A 2-step synthesis of Combretastatin A-4 and derivatives as potent tubulin assembly inhibitors.

A series of combretastatin derivatives were designed and synthesised by a two-step stereoselective synthesis by use of Wittig olefination followed by Suzuki cross-coupling. Interestingly, all new compounds (2a-2i) showed potent cell-based antiproliferative activities in nanomolar concentrations. Among the compounds, 2a, 2b and 2e were the most active across three cancer cell lines. In addition, these compounds inhibited the polymerisation of tubulin in vitro more efficiently than CA-4. They caused cell cycle arrest in G2/M phase further confirming their ability to inhibit tubulin polymerisation.

Circular and linear: a tale of aptamer selection for the activation of SIRT1 to induce death in cancer cells.

It is a challenge to select the right target to treat conditions without affecting non-diseased cells. Cancer belongs to the top 10 causes of death in the world and it remains difficult to treat. Amongst cancer emerging targets, silent information regulator 1 (SIRT1) - a histone deacetylase - has shown many roles in cancer, ageing and metabolism. Here we report novel SIRT1 ligands that bind and modulate the activity of SIRT1 within cells and enhance its enzymatic activity. We developed a modified aptamer capable of binding to and forming a complex with SIRT1. Our ligands are aptamers, they can be made of DNA or RNA oligonucleotides, their binding domain can recognise a target with very high affinity and specificity. We used the systematic evolution of ligands by exponential enrichment (SELEX) technique to develop circular and linear aptamers selectively binding to SIRT1. Cellular consequences of the interaction were monitored by fluorescence microscopy, cell viability assay, stability and enzymatic assays. Our results indicate that from our pool of aptamers, circular AC3 penetrates cancerous cells and is recruited to modulate the SIRT1 activity. This modulation of SIRT1 resulted in anticancer activity on different cancer cell lines. Furthermore, this modified aptamer showed no toxicity on one non-cancerous cell line and was stable in human plasma. We have demonstrated that aptamers are efficient tools for localisation of internal cell targets, and in this particular case, anticancer activity through modulation of SIRT1.

Structure-activity studies of the binding of the flavonoid scaffold to DNA.

BACKGROUND: Flavonoids have been shown to have a wide variety of biological activities and proven to be good scaffolds for the design of DNA-binding agents as anticancer therapeutics. MATERIALS AND METHODS: In structure-activity relationship studies, flavonoid derivatives were designed and synthesised through various organic synthesis protocols, resulting in novel or previously described molecules. These were studied by UV-Vis absorbance and fluorescence spectroscopy as well as competition dialysis for their binding to DNA isoforms. Their cytotoxic potential was assessed using MTS assays on MCF-7 breast cancer and CCRFCEM leukaemia cell lines. RESULTS AND CONCLUSION: Introduction of moieties such as chloride, nitrogen, acetoxy and methoxy groups did not help to improve binding affinity, but introduction of tertiary amines improved the binding 1,000-fold due to an improved interaction of the compound with the nucleic acid; replacement of oxygen by sulphur increased the binding 7-fold, possibly because sulphur being less electronegative than oxygen would allow the electrons of the molecule to interact more strongly with the nucleic acid. Inhibition of growth by 50% (IG(50)) values were moderate in breast and leukaemia cancer cell lines possibly due to the flavonoids interacting with other cellular components besides the nucleic acids.

The characterisation of flavone-DNA isoform interactions as a basis for anticancer drug development.

BACKGROUND: The interactions of small molecules with nucleic acids are of considerable interest for the design of novel anticancer compounds. The physical properties and sequence specificity observed in the interactions between a group of flavonoids with proven antitumour activity and various nucleic acid structures are summarised. MATERIALS AND METHODS: UV and fluorescence spectroscopy, together with competition dialysis, were used to assess the affinity of the drugs for the nucleic acid structures in the presence or absence of different metal ions. The effect of these compounds on breast and leukaemia cancer cell lines was evaluated using MTS and COMET assays and flow cytometry. RESULTS AND CONCLUSION: The flavonoids studied are weak duplex DNA-binding ligands and the binding of flavonoids to DNA is affected by metal ions. Baicalein and quercetin display stronger affinity for triplex and quadruplex than for double-stranded DNA and offer interesting scaffolds for the design of novel, high order DNA-binding agents.

Effect of O6-methylguanine on the stability of G-quadruplex DNA.

The effects of substitution of O6-methylguanine on the structure and stability of a human telomere quadruplex was studied by circular dichroism, thermal denaturation, analytical ultracentrifugation, and molecular dynamics simulations. The results show that, while quadruplex structures can form containing the modified base, they are much less stable than the normal unmodified structure. The extent of destabilization is critically dependent on the exact position of the modified base within the quadruplex structure.

Synthesis and biological evaluation of bisindenoisoquinolines as topoisomerase I inhibitors.

The indenoisoquinolines represent a class of non-camptothecin topoisomerase I (Top1) inhibitors that exert cytotoxicity by trapping the covalent complex formed between DNA and Top1 during relaxation of DNA supercoils. As an ongoing evaluation of Top1 inhibition and anticancer activity, indenoisoquinolines were linked via their lactam side chains to provide polyamines end-capped with intercalating motifs. The resulting bisindenoisoquinolines were evaluated for cytotoxicity in the National Cancer Institute's human cancer cell screen and for Top1 inhibition. Preliminary findings suggested that the 2-3-2 and 3-3-3 linkers, referring to the number of carbons between nitrogen atoms, were optimal for both potent Top1 inhibition and cytotoxicity. Using optimized linkers, bisindenoisoquinolines were synthesized with nitro and methoxy substituents on the aromatic rings. The biological results for substituted compounds revealed a disagreement between the structure-activity relationships of monomeric indenoisoquinolines and bisindenoisoquinolines as Top1 inhibitors, but cytotoxicity was maintained for both series of compounds.

Competition dialysis: a method for the study of structural selective nucleic acid binding.

Competition dialysis is a powerful new tool for the discovery of ligands that bind to nucleic acids with structural- or sequence-selectivity. The method is based on firm thermodynamic principles and is simple to implement. In the competition dialysis experiment, an array of nucleic acid structures and sequences is dialyzed against a common test ligand solution. After equilibration, the amount of ligand bound to each structure or sequence is determined by absorbance or fluorescence measurements. Since all structures and sequences are in equilibrium with the same free ligand concentration, the amount bound is directly proportional to the ligand binding affinity. Competition dialysis thus provides a direct and quantitative measure of selectivity, and unambiguously identifies which of the samples within the array are preferred by a particular ligand. We describe here the third generation implementation of the method, in which competition dialysis was adapted for use in a 96-well plate format. In this format, we have been able to greatly expand the array of nucleic acid structures studied, and now can routinely study the interactions of a ligand of interest with 46 different structures and sequences.

Circular dichroism to determine binding mode and affinity of ligand-DNA interactions.

Circular dichroism (CD) is a useful technique for an assessment of DNA-binding mode, being a more accessible, low-resolution complement to NMR and X-ray diffraction methods. Ligand-DNA interactions can be studied by virtue of the interpretation of induced ligand CD signals resulting from the coupling of electric transition moments of the ligand and DNA bases within the asymmetric DNA environment. This protocol outlines methods to determine the binding mode and affinity of ligand-DNA interactions and takes approximately 7.5 h.