Telomerase inhibition by new di-and trisubstituted acridine derivatives

V. V. Negrutska, I. V. Saraieva, V. G. Kostina © 2016 V. V. Negrutska et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited UDC 577.151.042+547.8


Introduction
Telomerase is a unique reverse transcriptase synthesizing the telomeres using its own RNA component as a template and single-stranded telomeric DNA as a primer [1].This enzyme is responsible for maintaining the telomere length and re-synthesizes the telomeric sequences partially lost upon each cellular division.Telomerase is active in a majority of tumor cells, which results in their immortalization.Increased telomerase activity was detected in 85-90 % of human tumors [2].In contrast, it is inactive in normal somatic cells.So telomerase inhibitors have a significant potential as anticancer drugs [2,3].
Guanine-rich telomeric DNA sequences can fold into specific four-stranded structures called G-quad ruplexes (G4) formed by the stacks of guanine quartets linked by non-canonical systems of hydrogen bonds [4][5][6].Compounds able to specifically bind and stabilize G4 structures were found to inhibit telomerase and demonstrated antitumor activity.A va riety of compounds, mostly containing a large heteroaromatic core, were shown to be G4-stabilizing ligands [4,6,7].
A number of known telomerase inhibitors are based on acridine scaffold.Most of them contain pairs of basic substituents, e.g. at C-3 and C-6 or C-4 and C-5 positions of the ring, and quite often an aromatic fragment at C-9 [4,[6][7][8][9][10][11].In this work we have studied a series of novel 4,9-di and 4,5,9-trisubstituted acridines as potential telomerase inhibitors.Their biological testing was performed using the modification of classic in vitro TRAP (Telomeric Repeat Amplification Protocol) assay [12].

Materials and Methods
Acridone carboxamides 1, 2 were obtained as previously described [13].Acridine derivatives with aliphatic basic 9-substituents were prepared from these compounds or their 5-methyl-substituted analogues (synthetic details will be reported in a separate paper).

Short Communication
TRAP analysis was performed according to TRAPeze protocol [TRAPeze ® Telomerase Detection Kit.Chemicon International, Temecula, CA, USA, 2005.39 p.] with deoxyoligonucleotide primers 5´-GTGCCCTTACCCTTACCCTTACCCTAA (CXext) [15] and 5´-AATCCGTCGAGCAGAGTT (TS) from Eurogentec (Belgium).Inhibitors were tested in a concentration range from 40 to 0.625 µM using a serial 2-fold dilution.After the telomerase extension reaction the tested compounds were removed from the mixture by phenol/chloroform extraction followed by ethanol precipitation.PCRamplified DNA products were analyzed by gel electrophoresis.20 µL sample was mixed with 2 µL of loading buffer (5x TBE buffer, 20 % sucrose, 0.2 % bromophenol blue) and loaded onto a standard 12 % non-denaturing polyacrylamide gel (19:1) prepared in 0.5x TBE.Electrophoresis was run for 0.5 h at 160 V and then for 0.5 h at 300 V. Gel was stained with 1x SYBR Green I for 30 min at room temperature.DNA bands were visualized on a ChemiDoc imager (Bio-Rad, USA), and images were processed with TotallLab v2.01 software (Nonlinear Dynamics, UK).
Inhibition activity was determined by comparing the amount of DNA produced in the test assay with that in a control reaction with no inhibitor added.For quantitative analysis, optical densities of the sets of DNA product bands on corresponding lanes of the gel were integrated.IC 50 values (compound concentrations required for achieving 50 % inhibition of enzymatic activity) were obtained from the concentration-activity plots.At least three independent experiments were performed for each compound.

Results and Discussion
We have recently found that acridone derivatives containing pyridyl groups at 4-carboxamide fragment inhibit telomerase at 100 µM concentration.Molecular modeling suggested the introduction of additional phosphate-binding group in these compounds to improve their G4 binding and thus inhibition activity [16].A series of new acridine derivatives with two aliphatic and aromatic basic fragments were synthesized.The structures of potential inhibitors (Table ) were designed taking into account that strongly basic group like N,N-dimethylami noal kyl introduced at C-9 position is easily protonated in water and thus could interact with G4 phosphate ani ons enhancing the ligand binding.In addition to phosphate binding, the basic pyridyl function at 4-carboxamide was expected to form hydrophobic bonds with G4 DNA bases further increasing the ligand affinity.
The level of telomerase inhibition was determined in TRAP assay.In this 2-step method [12], telomerase enzymatic reaction is followed by PCR amplification of its DNA products and analysis of the resulting reaction mixture by gel electrophoresis.Tumor cell lysate is used as the source of telomerase.We have employed TRAPeze protocol from Chemicon [TRAPeze ® Telomerase Detection Kit.Chemicon International, Temecula, CA, USA, 2005.39 p.], a well-established modification of the original method [12].The inhibitors were removed from the reaction mixture before the PCR step to exclude their possi- ble effect on Taq-DNA-polymerase.Alternative reverse PCR primer CXext developed to minimize analysis artifacts [15] was used instead of the standard CX.
The compounds were tested in a concentration range 40-0.625 µM (Fig. 1).The activity plots built by calculating the yield of enzymatic reaction products as a function of drug concentration allowed determining the IC 50 values of inhibitors (Table ).
The test results confirmed the idea of structure design to be correct.9-Acridone derivatives 1-2 with o-and m-pyridyl fragments were relatively inactive as telomerase inhibitors.However, the activity of all 9-substituted analogues bearing strongly basic functions sharply increased.The inhibition data indicated a higher activity of the compounds with N,N-di methylaminopropyl residue (5, 6) as compared to their analogues containing a shorter Me 2 NCH 2 CH 2 group (3, 4).As expected, electron-donating 5-me thyl substituent significantly enhanced the biological activity (compare the IC 50 values of compounds 7 and 5, 8 and 6), perhaps due to increasing the electronic density on acridine core which resulted in its more efficient π-π stacking with G4.

Conclusions 4 , 9 -
and 4,5,9-substiuted acridines containing two basic DNA-binding moieties efficiently inhibit telome rase in vitro at low micromolar concentrations.Trisubstituted derivative 7 with IC 50 = 2.6 µM is the most active inhibitor.The key structural feature determining the activity of compounds is a presence of highly basic N,N-dimethylaminoalkyl group at C-9 position.Further optimization would allow the development of more potent acridine-based telo merase inhibitors.