Synthetic calicheamicin mimics with novel initiation mechanisms: DNA cleavage, cytotoxicity, and apoptosis.

BACKGROUND: Calicheamicin gamma 1I is a bacterial product that is a prominent member of the enediyne class of antitumor antibiotics, and has been extensively studied. Calicheamicin gamma 1I binds to DNA, causing double-stranded breaks, and cells exposed to it eventually become apoptotic. It can now be made synthetically, and highly potent biological mimics have been designed. Such molecules have many potential clinical applications, but are complex to make. We therefore investigated whether simplified versions of these molecules are biologically active. RESULTS: We designed and synthesized a number of simple calicheamicin mimics and evaluated their biological activity. We also constructed mimics that are particularly suitable for conjugation to proteins, oligonucleotides, and other delivery systems. Several active mimics were found, and two in particular, which lack the trisulfide and oligosaccharide moieties of calicheamicin, had potent DNA-cleaving and cytotoxic activities. They caused chiefly single-stranded cuts in DNA, however, unlike the natural molecule, which causes double-stranded DNA cuts. Although they were able to induce apoptosis, they were less potent than the natural compound in this assay. CONCLUSIONS: The simple enediyne mimics were less potent than calicheamicin gamma 1I, presumably because they lack the oligosaccharide DNA-binding domain. Nevertheless, considering their relatively primitive structures, they have remarkable biological properties. They may be useful biological tools and are potential leads for the development of chemotherapeutic agents. We propose that the ability of the enediynes to induce apoptosis is related to their ability to make double-stranded cuts in DNA.

New fluorescent probes for the measurement of cell membrane viscosity.

BACKGROUND: Molecular rotors are fluorescent molecules that exhibit viscosity-dependent fluorescence quantum yield, potentially allowing direct measurements of cell membrane viscosity in cultured cells. Commercially available rotors, however, stain not only the cell membrane, but also bind to tubulin and migrate into the cytoplasm. We synthesized molecules related to 9-(dicyanovinyl)-julolidine (DCVJ), which featured hydrocarbon chains of different length to increase membrane compatibility. RESULTS: Longer hydrocarbon chains attached to the fluorescent rotor reduce the migration of the dye into the cytoplasm and internal compartments of the cell. The amplitude of the fluorescence response to fluid shear stress, known to decrease membrane viscosity, is significantly higher than the response obtained from DCVJ. Notably a farnesyl chain showed a more than 20-fold amplitude over DCVJ and allowed detection of membrane viscosity changes at markedly lower shear stresses. CONCLUSIONS: The modification of molecular rotors towards increased cell membrane association provides a new research tool for membrane viscosity measurements. The use of these rotors complements established methods such as fluorescence recovery after photobleaching with its limited spatial and temporal resolution and fluorescence anisotropy, which has low sensitivity and may be subject to other effects such as deformation.

The relationship of brevetoxin 'length' and A-ring functionality to binding and activity in neuronal sodium channels.

BACKGROUND: Brevetoxins are polyether ladder toxins that are ichthyotoxic at nanomolar concentrations. They bind to voltage-gated sodium channels, causing four distinct electrophysiological effects: (i) a shift of activation potential; (ii) occurrence of subconductance states; (iii) induction of longer mean open times of the channel; and (iv) inhibition of channel inactivation. We set out to determine whether these functions all require the same structural elements within the brevetoxin molecules. RESULTS: Several synthetically prepared structural analogs of brevetoxin B were examined in synaptosome receptor binding assays and by functional electrophysiological measurements. A truncated analog is not ichthyotoxic at micromolar concentrations, shows decreased receptor-binding affinity, and causes only a shift of activation potential without affecting mean open times or channel inactivation. An analog with the A-ring carbonyl removed binds to the receptor with nanomolar affinity, produces a shift of activation potential and inhibits inactivation, but does not induce longer mean open times. An analog in which the A-ring diol is reduced shows low binding affinity, yet populates five subconductance states. CONCLUSIONS: Our data are consistent with the hypothesis that binding to sodium channels requires an elongated cigar-shaped molecule, approximately 30 A long. The four electrophysiological effects of the brevetoxins are not produced by a single structural feature, however, since they can be decoupled by using modified ligands, which are shown here to be partial sodium channel agonists. We propose a detailed model for the binding of brevetoxins to the channel which explains the differences in the effects of the brevetoxin analogs. These studies also offer the potential for developing brevetoxin antagonists.

Enantioselective synthesis of the [6,6] spiroketal core of reveromycin A.

[structure: see text] Reveromycin A (1) belongs to a family of microbial polyketides with unusual structural features and biological activities. The structure of 1 is composed of a [6,6] spiroketal core decorated with highly unsaturated side chains. As a prelude to the synthesis of 1, we present herein a short, efficient, and enantioselective synthesis of the C9-C21 fragment 5 (spiroketal core) of reveromycin A.

Stereoselective synthesis of (-)-acanthoic acid.

[reaction: see text] The first stereoselective synthesis of (-)-acanthoic acid (1) has been designed and accomplished. Our synthetic plan departs from (-) Wieland-Miesher ketone (7) and calls upon a Diels-Alder cycloaddition reaction for the construction of the C ring of 1. The described synthesis confirms the proposed stereochemistry of 1 and represents an efficient entry into an unexplored class of biologically active diterpenes.

Effects of solvent polarity and solvent viscosity on the fluorescent properties of molecular rotors and related probes.

Fluorescent molecular rotors belong to a group of twisted intramolecular charge transfer complexes (TICT) whose photophysical characteristics depend on their environment. In this study, the influence of solvent polarity and viscosity on several representative TICT compounds (three Coumarin derivatives, 4,4-dimethylaminobenzonitrile DMABN, 9-(dicyanovinyl)-julolidine DCVJ), was examined. While solvent polarity caused a bathochromic shift of peak emission in all compounds, this shift was lowest in the case of molecular rotors. Peak intensity was influenced strongly by solvent viscosity in DMABN and the molecular rotors, but polarity and viscosity influences cannot be separated with DMABN. Coumarins, on the other hand, did not show viscosity sensitivity. This study shows the unique suitability of molecular rotors as fluorescent viscosity sensors.

Stereoselective total synthesis of reveromycin B and C19-epi-reveromycin B.

Our studies toward the total synthesis of the reveromycin family of natural products are described herein. Our synthetic approach is efficient, stereocontrolled, and convergent and has resulted in the first synthesis of reveromycin B (4) and C19-epi-reveromycin B (55). Key steps of this successful strategy include: a modified Negishi coupling (construction of C7-C8 bond) and a Kishi-Nozaki reaction (construction of C19-C20 bond), which were employed in the attachment of the target side chains. The key building blocks for the total synthesis were thus defined as vinyl iodide 6, alkyne 7, and alkyne 8. Our synthesis illustrates the utility of the modified Negishi coupling for the construction of complex dienes, confirms the proposed stereochemistry of reveromycins and paves the way for the preparation of designed analogues for biological study.

Investigation of the biological mode of action of clerocidin using whole cell assays.

Clerocidin, a diterpenoid natural product, has been shown in vitro to inhibit DNA religation following cleavage by topoisomerase II. Herein, we characterize the efficacy and specificity of clerocidin in HeLa cells. Our results suggest that clerocidin recognizes topoisomerase II as its main intracellular target and binds to this enzyme prior to formation of the 'cleavable complex' with DNA. These pharmacological features attest to the promising chemotherapeutic potential of this natural product.

Design, synthesis, and evaluation of N-aroyloxy-2-thiopyridones as DNA photocleaving reagents.

N-Benzoyloxy-2-thiopyridone (12) was shown to induce single-strand nicks in duplex DNA upon irradiation with visible light (lambda&350 nm). This finding led to the design of a series of compounds, in which an acridinyl nucleus was covalently linked to the N-benzoyloxy-2-thiopyridone unit. These conjugates (15, 16, 17 and 18) were synthesized and evaluated as novel DNA photocleaving reagents. Optimal photocleaving activity was observed for conjugate 16, in which a flexible polymethylene spacer of 4 carbons was used to connect the aminoacridine entity to the thiopyridone. This compound was shown to cleave DNA at low microM concentrations and was approximately two-orders of magnitude more efficient than the parent N-benzoyloxy-2-thiopyridone (12). Furthermore, the DNA cleavage ladders induced by 16 and 12 were found to be identical and of no significant sequence selectivity. These data suggest that the N-aroyloxy-2-thiopyridones can be used for the design of new DNA photocleaving reagents with potential use as 'photofootprinting agents' or as 'site-directed photonucleases'.

Enantioselective synthesis of the antiinflammatory agent (-)-acanthoic acid.

An enantioselective synthesis of the potent antiinflammatory agent (-)-acanthoic acid (1) is described. The successful strategy departs from (-)-Wieland-Miescher ketone (10), which is readily available in both enantiomeric forms and constitutes the starting point toward a fully functionalized AB ring system of 1. Conditions were developed for a regioselective double alkylation at the C4 center of the A ring, which produced compound 32 as a single stereoisomer. Construction of the C ring of 1 was accomplished via a Diels-Alder reaction between sulfur-containing diene 43 and methacrolein (36), which after desulfurization and further functionalization yielded synthetic acanthoic acid. The described synthesis confirms the proposed stereochemistry of the natural product and represents a fully stereocontrolled entry into an underexplored class of biologically active diterpenes.

Sequence-selective carbohydrate-DNA interaction: dimeric and monomeric forms of the calicheamicin oligosaccharide interfere with transcription factor function.

The synthetic oligosaccharide moiety of the antibiotic calicheamicin and the head-to-head dimer of this oligosaccharide are known to bind to the minor groove of DNA in a sequence-selective manner preferring distinct target sequences. We tested these carbohydrates for their ability to interfere with transcription factor function. The oligosaccharides inhibit binding of transcription factors to DNA in a sequence-selective manner, probably by inducing a conformational change in DNA structure. They also interfere with transcription by polymerase II in vitro. The effective concentrations of the oligosaccharides for inhibition of transcription factor binding and for transcriptional inhibition are in the micromolar range. The dimer is a significantly more active inhibitor than is the monomer.