosessiin säätelemällä noin puolta kaikista proteiineja koodaavista geeneistä. MikroRNA:n ilmentymisessä, eräs tärkeä vaihe on III tyypin RNaasin, Dicerin, suorittama pre-miRNA:n prosessointi valmiiksi mikroRNA-juosteeksi. Dicerin toiminnan ja ilmentymisen on mitattu heikentyvän ikääntymisen johdosta, sekä useissa eri taudeissa. Erityisesti keskushermostotautien ja mikroRNA prosessointiin liittyvien ongelmien välillä on löydetty yhteys. Tuoreimpien tutkimusten mukaan Dicerilla on tärkeä rooli myös dopaminergisten hermosolujen selviytymisen kannalta ja lisäksi Dicer muuntogeenisillä hiirillä mustatumakkeen dopamiinihermosolut kuolevat, joka on Parkinsonin taudin keskeisin patofysiologinen ilmiö. Dicerin aktiivisuuden tehostamisella, käyttäen enoksasiinia, on suojaava vaikutus dopamiinihermosoluille. Enoksasiini, joka on fluorokinoloneihin kuuluva antimikrobinen yhdiste, tehostaa Diceria vain suurilla pitoisuuksilla (10-100 μM). Lisäksi se on polyfarmakologinen voiden aiheuttaa paljon vakavia haittavaikutuksia, joten se ei ole optimaalinen lääkeaine Dicer-puutoksiin liitettyjen tautien hoitamiseen. Tämän erikoistyön tavoitteena oli kehittää solupohjainen, fluoresenssiin perustuva menetelmä, jolla voisi seuloa parempia Diceria aktivoivia yhdisteitä. Dicerin aktiivisuutta mittaavia menetelmiä on jo kehitetty aiemmin muutamia, mutta ne eivät ole optimaalisia Diceria-aktivoivien yhdisteiden seulomiseksi. Osa kokeista on entsyymipohjaisia eivätkä ne ota huomioon solunsisäistä endogeenistä Diceria. Kokeissa käytettävä RNA, jonka Dicer prosessoi, ei edusta yleisiä nisäkässolujen RNA-tyyppejä. Tietyissä kokeissa ei ole sisäistä suhteuttavaa tekijää (esimerkiksi toista fluoresoivaa proteiinia) tai niillä ei ole mahdollista suorittaa laajoja seulontoja. Työssä suunniteltiin ensiksi edellä mainitut puutteet huomioon ottaen reportteriplasmidi in silico. Plasmidi ilmentää kahta fluoresoivaa proteiinia, mCherry:ä (punainen) ja EGFP:tä,(vihreä) sekä mCherry:n ilmentymistä estävää siRNA-juostetta pre-miR155:n runkoon liitettynä, jonka Dicer prosessoi. Näin ollen, mittaamalla punaisen ja vihreän fluoresenssi-intensiteettien suhdetta, voidaan tutkia Dicerin aktiivisuutta. Plasmidissa on myös useita säätelyelementtejä ilmentymisen tasaamiseksi. Plasmidi valmistettiin molekyylikloonausmenetelmin ja sen toiminnollisuutta testattiin Dicerin aktiivisuuteen vaikuttavilla yhdisteillä. Seulontakoetta optimoitiin eri solulinjoilla ja olosuhdemuutoksilla, ja lisäksi valmistettiin stabiileja solulinjoja laajamittaisen seulonnan helpottamiseksi. Lopuksi suoritettiin pienen mittakaavan seulonta kymmenelle farmakologisesti aktiiviselle yhdisteelle. Ohimenevästi transfektoituna, Kiinanhamsterin munasarjasoluissa, mCherryn hiljentäminen oli niin tehokasta, että hiljentämisen tehostamista ei voitu luotettavasti mitata. Hallitsemalla ilmentymistä doksisykliinin avulla, Tet-On-systeemillä FLP-IN 293 T-Rex soluilla, saatiin ilmentymistä kontrolloitua ja mittaukset luotettaviksi. Seulontakoe saatiin toimimaan 72 tunnin ja 120 tunnin aikapisteillä käyttäen enoksasiinia positiivisena kontrollina. Seulonnasta ei löydetty mCherry/EGFP fluoresenssien suhdetta merkitsevästi vähentäviä yhdisteitä ja lisäksi enoksasiinin vaikutus ei ollut enää tilastollisesti merkitsevä. Tämän perusteella suoritettiin laajempi analyysi enoksasiinin vaikutuksista, missä havaittiin, että sen vaikutus mCherry/EGFP fluoresenssien suhteen vähentämisessä oli, vaikkakin tilastollisesti merkitsevä, hyvin vähäinen 72 tunnin kokeessa. Huomioitavaa pienen mittakaavan seulonnasta on, että metformiinin ja BDNF:n, joiden on aiemmin osoitettu lisäävän Dicerin ilmentymistä, vaikutukset olivat vastaavia enoksasiinin vaikutukseen. Seulontakokeen laatu laajamittaisen seulontakokeen suhteen määritettiin laskemalla kokeille Z-tekijän sekä hajonnan koeffisienttien arvot. Nämä osoittivat, että kokeen hajonta oli hyväksyttävä, mutta ero kontrollien välillä oli liian pieni, jotta koetta voisi käyttää luotettavasti seulomiseen. Tärkeimpinä johtopäätöksinä, metformiinin ja BDNF:n vaikutuksia Diceriin tulisi tutkia tarkemmin, ja koetta on optimoitava lisää, jotta laajamittaisia seulontoja voidaan suorittaa mahdollisimman vähillä resursseilla