Sequence-Specific Base Pair Mimics Are Efficient Topoisomerase IB Inhibitors

Topoisomerase IB controls DNA topology by cleaving DNA transiently. This property is used by inhibitors, such as camptothecin, that stabilize, by inhibiting the religation step, the cleavage complex, in which the enzyme is covalently attached to the 3′-phosphate of the cleaved DNA strand. These drugs are used in clinics as antitumor agents. Because three-dimensional structural studies have shown that camptothecin derivatives act as base pair mimics and intercalate between two base pairs in the ternary DNA–topoisomerase–inhibitor complex, we hypothesized that base pairs mimics could act like campthotecin and inhibit the religation reaction after the formation of the topoisomerase I–DNA cleavage complex. We show here that three base pair mimics, nucleobases analogues of the aminophenyl-thiazole family, once targeted specifically to a DNA sequence were potent topoisomerase IB inhibitors. The targeting was achieved through covalent linkage to a sequence-specific DNA ligand, a triplex-forming oligonucleotide, and was necessary to position and keep the nucleobase analogue in the cleavage complex. In the absence of triplex formation, only a weak binding to the DNA and topoisomerase I-mediated DNA cleavage was observed. The three compounds were equally active once conjugated, implying that the intercalation of the nucleobase upon triplex formation is the essential feature for the inhibition activity

Targeting the Production of Oncogenic MicroRNAs with Multimodal Synthetic Small Molecules

MicroRNAs (miRNAs) are a recently discovered category of small RNA molecules that regulate gene expression at the post-transcriptional level. Accumulating evidence indicates that miRNAs are aberrantly expressed in a variety of human cancers and revealed to be oncogenic and to play a pivotal role in initiation and progression of these pathologies. It is now clear that the inhibition of oncogenic miRNAs, defined as blocking their biosynthesis or their function, could find an application in the therapy of different types of cancer in which these miRNAs are implicated. Here we report the design, synthesis, and biological evaluation of new small-molecule RNA ligands targeting the production of oncogenic microRNAs. In this work we focused our attention on miR-372 and miR-373 that are implicated in the tumorigenesis of different types of cancer such as gastric cancer. These two oncogenic miRNAs are overexpressed in gastric cancer cells starting from their precursors pre-miR-372 and pre-miR-373, two stem-loop structured RNAs that lead to mature miRNAs after cleavage by the enzyme Dicer. The small molecules described herein consist of the conjugation of two RNA binding motives, i.e., the aminoglycoside neomycin and different natural and artificial nucleobases, in order to obtain RNA ligands with increased affinity and selectivity compared to that of parent compounds. After the synthesis of this new series of RNA ligands, we demonstrated that they are able to inhibit the production of the oncogenic miRNA-372 and -373 by binding their pre-miRNAs and inhibiting the processing by Dicer. Moreover, we proved that some of these compounds bear anti-proliferative activity toward gastric cancer cells and that this activity is likely linked to a decrease in the production of targeted miRNAs. To date, only few examples of small molecules targeting oncogenic miRNAs have been reported, and such inhibitors could be extremely useful for the development of new anticancer therapeutic strategies as well as useful biochemical tools for the study of miRNAs’ pathways and mechanisms. Furthermore, this is the first time that a design based on current knowledge about RNA targeting is proposed in order to target miRNAs’ production with small molecules

The sequence of the target duplex and the TFOs, and the chemical structure of the drug–TFO conjugates

<p><b>Copyright information:</b></p><p>Taken from "Molecular basis of the targeting of topoisomerase II-mediated DNA cleavage by VP16 derivatives conjugated to triplex-forming oligonucleotides"</p><p>Nucleic Acids Research 2006;34(6):1900-1911.</p><p>Published online 5 Apr 2006</p><p>PMCID:PMC1447649.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> The 77 bp duplex target sequence was inserted between the BamHI and EcoRI sites of pBSK. The TFO is complementary to the oligopurine strand of the duplex and binds parallel to it. The target site is in boldface for the 20 nt TFO and is underlined for the 16 nt TFOs. , 5-methyl-2′-deoxycytidine; , 5-propynyl-2′-deoxyuridine. The structures of the VP16 derivatives-TFO conjugates used in this study are shown. The nomenclature of the conjugates is described in the Materials and Methods

Quantification of topo II-mediated DNA cleavage in the presence of TFO–drug conjugates

<p><b>Copyright information:</b></p><p>Taken from "Molecular basis of the targeting of topoisomerase II-mediated DNA cleavage by VP16 derivatives conjugated to triplex-forming oligonucleotides"</p><p>Nucleic Acids Research 2006;34(6):1900-1911.</p><p>Published online 5 Apr 2006</p><p>PMCID:PMC1447649.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> The analysis was as in and the gels were quantified after normalization relative to total radioactivity loaded. () Scheme showing the enhanced cleavage site of each conjugate: the 5′ conjugates are depicted in green as the corresponding cleavage site a; the 3′ 16TFO conjugates are depicted in red as the corresponding cleavage site f; the 3′ 20TFO conjugates are depicted in blue as the corresponding cleavage site g. The other topo II-mediated DNA cleavage sites described in the text are also labeled with letters. () Quantification of the specific cleavage for 20TFO-L- on both strands compared to free 20TFO-L. The cleavage intensity was normalized to the cleavage intensity of the free drug (at 1 µM) on a logarithmic scale at each cleavage site. The oligopyrimidine strand is in gray and the oligopurine strand is in black, the conjugate in filled bars and the 20TFO-L alone in hatched bars. () Specific cleavage intensities of the conjugates on the oligopyrimidine-containing strand of the duplex (Y). The 5′ 16TFO conjugates are depicted in light green (hatched bars -L-16TFO, squares -L-16TFO, horizontal bars -L-16TFO, vertical bars -L-16TFO), the 5′ 20TFO conjugates are depicted in dark green (hatched bars -L-20TFO, vertical bars -L-20TFO), the 3′ 16TFO conjugates are in red [hatched bars 16TFO-L-, squares 16TFO-L-, horizontal bars 16TFO-L-, vertical bars 16TFO-L-, crosses 16TFO-L-(4)] and the 3′ 20TFO ones are in blue [hatched bars 20TFO-L-, vertical bars 20TFO-L-, crosses 20TFO-L-(4), dots 20TFO-L-(4)]