Ameliorative effect of α-tocopherol and tocotrienol-rich palm oil extract on menopause-associated mood disorder in ovariectomized mice.

Menopause-associated mood disorder is characterized by emotional depression, anxiety, and stress, which accompany hypogonadism in women in the menopausal phase. The current treatment for menopause-associated mood disorder provides only symptomatic relief and is associated with many side effects. Supplementation with vitamin E has been shown to be effective in ameliorating anxiety and depression. However, the effects of vitamin E and its underlying mechanism in ameliorating menopause-associated mood disorders remain uncertain. This work evaluated the effects of α-tocopherol and tocotrienol-rich palm oil extract on depressive and anxiety-related phenotypes induced by estrogen deficiency through ovariectomy in mice. Our study revealed that ovariectomized mice exhibited alterations in behavior indicative of depressive- and anxiety-like behaviors. The serum corticosterone level, a glucocorticoid hormone associated with stress, was found to be elevated in ovariectomized mice as compared to the sham group. Oral administration of α-tocopherol (50 and 100 mg/kg) and tocotrienol-rich palm oil extract (100 and 200 mg/kg) for 14 days alleviated these behavioral changes, as observed in open field, social interaction, and tail suspension tests. However, treatment with tocotrienol-rich palm oil extract, but not α-tocopherol, modulated the depressive- and anxiety-like responses in ovariectomized mice subjected to chronic restraint stress. Both treatments suppressed the elevated serum corticosterone level. Our findings suggested that α-tocopherol and tocotrienol-rich palm oil extract alleviated menopause-associated mood disorder, at least in part, by modulating the hypothalamic-pituitary-adrenal (HPA) axis. The findings of this study can provide a new foundation for the treatment of menopause-associated depressive- and anxiety-like phenotypes, for the betterment of psychological wellbeing.

Neuroprotective effects of Gypenosides: A review on preclinical studies in neuropsychiatric disorders.

Gynostemma pentaphyllum (Thunb.) Makino is a perennial creeping herb belonging to the Cucurbitaceae family that has a long history of usage in traditional oriental medicine. Gypenosides are the primary bioactive compounds in Gynostemma pentaphyllum. Because of the medicinal value of gypenosides, functional food and supplements containing gypenosides have been promoted and consumed with popularity, especially among Asian communities. This review presented the progress made in the research of pharmacological properties of gypenosides on diseases of the nervous system and their possible mechanism of action. To date, preclinical studies have demonstrated the therapeutic effects of gypenosides in alleviating neuropsychiatric disorders like depression, Parkinson's disease, Alzheimer's disease, secondary dementia, stroke, optic neuritis, etc. Pharmacological studies have discovered that gypenosides can modulate various major signaling pathways like NF-κB, Nrf2, AKT, ERK1/2, contributing to the neuroprotective properties. However, there is a dearth of clinical research on gypenosides, with current investigations on the compounds being mainly conducted in vitro and on animals. Future studies focusing on isolating and purifying novel gypenosides and investigations on exploring the potential molecular mechanism underlying their biological activities are warranted, which may serve as a foundation for further clinical trials for the betterment of human health.

Menopause-Associated Depression: Impact of Oxidative Stress and Neuroinflammation on the Central Nervous System-A Review.

Perimenopausal depression, occurring shortly before or after menopause, is characterized by symptoms such as emotional depression, anxiety, and stress, often accompanied by endocrine dysfunction, particularly hypogonadism and senescence. Current treatments for perimenopausal depression primarily provide symptomatic relief but often come with undesirable side effects. The development of agents targeting the specific pathologies of perimenopausal depression has been relatively slow. The erratic fluctuations in estrogen and progesterone levels during the perimenopausal stage expose women to the risk of developing perimenopausal-associated depression. These hormonal changes trigger the production of proinflammatory mediators and induce oxidative stress, leading to progressive neuronal damage. This review serves as a comprehensive overview of the underlying mechanisms contributing to perimenopausal depression. It aims to shed light on the complex relationship between perimenopausal hormones, neurotransmitters, brain-derived neurotrophic factors, chronic inflammation, oxidative stress, and perimenopausal depression. By summarizing the intricate interplay between hormonal fluctuations, neurotransmitter activity, brain-derived neurotrophic factors, chronic inflammation, oxidative stress, and perimenopausal depression, this review aims to stimulate further research in this field. The hope is that an increased understanding of these mechanisms will pave the way for the development of more effective therapeutic targets, ultimately reducing the risk of depression during the menopausal stage for the betterment of psychological wellbeing.

Finding Lead Compounds for Dengue Antivirals from a Collection of Old Drugs through In Silico Target Prediction and Subsequent In Vitro Validation.

Dengue virus (DENV) infection is one of the most widely spread flavivirus infections. Despite the fatality it could cause, no antiviral treatment is currently available to treat the disease. Hence, this study aimed to repurpose old drugs as novel DENV NS3 inhibitors. Ligand-based (L-B) and proteochemometric (PCM) prediction models were built using 62,354 bioactivity data to screen for potential NS3 inhibitors. Selected drugs were then subjected to the foci forming unit reduction assay (FFURA) and protease inhibition assay. Finally, molecular docking was performed to validate these results. The in silico studies revealed that both models performed well in the internal and external validations. However, the L-B model showed better accuracy in the external validation in terms of its sensitivity (0.671). In the in vitro validation, all drugs (zileuton, trimethadione, and linalool) were able to moderately inhibit the viral activities at the highest concentration tested. Zileuton showed comparable results with linalool when tested at 2 mM against the DENV NS3 protease, with a reduction of protease activity at 17.89 and 18.42%, respectively. Two new compounds were also proposed through the combination of the selected drugs, which are ziltri (zilueton + trimethadione) and zilool (zileuton + linalool). The molecular docking study confirms the in vitro observations where all drugs and proposed compounds were able to achieve binding affinity ≥ -4.1 kcal/mol, with ziltri showing the highest affinity at -7.7 kcal/mol, surpassing the control, panduratin A. The occupation of both S1 and S2 subpockets of NS2B-NS3 may be essential and a reason for the lower binding energy shown by the proposed compounds compared to the screened drugs. Based on the results, this study provided five potential new lead compounds (ziltri, zilool, zileuton, linalool, and trimethadione) for DENV that could be modified further.

Computational-aided design: minimal peptide sequence to block dengue virus transmission into cells.

Dengue virus (DV) infection is one of the main public health concerns, affecting approximately 390 million people worldwide, as reported by the World Health Organization. Yet, there is no antiviral treatment for DV infection. Therefore, the development of potent and nontoxic anti-DV, as a complement for the existing treatment strategies, is urgently needed. Herein, we investigate a series of small peptides inhibitors of DV antiviral activity targeting the entry process as the promising strategy to block DV infection. The peptides were designed based on our previously reported peptide sequence, DN58opt (TWWCFYFCRRHHPFWFFYRHN), to identify minimal effective inhibitory sequence through molecular docking and dynamics studies. The in silico designed peptides were synthesized using conventional Fmoc solid-phase peptide synthesis chemistry, purified by RP-HPLC and characterized using LCMS. Later, they were screened for their antiviral activity. One of the peptides, AC 001, was able to reduce about 40% of DV plaque formation. This observation correlates well with the molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) analysis - AC 001 showed the most favorable binding affinity through 60 ns simulations. Pairwise residue decomposition analysis has revealed four key residues that contributed to the binding of these peptides into the DV2 E protein pocket. This work identifies the minimal peptide sequence required to inhibit DV replication and explains the behavior observed on an atomic level using computational study.Communicated by Ramaswamy H. Sarma.

Rational drug discovery: Ellagic acid as a potent dual-target inhibitor against hepatitis C virus genotype 3 (HCV G3) NS3 enzymes.

Structure-based virtual screening (SBVS) has served as a popular strategy for rational drug discovery. In this study, we aimed to discover novel benzopyran-based inhibitors that targeted the NS3 enzymes (NS3/4A protease and NS3 helicase) of HCV G3 using a combination of in silico and in vitro approaches. With the aid of SBVS, six novel compounds were discovered to inhibit HCV G3 NS3/4A protease and two phytochemicals (ellagic acid and myricetin) were identified as dual-target inhibitors that inhibited both NS3/4A protease and NS3 helicase in vitro (IC50  = 40.37 ± 5.47 nm and 6.58 ± 0.99 µm, respectively). Inhibitory activities against the replication of HCV G3 replicons were further assessed in a cell-based system with four compounds showed dose-dependent inhibition. Compound P8 was determined to be the most potent compound from the cell-based assay with an EC50 of 19.05 µm. The dual-target inhibitor, ellagic acid, was determined as the second most potent (EC50  = 32.37 µm) and the most selective in its inhibitory activity against the replication of HCV replicons, without severely affecting the viability of the host cells (selectivity index > 6.18).

Endoplasmic reticulum: a focal point of Zika virus infection.

Zika virus (ZIKV) belongs to the Flavivirus genus of the Flaviviridae family. It is an arbovirus that can cause congenital abnormalities and is sexually transmissible. A series of outbreaks accompanied by unexpected severe clinical complications have captured medical attention to further characterize the clinical features of congenital ZIKV syndrome and its underlying pathophysiological mechanisms. Endoplasmic reticulum (ER) and ER-related proteins are essential in ZIKV genome replication. This review highlights the subcellular localization of ZIKV to the ER and ZIKV modulation on the architecture of the ER. This review also discusses ZIKV interaction with ER proteins such as signal peptidase complex subunit 1 (SPCS1), ER membrane complex (EMC) subunits, and ER translocon for viral replication. Furthermore, the review covers several important resulting effects of ZIKV infection to the ER and cellular processes including ER stress, reticulophagy, and paraptosis-like death. Pharmacological targeting of ZIKV-affected ER-resident proteins and ER-associated components demonstrate promising signs of combating ZIKV infection and rescuing host organisms from severe neurologic sequelae.

Discovery of Dengue Virus Inhibitors.

To date, there is still no approved anti-dengue agent to treat dengue infection in the market. Although the only licensed dengue vaccine, Dengvaxia is available, its protective efficacy against serotypes 1 and 2 of dengue virus was reported to be lower than serotypes 3 and 4. Moreover, according to WHO, the risk of being hospitalized and having severe dengue increased in seronegative individuals after they received Dengvaxia vaccination. Nevertheless, various studies had been carried out in search of dengue virus inhibitors. These studies focused on the structural (C, prM, E) and non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) of dengue virus as well as host factors as drug targets. Hence, this article provides an overall up-to-date review of the discovery of dengue virus inhibitors that are only targeting the structural and non-structural viral proteins as drug targets.

Small molecule grp94 inhibitors block dengue and Zika virus replication.

Two major flaviviruses, dengue virus (DENV) and Zika virus (ZIKV), cause severe health and economic burdens worldwide. Recently, genome-wide screenings have uncovered the importance of regulators of the Hrd1 ubiquitin ligase-mediated endoplasmic reticulum (ER)-associated degradation (ERAD) pathway for flavivirus replication in host cells. Here we report the identification of the compound Bardoxolone methyl (CDDO-me) as a potent inhibitor of the Hrd1 ubiquitin ligase-mediated ERAD, which possesses a broad-spectrum activity against both DENV and ZIKV. Cellular thermal shift assay (CETSA) suggested that CDDO-me binds to grp94, a key component of the Hrd1 pathway, at a low nanomolar concentration, whereas interaction was not detected with its paralog Hsp90. CDDO-me and the grp94 inhibitor PU-WS13 substantially suppressed DENV2 replication and the cytopathic effects caused by DENV and ZIKV infection. The antiviral activities of both compounds were demonstrated for all four DENV serotypes and four ZIKV strains in multiple human cell lines. This study defines grp94 as a crucial host factor for flavivirus replication and identified CDDO-me as a potent small molecule inhibitor of flavivirus infection. Inhibition of grp94 may contribute to the antiviral activity of CDDO-me. Further investigation of grp94 inhibitors may lead to a new class of broad-spectrum anti-flaviviral medications.

Carnosine exhibits significant antiviral activity against Dengue and Zika virus.

Dengue virus (DENV) and Zika virus (ZIKV) are flaviviruses transmitted to humans by their common vector, Aedes mosquitoes. DENV infection represents one of the most widely spread mosquito-borne diseases whereas ZIKV infection occasionally re-emerged in the past causing outbreaks. Although there have been considerable advances in understanding the pathophysiology of these viruses, no effective vaccines or antiviral drugs are currently available. In this study, we evaluated the antiviral activity of carnosine, an endogenous dipeptide (ß-alanyl-l-histidine), against DENV serotype 2 (DENV2) and ZIKV infection in human liver cells (Huh7). Computational studies were performed to predict the potential interactions between carnosine and viral proteins. Biochemical and cell-based assays were performed to validate the computational results. Mode-of-inhibition, plaque reduction, and immunostaining assays were performed to determine the antiviral activity of carnosine. Exogenous carnosine showed minimal cytotoxicity in Huh7 cells and rescued the viability of infected cells with EC50 values of 52.3 and 59.5 µM for DENV2 and ZIKV infection, respectively. Based on the mode-of-inhibition assays, carnosine inhibited DENV2 mainly by inhibiting viral genome replication and interfering with virus entry. Carnosine antiviral activity was verified with immunostaining assay where carnosine treatment diminished viral fluorescence signal. In conclusion, carnosine exhibited significant inhibitory effects against DENV2 and ZIKV replication in human liver cells and could be utilized as a lead peptide for the development of effective and safe antiviral agents against DENV and ZIKV.

Thioguanine-based DENV-2 NS2B/NS3 protease inhibitors: Virtual screening, synthesis, biological evaluation and molecular modelling.

Dengue virus Type 2 (DENV-2) is predominant serotype causing major dengue epidemics. There are a number of studies carried out to find its effective antiviral, however to date, there is still no molecule either from peptide or small molecules released as a drug. The present study aims to identify small molecules inhibitor from National Cancer Institute database through virtual screening. One of the hits, D0713 (IC50 = 62 µM) bearing thioguanine scaffold was derivatised into 21 compounds and evaluated for DENV-2 NS2B/NS3 protease inhibitory activity. Compounds 18 and 21 demonstrated the most potent activity with IC50 of 0.38 µM and 16 µM, respectively. Molecular dynamics and MM/PBSA free energy of binding calculation were conducted to study the interaction mechanism of these compounds with the protease. The free energy of binding of 18 calculated by MM/PBSA is -16.10 kcal/mol compared to the known inhibitor, panduratin A (-11.27 kcal/mol), which corroborates well with the experimental observation. Results from molecular dynamics simulations also showed that both 18 and 21 bind in the active site and stabilised by the formation of hydrogen bonds with Asn174.

Development of a NS2B/NS3 protease inhibition assay using AlphaScreen® beads for screening of anti-dengue activities.

BACKGROUND: Dengue infection is an endemic infectious disease and it can lead to dengue fever, dengue hemorrhagic fever, and/or dengue shock syndromes. Dengue NS2B/NS3 protease complex is essential for viral replication and is a primary target for anti-dengue drug development. In this study, a NS2B/NS3 protease inhibition assay was developed using AlphaScreen® beads and was used to screen compounds for their protease inhibition activities. METHODS: The assay system utilized a known NS2B/NS3 peptide substrate, a recombinant of NS2B/NS3 protease with proprietary StrepTactin® donor and nickel chelate acceptor beads in 384-well format. RESULTS: The optimized assay to screen for NS2B/NS3 protease inhibitors was demonstrated to be potentially useful with reasonable z' factor, coefficient variance and signal to background ratio. However, screening of synthesized thioguanine derivatives using the optimized AlphaScreen® assay revealed weak NS2B/NS3 inhibition activities. CONCLUSION: The AlphaScreen® assay to screen for NS2B/NS3 protease inhibitors is potentially applicable for high throughput screening.

A Microfluidic Lab-on-a-Disc (LOD) for Antioxidant Activities of Plant Extracts.

Antioxidants are an important substance that can fight the deterioration of free radicals and can easily oxidize when exposed to light. There are many methods to measure the antioxidant activity in a biological sample, for example 2,2-diphenyl-1-picrylhydrazyl (DPPH) antioxidant activity test, which is one of the simplest methods used. Despite its simplicity, the organic solvent that has been used to dilute DPPH is easily evaporated and degraded with respect to light exposure and time. Thus, it needs to be used at the earliest convenient time prior to the experiment. To overcome this issue, a rapid and close system for antioxidant activity is required. In this paper, we introduced the Lab-on-a-Disc (LoD) method that integrates the DPPH antioxidant activity test on a microfluidic compact disc (CD). We used ascorbic acid, quercetin, Areca catechu, Polygonum minus, and Syzygium polyanthum plant extracts to compare the results of our proposed LoD method with the conventional method. Contrasted to the arduous laborious conventional method, our proposed method offer rapid analysis and simple determination of antioxidant. This proposed LoD method for antioxidant activity in plants would be a platform for the further development of antioxidant assay.

Comparative proteomics reveals that YK51, a 4-Hydroxypandurantin-A analogue, downregulates the expression of proteins associated with dengue virus infection.

Dengue is endemic throughout tropical and subtropical regions of the world. Currently, there is no clinically approved therapeutic drug available for this acute viral infection. Although the first dengue vaccine Dengvaxia has been approved for use in certain countries, it is limited to those without a previous dengue infection while the safety and efficacy of the vaccine in those elderly and younger children still need to be identified. Therefore, it is becoming increasingly important to develop therapeutics/drugs to combat dengue virus (DENV) infection. YK51 is a synthetic analogue of 4-Hydroxypandurantin A (a compound found in the crude extract of the rhizomes of Boesenbergia rotunda) that has been extensively studied by our research group. It has been shown to possess outstanding antiviral activity due to its inhibitory activity against NS2B/NS3 DENV2 protease. However, it is not known how YK51 affects the proteome of DENV infected cells. Therefore, we performed a comparative proteomics analysis to identify changes in protein expression in DENV infected HepG2 cells treated with YK51. Classical two-dimensional gel electrophoresis followed by protein identification using tandem mass spectrometry was employed in this study. Thirty proteins were found to be down-regulated with YK51 treatment. In silico analysis predicted that the down-regulation of eight of these proteins may inhibit viral infection. Our results suggested that apart from inhibiting the NS2B/NS3 DENV2 protease, YK51 may also be causing the down-regulation of a number of proteins that may be responsible in, and/or essential to virus infection. However, functional characterization of these proteins will be necessary before we can conclusively determine their roles in DENV infection.

Sofosbuvir as treatment against dengue?

Dengvaxia® (CTD-TDV), the only licensed tetravalent dengue vaccine by Sanofi Pasteur, was made available since 2015. However, administration of CTD-TDV, in general, has not received the prequalification recommendation from the World Health Organization. Having a universal antidengue agent for treatment will therefore beneficial. Accordingly, the development of nucleoside inhibitors specific to dengue viral polymerase that perturb dengue infection has been studied by many. Alternatively, we have used a marketed anti-HCV prodrug sofosbuvir to study its in silico and in vitro effects against dengue. As a result, the active metabolite of sofosbuvir (GS-461203) was predicted to bind to the catalytic motif (Gly-Asp-Asp) of dengue viral polymerase with binding affinity of -6.9 kcal/mol. Furthermore, sofosbuvir demonstrated excellent in vitro viral inhibition with an EC90 of 0.4 µm. In addition, this study demonstrated the requirement of specific liver enzymes to activate the prodrug into GS-461203 to exert its antidengue potential. All in all, sofosbuvir should be subjected to in-depth studies to provide information of its efficacy toward dengue and its lead potential as DENV polymerase inhibitor in human subjects. In conclusion, we have expended the potential of the clinically available drug sofosbuvir as treatment for dengue.

The Importance of Some Plant Extracts as Skin Anti-aging Resources: A Review.

BACKGROUND: Skin is the largest and most visible organ of the body. Many of its functions include temperature regulation, immunity from microorganisms, maintaining electrolyte balance, and protection from physical injuries, chemical agents and ultraviolet (UV) radiation. Aging occurs in every layer of the skin, primarily due to the degradation of its components. Induction of degradative enzymes and the abundant production of reactive oxygen species lead to skin aging. Understanding the complexity of skin structure and factors contributing to the skin aging will help us impede the aging process. Applications of anti-aging products are a common method to prevent or repair damages that lead to aging. CONCLUSION: This review will provide information on the causes and indicators of skin aging as well as examine studies that have used plants to produce anti-aging products.

Conformational and energy evaluations of novel peptides binding to dengue virus envelope protein.

Effective novel peptide inhibitors which targeted the domain III of the dengue envelope (E) protein by blocking dengue virus (DENV) entry into target cells, were identified. The binding affinities of these peptides towards E-protein were evaluated by using a combination of docking and explicit solvent molecular dynamics (MD) simulation methods. The interactions of these complexes were further investigated by using the Molecular Mechanics-Poisson Boltzmann Surface Area (MMPBSA) and Molecular Mechanics Generalized Born Surface Area (MMGBSA) methods. Free energy calculations of the peptides interacting with the E-protein demonstrated that van der Waals (vdW) and electrostatic interactions were the main driving forces stabilizing the complexes. Interestingly, calculated binding free energies showed good agreement with the experimental dissociation constant (Kd) values. Our results also demonstrated that specific residues might play a crucial role in the effective binding interactions. Thus, this study has demonstrated that a combination of docking and molecular dynamics simulations can accelerate the identification process of peptides as potential inhibitors of dengue virus entry into host cells.

Subversion of immunoproteasome subunit expression in dengue virus serotype 2-infected HepG2 cells.

INTRODUCTION:: Infection with all serotypes of dengue virus (DV) results in augmented antigen presentation by MHC class I molecules. However, the upregulation of immunoproteasome subunits only results from infection with two serotypes. This study aims to elucidate changes in the expression of immunoproteasome subunits resulting from infection with DV, particularly DV serotype 2 (DV2). METHODS:: HepG2 cells were grown in various culture milieu. Total cellular RNA and proteins were extracted and quantified. RESULTS:: Results demonstrated sequestration of immunoproteasome subunits LMP2 and LMP7 in DV2-infected cells. CONCLUSIONS:: This study provides insights into the mechanisms underlying immune evasion by DV.

NMDA receptor antagonism with novel indolyl, 2-(1,1-Dimethyl-1,3-dihydro-benzo[e]indol-2-ylidene)-malonaldehyde, reduces seizures duration in a rat model of epilepsy.

N-methyl-D-aspartate receptors (NMDAR) play a central role in epileptogensis and NMDAR antagonists have been shown to have antiepileptic effects in animals and humans. Despite significant progress in the development of antiepileptic therapies over the previous 3 decades, a need still exists for novel therapies. We screened an in-house library of small molecules targeting the NMDA receptor. A novel indolyl compound, 2-(1,1-Dimethyl-1,3-dihydro-benzo[e]indol-2-ylidene)-malonaldehyde, (DDBM) showed the best binding with the NMDA receptor and computational docking data showed that DDBM antagonised the binding sites of the NMDA receptor at lower docking energies compared to other molecules. Using a rat electroconvulsive shock (ECS) model of epilepsy we showed that DDBM decreased seizure duration and improved the histological outcomes. Our data show for the first time that indolyls like DDBM have robust anticonvulsive activity and have the potential to be developed as novel anticonvulsants.

Subversion of immunoproteasome subunit expression in dengue virus serotype 2-infected HepG2 cells

Abstract: INTRODUCTION: Infection with all serotypes of dengue virus (DV) results in augmented antigen presentation by MHC class I molecules. However, the upregulation of immunoproteasome subunits only results from infection with two serotypes. This study aims to elucidate changes in the expression of immunoproteasome subunits resulting from infection with DV, particularly DV serotype 2 (DV2). METHODS: HepG2 cells were grown in various culture milieu. Total cellular RNA and proteins were extracted and quantified. RESULTS: Results demonstrated sequestration of immunoproteasome subunits LMP2 and LMP7 in DV2-infected cells. CONCLUSIONS: This study provides insights into the mechanisms underlying immune evasion by DV.