Repurposing of Nitroxoline Drug for the Prevention of Neurodegeneration.

Oxidative stress has been documented as one of the significant causes of neurodegenerative diseases. Therefore, antioxidant therapy for the prevention of neurodegenerative diseases seems to be an interesting strategy in drug discovery. The quinoline-based compound, namely 5-nitro-8-quinolinol (NQ), has shown excellent antimicrobial, anticancer, and anti-inflammatory activities. However, its neuroprotective effects and precise molecular mechanisms in human neuronal cells have not been elucidated. In this work, the effects of NQ on cell viability and morphology were evaluated by the MTT assay and microscopic observation. Moreover, the underlying mechanisms of this compound, inducing the survival rate of neuronal cells under oxidative stress, were investigated by reactive oxygen species (ROS) assay, flow cytometry, Western blotting, and immunofluorescence techniques. In addition, the molecular interaction of sirtuin1 (SIRT1) with NQ was constructed using the AutoDock 4.2 program. Interestingly, NQ protected SH-SY5Y cells against H2O2-induced neurotoxicity through scavenging ROS, upregulating the levels of SIRT1 and FOXO3a, increasing the levels of antioxidant enzymes (catalase and superoxide dismutase), promoting antiapoptotic BCL-2 protein expression, and reducing apoptosis. Besides, molecular docking also revealed that NQ interacted satisfactorily with the active site of SIRT1 similar to the resveratrol, which is the SIRT1 activator and strong antioxidant. These findings suggest that NQ prevents oxidative-stress-induced neurodegeneration because of its antioxidant capacity as well as antiapoptotic property through SIRT1-FOXO3a signaling pathway. Thus, NQ might be a drug that could be repurposed for prevention of neurodegeneration.

Production and Characterization of Recombinant Wild Type Uricase from Indonesian Coelacanth (L. menadoensis) and Improvement of Its Thermostability by In Silico Rational Design and Disulphide Bridges Engineering.

The ideal therapeutic uricase (UOX) is expected to have the following properties; high expression level, high activity, high thermostability, high solubility and low immunogenicity. The latter property is believed to depend largely on sequence identity to the deduced human UOX (dH-UOX). Herein, we explored L. menadoensis uricase (LM-UOX) and found that it has 65% sequence identity to dH-UOX, 68% to the therapeutic chimeric porcine-baboon UOX (PBC) and 70% to the resurrected ancient mammal UOX. To study its biochemical properties, recombinant LM-UOX was produced in E. coli and purified to more than 95% homogeneity. The enzyme had specific activity up to 10.45 unit/mg, which was about 2-fold higher than that of the PBC. One-litre culture yielded purified protein up to 132 mg. Based on homology modelling, we successfully engineered I27C/N289C mutant, which was proven to contain inter-subunit disulphide bridges. The mutant had similar specific activity and production yield to that of wild type (WT) but its thermostability was dramatically improved. Up on storage at -20 °C and 4 °C, the mutant retained ~100% activity for at least 60 days. By keeping at 37 °C, the mutant retained ~100% activity for 15 days, which was 120-fold longer than that of the wild type. Thus, the I27C/N289C mutant has potential to be developed for treatment of hyperuricemia.

Neuroprotective Effects of Phenolic and Carboxylic Acids on Oxidative Stress-Induced Toxicity in Human Neuroblastoma SH-SY5Y Cells.

An increase in oxidative stress is a key factor responsible for neurotoxicity induction and cell death leading to neurodegenerative diseases including Parkinson's and Alzheimer's diseases. Plant phenolics exert diverse bioactivities i.e., antioxidant, anti-inflammatory, and neuroprotective effects. Herein, phenolic compounds, namely protocatechuic aldehyde (PCA) constituents of Hydnophytum formicarum Jack. including vanillic acid (VA) and trans-ferulic acid (FA) found in Spilanthes acmella Murr., were explored for anti-neurodegenerative properties using an in vitro model of oxidative stress-induced neuroblastoma SH-SY5Y cells. Exposure of the neuronal cells with H2O2 resulted in the decrease of cell viability, but increasing in the level of reactive oxygen species (ROS) together with morphological changes and inducing cellular apoptosis. SH-SY5Y cells pretreated with 5 µM of PCA, VA, and FA were able to attenuate cell death caused by H2O2-induced toxicity, as well as decreased ROS level and apoptotic cells after 24 h of treatment. Pretreated SH-SY5Y cells with phenolic compounds also helped to upregulate H2O2-induced depletion of the expressions of sirtuin-1 (SIRT1) and forkhead box O (FoxO) 3a as well as induce the levels of antioxidant (superoxide dismutase (SOD) 2 and catalase) and antiapoptotic B-cell lymphoma 2 (Bcl-2) proteins. The findings suggest that these phenolics might be promising compounds against neurodegeneration.

Structural and biochemical characterization of two heme binding sites on α1-microglobulin using site directed mutagenesis and molecular simulation.

BACKGROUND: α1-Microglobulin (A1M) is a reductase and radical scavenger involved in physiological protection against oxidative damage. These functions were previously shown to be dependent upon cysteinyl-, C34, and lysyl side-chains, K(92, 118,130). A1M binds heme and the crystal structure suggests that C34 and H123 participate in a heme binding site. We have investigated the involvement of these five residues in the interactions with heme. METHODS: Four A1M-variants were expressed: with cysteine to serine substitution in position 34, lysine to threonine substitutions in positions (92, 118, 130), histidine to serine substitution in position 123 and a wt without mutations. Heme binding was investigated by tryptophan fluorescence quenching, UV-Vis spectrophotometry, circular dichroism, SPR, electrophoretic migration shift, gel filtration, catalase-like activity and molecular simulation. RESULTS: All A1M-variants bound to heme. Mutations in C34, H123 or K(92, 118, 130) resulted in significant absorbance changes, CD spectral changes, and catalase-like activity, suggesting involvement of these side-groups in coordination of the heme-iron. Molecular simulation support a model with two heme-binding sites in A1M involving the mutated residues. Binding of the first heme induces allosteric stabilization of the structure predisposing for a better fit of the second heme. CONCLUSIONS: The results suggest that one heme-binding site is located in the lipocalin pocket and a second binding site between loops 1 and 4. Reactions with the hemes involve the side-groups of C34, K(92, 118, 130) and H123. GENERAL SIGNIFICANCE: The model provides a structural basis for the functional activities of A1M: heme binding activity of A1M.

Understanding the molecular basis of EGFR kinase domain/MIG-6 peptide recognition complex using computational analyses.

BACKGROUND: Epidermal growth factor receptor (EGFR) signalling plays a major role in biological processes, including cell proliferation, differentiation and survival. Since the over-expression of EGFR causes human cancers, EGFR is an attractive drug target. A tumor suppressor endogenous protein, MIG-6, is known to suppress EGFR over-expression by binding to the C-lobe of EGFR kinase. Thus, this C-lobe of the EGFR kinase is a potential new target for EGFR kinase activity inhibition. In this study, molecular dynamics (MD) simulations and binding free energy calculations were used to investigate the protein-peptide interactions between EGFR kinase and a 27-residue peptide derived from MIG-6_s1 segment (residues 336-362). RESULTS: These 27 residues of MIG-6_s1 were modeled from the published MIG-6 X-ray structure. The binding dynamics were detailed by applying the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method to predict the binding free energy. Both van der Waals interactions and non-polar solvation were favorable driving forces for binding process. Six residues of EGFR kinase and eight residues of MIG-6_s1 residues were shown to be responsible for interface binding in which we investigated per residue free energy decomposition and the results from the computational alanine scanning approach. These residues also had higher hydrogen bond occupancies than other residues at the binding interface. The results from the aforementioned calculations reasonably agreed with the previous experimental mutagenesis studies. CONCLUSIONS: Molecular dynamics simulations were used to investigate the interactions of MIG-6_s1 to EGFR kinase domain. Our study provides an insight into such interactions that is useful in guiding the design of novel anticancer therapeutics. The information on our modelled peptide interface with EGFR kinase could be a possible candidate for an EGFR dimerization inhibitor.

Illuminating the origins of spectral properties of green fluorescent proteins via proteochemometric and molecular modeling.

Green fluorescent protein (GFP) has immense utility in biomedical imaging owing to its autofluorescent nature. In efforts to broaden the spectral diversity of GFP, there have been several reports of engineered mutants via rational design and random mutagenesis. Understanding the origins of spectral properties of GFP could be achieved by means of investigating its structure-activity relationship. The first quantitative structure-property relationship study for modeling the spectral properties, particularly the excitation and emission maximas, of GFP was previously proposed by us some years ago in which quantum chemical descriptors were used for model development. However, such simplified model does not consider possible effects that neighboring amino acids have on the conjugated π-system of GFP chromophore. This study describes the development of a unified proteochemometric model in which the GFP chromophore and amino acids in its vicinity are both considered in the same model. The predictive performance of the model was verified by internal and external validation as well as Y-scrambling. Our strategy provides a general solution for elucidating the contribution that specific ligand and protein descriptors have on the investigated spectral property, which may be useful in engineering novel GFP variants with desired characteristics.

Aminochalcones Attenuate Neuronal Cell Death under Oxidative Damage via Sirtuin 1 Activity.

Encouraged by the lack of effective treatments and the dramatic growth in the global prevalence of neurodegenerative diseases along with various pharmacological properties of chalcone pharmacophores, this study focused on the development of aminochalcone-based compounds, organic molecules characterized by a chalcone backbone (consisting of two aromatic rings connected by a three-carbon α,ß-unsaturated carbonyl system) with an amino group attached to one of the aromatic rings, as potential neuroprotective agents. Thus, the aminochalcone-based compounds in this study were designed by bearing a -OCH3 moiety at different positions on the ring and synthesized by the Claisen-Schmidt condensation. The compounds exhibited strong neuroprotective effects against hydrogen peroxide-induced neuronal death in the human neuroblastoma (SH-SY5Y) cell line (i.e., by improving cell survival, reducing reactive oxygen species production, maintaining mitochondrial function, and preventing cell membrane damage). The aminochalcone-based compounds showed mild toxicity toward a normal embryonic lung cell line (MRC-5) and a human neuroblastoma cell line, and were predicted to have preferable pharmacokinetic profiles with potential for oral administration. Molecular docking simulation indicated that the studied aminochalcones may act as competitive activators of the well-known protective protein, SIRT1, and provided beneficial knowledge regarding the essential key chemical moieties and interacting amino acid residues. Collectively, this work provides a series of four promising candidate agents that could be developed for neuroprotection.

Promising 8-Aminoquinoline-Based Metal Complexes in the Modulation of SIRT1/3-FOXO3a Axis against Oxidative Damage-Induced Preclinical Neurons.

The discovery of novel bioactive molecules as potential multifunctional neuroprotective agents has clinically drawn continual interest due to devastating oxidative damage in the pathogenesis and progression of neurodegenerative diseases. Synthetic 8-aminoquinoline antimalarial drug is an attractive pharmacophore in drug development and chemical modification owing to its wide range of biological activities, yet the underlying molecular mechanisms are not fully elucidated in preclinical models for oxidative damage. Herein, the neuroprotective effects of two 8-aminoquinoline-uracil copper complexes were investigated on the hydrogen peroxide-induced human neuroblastoma SH-SY5Y cells. Both metal complexes markedly restored cell survival, alleviated apoptotic cascades, maintained antioxidant defense, and prevented mitochondrial function by upregulating the sirtuin 1 (SIRT1)/3-FOXO3a signaling pathway. Intriguingly, in silico molecular docking and pharmacokinetic prediction suggested that these synthetic compounds acted as SIRT1 activators with potential drug-like properties, wherein the uracil ligands (5-iodoracil and 5-nitrouracil) were essential for effective binding interactions with the target protein SIRT1. Taken together, the synthetic 8-aminoquinoline-based metal complexes are promising brain-targeting drugs for attenuating neurodegenerative diseases.

Co-occurrence of mcr-2 and mcr-3 genes on chromosome of multidrug-resistant Escherichia coli isolated from healthy individuals in Thailand.

The aim of this study was to characterize three strains of colistin-resistant E. coli isolated from feces samples of healthy individuals in Thailand. The three strains, namely, SY_EC03, SY_EC07, and SY_EC10 were identified as ST165, ST1602, and ST34. All isolates exhibited multidrug-resistant phenotype, which is mediated by accumulation of various antimicrobial resistance genes. SY_EC03 contained mcr-1.1 while SY_EC07 co-harbored mcr-2.3 and mcr-3.4, and SY_EC10 co-harbored mcr-1.1 and mcr-3.5. Genomic analysis revealed that mcr-1.1 of the two strains were located on IncI2 plasmid with genetic environment of ISApl1-mcr-1.1-PAP2, which is a composite transposon Tn6330 with single-ended. Regarding mcr-2.3, the gene was identified within the composite transposon of ISKpn71-mcr-2.3-ISSpu2-ISKpn71, which was located on a novel mobile genetic element (MGE) that was integrated into the chromosome by phage integrase. For mcr-3.4 and mcr-3.5, the genes were confirmed to locate on the chromosome by S1-PFGE/DNA hybridization. Hence, to the best of our knowledge, this is the first report on co-occurrence of mcr-2 and mcr-3 on chromosome of E. coli. More interestingly, mcr-2 was found to locate on a novel MGE, which had never been described. In addition, we also report the co-occurrence of plasmidic mcr-1.1 and chromosomal mcr-3.5 which is extremely rare. Since all these bacteria were isolated from healthy individuals and the identified STs have been found in a variety of origins, all these clones may serve as reservoir for horizontal and vertical transmission of mcr genes. Strategic action plans to control and prevent the spread of mcr genes are urgently needed.

Structural analysis of cannabinoids against EGFR-TK leads a novel target against EGFR-driven cell lines.

Epidermal growth factor receptor (EGFR) is a member of the ErbB family of proteins and are involved in downstream signal transduction, plays prominent roles in cell growth regulation, proliferation, and the differentiation of many cell types. They are correlated with the stage and severity of cancer. Therefore, EGFRs are targeted proteins for the design of new drugs to treat cancers that overexpress these proteins. Currently, several bioactive natural extracts are being studied for therapeutic purposes. Cannabis has been reported in many studies to have beneficial medicinal effects, such as anti-inflammatory, analgesic, antibacterial, and anti-inflammatory effects, and antitumor activity. However, it is unclear whether cannabinoids reduce intracellular signaling by inhibiting tyrosine kinase phosphorylation. In this study, cannabinoids (CBD, CBG, and CBN) were simulated for binding to the EGFR-intracellular domain to evaluate the binding energy and binding mode based on molecular docking simulation. The results showed that the binding site was almost always located at the kinase active site. In addition, the compounds were tested for binding affinity and demonstrated their ability to inhibit kinase enzymes. Furthermore, the compounds potently inhibited cellular survival and apoptosis induction in either of the EGFR-overexpressing cell lines.

Identification of tripeptides against tyrosine kinase domain of EGFR for lung cancer cell inhibition by in silico and in vitro studies.

Epidermal growth factor receptor tyrosine kinase domain (EGFR-TK) has been one of the prominent targets for therapeutics of several human cancers, in particular non-small cell lung cancer. Although several small chemical compounds targeting EGFR-TK have been approved by FDA for treatment of such a cancer, the discovery of a new class of EGFR-TK inhibitors, for example, small peptides, is still desired. In this study, using molecular docking-based virtual screening, we selected five small peptides with high docking scores from eight thousand peptides as candidate compounds against EGFR-TK. Among five, the tripeptide WFF had the most potency to suppress the survival of non-small cell lung cancer cells but had the least toxicity to human liver cancer cells. Our in vitro kinase assays showed that WFF exhibited much lower inhibitory activity against purified EGFR-TK than the drug erlotinib (i.e., IC50  values of ≈ 0.62 µM vs ≈ 7.57 nM, respectively). The relative free binding energies estimated from molecular dynamic simulations were consistent with the in vitro experiments in which the WFF bound had a lower affinity than erlotinib bound to EGFR-TK (i.e., ΔGbind values of -20.3 kJ/mol vs ≈ -126.8 kJ/mol, respectively). In addition, the simulation analyses demonstrated the difference in EGFR binding preference between the drug and tripeptide in which erlotinib was stably bound in the ATP-binding pocket for 4-anilinoquinazoline class of inhibitors, while WFF moved out of that pocket to interact with polar amino acid residues on the αC-helix, activation loop, and substrate-binding region. Our findings suggest preferable interactions of the potential tripeptide on enzyme inhibition that are useful for further development of a new class of inhibitors targeting EGFR-TK.

Emergence of plasmid-mediated colistin resistance mcr-3.5 gene in Citrobacter amalonaticus and Citrobacter sedlakii isolated from healthy individual in Thailand.

Citrobacter spp. are Gram-negative bacteria commonly found in environments and intestinal tracts of humans and animals. They are generally susceptible to third-generation cephalosporins, carbapenems and colistin. However, several antibiotic resistant genes have been increasingly reported in Citrobacter spp., which leads to the postulation that Citrobacter spp. could potentially be a reservoir for spreading of antimicrobial resistant genes. In this study, we characterized two colistin-resistant Citrobacter spp. isolated from the feces of a healthy individual in Thailand. Based on MALDI-TOF and ribosomal multilocus sequence typing, both strains were identified as Citrobacter sedlakii and Citrobacter amalonaticus. Genomic analysis and S1-nuclease pulsed field gel electrophoresis/DNA hybridization revealed that Citrobacter sedlakii and Citrobacter amalonaticus harbored mcr-3.5 gene on pSY_CS01 and pSY_CA01 plasmids, respectively. Both plasmids belonged to IncFII(pCoo) replicon type, contained the same genetic context (Tn3-IS1-ΔTnAs2-mcr-3.5-dgkA-IS91) and exhibited high transferring frequencies ranging from 1.03×10-4 - 4.6×10-4 CFU/recipient cell Escherichia coli J53. Colistin-MICs of transconjugants increased ≥ 16-fold suggesting that mcr-3.5 on these plasmids can be expressed in other species. However, beside mcr, other major antimicrobial resistant determinants in multidrug resistant Enterobacterales were not found in these two isolates. These findings indicate that mcr gene continued to evolve in the absence of antibiotics selective pressure. Our results also support the hypothesis that Citrobacter could be a reservoir for spreading of antimicrobial resistant genes. To the best of our knowledge, this is the first report that discovered human-derived Citrobacter spp. that harbored mcr but no other major antimicrobial resistant determinants. Also, this is the first report that described the presence of mcr gene in C. sedlakii and mcr-3 in C. amalonaticus.

Extracellular Vesicle-Mediated IL-1 Signaling in Response to Doxorubicin Activates PD-L1 Expression in Osteosarcoma Models.

The expression of programmed cell death ligand 1 (PD-L1) in tumors is associated with tumor cell escape from T-cell cytotoxicity, and is considered a crucial effector in chemoresistance and tumor relapse. Although PD-L1 induction has been observed in patients after chemotherapy treatment, the mechanism by which the drug activates PD-L1 expression remains elusive. Here, we identified the extracellular vesicles (EVs) as a molecular mediator that determines the effect of doxorubicin on PD-L1 expression in osteosarcoma models. Mechanistically, doxorubicin dependently stimulates the release of extracellular vesicles, which mediate autocrine/paracrine signals in osteosarcoma cells. The recipient cells were stimulated by these EVs and acquired the ability to promote the expression of inflammatory cytokines interleukin (IL)-1ß and IL-6. In response to doxorubicin, IL-1ß, but not IL-6, allowed- osteosarcoma cells to promote the expression of PD-L1, and the elimination of IL-1ß/IL-1 receptor signaling with IL-1 receptor antagonist reduced PD-L1 expression. Together, these findings provided insights into the role of EV release in response to chemotherapy that mediates PD-L1 expression via the IL-1 signaling pathway, and suggested that the combination of a drug targeting IL-1 or PD-L1 with chemotherapy could be an effective treatment option for osteosarcoma patients.

Neuroprotective Properties of Bis-Sulfonamide Derivatives Against 6-OHDA-Induced Parkinson's Model via Sirtuin 1 Activity and in silico Pharmacokinetic Properties.

Parkinson's disease (PD) is considered one of the health problems in the aging society. Due to the limitations of currently available drugs in preventing disease progression, the discovery of novel neuroprotective agents has been challenged. Sulfonamide and its derivatives were reported for several biological activities. Herein, a series of 17 bis-sulfonamide derivatives were initially tested for their neuroprotective potential and cytotoxicity against the 6-hydroxydopamine (6-OHDA)-induced neuronal death in SH-SY5Y cells. Subsequently, six compounds (i.e., 2, 4, 11, 14, 15, and 17) were selected for investigations on underlying mechanisms. The data demonstrated that the pretreatment of selected compounds (5 µM) can significantly restore the level of cell viability, protect against mitochondrial membrane dysfunction, decrease the activity of lactate dehydrogenase (LDH), decrease the intracellular oxidative stress, and enhance the activity of NAD-dependent deacetylase sirtuin-1 (SIRT1). Molecular docking was also performed to support that these compounds could act as SIRT1 activators. In addition, in silico pharmacokinetic and toxicity profile prediction was also conducted for guiding the potential development. Thus, the six neuroprotective bis-sulfonamides were highlighted as potential agents to be further developed for PD management.

Modulatory Effects of Alpha-Mangostin Mediated by SIRT1/3-FOXO3a Pathway in Oxidative Stress-Induced Neuronal Cells.

BACKGROUND: alpha-Mangostin, a polyphenolic xanthone, is primarily found in the pericarp of mangosteen throughout Southeast Asia and is considered as the "Queen of Fruit" in Thailand. Nonetheless, it is not clarified how alpha-mangostin protects neuronal cells against oxidative stress. OBJECTIVE: In this study, molecular mechanisms underlying the neuroprotective effect of alpha-mangostin in defending hydrogen peroxide (H2O2)-induced neurotoxicity was explored. METHODS: cytotoxicity, reactive oxygen species (ROS) generation, apoptotic cascades, and protein expression profiles were performed incorporation of molecular docking. RESULTS: Human SH-SY5Y cells were pretreated with 1 µM alpha-mangostin for 3 h prior to exposure to 400 µM H2O2. alpha-Mangostin significantly inhibited oxidative stress-induced cell death in neuronal cells by reducing BAX protein, decreasing caspase-3/7 activation, and increasing anti-apoptotic BCL-2 protein. Collectively, alpha-mangostin was demonstrated to be a prominent ROS suppressor which reversed the reduction of antioxidant enzymes (CAT and SOD2). Surprisingly, alpha-mangostin significantly promoted the expression of the sirtuin family and the FOXO3a transcription factor exerting beneficial effects on cell survival and longevity. A molecular docking study predicted that alpha-mangostin is directly bound to the active site of SIRT1. CONCLUSION: Findings from this study suggest that alpha-mangostin potentially serves as a promising therapeutic compound against oxidative stress by activation of the SIRT1/3-FOXO3a pathway comparable to the effect of memantine, an anti-AD drug used for the treatment of moderate to severe dementia.

Computational analysis of binding between malarial dihydrofolate reductases and anti-folates.

BACKGROUND: Plasmodium falciparum readily develops resistance to the anti-folates pyrimethamine and proguanil via a characteristic set of mutations in the dihydrofolate reductase (PfDHFR) gene that leads to reduced competitive drug binding at the enzyme's active site. Analogous mutations can be found in the DHFR gene in isolates of Plasmodium vivax (PvDHFR) although anti-folates have not been widely used for the treatment of this infection. Here the interactions between DHFR inhibitors and modelled structures of the DHFR enzymes of Plasmodium malariae (PmDHFR) and Plasmodium ovale (PoDHFR) are described, along with an investigation of the effect of recently reported mutations within PmDHFR. METHODS: DHFR models for PmDHFR and PoDHFR were constructed using the solved PfDHFR-TS and PvDHFR structures respectively as templates. The modelled structures were docked with three DHFR inhibitors as ligands and more detailed interactions were explored via simulation of molecular dynamics. RESULTS: Highly accurate models were obtained containing sets of residues that mediate ligand binding which are highly comparable to those mediating binding in known crystal structures. Within this set, there were differences in the relative contribution of individual residues to inhibitor binding. Modelling of PmDHFR mutant sequences revealed that PmDHFR I170M was associated with a significant reduction in binding energy to all DHFR inhibitors studied, while the other predicted resistance mutations had lesser or no effects on ligand binding. CONCLUSIONS: Binding of DHFR inhibitors to the active sites of all four Plasmodium enzymes is broadly similar, being determined by an analogous set of seven residues. PmDHFR mutations found in field isolates influenced inhibitor interactions to a varying extent. In the case of the isolated I170M mutation, the loss of interaction with pyrimethamine suggests that DHFR-inhibitor interactions in P. malariae are different to those seen for DHFRs from P. falciparum and P. vivax.

Receptor-based virtual screening of EGFR kinase inhibitors from the NCI diversity database.

Epidermal growth factor receptor (EGFR) abnormalities have been associated with several types of human cancer. The crystal structures of its tyrosine kinase domain (EGFR-TK) complexed with small molecule inhibitors revealed the kinase inhibition modes, prompting us to search for novel anti-cancer drugs. A total of 1,990 compounds from the National Cancer Institute (NCI) diversity set with nonredundant structures have been tested to inhibit cancer cell lines with unknown mechanism. Cancer inhibition through EGFR-TK is one of the mechanisms of these compounds. In this work, we performed receptor-based virtual screening against the NCI diversity database. Using two different docking algorithms, AutoDock and Gold, combined with subsequent post-docking analyses, we found eight candidate compounds with high scoring functions that all bind to the ATP-competitive site of the kinase. None of these compounds belongs to the main group of the currently known EGFR-TK inhibitors. Binding mode analyses revealed that the way these compounds complexed with EGFR-TK differs from quinazoline inhibitor binding and the interaction mainly involves hydrophobic interactions. Also, the common kinase-inhibitor (NH---N and CO---HC) hydrogen bonds between the hinge region and the hit compounds are rarely observed. Our results suggest that these molecules could be developed as novel lead compounds in anti-cancer drug design.

Potential tripeptides against the tyrosine kinase domain of human epidermal growth factor receptor (HER) 2 through computational and kinase assay approaches.

An abnormal activation of human epidermal growth factor receptor (HER) 2 has been found to associate with several types of human cancer, and thus the protein is a prominent target for cancer therapy. Although several small chemical molecules targeting the tyrosine kinase (TK) of HER family have been identified, the development of a new class of inhibitors, i.e., small peptides inhibiting the function of tyrosine kinase is still promising. Here, we screened 8000 tripeptides for candidate potential inhibitors against HER2-TK using molecular docking. Our in vitro kinase assays showed that the candidate tripeptides had more than 50% relative inhibition to HER2-TK. Even though these tripeptides had much lower inhibitory activity than that of the drug Lapatinib, the tripeptides WWW exhibited high inhibitory activity with the IC50 of ≈283 µM, while FYW showed lower activity with the IC50 of ≈1723 µM. The relative binding free energies calculated by MM/PBSA method were comparable to the inhibition experiment in that Lapatinib binding was ≈-139 kJ/mol whereas the binding of WWW and FYW was ≈-112 kJ/mol and ≈-81 kJ/mol, respectively. Energy calculation also indicated that the HER2-TK/inhibitor interactions were dominated by van der Waals over electrostatic contributions. In addition, molecular interaction analyses revealed that several interacting residues with more negative binding free energy could mostly contribute the hydrophobic interaction. Therefore, we suggested preferable interactions for further development of potential tripeptides as a new anticancer peptide targeting HER2-TK.

Butein, isoliquiritigenin, and scopoletin attenuate neurodegeneration via antioxidant enzymes and SIRT1/ADAM10 signaling pathway.

Neuronal cell death is a key feature of neurodegenerative disorders such as Parkinson's and Alzheimer's diseases. Plant polyphenols, namely butein, isoliquiritigenin, and scopoletin, have been shown to exhibit various biological activities including anti-inflammatory, antimicrobial, and antioxidant activities. Herein, butein, isoliquiritigenin, and scopoletin were explored for their neuroprotective properties against oxidative stress-induced human dopaminergic SH-SY5Y cell death. The cells exposed to hydrogen peroxide (H2O2) revealed a reduction in cell viability and increases in apoptosis and levels of reactive oxygen species (ROS). Interestingly, pretreatment of SH-SY5Y cells with 5 µM of butein, isoliquiritigenin, or scopoletin protected against the cell death induced by H2O2, and decreased the levels of apoptotic cells and ROS. In addition, the levels of SIRT1, FoxO3a, ADAM10, BCL-2, and antioxidant enzymes (catalase and SOD2) were maintained in the cells pretreated with butein, isoliquiritigenin, or scopoletin before H2O2 treatment compared to cells without pretreatment and the reference (resveratrol). Molecular docking analysis revealed that the interactions between the activator-binding sites of SIRT1 and the phenolic compounds were similar to those of resveratrol. Taken together, the data suggest that these polyphenolic compounds could be potential candidates for prevention and/or treatment of neurodegeneration.

Yield improvement and enzymatic dissection of Plasmodium falciparum plasmepsin V.

To survive within a red blood cell (RBC), malaria parasites establish striking modifications to the permeability, rigidity and cytoadherence properties of the host cell. This is mediated by the export of hundreds of proteins from the parasite into the erythrocyte. Plasmodium falciparum plasmepsin V (PfPMV), is an ER resident aspartic protease that processes proteins for export into the host erythrocyte, plays a crucial role in parasite virulence and survival and is considered a potential malaria drug target. Most attempts at its heterologous expression in Escherichia coli have resulted in mainly the production of insoluble proteins. In this study, we employed a multipurpose fusion tag to improve the production of PfPMV in E. coli. Recombinant PfPMVm, comprising residues 84-521, was substantially obtained in soluble form and could be purified in a single step, yielding a 3.7-fold increase in purified PfPMVm compared to previous reports. Additionally, we have mutated the catalytic residues (D118N and D365N), individually and together, and the unpaired cysteine residue C178 to evaluate the effects on catalytic efficiency. Mutation of D365 had more pronounced effects on the catalytic efficiency than that of D118, suggesting that the D365 may act as a catalytic nucleophile to activate the water molecule. The importance of C178 was also confirmed by the inhibition by metal ions, indicating that C178 is partially involved in the substrate recognition. Collectively, our results describe an improved system to produce recombinant PfPMVm in E. coli and dissect the amino acids involved in catalysis and substrate recognition.