Cyclic Peptide Antibiotics

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Features of cyclic peptide antibiotics

Cyclic peptide antibiotics represent a category of antibiotics distinguished by a cyclic configuration, wherein the amino acid sequence creates a loop instead of a linear arrangement. This distinctive structure has numerous benefits, including improved stability and resistance to enzymatic breakdown, rendering them efficient against various pathogenic diseases. These antibiotics frequently have significant efficacy against multi-drug resistant infections, rendering them especially indispensable in contemporary treatment.

Bactenecin

The granules of bovine neutrophils contain bactenecin, a 12-residue cathelicidin antimicrobial peptide (AMP) with a cyclic structure formed by a disulfide link. It has wide-ranging antibacterial action and is the smallest cationic AMP that is known. Research has demonstrated that bacterenecin can penetrate bacterial membranes on both the outer and inner surfaces after binding to LPS. While bactenecin does show some action against Gram-positive bacteria, its effectiveness is far higher against Gram-negative bacteria.

Mechanism of action: Bactenecins primarily target microbial cell membranes owing to their cationic (positively charged) characteristics, which engage with the negatively charged microbial membranes. They can compromise cell membrane integrity by creating holes or by directly infiltrating and disrupting intracellular targets. Certain bactenecins, especially proline-rich variants such as Bac5 and Bac7, penetrate bacterial cells and impede protein synthesis by attaching to ribosomes.

Polymyxins

As a last resort in the treatment of illnesses caused by Gram-negative bacteria that have developed resistance to many drugs, polymyxin B-a natural cationic lipopeptide antibiotic—has been generated by Bacillus polymyxa. It is structurally composed of a peptide ring with a fatty acid tail and peptide side chains. Polymyxin B, similar to bactenecin, binds to the LPS of Gram-negative bacteria's outer membrane and permeabilizes both the outer and inner membranes, killing the bacterium.

Mechanism of action: Polymyxins' positively charged amino groups interact with the negatively charged LPS in Gram-negative bacterial outer membrane. This alters the membrane's arrangement. Polymyxins promote membrane permeability by substituting calcium and magnesium ions that stabilize the membrane, therefore promoting cell leakage and lysis. Essential chemicals leaked from the increasingly porous bacterial membrane causes the bacterial cell to die. Though it restricts their efficacy against Gram-positive bacteria, which lack an outer membrane, this membrane-targeting mechanism makes polymyxins especially powerful against Gram-negative bacteria.

Octapeptins

Derived from Bacillus circulans, octapeptins are a class of naturally occurring cyclic lipopeptide antibiotics with potent action against a wide variety of bacterial species. Octapeptidtins are still mostly unknown, even though they were found more than 40 years ago. The basic building block of octapeptin is a fatty acid tail and a D-amino acid connected to a cyclic peptide ring. Polymyxins and octapeptins have a common structural feature: a cyclic peptide core connected to an N-terminal fatty acid chain. A hydrophobic motif in the peptide ring is another distinguishing feature of both. Because octapeptins typically have less charges than polymyxins, they are more hydrophobic than the latter.

Mechanism of action: Octapeptins engage with the LPS layer of Gram-negative bacteria by adhering to LPS in the outer membrane. The lipid tail facilitates the antibiotic's penetration and disruption of the bacterial membrane, resulting in cell leakage, lysis, and subsequent death. Although this process resembles that of polymyxins, octapeptins exhibit subtle variations in their membrane contact, potentially enhancing their efficacy against specific polymyxin-resistant bacteria.

Capreomycin

Streptomyces capreolus produces the natural polypeptide antibiotic capreomycin, which has been used for managing tuberculosis. Its antibacterial properties are thought to be caused by its ability to impede the creation of ribosomal proteins. For example, research indicates that capreomycin suppresses Mycobacterium TB via binding to and interacting with the 16S rRNA of the 30S ribosomal subunit. Animoglycosides are commonly used to describe capreomycin since they both have nephrotoxic side effects and comparable intracellular action. Thus, it is postulated that capreomycin, like aminoglycosides, translocates through a cellular uptake pathway as they cannot freely traverse cell membranes and must enter cells via endocytosis. Indeed, new research has shown that Mycobacterium smegmatis becomes more sensitive to aminoglycosides and capreomycin when uptake regulators are disrupted.

Mechanism of action: Capreomycin impedes protein synthesis in Mycobacterium TB. Although its precise mechanism remains incompletely elucidated, it is recognized to attach to bacterial ribosomes, specifically obstructing their capacity to convert mRNA into functional proteins. This disruption inhibits bacterial growth and multiplication, producing a bacteriostatic impact instead of a bactericidal one. Capreomycin is efficacious against Mycobacterium TB and some atypical mycobacteria; nevertheless, it is mostly ineffective against the majority of other bacterial species due to its specificity for mycobacterial ribosomes.

Fig.1 Chemical structures of the common cyclic peptide antibiotics. (Lee M W., et al., 2020)

Pargamicins

Pargamicin A (PRG-A) was isolated from the fermentation broth of the soil actinomycete strain Amycolatopsis sp. ML1-hF4 while searching for antibiotics that might kill methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterobacteriaceae. In vitro, PRG-A demonstrates superior or equal antibacterial activity to that of other drugs on the market, such as vancomycin, thanks to its structurally distinct cyclic peptide composition of N-methyl-3-hydroxy valine, 4-hydroxy piperazic acid (4-OH-Pip), sarcosine, phenylalanine, N-hydroxy isoleucine (NOH-Ile), and piperazic acid (Pip). A mechanism different from daptomycin's led to PRG-A's fast bactericidal effect against staphylococci and enterococci; this method entailed damaging the target cells' membrane integrity. The culture broth of the PRG-A-producing strain Amycolatopsis sp. ML1-hF4 revealed the presence of novel active components PRG-B, -C, and -D while optimizing PRG-A production.

The antibacterial activity of PRG-B and PRG-D was poorer against Gram-positive bacteria, but PRG-A and PRG-C showed robust action against methicillin-resistant S. aureus and VRE. PRG-A and PRG-B shown equivalent antibacterial activity against staphylococci and enterococci, in contrast to PRG-C and PRG-D, which have a polar group in Pip's northern region and were four to eight times less effective against staphylococci than against enterococci. Polar groups in Pip's northern area may hinder contact with staphylococcal membrane, according to this. Despite the fact that all of the PRGs were effective against VRE, not a single one of the tested compounds-including vancomycin—had any effect on Gram-negative bacteria.

Mechanism of action: The hydrophobic tail of pargamicins penetrates the lipid bilayer of the bacterial cell membrane. This insertion is preferential for bacterial membranes owing to compositional changes, specifically in phospholipid concentration relative to mammalian cells. Upon integration into the membrane, pargamicins induce disruption of the cell membrane's architecture. This results in heightened permeability, permitting ions (such as potassium, calcium, and sodium) and tiny molecules to escape the cell. This leakage disturbs the ionic equilibrium of the cell, resulting in depolarization of the bacterial membrane. Maintaining an appropriate ionic gradient is essential for cellular functions; hence, this imbalance hinders bacterial activity and survival. Pargamicins not only induce membrane leakage but also disrupt membrane-associated activities essential for energy generation, nutrition transport, and several cellular functions. Pargamicins impede essential activities, hence obstructing bacterial growth and replication, ultimately leading to cell death.

Fig.2 Structure of pargamicin (PRG) A, B, C and D. (Hashizume H., et al., 2017)

Fig.3 Antimicrobial activities of pargamicins. (Hashizume H., et al., 2017)

Daptomycin and Amphomycin

One cyclic lipopeptide found in the fermentation broth of Streptomyces roseosporus in the early 1980s is daptomycin, officially identified as LY146032 by Eli Lilly. Daptomycin is effective against a wide variety of Gram-positive bacteria. In 2003, the FDA authorized daptomycin, the first of a new family of antibiotics, for the treatment of severe infections, in response to the rise of multidrug resistance. A54145, amphomycin, friulimicin, MX-2401, tsushimycin, and daptomycin are all members of the same lipopeptide antibiotic family with very similar chemical structures. A cyclic-core structure with 10 amino acids and a hydrophobic sidechain connected (gray oval) is one of the common structural features. The antibiotics daptomycin and amphomycin have a conserved Asp₄-X₅-Asp₆-Gly₇ motif, which binds calcium ions, and both show calcium-dependent activity. At position X₅, daptomycin has a d-Ala amino acid, while amphomycin has a Gly. A major structural distinction between the two antibiotics is that amphomycin has a peptide link between Dab1 and Pro10, whereas daptomycin is a cyclic depsipeptide with an ester bond between Thr₁ and Kyn₁₀.

Although amphomycin and daptomycin share structural similarities, their action mechanisms are very different. When amphomycin was added to Staphylococcus aureus during its mid-exponential growth, the bacteria's cell walls weakened and Park's nucleotide accumulated. Daptomycin, on the other hand, is mostly effective against bacterial membranes. The calcium-dependent binding of daptomycin to lipid vesicles has been demonstrated in in vitro experiments. The negatively charged phosphoglycerol headgroup of membrane lipids is bound with great affinity by the daptomycin-Ca²⁺ complex. It is proposed that membrane-bound daptomycin aggregates and allows potassium ions to seep out by penetrating the lipid bilayer with its hydrophobic sidechain. The strong bactericidal action of daptomycin is thought to be responsible for this depolarization of the membrane. Nevertheless, it has been demonstrated that significant quantities of daptomycin are necessary for the bacterial membrane depolarization to take place. The addition of daptomycin to B. subtilis causes cell development with abnormal cell wall shape and faulty septum formation, suggesting that daptomycin, even at low doses, interferes with cell wall production.

Mechanism of action: Daptomycin functions by attaching to the bacterial cell membrane in a calcium-dependent fashion, facilitating the formation of holes inside the membrane. The creation of pores results in ion leakage, primarily potassium, which disrupts the ionic gradients of the bacterial cell, leading to depolarization and subsequent suppression of DNA, RNA, and protein synthesis, ultimately causing cell death. Daptomycin exhibits bactericidal activity, effectively eliminating Gram-positive bacteria.

Unlike daptomycin, amphomycin primarily inhibits cell wall synthesis by lipid intermediates accumulation and the peptidoglycan synthesis, which prevents the transfer of peptidoglycan precursors across the cell membrane, which is essential for building the bacterial cell wall, leading to cell lysis and death.

Fig.4 Chemical structure of decapeptide cyclic antimicrobial peptides. (Rimal B., et al., 2023)

Rakicidin F and Asperpeptide A

A marine sponge-derived actinomycete strain, Streptomyces sp. GKU 220, produced the antibacterial cyclic depsipeptide rakicidin F. At 25 μg per disk, rakicidin F exhibited growth inhibitory action against Bacillus subtilis and Escherichia coli in the antimicrobial activity experiment. Aspergillus sp. XS-20090B15, a fungus isolated from gorgonian, produced a cyclic pentapeptide called asperpeptide A cyclo(-Pro-Ala-Ala-Tyr-5-OHAA). The antibacterial activity of asperpeptide A was shown against Bacillus cereus and Staphylococcus epidermidis, and the MIC value for both bacteria was 12.5 μM.

Mechanism of action: Rakicidin F is a cyclic depsipeptide characterized by a hydrophobic tail that facilitates its integration into bacterial cell membranes. This integration is believed to undermine membrane integrity, leading to the leakage of essential ions and molecules. This break compromises the selective permeability of the bacterial membrane, leading to cell death. Rakicidin F exhibits selective cytotoxicity, particularly under hypoxic circumstances, effectively targeting hypoxic tumor cells. Hypoxic areas within tumors frequently exhibit resistance to conventional treatment, rendering rakicidin F a viable candidate for cancer research.

Asperpeptide A demonstrates antibacterial and cytotoxic properties, since it can inhibit bacterial cell wall formation or damages cellular membranes, like to other cyclic peptides.

Fig.5 Antibacterial cyclic peptides including Rakicidin F and Asperpeptide A. (Abdalla M A., et al., 2018)

References

1. Lee M W., et al., How do cyclic antibiotics with activity against Gram-negative bacteria permeate membranes? A machine learning informed experimental study, Biochimica et Biophysica Acta (BBA)-Biomembranes, 2020, 1862(8): 183302.
2. Hashizume H., et al., Structure and antibacterial activities of new cyclic peptide antibiotics, pargamicins B, C and D, from Amycolatopsis sp. ML1-hF4, The Journal of Antibiotics, 2017, 70(5): 699-704.
3. Rimal B., et al., The effects of daptomycin on cell wall biosynthesis in Enterococcal faecalis, Scientific Reports, 2023, 13(1): 12227.
4. Abdalla M A., et al., Natural cyclic peptides as an attractive modality for therapeutics: A mini review, Molecules, 2018, 23(8): 2080.