Evaluation of the inhibitory potency of anti-dengue phytocompounds against DENV-2 NS2B-NS3 protease: virtual screening, ADMET profiling and molecular dynamics simulation investigations.

Dengue fever has been a worldwide concern, with 50-100 million new infections each year mainly due to five different serotypes of the Dengue virus (DENV). Designing a perfect anti-dengue agent that can inhibit all the serotypes by distinguishing antigenic differences is quite difficult. Previous anti-dengue researches have included chemical compounds screening against DENV enzymes. The ongoing analysis is meant for investigation of the plant-based compounds as antagonistic to DENV-2 focusing on the specific NS2B-NS3Pro target, a trypsin like serine protease that cuts the DENV polyprotein into separate proteins crucial for viral reproduction. Initially, a virtual library of more than 130 phytocompounds was prepared from previously published reports of plants with anti-dengue properties, which were then virtually screened and shortlisted against the WT, H51N and S135A mutant of DENV-2 NS2B-NS3Pro. The three top-most compounds were viewed as Gallocatechin (GAL), Flavokawain-C (FLV), and Isorhamnetin (ISO) showing docking scores of -5.8, -5.7, -5.7 kcal/mol for WT, -7.5, -6.8, -7.6 kcal/mol for the H51N, and -6.9, -6.5, -6.1 kcal/mol for the S135A mutant protease, respectively. 100 ns long MD simulations and MM-GBSA based free energy calculations were performed on the NS2B-NS3Pro complexes to witness the relative binding affinity of the compounds and favourable molecular interactions network. A comprehensive analysis of the study reveals some promising outcomes with ISO as the topmost compound with favourable pharmacokinetic properties for the WT and mutants (H51N and S135A) as well, suggesting as a novel anti-NS2B-NS3Pro agent with better adapting characters in both the mutants.Communicated by Ramaswamy H. Sarma.

Investigating Lycotoxin-An1a (An1a), a defense antiviral peptide from Alopecosa nagpag venom as prospective anti-dengue agent against DENV-2 NS2B-NS3 protease.

Dengue fever is a global health concern with no effective therapy. Screening synthetic chemicals, animal-originated compounds, and phytocompounds against Dengue virus (DENV) targets has failed to find dengue antivirals. The current study examines animal drugs as antagonists against NS2B-NS3Pro, one of DENV's most promising therapeutic targets for dengue fever. Antiviral-Lycotoxin-An1a (An1a), a defence antiviral peptide isolated from the venom of Alopecosa nagpag, a toxic spider. Based on prior in vitro research, it was discovered that the venom peptide suppresses the action of DENV-2 NS2B-NS3Pro. An1a peptide with NS2B-NS3Pro wild type (WT) and two mutants (H51N and S135A) was tested for anti-dengue characteristics using in silico analysis. The WT NS2B-NS3Pro has a catalytic triad of His51, Asp75, and Ser135 in the active site, but the mutants have N51 instead of His51 and Ala135 instead of Ser135. The dynamic sites of the three proteases (WT, H51N, S135A) and the peptide toxin (An1a) were taken into account to achieve molecular docking of An1a with WT NS2B-NS3Pro in conjunction with H51N and S135A. Cluspro-2 performs rigid-flexible docking to predict peptide binding affinity, effectiveness, and inhibitory consistency. Since the ligand had a higher binding affinity, docking score, and molecular interaction network, MD simulations and MM-GBSA free energy calculations were used to investigate the stability of the three protein-peptide complexes. The computer-aided screening and manufacture of spider venom-based anti-dengue medicines yielded intriguing results in the preliminary studies. This study is significant in defining the ideal therapeutic candidate against dengue infections.

Theoretical insights into the binding interaction of Nirmatrelvir with SARS-CoV-2 Mpro mutants (C145A and C145S): MD simulations and binding free-energy calculation to understand drug resistance.

Mpro, the main protease and a crucial enzyme in SARS-CoV-2 is the most fascinating molecular target for pharmacological treatment and is also liable for viral protein maturation. For antiviral therapy, no drugs have been approved clinically to date. Targeting the Mpro with a compound having inhibitory properties against it can hinder viral replication. The therapeutic potential of the antiviral compound Nirmatrelvir (NMV) against SARS-CoV-2 Mpro was investigated using a systematic approach of molecular docking, MD simulations, and binding free energy calculation based on the MM-GBSA method. NMV, a covalent inhibitor with a recently revealed chemical structure, is a promising oral antiviral clinical candidate with significant in vitro anti-SARS-CoV-2 action in third-phase clinical trials. To explore the therapeutic ability and possible drug resistance, the Mpro system was studied for WT and two of its primary mutants (C145A & C145S). The protein-ligand (Mpro/NMV) complexes were further examined through long MD simulations to check the possible drug resistance in the mutants. To understand the binding affinity, the MM-GBSA method was applied to the Mpro/NMV complexes. Moreover, PCA analysis confirms the detachment of the linker region from the major domains in C145S and C145A mutants allowing for conformational alterations in the active-site region. Based on the predicted biological activities and binding affinities of NMV to WT and mutant (C145A & C145S) Mpro, it can be stipulated that NMV may have conventional potency to act as an anti-viral agent against WT Mpro, while the catalytic-dyad mutations may show substantial mutation-induced drug resistance.Communicated by Ramaswamy H. Sarma.

Molecular surveillance of Kelch-13 gene in Plasmodium falciparum field isolates from Mayurbhanj District, Odisha, India, and in silico artemisinin-Kelch-13 protein interaction study.

The global malaria control and elimination program faces major threats due to the emergence and transmission of the anti-malarial drug-resistant strain of Plasmodium falciparum. Monitoring of artemisinin (ART) resistance marker Kelch-13 in the malaria-endemic region is essential in mitigating the disease's morbidity and mortality. The current study aimed to generate baseline information for further surveillance in the future. The current research was designed and conducted from July 2019 to June 2021 to monitor Pfkelch13 mutation at the molecular level in the eastern region of India. We also conducted an in silico study to understand the drug-protein interactions between ART and the protein crystal of PfKelch13 (KELCH) with PDB id:4ZGC. The kelch-13 gene was amplified by nested polymerase chain reaction (PCR) and sequenced through the Sanger sequencing method. Reference 3D7 clone (PF3D7_1343700) was used to align and probe all the sequences. The sequence analysis showed the absence of validated or associated mutation in the Kelch-13 propeller domain. The absence of natural selection in drug resistance was confirmed by the Tajima test. Further, in silico interaction studies between the drug ART and the Kelch propeller domain of P. falciparum were evaluated by structure predictions, molecular docking, molecular dynamics (MD) simulations, and estimations of binding free energies for the KELCH-ART complex. The results were compared with the apoprotein (KELCH-APO). The study confirmed the favorable binding of ART with the Kelch-13 propeller domain.

Molecular insights to the binding interactions of APNS containing HIV-protease inhibitors against SARS-CoV-2 Mpro: an in silico approach towards drug repurposing.

SARS-CoV-2 Mpro is one of the most vital enzymes of the new coronavirus-2 (SARS-CoV-2) and is a crucial target for drug discovery. Unfortunately, there is not any potential drugs available to combat the action of SARS-CoV-2 Mpro. Based on the reports HIV-protease inhibitors can be applied against the SARS by targeting the SARS-CoV-1 Mpro, we have chosen few clinically trialed experimental and allophenylnorstatine (APNS) containing HIV-protease inhibitors (JE-2147, JE-533, KNI-227, KNI-272 & KNI-1931), to examine their binding affinities with SARS-CoV-2 Mpro and to assess their potential to check for a possible drug candidate against the protease. Here, we have chosen a methodology to understand the binding mechanism of these five inhibitors to SARS-CoV-2 Mpro by merging molecular docking, molecular dynamics (MD) simulation and MM-PBSA based free energy calculations. Our estimations disclose that JE-2147 is highly effective (ΔGBind = -28.31 kcal/mol) due to an increased favorable van der Waals (ΔEvdw) interactions and decreased solvation (ΔGsolv) energies between the inhibitor and viral protease. JE-2147 shows a higher level of interactions as compared to JE-533 (-6.85 kcal/mol), KNI-227 (-18.36 kcal/mol), KNI-272 (-15.69 kcal/mol) and KNI-1931 (-21.59 kcal/mol) against SARS-CoV-2 Mpro. Binding contributions of important residues (His41, Met49, Cys145, His164, Met165, Glu166, Pro168, Gln189, etc.) from the active site or near the active site regions with ≥1.0 kcal/mol suggest a potent binding of the inhibitors. It is anticipated that the current study of binding interactions of these APNS containing inhibitors can pitch some valuable insights to design the significantly effective anti-SARS-CoV-2 Mpro drugs.Communicated by Ramaswamy H. Sarma.

Targeting the DENV NS2B-NS3 protease with active antiviral phytocompounds: structure-based virtual screening, molecular docking and molecular dynamics simulation studies.

Dengue fever has been a global health concern. Mitigation is a challenging problem due to non-availability of workable treatments. The most difficult objective is to design a perfect anti-dengue agent capable of inhibiting infections caused by all four serotypes. Various tactics have been employed in the past to discover dengue antivirals, including screening of chemical compounds against dengue virus enzymes. The objective of the current study is to investigate phytocompounds as anti-dengue remedies that target the non-structural 2B and non-structural 3 protease (NS2B-NS3pro), a possible therapeutic target for dengue fever. Initially, 300 + antiviral phytocompounds were collected from Duke's phytochemical and ethnobotanical database and 30 phytocompounds with anti-dengue properties were identified from previously reported studies, which were virtually screened against NS2B-NS3pro using molecular docking and toxicity evaluation. The top five most screened ligands were naringin, hesperidin, gossypol, maslinic acid and rhodiolin with binding affinities of - 8.7 kcal/mol, - 8.5 kcal/mol, - 8.5 kcal/mol, - 8.5 kcal/mol and - 8.1 kcal/mol, respectively. The finest docked compounds complexed with NS2B-NS3pro were subjected for molecular dynamics (MD) simulations and binding free energy estimations through molecular mechanics generalized born surface area-based calculations. The results of the study are intriguing in the context of computer-aided screening and the binding affinities of the phytocompounds, proposing maslinic acid (MAS) as a potent bioactive antiviral for the development of phytocompound-based anti-dengue agent.

Functionalized carbon nanotubes as an alternative to traditional anti-HIV-1 protease inhibitors: An understanding towards Nano-medicine development through MD simulations.

The Human Immunodeficiency Virus (HIV) has been the source of epidemic infection of AIDS for a longer period. One of the most difficult tasks is identifying novel medications that can help to decrease or control this global health hazard by overcoming drug resistance. In recent decades' nanoparticles are emerging as extremely relevant in drug delivery platforms. In the current study, the pristine (SWCNT) and hydroxyl functionalized (SWCNT-OH) versions of the SWCNT were investigated as inhibitors against the wild-type (WT) and three key mutants of HIV-1 protease (HIV-pr) (I50V, V82A, and I84V). Molecular docking of SWCNT in the catalytic domain and running all-atom MD simulations of all complexes are also part of this project. A thorough inspection of conformational dynamics from 50 ns trajectories reveals that both the pristine and SWCNT-OH can fit right to the pocket region of HIV-pr and govern flap dynamics. The binding affinity of the four HIV-pr-SWCNT/SWCNT-OH complexes was further investigated using MM-PBSA-dependent binding free energy studies. In most mutants and WT systems, SWCNT-OH was reported to bind proportionately many folds (kcal/mol) more than pristine SWCNTs. Hence, SWCNTs are possible HIV-pr inhibitors in terms of their stable existence in the pocket area, stronger binding to the protease, and regulation of flap dynamics in controlling the active site volume, which have vast potential for applications against drug resistance.

Deacetylation of LAMP1 drives lipophagy-dependent generation of free fatty acids by Abrus agglutinin to promote senescence in prostate cancer.

Therapy-induced senescence in cancer cells is an irreversible antiproliferative state, which inhibits tumor growth and is therefore a potent anti-neoplastic mechanism. In this study, low doses of Abrus agglutinin (AGG)-induced senescence through autophagy in prostate carcinoma cells (PC3) and inhibited proliferation. The inhibition of autophagy with 3-methyl adenine reversed AGG-induced senescence, thus confirming that AGG-triggered senescence required autophagy. AGG treatment also led to lipophagy-mediated accumulation of free fatty acids (FFAs), with a concomitant decrease in the number of lipid droplets. Lalistat, a lysosomal acid lipase inhibitor, abrogated AGG-induced lipophagy and senescence in PC3 cells, indicating that lipophagy is essential for AGG-induced senescence. The accumulation of FFAs increased reactive oxygen species generation, a known facilitator of senescence, which was also reduced in the presence of lalistat. Furthermore, AGG upregulated silent mating type information regulator 2 homolog 1 (SIRT1), while the presence of sirtinol reduced autophagy flux and the senescent phenotype in the AGG-treated cells. Mechanistically, AGG-induced cytoplasmic SIRT1 deacetylated a Lys residue on the cytoplasmic domain of lysosome-associated membrane protein 1 (LAMP1), an autolysosomal protein, resulting in lipophagy and senescence. Taken together, our findings demonstrate a novel SIRT1/LAMP1/lipophagy axis mediating AGG-induced senescence in prostate cancer cells.

TNF-α promoter polymorphisms (G-238A and G-308A) are associated with susceptibility to Systemic Lupus Erythematosus (SLE) and P. falciparum malaria: a study in malaria endemic area.

Tumor necrosis factor-α (TNF-α) is a proinflammatory cytokine associated with autoimmune and infectious diseases. Importance of TNF-α in P. falciparum malaria and systemic lupus erythematosus (SLE) have been demonstrated. However, association of functional promoter variants with SLE and malaria is lacking in malaria endemic population. A total of 204 female SLE patients and 224 age and sex matched healthy controls were enrolled in the study. Three hundred fourteen P. falciparum infected patients with different clinical phenotypes were included. TNF-α polymorphisms (G-238A & G-308A) were genotyped by PCR-RFLP. Plasma levels of TNF-α was quantified by ELISA. Heterozygous mutants and minor alleles of TNF-α (G-238A and G-308A) polymorphisms were significantly higher in SLE patients compared to healthy controls and associated with development of lupus nephritis. In addition, both promoter variants were associated with severe P. falciparum malaria. SLE patients demonstrated higher levels of plasma TNF-α compared to healthy controls. TNF-α (G-238A and G-308A) variants were associated with higher plasma TNF-α. In conclusion, TNF-α (G-238A & G-308A) variants are associated with higher plasma TNF-α levels in SLE patients residing in malaria endemic areas and could be a contributing factor in the development of SLE and susceptibility to severe P. falciparum malaria.

p73 induction by Abrus agglutinin facilitates Snail ubiquitination to inhibit epithelial to mesenchymal transition in oral cancer.

BACKGROUND: Epithelial-to-mesenchymal transition (EMT), a key step in oral cancer progression, is associated with invasion, metastasis, and therapy resistance, thus targeting the EMT represents a critical therapeutic strategy for the treatment of oral cancer metastasis. Our previous study showed that Abrus agglutinin (AGG), a plant lectin, induces both intrinsic and extrinsic apoptosis to activate the tumor inhibitory mechanism. OBJECTIVE: This study aimed to investigate the role of AGG in modulating invasiveness and stemness through EMT inhibition for the development of antineoplastic agents against oral cancer. METHODS: The EMT- and stemness-related proteins were studied in oral cancer cells using Western blot analysis and fluorescence microscopy. The potential mechanisms of Snail downregulation through p73 activation in FaDu cells were evaluated using Western blot analysis, immunoprecipitation, confocal microscopy, and molecular docking analysis. Immunohistochemical staining of the tumor samples of AGG-treated FaDu-xenografted nude mice was performed. RESULTS: At the molecular level, AGG-induced p73 suppressed Snail expression, leading to EMT inhibition in FaDu cells. Notably, AGG promoted the translocation of Snail from the nucleus to the cytoplasm in FaDu cells and triggered its degradation through ubiquitination. In this setting, AGG inhibited the interaction between Snail and p73 in FaDu cells, resulting in p73 activation and EMT inhibition. Moreover, in epidermal growth factor (EGF)-stimulated FaDu cells, AGG abolished the upregulation of extracellular signal-regulated kinase (ERK)1/2 that plays a pivotal role in the upregulation of Snail to regulate the EMT phenotypes. In immunohistochemistry analysis, FaDu xenografts from AGG-treated mice showed decreased expression of Snail, SOX2, and vimentin and increased expression of p73 and E-cadherin compared with the control group, confirming EMT inhibition as part of its anticancer efficacy against oral cancer. CONCLUSION: In summary, AGG stimulates p73 in restricting EGF-induced EMT, invasiveness, and stemness by inhibiting the ERK/Snail pathway to facilitate the development of alternative therapeutics for oral cancer.

Estrogen: The necessary evil for human health, and ways to tame it.

Estrogen is a pivotal enzyme for survival and health in both genders, though their quantum, tropism, tissue-specific distribution, and receptor affinity varies with different phases of life. Converted from androgen via aromatase enzyme, this hormone is indispensable to glucose homeostasis, immune robustness, bone health, cardiovascular health, fertility, and neural functions. However, estrogen is at the center of almost all human pathologies as well-infectious, autoimmune, metabolic to degenerative. Both hypo and hyper level of estrogen has been linked to chronic and acute diseases. While normal aging is supposed to lower its level, leading to tissue degeneration (bone, muscle, neural etc.), and metabolite imbalance (glucose, lipid etc.), the increment in inflammatory agents in day-to-day life are enhancing the estrogen (or estrogen mimic) level, fueling 'estrogen dominance'. The resultant excess estrogen is inducing an overexpression of estrogen receptors (ERα and ERß), harming tissues, leading to autoimmune diseases, and neoplasms. The unprecedented escalation in the polycystic ovary syndrome, infertility, breast cancer, ovary cancer, and gynecomastia cases are indicating that this sensitive hormone is getting exacerbated. This critical review is an effort to analyze the dual, and opposing facets of estrogen, via understanding its crosstalk with other hormones, enzymes, metabolites, and drugs. Why estrogen level correction is no trivial task, and how it can be restored to normalcy by a disciplined lifestyle with wise dietary and selective chemical usage choices has been discussed. Overall, our current state of knowledge does not disclose the full picture of estrogen's pleiotropic importance. Hence, this review should be a resource for general public as well as researchers to work in that direction.

PUMA dependent mitophagy by Abrus agglutinin contributes to apoptosis through ceramide generation.

PUMA, a BH3-only pro-apoptotic Bcl2 family protein, is known to translocate from the cytosol into the mitochondria in order to induce apoptosis. Interestingly, the induction of PUMA by p53 plays a critical role in DNA damage-induced apoptosis. In this study, we reported mitophagy inducing potential of PUMA triggered by phytolectin Abrus agglutinin (AGG) in U87MG glioblastoma cells and established AGG-induced ceramide acts as the chief mediator of mitophagy dependent cell death through activation of both mitochondrial ROS as well as ER stress. Importantly, AGG upregulates PUMA expression in U87MG cells with the generation of dysfunctional mitochondria, with gain and loss of function of PUMA is shown to alter mitophagy induction. At the molecular level, our study identified that the LC3 interacting region (LIR) located at the C-terminal end of PUMA interacts with LC3 in order to stimulate mitophagy. In addition, AGG is also found to trigger ubiquitination of PUMA which in turn interacted with p62 for prompting mitophagy suggesting that AGG turns on PUMA-mediated mitophagy in U87MG cells in both p62-dependent as well as in p62-independent manner. Interestingly, AGG-triggered ceramide production through activation of ceramide synthase-1 leads to induction of ER stress and ROS accumulation to promote mitochondrial damage as well as mitophagy. Further, upon pre-treatment with Mdivi-1, DRP1 inhibitor, AGG exposure results in suppression of apoptosis in U87MG cells indicating AGG-induced mitophagy switches to apoptosis that can be exploited for better cancer therapeutics.

In silico analysis of ChtBD3 domain to find its role in bacterial pathogenesis and beyond.

Chitin binding domain 3, known by the acronym ChtBD3, is a domain in the enzymes and proteins of several pathogenic virus, bacteria and fungi. As this domain is evolutionarily-conserved in virulence factors of these infectious agents, its detailed investigation is of clinical interest. In this regard, the current in silico study analyzed ChtBD3 domain distribution in bacterial proteins present in publicly-available SMART (simple modular architecture research tool) database. Also, the co-occurring domains of ChtBD3 in the studied proteins were mapped to understand positional rearrangement of the domain and consequent functional diversity. Custom-made scripts were used to interpret the data and to derive patterns. As expected, interesting results were obtained. ChtBD3 domain co-occurred with other critical domains like peptidase, glycol_hydrolase, kinase, hemagglutinin-acting, collagen-binding, among others. The findings are expected to be of clinical relevance.

Elimination of dysfunctional mitochondria through mitophagy suppresses benzo[a]pyrene-induced apoptosis.

Mitophagy, a special type of autophagy, plays an important role in the mitochondria quality control and cellular homeostasis. In this study, we examined the molecular mechanism of mitophagy induction with benzo[a]pyrene (B[a]P), a ubiquitous polycyclic aromatic hydrocarbon, which acts as a prosurvival response against apoptotic cell death. Our study showed that B[a]P displayed higher cytotoxicity in autophagy-deficient HaCaT cells as compared to control. Further, we showed that B[a]P triggered the Beclin-1-dependent autophagy through the mammalian target of rapamycin (mTOR)/AMP-activated protein kinase (AMPK) pathway. Moreover, our study indicated that the B[a]P-induced autophagy was initiated through the activation of cytochrome P450 1B1 (CYP1B1) and the aryl hydrocarbon receptor (AhR) in HaCaT cells. Intriguingly, the B[a]P-induced Beclin-1-mediated mitophagy was suppressed in CYP1B1 and AhR knockdown HaCaT cells, indicating a crucial role of B[a]P activation in the mitophagy induction to regulate cell death. B[a]P was shown to increase the mitochondrial dysfunction and decrease the mitochondrial membrane potential, resulting in depletion of ATP level along with the inhibition of the oxygen consumption rate in HaCaT cells. Importantly, the supplementation of methyl pyruvate compensated for the B[a]P-induced drop in the ATP level and mitigated the reactive oxygen species burden and autophagy. Mechanistically, B[a]P inhibited the manganese superoxide dismutase (MnSOD) activity and we found that the activated mitochondrial CYP1B1 interacted with MnSOD, inflicting mitophagy to protect from B[a]P-induced apoptosis. In summary, our study reveals mitophagy induction as a cellular protection mechanism against B[a]P-triggered toxicity and carcinogenesis.

A holistic review on the autoimmune disease vitiligo with emphasis on the causal factors.

Vitiligo is an idiopathic systemic autoimmune disease affecting skin, hair and oral mucosa. This genetic yet acquired disease characterized by melanin loss is a cause of morbidity across all races. Though thyroid disturbance has been recognized as a key trigger of this pathology, an array of other factors plays critical role in its manifestation. Multiple hormones (corticotropin-releasing hormone, adrenocorticotropic hormone, α-melanocyte-stimulating hormone, melatonin, calcitriol, testosterone, estrogen), genes (Human leukocyte antigen (HLA), Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), Forkhead box D3 (FOXD3), Cluster of differentiation 117 (CD117), Estrogen receptor (ESR) 1, Cyclooxygenase-2 (COX2), Vitiligo-associated protein 1 (VIT1)), and lifestyle choices (stress, diet, cosmetic products, and medications) have been suspected as drivers of this disorder. The pathological mechanisms have been understood in recent times, with the aid of genomic studies; however a universally-effective therapy is yet to be achieved. This review discusses these under-investigated facets of vitiligo onset and progression; hence, it is expected to enrich vitiligo research.

Abrus Agglutinin, a type II ribosome inactivating protein inhibits Akt/PH domain to induce endoplasmic reticulum stress mediated autophagy-dependent cell death.

Abrus agglutinin (AGG), a type II ribosome-inactivating protein has been found to induce mitochondrial apoptosis. In the present study, we documented that AGG-mediated Akt dephosphorylation led to ER stress resulting the induction of autophagy-dependent cell death through the canonical pathway in cervical cancer cells. Inhibition of autophagic death with 3-methyladenine (3-MA) and siRNA of Beclin-1 and ATG5 increased AGG-induced apoptosis. Further, inhibiting apoptosis by Z-DEVD-FMK and N-acetyl cysteine (NAC) increased autophagic cell death after AGG treatment, suggesting that AGG simultaneously induced autophagic and apoptotic death in HeLa cells. Additionally, it observed that AGG-induced autophagic cell death in Bax knock down (Bax-KD) and 5-FU resistant HeLa cells, confirming as an alternate cell killing pathway to apoptosis. At the molecular level, AGG-induced ER stress in PERK dependent pathway and inhibition of ER stress by salubrinal, eIF2α phosphatase inhibitor as well as siPERK reduced autophagic death in the presence of AGG. Further, our in silico and colocalization study showed that AGG interacted with pleckstrin homology (PH) domain of Akt to suppress its phosphorylation and consequent downstream mTOR dephosphorylation in HeLa cells. We showed that Akt overexpression could not augment GRP78 expression and reduced autophagic cell death by AGG as compared to pcDNA control, indicating Akt modulation was the upstream signal during AGG's ER stress mediated autophagic cell death. In conclusion, we established that AGG stimulated cell death by autophagy might be used as an alternative tumor suppressor mechanism in human cervical cancer. © 2016 Wiley Periodicals, Inc.

Glycosylation Effects on FSH-FSHR Interaction Dynamics: A Case Study of Different FSH Glycoforms by Molecular Dynamics Simulations.

The gonadotropin known as follicle-stimulating hormone (FSH) plays a key role in regulating reproductive processes. Physiologically active FSH is a glycoprotein that can accommodate glycans on up to four asparagine residues, including two sites in the FSHα subunit that are critical for biochemical function, plus two sites in the ß subunit, whose differential glycosylation states appear to correspond to physiologically distinct functions. Some degree of FSHß hypo-glycosylation seems to confer advantages toward reproductive fertility of child-bearing females. In order to identify possible mechanistic underpinnings for this physiological difference we have pursued computationally intensive molecular dynamics simulations on complexes between the high affinity site of the gonadal FSH receptor (FSHR) and several FSH glycoforms including fully-glycosylated (FSH24), hypo-glycosylated (e.g., FSH15), and completely deglycosylated FSH (dgFSH). These simulations suggest that deviations in FSH/FSHR binding profile as a function of glycosylation state are modest when FSH is adorned with only small glycans, such as single N-acetylglucosamine residues. However, substantial qualitative differences emerge between FSH15 and FSH24 when FSH is decorated with a much larger, tetra-antennary glycan. Specifically, the FSHR complex with hypo-glycosylated FSH15 is observed to undergo a significant conformational shift after 5-10 ns of simulation, indicating that FSH15 has greater conformational flexibility than FSH24 which may explain the more favorable FSH15 kinetic profile. FSH15 also exhibits a stronger binding free energy, due in large part to formation of closer and more persistent salt-bridges with FSHR.

Exploring the drug resistance of V32I and M46L mutant HIV-1 protease to inhibitor TMC114: flap dynamics and binding mechanism.

Inhibitors of HIV-1 protease (HIV-1-pr) generally only bind to the active site of the protease. However, for some mutants such as V32I and M46L the TMC114 can bind not only to the active cavity but also to the groove of the flexible flaps. Although the second binding site suggests the higher efficiency of the drug against HIV-1-pr, the drug resistance in HIV-1-pr due to mutations cannot be ignored, which prompts us to investigate the molecular mechanisms of drug resistance and behavior of double bound TMC114 (2T) to HIV-1-pr. The conformational dynamics of HIV-1-pr and the binding of TMC114 to the WT, V32I and M46L mutants were investigated with all-atom molecular dynamic (MD) simulation. The 20 ns MD simulation shows many fascinating effects of the inhibitor binding to the WT and mutant proteases. MM-PBSA calculations explain the binding free energies unfavorable for the M46L and V32I mutants as compared to the WT. For the single binding (1T) the less binding affinity can be attributed to the entropic loss for both V32I-1T and M46L-1T. Although the second binding of TMC114 with flap does increase binding energy for the mutants (V32I-2T and M46L-2T), the considerable entropy loss results in the lower binding Gibbs free energies. Thus, binding of TMC114 in the flap region does not help much in the total gain in binding affinity of the system, which was verified from this study and thereby validating experiments.