Research Progress on the Structural Classification and Functional Applications of Lasso Peptides

Lasso peptides, a class of ribosomally synthesized and post-translationally modified peptides (RiPPs), are named for their characteristic interlocked lasso topology. They possess a helical C-terminal tail that threads through an intramolecular lactam ring at the N-terminus, forming a lasso-like structure. The peptide ring at the N-terminus typically consists of 7 to 9 amino acids. While most lasso peptides have N-terminal amino acid residues such as glycine (Gly), serine (Ser), cysteine (Cys), or alanine (Ala), recent discoveries have also unveiled novel lasso peptides with leucine (Leu) at the N-terminus. The large peptide ring in lasso peptides is formed between the α-amino group of the N-terminal amino acid and the carboxylic acid side chain of either aspartic acid (Asp) or glutamic acid (Glu). This unique structure imparts lasso peptides with exceptional stability against chemical, thermal, and proteolytic degradation.

Structure and Classification of Lasso Peptides

Lasso peptides can be classified into four categories based on the number and positions of disulfide bonds they contain, as illustrated in the figure below. Lasso peptides where the C-terminal peptide chain is connected to the macrocyclic ring by two disulfide bonds are defined as Class I lasso peptides. If the peptide tail, after threading through the ring, is not connected to the macrocyclic ring by disulfide bonds but instead stabilized by spatial interactions to maintain the lasso topology, they are referred to as Class II lasso peptides. When only one disulfide bond is formed between the C-terminal sequence of the peptide tail and the macrocyclic ring, the lasso peptides are further divided into Class III or Class IV based on the position of the disulfide bond. Specifically, Class III lasso peptides have a disulfide bond connecting the macrocyclic ring to the peptide tail, while in Class IV, the disulfide bond exists solely within the peptide tail. Among these, Class II lasso peptides are the most common and characteristic of all known lasso peptides, typically originating from Proteobacteria. In contrast, Class I, III, and IV lasso peptides are usually derived from Actinomycetes. Class II lasso peptides sourced from Proteobacteria are often polar and hydrophilic, while Class I and II lasso peptides isolated from Streptomyces (such as MccJ25, propeptin, and anantin) are hydrophobic. These structural characteristics determine the diverse biological activities exhibited by lasso peptides.

Functional Applications of Lasso Peptides

To date, several lasso peptides, such as Siamycin derived from Streptomyces, have been reported to play roles in diabetes treatment and targeting receptors associated with cancer, such as endothelin β receptors. Additionally, Microcin J25 has also been documented to possess antibacterial and receptor antagonist bioactivities.

1. Antibacterial Activity

The majority of lasso peptides discovered thus far are synthesized in Proteobacteria and Actinomycetes, including lanthipeptides, microcins, lactocins, blue lantibiotics, and thiopeptides. Many of these lasso peptides exhibit antibacterial effects. Numerous lanthipeptides possess bactericidal activity against various Gram-positive bacteria, and some show potent activity against methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci, and oxacillin-resistant Gram-positive bacteria. Lasso peptide MccJ25 is effective against Gram-negative bacteria (e.g., Escherichia coli and Salmonella), neutralizes endotoxins, and inhibits the production of inflammatory cytokines through its immunomodulatory effects. Additionally, active peptide MccJ25 prevents intestinal damage and inflammation caused by enterotoxigenic Escherichia coli K88, making it a potential preventive agent against pathogen infections in animals, food, or humans. Lasso peptides marinopyrroles A-F, derived from Streptomyces sannurensis, also exhibit strong antibacterial activity against MRSA. The antibacterial mechanisms of lasso peptides are believed to operate through two primary pathways: firstly, by interacting with bacterial cell membranes, forming ion channels that lead to the leakage of intracellular contents and subsequent bacterial death; secondly, by entering cells without disrupting membranes, binding to intracellular targets, and inhibiting their metabolism, ultimately resulting in bacterial killing. For instance, MccJ25 binds to the bacterial outer membrane receptor protein FhuA, enters the cell, and inhibits RNA polymerase, thereby exerting its bactericidal effect.

2. Receptor Antagonists

Lasso peptide RES-701-1, derived from Streptomyces sp., is a selective antagonist of the endothelin receptor ETB. Currently, the therapeutic efficacy of some endothelin peptide antagonists is limited due to their degradation in the gastrointestinal tract. Consequently, modifying and designing peptide antagonists with increased proteolytic stability based on the RES-701-1 sequence of lasso peptides has become a research focus in recent years. Anantin, a lasso peptide produced by Streptomyces coulescens, is the first microbially-derived atrial natriuretic factor antagonist (ANP). Natriuretic peptides (NPs) are hormones involved in maintaining osmotic and vascular homeostasis, eliciting natriuretic, diuretic, and vasodilatory effects. They also exhibit anti-fibrotic, anti-proliferative, and anti-hyperplastic activities, participating in the remodeling of the cardiac and vascular systems. BI-32169, a lasso peptide sourced from Burkholderia thailandensis, is a potent antagonist of the glucagon receptor and is the first naturally occurring peptide antagonist with a sequence non-homologous to glucagon. Its primary structure differs significantly from that of glucagon, and thus, the mechanisms underlying BI-32169's antagonistic effects remain unclear. Given the generally low stability of peptide antagonists of the glucagon receptor, their therapeutic applications lag far behind small-molecule organics. However, the lasso structure and unique structural characteristics of lasso peptides offer potential for enhancing the stability of novel peptide antagonists, thereby advancing their development.

3. Enzyme Inhibitors

Lasso peptide MS-271, derived from Streptomyces griseoflavus, is an inhibitor of smooth muscle myosin light chain kinase (MLCK). Typically, peptide inhibitors exhibit superior specificity; however, their low stability in vivo has limited their applications. Lasso peptide inhibitors, with their unique structural stability, offer a novel approach for the development of MLCK inhibitor drugs. Propeptin, a lasso peptide produced by Microbispora sp. SNA-115, is a serine protease inhibitor. Studies have shown that propeptin, as a serine protease inhibitor, exhibits neuroprotective, anti-amnestic, and cognitive-enhancing effects in animal models.

MccJ25 and Capistruin are lasso peptides produced by Proteobacteria that inhibit bacteria by inhibiting RNA polymerase (RNAP) activity. Bacterial RNAP, crucial for bacterial growth and survival, serves as an important target for antibiotics. Its conservation in bacteria allows for selective targeting without affecting eukaryotic RNAP. Research by Mukhopadhyay et al. indicates that MccJ25 inhibits transcription by binding to the secondary channel of RNAP, thereby blocking substrate access to the active site.

4. Molecular Scaffolds

Currently, peptide-based drugs are widely used in clinical treatments due to their higher target specificity compared to small-molecule drugs and lower immunogenicity compared to traditional protein-based therapies. However, the application of conventional polypeptide drugs is often limited by their low solubility, poor bioavailability, and instability. To address these issues, the utilization of naturally occurring lasso peptides with stable structures as molecular scaffolds for epitope grafting to develop peptide drugs has emerged as a promising solution. In many tumor cells, the RGD (Arginine, Glycine, Aspartic) sequence in the αvβ3 subunit of integrins is overexpressed. By replacing G12, I13, and G14 in MccJ25 with RGD, a hybrid peptide can be created for cancer recognition and therapy. RES-701-1, with its endothelin B receptor antagonistic function, forms a hybrid peptide with endothelin that significantly enhances its affinity and antagonism towards ETB. Furthermore, incorporating the RGD sequence into RES-701-1 can improve its biological activity and structural stability. Modifications to the lasso loop and tail regions of lasso peptides can also confer new biological activities. Unfortunately, to date, lasso scaffolds cannot be obtained through chemical synthesis methods and rely solely on the action of lasso peptide biosynthetic modification enzymes. The difficulty in heterologous expression and purification of these enzymes poses a challenge for this application.

Lasso Peptides: A Unique Class of Ribosomally Synthesized and Post-translationally Modified Natural Peptides.Lasso peptides are a unique class of natural peptides that exhibit a lasso-like topology after ribosomal synthesis and post-translational modification. Their exceptional topological structure endows these peptides with remarkable resistance to physical and chemical degradation as well as protease hydrolysis. In recent years, advancements in technologies such as mass spectrometry analysis and the development of genomic sequencing have led to a comprehensive understanding of the sequence and structural diversity of lasso peptides. Furthermore, there has been a deeper insight into the biosynthetic mechanisms of these peptides. Nevertheless, the biosynthesis of lasso peptides and the heterologous expression and purification of their biosynthetic modification enzymes still pose significant challenges. If lasso peptides can be directionally modified and engineered through chemical synthesis and genetic recombination, it would further expand their applications in the medical field.

References

[1] Cheng Cheng, Hua Zichun. Lasso Peptides: A Versatile Carrier for Peptide Drug Design and Modification [J]. Pharmaceutical Progress, 2019, 43(10): 777-785.

[2] Li Xinrui, Lu Meiling. Research Progress on Lasso Peptides [J]. Pharmaceutical Biotechnology, 2020, 27(06): 587-593.

About the Author

Xiaonisha, a food technology professional holding a Master's degree in Food Science, is currently employed at a prominent domestic pharmaceutical research and development company. Her primary focus lies in the development and research of nutritional foods, where she contributes her expertise and passion to create innovative products.