Tacrine - Benzothiazoles: Novel class of potential multitarget anti-Alzheimers drugs dealing with cholinergic, amyloid and mitochondrial systems.

A series of tacrine - benzothiazole hybrids incorporate inhibitors of acetylcholinesterase (AChE), amyloid ß (Aß) aggregation and mitochondrial enzyme ABAD, whose interaction with Aß leads to mitochondrial dysfunction, into a single molecule. In vitro, several of 25 final compounds exerted excellent anti-AChE properties and interesting capabilities to block Aß aggregation. The best derivative of the series could be considered 10w that was found to be highly potent and selective towards AChE with the IC50 value in nanomolar range. Moreover, the same drug candidate exerted absolutely the best results of the series against ABAD, decreasing its activity by 23% at 100 µM concentration. Regarding the cytotoxicity profile of highlighted compound, it roughly matched that of its parent compound - 6-chlorotacrine. Finally, 10w was forwarded for in vivo scopolamine-induced amnesia experiment consisting of Morris Water Maze test, where it demonstrated mild procognitive effect. Taking into account all in vitro and in vivo data, highlighted derivative 10w could be considered as the lead structure worthy of further investigation.

Benzothiazolyl Ureas are Low Micromolar and Uncompetitive Inhibitors of 17ß-HSD10 with Implications to Alzheimer's Disease Treatment.

Human 17ß-hydroxysteroid dehydrogenase type 10 is a multifunctional protein involved in many enzymatic and structural processes within mitochondria. This enzyme was suggested to be involved in several neurological diseases, e.g., mental retardation, Parkinson's disease, or Alzheimer's disease, in which it was shown to interact with the amyloid-beta peptide. We prepared approximately 60 new compounds based on a benzothiazolyl scaffold and evaluated their inhibitory ability and mechanism of action. The most potent inhibitors contained 3-chloro and 4-hydroxy substitution on the phenyl ring moiety, a small substituent at position 6 on the benzothiazole moiety, and the two moieties were connected via a urea linker (4at, 4bb, and 4bg). These compounds exhibited IC50 values of 1-2 µM and showed an uncompetitive mechanism of action with respect to the substrate, acetoacetyl-CoA. These uncompetitive benzothiazolyl inhibitors of 17ß-hydroxysteroid dehydrogenase type 10 are promising compounds for potential drugs for neurodegenerative diseases that warrant further research and development.

Mitochondrial dysfunction and Alzheimer's disease: prospects for therapeutic intervention.

Alzheimer's disease (AD) is a multifactorial neurodegenerative disease and has become a major socioeconomic issue in many developed countries. Currently available therapeutic agents for AD provide only symptomatic treatments, mainly because the complete mechanism of the AD pathogenesis is still unclear. Although several different hypotheses have been proposed, mitochondrial dysfunction has gathered interest because of its profound effect on brain bioenergetics and neuronal survival in the pathophysiology of AD. Various therapeutic agents targeting the mitochondrial pathways associated with AD have been developed over the past decade. Although most of these agents are still early in the clinical development process, they are used to restore mitochondrial function, which provides an alternative therapeutic strategy that is likely to slow the progression of the disease. In this mini review, we will survey the AD-related mitochondrial pathways and their small-molecule modulators that have therapeutic potential. We will focus on recently reported examples, and also overview the current challenges and future perspectives of ongoing research. [BMB Reports 2020; 53(1): 47-55].

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-ß (Aß) neurotoxicity, the pathogenic protein implicated in AD. This is due in part to the interaction between Aß and amyloid-binding alcohol dehydrogenase (ABAD). This mitochondrial protein is a vital energy regulator that, following Aß 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 Aß-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 Aß-ABAD complex-mediated toxicity. Targeting the Aß-ABAD interaction has emerged as a novel therapeutic strategy for AD. Herein, we review the chemistry and pharmacology of reported ABAD inhibitors.

In Vitro Assay Development and HTS of Small-Molecule Human ABAD/17ß-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-ß peptide (Aß). Aß 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 Aß-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.

A chemical genomics approach to drug reprofiling in oncology: Antipsychotic drug risperidone as a potential adenocarcinoma treatment.

Drug reprofiling is emerging as an effective paradigm for discovery of cancer treatments. Herein, an antipsychotic drug is immobilised using the Magic Tag® chemical genomics tool and screened against a T7 bacteriophage displayed library of polypeptides from Drosophila melanogaster, as a whole genome model, to uncover an interaction with a section of 17-ß-HSD10, a proposed prostate cancer target. A computational study and enzyme inhibition assay with full length human 17-ß-HSD10 identifies risperidone as a drug reprofiling candidate. When formulated with rumenic acid, risperidone slows proliferation of PC3 prostate cancer cells in vitro and retards PC3 prostate cancer tumour growth in vivo in xenografts in mice, presenting an opportunity to reprofile risperidone as a cancer treatment.

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 (Aß). 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/17ß-HSD10 modulators for Alzheimer's disease treatment.

Amyloid-beta peptide (Aß) 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 17ß-hydroxysteroid dehydrogenase type 10 (17ß-HSD10). Altered enzyme function caused by the Aß-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.

High mitochondrial respiration and glycolytic capacity represent a metabolic phenotype of human tolerogenic dendritic cells.

Human dendritic cells (DCs) regulate the balance between immunity and tolerance through selective activation by environmental and pathogen-derived triggers. To characterize the rapid changes that occur during this process, we analyzed the underlying metabolic activity across a spectrum of functional DC activation states, from immunogenic to tolerogenic. We found that in contrast to the pronounced proinflammatory program of mature DCs, tolerogenic DCs displayed a markedly augmented catabolic pathway, related to oxidative phosphorylation, fatty acid metabolism, and glycolysis. Functionally, tolerogenic DCs demonstrated the highest mitochondrial oxidative activity, production of reactive oxygen species, superoxide, and increased spare respiratory capacity. Furthermore, assembled, electron transport chain complexes were significantly more abundant in tolerogenic DCs. At the level of glycolysis, tolerogenic and mature DCs showed similar glycolytic rates, but glycolytic capacity and reserve were more pronounced in tolerogenic DCs. The enhanced glycolytic reserve and respiratory capacity observed in these DCs were reflected in a higher metabolic plasticity to maintain intracellular ATP content. Interestingly, tolerogenic and mature DCs manifested substantially different expression of proteins involved in the fatty acid oxidation (FAO) pathway, and FAO activity was significantly higher in tolerogenic DCs. Inhibition of FAO prevented the function of tolerogenic DCs and partially restored T cell stimulatory capacity, demonstrating their dependence on this pathway. Overall, tolerogenic DCs show metabolic signatures of increased oxidative phosphorylation programing, a shift in redox state, and high plasticity for metabolic adaptation. These observations point to a mechanism for rapid genome-wide reprograming by modulation of underlying cellular metabolism during DC differentiation.

Determination of small molecule ABAD inhibitors crossing blood-brain barrier and pharmacokinetics.

A major obstacle to the development of effective treatment of Alzheimer's disease (AD) is successfully delivery of drugs to the brain. We have previously identified a series of benzothiazole phosphonate compounds that block the interaction of amyloid-ß peptide with amyloid-ß binding alcohol dehydrogenase (ABAD). A selective and sensitive method for the presence of three new benzothiazole ABAD inhibitors in mouse plasma, brain, and artificial cerebrospinal fluid has been developed and validated based on high performance liquid chromatography tandem mass spectrometry. Mass spectra were generated using Micromass Quattro Ultima "triple" quadrupole mass spectrometer equipped with an Electrospray Ionization interface. Good linearity was obtained over a concentration range of 0.05-2.5 µg/ml. The lowest limit of quantification and detection was found to be 0.05 µg/ml. All inter-day accuracies and precisions were within ± 15% of the nominal value and ± 20%, respectively, at the lower limit of quantitation. The tested compounds were stable at various conditions with recoveries >90.0% (RSD <10%). The method used for pharmacokinetic studies of compounds in mouse cerebrospinal fluid, plasma, and brain is accurate, precise, and specific with no matrix effect. Pharmacokinetic data showed that these compounds penetrate the blood-brain barrier (BBB) yielding 4-50 ng/ml peak brain concentrations and 2 µg/ml peak plasma concentrations from a 10 mg/kg dose. These results indicate that our newly synthesized small molecule ABAD inhibitors have a good drug properties with the ability to cross the blood-brain barrier, which holds a great potential for AD therapy.

Identification of human ABAD inhibitors for rescuing Aß-mediated mitochondrial dysfunction.

Amyloid beta (Aß) binding alcohol dehydrogenase (ABAD) is a cellular cofactor for promoting (Aß)-mediated mitochondrial and neuronal dysfunction, and cognitive decline in transgenic Alzheimer's disease (AD) mouse models. Targeting mitochondrial ABAD may represent a novel therapeutic strategy against AD. Here, we report the biological activity of small molecule ABAD inhibitors. Using in vitro surface plasmon resonance (SPR) studies, we synthesized compounds with strong binding affinities for ABAD. Further, these ABAD inhibitors (ABAD-4a and 4b) reduced ABAD enzyme activity and administration of phosphonate derivatives of ABAD inhibitors antagonized calcium-mediated mitochondrial swelling. Importantly, these compounds also abolished Aß-induced mitochondrial dysfunction as shown by increased cytochrome c oxidase activity and adenosine-5'-triphosphate levels, suggesting protective mitochondrial function effects of these synthesized compounds. Thus, these compounds are potential candidates for further pharmacologic development to target ABAD to improve mitochondrial function.

Structure-based design and synthesis of benzothiazole phosphonate analogues with inhibitors of human ABAD-Aß for treatment of Alzheimer's disease.

Amyloid binding alcohol dehydrogenase, a mitochondrial protein, is a cofactor facilitating amyloid-ß peptide (Aß) induced cell stress. Antagonizing Aß-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 Aß-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.

Hydroxysteroid (17ß) dehydrogenase X in human health and disease.

Hydroxysteroid (17ß) dehydrogenase 10 (HSD10), the HSD17B10 gene product, is a mitochondrial NAD(+)-dependent dehydrogenase. There are two outstanding features of this vital enzyme: (a) the versatility of its catalytic endowment is attributed to the flexibility of its active site to accommodate diverse substrates such as steroids, fatty acids, bile acid, and xenobiotics; (b) its capacity to bind other proteins and peptides. For example, it tightly binds with three identical subunits to compose a homotetramer. The homotetramer then binds with two other proteins, namely, RNA (guanine-9-)methyl-transferase domain containing-1 and KIAA0391, to form mitochondrial RNase P. Furthermore, various HSD10 functions are inhibited when the enzyme is bound by amyloid-ß peptide or estrogen receptor alpha. Missense mutations of HSD10 may cause neurodegeneration related to HSD10 deficiency, whereas a silent mutation of HSD10 results in mental retardation, choreoathetosis and abnormal behavior (MRXS10). The clinical condition of some HSD10 patients mimics mitochondrial disorders. Since normal HSD10 function is essential for brain cognitive activity, elevated levels of HSD10 found in brains of Alzheimer disease (AD) patients and mouse AD model might counterbalance the inhibition of HSD10 by amyloid-ß peptide. The investigation of HSD10 may lead to a better understanding of AD pathogenesis.

Inhibition of amyloid-beta (Abeta) peptide-binding alcohol dehydrogenase-Abeta interaction reduces Abeta accumulation and improves mitochondrial function in a mouse model of Alzheimer's disease.

Amyloid-ß (Aß) peptide-binding alcohol dehydrogenase (ABAD), an enzyme present in neuronal mitochondria, exacerbates Aß-induced cell stress. The interaction of ABAD with Aß exacerbates Aß-induced mitochondrial and neuronal dysfunction. Here, we show that inhibition of the ABAD-Aß interaction, using a decoy peptide (DP) in vitro and in vivo, protects against aberrant mitochondrial and neuronal function and improves spatial learning/memory. Intraperitoneal administration of ABAD-DP [fused to the transduction of human immunodeficiency virus 1-transactivator (Tat) protein and linked to the mitochondrial targeting sequence (Mito) (TAT-mito-DP) to transgenic APP mice (Tg mAPP)] blocked formation of ABAD-Aß complex in mitochondria, increased oxygen consumption and enzyme activity associated with the mitochondrial respiratory chain, attenuated mitochondrial oxidative stress, and improved spatial memory. Similar protective effects were observed in Tg mAPP mice overexpressing neuronal ABAD decoy peptide (Tg mAPP/mito-ABAD). Notably, inhibition of the ABAD-Aß interaction significantly reduced mitochondrial Aß accumulation. In parallel, the activity of mitochondrial Aß-degrading enzyme PreP (presequence peptidase) was enhanced in Tg mAPP mitochondria expressing the ABAD decoy peptide. These data indicate that segregating ABAD from Aß protects mitochondria/neurons from Aß toxicity; thus, ABAD-Aß interaction is an important mechanism underlying Aß-mediated mitochondrial and neuronal perturbation. Inhibitors of ABAD-Aß interaction may hold promise as targets for the prevention and treatment of Alzheimer's disease.

ABAD: a potential therapeutic target for Abeta-induced mitochondrial dysfunction in Alzheimer's disease.

Amyloid-beta-peptide (Abetabinding to mitochondrial Abeta-binding alcohol dehydrogenase (ABAD) enzyme triggers a series of events leading to mitochondrial dysfunction characteristic of Alzheimer's disease (AD). Thus this interaction may represent a novel target for treatment strategy against AD. In this review we summarize current findings regarding the ABAD-Abeta interaction, namely structural and biophysical data, available inhibitors and more recent data from proteomic studies.

Metabolic mechanisms in heart failure.

Although neurohumoral antagonism has successfully reduced heart failure morbidity and mortality, the residual disability and death rate remains unacceptably high. Though abnormalities of myocardial metabolism are associated with heart failure, recent data suggest that heart failure may itself promote metabolic changes such as insulin resistance, in part through neurohumoral activation. A detrimental self-perpetuating cycle (heart failure --> altered metabolism --> heart failure) that promotes the progression of heart failure may thus be postulated. Accordingly, we review the cellular mechanisms and pathophysiology of altered metabolism and insulin resistance in heart failure. It is hypothesized that the ensuing detrimental myocardial energetic perturbations result from neurohumoral activation, increased adverse free fatty acid metabolism, decreased protective glucose metabolism, and in some cases insulin resistance. The result is depletion of myocardial ATP, phosphocreatine, and creatine kinase with decreased efficiency of mechanical work. On the basis of the mechanisms outlined, appropriate therapies to mitigate aberrant metabolism include intense neurohumoral antagonism, limitation of diuretics, correction of hypokalemia, exercise, and diet. We also discuss more novel mechanistic-based therapies to ameliorate metabolism and insulin resistance in heart failure. For example, metabolic modulators may optimize myocardial substrate utilization to improve cardiac function and exercise performance beyond standard care. The ultimate success of metabolic-based therapy will be manifest by its capacity further to lessen the residual mortality in heart failure.

Roles of type 10 17beta-hydroxysteroid dehydrogenase in intracrinology and metabolism of isoleucine and fatty acids.

Human type 10 17beta-hydroxysteroid dehydrogenase (HSD) is a homotetrameric protein located in mitochondria. This enzyme was alternatively named short chain L-3-hydroxyacyl-CoA dehydrogenase (SCHSD). This NAD(H)-dependent dehydrogenase is essential for the metabolism of branched-chain fatty acids and isoleucine, and is expressed in a variety of tissues, e.g., prostate, brain, liver, and heart. This enzyme inactivates 17beta-estradiol and exhibits a strong oxidative 3alpha-HSD activity to convert 5alpha-androstanediol and allopregnanolone into 5alpha-dihydrotestosterone (5alpha-DHT) and 5alpha-dihydroprogesterone, respectively, in living cells. Certain malignant prostatic epithelial cells and activated astrocytes in Alzheimer's disease patient's brain contain extraordinarily high levels of this enzyme. This mitochondrial dehydrogenase enables prostate cancer cells to generate 5alpha-DHT in the absence of testosterone. Its inactivation of allopregnanolone is important to the modulation of GABA(A) receptor. Among steroidogenic enzymes 17beta-HSD10 plays a significant part in the intracrinology. Although this protein has an affinity for amyloid-beta peptide, its role in the pathogenesis of Alzheimer's disease is far from clear. Additional knowledge of this versatile enzyme would provide the foundation for designing new drugs aimed at treating some neurological diseases and certain types of cancers.

Physicochemical characteristics of soluble oligomeric Abeta and their pathologic role in Alzheimer's disease.

Extracellular fibrillar amyloid deposits are prominent and universal Alzheimer's disease (AD) features, but senile plaque abundance does not always correlate directly with the degree of dementia exhibited by AD patients. The mechanism(s) and dynamics of Abeta fibril genesis and deposition remain obscure. Enhanced Abeta synthesis rates coupled with decreased degradative enzyme production and accumulating physical modifications that dampen proteolysis may all enhance amyloid deposit formation. Amyloid accumulation may indirectly exert the greatest pathologic effect on the brain vasculature by destroying smooth muscle cells and creating a cascade of negative impacts on cerebral blood flow. The most visible manifestation of amyloid dis-equilibrium could actually be a defense mechanism employed to avoid serious vascular wall degradation while the major toxic effects to the gray and white matter neurons are mediated by soluble oligomeric Abeta peptides with high beta-sheet content. The recognition that dynamic soluble oligomeric Abeta pools exist in AD and are correlated to disease severity led to neurotoxicity and physical conformation studies. It is now recognized that the most basic soluble Abeta peptides are stable dimers with hydrophobic regions sequestered from the aqueous environment and are capable of higher order aggregations. Time course experiments employing a modified ELISA method able to detect Abeta oligomers revealed dynamic intermolecular interactions and additional experiments physically confirmed the presence of stable amyloid multimers. Amyloid peptides that are rich in beta-sheet structure are capable of creating toxic membrane ion channels and a capacity to self-assemble as annular structures was confirmed in vitro using atomic force microscopy. Biochemical studies have established that soluble Abeta peptides perturb metabolic processes, provoke release of deleterious reactive compounds, reduce blood flow, induce mitochondrial apoptotic toxicity and inhibit angiogenesis. While there is no question that gross amyloid deposition does contribute to AD pathology, the destructive potential now associated with soluble Abeta suggests that treatment strategies that target these molecules may be efficacious in preventing some of the devastating effects of AD.

Supplementation with alpha-linolenic acid-rich diacylglycerol suppresses fatty liver formation accompanied by an up-regulation of beta-oxidation in Zucker fatty rats.

Insulin resistance-related obesity and diabetes mellitus are the predominant causes of fatty liver disease. Here we examine the effects of dietary diacylglycerol (DG), which is a minor component of plant oils, on lipid accumulation and the expression of genes involved in lipid metabolism in the liver. The animals were fed diets containing either 10% triacylglycerol (TG), 10% TG + 4% alpha-linolenic acid-rich TG (ALATG) or 10% TG + 4% alpha-linolenic acid-rich diacylglycerol (ALADG) for a period of 1 month. Supplementation with ALADG significantly inhibited hepatic triglyceride accumulation; this was accompanied by the up-regulation of beta-oxidation activity, and acyl-CoA oxidase (ACO) and medium-chain acyl-CoA dehydrogenase (MCAD) mRNA levels. By contrast, no significant changes were observed in the levels of peroxisome proliferator-activated receptor-alpha (PPARalpha) and sterol regulatory element-binding protein-1 (SREBP-1) mRNAs. These results indicate that ALADG might be useful in the prevention of fatty liver formation; this effect could be closely related to the stimulation of lipid catabolism in the liver. In addition, our findings suggest that both acylglycerol structure (that is, the structural difference between TG and DG) and fatty-acid species affect the nutritional behaviour of dietary lipids.