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Oncotarget Oct 2015Preclinical evaluation of Retrocyclins (RC-100, RC-101) and Protegrin-1 (PG-1) antimicrobial peptides (AMPs) is important because of their therapeutic potential against...
Preclinical evaluation of Retrocyclins (RC-100, RC-101) and Protegrin-1 (PG-1) antimicrobial peptides (AMPs) is important because of their therapeutic potential against bacterial, fungal and viral infections. Human mast cells (HMCs) play important roles in host defense and wound healing but the abilities of retrocyclins and protegrin-1 to harness these functions have not been investigated. Here, we report that chemically synthesized RC-100 and PG-1 caused calcium mobilization and degranulation in HMCs but these responses were not blocked by an inhibitor of formyl peptide receptor-like 1 (FPRL1), a known receptor for AMPs. However, RC-100 and PG-1 induced degranulation in rat basophilic leukemia (RBL-2H3) cells stably expressing Mas related G protein coupled receptor X2 (MrgX2). Chemical synthesis of these AMPs is prohibitively expensive and post-synthesis modifications (cyclization, disulfide bonds, folding) are inadequate for optimal antimicrobial activity. Indeed, we found that synthetic RC-100, which caused mast cell degranulation via MrgX2, did not display any antimicrobial activity. Green-fluorescent protein (GFP)-tagged RC-101 (analog of RC-100) and GFP-tagged PG-1 purified from transgenic plant chloroplasts killed bacteria and induced mast cell degranulation. Furthermore, GFP-PG1 bound specifically to RBL-2H3 cells expressing MrgX2. These findings suggest that retrocyclins and protegrins activate HMCs independently of FPRL1 but via MrgX2. Harnessing this novel feature of AMPs to activate mast cell's host defense/wound healing properties in addition to their antimicrobial activities expands their clinical potential. Low cost production of AMPs in plants should facilitate their advancement to the clinic overcoming major hurdles in current production systems.
Topics: Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Bacteria; Calcium Signaling; Cell Degranulation; Cell Line, Tumor; Defensins; Dose-Response Relationship, Drug; Humans; Immunologic Factors; Mast Cells; Microbial Viability; Nerve Tissue Proteins; Plants, Genetically Modified; Rats; Receptors, G-Protein-Coupled; Receptors, Neuropeptide; Recombinant Fusion Proteins; Signal Transduction; Time Factors; Transfection
PubMed: 26378047
DOI: 10.18632/oncotarget.5611 -
Computational and Structural... 2023SARS-CoV-2 variants continue to spread throughout the world and cause waves of COVID-19 infections. It is important to find effective antiviral drugs to combat...
BACKGROUND
SARS-CoV-2 variants continue to spread throughout the world and cause waves of COVID-19 infections. It is important to find effective antiviral drugs to combat SARS-CoV-2 and its variants. The main protease (M) of SARS-CoV-2 is a promising therapeutic target due to its crucial role in viral replication and its conservation in all the variants. Therefore, the aim of this work was to identify an effective inhibitor of M.
METHODS
We studied around 200 antimicrobial peptides using methods including molecular docking and allergenicity and toxicity prediction. One selected antiviral peptide was studied experimentally using a Bioluminescence Resonance Energy Transfer (BRET)-based M biosensor, which reports M activity through a decrease in energy transfer.
RESULTS
Molecular docking identified one natural antimicrobial peptide, Protegrin-2, with high binding affinity and stable interactions with M allosteric residues. Furthermore, free energy calculations and molecular dynamics simulation illustrated a high affinity interaction between the two. We also determined the impact of the binding of Protegrin-2 to M using a BRET-based assay, showing that it inhibits the proteolytic cleavage activity of M.
CONCLUSIONS
Our and experimental studies identified Protegrin-2 as a potent inhibitor of M that could be pursued further towards drug development against COVID-19 infection.
PubMed: 37576748
DOI: 10.1016/j.csbj.2023.07.020 -
Molecules (Basel, Switzerland) Aug 2022Glioblastoma (GBM) is one of the most aggressive and lethal malignancy of the central nervous system. Temozolomide is the standard of care for gliomas, frequently...
Anticancer Effect of Cathelicidin LL-37, Protegrin PG-1, Nerve Growth Factor NGF, and Temozolomide: Impact on the Mitochondrial Metabolism, Clonogenic Potential, and Migration of Human U251 Glioma Cells.
Glioblastoma (GBM) is one of the most aggressive and lethal malignancy of the central nervous system. Temozolomide is the standard of care for gliomas, frequently results in resistance to drug and tumor recurrence. Therefore, further research is required for the development of effective drugs in order to guarantee specific treatments to succeed. The aim of current study was to investigate the effects of nerve growth factor (NGF), human cathelicidin (LL-37), protegrin-1 (PG-1), and temozolomide on bioenergetic function of mitochondria, clonogenicity, and migration of human U251 glioma cells. Colony formation assay was used to test the ability of the glioma cells to form colonies in vitro. The U251 glioma cells migration was evaluated using wound-healing assay. To study the mitochondrial metabolism in glioma cells we measured oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) using a Seahorse XF cell Mito stress test kit and Seahorse XF cell Glycolysis stress kit, respectively. We revealed that LL-37, NGF, and TMZ show strong anti-tumorigenic activity on GMB. LL-37 (4 μM), TMZ (155 μM), and NGF (7.55 × 10 μM) inhibited 43.9%-60.3%, 73.5%-81.3%, 66.2% the clonogenicity of glioma U251 cells for 1-2 days, respectively. LL-37 (4 μM), and NGF (7.55 × 10 μM) inhibited the migration of U251 glioma cells on the third and fourth days. TMZ also inhibited the migration of human glioma U251 cells over 1-3 days. In contrast, PG-1 (16 μM) stimulated the migration of U251 glioma cells on the second, fourth, and sixth days. Anti-mitogenic and anti-migration activities of NGF, LL-37, and TMZ maybe are relation to their capacity to reduce the basal OCR, ATP-synthetase, and maximal respiration of mitochondria in human glioma U251 cells. Glycolysis, glycolytic capacity and glycolytic spare in glioma U251 cells haven`t been changed under the effect of NGF, LL-37, PG-1, and TMZ in regard to control level. Thus, LL-37 and NGF inhibit migration and clonogenicity of U251 glioma cells, which may indicate that these compounds have anti-mitogenic and anti-migration effects on human glioma cells. The study of the mechanisms of these effects may contribute in the future to the use of NGF and LL-37 as therapeutic agents for gliomas.
Topics: Antimicrobial Cationic Peptides; Antineoplastic Agents, Alkylating; Apoptosis; Brain Neoplasms; Cell Line, Tumor; Dacarbazine; Drug Resistance, Neoplasm; Glioma; Humans; Mitochondria; Neoplasm Recurrence, Local; Nerve Growth Factor; Temozolomide; Cathelicidins
PubMed: 35956937
DOI: 10.3390/molecules27154988 -
The Journal of Membrane Biology Jun 2020Protegrin-1 (PG-1), an 18-residue β-hairpin stabilized by two disulfide bonds, is a member of a family of powerful antimicrobial peptides which are believed to act...
Protegrin-1 (PG-1), an 18-residue β-hairpin stabilized by two disulfide bonds, is a member of a family of powerful antimicrobial peptides which are believed to act through membrane permeabilization. Here we used a combination of experimental and computational approaches to characterize possible structural arrangements of PG-1 in lipid bilayers mimicking bacterial membranes. We have measured the dose-response function of the PG-1-induced leakage of markers of various sizes from vesicles and found it to be consistent with the formation of pores of two different sizes. The first one allows the release of small dyes and occurs at peptide:lipid ratios < 0.006. Above this ratio, larger pores are observed through which the smallest of dextrans FD4 can be released. In parallel with pore formation, we observe a general large-scale destabilization of vesicles which is probably related to complete rupture of some vesicles. The population of vesicles that are completely ruptured depends linearly on PG-1:lipid ratio. Neither pore size, nor vesicle rupture are influenced by the formation of disulfide bonds. Previous computational work on oxidized protegrin is complemented here by all-atom MD simulations of PG-1 with reduced disulfide bonds both in solution (monomer) and in a bilayer (dimer and octamer). The simulations provide molecular insights into the influence of disulfide bonds on peptide conformation, aggregation, and oligomeric structure.
Topics: Algorithms; Antimicrobial Cationic Peptides; Lipid Bilayers; Models, Molecular; Models, Theoretical; Molecular Conformation; Structure-Activity Relationship
PubMed: 32500172
DOI: 10.1007/s00232-020-00124-3 -
International Journal of Molecular... Apr 2022Global rise of infections and deaths caused by drug-resistant bacterial pathogens are among the unmet medical needs. In an age of drying pipeline of novel antibiotics to... (Review)
Review
Global rise of infections and deaths caused by drug-resistant bacterial pathogens are among the unmet medical needs. In an age of drying pipeline of novel antibiotics to treat bacterial infections, antimicrobial peptides (AMPs) are proven to be valid therapeutics modalities. Direct in vivo applications of many AMPs could be challenging; however, works are demonstrating encouraging results for some of them. In this review article, we discussed 3-D structures of potent AMPs e.g., polymyxin, thanatin, MSI, protegrin, OMPTA in complex with bacterial targets and their mode of actions. Studies on human peptide LL37 and de novo-designed peptides are also discussed. We have focused on AMPs which are effective against drug-resistant Gram-negative bacteria. Since treatment options for the infections caused by super bugs of Gram-negative bacteria are now extremely limited. We also summarize some of the pertinent challenges in the field of clinical trials of AMPs.
Topics: Anti-Bacterial Agents; Antimicrobial Peptides; Bacteria; Gram-Negative Bacteria; Humans
PubMed: 35562950
DOI: 10.3390/ijms23094558 -
Pharmaceutics Jul 2023Protegrin-1 (PG-1) is a cationic β-hairpin pore-forming antimicrobial peptide having a membranolytic mechanism of action. It possesses in vitro a potent antimicrobial...
Protegrin-1 (PG-1) is a cationic β-hairpin pore-forming antimicrobial peptide having a membranolytic mechanism of action. It possesses in vitro a potent antimicrobial activity against a panel of clinically relevant MDR ESKAPE pathogens. However, its extremely high hemolytic activity and cytotoxicity toward mammalian cells prevent the further development of the protegrin-based antibiotic for systemic administration. In this study, we rationally modulated the PG-1 charge and hydrophobicity by substituting selected residues in the central β-sheet region of PG-1 to design its analogs, which retain a high antimicrobial activity but have a reduced toxicity toward mammalian cells. In this work, eight PG-1 analogs with single amino acid substitutions and five analogs with double substitutions were obtained. These analogs were produced as thioredoxin fusions in . It was shown that a significant reduction in hemolytic activity without any loss of antimicrobial activity could be achieved by a single amino acid substitution, V16R in the -terminal β-strand, which is responsible for the PG-1 oligomerization. As the result, a selective analog with a ≥30-fold improved therapeutic index was obtained. FTIR spectroscopy analysis of analog, [V16R], revealed that the peptide is unable to form oligomeric structures in a membrane-mimicking environment, in contrast to wild-type PG-1. Analog [V16R] showed a reasonable efficacy in septicemia infection mice model as a systemic antibiotic and could be considered as a promising lead for further drug design.
PubMed: 37631261
DOI: 10.3390/pharmaceutics15082047 -
Frontiers in Pharmacology 2019Cathelicidins, a class of antimicrobial peptides, have been widely studied for their antimicrobial role in innate immune responses during infection and inflammation. At...
Cathelicidins, a class of antimicrobial peptides, have been widely studied for their antimicrobial role in innate immune responses during infection and inflammation. At sub-antimicrobial concentrations, various cathelicidins from different species have been reported to exert chemotactic activity on neutrophils, monocytes, dendritic cells and T-cells, and also enhance angiogenesis and wound healing. To date, the role of the pig cathelicidin, protegrin-1 (PG-1), in immune modulation and tissue repair in the intestinal tract has not been investigated. The aim of the present study was to examine the potential protective effects of recombinant PG-1 in a mouse dextran sodium sulfate (DSS)-induced colitis inflammation model. This is the first report showing the protective effects of PG-1 in its various forms (pro-, cathelin-, and mature-forms) in attenuating significant body weight loss associated with DSS-induced colitis ( < 0.05). PG-1 treatment improved histological scores ( < 0.05) and influenced the gene expression of inflammatory mediators and tissue repair factors such as trefoil factor 3 (TFF3) and mucin (MUC-2). Protegrin treatment also altered the metabolite profile, returning the metabolite levels closer to untreated control levels. These findings lay the foundation for future oral application of recombinant PG-1 to potentially treat intestinal damage and inflammation.
PubMed: 30873029
DOI: 10.3389/fphar.2019.00156 -
Journal of Peptide Science : An... Apr 2017Protegrin-1 is a widely studied 18-residue β-hairpin antimicrobial peptide. Evidence suggests that it acts via a β-barrel pore formation mechanism, but the exact...
Protegrin-1 is a widely studied 18-residue β-hairpin antimicrobial peptide. Evidence suggests that it acts via a β-barrel pore formation mechanism, but the exact number of peptides comprising the pore state is unknown. In this study, we performed molecular dynamics simulations of β-barrels of protegrin and three related mutants (v14v16l, v14v16a, and r4n) in NCNC parallel topology in implicit membrane pores of varying radius and curvature for oligomeric numbers 6-14. We then identified the optimal pore radius and curvature values for all constructs and determined the total effective energy and the translational and rotational entropic losses. These, along with an estimate of membrane deformation free energy from experimental line tension values, provided an estimate of the overall energetics of formation of each pore state. The results indicated that oligomeric numbers 7-13 are generally stable, allowing the possibility of a heterogeneous pore state. The optimal oligomeric state for protegrin is the nonamer, shifting to higher numbers for the mutants. Protegrin, v14v16l, and r4n are stable as membrane-inserted β-barrels, but v14v16a seems much less so because of its decreased hydrophobicity. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.
Topics: Antimicrobial Cationic Peptides; Molecular Dynamics Simulation
PubMed: 28382709
DOI: 10.1002/psc.2992 -
International Journal of Molecular... Aug 2021Infectious intestinal colitis, manifesting as intestinal inflammation, diarrhea, and epithelial barrier disruption, affects millions of humans worldwide and, without...
Infectious intestinal colitis, manifesting as intestinal inflammation, diarrhea, and epithelial barrier disruption, affects millions of humans worldwide and, without effective treatment, can result in death. In addition to this, the significant rise in antibiotic-resistant bacteria poses an urgent need for alternative anti-infection therapies for the treatment of intestinal disorders. Antimicrobial peptides (AMPs) are potential therapies that have broad-spectrum antimicrobial activity due to their (1) unique mode of action, (2) broad-spectrum antimicrobial activity, and (3) protective role in GI tract maintenance. Protegrin-1 (PG-1) is an AMP of pig origin that was previously shown to reduce the pathological effects of chemically induced digestive tract inflammation (colitis) and to modulate immune responses and tissue repair. This study aimed to extend these findings by investigating the protective effects of PG-1 on pathogen-induced colitis in an infection study over a 10-day experimental period. The oral administration of PG-1 reduced intestinal infection in mice as evidenced by reduced histopathologic change in the colon, prevention of body weight loss, milder clinical signs of disease, and more effective clearance of bacterial infection relative to challenged phosphate-buffered saline (PBS)-treated mice. Additionally, PG-1 treatment altered the expression of various inflammatory mediators during infection, which may act to resolve inflammation and re-establish intestinal homeostasis. PG-1 administered in its mature form was more effective relative to the pro-form (ProPG-1). To our knowledge, this is the first study demonstrating the protective effects of PG-1 on infectious colitis.
Topics: Animals; Anti-Inflammatory Agents; Antimicrobial Cationic Peptides; Citrobacter rodentium; Colitis; Colon; Cytokines; Disease Models, Animal; Enterobacteriaceae Infections; Female; Inflammation; Intestinal Mucosa; Intestines; Mice; Mice, Inbred C57BL; Pore Forming Cytotoxic Proteins
PubMed: 34502403
DOI: 10.3390/ijms22179494 -
The Journal of Physical Chemistry. B Oct 2017Protegrin-1 is an 18-residue β-hairpin antimicrobial peptide (AMP) that has been suggested to form transmembrane β-barrels in biological membranes. However,...
Protegrin-1 is an 18-residue β-hairpin antimicrobial peptide (AMP) that has been suggested to form transmembrane β-barrels in biological membranes. However, alternative structures have also been proposed. Here, we performed multimicrosecond, all-atom molecular dynamics simulations of various protegrin-1 oligomers on the membrane surface and in transmembrane topologies. The membrane surface simulations indicated that protegrin dimers are stable, whereas trimers and tetramers break down. Tetrameric arcs remained stably inserted in lipid membranes, but the pore water was displaced by lipid molecules. Unsheared protegrin β-barrels opened into β-sheets that surrounded stable aqueous pores, whereas tilted barrels with sheared hydrogen bonding patterns were stable in most topologies. A third type of observed pore consisted of multiple small oligomers surrounding a small, partially lipidic pore. We also considered the β-hairpin AMP tachyplesin, which showed less tendency to oligomerize than protegrin: the octameric bundle resulted in small pores surrounded by six peptides as monomers and dimers, with some peptides returning to the membrane surface. The results imply that multiple configurations of protegrin oligomers may produce aqueous pores and illustrate the relationship between topology and putative steps in protegrin-1's pore formation. However, the long-term stability of these structures needs to be assessed further.
Topics: Anti-Infective Agents; Antimicrobial Cationic Peptides; Cell Membrane; Cell-Penetrating Peptides; Models, Biological; Peptides
PubMed: 28879767
DOI: 10.1021/acs.jpcb.7b06591