New liposidomycin congeners produced by *Streptomyces* sp. TMPU-20A065, anti-*Mycobacterium avium* complex agents with therapeutic efficacy in a silkworm infection model

Mycobacterium avium complex (MAC) infections, primarily caused by *M. avium* and *M. intracellulare*, are increasingly prevalent nontuberculous mycobacterial pulmonary disorders, surpassing tuberculosis in incidence in developed countries. Symptoms mimic tuberculosis (weight loss, fever, fatigue, night sweats) but progress slower with a poorer prognosis. Clarithromycin (CAM), often combined with rifampicin and ethambutol, is a first-line treatment, but its efficacy is limited, requiring prolonged administration (over a year) and leading to drug resistance. While amikacin liposome inhalation suspension gained approval in 2018, its use is restricted to patients unresponsive to conventional therapies. The urgent need for novel anti-MAC agents with different mechanisms of action necessitates the discovery and development of new drug candidates. This study employed an *in vivo*-mimic silkworm infection model to screen for anti-MAC agents from microbial resources. This approach, based on previous research by the authors, led to the identification of potent anti-MAC compounds from *Streptomyces* sp. TMPU-20A065: three new liposidomycin congeners (1, 2, and 4) and 14 known liposidomycins (3 and 5–17). This paper details the fermentation, isolation, structural elucidation, and *in vitro* and *in vivo* antimycobacterial activities of these liposidomycins.

The introduction cites several previous publications (references [1-4]) highlighting the increasing prevalence and challenges in treating MAC infections with existing therapies. The limitations of current first-line treatments like clarithromycin, including the emergence of drug resistance due to prolonged treatment duration, are emphasized. The authors also reference their own prior work ([5-13]) establishing the *in vivo* silkworm infection model used in this study for anti-MAC agent screening. This demonstrates a strong foundation in the methodology used and a clear understanding of the current gaps in MAC treatment strategies. The cited literature on liposidomycins ([16-19]) provides context for the structural elucidation of the isolated compounds.

The study involved several key methodological steps: 1. **Microorganism and Fermentation:** The liposidomycin-producing *Streptomyces* sp. TMPU-20A065 was isolated from soil and identified via 16S rDNA sequencing. Fermentation was carried out in a production medium under specific conditions (temperature, shaking speed, duration). 2. **Isolation and Purification:** An anti-MAC activity-guided isolation process was employed, using a Diaion HP-20 column followed by multiple rounds of medium-pressure liquid chromatography (MPLC) and preparative high-performance liquid chromatography (HPLC) to separate and purify the compounds. Specific columns, mobile phases (e.g., methanol, acetone, acetonitrile, TFA), flow rates, and UV detection wavelengths were utilized. The process resulted in the isolation of three new compounds (1, 2, and 4) and fourteen known liposidomycins (3 and 5-17). 3. **Structural Elucidation:** The structures of the known liposidomycins (3 and 5-17) were confirmed by comparing their spectroscopic data (NMR and MS) with literature values. The structures of the new congeners (1, 2, and 4) were elucidated using various spectroscopic techniques including UV, IR, HR-FAB-MS, and detailed 1D and 2D NMR analysis (COSY) to determine the connectivity and stereochemistry. The molecular formulas and key structural features such as the acyl side chains were determined. 4. **Antimycobacterial Activity Assay (*in vitro*):** A broth microdilution method was used to determine the minimum inhibitory concentrations (MICs) of compounds 1-17 against *M. avium*, *M. intracellulare*, *M. bovis* BCG Pasteur, and *M. smegmatis*. The MIC was defined as the lowest concentration inhibiting 90% of bacterial growth. 5. **Antimycobacterial Activity Assay (*in vivo*):** A silkworm infection model was used to assess the therapeutic efficacy of compounds 1-17. Fourth-instar silkworm larvae were infected with *M. avium* or *M. intracellulare*, followed by treatment with the test compounds. The effective dose 50 (ED₅₀) values, representing the dose required for 50% survival, were determined. 6. **Antibacterial Activity Assay:** A broth microdilution method (CLSI document M07-A09) was employed to determine the MIC values against various bacterial strains (*B. subtilis*, *S. aureus*, *E. coli*, and *P. aeruginosa*).

The study successfully isolated and characterized three new liposidomycin congeners (1, 2, and 4) and 14 known liposidomycins (3 and 5-17) from *Streptomyces* sp. TMPU-20A065. The structures of the new congeners were fully elucidated using spectroscopic techniques, revealing variations in their acyl side chains. Compound 1 has a decanoic acid moiety, compound 2 has a tetradec-5,8-dienoic acid moiety (with two cis double bonds), and compound 4 contains a 7-tetradecenoic acid moiety. All 17 compounds (1-17) exhibited *in vitro* activity against *M. avium* and *M. intracellulare*, with MIC values ranging from 2.0 to 64 µg/ml. Importantly, all 17 compounds demonstrated significant therapeutic efficacy in the *in vivo* silkworm infection model, with ED₅₀ values ranging from 0.12 to 3.7 µg larva⁻¹ g⁻¹. The *in vivo* activity suggests potential for the development of new drugs for the treatment of MAC infections. The detailed structural elucidation and biological activity data provide valuable information for structure-activity relationship (SAR) studies and further optimization of these compounds as anti-MAC agents.

This research successfully identified a novel group of anti-MAC agents, expanding the repertoire of potential therapeutic options. The *in vivo* efficacy observed in the silkworm model is promising, particularly given the limitations of existing treatments. The variation in acyl side chains among the liposidomycin congeners suggests avenues for future SAR studies to optimize potency and possibly reduce toxicity. The findings address the research question by demonstrating the potential of liposidomycins as effective anti-MAC agents. The development of these compounds could contribute significantly to the treatment of MAC infections, offering a new approach to combat this increasingly prevalent disease. Further studies are needed to evaluate their pharmacokinetic and pharmacodynamic properties, as well as their safety and efficacy in mammalian models before clinical trials.

This study successfully identified three new liposidomycin congeners and 14 known ones as potent anti-MAC agents, exhibiting both *in vitro* and *in vivo* efficacy. The structural characterization of the new congeners provides insights for future SAR studies. This discovery holds promise for developing novel treatments against MAC infections, addressing a critical unmet medical need. Further investigations into the compounds' pharmacokinetics, pharmacodynamics, and potential toxicity in higher organisms are warranted.

The study used a silkworm infection model, which may not perfectly mimic human MAC infection. Further studies in mammalian models are necessary to confirm the efficacy and safety of these compounds. The limited quantities of isolated compounds, especially the new congeners, restricted comprehensive pharmacological investigations.