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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 -
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 -
International Journal of Molecular... 2012Antimicrobial peptides (AMPs) are naturally-occurring molecules that exhibit strong antibiotic properties against numerous infectious bacterial strains. Because of their... (Review)
Review
Antimicrobial peptides (AMPs) are naturally-occurring molecules that exhibit strong antibiotic properties against numerous infectious bacterial strains. Because of their unique mechanism of action, they have been touted as a potential source for novel antibiotic drugs. We present a summary of computational investigations in our lab aimed at understanding this unique mechanism of action, in particular the development of models that provide a quantitative connection between molecular-level biophysical phenomena and relevant biological effects. Our work is focused on protegrins, a potent class of AMPs that attack bacteria by associating with the bacterial membrane and forming transmembrane pores that facilitate the unrestricted transport of ions. Using fully atomistic molecular dynamics simulations, we have computed the thermodynamics of peptide-membrane association and insertion, as well as peptide aggregation. We also present a multi-scale analysis of the ion transport properties of protegrin pores, ranging from atomistic molecular dynamics simulations to mesoscale continuum models of single-pore electrodiffusion to models of transient ion transport from bacterial cells. Overall, this work provides a quantitative mechanistic description of the mechanism of action of protegrin antimicrobial peptides across multiple length and time scales.
Topics: Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Cell Membrane; Gram-Negative Bacteria; Ion Channels; Ion Transport; Microbial Sensitivity Tests; Models, Molecular; Models, Theoretical; Molecular Dynamics Simulation; Thermodynamics
PubMed: 23109834
DOI: 10.3390/ijms130911000 -
Molecular Pharmaceutics Apr 2012More than two dozen clinical syndromes known as amyloid diseases are characterized by the buildup of extended insoluble fibrillar deposits in tissues. These amorphous... (Review)
Review
More than two dozen clinical syndromes known as amyloid diseases are characterized by the buildup of extended insoluble fibrillar deposits in tissues. These amorphous Congo red staining deposits known as amyloids exhibit a characteristic green birefringence and cross-β structure. Substantial evidence implicates oligomeric intermediates of amyloids as toxic species in the pathogenesis of these chronic disease states. A growing body of data has suggested that these toxic species form ion channels in cellular membranes causing disruption of calcium homeostasis, membrane depolarization, energy drainage, and in some cases apoptosis. Amyloid peptide channels exhibit a number of common biological properties including the universal U-shape β-strand-turn-β-strand structure, irreversible and spontaneous insertion into membranes, production of large heterogeneous single-channel conductances, relatively poor ion selectivity, inhibition by Congo red, and channel blockade by zinc. Recent evidence has suggested that increased amounts of amyloids not only are toxic to its host target cells but also possess antimicrobial activity. Furthermore, at least one human antimicrobial peptide, protegrin-1, which kills microbes by a channel-forming mechanism, has been shown to possess the ability to form extended amyloid fibrils very similar to those of classic disease-forming amyloids. In this paper, we will review the reported antimicrobial properties of amyloids and the implications of these discoveries for our understanding of amyloid structure and function.
Topics: Amyloid; Amyloid beta-Peptides; Animals; Anti-Infective Agents; Electrophysiology; Humans
PubMed: 22081976
DOI: 10.1021/mp200419b -
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 Physical Chemistry. B Mar 2010The free energies of adsorption of the monomer or dimer of the cationic beta-hairpin antimicrobial peptide protegrin-1 (PG1) in a specific binding orientation on a lipid...
The free energies of adsorption of the monomer or dimer of the cationic beta-hairpin antimicrobial peptide protegrin-1 (PG1) in a specific binding orientation on a lipid bilayer are determined using molecular dynamics (MD) simulations and Poisson-Boltzmann calculations. The bilayer is composed of anionic palmitoyl-oleoyl-phosphatidylglycerol (POPG) and palmitoyl-oleoyl-phosphatidylethanolamine (POPE) with ratio 1:3 (POPG/POPE). PG1 is believed to kill bacteria by binding on their membranes. There, it forms pores that lyse the bacteria. Herein we focus on the thermodynamics of binding. In particular, we explore the role of counterion release from the lipid bilayer upon adsorption of either the monomeric or the dimeric form of PG1. Twenty-two 4-ns-long MD trajectories of equilibrated systems are generated to determine the free energy profiles for the monomer and dimer as a function of the distance between the peptide(s) and the membrane surface. The MD simulations are conducted at 11 different separations from the membrane for each of the two systems, one with PG1, the second with a PG1 dimer of only a specific orientation of the monomer and dimer without taking into account the change of entropy for the peptide. To calculate the potential of mean force for each peptide/membrane system, a variant of constrained MD and thermodynamic integration is used. We observed that PG1 dimer binds more favorably to the POPG/POPE membrane. A simple method for relating the free energy profile to the PG1-membrane binding constant is employed to predict a free energy of adsorption of -2.4 +/- 0.8 kcal/mol. A corresponding PG1-dimer-membrane binding constant is calculated as -3.5 +/- 1.1 kcal/mol. Free energy profiles from MD simulation were extensively analyzed and compared with results of Poisson-Boltzmann theory. We find the peptide-membrane attraction to be dominated by the entropy increase due to the release of counterions in a POPG/POPE lipid bilayer.
Topics: Adsorption; Antimicrobial Cationic Peptides; Dimerization; Lipid Bilayers; Models, Chemical; Molecular Dynamics Simulation; Phosphatidylethanolamines; Phosphatidylglycerols; Thermodynamics
PubMed: 20136112
DOI: 10.1021/jp909640g -
European Journal of Biochemistry Feb 2002Dendrimeric peptides selective for microbial surfaces have been developed to achieve broad antimicrobial activity and low hemolytic activity to human erythrocytes. The...
Dendrimeric peptides selective for microbial surfaces have been developed to achieve broad antimicrobial activity and low hemolytic activity to human erythrocytes. The dendrimeric core is an asymmetric lysine branching tethered with two to eight copies of a tetrapeptide (R4) or an octapeptide (R8). The R4 tetrapeptide (RLYR) contains a putative microbial surface recognition BHHB motif (B = basic, H = hydrophobic amino acid) found in protegrins and tachyplesins whereas the octapeptide R8 (RLYRKVYG) consists of an R4 and a degenerated R4 repeat. Antimicrobial assays against 10 organisms in high- and low-salt conditions showed that the R4 and R8 monomers as well as their divalent dendrimers contain no to low activity. In contrast, the tetra- and octavalent R4 and R8 dendrimers are broadly active under either conditions, exhibiting relatively similar potency with minimal inhibition concentrations < 1 microm against both bacteria and fungi. Based on their size and charge similarities, the potency and activity spectrum of the tetravalent R4 dendrimer are comparable to protegrins and tachyplesins, a family of potent antimicrobials containing 17-19 residues. Compared with a series of linearly repeating R4 peptides, the R4 dendrimers show comparable antimicrobial potency, but are more aqueous soluble, more stable to proteolysis, less toxic to human cells and more easily synthesized chemically. These results suggest repeating peptides that cluster the charge and hydrophobic residues may represent a primitive form of microbial pattern-recognition. Incorporating such knowledge in a dendrimeric design therefore presents an attractive approach for developing novel peptide antibiotics.
Topics: Anti-Bacterial Agents; Biochemistry; Chymotrypsin; Drug Design; Hemolysis; Humans; Microbial Sensitivity Tests; Peptides; Structure-Activity Relationship; Trypsin
PubMed: 11846794
DOI: 10.1046/j.0014-2956.2001.02728.x -
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 -
Plant Biotechnology Journal Jan 2011Retrocyclin-101 (RC101) and Protegrin-1 (PG1) are two important antimicrobial peptides that can be used as therapeutic agents against bacterial and/or viral infections,...
Retrocyclin-101 (RC101) and Protegrin-1 (PG1) are two important antimicrobial peptides that can be used as therapeutic agents against bacterial and/or viral infections, especially those caused by the HIV-1 or sexually transmitted bacteria. Because of their antimicrobial activity and complex secondary structures, they have not yet been produced in microbial systems and their chemical synthesis is prohibitively expensive. Therefore, we created chloroplast transformation vectors with the RC101 or PG1 coding sequence, fused with GFP to confer stability, furin or Factor Xa cleavage site to liberate the mature peptide from their fusion proteins and a His-tag to aid in their purification. Stable integration of RC101 into the tobacco chloroplast genome and homoplasmy were confirmed by Southern blots. RC101 and PG1 accumulated up to 32%-38% and 17%∼26% of the total soluble protein. Both RC101 and PG1 were cleaved from GFP by corresponding proteases in vitro, and Factor Xa-like protease activity was observed within chloroplasts. Confocal microscopy studies showed location of GFP fluorescence within chloroplasts. Organic extraction resulted in 10.6-fold higher yield of RC101 than purification by affinity chromatography using His-tag. In planta bioassays with Erwinia carotovora confirmed the antibacterial activity of RC101 and PG1 expressed in chloroplasts. RC101 transplastomic plants were resistant to tobacco mosaic virus infections, confirming antiviral activity. Because RC101 and PG1 have not yet been produced in other cell culture or microbial systems, chloroplasts can be used as bioreactors for producing these proteins. Adequate yield of purified antimicrobial peptides from transplastomic plants should facilitate further preclinical studies.
Topics: Anti-Infective Agents; Antimicrobial Cationic Peptides; Chloroplasts; Defensins; Gene Expression Regulation, Plant; Genetic Vectors; Pectobacterium carotovorum; Plants, Genetically Modified; Recombinant Proteins; Nicotiana; Tobacco Mosaic Virus; Transgenes
PubMed: 20553419
DOI: 10.1111/j.1467-7652.2010.00538.x -
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