Protegrin-1 Inhibits Dengue NS2B-NS3 Serine Protease and Viral Replication in MK2 Cells

Dengue diseases have an economic as well as social burden worldwide. In this study, the antiviral activity of protegrin-1 (PG-1, RGGRLCYCRRRFCVCVGR) peptide towards dengue NS2B-NS3pro and viral replication in Rhesus monkey kidney (MK2) cells was investigated. The peptide PG-1 was synthesized by solid-phase peptide synthesis, and disulphide bonds formation followed by peptide purification was confirmed by LC-MS and RPHPLC. Dengue NS2B-NS3pro was produced as a single-chain recombinant protein inE. coli. The NS2B-NS3pro assay was carried out by measuring the florescence emission of catalyzed substrate. Real-time PCR was used to evaluate the inhibition potential of PG-1 towards dengue serotype-2 (DENV-2) replication in MK2 cells. The results showed that PG-1 inhibited dengue NS2B-NS3pro at IC50of 11.7 μM. The graded concentrations of PG-1 at nontoxic range were able to reduce viral replication significantly (P<0.001) at 24, 48, and 72 hrs after viral infection. However, the percentage of inhibition was significantly (P<0.01) higher at 24 hrs compared to 48 and 72 hrs. These data show promising therapeutic potential of PG-1 against dengue infection, hence it warrants further analysis and improvement of the peptide features as a prospective starting point for consideration in designing attractive dengue virus inhibitors.

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Human Monoclonal scFvs that Neutralize Fribrinogenolytic Activity of Kaouthiagin, a Zinc-Metalloproteinase in Cobra (Naja kaouthia) Venom

Toxins ◽

10.3390/toxins10120509 ◽

2018 ◽

Vol 10 (12) ◽

pp. 509

Author(s):

Jirawat Khanongnoi ◽

Siratcha Phanthong ◽

Onrapak Reamtong ◽

Anchalee Tungtronchitr ◽

Wanpen Chaicumpa ◽

...

Keyword(s):

Therapeutic Potential ◽

Gel Filtration ◽

Peptide Bond ◽

Phage Display Library ◽

Single Step ◽

Single Chain ◽

Single Chain Antibody ◽

Venom Component ◽

E Coli ◽

Naja Kaouthia

Snake venom-metalloproteinases (SVMPs) are the primary factors that disturb hemostasis and cause hemorrhage in the venomous snake bitten subjects. Kaouthiagin is a unique SVMP that binds and cleaves von Willebrand factor (vWF) at a specific peptide bond leading to inhibition of platelet aggregation, which enhances the hemorrhage. Kaouthiagin is a low abundant venom component of Thai cobra (Naja kaouthia); thus, most horse-derived antivenins used for cobra bite treatment do not contain adequate anti-kaouthiagin. This study aimed to produce human single-chain antibody variable fragments (HuscFvs) that bind to and interfere with kaouthiagin activity for further clinical use. Kaouthiagin was purified from N. kaouthia-holovenom by a single-step gel-filtration chromatography. The purified venom component was used in phage-biopanning to select the kaouthiagin-bound HuscFv-displayed-phage clones from a HuscFv-phage display library. The selected phages were used to infect Escherichia coli bacteria. Soluble HuscFvs expressed by three phage-transformed-E. coli clones interfered with cobra kaouthiagin binding to human vWF. Computerized simulation indicated that HuscFv of two phage-transformed E. coli clones formed contact interface with kaouthiagin residues at or near catalytic site and effectively inhibited fibrinogenolytic activity of the kaouthiagin. The HuscFvs have therapeutic potential as an adjunct of antivenins in treatment of bleeding caused by venomous snakebites.

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Peptide Design Considerations

Synthetic Peptides ◽

10.1093/oso/9780195132618.003.0005 ◽

2002 ◽

Author(s):

Michael L. Moore ◽

Gregory A. Grant

Keyword(s):

Therapeutic Potential ◽

Solid Phase ◽

Proteolytic Enzymes ◽

Solid Phase Peptide Synthesis ◽

Biological Action ◽

Hiv Protease ◽

Affinity Labels ◽

Fibrinogen Receptor ◽

Routine Basis ◽

Synthetic Antigens

Peptides have become an increasingly important class of molecules in biochemistry, medicinal chemistry, and physiology. Many naturally occurring, physiologically relevant peptides function as hormones, neurotransmitters, cytokines, and growth factors. Peptide analogs that possess agonist or antagonist activity are useful as tools to study the biochemistry, physiology, and pharmacology of these peptides, to characterize their receptor(s), and to study their biosynthesis, metabolism, and degradation. Radiolabeled analogs and analogs bearing affinity labels have been used for receptor characterization and isolation. Peptide substrates of proteases, kinases, phosphatases, and aminoacyl or glycosyl transferases are used to study enzyme kinetics, mechanism of action, and biochemical and physiological roles and to aid in the isolation of enzymes and in the design of inhibitors. Peptides are also used as synthetic antigens for the preparation of polyclonal or monoclonal antibodies targeted to specific sequences. Epitope mapping with synthetic peptides can be used to identify specific antigenic peptides for the preparation of synthetic vaccines, to determine protein sequence regions that are important for biological action, and to design small peptide mimetics of protein structure or function. A number of peptide hormones or analogs thereof, including arginine vasopressin, oxytocin, luteinizing hormone releasing hormone (LHRH), adrenocorticotropic hormone (ACTH), and calcitonin, have already found use as therapeutic agents, and many more are being investigated actively. Peptide-based inhibitors of proteolytic enzymes, such as angiotensin converting enzyme (ACE) and human immunodeficiency virus (HIV) protease, have widespread clinical use, and inhibitors of renin and elastase are also being investigated for therapeutic use. Finally, peptides designed to block the interaction of protein molecules by mimicking the combining site of one of the proteins, such as the fibrinogen receptor antagonists, show great therapeutic potential as well. With the development of solid-phase peptide synthesis by Bruce Merrifield (1963) and the optimization of supports, protecting groups, and coupling and deprotection chemistries by a large number of researchers, it has become possible to obtain useful amounts of peptides on a more or less routine basis.

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Preparation of single-chain Fv antibodies in the cytoplasm of Escherichia coli by simplified and systematic chaperone optimization

The Journal of Biochemistry ◽

10.1093/jb/mvz059 ◽

2019 ◽

Vol 166 (6) ◽

pp. 455-462 ◽

Cited By ~ 3

Author(s):

Chenjiang Liu ◽

Yoshihiro Kobashigawa ◽

Soichiro Yamauchi ◽

Yuya Toyota ◽

Manaka Teramoto ◽

...

Keyword(s):

Escherichia Coli ◽

Tertiary Structure ◽

Guanidine Hydrochloride ◽

Molecular Size ◽

Soluble Form ◽

Single Chain ◽

Variable Regions ◽

E Coli ◽

Disulphide Bonds ◽

Single Chain Fv

Abstract A single-chain variable fragment (scFv) antibody is a recombinant protein in which a peptide linker connects the variable regions of the heavy chain and light chain. Due to its smaller molecular size, an scFv can be expressed using Escherichia coli. The presence of two disulphide bonds in the molecule often prevents expression of correctly folded scFv in the E. coli cytoplasm, making a refolding process necessary to regenerate scFv activity. The refolding process is time-consuming and requires large amounts of expensive reagents, such as guanidine hydrochloride, l-arginine and glutathione. Here, to conveniently obtain scFv proteins, we devised a simple and systematic method to optimize the co-expression of chaperone proteins and to combine them with specially engineered E. coli strains that permit the formation of stable disulphide bonds within the cytoplasm. Several scFv proteins were successfully obtained in a soluble form from E. coli cytoplasm. Thermal denaturation experiments and/or surface plasmon resonance measurements revealed that the thus-obtained scFvs possessed a stable tertiary structure and antigen-binding activity. The combined use of engineered E. coli with the simplified and systematic chaperone optimization can be useful for the production of scFv proteins.

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C-Terminal Modifications Broaden Activity of the Proline-Rich Antimicrobial Peptide, Chex1-Arg20

Australian Journal of Chemistry ◽

10.1071/ch15169 ◽

2015 ◽

Vol 68 (9) ◽

pp. 1373 ◽

Cited By ~ 9

Author(s):

Wenyi Li ◽

Julien Tailhades ◽

M. Akhter Hossain ◽

Neil M. O'Brien-Simpson ◽

Eric C. Reynolds ◽

...

Keyword(s):

Antibacterial Activity ◽

Antimicrobial Peptide ◽

Peptide Synthesis ◽

Mammalian Cells ◽

Solid Phase ◽

Solid Phase Peptide Synthesis ◽

Gram Negative Bacteria ◽

Gram Negative ◽

E Coli ◽

Chemical Strategies

A series of N- and C-terminal modifications of the monomeric proline-rich antimicrobial peptide, Chex1-Arg20, was obtained via different chemical strategies using Fmoc/tBu solid-phase peptide synthesis in order to study their effects on a panel of Gram-negative bacteria. In particular, C-terminal modifications with hydrazide or alcohol functions extended their antibacterial activity from E. coli and K. pneumoniae to other Gram-negative species, A. baumannii and P. aeruginosa. Furthermore, these analogues did not show cytotoxicity towards mammalian cells. Hence, such modifications may aid in the development of more potent proline-rich antimicrobial peptides with a greater spectrum of activity against Gram-negative bacteria than the parent peptide.

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Total Synthesis and Antimicrobial Evaluation of Pagoamide A

Frontiers in Chemistry ◽

10.3389/fchem.2021.741290 ◽

2021 ◽

Vol 9 ◽

Author(s):

Cheng-Han Wu ◽

John Chu

Keyword(s):

Total Synthesis ◽

Solid Phase ◽

Solid Phase Peptide Synthesis ◽

Building Blocks ◽

Synthetic Methods ◽

Marine Natural Product ◽

Moderate Activity ◽

Starting Point ◽

Antimicrobial Evaluation ◽

Synthetic Sequence

Natural products are often the starting point for drug development and also the testing ground for synthetic methods. Herein we describe the total synthesis and antimicrobial evaluation of a marine natural product, pagoamide A, which is a macrocyclic depsipeptide with two backbone thiazole units and a dimethylated N-terminus. The two thiazole building blocks were synthesized from commercially available materials in four or fewer steps and employed directly in solid-phase peptide synthesis (SPPS) to afford pagoamide A. The use of SPPS ensured that the synthetic sequence is operationally straightforward and, if needed, permits modular substitution of building blocks to easily access diverse structural analogs. Our antimicrobial assays showed that pagoamide A has moderate activity against Bacillus subtilis.

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Synthesis of a tumorspecific single-chain antibody and derivatives in E. coli and COS-1 cells

Immunology Letters ◽

10.1016/s0165-2478(97)87603-1 ◽

1997 ◽

Vol 56 (1-3) ◽

pp. 191

Author(s):

S Dincq

Keyword(s):

Single Chain ◽

Single Chain Antibody ◽

E Coli

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Co-expression of recombinant single chain variable fragment recognizing blood antigen fused with sumo and chaperones in Escherichia coli

ACADEMIA JOURNAL OF BIOLOGY ◽

10.15625/2615-9023/v40n4.11689 ◽

2018 ◽

Vol 40 (4) ◽

Author(s):

Dang Thi Ngoc Ha ◽

Le Thi Thu Hong ◽

Truong Nam Hai

Keyword(s):

Recombinant Antibody ◽

Blood Type ◽

Soluble Form ◽

Supernatant Fraction ◽

Blood Group Antigens ◽

Single Chain ◽

E Coli ◽

Recombinant Scfv ◽

Single Chain Variable Fragments

Single chain variable fragments (scFv) have widely been used in research, diagnosis and treatment, but the scFv is considered as difficult protein for expression in E. coli. In previous studies, we expressed a construction of recombinant single chain variable fragments again antigen specific for blood type A (antiA-scFv) individually or fused with Trx or SUMO. However, soluble fraction was low abandant and only approximately 40% when fused with Trx, the other cases were expressed in form of inclusion body. Therefore, it was difficult for purification, refolding and activity assesment. In thispaper, we demonstrated a suitable construction for soluble production of antiA-scFv fused with SUMO (SM/antiA-scFv) in presence of chaparones. Under fermentation with 0.1 mM IPTG at 20oC, the SM/antiA-scFv was entirely expressed in soluble form. Importantly, after cleavage from SUMO with SUMOprotease, antiA-scFv was still maintained in the supernatant fraction. Therefore, it can help ensure bioactivity and is useful for purification process. To the best of our knowledge, this is the first report showing soluble recombinant scFv fused with SUMO in presence of chaperone for determination of blood group antigens. Thus, this result facilitates the optimal study of soluble expression, purification and bioactivity determination of the antiA-scFv recombinant antibody.

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A Strategy for Thioamide Incorporation During Fmoc Solid-Phase Peptide Synthesis with Robust Stereochemical Integrity

10.26434/chemrxiv.8206247.v1 ◽

2019 ◽

Cited By ~ 1

Author(s):

luis camacho III ◽

Bryan J. Lampkin ◽

Brett VanVeller

Keyword(s):

Amino Acid ◽

Functional Groups ◽

Peptide Synthesis ◽

Solid Phase ◽

Solid Phase Peptide Synthesis ◽

Side Chains ◽

Amino Acid Side Chains ◽

Peptide Elongation

We describe a method to protect the sensitive stereochemistry of the thioamide—in analogy to the protection of the functional groups of amino acid side chains—in order to preserve the thioamide moiety during peptide elongation.<br>

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Antimicrobial Activity of Silver Nanoparticles Prepared Under an Ultrasonic Field

International Journal of Pharmaceutical Sciences and Nanotechnology ◽

10.37285/ijpsn.2011.4.3.8 ◽

2011 ◽

Vol 4 (3) ◽

pp. 1491-1496

Author(s):

Umadevi M ◽

Rani T ◽

Balakrishnan T ◽

Ramanibai R

Keyword(s):

Escherichia Coli ◽

Antimicrobial Activity ◽

Silver Nanoparticles ◽

Reduction Method ◽

Therapeutic Potential ◽

Chemical Reduction ◽

Ultrasonic Field ◽

Great Promise ◽

Chemical Reduction Method ◽

E Coli

Nanotechnology has great promise for improving the therapeutic potential of medicinal molecules and related agents. In this study, silver nanoparticles of different sizes were synthesized in an ultrasonic field using the chemical reduction method with sodium borohydride as a reducing agent. The size effect of silver nanoparticles on antimicrobial activity were tested against the microorganisms Staphylococcus aureus (MTCC No. 96), Bacillus subtilis (MTCC No. 441), Streptococcus mutans (MTCC No. 497), Escherichia coli (MTCC No. 739) and Pseudomonas aeruginosa (MTCC No. 1934). The results shows that B. subtilis, and E. coli were more sensitive to silver nanoparticles and its size, indicating the superior antimicrobial efficacy of silver nanoparticles.

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