A Combined Effect of G-Quadruplex and Neuro-Inducers as an Alternative Approach to Human Glioblastoma Therapy.

Cancer cell reprogramming based on treatment with G-quadruplex, having antiproliferative power, along with small molecules able to develop iPSCs into neurons, could create a novel approach to diminish the chance of glioblastoma recurrence and circumvent tumor resistance to conventional therapy. In this research, we have tested several combinations of factors to affect both total cell cultures, derived from tumor tissue of patients after surgical resection and two subfractions of this cell culture after dividing them into CD133-enriched and CD133-depleted populations (assuming CD133 to be a marker of glioblastoma stem-like cells). CD133+ and CD133- cells exhibit different responses to the same combinations of factors; CD133+ cells have stem-like properties and are more resistant. Therefore, the ability to affect CD133+ cells provides a possibility to circumvent resistance to conventional therapy and to build a promising strategy for translation to improve the treatment of patients with glioblastoma.

Covalent Bi-Modular Parallel and Antiparallel G-Quadruplex DNA Nanocostructs Reduce Viability of Patient Glioma Primary Cell Cultures.

G-quadruplex oligonucleotides (GQs) exhibit specific anti-proliferative activity in human cancer cell lines, and they can selectively inhibit the viability/proliferation of cancer cell lines vs. non-cancer ones. This ability could be translated into a cancer treatment, in particular for glioblastoma multiform (GBM), which currently has a poor prognosis and low-efficiency therapeutic treatments. A novel bi-modular GQ, bi-(AID-1-T), a twin of the previously described three-quartet AID-1-T, was designed and studied in terms of both its structure and function. A covalent conjugation of two AID-1-Ts via three thymidine link, TTT, did not interfere with its initial GQ structure. A comparison of bi-(AID-1-T) with its mono-modular AID-1-T, mono-modular two-quartet HD1, and bi-modular bi-HD1, as well as conventional two-quartet AS1411, was made. Among the five GQs studied, bi-(AID-1-T) had the highest anti-proliferative activity for the neural cancer cell line U87, while not affecting the control cell line, human embryonic fibroblasts. GQs, for the first time, were tested on several primary glioma cultures from patient surgical samples. It turned out that the sensitivity of the patient primary glioma cultures toward GQs varied, with an apparent IC50 of less than 1 µM for bi-(AID-1-T) toward the most sensitive G11 cell culture (glioma, Grade III).

Bimodular Antiparallel G-Quadruplex Nanoconstruct with Antiproliferative Activity.

Oligonucleotides with an antiproliferative activity for human cancer cells have attracted attention over the past decades; many of them have a G-quadruplex structure (GQ), and a cryptic target. In particular, DNA oligonucleotide HD1, a minimal GQ, could inhibit proliferation of some cancer cell lines. The HD1 is a 15-nucleotide DNA oligonucleotide that folds into a minimal chair-like monomolecular antiparallel GQ structure. In this study, for eight human cancer cell lines, we have analyzed the antiproliferative activities of minimal bimodular DNA oligonucleotide, biHD1, which has two HD1 modules covalently linked via single T-nucleotide residue. Oligonucleotide biHD1 exhibits a dose-dependent antiproliferative activity for lung cancer cell line RL-67 and cell line of central nervous system cancer U87 by MTT-test and Ki-67 immunoassay. The study of derivatives of biHD1 for the RL-67 and U87 cell lines revealed a structure-activity correlation of GQ folding and antiproliferative activity. Therefore, a covalent joining of two putative GQ modules within biHD1 molecule provides the antiproliferative activity of initial HD1, opening a possibility to design further GQ multimodular nanoconstructs with antiproliferative activity-either as themselves or as carriers.

The Evaluation of Pharmacodynamics and Pharmacokinetics of Anti-thrombin DNA Aptamer RA-36.

Anticoagulants are a vital class of drugs, which are applied for short-term surgical procedures, and for long-term treatments for thrombosis prevention in high risk groups. Several anticoagulant drugs are commercially available, but all have intrinsic disadvantages, e.g., bleeding risks, as well as specific ones, e.g., immune response to peptide/protein drugs. Therefore, the search for novel, efficient and safe anticoagulants is essential. Nucleic acid aptamers are an emerging class of contemporary pharmaceuticals which are fully biocompatible and biodegradable; they have low toxicity, and are as efficient as many protein-based drugs. The anti-thrombin DNA aptamer RA-36 has been created using a combination of rational design and molecular dynamics, showing several extra-features over existing aptamers. Aptamer RA-36 has a bimodular structure; the first G-quadruplex binds and inhibits thrombin, whereas the second G-quadruplex varies the properties of the first. This bimodular structure provides a favorable dose-effect dependence allowing the risk of bleeding to be potentially decreased. Here, the results of efficiency trials of the aptamer are presented. The aptamer RA-36 has a distinctive species specificity; therefore, the careful selection of experimental animals was required. The anticoagulant activity was characterized in rats and monkeys in vivo. Antithrombotic activity was evaluated in the live murine model of the induced thrombosis. Pharmacokinetics was estimated by tracking radionuclide labeled aptamer in rats. The aptamer was thoroughly characterized using bivalirudin as a reference drug. Despite the different profiles of anticoagulant activity, these two compounds could refer to each other, and the corresponding doses could be estimated. Bivalirudin turned out to have 10-fold higher anticoagulant and antithrombotic activity. The difference in activity is easy to explain due to the pharmacokinetic profiles of the substances: the aptamer RA-36 has 20-fold faster elimination from blood with a half-life of 1 min. The entire dataset revealed that the non-modified DNA aptamer could be an alternative to the currently used bivalent peptide inhibitor; the dosage profile could be improved by manipulating aptamer pharmacokinetics. The study has revealed aptamer RA-36 to be one of the most promising candidates for further development as a new generation of anticoagulants.

Evaluation of antithrombotic activity of thrombin DNA aptamers by a murine thrombosis model.

Aptamers are nucleic acid based molecular recognition elements with a high potential for the theranostics. Some of the aptamers are under development for therapeutic applications as promising antithrombotic agents; and G-quadruplex DNA aptamers, which directly inhibit the thrombin activity, are among them. RA-36, the 31-meric DNA aptamer, consists of two thrombin binding pharmacophores joined with the thymine linker. It has been shown earlier that RA-36 directly inhibits thrombin in the reaction of fibrinogen hydrolysis, and also it inhibits plasma and blood coagulation. Studies of both inhibitory and anticoagulation effects had indicated rather high species specificity of the aptamer. Further R&D of RA-36 requires exploring its efficiency in vivo. Therefore the development of a robust and adequate animal model for effective physiological studies of aptamers is in high current demand. This work is devoted to in vivo study of the antithrombotic effect of RA-36 aptamer. A murine model of thrombosis has been applied to reveal a lag and even prevention of thrombus formation when RA-36 was intravenous bolus injected in high doses of 1.4-7.1 µmol/kg (14-70 mg/kg). A comparative study of RA-36 aptamer and bivalirudin reveals that both direct thrombin inhibitors have similar antithrombotic effects for the murine model of thrombosis; though in vitro bivalirudin has anticoagulation activity several times higher compared to RA-36. The results indicate that both RA-36 aptamer and bivalirudin are direct thrombin inhibitors of different potency, but possible interactions of the thrombin-inhibitor complex with other components of blood coagulation cascade level the physiological effects for both inhibitors.