Computational Investigation of Novel Compounds as Dual Inhibitors of AChE and GSK-3ß for the Treatment of Alzheimer's Disease.

BACKGROUND: Alzheimer's disease (AD) stands out as one of the most devastating and prevalent neurodegenerative disorders known today. Researchers have identified several enzymatic targets associated with AD among which Glycogen synthase kinase-3ß (GSK-3ß) and Acetylcholinesterase (AChE) are prominent ones. Unfortunately, the market offers very few drugs for treating or managing AD, and none have shown significant efficacy against it. OBJECTIVES: To address this critical issue, the design and discovery of dual inhibitors will represent a potential breakthrough in the fight against AD. In the pursuit of designing novel dual inhibitors, we explored molecular docking and dynamics analyses of tacrine and amantadine uredio-linked amide analogs such as GSK-3ß and AChE dual inhibitors for curtailing AD. Tacrine and adamantine are the FDA-approved drugs that were structurally modified to design and develop novel drug candidates that may demonstrate concurrently dual selectivity towards GSK-3ß and AChE. METHODS: In the following study, molecular docking was executed by employing AutoDock Vina, and molecular dynamics and ADMET predictions were made using Desmond, Qikprop modules of Schrödinger. RESULTS: Our findings revealed that compounds DST2 and DST11 exhibited remarkable molecular interactions with active sites of GSK-3ß and AChE, respectively. These compounds effectively interacted with key amino acids, namely Lys85, Val135, Asp200, and Phe295, resulting in highly favourable docking energies of -9.7 and -12.7 kcal/mol. Furthermore, through molecular dynamics simulations spanning a trajectory of 100 ns, we confirmed the stability of ligands DST2 and DST11 within the active cavities of GSK-3ß and AChE. The compounds exhibiting the most promising docking results also demonstrated excellent ADMET profiles. Notably, DST21 displayed an outstanding human oral absorption rate of 76.358%, surpassing the absorption rates of other molecules. CONCLUSION: Overall, our in-silico studies revealed that the designed molecules showed potential as novel anti-Alzheimer agents capable of inhibiting both GSK-3ß and AChE simultaneously. So, in the future, the designing and development of dual inhibitors will harbinger a new era of drug design in AD treatment.

Comparison of Preemptive Effect of Dexamethasone and Methylprednisolone After Third Molar Surgery: A Split-Mouth Randomized Triple-Blind Clinical Trial.

AIM: The study aimed to compare the efficacy of methylprednisolone and dexamethasone injected into masseter muscle preoperatively in surgical extraction of lower third molars. MATERIALS AND METHODS: This study was carried out on 20 patients who reported to the department of Oral and Maxillofacial surgery, Sri Rajiv Gandhi College of Dental Sciences and Hospital Bangalore, requiring surgical removal of bilateral mandibular third molars. The efficacy of corticosteroid was evaluated based on its ability to reduce pain, swelling and trismus following the surgical extraction of impacted lower third molars. RESULTS: There was no statistical difference between the two steroids with both of them achieving equal level of pain control. There was a statistically significant difference on the second postoperative day with dexamethasone showing clinically superior result. The difference in oral aperture was found to be statistically significant with dexamethasone showing a decreased reduction in postoperative mouth opening on both second and seventh day. CONCLUSION: This study conclusively proves that patient comfort levels are far better with the use of dexamethasone.

Fever, Fasting, and Rhabdomyolysis in an Adult Male.

A 34-year-old man presents with recurrent episodes of acute reversible muscle weakness, soreness, pain, cramps and myoglobinuria with elevated creatine kinase. Symptoms were triggered by fasting, sustained long duration exercise and viral infection. A metabolic myopathy was suspected. Genetic testing showed a homozygous pathogenic variant in CPT2 gene resulting in deficiency of Carnitine Pamitoyl transferase II, an enzyme in the carnitine cycle. The cycle plays a vital role in transport of long chain hydrophobic fatty acids from the cytosol into the mitochondrial matrix for the production of energy via ß-oxidation. Carnitine Pamitoyl transferase II deficiency is the most common inherited disorder of lipid metabolism affecting the skeletal muscle of adults. It is also the most frequent cause of hereditary myoglobinuria across all ages. Our case presents an analysis of important clinical features of carbohydrate and lipid metabolism disorders. It highlights how thermolability of the mutant enzyme, rather than its actual deficiency, explains triggering of muscle symptoms by prolonged exercise, infections, febrile episodes, or exposure to cold.

Facial Nerve Injury in Temporomandibular Joint Approaches.

Facial paralysis can be a devastating consequence resulting from blunt and penetrating trauma to the head and neck, as well as surgical injury, either accidental or due to involvement by tumor. In addition, the etiology can be attributed to a variety of other causes, ranging from infectious to metabolic, and is frequently idiopathic in nature. The incidence of facial nerve injury during temporomandibular joint (TMJ) surgeries varies among surgeons. There are many factors that could contribute to the injury of the temporal and zygomatic branches of the facial nerve. These nerves lie in a confluence of superficial fascia, temporalis fascia, and periosteum and may be injured by any dissection technique that attempts to violate the integrity of these regions. Excessive or heavy-handed retraction causes compression and/or stretching of nerve fibers resulting in neuropraxia. The facial nerve then enters the parotid gland, where the main trunk branches into the upper and lower divisions at the pes anserinus. The nerve further divides into five main branches: the temporal, zygomatic, buccal, marginal mandibular, and cervical. The temporal branch lies within the superficial muscular aponeurotic system at the level of the zygomatic arch. In this paper, we evaluate the facial nerve function based on the House-Brackmann grading index after the preauricular approach for the treatment of condylar fractures, pathologies, and TMJ ankylosis cases. The nerve functional regeneration postfacial nerve injury has been evaluated and reported in this retrospective study.

Activation of GR but not PXR by dexamethasone attenuated acetaminophen hepatotoxicities via Fgf21 induction.

Glucocorticoid receptor (GR) signaling is indispensable for cell growth and development, and plays important roles in drug metabolism. Fibroblast growth factor (Fgf) 21, an important regulator of glucose, lipid, and energy metabolism, plays a cytoprotective role by attenuating toxicities induced by chemicals such as dioxins, acetaminophen (APAP), and alcohols. The present study investigates the impact of dexamethasone (DEX)-activated GR on Fgf21 expression and how it affects the progression of APAP-induced hepatotoxicity. Our results showed that DEX dose/concentration- and time-dependently increased Fgf21 mRNA and protein expression in mouse liver as well as cultured mouse and human hepatoma cells. By using PXR-null mouse model, we demonstrated that DEX induced Fgf21 expression by a PXR-independent mechanism. In cultured mouse and human hepatoma cells, inhibition of GR signaling, by RU486 (Mifepristone) or GR silencing using GR-specific siRNA, attenuated DEX-induced Fgf21 expression. In addition, DEX increased luciferase reporter activity driven by the 3.0-kb mouse and human Fgf21/FGF21 gene promoter. Further, ChIP-qPCR assays demonstrated that DEX increased the binding of GR to the specific cis-regulatory elements located in the 3.0-kb mouse and human Fgf21/FGF21 gene promoter. Pretreatment of 2mg/kg DEX ameliorated APAP-induced liver injury in wild-type but not Fgf21-null mice. In conclusion, via GR activation, DEX induced Fgf21 expression in mouse liver and human hepatoma cells.

Assessment of systemic inflammatory response after total extraperitoneal repair and Lichtenstein repair for inguinal hernia.

PURPOSE: The aim of the study was assessment of the systemic inflammatory response (SIR) intensity by measuring the blood serum levels of high sensitivity C-reactive protein (hsCRP), Interleukin-6 (IL-6) and Total Leukocyte Counts of patients. The estimations were done before and after the patient underwent either open Lichtenstein or endoscopic TEP inguinal hernia repair. This is a prospective observational type of study. METHODS: Sixty patients with a diagnosis of unilateral uncomplicated inguinal hernia were included in the study. Patients were divided into two groups. In the first group, endoscopic total extraperitoneal repair (TEP) was done, while the other group underwent Lichtenstein repair. The patient selection was random. Serum markers for SIR were measured prior to and 24 h post-surgery. RESULTS: Total extraperitoneal repair (TEP) and open Lichtenstein inguinal hernia repair both cause a significant Systemic Inflammatory Response in the body. The rise in serum markers for SIR post-surgery was statistically significant in both the groups. The rise in serum hsCRP and IL-6 concentrations was observed to be equivocal among the two groups. Statistically significant difference was observed in serum TLC rise: Lichtenstein repair group having a higher value. CONCLUSION: Both, open and endoscopic surgical techniques incite a systemic inflammatory response in the body. However, it cannot be conclusively stated that TEP is associated with lesser SIR compared to the Lichtenstein repair on the basis of this study.

Targeting cancer metabolism by simultaneously disrupting parallel nutrient access pathways.

Oncogenic mutations drive anabolic metabolism, creating a dependency on nutrient influx through transporters, receptors, and macropinocytosis. While sphingolipids suppress tumor growth by downregulating nutrient transporters, macropinocytosis and autophagy still provide cancer cells with fuel. Therapeutics that simultaneously disrupt these parallel nutrient access pathways have potential as powerful starvation agents. Here, we describe a water-soluble, orally bioavailable synthetic sphingolipid, SH-BC-893, that triggers nutrient transporter internalization and also blocks lysosome-dependent nutrient generation pathways. SH-BC-893 activated protein phosphatase 2A (PP2A), leading to mislocalization of the lipid kinase PIKfyve. The concomitant mislocalization of the PIKfyve product PI(3,5)P2 triggered cytosolic vacuolation and blocked lysosomal fusion reactions essential for LDL, autophagosome, and macropinosome degradation. By simultaneously limiting access to both extracellular and intracellular nutrients, SH-BC-893 selectively killed cells expressing an activated form of the anabolic oncogene Ras in vitro and in vivo. However, slower-growing, autochthonous PTEN-deficient prostate tumors that did not exhibit a classic Warburg phenotype were equally sensitive. Remarkably, normal proliferative tissues were unaffected by doses of SH-BC-893 that profoundly inhibited tumor growth. These studies demonstrate that simultaneously blocking parallel nutrient access pathways with sphingolipid-based drugs is broadly effective and cancer selective, suggesting a potential strategy for overcoming the resistance conferred by tumor heterogeneity.

Azacyclic FTY720 Analogues That Limit Nutrient Transporter Expression but Lack S1P Receptor Activity and Negative Chronotropic Effects Offer a Novel and Effective Strategy to Kill Cancer Cells in Vivo.

FTY720 sequesters lymphocytes in secondary lymphoid organs through effects on sphingosine-1-phosphate (S1P) receptors. However, at higher doses than are required for immunosuppression, FTY720 also functions as an anticancer agent in multiple animal models. Our published work indicates that the anticancer effects of FTY720 do not depend on actions at S1P receptors but instead stem from FTY720s ability to restrict access to extracellular nutrients by down-regulating nutrient transporter proteins. This result was significant because S1P receptor activation is responsible for FTY720s dose-limiting toxicity, bradycardia, that prevents its use in cancer patients. Here, we describe diastereomeric and enantiomeric 3- and 4-C-aryl 2-hydroxymethyl pyrrolidines that are more active than the previously known analogues. Of importance is that these compounds fail to activate S1P1 or S1P3 receptors in vivo but retain inhibitory effects on nutrient transporter proteins and anticancer activity in solid tumor xenograft models. Our studies reaffirm that the anticancer activity of FTY720 does not depend upon S1P receptor activation and uphold the promise of using S1P receptor-inactive azacyclic FTY720 analogues in human cancer patients.

Nitric Oxide Synthase as a Target for Methicillin-Resistant Staphylococcus aureus.

Bacterial infections associated with methicillin-resistant Staphylococcus aureus (MRSA) are a major economic burden to hospitals, and confer high rates of morbidity and mortality among those infected. Exploitation of novel therapeutic targets is thus necessary to combat this dangerous pathogen. Here, we report on the identification and characterization, including crystal structures, of two nitric oxide synthase (NOS) inhibitors that function as antimicrobials against MRSA. These data provide the first evidence that bacterial NOS (bNOS) inhibitors can work synergistically with oxidative stress to enhance MRSA killing. Crystal structures show that each inhibitor contacts an active site Ile residue in bNOS that is Val in the mammalian NOS isoforms. Mutagenesis studies show that the additional nonpolar contacts provided by the Ile in bNOS contribute to tighter binding toward the bacterial enzyme.

Esters of the marine-derived triterpene sipholenol A reverse P-GP-mediated drug resistance.

Our previous studies showed that several sipholane triterpenes, sipholenol A, sipholenone E, sipholenol L and siphonellinol D, have potent reversal effect for multidrug resistance (MDR) in cancer cells that overexpressed P-glycoprotein (P-gp/ABCB1). Through comparison of cytotoxicity towards sensitive and multi-drug resistant cell lines, we identified that the semisynthetic esters sipholenol A-4-O-acetate and sipholenol A-4-O-isonicotinate potently reversed P-gp-mediated MDR but had no effect on MRP1/ABCC1 and BCRP/ABCG2-mediated MDR. The results from [3H]-paclitaxel accumulation and efflux studies suggested that these two triterpenoids were able to increase the intracellular accumulation of paclitaxel by inhibiting its active efflux. In addition, western blot analysis revealed that these two compounds did not alter the expression levels of P-gp when treated up to 72 h. These sipholenol derivatives also stimulated the ATPase activity of P-gp membranes, which suggested that they might be substrates of P-gp. Moreover, in silico molecular docking studies revealed the virtual binding modes of these two compounds into human homology model of P-gp. In conclusion, sipholenol A-4-O-acetate and sipholenol A-4-O-isonicotinate efficiently inhibit the P-gp and may represent potential reversal agents for the treatment of multidrug resistant cancers.

Hunter's syndrome: a case report.

Hunter's syndrome or mucopolysaccharidosis (MPS) type II is an X-linked recessive mucopolysaccharide disorder caused by a defect in the metabolism of glycosaminoglycans (GAGs) characterized by involvement of nervous, cardiovascular, respiratory, and mucoskeletal systems along with numerous oral manifestations. This is a case report of a 13-year-old boy referred to the Department of Pediatric Dentistry with a chief complaint of irregularly placed teeth from a general physician. Here we highlight the pivotal role of pediatric dentists in diagnosis and treatment planning for patients diagnosed with such systemic conditions and the provision of advanced dental care in the management of the same.

Fibroblast growth factor (Fgf) 21 is a novel target gene of the aryl hydrocarbon receptor (AhR).

The toxic effects of dioxins, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), mainly through activation of the aryl hydrocarbon receptor (AhR) are well documented. Fibroblast growth factor (Fgf) 21 plays critical roles in metabolic adaptation to fasting by increasing lipid oxidation and ketogenesis in the liver. The present study was performed to determine whether activation of the AhR induces Fgf21 expression. In mouse liver, TCDD increased Fgf21 mRNA in both dose- and time-dependent manners. In addition, TCDD markedly increased Fgf21 mRNA expression in cultured mouse and human hepatocytes. Moreover, TCDD increased mRNA (in liver) and protein levels (in both liver and serum) of Fgf21 in wild-type mice, but not in AhR-null mice. Chromatin immunoprecipitation assays showed that TCDD increased AhR protein binding to the Fgf21 promoter (-105/+1 base pair). Fgf21-null mice administered 200µg/kg of TCDD died within 20days, whereas wild-type mice receiving the same treatment were still alive at one month after administration. This indicates that TCDD-induced Fgf21 expression protects against TCDD toxicity. Diethylhexylphthalate (DEHP) pretreatment attenuated TCDD-induced Fgf21 expression in mouse liver and white adipose tissue, which may explain a previous report that DEHP pretreatment decreases TCDD-induced wasting. In conclusion, Fgf21 appears to be a target gene of AhR-signaling pathway in mouse and human liver.

Nutritional and hormonal regulation of the TOR effector 4E-binding protein (4E-BP) in the mosquito Aedes aegypti.

Mosquitoes require blood for egg development, and, as a consequence, they transmit pathogens of devastating diseases. Target of rapamycin (TOR) signaling is a key pathway linking blood feeding and egg development in the mosquito Aedes aegypti. We show that the regulation of the TOR effector translational repressor 4E-BP is finely tuned to the nutritional requirements of the female mosquito, and it occurs at transcriptional and post-translational levels. Immediately after blood feeding, 4E-BP became hyperphosphorylated, suggesting rapid inhibition of its translational repression function. 4E-BP was highly phosphorylated after in vitro incubation of the fat body in the presence of amino acids; this phosphorylation was rapamycin insensitive, in contrast to another TOR target, S6K, phosphorylation of which was rapamycin sensitive. A high level of 4E-BP phosphorylation was also elicited by insulin. Rapamycin and the PI3K inhibitor LY294002 blocked insulin-mediated 4E-BP phosphorylation. RNA-interference depletion of the insulin receptor or Akt resulted in severe reduction of 4E-BP phosphorylation. Phosphorylation and stability of 4E-BP was dependent on its partner eIF4E translation initiation factor. Silencing of 4E-BP resulted in reduction of the life span of adult female mosquitoes. This study demonstrates a dual nutritional and hormonal control of 4E-BP and its role in mosquito egg development.

The small GTPase Rheb is a key component linking amino acid signaling and TOR in the nutritional pathway that controls mosquito egg development.

Mosquitoes transmit numerous devastating human diseases because they require blood feeding for egg development. Previously, we have shown that the nutritional Target-of-Rapamycin (TOR) pathway mediates blood-meal activation of mosquito reproductive cycles. Blood-derived amino acid (AA) signaling through the nutrient-sensitive TOR kinase is critical for the transcriptional activation of the major yolk protein precursor (YPP) gene, vitellogenin (Vg), initiation of vitellogenesis and egg development. In this study, we provide in vitro and in vivo evidence that the Rheb GTPase (Ras Homologue Enriched in Brain), which is an upstream activator of TOR, is required for AA-mediated activation of the TOR pathway in the fat body of the mosquito Aedes aegypti. Using RNA interference (RNAi) methods, we showed that Rheb was indispensable in AA-induced phosphorylation of S6 kinase, a key downstream substrate of TOR activation. Rheb RNAi depletion resulted in significant downregulation of Vg transcription and translation in the mosquito fat body, which was monitored in vivo after blood meal or in vitro organ culture after AA stimulation. Egg development was severely hindered in mosquitoes with a Rheb RNAi depletion background. This study represents a notable step in deciphering molecular pathways controlling reproduction of this important vector of human diseases.

Effect of insulin and 20-hydroxyecdysone in the fat body of the yellow fever mosquito, Aedes aegypti.

In mosquitoes, yolk protein precursor (YPP) gene expression is activated after a blood meal through the synergistic action of a steroid hormone and the amino acid/target of rapamycin (TOR) signaling pathway in the fat body. We investigated the role of insulin signaling in the regulation of YPP gene expression. The presence of mosquito insulin receptor (InR) and the Protein kinase B (PKB/Akt) in the adult fat body of female mosquitoes was confirmed by means of the RNA interference (RNAi). Fat bodies stimulated with insulin were able to promote the phosphorylation of ribosomal S6 Kinase, a key protein of the TOR signaling pathway. Importantly, insulin in combination with 20-hydroxyecdysone activated transcription of the YPP gene vitellogenin (Vg), and this process was sensitive to the phosphoinositide-3 kinase (PI-3k) inhibitor LY294002 as well as the TOR inhibitor rapamycin. RNAi-mediated knockdown of the mosquito InR, Akt, and TOR inhibited insulin-induced Vg gene expression as well as S6 Kinase phosphorylation in in vitro fat body culture assays.

Target of rapamycin-dependent activation of S6 kinase is a central step in the transduction of nutritional signals during egg development in a mosquito.

Female mosquitoes are effective disease vectors, because they take blood from vertebrate hosts to obtain nutrients for egg development. Amino acid signaling via the target of rapamycin (TOR) pathway has been identified as a key requirement for the activation of egg development after a blood meal. We report the characterization of the TOR kinase and one of its major downstream targets, S6 kinase, of the yellow fever mosquito Aedes aegypti during egg development in adult females. Both TOR and S6K mRNA are expressed at high levels in the ovaries and in lower levels in fat body and other tissues. After a blood meal, the subcellular localization of TOR shifts from the cytoplasm to the plasma membrane of fat body cells. By detecting phosphothreonine 388 of mosquito S6 kinase, we show that TOR activity strongly increases in fat body and ovaries after a blood meal in vivo. Furthermore, phosphorylation of S6 kinase increases in in vitro cultured fat bodies after stimulation with amino acids. This increase is sensitive to the TOR inhibitor rapamycin in a concentration-dependent manner but not to the phosphatidylinositol 3-kinase/phosphatidylinositol 3-kinase-related kinase inhibitor LY294002, the MAPK inhibitor PD98059, or the translational inhibitor cycloheximide. RNA interference-mediated reduction of S6 kinase strongly inhibits the amino acid-induced up-regulation of the major yolk protein vitellogenin in vitro and effectively disrupts egg development after a blood meal in vivo. Our data show that TOR-dependent activation of S6 kinase is a central step in the transduction of nutritional information during egg development in mosquitoes.