Combinatorial metabolic engineering of Streptomyces sp. CB03234-S for the enhanced production of anthraquinone-fused enediyne tiancimycins.

BACKGROUND: Anthraquinone-fused enediynes (AFEs) are excellent payloads for antibody-drug conjugates (ADCs). The yields of AFEs in the original bacterial hosts are extremely low. Multiple traditional methods had been adopted to enhance the production of the AFEs. Despite these efforts, the production titers of these compounds are still low, presenting a practical challenge for their development. Tiancimycins (TNMs) are a class of AFEs produced by Streptomyces sp. CB03234. One of their salient features is that they exhibit rapid and complete cell killing ability against various cancer cell lines. RESULTS: In this study, a combinatorial metabolic engineering strategy guided by the CB03234-S genome and transcriptome was employed to improve the titers of TNMs. First, re-sequencing of CB03234-S (Ribosome engineered mutant strains) genome revealed the deletion of a 583-kb DNA fragment, accounting for about 7.5% of its genome. Second, by individual or combined inactivation of seven potential precursor competitive biosynthetic gene clusters (BGCs) in CB03234-S, a double-BGC inactivation mutant, S1009, was identified with an improved TNMs titer of 28.2 ± 0.8 mg/L. Third, overexpression of five essential biosynthetic genes, including two post-modification genes, and three self-resistance auxiliary genes, was also conducted, through which we discovered that mutants carrying the core genes, tnmE or tnmE10, exhibited enhanced TNMs production. The average TNMs yield reached 43.5 ± 2.4 mg/L in a 30-L fermenter, representing an approximately 360% increase over CB03234-S and the highest titer among all AFEs to date. Moreover, the resulting mutant produced TNM-W, a unique TNM derivative with a double bond instead of a common ethylene oxide moiety. Preliminary studies suggested that TNM-W was probably converted from TNM-A by both TnmE and TnmE10. CONCLUSIONS: Based on the genome and transcriptome analyses, we adopted a combined metabolic engineering strategy for precursor enrichment and biosynthetic pathway reorganization to construct a high-yield strain of TNMs based on CB03234-S. Our study establishes a solid basis for the clinical development of AFE-based ADCs.

Discovery of a DNA Topoisomerase I Inhibitor Huanglongmycin N and Its Congeners from Streptomyces sp. CB09001.

Huanglongmycin (HLM) congeners G-N (7-14) were isolated from Streptomyces sp. CB09001. Among them, 10-12 possesses a tricyclic scaffold with benzene-fused pyran/pyrone, confirmed by X-ray single crystal diffraction analysis of 12. The structure-activity relationship study of 1, 13, and 14 revealed not only the stronger cytotoxicity of 14 against tested cancer cells but also the critical role of the C-7 ethyl group of 14 in its binding to the DNA-topoisomerase I complex.

Synthesis and biological evaluation of platensic alcohol as an adamantane surrogate in antitumor drug lead adaphostin.

Adamantane has been widely used as a "lipophilic bullet" in drug discovery and development, due to its unique diamond-like architecture with benign pharmacological/ pharmaceutical properties. Platensimycin is a natural product isolated from a soil streptomycete, which contains an adamantane-like moiety extensively modified from a diterpenoid precursor. In the current study, platensic alcohol was semisynthesized from platensimycin and used as an adamantane surrogate in anticancer drug lead adaphostin. The resulting hybrid platensic alcohol/adaphostin compounds, eg. 4a and 4b, exhibited similar cytotoxic activity with adaphostin against the tested cancer cell lines. In particular, 4b generates significantly more reactive oxygen species (ROS) and shows stronger synergy with the clinically used histone deacetylase inhibitor vorinostat than adaphostin, probably due to the presence of two hydroquinone groups. Density functional theory calculation supports that there could be certain π-π stacking interaction in 4b in aqueous solution, which might explain that 4b has similar serum stability with adaphostin. Our study not only leads to the identification of 4b as a potent ROS generating agent, but showcases a simple scaffold hopping strategy to harvest lipophilic scaffolds from natural products.

Stereoselective functionalization of platensimycin and platencin by sulfa-Michael/aldol reactions.

Bioinspired sulfa-Michael/aldol cascade reactions have been developed for the semisynthesis of sulfur-containing heterocyclic derivatives of platensimycin and platencin, with three newly formed contiguous stereogenic centers. Density functional theory calculations revealed the mechanism for the stereochemistry control. This method was used in a synthesis of a platensimycin thiophene analogue with potent antibacterial activities against Staphylococcus aureus.

Yangpumicins F and G, Enediyne Congeners from Micromonospora yangpuensis DSM 45577.

Enediyne natural products are among the most cytotoxic small molecules and thus excellent payload candidates for the development of antibody-drug conjugates (ADCs). Here we report the isolation and structural elucidation of two new 10-membered anthraquinone-fused enediynes, yangpumicins (YPM) F (6) and G (7), together with five known congeners, YPM A-E (1-5), from Micromonospora yangpuensis DSM 45577. YPM F (6) and G (7) showed strong cytotoxicity against the tested human cancer cell lines, as well as activity against several Gram-positive and Gram-negative pathogens. The 1,2-diols in 6 and 7 promise to enable new linker chemistry for the development of YPM-based ADCs.

Biomimetic Stereoselective Sulfa-Michael Addition Leads to Platensimycin and Platencin Sulfur Analogues against Methicillin-Resistant Staphylococcus aureus.

Several sulfur-containing platensimycin (PTM) and platencin (PTN) analogues, with activities comparable to the parent natural products, have recently been discovered from microorganisms, implying a biomimetic route to diversify the PTM and PTN scaffolds for structure-activity relationship study. We present here a substrate-directed and scaleable semisynthetic strategy to make PTM and PTN sulfur analogues with excellent diasteroselectivity, without using any chiral catalysts. Most of the sulfur analogues showed strong activities against clinical Staphylococcus aureus isolates, with minimum inhibitory concentrations of 0.5-2 µg mL-1. Density functional theory calculations were in agreement with the observed selectivity for these analogues and suggest that the conformation restraints of the terpene cages of PTM and PTN on the transition states determine the si-face attack selectivity.

A nordehydroabietyl amide-containing chiral diene for rhodium-catalysed asymmetric arylation to nitroolefins.

A highly enantioselective rhodium catalysed asymmetric arylation (RCAA) of nitroolefins with arylboronic acids is presented using a newly developed, C1-symmetric, non-covalent interacted, phellandrene derived, nordehydroabietyl amide-containing chiral diene under mild conditions. Stereoelectronic effects were studied, suggesting an activation of the bound substrate through the secondary amide as a hydrogen-bond donor.

Anti-HER2 Immunoliposomes: Antitumor Efficacy Attributable to Targeted Delivery of Anthraquinone-Fused Enediyne.

Although natural products are essential sources of small-molecule antitumor drugs, some can exert substantial toxicities, limiting their clinical utility. Anthraquinone-fused enediyne natural products are remarkably potent antitumor drug candidates, and uncialamycin and tiancimycin (TNM) A are under development as antibody-drug conjugates. Herein, a novel drug delivery system is introduced for TNM A using anti-human epidermal growth factor receptor 2 (HER2) immunoliposomes (ILs). Trastuzumab-coated TNM A-loaded ILs (HER2-TNM A-ILs) is engineered with an average particle size of 182.8 ± 2.1 nm and a zeta potential of 1.75 ± 0.12 mV. Compared with liposomes lacking trastuzumab, HER2-TNM A-ILs exhibited selective toxicity against HER2-positive KPL-4 and SKBR3 cells. Coumarin-6, a fluorescent TNM A surrogate, is encapsulated within anti-HER2 ILs; the resultant ILs have enhanced cellular uptake in KPL-4 and SKBR3 cells when compared with control liposomes. Furthermore, ILs loaded with more Cy5.5 accumulated in KPL-4 mouse tumors. A single HER2-TNM A-IL dose (0.02 mg kg-1) suppressed the growth of HER2-positive KPL-4 mouse tumors without apparent toxicity. This study not only provides a straightforward method for the effective delivery of TNM A against HER2-positive breast tumors but also underscores the potential of IL-based drug delivery systems when employing highly potent cytotoxins as payloads.

DNA Interaction and Cleavage Modes of Anthraquinone-Fused Enediynes: A Study on Tiancimycins, Yangpumicins, and Their Semisynthetic Analogues.

Dynemicin A has been the sole prototypical anthraquinone-fused enediyne (AFE) explored since its discovery in 1989. This study investigates the distinct DNA binding and cleavage mechanisms of emerging AFEs, represented by tiancimycins and yangpumicins, along with semisynthetic analogues. Our findings reveal their potent cytotoxicity against various tumor cell lines, while 18-methoxy tiancimycin A treatment could significantly suppress breast tumor growth with minimal toxicity. One of the most potent AFEs, i.e., tiancimycin A, preferentially targets DNA sequences 5'-ATT, 5'-CTT, 5'-GAA, 5'-GAT, and 5'-TTA. Molecular dynamics simulations suggest that emerging AFEs intercalate deeper into AT-rich DNA base pairs compared to dynemicin A. Importantly, tiancimycin A may equilibrate between insertional and intercalative modes without deintercalation, enabling selective cleavage of T and A bases. This study underscores how subtle structural variations among AFEs significantly influence their DNA recognition and cleavage, facilitating future design of novel AFEs as potent and selective payloads for antibody-drug conjugates.

Morphing Natural Product Platensimycin via Heck, Sonogashira, and One-Pot Sonogashira/Cycloaddition Reactions to Produce Antibiotics with In Vivo Activity.

Type II fatty acid synthases are promising drug targets against major bacterial pathogens. Platensimycin (PTM) is a potent inhibitor against ß-ketoacyl-[acyl carrier protein] synthase II (FabF) and ß-ketoacyl-[acyl carrier protein] synthase I (FabB), while the poor pharmacokinetics has prevented its further development. In this work, thirty-two PTM derivatives were rapidly prepared via Heck, Sonogashira, and one-pot Sonogashira/cycloaddition cascade reactions based on the Gram-scale synthesis of 6-iodo PTM (4). About half of the synthesized compounds were approximately equipotent to PTM against the tested Staphylococcus aureus strains. Among them, the representative compounds 4, A4, and B8 exhibited different plasma protein binding affinity or stability in the human hepatic microsome assay and showed improved in vivo efficacy over PTM in a mouse peritonitis model. In addition, A4 was also effective in an S. aureus-infected skin mouse model. Our study not only significantly expands the known PTM derivatives with improved antibacterial activities in vivo, but showcased that C-C cross-coupling reactions are useful tools to functionalize natural product drug leads.

Semisynthesis and Biological Evaluation of Platencin Thioether Derivatives: Dual FabF and FabH Inhibitors against MRSA.

The discovery and clinical use of multitarget monotherapeutic antibiotics is regarded as a promising approach to reduce the development of antibiotic resistance. Platencin (PTN), a potent natural antibiotic initially isolated from a soil actinomycete, targets both FabH and FabF, the initiation and elongation condensing enzymes for bacterial fatty acid biosynthesis. However, its further clinical development has been hampered by poor pharmacokinetics. Herein we report the semisynthesis and biological evaluation of platencin derivatives 1-15 with potent antibacterial activity against methicillin-resistant Staphylococcus aureus in vitro. Some of these PTN analogues showed similar yet distinct interactions with FabH and FabF, as shown by molecular docking, differential scanning fluorometry, and isothermal titration calorimetry. Compounds 3, 8, 10, and 14 were further evaluated in a mouse peritonitis model, among which 8 showed in vivo antibacterial activity comparable to that of PTN. Our results suggest that semisynthetic modification of PTN is a rapid route to obtain active PTN derivatives that might be further developed as promising antibiotics against drug-resistant major pathogens.

Syn-2, 3-diols and anti-inflammatory indole derivatives from Streptomyces sp. CB09001.

Two new natural diols, (2S, 3S, 4S)-4-methyl-1-phenylhexane-2,3-diol (1) and (2S, 3S)-4-methyl-1-phenylpentane-2,3-diol (2), together with five known compounds, xenocyloins B-D (3-5), lumichrome (6) and thymidine (7) were isolated from Streptomyces sp. CB09001. The absolute configurations of 1 and 2 were established by crystallographic structure analysis. The anti-inflammatory effects of 1-7 were also investigated in RAW246.7 murine macrophage cells stimulated by lipopolysaccharide. The indole derivative xenocyloin B (3) significantly inhibited inducible nitric oxide synthase expression in RAW264.7 cells and could be a potential anti-inflammatory drug lead.

Late-Stage Functionalization of Platensimycin Leading to Multiple Analogues with Improved Antibacterial Activity in Vitro and in Vivo.

Bacterial fatty acid synthases are promising antibacterial targets against multidrug-resistant pathogens. Platensimycin (PTM) is a potent FabB/FabF inhibitor, while its poor pharmacokinetics hampers the clinical development. In this study, a focused library of PTM derivatives was prepared through thiolysis of PTM oxirane (1), followed by various C-C cross-coupling reactions in high yields. Antibacterial screening of these compounds in vitro yielded multiple hits with improved anti-Staphylococcus activities over PTM. Among them, compounds A1, A3, A17, and A28 exhibited improved antibacterial activities over PTM against methicillin-resistant Staphylococcus aureus (MRSA) in a mouse peritonitis model. Compound A28 was further shown to be effective against MRSA infection in a mouse wound model, in comparison to mupirocin. Therefore, the facile preparation and screening of these PTM derivatives, together with their potent antibacterial activities in vivo, suggest a promising strategy to improve the antibacterial activity and pharmacokinetic properties of PTM.

Semisynthesis of Platensimycin Derivatives with Antibiotic Activities in Mice via Suzuki-Miyaura Cross-Coupling Reactions.

Platensimycin (PTM), originally isolated from soil bacteria Streptomyces platensis, is a potent FabF inhibitor against many Gram-positive pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci. However, the further clinical development of PTM is hampered by its poor pharmacokinetic properties. In this study, 20 PTM derivatives were prepared by Suzuki-Miyaura cross-coupling reactions catalyzed by Pd (0)/C. Compared to PTM, 6-pyrenyl PTM (6t) showed improved antibacterial activity against MRSA in a mouse peritonitis model. Our results support the strategy to target the essential fatty acid synthases in major pathogens, in order to discover and develop new generations of antibiotics.