Peptide inhibitors of human HMG-CoA reductase as potential hypocholesterolemia agents.

Hypercholesterolemia may lead to obesity and cardiovascular diseases. To prevent hypercholesterolemia, many drugs have been developed while searching for better drugs to treat hypercholesterolemia has never been ended. Other than small molecule drugs, peptide drugs are gaining more visibilities in many therapeutic areas. In the present study, we employed phage-display techniques to screen peptide inhibitors against human HMG-CoA reductase. The results indicate that the tetrapeptide PMAS inhibits hHMGR effectively (IC50=68 µM), could be a lead compound to develop hypocholesterolemic agents.

Syntheses of some α-cyclic tripeptides as potential inhibitors for HMG-CoA Reductase.

α-Cyclic tripeptides (CtPs) are the most rigid members of the cyclic peptide family. However, due to their synthetic difficulty, biological activity has remained undisclosed. The incorporation of side-chain-protected natural amino acids into functional CtPs was performed to explore the potential biological functions. Several novel CtPs that consist of protected serine (S(Bn)) and/or glutamate (E(OBn)) were prepared from corresponding linear tripeptides by chemical synthesis. There is a strong possibility for CtPs that contain 3 phenyl groups to correlate with atorvastatin structure. The binding effects in human HMG-CoA reductase (hHMGR) activities were first evaluated by molecular docking. High docking scores were received with these CtPs for enzyme. Therefore, enzymatic assays were carried out and the compound cyclo(S(Bn))3 was indeed able to moderately inhibit hHMGR (IC50 = 110 µM).

Immunomodulatory effect of marine cembrane-type diterpenoids on dendritic cells.

Dendritic cells (DCs) are antigen presenting cells, which can present antigens to T-cells and play an important role in linking innate and adaptive immunity. DC maturation can be induced by many stimuli, including pro-inflammatory cytokines and bacterial products, such as lipopolysaccharides (LPS). Here, we examined the immunomodulatory effects of marine cembrane compounds, (9E,13E)-5-acetoxy-6-hydroxy-9,13-dimethyl-3- methylene-3,3a,4,5,6,7,8,11,12,14a-decahydro-2H-cyclotrideca[b]furan-2-one (1), (9E,13E)- 5-acetoxy-6-acetyl-9,13-dimethyl-3-methylene-3,3a,4,5,6,7,8,11,12,14a-decahydro-2H-cyclotrideca[b]furan-2-one (2), lobocrassin B (3), (-)14-deoxycrassin (4), cembranolide B (5) and 13-acetoxysarcocrassolide (6) isolated from a soft coral, Lobophytum crassum, on mouse bone marrow-derived dendritic cells (BMDCs). The results revealed that cembrane-type diterpenoids, especially lobocrassin B, effectively inhibited LPS-induced BMDC activation by inhibiting the production of TNF-α. Pre-treatment of BMDCs with Lobocrassin B for 1 h is essential to prohibit the following activation induced by various toll-like receptor (TLR) agonists, such as LPS, zymosan, lipoteichoic acid (LTA) and Pam2CSK4. Inhibition of NF-κB nuclear translocation by lobocrassin B, which is a key transcription factor for cytokine production in TLR signaling, was evident as assayed by high-content image analysis. Lobocrassin B attenuated DC maturation and endocytosis as the expression levels of MHC class II and the co-stimulatory molecules were downregulated, which may affect the function of DCs to initiate the T-cell responses. Thus, lobocrassin B may have the potential in treatment of immune dysregulated diseases in the future.

A dodecapeptide (YQVTQSKVMSHR) exhibits antibacterial effect and induces cell aggregation in Escherichia coli.

Antimicrobial peptides play an important role in the innate immune response and host defense mechanism. In the present study, we employed phage display technique to screen for inhibitors which may block the phosphoenolpyruvatedependent phosphotransferase system (PTS) pathway and hence retard cell growth. The recombinant histidine-containing phosphocarrier HPr protein was prepared as the target to screen for the tight binders from the phage-displayed random peptide library Ph.D.-12. The biopanning processes were performed and the binding capabilities of the selected phage were further estimated by enzyme-linked immunosorbent assay (ELISA). The single-stranded DNAs of the 20 selected phages were isolated, sequenced, and five corresponding peptides were synthesized. Only one of the five peptides, AP1 (YQVTQSK VMSHR) was found to inhibit the growth of Escherichia coli cells efficiently (IC50~50 µM). Molecular modeling reveals that AP1 may block the EI-HPr interaction and phosphotransfer. Interestingly, AP1 was also found to induce cell aggregation in a concentration-dependent manner. Since glycogen accumulation has been attributed to biofilm formation, the effects of AP1 on the intracellular glycogen levels were measured. The results strongly indicate that the cell aggregation may be caused by the binding of peptide AP1 with HPr to block the interaction of dephosphorylated HPr with glycogen phosphorylase (GP). Because glycogen phosphorylase activity can be activated by HPr-GP interaction, the binding of AP1 to HPr would cause a decreasing rate of glycogen breakdown in M9 medium and accumulation of glycogen, which may lead to eventual cell aggregation. To the best of our knowledge, this is the first study to demonstrate that an inhibitor bound to a dephosphorylated HPr can decouple its regulatory function and induce cell aggregation.

Inhibitory effect of n-butylidenephthalide on neointimal hyperplasia in balloon injured rat carotid artery.

This investigation was designed to determine the inhibitory effects and mechanisms of n-butylidenephthalide (BP) from Angelica sinensis on smooth muscle cell (SMC) proliferation in vitro and in balloon injured rat carotid artery. Treatment of cultured rat aorta SMC-derived A7r5 cells with 25-100 µg/mL BP significantly inhibited the proliferation and arrested the cell cycle in G(0)/G(1) phase. BP induced the expression and migration of Nur77 from the nucleus to the cytoplasm. Among signal pathways, JNK and p38 MAPK were phosphorylated after BP treatment. In vivo, the neointimal area of common carotid artery 2 weeks after balloon injury reduced significantly in Sprague-Dawley rats treated with 150-300 mg/kg BP compared with the control. The proliferative activity indicated by immunohistochemical detection of Ki-67 positive cells in the neointima was significantly decreased in the 60-300 mg/kg BP treatment groups. The apoptotic activity indicated by cleaved caspase-3 positive cells and Nur77 positive cells in the neointima was significantly increased in rats treated with 60-300 mg/kg BP. This study demonstrated BP inhibited neointimal hyperplasia in balloon injured rat carotid artery due to its dual effects of proliferative inhibition and apoptotic induction on SMCs. Up-regulation of Nur77 gene may partly explain the antihyperplasia activity of BP on the neointima.

Proteomic comparative analysis of pathogenic strain 232 and avirulent strain J of Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae is an important pathogen of pigs causing enzootic pneumonia of swine. The pathogen remains largely enigmatic as far as the host-pathogen interactions are concerned. In the present study, the protein profiles of two strains of M. hyopneumoniae were compared by two-dimensional gel electrophoresis and mass spectrometry. The results indicate that the major adhesin P97, the 50-kDa protein derived from P159 adhesin, and the 43-kDa cleavage product of P102 are expressed at much higher levels in the pathogenic strain 232. In contrast, the avirulent strain J switches its focus to metabolism and expresses more glyceraldehyde 3-phosphate dehydrogenase in gluconeogenesis and lactate dehydrogenase, pyruvate dehydrogenase, and phosphate acetyltransferase in the pyruvate metabolism pathway. We speculate that the avirulent strain may have developed better capabilities to cope with the rich environment during repeated inoculations. Simultaneously, the capability to infect host cells may become less important so that the adhesion-related protein genes are down-regulated.

Mycoplasmas regulate the expression of heat-shock protein genes through CIRCE-HrcA interactions.

Mycoplasmas in general are rarely exposed to severe environmental changes except during its colonization and infection processes. Genomic analysis indicates that Mycoplasma hyopneumoniae possesses the genes of a single sigma factor and the HrcA repressor of negative regulation of the heat-shock response. A perfect inverted repeat sequence (5'-CTGGCACTT-N(9)-AAGTGCCAA-3') upstream of the DnaK gene has also been identified. In the present study, we demonstrate the functionality of HrcA-CIRCE interactions using the gel electrophoretic mobility shift assay. The presence of the unique sigma factor, HrcA repressor, and the CIRCE-like sequences reveals that mycoplasmal species may all use the negative regulatory mechanism in the heat-shock response. It is conceivable that mycoplasmas may have evolved a single HrcA repressor-based mechanism which might be the most simple and economical way of controlling HSP gene expression.

Identification and the molecular mechanism of a novel myosin-derived ACE inhibitory peptide.

The objective of this work was to identify a novel ACE inhibitory peptide from myosin using a number of in silico methods. Myosin was evaluated as a substrate for use in the generation of ACE inhibitory peptides using BIOPEP and ExPASy PeptideCutter. Then the ACE inhibitory activity prediction of peptides in silico was evaluated using the program peptide ranker, following the database search of known and unknown peptides using the program BIOPEP. In addition, the interaction mechanisms of the peptide and ACE were evaluated by DS. All of the tripeptides were predicted to be nontoxic. Results suggested that the tripeptide NCW exerted potent ACE inhibitory activity with an IC50 value of 35.5 µM. Furthermore, the results suggested that the peptide NCW comes into contact with Zn 701, Tyr 523, His 383, Glu 384, Glu 411, and His 387. The potential molecular mechanism of the NCW/ACE interaction was investigated. Results confirmed that the higher inhibitory potency of NCW might be attributed to the formation of more hydrogen bonds with the ACE's active site. Therefore, the in silico method is effective to predict and identify novel ACE inhibitory peptides from protein hydrolysates.

Anti-Alzheimers activity and molecular mechanism of albumin-derived peptides against AChE and BChE.

Alzheimer's disease (AD) is a global health issue affecting millions of elderly people worldwide. The aim of the present study was to identify novel anti-AD peptides isolated from albumin. Anti-AD activities of the peptides were evaluated via inhibitory activities on acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Furthermore, the potential molecular mechanisms of the KLPGF/AChE were investigated by CDOCKER of Discovery studio 2017. The results revealed that peptide KLPGF could effectively inhibit AChE with an inhibition rate of 61.23% at a concentration of 50 µg mL-1. In addition, the peptide KLPGF came in contact with acylation sites and peripheral anion sites of AChE. The present study demonstrates that the peptide KLPGF could become a potential functional food intervention in AD.

Protective effects of a freeze-dried extract of vegetables and fruits on the hydroxyl radical-mediated oxidative damage of DNA and decrease of erythrocytes deformability.

The protective effects of a freeze-dried extracts of vegetables and fruits (BauYuan; BY) on the hydroxyl radical-mediated DNA strand breakages and the structural integrity of human red blood cells (RBCs) were investigated. First, the supercoiled plasmid (pEGFP-C1) DNA was subjected to oxidation damage by an ascorbate-fortified Fenton reaction and the protective effects were analyzed by agarose gel electrophoresis. In the absence of BY extracts, exposure of the high-throughput .OH-generating system (Fe2+ concentration >1.0 microM) caused a complete fragmentation of DNA. Supplementation of BY extract (1 mg/mL) to the plasmid DNA prior to the exposure could prevent it significantly. In contrast, as the plasmid exposed to a low-grade .OH-generating system (Fe2+<0.1 microM), the BY extract (1 mg/mL) provided an almost complete protection. Next, the cell deformabilities were measured to assess the protection effects of various BY extracts on human erythrocytes exposed to the oxidative insults. We found that both the aqueous extract and the organic solvent-derived extracts could strongly protect human RBCs from the reactive oxygen species (ROS)-mediated decrease in the deformability indices. The results implicated that the BY extracts could effectively protect the cell membrane integrity via scavenging ROS which enabling RBCs to maintain a balance of water content and surface area to prevent the drop of cell deformability.

Discovering Peptide Inhibitors of Human Squalene Synthase Through Screening the Phage-Displayed Cyclic Peptide c7c Library.

Many drugs for the treatment of hypercholesterolemia are targeting the enzymes involved in human cholesterol biosynthesis pathway. Squalene synthase, the rate-limiting enzyme located at the downstream of cholesterol synthesis pathway, has become a better candidate to develop next-generation hypocholesterolemia drugs. In the present study, we cloned and expressed the recombinant human squalene synthase (hSQS) as the lure to isolate potential peptide inhibitors from screening the conformation-constrained phage-displayed cyclic peptide c7c library. Their binding capabilities were further estimated by ELISA. Their pharmaceutical potentials were then analyzed through molecular modeling and the ADMET property evaluations. Four ennea-peptides and nine tetra-peptides were finally synthesized to evaluate their inhibitory potentials toward hSQS. The results indicate that the ennea-peptide CLSPHSMFC, tetra-peptides SMFC, CKTE, and WHQW can effectively inhibit hSQS activities (IC50 values equal to 64, 76, 87, and 90 µM, respectively). These peptides may have potentials to develop future cholesterol-lowering therapeutics. The ligand-protein interaction analysis also reveals that the inner hydrophobic pocket could be a more critical site of hSQS.

Exploration of Peptide Inhibitors of Human Squalene Synthase through Molecular Modeling and Phage Display Technique.

Many studies have demonstrated the role of elevated levels of serum cholesterol in the pathogenesis of atherosclerosis and coronary heart disease. Various drugs targeting the key enzymes involved in the cholesterol biosynthesis pathway have been investigated for the treatment of hypercholesterolemia. Human squalene synthase has been one of the most important targets for therapeutic intervention. In the present study, we used the recombinant human squalene synthase as the lure for screening the peptide inhibitors from phage-displayed random peptide library. The tightly bound phages and their derived peptides were further evaluated based on their potential binding capabilities, molecular modeling characteristics and predicted absorption, distribution, metabolism, excretion, toxicity (ADMET) properties. Several hexa-peptides and tetra-peptides were finally synthesized to assay their inhibitory effects toward the recombinant human squalene synthase. The results demonstrated that the hexa-peptide FTACNW and tetra-peptide VACL can inhibit human squalene synthase effectively (with IC50 values near 100 µM) and may have potential to develop further as future hypocholesterolemia agents.

Epitope mapping of Mycoplasma hyopneumoniae using phage displayed peptide libraries and the immune responses of the selected phagotopes.

Phage display techniques have been widely employed to map the epitope structures which served as the basis for developing molecular vaccines. In the present study, we applied this technique to map the epitopes of Mycoplasma hyopneumoniae, the etiologic agent causing swine enzootic pneumonia, and evaluated directly the immune responses in mice of the selected phage-displayed epitopes (phagotopes). Two phage-displayed random peptide libraries were biopanned with the protein A-purified IgG of the rabbit anti-M. hyopneumoniae hyperimmune serum and the selected phage clones were sequenced and analyzed. Some of the inserts of the selected phagotopes showed a good match with the known proteins of M. hyopneumoniae. Others, which did not match with any known proteins, but shared extensive homology with each other, were clustered and classified as the conformational epitopes of M. hyopneumoniae. To evaluate the potential of using these phagotopes as effective vaccines, several phage clones were chosen to immunize mice. IgA coproantibody, IgA in bronchoalveolar lavage fluid and serum IgG responses were assayed. The serum raised by the phage clones clearly recognized several major mycoplasmal proteins indicating that the phagotope-induced immune responses were antigen-specific. The stronger IgG1 response revealed that the immune responses of the epitope-displaying phage were mainly through Th2 activation. The growth inhibition assay showed that the selected phage clones CS4 and varphi58 are potential vaccine candidates and suggested that the mycoplasmal 97 kDa, 56 kDa, 30 kDa and 23 kDa proteins may play important roles in the immune responses. The present work demonstrates that the whole epitope profile of a microorganism can be obtained through screening the phage displayed peptide libraries with the hyperimmune serum and reveals the potential of using epitope-displaying phages as peptide vaccines.

Exploration of natural product ingredients as inhibitors of human HMG-CoA reductase through structure-based virtual screening.

Cholesterol plays an important role in living cells. However, a very high level of cholesterol may lead to atherosclerosis. HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase is the key enzyme in the cholesterol biosynthesis pathway, and the statin-like drugs are inhibitors of human HMG-CoA reductase (hHMGR). The present study aimed to virtually screen for potential hHMGR inhibitors from natural product to discover hypolipidemic drug candidates with fewer side effects and lesser toxicities. We used the 3D structure 1HWK from the PDB (Protein Data Bank) database of hHMGR as the target to screen for the strongly bound compounds from the traditional Chinese medicine database. Many interesting molecules including polyphenolic compounds, polisubstituted heterocyclics, and linear lipophilic alcohols were identified and their ADMET (absorption, disrtibution, metabolism, excretion, toxicity) properties were predicted. Finally, four compounds were obtained for the in vitro validation experiments. The results indicated that curcumin and salvianolic acid C can effectively inhibit hHMGR, with IC50 (half maximal inhibitory concentration) values of 4.3 µM and 8 µM, respectively. The present study also demonstrated the feasibility of discovering new drug candidates through structure-based virtual screening.

Honokiol, a Lignan Biphenol Derived from the Magnolia Tree, Inhibits Dengue Virus Type 2 Infection.

Dengue is the most widespread arbovirus infection and poses a serious health and economic issue in tropical and subtropical countries. Currently no licensed vaccine or compounds can be used to prevent or manage the severity of dengue virus (DENV) infection. Honokiol, a lignan biphenol derived from the Magnolia tree, is commonly used in Eastern medicine. Here we report that honokiol has profound antiviral activity against serotype 2 DENV (DENV-2). In addition to inhibiting the intracellular DENV-2 replicon, honokiol was shown to suppress the replication of DENV-2 in baby hamster kidney (BHK) and human hepatocarcinoma Huh7 cells. At the maximum non-toxic dose of honokiol treatment, the production of infectious DENV particles was reduced >90% in BHK and Huh7 cells. The underlying mechanisms revealed that the expression of DENV-2 nonstructural protein NS1/NS3 and its replicating intermediate, double-strand RNA, was dramatically reduced by honokiol treatment. Honokiol has no effect on the expression of DENV putative receptors, but may interfere with the endocytosis of DENV-2 by abrogating the co-localization of DENV envelope glycoprotein and the early endosomes. These results indicate that honokiol inhibits the replication, viral gene expression, and endocytotic process of DENV-2, making it a promising agent for chemotherapy of DENV infection.

Plaque reduction test: an alternative method to assess specific antibody response to pIII-displayed peptide of filamentous phage M13.

Phage-displayed peptide systems have been used to identify the immunogenic epitopes and to develop the design of peptide-based or peptide-displaying phages themselves as vaccine candidates. To estimate the humoral immunity of phage-based vaccine, it is necessary to evaluate the antibody response specifically directed at the displayed peptide. Enzyme-linked immunosorbent assays (ELISAs) and Western blot analysis are commonly used for this purpose. However, using these methods, it is not easy to distinguish the antibody response against phage coat protein or the antibody response specific to the displayed peptide. The purified anti-Mycoplasma hyopneumoniae IgG was used to screen heptapeptides displaying on the pIII coat protein of M13 phage. Four selected phage clones were chosen to immunize mice. In order to evaluate the specific antibody response that is directed against heptapeptides, advantage was taken of the natural property of M13 phage to infect Escherichia coli, which is mediated by the pIII coat protein binding with the F pili of E. coli, and plaque reduction tests were performed to assess the specificity of antibody response. By comparing the number of plaques produced by the different phages (which are the same except for the displayed peptides) neutralized by the antiserum, we could demonstrate that the specificity of antibody response is directed against the peptide displayed on pIII coat protein. The results described here indicate that plaque reduction test is a convenient and more precise method to detect the antibody against the phage-displayed peptide.

Monoclonal antibodies for diagnosis of enterovirus 71.

Enterovirus 71 (EV71), one of the major causative agents of hand, foot, and mouth disease (HFMD), is now recognized as an emerging neurotropic virus in Asia and may cause severe neurologic complications and mortalities. Laboratory diagnosis of EV71 infection must be efficient and accurate, which could be accomplished by various immunoassays. In this study we use a live EV71 isolate, Tainan/4643/98, with genotype C2 as an immunogen to sensitize BALB/c (H-2(d)) mice and then generate the EV71-specific murine monoclonal antibodies. Five hybridoma clones were established and their monoclonal antibodies were characterized. All five clones are applicable in immunofluorescence staining but with different sensitivities-that is, MAbs 22, 24, and 27 were sensitive in IFA detection, and MAbs 22 and 24 were also confirmed in flow cytometry. None of these cross-reacted with coxsackievirus A16 (CVA16) or Echovirus type 6 (ECHO6), but each varied in binding to different EV71 subgenogroups (B1, B4, B5, C2, and C4). Western blot analysis revealed that all of these MAbs reacted with EV71 VP1 capsid proteins, and in addition MAbs 22 and 24 exhibited potent neutralizing activities against EV71 and protected cells from infection. Further, mapping the epitopes for each MAb revealed that only MAb 27 showed positive for the linear epitope DVIESSIGDSVSRAL, which was located at the N-terminus (a.a. 6-20) of EV71 VP1 and highly conserved among all EV71 subgenotypes. Thus, these MAbs may provide valuable tools for the laboratory diagnosis of EV71 infection and for vaccine development.

Xanthone derivatives could be potential antibiotics: virtual screening for the inhibitors of enzyme I of bacterial phosphoenolpyruvate-dependent phosphotransferase system.

The phosphoenolpyruvate phosphotransferase system (PTS) is ubiquitous in eubacteria and absent from eukaryotes. The system consists of two phosphoryl carriers, enzyme I (EI) and the histidine-containing phosphoryl carrier protein (HPr), and several PTS transporters, catalyzing the concomitant uptake and phosphorylation of several carbohydrates. Since a deficiency of EI in bacterial mutants lead to severe growth defects, EI could be a drug target to develop antimicrobial agents. We used the 3D structure PDB 1ZYM of Escherichia coli EI as the target to virtually screen the potential tight binders from NPPEDIA (Natural Product Encyclopedia), ZINC and Super Natural databases. These databases were screened using the docking tools of Discovery Studio 2.0 and the Integrated Drug Design System IDDS. Among the many interesting hits, xanthone derivatives with reasonably high Dock scores received more attentions. Two of the xanthone derivatives were obtained to examine their capabilities to inhibit cell growth of both Gram-positive and Gram-negative bacterial strains. The results indicate that they may exert the inhibition effects by blocking the EI activities. We have demonstrated for the first time that the xanthone derivatives have high potential to be developed as future antibiotics.

Proteomic analysis of the interactions between Mycoplasma hyopneumoniae and porcine tracheal ciliated cells.

Mycoplasma hyopneumoniae colonizes at the porcine respiratory-ciliated epithelial cells and causes the enzootic pneumonia of swine. The adhesion step is crucial in the colonization process. A few adhesion molecules have been characterized, and the concurrent receptors from the porcine ciliated cells have also been suggested to recognize the adhesion molecules. In the present study, the interactions between M. hyopneumoniae and porcine tracheal ciliated cells were investigated by employing the Far-Western blotting method. The results indicate that aconitase, lamin A/C, and peroxiredoxin of the porcine tracheal ciliated cell may interact specifically with the mycoplasmal proteins. We speculate that these mycoplasmal proteins could be secreted cleavage products, and their relative small size may enable them to penetrate into ciliated cells interfering with important metabolic pathways and other critical cellular processes.

Characterization of the interactions of lysozyme with DNA by surface plasmon resonance and circular dichroism spectroscopy.

Association with nucleic acid has been recognized as a unique role of lysozyme and may explain why lysozyme was called a killer protein against HIV infection. In the present study, we characterized the interactions of lysozyme and its derived peptides with a biotin-labeled pUC19 plasmid DNA. Real-time detection of the macromolecular interaction was performed using the SPR (surface plasmon resonance) spectroscopy. The SPR sensorgrams were analyzed and the association and dissociation rate constants as well as the dissociation equilibrium constant KD were, thus, estimated. The results reveal that other than the electrostatic interactions between the basic protein and the nucleotide sequences carrying negative charges, the specific DNA-binding motifs at the N- and C-termini of lysozyme were also involved in the interactions. The nonapeptide RAWVAWRNR (aa 107-115 of lysozyme) reported previously to block HIV-1 viral entrance and replication was also able to bind DNA with its KD value comparable to that of histones. The possibilities of ligand-binding-induced conformational changes were investigated using the circular dichroism spectroscopy. The CD spectra (200-320 nm) reveal that the conformational changes indeed occur as the spectra of lysozyme-DNA interactions are much less at the major trough region than the sum of individual spectra. The interaction of lysozyme with DNA molecules may interfere with DNA replication, modulate gene expression, and block bacterial and viral infections. These all suggest that human lysozyme may represent part of the innate immune system with a very broad protective spectrum.