Cytotoxic and antibacterial diketopiperazine cyclic dipeptides from Streptomyces canescens associated with marine sponge Haliclona caerulea

Technical Summary: Cytotoxic and Antibacterial Diketopiperazine Cyclic Dipeptides from Streptomyces canescens

Problem Statement
The rising global threat of antibiotic resistance and the urgent need for sustainable sources of novel bioactive compounds have driven the exploration of marine microbial ecosystems. Marine sponges, particularly those of the genus Haliclona, are known to harbor diverse bacterial communities, including Streptomyces species, which serve as prolific producers of secondary metabolites. While Haliclona sponges have yielded various alkaloids, proline-based 2,5-diketopiperazines (DKPs) derived specifically from Streptomyces symbionts of Haliclona caerulea remain underexplored. This study addresses the gap in knowledge regarding the chemical diversity and bioactivity of DKPs from this specific marine symbiotic relationship, aiming to identify potential leads for antibacterial and anticancer therapies.

Methodology
The research employed a multi-disciplinary approach combining microbiology, natural product chemistry, and computational biology:

1. Sample Collection and Isolation: Marine sponge Haliclona caerulea was collected from Larak Island, Persian Gulf. Associated bacteria were isolated using standard microbiological techniques on starch-yeast extract peptone agar (SYPA) and marine sponge agar (MSA).
2. Strain Identification: The most potent isolate, designated AHL19, was characterized through morphological, biochemical, and physiological tests. Molecular identification was confirmed via 16S rRNA gene sequencing and phylogenetic analysis, identifying the strain as Streptomyces canescens.
3. Fermentation and Extraction: The strain was cultivated in optimized fermentation media. Crude extracts were obtained using ethyl acetate. To isolate specific compounds, the crude extract underwent liquid-liquid extraction (LLE) partitioning into n-hexane, dichloromethane (DCM), and aqueous methanol fractions.
4. Bioactivity Screening:
   • Antibacterial: Minimum Inhibitory Concentration (MIC) assays were performed against Staphylococcus aureus, Micrococcus luteus, Escherichia coli, and Pseudomonas aeruginosa.
   • Cytotoxicity: The MTT reduction assay was used to evaluate cytotoxicity against the human colorectal adenocarcinoma cell line (HT29).
5. Structural Elucidation: The bioactive DCM fraction was subjected to semi-preparative HPLC. Six compounds were isolated and characterized using LC-ESI-MS and extensive 1D and 2D NMR spectroscopy (1H, 13C, COSY, HSQC, HMBC).
6. Computational Studies:
   • Molecular Docking: The isolated DKPs were docked against bacterial targets (DNA gyrase A, DNA gyrase B, Dihydrofolate reductase [DHFR]) and a cancer-related target (β-tubulin) to predict binding affinities and interaction modes.
   • Molecular Dynamics (MD) Simulation: 100-ns MD simulations were conducted to assess the structural stability and interaction persistence of the top-performing compound (Compound 5) with DHFR and β-tubulin, comparing it to the standard antibiotic streptomycin.

Key Contributions and Results

• Isolation of Novel Bioactive Metabolites: The study successfully isolated and characterized six proline-based 2,5-diketopiperazines (DKPs) from the DCM fraction of S. canescens AHL19:

  1. Cyclo (L-Pro-L-Val)
  2. Cyclo (L-Pro-L-Ile)
  3. Cyclo (L-Pro-L-Leu)
  4. Cyclo (L-Pro-L-Phe)
  5. Cyclo (L-Pro-L-Tyr)
  6. Cyclo (4-hydroxy-L-Pro-L-Phe)
     This represents the first report of these specific DKPs from H. caerulea-associated Streptomyces.

• Antibacterial Activity: The ethyl acetate crude extract demonstrated broad-spectrum antibacterial activity with MIC values ranging from 64 to 256 µg/mL against Gram-positive and Gram-negative pathogens. The DCM fraction, containing the isolated DKPs, was selected for further study due to its potent cytotoxicity.

• Cytotoxicity: The DCM fraction exhibited potent cytotoxicity against HT29 cells with an IC50 value of 4.0 ± 0.7 µg/mL. The aqueous methanol fraction showed even higher potency (IC50 1.9 ± 0.3 µg/mL), though the DCM fraction was prioritized for compound isolation based on its chemical profile.

• Computational Insights:

  • Target Selection: Molecular docking revealed that the isolated DKPs did not show significant binding affinity for DNA gyrase A or B. However, they showed promising interactions with DHFR and β-tubulin.
  • Compound 5 Superiority: Cyclo (L-Pro-L-Tyr) (Compound 5) demonstrated the highest binding affinity among the tested compounds. It achieved a docking score of -8.108 kcal/mol against DHFR and -9.599 kcal/mol against β-tubulin, outperforming streptomycin in the DHFR assay.
  • Role of Hydroxyl Groups: The presence of a hydroxyl group on the phenyl ring of Compound 5 was identified as critical for enhanced binding, forming additional hydrogen bonds with residues Thr122 (in DHFR) and Gln134 (in β-tubulin).
  • MD Simulation Stability: MD simulations confirmed that the Compound 5-DHFR complex exhibited greater structural stability (lower RMSD fluctuations) and more persistent key interactions compared to the streptomycin-DHFR complex over a 100-ns trajectory. Similar stability was observed for the Compound 5-β-tubulin complex.

Significance and Claims
The authors claim this study as the first report of proline-based DKPs isolated from Streptomyces canescens associated with the marine sponge Haliclona caerulea. The research underscores the untapped biodiversity of marine symbionts as a sustainable source for drug discovery.

The paper posits that these proline-based DKPs, characterized by their conformational rigidity and varying degrees of antibacterial and cytotoxic effects, are promising drug leads. Specifically, Compound 5 is highlighted as a potent natural compound capable of effectively targeting bacterial DHFR and cancer-related β-tubulin. The study concludes that these findings support the potential of marine microbial ecosystems in addressing antibiotic resistance and neoplastic diseases, paving the way for further investigations into the mechanisms, semisynthesis, and clinical applications of these cyclic dipeptides.