Andrew W. Robertson

Eight-membered ring-containing jadomycins: implications for non-enzymatic natural products biosynthesis.

Jadomycin Oct (1) was isolated from Streptomyces venezuelae ISP5230 and characterized as a structurally unique eight-membered l-ornithine ring-containing jadomycin. The structure was elucidated through the semisynthetic derivatization of starting material via chemoselective acylation of the l-ornithine α-amino group using activated succinimidyl esters. Incorporation of 5-aminovaleric acid led to jadomycin AVA, a second eight-membered ring-containing jadomycin. These natural products illustrate the structural diversity permissible from a non-enzymatic step within a biosynthetic pathway and exemplifies the potential for discovery of novel scaffolds.

Isolation and Synthetic Diversification of Jadomycin 4-Amino-l-phenylalanine.

Streptomyces venezuelae ISP5230 was grown in the presence of phenylalanine analogues to observe whether they could be incorporated into novel jadomycin structures. It was found that the bacteria successfully produced jadomycins incorporating 4-aminophenylalanine enantiomers. Upon isolation and characterization of jadomycin 4-amino-l-phenylalanine (1), it was synthetically derivatized, using activated succinimidyl esters, to yield a small jadomycin amide library. These are the first examples of oxazolone-ring-containing jadomycins that have incorporated an amino functionality subsequently used for derivatization.

Jadomycin breast cancer cytotoxicity is mediated by a copper-dependent, reactive oxygen species–inducing mechanism

Jadomycins are natural products biosynthesized by the bacteria Streptomyces venezuelae which kill drug‐sensitive and multidrug‐resistant breast cancer cells in culture. Currently, the mechanisms of jadomycin cytotoxicity are poorly understood; however, reactive oxygen species (ROS)–induced DNA cleavage is suggested based on bacterial plasmid DNA cleavage studies. The objective of this study was to determine if and how ROS contribute to jadomycin cytotoxicity in drug‐sensitive MCF7 (MCF7‐CON) and taxol‐resistant MCF7 (MCF7‐TXL) breast cancer cells. As determined using an intracellular, fluorescent, ROS‐detecting probe, jadomycins B, S, SPhG, and F dose dependently increased intracellular ROS activity 2.5‐ to 5.9‐fold. Cotreatment with the antioxidant N‐acetyl cysteine lowered ROS concentrations to below baseline levels and decreased the corresponding cytotoxic potency of the four jadomycins 1.9‐ to 3.3‐fold, confirming a ROS‐mediated mechanism. Addition of CuSO4 enhanced, whereas addition of the Cu(II)‐chelator d‐penicillamine reduced, the ROS generation and cytotoxicity of each jadomycin. Specific inhibitors of the antioxidant enzymes, superoxide dismutase 1, glutathione S‐transferase, and thioredoxin reductase, but not catalase, enhanced jadomycin‐mediated ROS generation and anticancer activity. In conclusion, the results indicate that jadomycin cytotoxicity involves the generation of cytosolic superoxide via a Cu(II)‐jadomycin reaction, a mechanism common to all jadomycins tested and observed in MCF7‐CON and drug‐resistant MCF7‐TXL cells. The superoxide dismutase 1, glutathione, and peroxiredoxin/thioredoxin cellular antioxidant enzyme pathways scavenged intracellular ROS generated by jadomycin treatment. Blocking these antioxidant pathways could serve as a strategy to enhance jadomycin cytotoxic potency in drug‐sensitive and multidrug‐resistant breast cancers.

Jadomycins Inhibit Type II Topoisomerases and Promote DNA Damage and Apoptosis in Multidrug-Resistant Triple-Negative Breast Cancer Cells

Jadomycins are natural products that kill drug-sensitive and multidrug-resistant (MDR) breast cancer cells. To date, the cytotoxic activity of jadomycins has never been tested in MDR breast cancer cells that are also triple negative. Additionally, there is only a rudimentary understanding of how jadomycins cause cancer cell death, which includes the induction of intracellular reactive oxygen species (ROS). We first created a paclitaxel-resistant, triple-negative breast cancer cell line [paclitaxel-resistant MDA-MB-231 breast cancer cells (231-TXL)] from drug-sensitive control MDA-MB-231 cells (231-CON). Using thiazolyl blue methyltetrazolium bromide cell viability–measuring assays, jadomycins B, S, and F were found to be equipotent in drug-sensitive 231-CON and MDR 231-TXL cells; and using ROS-detecting assays, these jadomycins were determined to increase ROS activity in both cell lines by up to 7.3-fold. Jadomycins caused DNA double-strand breaks in 231-CON and 231-TXL cells as measured by γH2AX Western blotting. Coincubation with the antioxidant N-acetyl cysteine or pro-oxidant auranofin did not affect jadomycin-mediated DNA damage. Jadomycins induced apoptosis in 231-CON and 231-TXL cells as measured by annexin V affinity assays, a process that was retained when ROS were inhibited. This indicated that jadomycins are capable of inducing MDA-MB-231 apoptotic cell death independently of ROS activity. Using quantitative polymerase chain reaction, Western blotting, and direct topoisomerase inhibition assays, it was determined that jadomycins inhibit type II topoisomerases and that jadomycins B and F selectively poison topoisomerase IIβ. We therefore propose novel mechanisms through which jadomycins induce breast cancer cell death independently of ROS activity, through inhibition or poisoning of type II topoisomerases and the induction of DNA damage and apoptosis.

Post Polyketide Synthase Carbon–Carbon Bond Formation in Type-II PKS-Derived Natural Products from Streptomyces venezuelae

Polyketide synthase (PKS) derived natural products are biosynthesized by head-to-tail addition of acetate and malonate extender units resulting in linear extended-polyketide chains. Despite the well-documented structural diversity associated with PKS-derived natural products, C-C chain branching deviating from the usual linear pattern is relatively rare. Herein, type-II PKS angucyclic natural products containing a hemiaminal functionality were identified and proposed as the parent of a series of C-C-branched analogues. These C-C linked acetate or pyruvate branching units were located at the α-positions on the extended polyketide chains of jadomycins incorporating 3- and 4-aminomethylbenzoic acids. Labeling studies utilizing [1-13C]-d-glucose provided mechanistic evidence that the C-C bond formation occurred as a result of a previously unidentified post-PKS processing, additional to the enzymes encoded within the biosynthetic gene cluster. Selected compounds were evaluated in cytotoxic or antimicrobial assays.

JadX is a Disparate Natural Product Binding Protein

We report that JadX, a protein of previously undetermined function coded for in the jadomycin biosynthetic gene cluster of Streptomyces venezuelae ISP5230, affects both chloramphenicol and jadomycin production levels in blocked mutants. Characterization of recombinant JadX through protein-ligand interactions by chemical shift perturbation and WaterLOGSY NMR spectroscopy resulted in the observation of binding between JadX and a series of jadomycins and between JadX and chloramphenicol, another natural product produced by S. venezuelae ISP5230. These results suggest JadX to be an unusual class of natural product binding protein involved in binding structurally disparate natural products. The ability for JadX to bind two different natural products in vitro and the ability to affect production of these secondary metabolites in vivo suggest a potential role in regulation or signaling. This is the first example of functional characterization of these JadX-like proteins, and provides insight into a previously unobserved regulatory process.

Characterization of l-Digitoxosyl-phenanthroviridin from Streptomyces venezuelae ISP5230.

The jadomycin-derived compound l-digitoxosyl-phenanthroviridin was isolated from fermentations of Streptomyces venezuelae ISP5230 grown in nutrient-deficient media with l-lysine as the sole nitrogen source. Structural elucidation was accomplished using a combination of high-resolution MS, LC-MS/MS, and 1D- and 2D-NMR. The compound was evaluated against the National Cancer Institute (NCI) 60 human tumor cell line screen in both the one-dose and five-dose screens, and cytotoxicity was compared to a small library of jadomycin analogues to probe the structure-activity relationship.

Furan and lactam jadomycin biosynthetic congeners Isolated from Streptomyces venezuelae ISP5230 cultured with Nε-Trifluoroacetyl-l-lysine

Angucycline antibiotics are composed of a classical four-ring angularly linked polyaromatic backbone. Differential cyclization chemistry of the A- and B-rings in jadomycin biosynthesis led to the discovery of two new furan analogues, while oxidation led to a ring-opened form of the jadomycin Nε-trifluoroacetyl-l-lysine (TFAL) congener. The compounds were isolated from Streptomyces venezuelae ISP5230 cultures grown with TFAL. Biosynthetic incorporation using d-[1-13C]-glucose in cultures enabled the unambiguous assignment of the aldehyde, alcohol, and amide functionalities present in these new congeners through NMR spectroscopy. Tandem mass spectrometry analysis of cultures grown with 15Nα- or 15Nε-lysine demonstrated the incorporation of Nα exclusively into the angucycline backbone, contrasting results with ornithine [J. Am. Chem. Soc. 2015, 137, 3271]. Compounds were evaluated against antimicrobial and cancer cell panels and found to possess good activity against Gram-positive bacteria.

Mortiamides A–D, Cyclic Heptapeptides from a Novel Mortierella sp. Obtained from Frobisher Bay

Four new cyclic heptapeptides, mortiamides A-D (1-4), were obtained from a novel Mortierella sp. isolate obtained from marine sediment collected from the intertidal zone of Frobisher Bay, Nunavut, Canada. The structures of the compounds were elucidated by NMR spectroscopy and tandem mass spectrometry. The absolute configurations of the amino acids were determined using Marfeys method. Localization of l and d amino acids within each compound was ascertained by retention time comparison of the partial hydrosylate products of each compound to synthesized dipeptide standards using LC-HRMS. Compounds 1-4 did not exhibit any significant antimicrobial or cytotoxic activity.

Isolation of a jadomycin incorporating l -ornithine, analysis of antimicrobial activity and jadomycin reactive oxygen species (ROS) generation in MDA-MB-231 breast cancer cells

Herein, we report the characterization and antimicrobial activity of a previously unreported jadomycin (1) obtained from a culture of S. venezuelae ISP5230 with l-ornithine (Orn). 1 arises from the rearrangement of a putative five-membered ring containing jadomycin incorporating Orn, whereby intramolecular attack of the E-ring carbonyl from the δ-NH2 group of the Orn side chain results in collapse of the oxazolone ring and formation of a stable six-membered lactam. This rearrangement produces a jadomycin with a 3a hemiaminal position that is susceptible to solvolysis. A structure–activity relationship is discussed based on the antimicrobial activity of 1 compared to previously reported jadomycins, providing evidence that the presence of a 3a hemiaminal enhances activity against Gram-positive bacteria. Additionally, assays to quantify reactive oxygen species (ROS) generation and cell viability were performed using a series of nine jadomycins. Compound 1 was found to produce the highest ROS activity and to possess the greatest cytotoxicity against MDA-MB-231 breast cancer cells.