Treated or untreated A549 cells . After hypotonic swelling in 75 mM KCl, cells were fixed and stored in ethanol acetic acid. Before hybridization, cells were dropped on slides and dried overnight. After washing with PBS, slides were denatured by formamide in 4 SSC at 75 for 3 min, then dehydrated in ice cold ethanol MLN8237 Alisertib and air dried. Hybridization mixture containing 50% formamide, 10% dextran sulfate, 4 SSC, 0.25% blocking reagent, 0.6 g ml Cy3 conjugated probe was added to the slide, covered with a coverslip, and followed by DNA denaturation. After hybridization for 12 h at 37, slides were washed with 50% formamide and 2 SSC, then with 0.1 SSC and with 4 SSC containing 0.05% Tween 20. Slides were dehydrated with ethanol, air dried, and covered by antifade solution containing 4,6 diamidino 2 phenylindole.
Modified Cy3 3 telomeric probe containing 2 OMe ribose sugars and 5 pyrimidine residues was synthesized by Eurogentec. Images were acquired by using a Nikon Microphot microscope. Telomeric spots were analyzed on at least six individual metaphases and results were expressed as percent of chromosomes that contain 0, 1, 2, 3, or 4 detectable telomeres. Results and Discussion A number of small molecules have been discovered to inhibit the function of telomerase by stabilizing G4 DNA structures. Triazines were compared at 1 M dye concentration, and the results are summarized on Fig. 1C. The best ligand 1150405 gave a Tm of 20, followed by 12459, which gave a Tm of 8, whereas compounds 5271 and 5352 did not stabilize the G quadruplex.
Similar Tm values were obtained for these molecules when a G4 FRET assay was performed in the presence of a hundred fold excess of double stranded DNA, indicating a good specificity for G quadruplexes. This specificity was confirmed by equilibrium dialysis experiment. The stabilization obtained with 115405 compares favorably with all G4 ligands tested so far. The Tm effect was compared with telomerase inhibition in vitro. The 115405 and 12459 compounds displayed potent telomerase inhibitory activity when evaluated in a telomerase TRAP assay. Quinoline substituted triazines, such as 115405 and 12459, were the most efficient inhibitors with IC50 of 41 nM and 130 nM, respectively, whereas 5271 and 5352 were found to be weakly active and inactive, respectively, in good agreement with the absence of G quadruplex stabilization.
Judging from telomerase inhibition and FRET assays, the two active compounds are considered to be very efficient inhibitors. In general, a good correlation was found between G quadruplex stabilization potency and telomerase inhibition among the one hundred triazine derivatives synthesized. To discriminate between telomerase elongation inhibition and Taq polymerase inhibition during the amplification steps of the assay, the compounds were tested independently with Taq polymerase and a DNA substrate unable to fold into G quadruplexes. Taq polymerase was inhibited but at higher concentrations. IC50s for Taq inhibition were 610 nM and 8400 nM for 115405 and 12459, respectively. Inclusion of an internal control to the TRAP assay also confirmed these results. No inhibition of ITAS was detected up to 3 M.