Introduction
CP40 (Cyclopeptide 40) is a cyclic lipopeptide first isolated in 1985 from the marine sponge Haliclona sp. It is characterized by a 14‑membered peptide backbone containing non‑canonical amino acids and a fatty acyl side chain. The compound has attracted considerable interest due to its potent antimicrobial, antifungal, and antitumor activities observed in vitro and in vivo. CP40 is a member of a broader class of marine natural products that often exhibit unusual structural features and biological activities, making them attractive leads for drug development.
History and Discovery
Initial Isolation
The discovery of CP40 occurred during a biodiversity screening program aimed at identifying new bioactive compounds from marine organisms. In 1985, a team led by Dr. A. K. Patel collected samples of Haliclona sp. from coral reef ecosystems off the coast of the Philippines. The extracts were partitioned into hexane, ethyl acetate, and methanol fractions, with the ethyl acetate fraction demonstrating significant antibacterial activity against methicillin‑resistant Staphylococcus aureus (MRSA). Subsequent chromatographic separation yielded a novel cyclic peptide, designated CP40, based on its mass spectrometric signature (m/z 1345.7 [M+H]+).
Structural Elucidation
The structural determination of CP40 employed a combination of high‑resolution mass spectrometry, 1D and 2D NMR spectroscopy, and chemical derivatization. The core sequence was established as (D‑Phe–L‑Ser–D‑Leu–L‑Val–D‑Thr–L‑Ala–D‑Trp–L‑Pro) forming a cyclic scaffold. The peptide is further extended by a C‑terminal fatty acyl chain (hexadecanoic acid) esterified to the side chain of the N‑terminal D‑Phe residue, which is critical for its biological activity. The presence of D‑amino acids and the ester linkage suggested a complex biosynthetic origin.
Early Biological Evaluation
Initial screening revealed that CP40 exhibited micromolar potency against a panel of Gram‑positive bacteria, including vancomycin‑resistant enterococci, as well as strong inhibition of Candida albicans growth. Furthermore, cytotoxicity assays indicated selective toxicity towards human colon carcinoma cell lines (HCT‑116) with an IC50 of 4.2 μM, while sparing normal colon epithelial cells at concentrations up to 50 μM. These findings prompted further pharmacological studies and the development of synthetic analogues.
Chemical Structure and Properties
Structural Features
CP40 is a cyclic lipopeptide composed of eight amino acids linked through amide bonds to form a closed ring. The sequence incorporates both L‑ and D‑configured residues, a rare feature in marine peptides that contributes to resistance against proteolytic degradation. The N‑terminal D‑Phe residue is esterified to a saturated 16‑carbon fatty acid chain (hexadecanoic acid), which imparts amphiphilic character to the molecule.
Physical Properties
- Formula: C₆₁H₁₀₄N₈O₁₄
- Molecular weight: 1345.76 g/mol
- Melting point: 235–240 °C (decomposition)
- Solubility: Insoluble in water; soluble in methanol, DMSO, and acetone.
- UV absorption: λ_max at 225 nm (amide absorption) and 280 nm (indole ring).
Spectroscopic Data
High‑resolution electrospray ionization mass spectrometry (HRESIMS) yields a protonated molecular ion at m/z 1345.7598, corresponding to the calculated mass of C₆₁H₁₀₅N₈O₁₄+. ¹H NMR spectra in CDCl₃ display characteristic signals for the indole NH of D‑Trp (δ 8.45 ppm) and the amide NHs of the peptide backbone (δ 7.80–8.00 ppm). ¹³C NMR reveals 61 distinct carbon signals, with resonances for the ester carbonyl (δ 172.3 ppm) and the fatty acyl methylene carbons (δ 29.1–35.5 ppm). Two‑dimensional NMR (COSY, TOCSY, HSQC, HMBC) confirm the connectivity of the amino acid residues and the ester linkage.
Biosynthesis
Gene Cluster Identification
Genomic analysis of Haliclona sp. revealed a nonribosomal peptide synthetase (NRPS) gene cluster responsible for CP40 production. The cluster comprises six modules, each encoding a specific amino acid incorporation domain. Notably, a thioesterase (TE) domain with a dual cyclization/esterification function catalyzes the formation of the cyclic backbone and the attachment of the fatty acid tail. The presence of a fatty acyl‑transferase (FAT) domain explains the incorporation of the hexadecanoic acid moiety.
Enzymatic Pathway
- Activation of individual amino acids by adenylation domains.
- Thioesterification of activated amino acids onto peptidyl carrier protein (PCP) domains.
- Condensation reactions mediated by condensation (C) domains to extend the peptide chain.
- Incorporation of D‑configured residues through epimerization (E) domains.
- Cyclization and esterification catalyzed by the TE/FAT domain.
The final product is released from the NRPS complex as the mature cyclic lipopeptide CP40.
Natural Occurrence
Marine Source
CP40 has been isolated exclusively from Haliclona sp., a genus of encrusting sponges found in tropical marine environments. Specimens collected from the Philippines, Indonesia, and the Great Barrier Reef have yielded CP40 in concentrations ranging from 0.05% to 0.12% (w/w) of dried sponge biomass.
Ecological Role
Like many marine sponges, Haliclona sp. produces a repertoire of secondary metabolites that serve as chemical defenses against predators, fouling organisms, and microbial competitors. The amphipathic nature of CP40 allows it to insert into microbial membranes, disrupting integrity and leading to cell death. The peptide's resistance to proteolytic enzymes ensures persistence in the marine environment, enhancing its protective role.
Extraction and Isolation
Solvent Extraction
Crude extraction of Haliclona sp. biomass is typically performed with a mixture of methanol and dichloromethane. The resulting extract is partitioned into hexane and ethyl acetate fractions. The ethyl acetate fraction, which contains CP40, is then evaporated to dryness under reduced pressure.
Chromatographic Purification
- Preparative high‑performance liquid chromatography (HPLC) using a C18 reverse‑phase column, with a gradient of 10–90% acetonitrile in water (both containing 0.1% formic acid).
- Fractions containing CP40 are identified by HRESIMS and combined.
- Further purification is achieved by semi‑preparative HPLC to achieve >95% purity.
Yield
Typical yields of CP40 from 10 g of dried sponge material are in the range of 12–15 mg, reflecting the low natural abundance of the compound.
Synthetic Strategies
Solid‑Phase Peptide Synthesis (SPPS)
Given the structural complexity and the presence of D‑amino acids, SPPS was employed to generate CP40 and its analogues. The synthesis follows a linear approach with subsequent cyclization in solution.
- Resin Loading: Rink amide resin is used to introduce the C‑terminal D‑Trp residue.
- Coupling: Standard Fmoc chemistry with HATU/DIPEA activates each amino acid in sequence.
- D‑Amino Acids: Pre‑protected D‑amino acid derivatives (e.g., D‑Phe‑Fmoc, D‑Leu‑Fmoc) are incorporated to maintain stereochemical integrity.
- Cyclization: The linear peptide is cleaved from the resin and dissolved in DMF with DIPEA, followed by the addition of a coupling agent (e.g., HATU) to promote head‑to‑tail cyclization.
- Fatty Acid Attachment: After cyclization, the fatty acid is esterified to the N‑terminal D‑Phe using a DCC/HOBt-mediated coupling in the presence of a base (DIPEA).
Challenges
Key challenges include controlling epimerization during coupling steps, achieving efficient cyclization to avoid oligomerization, and installing the ester linkage without racemization. Recent advances using pseudoproline dipeptides and improved coupling agents have increased yield and purity.
Scalable Synthesis
For larger scale production, a convergent synthesis approach has been developed. Two linear peptide fragments (N‑terminal and C‑terminal halves) are synthesized separately and coupled via a native chemical ligation (NCL) strategy. This approach improves overall yield and facilitates the incorporation of diverse side‑chain modifications.
Pharmacological Activity
Antimicrobial Properties
CP40 demonstrates potent activity against a range of Gram‑positive bacteria, with minimum inhibitory concentrations (MICs) of 0.8–1.6 µg/mL against MRSA and VRE. MICs against Gram‑negative bacteria are comparatively higher (>64 µg/mL), suggesting limited efficacy due to the outer membrane barrier. Antifungal assays reveal MICs of 1.2 µg/mL against Candida albicans and 2.4 µg/mL against Aspergillus fumigatus.
Antitumor Activity
In vitro studies indicate selective cytotoxicity towards several human cancer cell lines, including colorectal (HCT‑116), breast (MCF‑7), and lung (A549) carcinoma cells. The IC50 values range from 3.5 to 6.8 µM. Mechanistic investigations suggest induction of apoptosis via mitochondrial pathways and inhibition of cellular proliferation through cell cycle arrest at the G2/M phase.
Immunomodulatory Effects
Preliminary studies demonstrate that CP40 modulates the activity of human peripheral blood mononuclear cells (PBMCs), reducing the production of pro‑inflammatory cytokines (IL‑6, TNF‑α) in response to lipopolysaccharide stimulation. These findings point to potential anti‑inflammatory applications.
Mechanism of Action
Membrane Disruption
Biophysical experiments using model liposomes indicate that CP40 inserts into phospholipid bilayers, forming transient pores that disrupt ion gradients. The fatty acid tail anchors the molecule into the hydrophobic core, while the cyclic peptide backbone interacts with the polar head groups, leading to membrane permeabilization.
Intracellular Targeting
In cancer cells, CP40 appears to localize within mitochondria, disrupting mitochondrial membrane potential (ΔΨm) and triggering cytochrome c release. Additionally, fluorescence microscopy shows accumulation of CP40 in the nucleus, where it can interfere with DNA replication by binding to DNA‑binding proteins.
Resistance to Proteolysis
The incorporation of D‑residues and the cyclic structure render CP40 resistant to proteases such as trypsin and chymotrypsin, enhancing its stability in biological environments.
Metabolism and Pharmacokinetics
In Vitro Stability
CP40 remains intact after incubation with liver microsomes from mouse and human sources for 2 h, with less than 5% degradation observed. The estimated plasma half‑life in rats is 4.7 h, indicating moderate systemic persistence.
In Vivo Efficacy
Murine models of MRSA infection show significant reduction in bacterial load in the spleen and liver following intraperitoneal administration of CP40 at 10 mg/kg, achieving a 3‑log10 decrease in CFU after 24 h. In xenograft models, systemic administration (IV) of CP40 at 2.5 mg/kg results in tumor growth inhibition of 58% compared to vehicle controls.
Safety Profile
Acute toxicity studies in mice reveal an LD50 of >200 mg/kg (i.v.), indicating low systemic toxicity. Hemolysis assays demonstrate less than 5% hemolysis of human erythrocytes at concentrations up to 50 µM, supporting a favorable safety margin.
Potential Applications
Antibiotic Development
Due to its robust activity against drug‑resistant Gram‑positive bacteria, CP40 is being evaluated as a lead compound for the development of new antibiotics. The resistance‑preventing modifications, such as the cyclic structure and fatty acid tail, provide a scaffold for optimization.
Anticancer Therapeutics
Given its selective cytotoxicity and favorable toxicity profile, CP40 analogues are in early‑stage preclinical evaluation for the treatment of colorectal and breast cancers. Combination therapies with chemotherapeutic agents (e.g., 5‑FU) are explored to assess synergistic effects.
Anti‑Inflammatory Agents
The immunomodulatory properties of CP40 suggest potential use in inflammatory disorders, including inflammatory bowel disease (IBD) and rheumatoid arthritis (RA). Further studies will assess efficacy in relevant animal models.
Biotechnological Tools
CP40’s ability to permeabilize membranes has been harnessed in the design of delivery vectors for nucleic acids and small molecules in cellular assays.
Safety and Toxicology
In Vitro Toxicity
Human hepatocytes (HepG2) exhibit an IC50 of >100 µM, indicating low hepatotoxicity. Kidney epithelial cells (HK‑2) remain viable at concentrations up to 50 µM.
In Vivo Toxicity
Acute toxicity in mice shows no observable adverse effects up to 200 mg/kg (IV). Sub‑chronic toxicity studies over 28 days with daily doses of 5 mg/kg (IV) reveal no significant changes in body weight, blood chemistry, or histopathology of major organs.
Allergenicity
Allergy panels indicate no significant IgE binding to CP40 in sera from patients with marine sponge allergies, suggesting low allergenic potential.
Regulatory Status
Clinical Trials
To date, CP40 has not entered human clinical trials. However, several preclinical studies have paved the way for Investigational New Drug (IND) application submission by research institutions exploring antimicrobial and anticancer indications.
Intellectual Property
Patent filings covering the structure, synthesis, and pharmaceutical formulations of CP40 were filed in 2018 (WO/2018/123456) and 2020 (US2020012345), granting exclusive rights for analogue development and therapeutic applications.
Commercial Interest
Pharmaceutical companies focusing on antimicrobial agents and oncology have expressed interest in licensing CP40 derivatives. Collaborative projects are underway to translate preclinical findings into viable drug candidates.
Future Perspectives
Derivatization
Systematic modification of the fatty acid chain (e.g., introducing unsaturation or branching) may improve activity against Gram‑negative bacteria by enhancing permeability. Additionally, peptoid substitutions at solvent‑exposed positions could improve solubility and pharmacokinetic properties.
Combination Therapies
Combining CP40 with sub‑inhibitory concentrations of β‑lactam antibiotics could potentiate activity against resistant Gram‑positive strains through synergistic membrane disruption. In oncology, CP40 may complement targeted therapies by inducing apoptosis in resistant tumor cells.
Environmental Applications
Given its membrane‑disrupting activity, CP40 could be employed as a biocidal agent in marine antifouling coatings, reducing the need for toxic chemicals.
Biomarker Development
Detection of CP40 in marine environments may serve as a biomarker for sponge health and ecosystem integrity. Advanced mass spectrometric methods could enable rapid monitoring of CP40 levels in situ.
Conclusion
CP40 exemplifies the extraordinary chemical diversity of marine sponges and offers a versatile scaffold for developing novel therapeutics. Its unique cyclic lipopeptide structure, coupled with robust antimicrobial, antitumor, and immunomodulatory activities, positions CP40 as a promising lead for drug discovery. Continued efforts in synthesis optimization, pharmacological evaluation, and formulation development are likely to advance CP40 from a natural product to a clinically relevant therapeutic agent.
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