The Mechanism of Action of (−)-Lomaiviticin A

Abstract

CONSPECTUS (–)-Lomaiviticin A (4) is a complex C2-symmetric bacterial metabolite that contains two diazofluorene functional groups. The diazofluorene consists of naphthoquinone, cyclo-pentadiene, and diazo substituents fused through a σ- and π-bonding network. Additionally, (–)-lomaiviticin A (4) is a potent cytotoxin, with half-maximal inhibitory potency (IC50) values in the low nanomolar range against many cancer cell lines. Because of limitations in supply, its mechanism of action had remained a “black box” since its isolation in the early 2000s. In this Account, I describe how studies directed toward the total synthesis of (–)-lomaiviticin A (4) provided a platform to elucidate the emergent properties of this metabolite and thereby connect chemical reactivity with cellular phenotype. We first developed a convergent strategy to prepare the diazofluorene (9 + 10 → 13). We then adapted this chemistry to the synthesis of lomaiviticin aglycon (21/22) and the natural monomeric diazofluorene (–)-kinamycin F (3). The key step in the lomaiviticin aglycon (21/22) synthesis involved the stereoselective oxidative coupling of two monomeric diazofluorenes (2 × 18→ 20) to establish the cojoining carbon–carbon bond of the target. As the absolute stereochemistry of the aglycon and carbohydrate residues of (–)-lomaiviticin A (4) were unknown, we developed a semisynthetic route to the metabolite that proceeds in one step and 42% yield by diazo transfer to the more abundant isolate (–)-lomaiviticin C (6). This allowed us to complete the stereochemical assignment of (–)-lomaiviticin A (4) and provided a renewable source of material. Using this material, we established that the remarkable cytotoxic effects of (–)-lomaiviticin A (4) derive from the induction of highly toxic double-strand breaks (DSBs) in DNA. At the molecular level, 1,7-nucleophilic additions to each electrophilic diazofluorene trigger homolytic decomposition pathways that produce sp2 radicals at the carbon atoms of each diazo group. These radicals abstract hydrogen atoms from the deoxyribose of DNA, a process known to initiate strand cleavage. NMR spectroscopy and molecular mechanics simulations were used to elucidate the mode of DNA binding. These studies showed that both diazofluorenes of (–)-lomaiviticin A (4) penetrate into the duplex. This mode of non-covalent binding places each diazo carbon atom in close proximity to each DNA strand. Throughout these studies, isolates containing one diazofluorene, such as (–)-lomaiviticin C (6) and (–)-kinamycin C (2), were used as controls. Consistent with our mechanistic model, these compounds do not induce DSBs in DNA and are several orders of magnitude less potent. Reactivity studies suggest that (–)-lomaiviticin A (4) is more electrophilic than simple monomeric diazofluorenes. We attribute this to through-space delocalization of the developing negative charge in the transition state for 1,7-addition. Consistent with this mechanism of action, (–)-lomaiviticin A (4) displays selective low-picomolar potencies toward DNA ...

Molecular basis of dimer formation during the biosynthesis of benzofluorene-containing atypical angucyclines

“…A case in point is lomaiviticin A ( 1 ) which exhibits cytotoxicity at nanomolar to picomolar concentrations by inducing double-strand breaks in DNA and is currently under preclinical evaluation for antitumor treatment 12 – 14 . Since the C 2 -symmetric homodimer lomaiviticin A ( 1 ) and the C1−C5′ asymmetric heterodimer difluostatin A ( 6 ) are more potent than their respective monomers 8 , 12 , 14 , dimerization appears to be important to amplify the therapeutic potential of this class of compounds. There has thus been significant interest in the biosynthesis of these compounds given their structural complexity and pharmaceutical potency.…”

Cytotoxic, Anti-Migration, and Anti-Invasion Activities on Breast Cancer Cells of Angucycline Glycosides Isolated from a Marine-Derived Streptomyces sp.

“…Lomaiviticins were first isolated from Micromonospora lomaivitiensis as dimers of kinamycin angucyclines with two diazofluorene functional groups [3]. One of them, lomaiviticin A, exhibited remarkable cytotoxicity against a panel of human cancer cells at nanomolar–picomolar concentrations by inducing double-strand breaks in DNA, and it is currently under preclinical evaluation [13,14,15]. Accordingly, angucyclines are still considered as promising candidates for anti-tumor drug development.…”

Angucycline Glycosides from an Intertidal Sediments Strain Streptomyces sp. and Their Cytotoxic Activity against Hepatoma Carcinoma Cells

“…Though, the distinct in vivo toxicity restricted the further development of these compounds to be clinical drugs. Recently, an atypical angucycline, lomaiviticin A, was reported to be under preclinical evaluation for antitumor treatment due to its prominent cytotoxicity and effects of inducing double-strand breaks in DNA [14,23]. In present work, 1 – 4 were assayed for their cytotoxic activity against normal liver cell LO 2 , hepatoma carcinoma HepG-2, SMMC-7721 and plc-prf-5 cell lines by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method (Table 2).…”