Amyloid-binding Alcohol Dehydrogenase Research Articles

Functionalized Allopurinols Targeting Amyloid-Binding Alcohol Dehydrogenase Rescue Aβ-Induced Mitochondrial Dysfunction.

Alzheimer's disease (AD) is the most common dementia affecting one in nine people over 65. Only a handful of small-molecule drugs and the anti-β amyloid (Aβ) antibody aducanumab are approved to treat AD. However, they only serve to reduce symptoms of advanced disease. Novel treatments administered early in disease progression before the accumulation of Aβ and tau reaches the threshold where neuroinflammation is triggered and irreversible neuronal damage occurs are more likely to provide effective therapy. There is a growing body of evidence implying that mitochondrial dysfunction occurs at an early stage of AD pathology. The mitochondrial enzyme amyloid-binding alcohol dehydrogenase (ABAD) binds to Aβ potentiating toxicity. Moreover, ABAD has been shown to be overexpressed in the same areas of the brain most affected by AD. Inhibiting the Aβ-ABAD protein-protein interaction without adversely affecting normal enzyme turnover is hypothesized to be a potential treatment strategy for AD. Herein, we conduct structure-activity relationship studies across a series of functionalized allopurinol derivatives to determine their ability to inhibit Aβ-mediated reduction of estradiol production from ABAD. The lead compound resulting from these studies possesses potent activity with no toxicity up to 100 μM, and demonstrates an ability to rescue defective mitochondrial metabolism in human SH-SY5Y cells and rescue both defective mitochondrial metabolism and morphology ex vivo in primary 5XFAD AD mouse model neurons.

Novel Benzothiazole-based Ureas as 17β-HSD10 Inhibitors, A Potential Alzheimer's Disease Treatment.

It has long been established that mitochondrial dysfunction in Alzheimer’s disease (AD) patients can trigger pathological changes in cell metabolism by altering metabolic enzymes such as the mitochondrial 17β-hydroxysteroid dehydrogenase type 10 (17β-HSD10), also known as amyloid-binding alcohol dehydrogenase (ABAD). We and others have shown that frentizole and riluzole derivatives can inhibit 17β-HSD10 and that this inhibition is beneficial and holds therapeutic merit for the treatment of AD. Here we evaluate several novel series based on benzothiazolylurea scaffold evaluating key structural and activity relationships required for the inhibition of 17β-HSD10. Results show that the most promising of these compounds have markedly increased potency on our previously published inhibitors, with the most promising exhibiting advantageous features like low cytotoxicity and target engagement in living cells.

ABAD/17β-HSD10 reduction contributes to the protective mechanism of huperzine a on the cerebral mitochondrial function in APP/PS1 mice

Huperzine A (HupA) is a kind of Lycopodium alkaloid with potential disease-modifying qualities that has been reported to protect against β-amyloid (Aβ)-mediated mitochondrial damage in Alzheimer's disease. However, the fundamental molecular mechanism underlying the protective action of HupA against Aβ-mediated mitochondrial malfunction is not completely understood. Recently, the mitochondrial enzyme amyloid-binding alcohol dehydrogenase (ABAD) protein has been reported to facilitate Aβ-induced mitochondrial damage, resulting in mitochondrial malfunction and cell death. Our study found that HupA, but not the acetylcholinesterase inhibitor tacrine, reduced the deposition of Aβ and the ABAD level, and further reduced Aβ–ABAD complexes, thereby improving cerebral mitochondrial function in APP/PS1 mice. This was accompanied by attenuated reactive oxygen species overload, as well as increases adenosine triphosphate levels. Moreover, HupA decreased the release of cytochrome-c from mitochondria and the level of cleaved caspase-3, thereby increasing dissociated brain cell viability in APP/PS1 mice. Thus, our study demonstrated that a reduction in ABAD was involved in the protective mechanism of HupA on the cerebral mitochondrial function in APP/PS1 mice.

Amyloid-Binding Alcohol Dehydrogenase (ABAD) Inhibitors for the Treatment of Alzheimer's Disease.

Alzheimer's disease (AD) is the most common dementia. No cure exists, and current treatment only manages early symptoms. Mitochondrial dysfunction is a hallmark of amyloid-β (Aβ) neurotoxicity, the pathogenic protein implicated in AD. This is due in part to the interaction between Aβ and amyloid-binding alcohol dehydrogenase (ABAD). This mitochondrial protein is a vital energy regulator that, following Aβ binding, activates signaling cascades that lead to neuronal death. One of the most significant roles of ABAD is to maintain the balance of estradiol/estrone in neurons. However, the Aβ-ABAD interaction disrupts this balance and leads to a reduction in levels of estradiol, thus leading to an increase in reactive oxygen species levels and to apoptosis. Two additional proteins, peroxiredoxin-2 and endophilin-1, are implicated in Aβ-ABAD complex-mediated toxicity. Targeting the Aβ-ABAD interaction has emerged as a novel therapeutic strategy for AD. Herein, we review the chemistry and pharmacology of reported ABAD inhibitors.

IN VITRO EVALUATION OF BENZOTHIAZOLE-BASED UREAS AS POTENTIAL ABAD/17β-HSD10 MODULATORS FOR ALZHEIMER'S DISEASE THERAPEUTICS

The mitochondrial enzyme, amyloid binding alcohol dehydrogenase (ABAD), has been shown to mediate the cytotoxic effects of Amyloid-β within the Alzheimer's diseased brain. Mutational studies have shown that ABAD must be catalytically active for cytotoxicity to be observed and therefore the direct inhibition of ABAD may offer a novel therapeutic strategy to treat the disease. In 2006, Xie et al.1 identified benzothiazole urea analogues capable of perturbing the ABAD- Aβ interaction. We hypothesised that the capability of these analogues to disrupt the interaction may infer the ability to bind to ABAD, which in turn may inhibit ABAD activity. Consequently we have generated many series of benzothiazole urea derivatives and assessed their ability to inhibit ABAD; identifying and characterising several potent inhibitors and we have now made several bioisostere modifications to these molecules2,3,4. To assess the compounds therapeutic potential we are using many different techniques, but our primary screening strategy utilises recombinant ABAD enzyme extract to measure ABAD activity, followed by cell based and biophysical de-selection assays to verify the inhibitory nature, the cell permeability and cytotoxicity of these compounds. Medicinal chemistry is utilised to optimise the molecules pharmacokinetic properties and potency, whilst in sillico physiochemical prediction software and molecular modelling aim to optimise the chemical space available. We have characterised several different types of benzothiazole molecules at the recombinant protein level2,3,4 and now at the cellular level. These molecules exhibit nM potency in the protein assay and low μM potency in the cellular assays. These molecules exhibit promise as ABAD inhibitors and with further modifications and in vivo screening these compounds have the potential to be used as therapeutics in AD.

In Vitro Assay Development and HTS of Small-Molecule Human ABAD/17β-HSD10 Inhibitors as Therapeutics in Alzheimer’s Disease

A major hallmark of Alzheimer’s disease (AD) is the formation of neurotoxic aggregates composed of the amyloid-β peptide (Aβ). Aβ has been recognized to interact with numerous proteins, resulting in pathological changes to the metabolism of patients with AD. One such mitochondrial metabolic enzyme is amyloid-binding alcohol dehydrogenase (ABAD), where altered enzyme function caused by the Aβ-ABAD interaction is known to cause mitochondrial distress and cytotoxic effects, providing a feasible therapeutic target for AD drug development. Here we have established a high-throughput screening platform for the identification of modulators to the ABAD enzyme. A pilot screen with a total of 6759 compounds from the NIH Clinical Collections (NCC) and SelleckChem libraries and a selection of compounds from the BioAscent diversity collection have allowed validation and robustness to be optimized. The pilot screen revealed 16 potential inhibitors in the low µM range against ABAD with favorable physicochemical properties for blood-brain barrier penetration.

Synthesis and evaluation of frentizole-based indolyl thiourea analogues as MAO/ABAD inhibitors for Alzheimer’s disease treatment

Alzheimer’s disease (AD) is a neurodegenerative disorder associated with an excessive accumulation of amyloid-beta peptide (Aβ). Based on the multifactorial nature of AD, preparation of multi-target-directed ligands presents a viable option to address more pathological events at one time. A novel class of asymmetrical disubstituted indolyl thioureas have been designed and synthesized to interact with monoamine oxidase (MAO) and/or amyloid-binding alcohol dehydrogenase (ABAD). The design combines the features of known MAO inhibitors scaffolds (e.g. rasagiline or ladostigil) and a frentizole moiety with potential to interact with ABAD. Evaluation against MAO identified several compounds that inhibited in the low to moderate micromolar range. The most promising compound (19) inhibited human MAO-A and MAO-B with IC50 values of 6.34μM and 0.30μM, respectively. ABAD activity evaluation did not show any highly potent compound, but the compound series allowed identification of structural features to assist the future development of ABAD inhibitors. Finally, several of the compounds were found to be potent inhibitors of horseradish peroxidase (HRP), preventing the use of the Amplex™ Red assay to detect hydrogen peroxide produced by MAO, highlighting the need for serious precautions when using an enzyme-coupled assay.

Design, synthesis and in vitro evaluation of benzothiazole-based ureas as potential ABAD/17β-HSD10 modulators for Alzheimer’s disease treatment

Amyloid-beta peptide (Aβ) has been recognized to interact with numerous proteins, which may lead to pathological changes in cell metabolism of Alzheimer’s disease (AD) patients. One such known metabolic enzyme is mitochondrial amyloid-binding alcohol dehydrogenase (ABAD), also known as 17β-hydroxysteroid dehydrogenase type 10 (17β-HSD10). Altered enzyme function caused by the Aβ-ABAD interaction, was previously shown to cause mitochondrial distress and a consequent cytotoxic effect, therefore providing a feasible target in AD drug development. Based on previous frentizole derivatives studies, we report two novel series of benzothiazolyl ureas along with novel insights into the structure and activity relationships for inhibition of ABAD. Two compounds (37, 39) were identified as potent ABAD inhibitors, where compound 39 exhibited comparable cytotoxicity with the frentizole standard; however, one-fold higher cytotoxicity than the parent riluzole standard. The calculated and experimental physical chemical properties of the most potent compounds showed promising features for blood–brain barrier penetration.

Methylene Blue Improves Brain Mitochondrial ABAD Functions and Decreases Aβ in a Neuroinflammatory Alzheimer's Disease Mouse Model.

Methylene blue (MB) phase II clinical trials reported improvements in cognitive functions of Alzheimer's disease (AD) patients. Regarding MB mechanism of action, its antioxidant and mitochondrial protective effects have been previously described. In relation to AD, it has been recently reported that MB reduced amyloid beta (Aβ) levels in AD models. The mitochondrial enzyme amyloid-binding alcohol dehydrogenase (ABAD) has been shown to bind Aβ inducing mitochondrial dysfunction, providing a direct relation between Aβ toxicity and mitochondrial dysfunction occurring in AD. Since it has been reported that inhibiting ABAD protects mitochondrial functions and prevents Aβ-induced toxicity, the aim of the current study was to investigate if the protective effects of MB could be associated with an effect on ABAD levels and functions. The current study shows that MB is able to enhance cell viability, reduce both reactive oxygen species levels and importantly Aβ oligomers in a lipopolysaccharide (LPS) mouse model. Interestingly, ABAD levels were increased in the brains of the LPS mouse model and MB treatment was able to reduce its levels. Given that regulation of the estradiol level is a well-established function of ABAD, brain estradiol level was compared in LPS mouse model and in MB-treated mice. The results of the current study show that MB treatment is able to improve significantly the LPS-induced decrease of estradiol levels in mice brains, indicating its ability to modulate both levels and function of ABAD. These results give a new insight to possible mechanisms of MB in AD.

Molecular docking of fisetin with AD associated AChE, ABAD and BACE1 proteins.

Alzheimer׳s disease (AD) is one of the most common dementias showing slow progressive cognitive decline. Progression of intracerebral accumulation of beta amyloid (Aβ) peptides by the action of amyloid binding alcohol dehydrogenase (ABAD), a mitochondrial enzyme and β-site amyloid precursor protein cleaving enzyme 1 (BACE1) and the degradation of Acetylcholinesterase (AChE) the main pathological characteristics of AD. Therefore, it is of interest to evaluate the importance of fisetin (a flavonol that belongs to the flavonoid group of polyphenols) binding with AChE, ABAD and BACE1 proteins. Docking experiment of fisetin with these proteins using two different tools namely iGEMDOCK and FlexX show significant binding with acceptable binding values. Thus, the potential inhibitory role of fisetin with AD associated proteins is documented.

Methylene blue decreases brain mitochondrial ABAD and amyloid beta levels protecting mitochondrial functions in LPS-mouse model

Background Methylene blue (MB) is lately being proposed to be effective in treating Alzheimer’s disease (AD). Phase 2 clinical trials reported improvements in cognitive functions of AD patients after MB treatment. One of the main mechanisms of action that has been described for MB is inhibition of Tau aggregation [1]. Moreover, its antioxidant and mitochondrial protection have been previously described [2]. Only recently, a study using a triple transgenic AD mouse model has tested the mechanism of MB in vivo, showing improved cognition and reduced Ab levels after MB treatment [3]. Recently, the mitochondrial enzyme Amyloid binding alcohol dehydrogenase (ABAD) has been shown to bind Ab inducing mitochondrial dysfunction, providing a direct relation between Ab and mitochondrial dysfunction occurring in AD. Previous studies have shown that inhibiting ABAD protects mitochondrial functions and prevented Ab-induced toxicity [4][5]. Taking into consideration the mitochondrial protective effect of MB and the recent data suggesting its ability to reduce Ab levels, our aim was to investigate the effect of MB on ABAD and mitochondrial function in an LPS mouse model that has been previously described to induce memory impairment, with Ab accumulation in hippocampus and cerebral cortex [6].

Is Amyloid Binding Alcohol Dehydrogenase a Drug Target for Treating Alzheimer's Disease?

Current strategies for the treatment of Alzheimer's disease (AD) involve tackling the formation or clearance of the amyloid-beta peptide (Aβ) and/or hyper-phosphorylated tau, or the support and stabilization of the remaining neuronal networks. However, as we gain a clearer idea of the large number of molecular mechanisms at work in this disease, it is becoming clearer that the treatment of AD should take a combined approach of dealing with several aspects of the pathology. The concept that we also need to protect specific sensitive targets within the cell should also be considered. In particular the role of protecting the function of a specific mitochondrial protein, amyloid binding alcohol dehydrogenase (ABAD), will be the focus of this review. Mitochondrial dysfunction is a well-recognized fact in the progression of AD, though until recently the mechanisms involved could only be loosely labeled as changes in 'metabolism'. The discovery that Aβ can be present within the mitochondria and specifically bind to ABAD, has opened up a new area of AD research. Here we review the evidence that the prevention of Aβ binding to ABAD is a drug target for the treatment of AD.

Is Amyloid Binding Alcohol Dehydrogenase a Drug Target for Treating Alzheimer’s Disease?

Current strategies for the treatment of Alzheimer’s disease (AD) involve tackling the formation or clearance of the amyloid-beta peptide (Aβ) and/or hyper-phosphorylated tau, or the support and stabilization of the remaining neuronal networks. However, as we gain a clearer idea of the large number of molecular mechanisms at work in this disease, it is becoming clearer that the treatment of AD should take a combined approach of dealing with several aspects of the pathology. The concept that we also need to protect specific sensitive targets within the cell should also be considered. In particular the role of protecting the function of a specific mitochondrial protein, amyloid binding alcohol dehydrogenase (ABAD), will be the focus of this review. Mitochondrial dysfunction is a well-recognized fact in the progression of AD, though until recently the mechanisms involved could only be loosely labeled as changes in ‘metabolism’. The discovery that Aβ can be present within the mitochondria and specifically bind to ABAD, has opened up a new area of AD research. Here we review the evidence that the prevention of Aβ binding to ABAD is a drug target for the treatment of AD.

Structure‐Based Design and Synthesis of Benzothiazole Phosphonate Analogues with Inhibitors of Human ABAD‐Aβ for Treatment of Alzheimer’s Disease

Amyloid binding alcohol dehydrogenase, a mitochondrial protein, is a cofactor facilitating amyloid-β peptide (Aβ) induced cell stress. Antagonizing Aβ-ABAD interaction protects against aberrant mitochondrial and neuronal function and improves learning memory in the Alzheimer's disease mouse model. Therefore, it offers a potential target for Alzheimer's drug design, by identifying potential inhibitors of Aβ-ABAD interaction. 2D QSAR methods were applied to novel compounds with known IC(50) values, which formed a training set. A correlation analysis was carried out comparing the statistics of the measured IC(50) with predicted values. These selectivity-determining descriptors were interpreted graphically in terms of principle component analyses, which are highly informative for the lead optimization process with respect to activity enhancement. A 3D pharmacophore model also was created. The 2D QSAR and 3D pharmacophore models will assist in high-throughput screening. In addition, ADME descriptors were also determined to study their pharmacokinetic properties. Finally, amyloid binding alcohol dehydrogenase molecular docking study of these novel molecules was undertaken to determine whether these compounds exhibit significant binding affinity with the binding site. We have synthesized only the compounds that have shown the best drug-like properties as candidates for further studies.

Mitochondrial β-amyloid in Alzheimer's disease

It is well established that the intracellular accumulation of Aβ (amyloid β-peptide) is associated with AD (Alzheimer's disease) and that this accumulation is toxic to neurons. The precise mechanism by which this toxicity occurs is not well understood; however, identifying the causes of this toxicity is an essential step towards developing treatments for AD. One intracellular location where the accumulation of Aβ can have a major effect is within mitochondria, where mitochondrial proteins have been identified that act as binding sites for Aβ, and when binding occurs, a toxic response results. At one of these identified sites, an enzyme known as ABAD (amyloid-binding alcohol dehydrogenase), we have identified changes in gene expression in the brain cortex, following Aβ accumulation within mitochondria. Specifically, we have identified two proteins that are up-regulated not only in the brains of transgenic animal models of AD but also in those of human sufferers. The increased expression of these proteins demonstrates the complex and counteracting pathways that are activated in AD. Previous studies have identified approximate contact sites between ABAD and Aβ; on basis of these observations, we have shown that by using a modified peptide approach it is possible to reverse the expression of these two proteins in living transgenic animals and also to recover mitochondrial and behavioural deficits. This indicates that the ABAD-Aβ interaction is potentially an interesting target for therapeutic intervention. To explore this further we used a fluorescing substrate mimic to measure the activity of ABAD within living cells, and in addition we have identified chemical fragments that bind to ABAD, using a thermal shift assay.

(−)-CHANA, a Fluorogenic Probe for Detecting Amyloid Binding Alcohol Dehydrogenase HSD10 Activity in Living Cells

The association of 17β-hydroxysteroid dehydrogenase 10 (HSD10) with β-amyloid in the brain is known to contribute to the progression of Alzheimer's disease. Further, it has been shown that the interaction between the purified HSD10 and β-amyloid inhibits its enzymatic activity. However, to date no system has been developed to enable the study of HSD10 activity in intact living cells. To address this significant shortcoming, we have developed a novel fluorogenic probe, (-)-cyclohexenyl amino naphthalene alcohol [(-)-CHANA], to observe and measure the activity of HSD10 in living cells. The oxidation of (-)-CHANA by HSD10 results in the production and accumulation of a fluorescent product, which can be measured using real-time fluorescence microscopy. This compound permits the measurement of mitochondrial HSD10 activity and its inhibition by both a small molecule HSD10 inhibitor and by β-amyloid, in living cells. Herein, we define the parameters under which this probe can be used. This compound is likely to prove useful in future investigations aimed at developing therapeutic compounds targeting the HSD10-β-amyloid association.

The consequences of mitochondrial amyloid β-peptide in Alzheimer's disease

The Abeta (amyloid-beta peptide) has long been associated with Alzheimer's disease, originally in the form of extracellular plaques. However, in the present paper we review the growing evidence for the role of soluble intracellular Abeta in the disease progression, with particular reference to Abeta found within the mitochondria. Once inside the cell, Abeta is able to interact with a number of targets, including the mitochondrial proteins ABAD (amyloid-binding alcohol dehydrogenase) and CypD (cyclophilin D), which is a component of the mitochondrial permeability transition pore. Interference with the normal functions of these proteins results in disruption of cell homoeostasis and ultimately cell death. The present review explores the possible mechanisms by which cell death occurs, considering the evidence presented on a molecular, cellular and in vivo level.

Endophilin I Expression Is Increased in the Brains of Alzheimer Disease Patients

Alzheimer patients have increased levels of both the 42 amyloid-beta-peptide (Abeta) and the amyloid binding alcohol dehydrogenase (ABAD), which is an intracellular binding site for Abeta. The overexpression of Abeta and ABAD in transgenic mice has shown that the binding of Abeta to ABAD results in amplified neuronal stress and impairment of learning and memory. From a proteomic analysis of the brains from these animals, we have identified for the first time that the protein endophilin I increases in Alzheimer diseased brain. The increase in endophilin I levels in neurons is linked to an increase in the activation of the stress kinase c-Jun N-terminal kinase with the subsequent death of the neurons. We also demonstrate in living animals that the expression level of endophilin I is an indicator for the interaction of ABAD and Abeta as its expression levels return to normal if this interaction is perturbed. Therefore this identifies endophilin I as a new indicator of the progression of Alzheimer disease.

Interaction of amyloid binding alcohol dehydrogenase/Aβ mediates up-regulation of peroxiredoxin II in the brains of Alzheimer’s disease patients and a transgenic Alzheimer’s disease mouse model

Alzheimer’s patients have increased levels of both the 42 beta amyloid-β-peptide (Aβ) and amyloid binding alcohol dehydrogenase (ABAD) which is an intracellular binding site for Aβ. The over-expression of Aβ and ABAD in transgenic mice has shown that the binding of Aβ to ABAD results in exaggerating neuronal stress and impairment of learning and memory. From a proteomic analysis of the brains from these animals we identified that peroxiredoxin II levels increase in Alzheimer’s diseased brain. This increase in peroxiredoxin II levels protects neurons against Aβ induced toxicity. We also demonstrate, for the first time in living animals, that the expression level of peroxiredoxin II is an indicator for the interaction of ABAD and Aβ as its expression levels return to normal if this interaction is perturbed. Therefore this indicates the possibility of reversing changes observed in Alzheimer’s disease and that the Aβ–ABAD interaction is a suitable drug target.

Mitochondrial Dysfunction and Alzheimers Disease

Mitochondrial dysfunction has been implicated in causing metabolic abnormalities in Alzheimer's disease (AD). The searches for mitochondrial DNA variants associated with AD susceptibility have generated conflicting results. The age-related accumulation of somatic mitochondrial DNA deletion has been suggested to play a pathogenic role in the development of AD. Recent studies have demonstrated that amyloid-beta peptide (Abeta) progressively accumulates in mitochndrial matrix, as demonstrated in both transgenic mice over-expressing mutant amyloid precursor protein (APP) and autopsy brain from AD patients. Abeta-mediated mitochondrial stress was evidenced by impaired oxygen consumption and decreased respiratory chain complexes III and IV activities in brains from AD patients and AD-type transgenic mouse model. Furthermore, our studies indicated that interaction of intramitochondrial Abeta with a mitochondrial enzyme, amyloid binding alcohol dehydrogenase (ABAD), inhibits its enzyme activity, enhances generation of reactive oxygen species (ROS), impairs energy metabolism, and exaggerates Abeta-induced spatial learning/memory deficits and neuropathological changes in transgenic AD-type mouse model. Interception of ABAD-Abeta interaction may be a potential therapeutic strategy for Alzheimer's disease.