In Silico Study Identified Methotrexate Analog as Potential Inhibitor of Drug Resistant Human Dihydrofolate Reductase for Cancer Therapeutics.

Drug resistance is a core issue in cancer chemotherapy. A known folate antagonist, methotrexate (MTX) inhibits human dihydrofolate reductase (hDHFR), the enzyme responsible for the catalysis of 7,8-dihydrofolate reduction to 5,6,7,8-tetrahydrofolate, in biosynthesis and cell proliferation. Structural change in the DHFR enzyme is a significant cause of resistance and the subsequent loss of MTX. In the current study, wild type hDHFR and double mutant (engineered variant) F31R/Q35E (PDB ID: 3EIG) were subject to computational study. Structure-based pharmacophore modeling was carried out for wild type (WT) and mutant (MT) (variant F31R/Q35E) hDHFR structures by generating ten models for each. Two pharmacophore models, WT-pharma and MT-pharma, were selected for further computations, and showed excellent ROC curve quality. Additionally, the selected pharmacophore models were validated by the Guner-Henry decoy test method, which yielded high goodness of fit for WT-hDHFR and MT-hDHFR. Using a SMILES string of MTX in ZINC15 with the selections of 'clean', in vitro and in vivo options, 32 MTX-analogs were obtained. Eight analogs were filtered out due to their drug-like properties by applying absorption, distribution, metabolism, excretion, and toxicity (ADMET) assessment tests and Lipinski's Rule of five. WT-pharma and MT-pharma were further employed as a 3D query in virtual screening with drug-like MTX analogs. Subsequently, seven screening hits along with a reference compound (MTX) were subjected to molecular docking in the active site of WT- and MT-hDHFR. Through a clustering analysis and examination of protein-ligand interactions, one compound was found with a ChemPLP fitness score greater than that of MTX (reference compound). Finally, a simulation of molecular dynamics (MD) identified an MTX analog which exhibited strong affinity for WT- and MT-hDHFR, with stable RMSD, hydrogen bonds (H-bonds) in the binding site and the lowest MM/PBSA binding free energy. In conclusion, we report on an MTX analog which is capable of inhibiting hDHFR in wild type form, as well as in cases where the enzyme acquires resistance to drugs during chemotherapy treatment.

Comparative Gene-Expression Analysis of Alzheimer's Disease Progression with Aging in Transgenic Mouse Model.

Alzheimer's disease (AD) is a multifactorial neurodegenerative disorder characterized by progressive memory dysfunction and a decline in cognition. One of the biggest challenges to study the pathological process at a molecular level is that there is no simple, cost-effective, and comprehensive gene-expression analysis tool. The present study provides the most detailed (Reverse transcription polymerase chain reaction) RT-PCR-based gene-expression assay, encompassing important genes, based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) disease pathway. This study analyzed age-dependent disease progression by focusing on pathological events such as the processing of the amyloid precursor protein, tau pathology, mitochondrial dysfunction, endoplasmic reticulum stress, disrupted calcium signaling, inflammation, and apoptosis. Messenger RNA was extracted from the cortex and hippocampal region of APP/PS1 transgenic mice. Samples were divided into three age groups, six-, nine-, and 12-month-old transgenic mice, and they were compared with normal C57BL/6J mice of respective age groups. Findings of this study provide the opportunity to design a simple, effective, and accurate clinical analysis tool that can not only provide deeper insight into the disease, but also act as a clinical diagnostic tool for its better diagnosis.

Adiponectin-mimetic novel nonapeptide rescues aberrant neuronal metabolic-associated memory deficits in Alzheimer's disease.

BACKGROUND: Recently, we and other researchers reported that brain metabolic disorders are implicated in Alzheimer's disease (AD), a progressive, devastating and incurable neurodegenerative disease. Hence, novel therapeutic approaches are urgently needed to explore potential and novel therapeutic targets/agents for the treatment of AD. The neuronal adiponectin receptor 1 (AdipoR1) is an emerging potential target for intervention in metabolic-associated AD. We aimed to validate this hypothesis and explore in-depth the therapeutic effects of an osmotin-derived adiponectin-mimetic novel nonapeptide (Os-pep) on metabolic-associated AD. METHODS: We used an Os-pep dosage regimen (5 µg/g, i.p., on alternating days for 45 days) for APP/PS1 in amyloid ß oligomer-injected, transgenic adiponectin knockout (Adipo-/-) and AdipoR1 knockdown mice. After behavioral studies, brain tissues were subjected to biochemical and immunohistochemical analyses. In separate cohorts of mice, electrophysiolocal and Golgi staining experiments were performed. To validate the in vivo studies, we used human APP Swedish (swe)/Indiana (ind)-overexpressing neuroblastoma SH-SY5Y cells, which were subjected to knockdown of AdipoR1 and APMK with siRNAs, treated with Os-pep and other conditions as per the mechanistic approach, and we proceeded to perform further biochemical analyses. RESULTS: Our in vitro and in vivo results show that Os-pep has good safety and neuroprotection profiles and crosses the blood-brain barrier. We found reduced levels of neuronal AdipoR1 in human AD brain tissue. Os-pep stimulates AdipoR1 and its downstream target, AMP-activated protein kinase (AMPK) signaling, in AD and Adipo-/- mice. Mechanistically, in all of the in vivo and in vitro studies, Os-pep rescued aberrant neuronal metabolism by reducing neuronal insulin resistance and activated downstream insulin signaling through regulation of AdipoR1/AMPK signaling to consequently improve the memory functions of the AD and Adipo-/- mice, which was associated with improved synaptic function and long-term potentiation via an AdipoR1-dependent mechanism. CONCLUSION: Our findings show that Os-pep activates AdipoR1/AMPK signaling and regulates neuronal insulin resistance and insulin signaling, which subsequently rescues memory deficits in AD and adiponectin-deficient models. Taken together, the results indicate that Os-pep, as an adiponectin-mimetic novel nonapeptide, is a valuable and promising potential therapeutic candidate to treat aberrant brain metabolism associated with AD and other neurodegenerative diseases.

Oral Administration of Alpha Linoleic Acid Rescues Aß-Induced Glia-Mediated Neuroinflammation and Cognitive Dysfunction in C57BL/6N Mice.

In this work, we evaluated the effects of alpha linoleic acid (ALA), an omega-3 polyunsaturated fatty acid, on amyloid-beta-induced glial-cell-mediated neuroinflammation, amyloidogenesis, and cognitive dysfunction in mice. After an infusion of Aß1-42 (Aß1-42, 5 µL/5 min/mouse, intracerebroventricular injection (i.c.v), and respective treatments of ALA (60 mg/kg per oral for six weeks), neuroinflammation, apoptotic markers, and synaptic markers were evaluated by Western blot and immunofluorescence analyses. According to our findings, the infusion of Aß1-42 activated Toll-like receptor 4 (TLR4), glial fibrillary acidic protein (GFAP), and ionized calcium adaptor molecule 1 (Iba-1) in the frontal cortices and hippocampi of the Aß1-42-injected mice to a greater extent than the Aß1-42 + ALA-cotreated mice. Similarly, there was an elevated expression of phospho-c-Jun-N-terminal kinase (p-JNK), phospho-nuclear factor-kB p65 (p-NF-kB p65 (Ser536)), and tissue necrosis factor (TNF) in the Aß1-42 infused mouse brains; interestingly, these markers were significantly reduced in the Aß + ALA-cotreated group. The elevated expression of pro-apoptotic markers was observed during apoptotic cell death in the Aß1-42-treated mouse brains, whereas these markers were markedly reduced in the Aß + ALA-cotreated group. Moreover, Aß1-42 infusion significantly increased amyloidogenesis, as assessed by the enhanced expression of the amyloid precursor proteins (APP) beta-amyloid cleaving enzyme-1 (BACE-1) and amyloid-beta (Aß1-42) in the mouse brains, whereas these proteins were markedly reduced in the Aß + ALA-cotreated group. We also checked the effects of ALA against Aß-triggered synaptic dysfunction and memory dysfunction, showing that ALA significantly improved memory and synaptic functions in Aß-treated mouse brains. These results indicated that ALA could be an applicable intervention in neuroinflammation, apoptotic cell loss, amyloidogenesis, and memory dysfunction via the inhibition of TLR4 and its downstream targets in Aß + ALA-cotreated mouse brains.

Adiponectin homolog novel osmotin protects obesity/diabetes-induced NAFLD by upregulating AdipoRs/PPARα signaling in ob/ob and db/db transgenic mouse models.

BACKGROUND: In metabolic disorders, adiponectin and adiponectin receptors (AdipoR1/R2) signaling has a key role in improving nonalcoholic fatty liver disease (NAFLD) in obesity-associated diabetes. OBJECTIVE: To the best of our knowledge, here, we reported for the first time the underlying mechanistic therapeutic efficacy of the novel osmotin, a homolog of mammalian adiponectin, against NAFLD in leptin-deficient ob/ob and db/db mice. METHODS: The ob/ob and db/db mice were treated with osmotin at a dose of 5 µg/g three times a week for two weeks. To co-relate the in vivo results we used the human liver carcinoma HepG2 cells, subjected to knockdown with small siRNAs of AdipoR1/R2 and PPARα genes and treated with osmotin and palmitic acid (P.A.). MTT assay, Western blotting, immunohistofluorescence assays, and plasma biochemical analyses were applied. RESULTS: Osmotin stimulated AdipoR1/R2 and its downstream APPL1/PPAR-α/AMPK/SIRT1 pathways in ob/ob and db/db mice, and HepG2 cells exposed to P.A. Mechanistically, we confirmed that knockdown of AdipoR1/R2 and PPARα by their respective siRNAs abolished the osmotin activity in HepG2 cells exposed to P.A. Overall, the in vivo and in vitro results suggested that osmotin protected against NAFLD through activation of AdipoR1/R2 and its downstream APPL1/PPAR-α/AMPK/SIRT1 pathways as shown by the reduced body weight, blood glucose level and glycated hemoglobin, improved glucose tolerance, attenuated insulin resistance and hepatic glucogenesis, regulated serum lipid parameters, and increased fatty acid oxidation and mitochondrial functions. CONCLUSION: Our findings strongly suggest that novel osmotin might be a potential novel therapeutic tool against obesity/diabetes-induced NAFLD and other metabolic disorders.

Molecular Cloning and Expression of Osmotin in a Baculovirus-Insect System: Purified Osmotin Mitigates Amyloid-beta Deposition in Neuronal Cells.

Osmotin is a pathogenesis-related plant protein, have gained focus of research because of its homology with mammalian adiponectin. The therapeutic properties of osmotin have been explored in recent years as it exhibits neuroprotective effects against amyloid beta-, glutamate- and ethanol-induced synaptic dysfunction and neurodegeneration. In the present study, the full-length gene of the tobacco plant osmotin was cloned and expressed in the Sf9 insect cell line using the baculovirus expression system. In vitro analysis of purified Osmotin protein showed excellent cell viability, p-AMPK activation and a reduction in amyloid-beta deposition. Immunofluorescent analysis showed significant reduction in amyloid beta deposition in APP over expressing neuronal cells. Osmotin inhibited amyloid beta deposition by influencing expression of APP processing genes including APP, ADAM 10 and BACE 1. Purified Osmotin showed reduction in amyloid beta deposition in different in vitro models as well. Osmotin showed similar mechanism when compared with mammalian adiponectin in different in vitro models. The present method will be an excellent approach for the efficient and cost-effective production of the functional protein to be utilized for therapeutic purposes. Reduction in amyloid beta deposition by activation of p-AMPK influencing APP processing genes makes osmotin a potent therapeutic candidate for neurodegenerative diseases.

Suppression of adiponectin receptor 1 promotes memory dysfunction and Alzheimer's disease-like pathologies.

Recent studies on neurodegeneration have focused on dysfunction of CNS energy metabolism as well as proteinopathies. Adiponectin (ADPN), an adipocyte-derived hormone, plays a major role in the regulation of insulin sensitivity and glucose homeostasis in peripheral organs via adiponectin receptors. In spite of accumulating evidence that adiponectin has neuroprotective properties, the underlying role of adiponectin receptors has not been illuminated. Here, using gene therapy-mediated suppression with shRNA, we found that adiponectin receptor 1 (AdipoR1) suppression induces neurodegeneration as well as metabolic dysfunction. AdipoR1 knockdown mice exhibited increased body weight and abnormal plasma chemistry and also showed spatial learning and memory impairment in behavioural studies. Moreover, AdipoR1 suppression resulted in neurodegenerative phenotypes, diminished expression of the neuronal marker NeuN, and increased expression and activity of caspase 3. Furthermore, AD-like pathologies including insulin signalling dysfunction, abnormal protein aggregation and neuroinflammatory responses were highly exhibited in AdipoR1 knockdown groups, consistent with brain pathologies in ADPN knockout mice. Together, these results suggest that ADPN-AdipoR1 signalling has the potential to alleviate neurodegenerative diseases such as Alzheimer's diseases.

Co-Treatment with Anthocyanins and Vitamin C Ameliorates Ethanol- Induced Neurodegeneration via Modulation of GABAB Receptor Signaling in the Adult Rat Brain.

Chronic ethanol exposure is known to cause neuronal damage in both humans and experimental animal models. Ethanol treatment induces neurotoxicity via the generation of reactive oxygen species (ROS), while anthocyanins (extracted from black soybean) and ascorbic acid (vitamin C) are free radical scavengers that can be used as neuroprotective agents against ROS. In this study the underlying neuroprotective potential of black soybean anthocyanins and vitamin C was determined. For this purpose, adult rats were exposed to 10% (v/v) ethanol for 8 weeks, followed by co-treatment with anthocyanins (24 mg/kg) and vitamin C (100 mg/kg) during the last 4 weeks. Our results showed that ethanol administration increased the expression of γ -aminobutyric acid B1 receptor (GABAB1R) and induced neuronal apoptosis via alterations to the Bax/Bcl-2 ratio, release of cytochrome C and activation of caspase-3 and caspase-9. Anthocyanins alone and supplementation with vitamin C showed an additive effect in reversing the trend of apoptotic signals induced by ethanol in the cortex and hippocampus. Consequently, anthocyanins also decreased the expression of poly (ADP ribose) polymerase-1 induced by ethanol and prevented DNA damage. Furthermore, anthocyanins and vitamin C reversed the ethanol-induced expression of GABAB1R and its downstream signaling molecule phospho-cAMP response element binding protein. Moreover, histopathology and immunohistochemistry results showed that anthocyanins and vitamin C significantly reduced ethanol-induced neuronal cell death. Our study revealed a neuroprotective role of anthocyanins and vitamin C via modulation of GABAB1R expression in the adult brain. Hence, we suggest that anthocyanins or co-treatment with anthocyanins and vitamin C may be a new and potentially effective neuroprotective agent for alcohol abuse.

Recent Developments in Nanomedicines for Management of Various Health Issues Via Metabolism and Physico-Chemical Properties.

During the last decade the nanotechnologists began research on applications of nanomaterials for medicine and therapeutics. Various nanoparticles (nanomedicines) are being used worldwide for the diagnosis and management in a number of disorders including cancer and neurodegenerative disorders. The successful non-viral gene therapy is now possible with the advancements in nanotechnology. Mostly nanoparticles are divided into two main classes: organic and inorganic nanoparticles. Diverse features of nanomedicines with surface modification help to make them biocompatible with addition of varying polymer that facilitates targeted delivery of drug and its controlled release into the cells, tissues and organs. Liposomes, quantum dots, silver and gold nanoparticles are the most common examples of nanomedicines.