Anthraquinone-fused enediynes: discovery, biosynthesis and development.

Covering: up to the end of July, 2021Anthraquinone-fused enediynes (AFEs) are a subfamily of enediyne natural products. Dynemicin A (DYN A), the first member of the AFE family, was discovered more than thirty years ago. Subsequently, extensive studies have been reported on the mode of action and the interactions of AFEs with DNA using DYN A as a model. However, progress in the discovery, biosynthesis and clinical development of AFEs has been limited for a long time. In the past five years, four new AFEs have been discovered and significant progress has been made in the biosynthesis of AFEs, especially on the biogenesis of the anthraquinone moiety and their tailoring steps. Moreover, the streamlined total synthesis of AFEs and their analogues boosts the preparation of AFE-based linker-drugs, thus enabling the development of AFE-based antibody-drug conjugates (ADCs). This review summarizes the discovery, mechanism of action, biosynthesis, total synthesis and preclinical studies of AFEs.

Synthesis and biological properties of maleimide-based macrocyclic lactone enediynes.

Natural enediyne antibiotics are powerful DNA-cleavage agents due to the presence of the highly reactive hex-3-ene-1,5-diyne units. However, the complicated chemical structure and thermal instability make their synthesis, derivatization, and storage challenging. Heterocycle-fused enediynes, which exhibit strong antineoplastic activity, are promising analogues of natural enediynes for medicinal applications. To this end, a series of maleimide-based enediynes with macrocyclic lactone moieties were synthesized through the Sonagashira coupling reaction. Differential scanning calorimetry and electron paramagnetic resonance results showed that these macrocyclic enediynes exhibited a rather low onset temperature and the ability to generate radicals at physiological temperature. In addition, the structure-activity relationship of enediynes was analyzed by changing the ring size and the substituents on the propargyl group. Cellular experiments indicated that the diradicals produced by these enediynes efficiently cleaved DNA and disrupted the cell cycle distribution, and consequently induced tumor cell death via an apoptosis pathway at low half inhibitory concentrations. Computational studies suggested that the maleimide moiety promoted the propargyl-allenyl rearrangement of the cyclic enediyne, enabling the generation of diradical species through the Myers-Saito cyclization, and then abstracted hydrogen atoms from the H-donors.

Characterization and initial demonstration of in vivo efficacy of a novel heat-activated metalloenediyne anti-cancer agent.

BACKGROUND: Enediynes are anti-cancer agents that are highly cytotoxic due to their propensity for low thermal activation of radical generation. The diradical intermediate produced from Bergman cyclization of the enediyne moiety may induce DNA damage and cell lethality. The cytotoxicity of enediynes and difficulties in controlling their thermal cyclization has limited their clinical use. We recently showed that enediyne toxicity at 37 °C can be mitigated by metallation, but cytotoxic effects of 'metalloenediynes' on cultured tumor cells are potentiated by hyperthermia. Reduction of cytotoxicity at normothermia suggests metalloenediynes will have a large therapeutic margin, with cell death occurring primarily in the heated tumor. Based on our previous in vitro findings, FeSO4-PyED, an Fe co-factor complex of (Z)-N,N'-bis[1-pyridin-2-yl-meth-(E)-ylidene]oct-4-ene-2,6-diyne-1,8-diamine, was prioritized for further in vitro and in vivo testing in normal human melanocytes and melanoma cells. METHODS: Clonogenic survival, apopotosis and DNA binding assays were used to determine mechanisms of enhancement of FeSO4-PyED cytotoxicity by hyperthermia. A murine human melanoma xenograft model was used to assess in vivo efficacy of FeSO4-PyED at 37 or 42.5 °C. RESULTS: FeSO4-PyED is a DNA-binding compound. Enhancement of FeSO4-PyED cytotoxicity by hyperthermia in melanoma cells was due to Bergman cyclization, diradical formation, and increased apoptosis. Thermal enhancement, however, was not observed in melanocytes. FeSO4-PyED inhibited tumor growth when melanomas were heated during drug treatment, without inducing normal tissue damage. CONCLUSION: By leveraging the unique thermal activation properties of metalloenediynes, we propose that localized moderate hyperthermia can be used to confine the cytotoxicity of these compounds to tumors, while sparing normal tissue.

Antifeedant and Antifungal Activities of Metabolites Isolated from the Coculture of Endophytic Fungus Aspergillus tubingensis S1120 with Red Ginseng.

A new globoscinic acid derivative, aspertubin A (1) along with four known compounds, were obtained from the co-culture of Aspergillus tubingensis S1120 with red ginseng. The chemical structures of compounds were characterized by using spectroscopic methods, the calculated and experimental electronic circular dichroism. Panaxytriol (2) from red ginseng, and asperic acid (4) showed significant antifeedant effect with the antifeedant rates of 75 % and 80 % at the concentrations of 50 µg/cm2 . Monomeric carviolin (3) and asperazine (5) displayed weak attractant activity on silkworm. All compounds were assayed for antifungal activities against phytopathogens A. tubingensis, Nigrospora oryzae and Phoma herbarum and the results indicated that autotoxic aspertubin A (1) and panaxytriol (2) possessed selective inhibition against A. tubingensis with MIC values at 8 µg/mL. The co-culture extract showed higher antifeedant and antifungal activities against P. herbarum than those of monoculture of A. tubingensis in ordinary medium. So the medicinal plant and endophyte showed synergistic effect on the plant disease resistance by active compounds from the coculture of A. tubingensis S1120 and red ginseng.

Real-time monitoring and effector screening of APE1 based on rGO assisted DNA nanoprobe.

Human apurinic/pyrimidine endonuclease 1 (APE1) played a critical role in the occurrence, progress and prognosis of tumors through overexpression and subcellular localization. Thus, it has become an important target for enhancing the sensitivity of tumor cells to radiotherapy and chemotherapy. Therefore, detecting and imaging its intracellular activity is of great significance for inhibitor discovery, cancer diagnosis and therapy. In this work, using DNA-based nanoprobe, we developed a new method for monitor intracellular APE1 activity. The detecting system was consisted by single fluorophore labeled hairpin probe and reduced graphene oxide (rGO). The in vitro result showed that a liner response of the detection method ranged from 0.02 U/mL to 2 U/mL with a limit of detection of 0.02 U/mL. Furthermore, this strategy possessing high specificity was successfully applied for APE1-related inhibitor screening using intracellular fluorescence imaging. Panaxytriol, an effective inhibitor of APE1 activity, was screened from traditional Chinese medicine (TCM) and its effect on APE1 activity was monitored in real time in A549 cells. In summary, this sensitive and specific APE1 detection technology is expected to provide an assistance for APE1-related inhibitor screening and diseases diagnosis.

Inhibitory effect of panaxytriol on BV-2 microglial cell activation.

Activated microglia induce brain inflammation and neuronal death. Panaxytriol, ((3R,9R,10R)-Heptadec-1-en-4,6-diyne-3,9,10-triol), is a component of Panax ginseng C. A. Meyer extracts and activates the Nrf2-ARE signaling pathway. However, little is known about its effects on activated microglia in the brain. In this study, we investigated the effect of panaxytriol on lipopolysaccharide (LPS)-induced activated microglia in BV-2 cells. Panaxytriol suppressed LPS-induced NO production and inhibited the increase in iNOS protein expression in BV-2 cells. Besides, panaxytriol inhibited the mRNA expression of proinflammatory cytokines such as TNF-α, IL-1ß, and IL-6. The inhibitory effect of panaxytriol on microglia activation did not affect the Nrf2-ARE pathway and the MAPK pathway. However, panaxytriol suppressed LPS-induced NF-κB nuclear translocation. These results suggest that panaxytriol inhibits the LPS-induced activation of microglia via the inhibition of NF-κB signaling pathway.

Panaxytriol Inhibits Lipopolysaccharide-Induced Microglia Activation in Brain Inflammation in Vivo.

Brain inflammation is a pathological characteristic of neurodegenerative diseases. In this condition, excessively activated microglia elevate proinflammatory mediator levels. We previously reported that panaxytriol inhibited lipopolysaccharide (LPS)-induced microglia activation in vitro. However, the effects of panaxytriol on microglia activation in vivo require confirmation. In the present study, we found that panaxytriol suppressed both microglia and astrocyte activation by injected LPS intracerebrally to mice with LPS-induced brain inflammation. Panaxytriol was more effective on microglia than astrocytes. Moreover, panaxytriol tended to reduce LPS-induced spontaneous motor activity dysfunction. These results suggested that panaxytriol could improve brain health by suppressing microglia activation in neurodegenerative diseases.

Total Synthesis and Biological Evaluation of Tiancimycins A and B, Yangpumicin A, and Related Anthraquinone-Fused Enediyne Antitumor Antibiotics.

The family of anthraquinone-fused enediyne antitumor antibiotics was established by the discovery of dynemicin A and deoxy-dynemicin A. It was then expanded, first by the isolation of uncialamycin, and then by the addition to the family of tiancimycins A-F and yangpumicin A. This family of natural products provides opportunities in total synthesis, biology, and medicine due to their novel and challenging molecular structures, intriguing biological properties and mechanism of action, and potential in targeted cancer therapies. Herein, the total syntheses of tiancimycins A and B, yangpumicin A, and a number of related anthraquinone-fused enediynes are described. Biological evaluation of the synthesized compounds revealed extremely potent cytotoxicities against a number of cell lines, thus enriching the structure-activity relationships within this class of compounds. The findings of these studies may facilitate future investigations directed toward antibody-drug conjugates for targeted cancer therapies and provide inspiration for further advances in total synthesis and chemical biology.

Synthesis and Biological Evaluation of Shishijimicin A-Type Linker-Drugs and Antibody-Drug Conjugates.

Our previous studies with shishijimicin A resulted in the total synthesis of this scarce marine natural product and a number of its simpler analogues endowed with picomolar potencies against certain cancer cell lines. Herein, we describe the design, synthesis, and biological evaluation of four linker-drugs, anticipating the construction of antibody-drug conjugates (ADCs) as the ultimate goal of this research program. Using a common payload, the assembly of these linker-drugs utilized different linkers and attachment points, providing opportunities to probe the optimal molecular design of the intended ADCs as targeted cancer therapies. In the course of ADC generation and in vitro evaluation, we identified two linker-drugs with a promising in vitro plasma stability profile and excellent targeted cytotoxicity and specificity. Conjugation of shishijimicin A enediyne payloads through their phenolic moiety represents a novel approach to enediyne ADC creation, while the pharmacological profiles of at least two of the generated ADCs compare well with the profiles of the corresponding clinically approved ADC Kadcyla.

The Outliers: Metal-Mediated Radical Reagents for Biological Substrate Degradation.

The predictable and controllable interaction of small organic or peptidic molecules with biological substrates is the primary reason most pharmaceuticals are narrowly decorated carbon frameworks. The inhibition or activation binding models are measurable and without side reactions that can cause pathological angst. Yet many diseases, especially those involving rapid proliferation of cells (i.e., cancer) or aggregation of peptides (e.g., heart disease, Alzheimer's disease) have not yet been cured by inhibition therapeutics. Additionally, interventional medicine is often required to alleviate such maladies by physical removal first, followed by molecular-level therapy as a second stage. Thus, there appears to be a niche for more aggressive therapeutics that may employ harsher chemical processes to realize clinical efficacy, albeit without causing catastrophic side effects. Molecules that may be considered for this challenge are not typically biomimetic, nor do they fit the traditional pharmaceutical paradigm. They may have unusual modes of action or undesired reactivity that can be lethal if not controlled. These are the outliers; potential pharmacophores that biology does not know how to manage or adapt to. This is why they may be an intriguing class of agents that needs continuous development. In this Account, we connect the under-developed enediyne family of compounds and our metalloenediyne derivatives to existing radical-based therapeutics such as bleomycin and doxorubicin to illustrate that controlled diradical reactivity, although an outlier mechanism, has a place in the therapeutic portfolio. This is self-evident in that of the 11 natural product enediynes known, 2 have clinical impact, a strong ratio. We expand on the chemical diversity of potential enediyne constructs and focus on the accessible trigger mechanisms to activate diradical formation as a method to control toxicity. Moreover, we further illustrate how electromagnetic fields can be employed to activate both molecular and larger nanomaterial constructs that carry highly concentrated payloads of reactive reagent. Finally, we describe how controlled diradical reactivity can reach beyond traditional therapeutic targets such as DNA, to peptide aggregates found in blood clots, neural fibrils, and membrane scaffolds. It is our belief that cleverly constructed frameworks with well-designed and controlled activation/reaction schemes can lead to novel therapeutics that can challenge evolving viral and bacterial invaders. From this evangelical perspective, our hope is that the conceptual framework, if not the specific designs in this Account, stimulate the readership to develop out-of-the-box therapeutic designs that may combat resistant disease targets.

Triggering the Antitumor Activity of Acyclic Enediyne through Maleimide-Assisted Rearrangement and Cycloaromatization.

Acyclic enediynes are generally inactive under physiological conditions to be used as antitumor agents like their natural enediyne counterparts. A new mechanism named as maleimide-assisted rearrangement and cycloaromatization (MARACA) is uncovered to trigger the reactivity of acyclic enediynes. Through this mechanism, cascade 1,3-proton transfer processes are accelerated with the maleimide moiety at the ene position to enable the acyclic enediynes to undergo cycloaromatization and generate reactive radicals under physiological conditions. Computational studies suggest that the highest energy barrier for MARACA is 26.0 kcal/mol, much lower than that of Bergman cyclization pathway (39.6 kcal/mol). Experimental results show that maleimide-based enediynes exhibit low onset temperature, fast generation of radical species at 37 °C, and much faster reaction in aqueous solution than in nonpolar solvent, which is beneficial to achieve both high reactivity in physiological environment and high stability for storage and delivery in nonpolar media. The generated radical species are capable of causing high percentage of double-strand (ds) DNA cleavage, leading to significant cytotoxicity toward a panel of cancer cell lines with half inhibition concentration down to submicromolar level. Overall, the discovery of the MARACA mechanism provides a platform for designing novel acyclic enediynes with high potency for antitumor applications.

Effect of panaxytriol on cytochrome P450 3A4 via the pregnane X receptor regulatory pathway.

Panaxytriol (PXT) is one of the major effective components of red ginseng and Shenmai injection. The present study aimed to explore the effect of PXT on cytochrome P450 3A4 (CYP3A4) based on the pregnane X receptor (PXR)-CYP3A4 regulatory pathway in HepG2 cells and hPXR-overexpressing HepG2 cells treated with PXT for different time periods using quantitative polymerase chain reaction, Western blot, and dual-luciferase reporter gene assays. PXT could upregulate the levels of PXR and CYP3A4 mRNA in HepG2 cells treated with PXT for 1 hr, with no impact on the expression of their protein levels. The expression levels of both PXR and CYP3A4 mRNA and protein in HepG2 cells treated with PXT for 24 hr increased in a concentration-dependent manner. The effects of PXT on the expression of PXR and CYP3A4 mRNA and protein in hPXR-overexpressing HepG2 cells were similar to those in HepG2 cells. Moreover, the influence trend of PXT on CYP3A4 was consistent with that of PXR in HepG2 cells and hPXR-overexpressing HepG2 cells. The dual-luciferase reporter gene assay in HepG2 cells further demonstrated that PXT treatment for specific time periods could significantly induce the expression of CYP3A4 mediated by the PXR regulatory pathway.

Streamlined Total Synthesis of Shishijimicin A and Its Application to the Design, Synthesis, and Biological Evaluation of Analogues thereof and Practical Syntheses of PhthNSSMe and Related Sulfenylating Reagents.

Shishijimicin A is a scarce marine natural product with highly potent cytotoxicities, making it a potential payload or a lead compound for designed antibody-drug conjugates. Herein, we describe an improved total synthesis of shishijimicin A and the design, synthesis, and biological evaluation of a series of analogues. Equipped with appropriate functionalities for linker attachment, a number of these analogues exhibited extremely potent cytotoxicities for the intended purposes. The synthetic strategies and tactics developed and employed in these studies included improved preparation of previously known and new sulfenylating reagents such as PhthNSSMe and related compounds.

Photochemical Activation of Enediyne Warheads: A Potential Tool for Targeted Antitumor Therapy.

Spatial and temporal control over DNA cleavage by photoactivated enediynes can be complemented by additional factors such as the release of internal strain, chelation, pH changes, intramolecular H-bonds, and substituent effects. This review presents design and reactivity of photoactivated enediynes/enynes and analyses the chemical, biological, and photophysical challenges in their applications.

Relative Reactivity of Benzothiophene-Fused Enediynes in the Bergman Cyclization.

To find promising analogues of naturally occurring enediyne antibiotics with a sufficient reactivity in the Bergman cyclization and moderately stable under isolation and storage, a scale of relative enediynes reactivity was created on the basis of calculated free activation energies for the Bergman cyclization within 12 known and new benozothiophene, benzene, and cinnoline annulated 9- and 10-membered enediynes. To verify the predicted reactivity/stability balance, three new carbocyclic enediynes fused to a benzothiophene core bearing 3,4,5-trimethoxybenzene, fluoroisopropyl, and isopropenyl substituents were synthesized using the Nicholas-type macrocyclization. It was confirmed that annulation of a 3,4,5-trimethoxybenzene moiety to a 10-membered enediyne macrocycle imparts high reactivity to an enediyne while also conferring instability under ambient temperature. Fluoroisopropyl-substituted 10-membered enediyne from the opposite end of the scale was found to be stable while moderately reactive in the Bergman cyclization. Along with the experimentally confirmed moderate reactivity (DSC kinetic studies), (fluoroisopropyl)enediyne showed a significant DNA damaging activity in plasmid cleavage assays comparable with the known anticancer drug Zeocin.

Enediyne-based protein capture agents: demonstration of an enediyne moiety acting as a photoaffinity label.

Two enediyne based protein-capture compounds 1 and 2 were synthesized. Both these molecules have an aryl sulfonamide for reversible binding with Human Carbonic Anhydrase II (HCA II) and a pyrene moiety for the visualization of a capture event. While compound 1 has an aryl azide as a photo cross-linking agent, compound 2 lacks the azide moiety. Capture experiments with HCA II however show that both 1 and 2 can photo cross-link with the protein as indicated in gel electrophoresis as well as MALDI analysis after tryptic digestion of HCA II. This observation demonstrates the ability of the enediyne moiety to act as a photo-affinity label possibly via the addition of nucleophilic amino acids to the partially zwitterionic singlet form of the diradical generated by photo Bergman cyclization.

Alkoxy and Enediyne Derivatives Containing 1,4-Benzoquinone Subunits-Synthesis and Antitumor Activity.

The compounds produced by a living organism are most commonly as medicinal agents and starting materials for the preparation of new semi-synthetic derivatives. One of the largest groups of natural compounds consists of products containing a 1,4-benzoquinone subunit. This fragment occurs in three enediyne antibiotics, dynemicin A, deoxydynemicin A, and uncilamicin, which exhibit high biological activity. A series of alkoxy derivatives containing 1,4-naphthoquinone, 5,8-quinolinedione, and 2-methyl-5,8-quinolinedione moieties was synthesized. Moreover, the 1,4-benzoquinone subunit was contacted with an enediyne fragment. All obtained compounds were characterized by spectroscopy and spectrometry methods. The resulting alkane, alkene, alkyne and enediyne derivatives were tested as antitumor agents. They showed high cytotoxic activity depending on the type of 1,4-benzoquinone subunit and the employed tumor cell lines. The synthesized derivatives fulfill the Lipinski Rule of Five and have low permeability through the blood-brain barrier.

Engineered production of cancer targeting peptide (CTP)-containing C-1027 in Streptomyces globisporus and biological evaluation.

Conjugation of cancer targeting peptides (CTPs) with small molecular therapeutics has emerged as a promising strategy to deliver potent (but typically nonspecific) cytotoxic agents selectively to cancer cells. Here we report the engineered production of a CTP (NGR)-containing C-1027 and evaluation of its activity against selected cancer cell lines. C-1027 is an enediyne chromoprotein produced by Streptomyces globisporus, consisting of an apo-protein (CagA) and an enediyne chromophore (C-1027). NGR is a CTP that targets CD13 in tumor vasculature. S. globisporus SB1026, a recombinant strain engineered to encode CagA with the NGR sequence fused at its C-terminus, directly produces the NGR-containing C-1027 that is equally active as the native C-1027. Our results demonstrate the feasibility to produce CTP-containing enediyne chromoproteins by metabolic pathway engineering and microbial fermentation and will inspire efforts to engineer other CTP-containing drug binding proteins for targeted delivery.

[Advances in studies on chemical compositions of Atractylodes lancea and their biological activities].

This article mainly summarises the results of the chemical compositions and its pharmacological activities of Atractylodes Radix. The chemistry components isolated from Atractylodes Radix are mainly sesquiterpenoids, enediynes, triterpenoids, aromatic glycosides, and etc. Pharmacological results showed that Atractylodes Radix has inhibition of gastric acid secretion, promoting gastrointestinal movement and gastric emptying, hpyerglycemic, antibacterial, anti-inflammatory, cardiovascular protection and nervous system activity, etc. This article hopefully to provide a reference for further research, development and utilization of Atractylodes Radix.

FUS is phosphorylated by DNA-PK and accumulates in the cytoplasm after DNA damage.

Abnormal cytoplasmic accumulation of Fused in Sarcoma (FUS) in neurons defines subtypes of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). FUS is a member of the FET protein family that includes Ewing's sarcoma (EWS) and TATA-binding protein-associated factor 2N (TAF15). FET proteins are predominantly localized to the nucleus, where they bind RNA and DNA to modulate transcription, mRNA splicing, and DNA repair. In ALS cases with FUS inclusions (ALS-FUS), mutations in the FUS gene cause disease, whereas FTLD cases with FUS inclusions (FTLD-FUS) do not harbor FUS mutations. Notably, in FTLD-FUS, all FET proteins accumulate with their nuclear import receptor Transportin 1 (TRN1), in contrast ALS-FUS inclusions are exclusively positive for FUS. In the present study, we show that induction of DNA damage replicates several pathologic hallmarks of FTLD-FUS in immortalized human cells and primary human neurons and astrocytes. Treatment with the antibiotic calicheamicin γ1, which causes DNA double-strand breaks, leads to the cytoplasmic accumulation of FUS, TAF15, EWS, and TRN1. Moreover, cytoplasmic translocation of FUS is mediated by phosphorylation of its N terminus by the DNA-dependent protein kinase. Finally, we observed elevated levels of phospho-H2AX in FTLD-FUS brains, indicating that DNA damage occurs in patients. Together, our data reveal a novel regulatory mechanism for FUS localization in cells and suggest that DNA damage may contribute to the accumulation of FET proteins observed in human FTLD-FUS cases, but not in ALS-FUS.