Classification and Enzymatic Synthesis of Non-Ribosomal Peptides
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Classification and Enzymatic Synthesis of Non-Ribosomal Peptides

Natural products are an important source for drug development, among which peptide compounds are particularly notable, such as penicillins, cephamycins, and cyclosporine, which have become powerful tools for treating fungal or bacterial infections and immunosuppression. Peptide drugs have many advantages: small molecule peptides (less than 1000 Da) can precisely bind to targets, thus achieving highly specific pharmacological effects; large molecule peptides larger than 1000 Da can exhibit protein-like interactions in vivo, thereby achieving diverse biological effects; peptide drugs generally have good compatibility with the human body and pharmacokinetic properties, thus enabling better utilization and absorption; the side chains of peptide compounds provide numerous modification sites for chemical and biosynthesis, which can improve the properties of drugs and broaden their application range.

According to the biosynthetic mechanism, peptides are divided into two main categories: ribosomally synthesized and post-translationally modified peptides (RiPPs) and non-ribosomal peptides (NRPs). In drug development, NRPs have unique advantages compared to RiPPs, mainly manifested in structural diversity and rich post-modifications. Additionally, NRPs have higher protease stability, membrane permeability, and target binding ability.

The framework of NRPs is catalyzed by non-ribosomal peptide synthetases (NRPSs), and its assembly process includes loading of structural units, peptide chain elongation and product release, as well as on-line modifications. Post-modifications such as dimerization, oxidation, and cyclization further enrich the structural types of products. In addition to synthesizing peptide frameworks alone, NRPSs can hybridize with various biosynthetic pathways such as polyketide synthases (PKSs) or terpene cyclases (TCs) to form more complex structural types.

Classification of Non-Ribosomal Peptides

NRPs exhibit a wide range of pharmacological activities including anti-inflammatory, antifungal, antibacterial, antitubercular, and anticancer properties. Based on their sources, NRPs can be roughly categorized into bacterial NRPs, fungal NRPs, and semi-synthetic NRPs. Clinically, numerous peptide drugs sourced from bacteria are available, such as the lipopeptide antibiotic daptomycin for Gram-positive bacteria, the glycopeptide antibiotic bleomycin A2 for tumors, and the cyclic peptide antibiotic gramicidin S for Gram-positive bacteria. Fungal NRPs have been extensively used in the treatment and prevention of various diseases, such as β-lactam antibiotics. Penicillin G, a representative natural product of the penicillin class, mainly sourced from Penicillium, structurally resembles the terminal structure of bacterial cell wall's pentapeptide end, thus it can replace alanine-alamine to bind with transpeptidase, thereby inhibiting bacterial cell wall synthesis and causing bacterial death. β-lactam antibiotics are typical chemically semi-synthetic peptide drugs, such as ceftobiprole, which exhibits good inhibition against methicillin-resistant Staphylococcus aureus (MRSA).

  • Cyclosporins

Cyclosporine A (CsA), a cyclic peptide antibiotic, was first isolated and identified from Tolypocladium inflatum. CsA contains eleven hydrophobic amino acid residues, with seven of them methylated at N-terminus. In 1983, CsA was approved by the FDA for immunosuppressive therapy post-organ transplantation. As one of the most widely used immunosuppressants, CsA primarily targets cyclophilin, which catalyzes the cis-trans isomerization of proline peptide bonds in T cells. After binding to cyclophilin, CsA inhibits the activity of calcineurin, thereby inhibiting the production and release of interleukin 2 (IL-2), blocking the activation and proliferation of T cells for immunosuppression. Additionally, CsA exhibits various pharmacological activities such as antiviral, antifungal, antiparasitic, anti-inflammatory, and anticancer properties. Voclosporin, a chemical derivative based on the CsA scaffold, possesses stronger efficacy and metabolism rates, used for treating lupus nephritis.

CatalogProduct NameCAS NumberCategory
BAT-009200Cyclosporin A-Derivative 11487360-85-9Others
BAT-009201Cyclosporin A-Derivative 1 Free base286852-20-8Others
BAT-009202Cyclosporin A-Derivative 2156047-45-9Others
BAT-010028Cyclosporin A59865-13-3Peptide Inhibitors
  • Cyclodepsipeptides

Enniatins (ENNs) and beauvericin (BEA) belong to non-ribosomal hexadepsipeptide fungal toxins, characterized by hydroxylated nonproteinogenic amino acids and a fatty acid chain. The structure of ENNs consists of D-hydroxy acids and N-methyl-L-amino acids forming amide and lactone bonds alternately. The first ENN was reported in 1947 from Fusarium orthoceras. Common natural ENNs include enniatin A, A1, B, and B1. Enniatin B exhibits various biological activities including facilitating K+ and Na+ transmembrane transport, in vitro lipid-lowering activity, antibacterial, insecticidal, and herbicidal activities. Fusafungine, composed of multiple ENN components, down-regulates intercellular cell adhesion molecule-1 (ICAM-1) and inhibits the production of pro-inflammatory cytokines, thus used in the treatment of a range of upper respiratory tract diseases. BEA's structure was first isolated and identified from B. bassiana in 1969. BEA and its analogs, exceeding twenty, possess biological activities including insecticidal, anticancer, antibacterial, antiviral, anticonvulsant, and antitubercular activities.

  • Ergot Alkaloids

Ergot alkaloids (EAS) are structurally complex indole derivatives produced by ascomycetes. EAS are characterized by a tetracyclic ergoline moiety, where A and B rings originate from L-tryptophan (L-Trp) and C and D rings originate from dimethylallyl pyrophosphate (DMAPP) and cyclization of L-Trp, such as ergometrine. EAS structures are primarily divided into ergoclavines, ergoamides (simple ergot acid amide derivatives), and ergopeptines (ergot acid peptide derivatives). Natural EAS are widely used clinically due to their significant pharmacological activities, such as fumigaclavine C for treating inflammatory bowel disease and atherosclerosis, ergometrine for postpartum uterine bleeding, and ergotamine for migraine treatment.

Mechanism of Non-Ribosomal Peptide Synthetases

The core skeleton of natural NRPs is catalytically synthesized by NRPSs. NRPSs are multifunctional enzymes composed of multiple modules, where different domains within each module catalyze various biochemical reactions, including substrate recognition and loading, peptide chain elongation, and product release, thereby assembling simple building blocks into complex peptide products. The basic skeleton of NRPs comprises 20 natural L-amino acids, nonproteinogenic amino acids, fatty acids, α-hydroxy acids, α-keto acids, and heterocycles, while diverse modifications (such as reduction, oxidation, and methylation) further enrich the structural types of products. The basic modules of NRPSs include three catalytic domains: adenylation domain (A domain), thiolation domain (T domain)/peptidyl carrier protein (PCP domain), and condensation domain (C domain).

The basic enzymatic catalysis mechanism of peptide synthesis: The A domain is used for substrate recognition and activation. Under the action of adenosine triphosphate (ATP), this domain selectively recognizes amino acids and converts them into aminoacyl-adenylate intermediates. The T domain transitions from an unactivated apo form to an activated holo form, then utilizes its terminal thiol on the phosphopantetheine arm to covalently bind aminoacyl substrates via a thioester bond. The C domain is typically located at the N-terminus of each elongation module. This domain catalyzes the nucleophilic attack of the downstream receptor aminoacyl substrate's α-amino group on the thioester group of the upstream donor aminoacyl, forming an amide bond. Besides the three basic domains, NRPSs possess other modifying domains such as epimerization domain (E domain), methylation domain (MT domain), cyclization domain (Cy domain), oxidation domain (Ox domain), and formylation domain (F domain), which catalyze isomerization from L to D amino acids, methylation on N or C, formation of thiazole or oxazole rings, oxidation of heterocycles, and formylation reactions, respectively.

In the assembly process of NRPs, termination and release of peptide chain elongation are catalyzed by terminal thioester domains (TE domain), reduction domains (R domain), or C domains. The TE domain catalyzes peptide release to form free acids, lactones, or lactams. The R domain typically catalyzes the formation of alcohols or aldehydes. Besides catalyzing the formation of conventional amide bonds, the C domain also possesses diverse functionalities such as formation of β-lactams, dehydration, hydrolysis, cyclization, Pictet-Spengler cyclization, and Dieckmann condensation. The diverse domains and module numbers of NRPSs contribute to the structural diversity of peptides, thus providing ample space for the development of naturally sourced active NRPs.

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