Tunable and Photoactivatable Mimics of Calicheamicin γ1 for DNA Cleavage.

Calicheamicin γ1 and related natural products are renowned for their potency in DNA cleavage, serving as the warheads in commercial ADCs used for treating leukemia. Their mechanism of action involves the formation of aryl radicals, which abstract hydrogen atoms from nucleic acids. However, the complex strained enediyne structure of calicheamicin γ1 presents significant challenges in synthesis, resulting in high production costs and limited structural and activity modularity for tuning the therapeutic window. This report describes the development of simple molecular mimics based on diazonium salts, synthesized in fewer than 3 steps, capable of generating aryl radicals upon green or red light irradiation. SAR studies conducted on over 30 analogues reveal a wide range of potencies in DNA cleavage, with EC50 values ranging from low nanomolar to micromolar. Forming benzenoid diradicals does not appear to be necessary for potent DNA cleavage; instead, DNA cleavage can be achieved with radicals distributed among different arenes when connected with proper linkages. The potency is influenced by electronic effects, stereochemistry, orbital orientations, the distance between multiradicals, and the number of diazonium motifs within the molecule. In addition to providing a more cost-effective, efficient, and modular alternative to calicheamicin γ1, this technology offers the potential for enhanced specificity through spatiotemporal control.

Genome mining for new enediyne antibiotics.

Enediyne antibiotics epitomize nature's chemical creativity. They contain intricate molecular architectures that are coupled with potent biological activities involving double-stranded DNA scission. The recent explosion in microbial genome sequences has revealed a large reservoir of novel enediynes. However, while hundreds of enediyne biosynthetic gene clusters (BGCs) can be detected, less than two dozen natural products have been characterized to date as many clusters remain silent or sparingly expressed under standard laboratory growth conditions. This review focuses on four distinct strategies, which have recently enabled discoveries of novel enediynes: phenotypic screening from rare sources, biosynthetic manipulation, genomic signature-based PCR screening, and DNA-cleavage assays coupled with activation of silent BGCs via high-throughput elicitor screening. With an abundance of enediyne BGCs and emerging approaches for accessing them, new enediyne natural products and further insights into their biogenesis are imminent.

Enediyne natural product biosynthesis unified by a diiodotetrayne intermediate.

Enediyne natural products are renowned for their potent cytotoxicities but the biosynthesis of their defining 1,5-diyne-3-ene core moiety remains largely enigmatic. Since the discovery of the enediyne polyketide synthase cassette in 2002, genome sequencing has revealed thousands of distinct enediyne biosynthetic gene clusters, each harboring the conserved enediyne polyketide synthase cassette. Here we report that (1) the products of this cassette are an iodoheptaene, a diiodotetrayne and two pentaynes; (2) the diiodotetrayne represents a common biosynthetic intermediate for all known enediynes; and (3) cryptic iodination can be exploited to increase enediyne titers. These findings establish a unified biosynthetic pathway for the enediynes, set the stage to further advance enediyne core biosynthesis and enable fundamental breakthroughs in chemistry, enzymology and translational applications of enediyne natural products.

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.

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.

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.

Cofactorless oxygenases guide anthraquinone-fused enediyne biosynthesis.

The anthraquinone-fused enediynes (AFEs) combine an anthraquinone moiety and a ten-membered enediyne core capable of generating a cytotoxic diradical species. AFE cyclization is triggered by opening the F-ring epoxide, which is also the site of the most structural diversity. Previous studies of tiancimycin A, a heavily modified AFE, have revealed a cryptic aldehyde blocking installation of the epoxide, and no unassigned oxidases could be predicted within the tnm biosynthetic gene cluster. Here we identify two consecutively acting cofactorless oxygenases derived from methyltransferase and α/ß-hydrolase protein folds, TnmJ and TnmK2, respectively, that are responsible for F-ring tailoring in tiancimycin biosynthesis by comparative genomics. Further biochemical and structural characterizations reveal that the electron-rich AFE anthraquinone moiety assists in catalyzing deformylation, epoxidation and oxidative ring cleavage without exogenous cofactors. These enzymes therefore fill important knowledge gaps for the biosynthesis of this class of molecules and the underappreciated family of cofactorless oxygenases.

Synthesis of an enediyne carbon-allotrope surface for photo-thermal degradation of DNA.

The improper disposal of hospital waste products containing genetic materials poses a serious safety threat. We present herein an environmentally friendly technology using a graphene-based novel carbon-allotropic surface to remediate such wastes. The used carbon-allotrope is decorated with an enediyne (EDE-1) enriched aromatic pi-conjugated structure to create an efficient and active surface for cleaving DNA strands. Under controlled exposure of ultraviolet (UV) radiation and heat, the developed surface influences genetic degradation without disturbing the bacterial populations present downstream of the water treatment system. The designed material has been extensively characterized using physicochemical and biological tools. Our results indicate that this approach can possibly be introduced in large scale hospital waste disposal streams for remediating genetic hazards and thereby developing a portable self-contained system.

Triggered Crosslinking of Main-Chain Enediyne Polyurethanes via Bergman Cyclization.

Crosslinking chemistries occupy an important position in polymer modification with a particular importance when triggered in response to external stimuli. Enediyne (EDY) moieties are used as functional entities in this work, known to undergo a pericyclic Bergman cyclization (BC) to induce a triggered crosslinking of polyurethanes (PU) via the intermediately formed diradicals. Diamino-EDYs, where the distance between the enyne-moieties is known to be critical to induce a BC, are placed repetitively as main-chain structural elements in isophorone-based PUs to induce reinforcement upon heating, compression, or stretching. A 7-day compression under room temperature results in a ≈69% activation of the BC, together with the observation of an increase in tensile strength by 62% after 25 stretching cycles. The occurrence of BC is further proven by the decreased exothermic values in differential scanning calorimetry, together with characteristic peaks of the formed benzene moieties via IR spectroscopy. Purely heat-induced crosslinking contributes to 191% of the maximum tensile strength in comparison to the virgin PU. The BC herein forms an excellent crosslinking strategy, triggered by heat or force in PU materials.

Targeted Discovery of Cryptic Enediyne Natural Products via FRET-Coupled High-Throughput Elicitor Screening.

Enediyne antibiotics are a striking family of DNA-cleaving natural products with high degrees of cytotoxicity and structural complexity. Microbial genome sequences, which have recently accumulated, point to an untapped trove of "cryptic" enediynes. Most of the cognate biosynthetic gene clusters (BGCs) are sparingly expressed under standard growth conditions, making it difficult to characterize their products. Herein, we report a fluorescence-based DNA cleavage assay coupled with high-throughput elicitor screening for the rapid, targeted discovery of cryptic enediyne metabolites. We applied the approach to Streptomyces clavuligerus, which harbors two such BGCs with unknown products, identified steroids as effective elicitors, and characterized 10 cryptic enediyne-derived natural products, termed clavulynes A-J with unusual carbonate and terminal olefin functionalities, with one of these congeners matching the recently reported jejucarboside. Our results contribute to the growing repertoire of enediynes and provide a blueprint for identifying additional ones in the future.

Jejucarbosides B-E, Chlorinated Cycloaromatized Enediynes, from a Marine Streptomyces sp.

Four new chlorinated cycloaromatized enediyne compounds, jejucarbosides B-E (1-4), were discovered together with previously-identified jejucarboside A from a marine actinomycete strain. Compounds 1-4 were identified as new chlorinated cyclopenta[a]indene glycosides based on 1D and 2D nuclear magnetic resonance, high-resolution mass spectrometry, and circular dichroism (CD) spectra. Jejucarbosides B and E bear a carbonate functional group whereas jejucarbosides C and D are variants possessing 1,2-diol by losing the carbonate functionality. It is proposed that the production of 1-4 occurs via Bergman cycloaromatization capturing Cl- and H+ in the alternative positions of a p-benzyne intermediate derived from a 9-membered enediyne core. Jejucarboside E (4) displayed significant cytotoxicity against human cancer cell lines including SNU-638, SK-HEP-1, A549, HCT116, and MDA-MB-231, with IC50 values of 0.31, 0.40, 0.25, 0.29, and 0.48 µM, respectively, while jejucarbosides B-D (1-3) showed moderate or no cytotoxic effects.

Metagenomic data reveals type I polyketide synthase distributions across biomes.

Microbial polyketide synthase (PKS) genes encode the biosynthesis of many biomedically or otherwise commercially important natural products. Despite extensive discovery efforts, metagenomic analyses suggest that only a small fraction of nature's polyketide biosynthetic potential has been realized. Much of this potential originates from type I PKSs (T1PKSs), which can be further delineated based on their domain organization and the structural features of the compounds they encode. Notably, phylogenetic relationships among ketosynthase (KS) domains provide an effective method to classify the larger and more complex T1PKS genes in which they occur. Increased access to large metagenomic data sets from diverse habitats provides opportunities to assess T1PKS biosynthetic diversity and distributions through their smaller and more tractable KS domain sequences. Here, we used the web tool NaPDoS2 to detect and classify over 35,000 type I KS domains from 137 metagenomic data sets reported from eight diverse, globally distributed biomes. We found biome-specific separation with soils enriched in KSs from modular cis-acetyltransferase (AT) and hybrid cis-AT KSs relative to other biomes and marine sediments enriched in KSs associated with polyunsaturated fatty acid and enediyne biosynthesis. We linked the phylum Actinobacteria to soil-derived enediyne and cis-AT KSs while marine-derived KSs associated with enediyne and monomodular PKSs were linked to phyla from which the compounds produced by these biosynthetic enzymes have not been reported. These KSs were phylogenetically distinct from those associated with experimentally characterized PKSs suggesting they may be associated with novel structures or enzyme functions. Finally, we employed our metagenome-extracted KS domains to evaluate the PCR primers commonly used to amplify type I KSs and identified modifications that could increase the KS sequence diversity recovered from amplicon libraries. IMPORTANCE Polyketides are a crucial source of medicines, agrichemicals, and other commercial products. Advances in our understanding of polyketide biosynthesis, coupled with the increased availability of metagenomic sequence data, provide new opportunities to assess polyketide biosynthetic potential across biomes. Here, we used the web tool NaPDoS2 to assess type I polyketide synthase (PKS) diversity and distributions by detecting and classifying ketosynthase (KS) domains across 137 metagenomes. We show that biomes are differentially enriched in type I KS domains, providing a roadmap for future biodiscovery strategies. Furthermore, KS phylogenies reveal biome-specific clades that do not include biochemically characterized PKSs, highlighting the biosynthetic potential of poorly explored environments. The large metagenome-derived KS data set allowed us to identify regions of commonly used type I KS PCR primers that could be modified to capture a larger extent of environmental KS diversity. These results facilitate both the search for novel polyketides and our understanding of the biogeographical distribution of PKSs across Earth's major biomes.

Normal and Aberrant Methyltransferase Activities Give Insights into the Final Steps of Dynemicin A Biosynthesis.

The naturally occurring enediynes are notable for their complex structures, potent DNA cleaving ability, and emerging usefulness in cancer chemotherapy. They can be classified into three distinct structural families, but all are thought to originate from a common linear C15-heptaene. Dynemicin A (DYN) is the paradigm member of anthraquinone-fused enediynes, one of the three main classes and exceptional among them for derivation of both its enediyne and anthraquinone portions from this same early biosynthetic building block. Evidence is growing about how two structurally dissimilar, but biosynthetically related, intermediates combine in two heterodimerization reactions to create a nitrogen-containing C30-coupled product. We report here deletions of two genes that encode biosynthetic proteins that are annotated as S-adenosylmethionine (SAM)-dependent methyltransferases. While one, DynO6, is indeed the required O-methyltransferase implicated long ago in the first studies of DYN biosynthesis, the other, DynA5, functions in an unanticipated manner in the post-heterodimerization events that complete the biosynthesis of DYN. Despite its removal from the genome of Micromonospora chersina, the ΔdynA5 strain retains the ability to synthesize DYN, albeit in reduced titers, accompanied by two unusual co-metabolites. We link the appearance of these unexpected structures to a substantial and contradictory body of other recent experimental data to advance a biogenetic rationale for the downstream steps that lead to the final formation of DYN. A sequence of product-forming transformations that is in line with new and existing experimental results is proposed and supported by a model reaction that also encompasses the formation of the crucial epoxide essential for the activation of DYN for DNA cleavage.

A discrete intermediate for the biosynthesis of both the enediyne core and the anthraquinone moiety of enediyne natural products.

The enediynes are structurally characterized by a 1,5-diyne-3-ene motif within a 9- or 10-membered enediyne core. The anthraquinone-fused enediynes (AFEs) are a subclass of 10-membered enediynes that contain an anthraquinone moiety fused to the enediyne core as exemplified by dynemicins and tiancimycins. A conserved iterative type I polyketide synthase (PKSE) is known to initiate the biosynthesis of all enediyne cores, and evidence has recently been reported to suggest that the anthraquinone moiety also originates from the PKSE product. However, the identity of the PKSE product that is converted to the enediyne core or anthraquinone moiety has not been established. Here, we report the utilization of recombinant E. coli coexpressing various combinations of genes that encode a PKSE and a thioesterase (TE) from either 9- or 10-membered enediyne biosynthetic gene clusters to chemically complement ΔPKSE mutant strains of the producers of dynemicins and tiancimycins. Additionally, 13C-labeling experiments were performed to track the fate of the PKSE/TE product in the ΔPKSE mutants. These studies reveal that 1,3,5,7,9,11,13-pentadecaheptaene is the nascent, discrete product of the PKSE/TE that is converted to the enediyne core. Furthermore, a second molecule of 1,3,5,7,9,11,13-pentadecaheptaene is demonstrated to serve as the precursor of the anthraquinone moiety. The results establish a unified biosynthetic paradigm for AFEs, solidify an unprecedented biosynthetic logic for aromatic polyketides, and have implications for the biosynthesis of not only AFEs but all enediynes.

Examining Heterodimerization by Aryl C-N Coupling in Dynemicin Biosynthesis.

Distinct among the enediyne antitumor antibiotics, the dynemicin subgroup is comprised of two discrete halves, an enediyne and an anthraquinone, but each is ultimately derived from the same linear ß-hydroxyhexaene precursor. The linkage of these two halves by an aryl C-N bond is examined here using a variety of experimental approaches. We demonstrate that this heterodimerization is specific for anthracenyl iodide as the corresponding bromo- and amino-substituted anthracenes do not support dynemicin biosynthesis. Furthermore, biochemical experiments and chemical model reactions support an SRN1 mechanism for the aryl C-N coupling in which electron transfer occurs to the iodoanthracene, followed by loss of an anthracenyl iodide and partition of the resulting aryl radical between C-N coupling and reduction by hydrogen abstraction. An enzyme pull-down experiment aiming to capture the protein(s) involved in the coupling reaction is described in which two proteins, Orf14 and Orf16, encoded by the dynemicin biosynthetic gene cluster, are specifically isolated. Deletion of orf14 from the genome abolished dynemicin production accompanied by a 3-fold increased accumulation of the iodoanthracene coupling partner, indicating the plausible involvement of this protein in the heterodimerization process. On the other hand, the deletion of orf16 only reduced dynemicin production to 55%, implying a noncatalytic, auxiliary role of the protein. Structural comparisons using AlphaFold imply key similarities between Orf14 and X-ray crystal structures of several proteins from enediyne BGCs believed to bind hydrophobic polyene or enediyne motifs suggest Orf14 templates aryl C-N bond formation during the central heterodimerization in dynemicin biosynthesis.

Intramolecular C-C Bond Formation Links Anthraquinone and Enediyne Scaffolds in Tiancimycin Biosynthesis.

First discovered in 1989, the anthraquinone-fused enediynes are a class of DNA-cleaving bacterial natural products composed of a DNA-intercalating anthraquinone moiety and a 10-membered enediyne warhead. However, until recently, there has been a lack of genetically amenable hosts and sequenced biosynthetic gene clusters available for solving the biosynthetic questions surrounding these molecules. Herein, we have identified and biochemically and structurally characterized TnmK1, a member of the α/ß-hydrolase fold superfamily responsible for the C-C bond formation linking the anthraquinone moiety and enediyne core together in tiancimycin (TNM) biosynthesis. In doing so, two intermediates, TNM H and TNM I, in anthraquinone-fused enediyne biosynthesis, containing an unprecedented cryptic C16 aldehyde group, were identified. This aldehyde plays a key role in the TnmK1-catalyzed C-C bond formation via a Michael addition, representing the first example of this chemistry for the α/ß-hydrolase fold superfamily. Additionally, TNM I shows sub-nanomolar cytotoxicity against selected cancer cell lines, indicating a new mechanism of action compared to previously known anthraquinone-fused enediynes. Together, the findings from this study are expected to impact enzymology, natural product biosynthesis, and future efforts at enediyne discovery and drug development.

Targeted Discovery of an Enediyne-Derived Cycloaromatized Compound, Jejucarboside A, from a Marine Actinomycete.

A genomic and spectroscopic signature-based search revealed a cycloaromatized enediyne, jejucarboside A (1), from a marine actinomycete strain. The structure of 1 was determined as a new cyclopenta[a]indene glycoside bearing carbonate functionality by nuclear magnetic resonance, high-resolution mass spectrometry (MS), MS/MS, infrared spectroscopy, and a modified Mosher's method. An iterative enediyne synthase pathway has been proposed for the putative biosynthesis of 1 by genomic analysis. Jejucarboside A exhibited cytotoxicity against the HCT116 colon carcinoma cells.

Functionalized 10-Membered Aza- and Oxaenediynes through the Nicholas Reaction.

The scope and limitations of the Nicholas-type cyclization for the synthesis of 10-membered benzothiophene-fused heterocyclic enediynes with different functionalities were investigated. Although the Nicholas cyclization through oxygen could be carried out in the presence of an ester group, the final oxaenediyne was unstable under storage. Among the N-type Nicholas reactions, cyclization via an arenesulfonamide functional group followed by mild Co-deprotection was found to be the most promising, yielding 10-membered azaendiynes in high overall yields. By contrast, the Nicholas cyclization through the acylated nitrogen atom did not give the desired 10-membered cycle. It resulted in the formation of a pyrroline ring, whereas cyclization via an alkylated amino group resulted in a poor yield of the target 10-membered enediyne. The acylated 4-aminobenzenesulfonamide nucleophilic group was found to be the most convenient for the synthesis of functionalized 10-membered enediynes bearing a clickable function, such as a terminal triple bond. All the synthesized cyclic enediynes exhibited moderate activity against lung carcinoma NCI-H460 cells and had a minimal effect on lung epithelial-like WI-26 VA4 cells and are therefore promising compounds in the search for novel antitumor agents that can be converted into conjugates with tumor-targeting ligands.

An EGFR/HER2-targeted conjugate sensitizes gemcitabine-sensitive and resistant pancreatic cancer through different SMAD4-mediated mechanisms.

Chemoresistance limits its clinical implementation for pancreatic ductal adenocarcinoma (PDAC). We previously generated an EGFR/HER2 targeted conjugate, dual-targeting ligand-based lidamycin (DTLL), which shows a highly potent antitumor effect. To overcome chemoresistance in PDAC, we aim to study DTLL efficacy when combined with gemcitabine and explore its mechanisms of action. DTLL in combination with gemcitabine show a superior inhibitory effect on the growth of gemcitabine-resistant/sensitive tumors. DTLL sensitizes gemcitabine efficacy via distinct action mechanisms mediated by mothers against decapentaplegic homolog 4 (SMAD4). It not only prevents neoplastic proliferation via ATK/mTOR blockade and NF-κB impaired function in SMAD4-sufficient PDACs, but also restores SMAD4 bioactivity to trigger downstream NF-κB-regulated signaling in SMAD4-deficient tumors and to overcome chemoresistance. DTLL seems to act as a SMAD4 module that normalizes its function in PDAC, having a synergistic effect in combination with gemcitabine. Our findings provide insight into a rational SMAD4-directed precision therapy in PDAC.