Changes induced in mouse lipid metabolism by simultaneous impact of antisense oligonucleotide derivatives to <i>apoB</i>, <i>PCSK9</i>, and <i>apoCIII</i> mRNAs

Development of new drugs able to decrease the level of “bad” cholesterol, in particular, based on antisense oligonucleotide derivatives (ASOs), remains relevant for the patients with familial hypercholesterolemia and/or intolerant to statins. The goal of the work was to assess the changes in the lipid metabolism caused by variants of joint impact of the ASOs targeted to the mRNAs of its key genes: apoB, PCSK9, and apoCIII. Female C57BL/6J mice; nuclease-protected 13- and 20-nucleotide ASOs, and standard protocols for quantification of lipoproteins (HDL CHL, non-HDL CHL, and total CHL) and ALT in the blood serum were used in the work. The following combinations of ASOs were four times injected to the mouse caudal vein: 1) ASO to apoB, 2) ASO to apoCIII, 3) ASO to apoB and ASO to PCSK9, 4) ASO to apoB, ASO to PCSK9, and ASO to apoCIII, 5) ASO to apoB (three doses), ASO to PCSK9, and ASO to apoCIII (two doses), 6) ASO to PCSK9 and (ASO to apoCIII – only in the fourth administration). Triple weekly administration of these ASO combinations resulted in a decrease in non-HDL CHL by 25, 16, 35, 47, 60, and 7 %, respectively, as compared with the control and 1.8-, 1.5-, 1.9-, 2.4-, 3.1, and 1.24-fold higher HDL CHL/ non-HDL CHL ratio. The subsequent ASO injection with concurrent switching to a high-fat diet after 1 week resulted in a decrease in the non-HDL CHL by 28, 2, 28, 70, 33, and 49 % for ASOs (1–6), respectively, as compared with the control; the HDL CHL/non-HDL CHL ratio was 1.5-, 1.1-, 2-, 3.7-, 1.9-, and 2-fold better. The ALT concentration for all ASO combinations remained within the norm for the control animals, demonstrating the absence of any hepatotoxic effect. The best efficiency of ASOs requires selection of concentrations for single ASOs and their combinations as well as of the order and timing of administration. Thus, a new antisense approach is proposed

Imatinib mesylate (Gleevec) downregulates telomerase activity and inhibits proliferation in telomerase-expressing cell lines

Imatinib mesylate (IM) is a tyrosine kinase inhibitor, which inhibits phosphorylation of downstream proteins involved in BCR-ABL signal transduction. It has proved beneficial in treating patients with chronic myeloid leukaemia (CML). In addition, IM demonstrates activity against malignant cells expressing c-kit and platelet-derived growth factor receptor (PDGF-R). The activity of IM in the blastic crisis of CML and against various myeloma cell lines suggests that this drug may also target other cellular components. In the light of the important role of telomerase in malignant transformation, we evaluated the effect of IM on telomerase activity (TA) and regulation in various malignant cell lines. Imatinib mesylate caused a dose-dependent inhibition of TA (up to 90% at a concentration of 15 μM IM) in c-kit-expressing SK-N-MC (Ewing sarcoma), SK-MEL-28 (melanoma), RPMI 8226 (myeloma), MCF-7 (breast cancer) and HSC 536/N (Fanconi anaemia) cells as well as in ba/F3 (murine pro-B cells), which do not express c-kit, BCR-ABL or PDGF-R. Imatinib mesylate did not affect the activity of other DNA polymerases. Inhibition of TA was associated with 50% inhibition of proliferation. The inhibition of proliferation was associated with a decrease in the S-phase of the cell cycle and an accumulation of cells in the G2/M phase. No apoptosis was observed. Inhibition of TA was caused mainly by post-translational modifications: dephosphorylation of AKT and, to a smaller extent, by early downregulation of hTERT (the catalytic subunit of the enzyme) transcription. Other steps of telomerase regulation were not affected by IM. This study demonstrates an additional cellular target of IM, not necessarily mediated via known tyrosine kinases, that causes inhibition of TA and cell proliferation

A route to RNA with an alkylating group at the 5'-triphosphate residue.

Reaction of ATP with N,N,N'-tris(2-chloroethyl), N' (p-formyl-phenyl)propylenediamine-1,3 (abbreviation C13R) afforded a gamma-ester of ATP (abbreviation C1RpppA) - the product of alkylation by an aliphatic nitrogen mustard residue of C13R. The alkylating activity of the aromatic nitrogen mustard residue of C1RpppA is suppressed by the electron-acceptor effect of the p-formyl group. C1RpppA is a substrate of RNA-polymerase, and affords RNA with C1RpppA-residues at the 5'-termini