Terminally modified, short phosphorothioate oligonucleotides as inhibitors of gene expression in cells.

Phosphorothioates are excellent antisense inhibitors, which are active both in cells and in vivo. Since their affinity to complementary ribonucleic acids is rather low, long strands (⩾20-mers) are typically required to achieve the desired biological activity. However, mismatch discrimination of long inhibitors is reduced. In contrast, shorter phosphorothioates exhibit better sequence specificity, but have in most cases too low affinity for practical applications in cells. We screened a range of terminal modifiers of a 14-mer phosphorothioate sequence, which is complementary to mRNA of a representative gene, whose protein product is fluorescent (DsRed2) and easy to monitor in cells. We found that optimal combinations of 5'- and 3'-modifications include 5'-trimethoxystilbene with 3'-uracil(anthraquinone)-cap, 5'-chloic acid derivative with 3'-uracyl(anthraquinone)-cap and 5'-cholic acid derivative with three 3'-LNA moieties. In contrast to the LNA, stabilizing and activity-enhancing effects of other mentioned modifiers for PTO/RNA duplexes have not been previously reported. We observed that the 14-mer inhibitor carrying 5'-cholic acid derivative with three 3'-LNA moieties inhibits expression of DsRed2 in cells stronger than the unmodified 21-mer. Mismatch discrimination of this inhibitor was found to be comparable to that of the unmodified 14-mer.

4-Azidobenzyl ferrocenylcarbamate as an anticancer prodrug activated under reductive conditions.

Aminoferrocene-based prodrugs are activated in the presence of cancer-specific amounts of reactive oxygen species, e.g. H2O2, with the formation of products of two types: Fe-containing complexes, which catalyze generation of HO and O2(-), and quinone methides, which alkylate glutathione and inhibit the antioxidative system of the cell. Both processes act synergistically by increasing the oxidative stress in cancer cells thereby leading to their death. However, in the activation step including the cleavage of a B-C bond one molecule of H2O2 is consumed that counteracts the desired effect of the products released from aminoferrocenes. We replaced an H2O2-sensitive trigger in original prodrugs with an azide group. This trigger is slowly reduced in the presence of glutathione with the formation of an unstable arylamine intermediate, which decomposes with the release of iron ions and iminoquinone methides. These products induce strong oxidative stress in cells as we confirmed using 2',7'-dichlorodihydrofluorescin diacetate reagent in combination with flow cytometry. In this case the activation process does not consume H2O2. Correspondingly, we observed that the azide-containing prodrug is substantially more toxic towards human promyelocytic leukemia cell line HL-60 (IC50=27±4µM) than its H2O2-responsive analogue (IC50>50µM).

Short, terminally modified 2'-OMe RNAs as inhibitors of microRNA.

We applied 14-mer 2'-OMe RNAs as inhibitors of selected micro RNAs. To improve their properties, we introduced a trimethoxystilbene residue at the 5'-terminus and three 2'-fluoro-2'-deoxynucleotides at the 3'-terminus to obtain potent inhibitors, whose mismatch discrimination is substantially better than that of typically applied >18-mers.

Aminoferrocene-based prodrugs and their effects on human normal and cancer cells as well as bacterial cells.

Aminoferrocene-based prodrugs are activated under cancer-specific conditions (high concentration of reactive oxygen species, ROS) with the formation of glutathione scavengers (p-quinone methide) and ROS-generating iron complexes. Herein, we explored three structural modifications of these prodrugs in an attempt to improve their properties: (a) the attachment of a -COOH function to the ferrocene fragment leads to the improvement of water solubility and reactivity in vitro but also decreases cell-membrane permeability and biological activity, (b) the alkylation of the N-benzyl residue does not show any significant affect, and (c) the attachment of the second arylboronic acid fragment improves the toxicity (IC50) of the prodrugs toward human promyelocytic leukemia cells (HL-60) from 52 to 12 µM. Finally, we demonstrated that the prodrugs are active against primary chronic lymphocytic leukemia (CLL) cells, with the best compounds exhibiting an IC50 value of 1.5 µM. The most active compounds were found to not affect mononuclear cells and representative bacterial cells.

Aminoferrocene-based prodrugs activated by reactive oxygen species.

Cancer cells generally generate higher amounts of reactive oxygen species than normal cells. On the basis of this difference, prodrugs have been developed (e.g., hydroxyferrocifen), which remain inactive in normal cells, but become activated in cancer cells. In this work we describe novel aminoferrocene-based prodrugs, which, in contrast to hydroxyferrocifen, after activation form not only quinone methides (QMs), but also catalysts (iron or ferrocenium ions). The released products act in a concerted fashion. In particular, QMs alkylate glutathione, thereby inhibiting the antioxidative system of the cell, whereas the iron species induce catalytic generation of hydroxyl radicals. Since the catalysts are formed as products of the activation reaction, it proceeds autocatalytically. The most potent prodrug described here is toxic toward cancer cells (human promyelocytic leukemia (HL-60), IC(50) = 9 µM, and human glioblastoma-astrocytoma (U373), IC(50) = 25 µM), but not toxic (up to 100 µM) toward representative nonmalignant cells (fibroblasts).